The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell-type-specfic target genes

The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell-type-specfic target genes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell-type-specfic target genes Naman Gupta, Mallika Somayajulu, Katherine Gurdziel, Giovanni LoGrasso, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3678621/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2024 Read the published version in Scientific Reports → Version 1 posted 3 You are reading this latest preprint version Abstract The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contribute to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3’RNA sequencing (Seq) in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation. Biological sciences/Cell biology Biological sciences/Computational biology and bioinformatics Biological sciences/Immunology Biological sciences/Molecular biology Biological sciences/Neuroscience Biological sciences/Systems biology Health sciences/Medical research/Experimental models of disease Health sciences/Diseases/Eye diseases/Corneal diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The cornea is an avascular, transparent tissue between the eye and the external environment. It provides visual clarity and two thirds of the refractive power of the eye 1–3 . It also serves as first-line defense against microbial infection and other insults 1–3 . Multiple cell types of different origin, including corneal epithelium, stromal keratocytes, endothelial cells, resident immune cells (CRICs) and nerves 1–4 , works in sync to confer the unique architecture and functionalities of the cornea 1–4 . Although rare (1.2–5% in mouse 5–7 ), CRICs are a diverse population and have essential roles in almost every aspect of the development and functions of the cornea under both physiological and pathological conditions 8–16 . The cornea is the most densely sensory-innerved tissue in the body 17–19 . Sensory innervation of the cornea not only provides bodily sensation to various stimuli but also plays important roles in the homeostasis of the cornea as well as the pathogenesis of corneal diseases through neuroimmune interactions 18–33 . microRNAs (miRNAs) are small, non-coding RNAs and are post-transcriptional regulators of gene expression 34–37 . They play an important role in human diseases 38–45 and are viable therapeutic targets 46–49 . However, their roles in the neuroimmune interaction in health and diseases of the cornea are still largely unknown. The evolutionarily-conserved, paralogous miRNA cluster, miR-183/96/182 cluster (referred to as the miR-183C from here on) is highly, specifically expressed and required for the normal development and functions of sensory neurons 50–52 . Inactivation of miR-183C in conventional knockout (KO) mouse models results in multisensory defects 51,53–55 . Point mutations in the seed sequence of miR-96 results in non-syndromic hearing loss in both mouse 56 and human 57 . In addition, this miRNA cluster is also expressed and plays important roles in both innate 4,7,58–62 and adaptive immune cells 63–70 . In the cornea, we demonstrated that inactivation of miR-183C in a conventional KO mouse model results in decreased nerve density and reduced expression of capsaicin receptor TRPV1 and pro-inflammatory neuropeptide substance P (sP) precursor gene Tac1 60 . Inactivation or knockdown of miR-183C in innate immune cells, e.g. Mφ and neutrophils, reduces their production of pro-inflammatory cytokines, however, enhances their phagocytosis and intracellular bacterial killing capacity 60,61 . These result in a reduced inflammatory response to bacterial infection, e.g. Pseudomonas aeruginosa (PA) and contribute to a decreased severity of PA keratitis 60,61,71 . In addition to its effect on peripheral Mφ and neutrophils, we showed that inactivation of the miR-183C results in increased number of steady-state CRMCs in naïve mice 4,7 and myeloid cell infiltration into the cornea in the event of PA infection 7 . Collectively, these findings suggest a pivotal regulatory role for miR-183C in fine-tuning neuroimmune interaction in the cornea. However, since in the conventional KO mice, miR-183C is simultaneously inactivated in all miR-183C-expressing cells, including both CSN and CRMCs 7,60 , the phenotype observed reflects a composite effect of loss-of-function of miR-183C in both cell types. This precludes distinguishing whether the changes observed in CSN and CRMCs are intrinsic functions of miR-183C in sensory nerves and myeloid cells, respectively, or an extrinsic effect through neuroimmune interaction. To resolve this conundrum, we produced sensory neuron-specific (SNS) and myeloid cell-specific (MS) conditional knockout mice (CKO). Here we report the first characterization of these CKO mice in comparison to their corresponding wild type (WT) littermate control mice at their steady state. Our results reveal sensory neuron- and myeloid cell-specific functions of miR-183C and their impact on the homeostasis of the cornea. Methods Mice. All experiments and procedures involving animals and their care were pre-reviewed and approved by the Wayne State University Institutional Animal Care and Use Committee and carried out in accordance with National Institute of Health and Association for Research in Vision and Ophthalmology (ARVO) guidelines (Approved protocol number: IACUC-22-05-4618). The study is reported in accordance with ARRIVE guidelines. Euthanasia was performed by cervical dislocation under anesthesia with isoflurane followed by thoracotomy. The miR-183C KO – the miR-183C GT/GT mice, are on a 129S2/BL6-mixed background 51 and were originally derived from a gene-trap (GT) embryonic stem cell clone 51,72,73 . Csf1r-EGFP or MacGreen mice 74 were purchased from the Jackson lab (Stock number. 018549). In this strain, the EGFP transgene is under the control of the 7.2-kb mouse colony stimulating factor 1 receptor (Csf1r ) promoter, allowing specific expression of EGFP in the mononuclear phagocyte system (MPS) myeloid cells, including monocytes, macrophages and dendritic cells 74,75 . The Csf1r-EGFP mice were bred with miR-183C GT/+ to produce miR-183C KO [Csf1r-EGFP(+);miR-183C GT/GT ] and WT mice [Csf1r-EGFP(+);miR-183C +/+ ] on the background of Csf1r-EGFP as described previously 7 . Mice with miR-183C CKO allele, the miR-183C f/f , were provided by Dr. Patrick Ernfors, Karolinska Institutet, Sweden through the European Mouse Mutant Archive (EMMA. ID: EM12387). The miR183C f allele has two loxP sites flanking the 5’ and 3’ ends of the miR-183C for robust Cre-mediated miR-183C CKO 76 . The myeloid-specific, LysM-Cre mice 77 were purchased from The Jackson Laboratory (Stock number: 004781). The LysM-Cre knock-in/knock-out allele has a nuclear-localized (NLS)-Cre recombinase inserted into the first coding ATG of the lysozyme 2 gene ( Lyz2 ), both abolishing endogenous Lyz2 gene function and placing NLS-Cre expression under the control of the endogenous Lyz2 promoter/enhancer elements. Therefore, the LysM-Cre allows myeloid cell-specific expression of Cre recombinase. The sensory nerve specific Nav1.8-Cre mice 78 were kindly provided by Dr. John N. Wood, University College London, through Dr. Theodore J. Price, University of Texas Dallas. This strain is now available at the Jackson Laboratory (Stock Number: 036564). The voltage-gated sodium channel Na v 1.8 (encoded by the Scn10a gene) is one of the signature genes of the majority of nociceptive sensory neurons in the TG and dorsal root ganglia (DRG) 33,79–81 . Na v 1.8 promoter-driven Cre recombinase (Na v 1.8-Cre) is expressed in nearly all corneal nociceptive sensory nerves 33,78,82 . The reporter strain, R26 LSL − RFP(+/+) mice 83 , also known as Ai14 (Stock number: 007914, the Jackson Laboratory) has a loxP-flanked STOP cassette (LSL) in front of a tdTomato RFP cassette, all of which are inserted into the ROSA26 locus 83 . The LSL prevents the transcription of tdTomato RFP; however, when Cre recombinase is present, the LSL cassette will be excised to allow the expression of tdTomato RFP. miR-183C MS-CKO mice [LysM-Cre(+/-);miR-183C f/f ;Csf1r-EGFP(+/+)], in which MPS myeloid cells are labeled with EGFP, were produced by breeding of the LysM-Cre(+/-), miR-183C f/f and Csf1r-EGF(+/-) mice. Their littermates [LysM-Cre(-/-);miR-183C f/f ;Csf1r-EGFP(+/+)] are used as WT mice. SNS-CKO mice [Nav1.8-Cre(+/-);miR-183C f/f ;R26 LSL − RFP(+/+) ;Csf1r-EGFP(+/+)] and their WT littermates [Nav1.8-Cre(+/-);miR-183C +/+ ;R26 LSL − RFP(+/+) ;Csf1r-EGFP(+/+)] were produced by breeding of Nav1.8-Cre (+/-), miR-183C f/f , R26 LSL − RFP and Csf1r-EGF(+/-) mice. All breeding showed a normal mendelian inheritance pattern. Sex is considered as a biological variance in all studies. 8–12 weeks old, male and female mice were used as separate groups in all experiments. The age, sex and number of the mice in each experiment are specified in the figure legends and/or the text. Hematoxylin and eosin (H&E) staining of paraffin sections of mouse cornea . Mouse eyes were enucleated and fixed in 4% formalin. The embedding, sectioning and H&E staining were performed by Excalibur Pathology Inc (Oklahoma City, OK). Comparable sagittal sections through the optic nerves were imaged under a DM4000b brightfield microscope (Leica). Fluorescence-activated cell sorting (FACS). 8–12 weeks old, naïve MS-CKO and KO mice and age- and sex matched WT littermate controls were used to isolate Csf1r-EGFP + myeloid cells from the cornea and spleen for DNA and RNA preparations (see below). For corneal samples, corneas anterior to the limbi from 6–7 mice/genotype were carefully dissected and pooled for single cell preparation as described before 4,7 . For spleen cells, mononuclear cells were isolated following a standard protocol 84 . FACS was conducted on a Sony SY3200 cell sorter at the Microscopy, Imaging and Cytometry Resources Core (MICR), Wayne State University (WSU) to isolate Csf1r-EGFP + myeloid cells as we described previously 4,7 . EGFP- corneal and spleen mononuclear cells were used as negative controls to optimize the gating. DNA and genotyping PCR. Genomic DNA was prepared from mouse tail, cornea, TG, FACS-sorted Csf1-EGFP + myeloid cells using the gMax DNA mini kit (IBI Scientific) following manufacturer’s instruction as described previously 85 . Subsequently, DNA concentration and quality were assayed on the Nanodrop 2000 (ThermoFisher Scientific). 100 ng (from mouse cornea, TG and tails) or 4 ng (from FACS-sorted myeloid cells of the cornea and spleen) genomic DNA was used for genotyping PCR to detect tissue and/or cell type-specific recombination at genomic DNA level as we described previously 51,85 . PCR of 18s rRNA was amplified and used as a loading control. The genomic structure and primers are illustrated in Fig. S1 . The sequences of the primers are: 7Fintron1: 5’-TACCCTGAGTGTGTCTCAATC-3’; CKO-R: 5’-GCAGAGTCACAAACATGTGTAGC-3’; 18s rRNA Forward: 5’-GTAACCCGTTGAACCCCATT-3’; 18s rRNA Reverse: 5’-CCATCCAATCGGTAGTAG CG-3’. RNA preparation and qRT-PCR. Total RNA was prepared using the miRVana miRNA isolation kit (Life Technologies, Foster City, CA, USA) or the RNeasy (Qiagen, Frederick, MD, USA) for miRNA or mRNA studies, respectively, as described previously 50,86,87 . qRT-PCR for miRNAs was performed using Taqman miRNA primers and RT-PCR kit (Life Technologies) with snRNA U6 as an endogenous control as described before 50,87 . For protein-coding genes, qRT-PCR was performed using a QuantiFast SYBR Green RT-PCR kit and QuantiTect primers (Qiagen) with 18s rRNA as endogenous controls 50,51,87 . Tear volume measurement. Tear volume was measured by the Zone-Quick Phenol red thread (PRT) test (Yokota Co. Ltd). Manufacturer’s instruction was followed with modifications. Briefly, under light anesthesia by isoflurane, the lower eyelid was pulled down slightly; the PRT is placed between the palpebral conjunctiva of the lower eyelid and the eyeball at a point approximately 1/3 of the distance from the lateral canthus for 15 seconds. The length of the thread which turned red was measured. Corneal sensitivity test . Corneal sensitivity to mechanical stimuli was measured by a blink threshold test using a Cochet and Bonnet aesthesiometer (Western Ophthalmics). Briefly, the tip of a nylon filament, starting from 6 cm, was applied perpendicularly to the central cornea. The length at which the mouse blinks was registered as the blink threshold. Corneal flatmount, immunofluorescence (IF), confocal microscopy, and quantification of Csf1r-EGFP + corneal resident MPS cells and corneal nerve density. Corneal flatmount and IF was performed as described previously 7,71 . Briefly, mice were euthanized; eyes were enucleated and transferred to cold phosphate buffered saline (PBS). Under a dissecting scope (VWR International, Radnor, PA), the cornea anterior to the limbus was carefully dissected out. The corneas were transferred to cold 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4 for 1 h at 4°C. For direct confocal microscopy, the cornea was flattened by six evenly spaced cuts from the periphery toward the center and mounted in Vectashield media with DAPI (Vector Laboratories, Burlingame, CA) on Superfrost Plus slides (Fisherbrand). For IF, after fixation, the corneas were incubated in a blocking buffer with 2% normal goat serum (NGS) (Vector labs) in PBS for 30 min at room temperature (RT); then, permeabilized with 0.1% Triton X-100 in the blocking buffer for 30 min at RT. Subsequently, corneal tissues were incubated with mouse anti- β -Tubulin III (1/600 dilution. Cat. No. 801201, BioLegend, San Diego, CA, USA) antibodies in the blocking buffer for 72 hours at 4°C. After washes with PBS, the corneas were incubated with AF546–conjugated goat anti-mouse IgG (1/1000 dilution. Cat. No. A-11003, Thermo Fisher Scientific) for overnight at 4°C. After washing, the corneas were flattened and mounted on slides. Negative controls were treated similarly with omission of the primary antibody. All slides were imaged using a TCS SP8 laser confocal microscope (Leica Microsystems Inc. Buffalo Grove, IL). To capture the images of the entire cornea, a series of Z-stacked images under 10x objective were taken across the entire cornea and stitched together. These stitched Z-stacked images were merged/flattened for cell counting or nerve density quantification using Adobe Photoshop CS6 (64 bit) and ImageJ 1.52p software ( http://imagej.nih.gov/ij . NIH, Bethesda, MD, USA) as described previously 7 . Briefly, the raw image was first converted to the 16-bit grayscale; then the black & white binary image is optimized for the threshold to faithfully represent the original image and the cell density. The EGFP + cells in the entire cornea were counted. To quantify nerve density, the mean value of pixels in a 500-µm 2 square covering the center of the whorl-like subbasal plexus was quantified as the nerve density of the center; the ones in three 500-µm 2 squares randomly placed in the periphery of the cornea were measured, the average of which was recorded as nerve density of the peripheral cornea. Corneas from at least n = 3 mice/sex/genotype were quantified. 3’ RNA sequencing and data analysis : mRNAs were isolated from TG, corneas or FACS-sorted Csf1r-EGFP + myeloid cells of young adult (8–12 weeks old) male mice. For miR-183C KO strain, n = 3 for both KO and WT controls; for SNS-CKO strain, n = 3 for SNS-CKO mice, n = 4 for WT controls; for MS-CKO strain, n = 5 for both MS-CKO and WT controls were used. To isolate Csf1r-EGFP + myeloid cells, 12 corneas of 6 KO and 14 corneas of 7 WT control mice or 20 corneas of 10 MS-CKO or 10 WT control mice were pooled for FACS isolation. Subsequently, 3’ mRNA-Seq libraries were prepared by the Genome Sciences Core (GSC), WSU using the QuantSeq 3’ mRNA-Seq Library Prep kit FWD (Lexogen, Greenland, NH) 88 . High sensitivity D1000 ScreenTape peak range between 100–700 bp were in accordance with Lexogen guidelines. The libraries were sequenced on NovaSeq sequencer (Illumina). Reads were aligned to the mouse genome (Build mm10) using a public available software STAR 89 and tabulated for each gene region using HTSeq 90 . Differential expressed genes (DEGs) between KO or CKO vs WT controls were identified by edgeR 91 . miR-183C predicted target genes were identified by TargetScan algorithm (Targetscan.org) 92–96 in the upregulated genes in CKO or KO vs WT control and are recognized as miR-183C target genes in these tissues. Statistical significance of enrichment of miR-183C targets in upregulated genes was analyzed by Chi-square with Yates’ correction using the Analyze a 2x2 Contingency Table (GraphPad. https://www.graphpad.com/quickcalcs/contingency1/ ). Functional annotation analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) 97 as described before 98 . Statistical analysis. When the comparison was made among more than 2 conditions, one-way ANOVA with Bonferroni’s multiple comparison test was employed (GraphPad Prism); adjusted p < 0.05 was considered significant. Otherwise, a two-tailed Student’s t test was used to determine the significance; p < 0.05 was considered significant. Each experiment was repeated at least once to ensure reproducibility and data from a representative experiment are shown. Quantitative data is expressed as the mean ± SEM. Results Sensory nerve- and myeloid cell-specific knockout of miR-183C results in no major morphological changes of the cornea. To uncover tissue-specific functions of the miR-183C, we produced sensory neuron- and myeloid cell-specific miR-183C CKO mice ( Fig. S1 ). To detect whether inactivation of miR-183C in sensory nerves and/or myeloid cells causes major histological and morphological changes in the cornea, we performed H&E staining in cross-sections of the corneas of SNS-CKO, MS-CKO and KO mice and their WT control littermates. No gross morphological changes were observed in the cornea of miR-183C SNS-CKO, MS-CKO and conventional KO mice, when compared to their WT controls (Fig. 1 ). The total thickness (Fig. 1 ) and thickness of the epithelial and stromal layers (not shown) had no significant differences between the CKOs and KO vs their age- and sex-matched WT controls. Inactivation of miR-183C in either sensory neurons or myeloid cells results in increased number of CRMCs Quantification of Csf1r-GFP + CRMCs of young adult, naïve mice showed that the numbers of Csf1r-EGFP + resident myeloid cells in MS-miR-183C CKO mice [3278 ± 268/cornea (n = 3) in male; 2234 ± 80/cornea (n = 3) in female] were significantly increased in both male and female mice, when compared to age-matched WT controls [1697 ± 225/cornea (n = 3) in male, 1507 ± 107/cornea (n = 3) in female] (Fig. 2 A), suggesting miR-183C intrinsically regulates the number of CRMCs under homeostatic condition. In WT controls, male and female mice showed no difference in the number of CRMCs, however, in MS-CKO, the male vs female mice showed increased number of CRMCs (Fig. 2 A), suggesting a potential sex-related modulation. To test whether miR-183C in CSN has an extrinsic regulation on the CRMC population, we quantified the number of CRMCs in the SNS-CKO mice. Intriguingly, sensory nerve-specific inactivation of miR-183C also resulted in an increased number of CRMCs when compared to age- and sex-matched WT controls (Fig. 2 B). In male, it was increased by ~ 35% [3197 ± 151/cornea in SNS-CKO (n = 3) vs 2335 ± 121/cornea in WT controls (n = 4)]; while in the female by ~ 56% in female [3197 ± 151/cornea in SNS-CKO (n = 3) vs 2772 ± 298/cornea in WT controls (n = 4)](Fig. 2 B). The numbers of CRMCs showed no significant differences between male and female in both WT or SNS-CKO mice. Inactivation of miR-183C in sensory neurons, but not in CRMCs, results in decreased sensory nerve density in the epithelial layer. Previously, we showed that corneal nerve density is significantly decreased in the miR-183C KO mice 60 . To dissect the contribution of miR-183C in corneal sensory nerves or resident myeloid cells to this phenotype, we quantified corneal nerve density in the SNS-CKO and MS-CKO mice. β-III tubulin IF of flatmount cornea showed no difference in corneal nerve density of the MS-CKO vs their WT littermate control mice in both male and female (Fig. 3 A), suggesting loss of miR-183C in CRMCs had no contribution to the decreased corneal nerve density in the miR-183C KO mice 60 . However, the corneal nerve density was significantly decreased in the SNS-CKO vs WT littermate control mice in both male and female (Fig. 3 B), in both the whorl center and peripheral regions of the cornea, suggesting that decreased nerve density in the miR-183C conventional KO mice 60 is a result of loss of miR-183C in corneal sensory nerves; miR-183C imposes an intrinsic regulation of corneal sensory innervation. To further probe into the reduction of sensory nerve density, we quantified the sensory nerve density in the epithelial and stromal layers separately (Fig. 4 ). The layer-specific analyses showed that, in both the whorl center and peripheral regions of male mice, it is the density of the fine terminal nerves in the epithelial layer that is significantly decreased, but not the large nerves in the stroma layer (Fig. 4 ), suggesting that miR-183C regulates the terminal differentiation and/or extension of sensory nerve in the epithelial layer of the cornea, but has no significant impact on the establishment of large sensory nerves in the stromal layer. Similar effect was observed in female mice ( Fig.S2 ). Inactivation of miR-183C in sensory nerves results in decreased corneal sensitivity to mechanical stimuli Since inactivation of miR-183C in sensory neurons resulted in decreased sensory nerve density (Fig. 3 & 4 ), we hypothesize that the functions of the sensory nerve are compromised in the cornea. To test this hypothesis, we first tested corneal sensitivity to mechanical stimuli using a Cochet and Bonnet aesthesiometer. Our result showed in both miR-183C KO (Fig. 5 A) and SNS-CKO mice (Fig. 5 B), corneal sensitivity was significantly decreased, when compared to their WT littermate. In the conventional KO, corneal sensitivity was decreased by ~ 23% and 33% in male and female mice, respectively. In the SNS-CKO, corneal sensitivity was significantly reduced by ~ 10 and 11% in male and female mice, respectively. However, in MS-CKO mice, inactivation of miR-183C showed no impact on corneal sensitivity. These data suggest that miR-183C in corneal sensory nerves intrinsically regulate its function. Decreased corneal sensitivity in miR-183C KO is a result of loss of miR-183C in the sensory nerve. Inactivation of miR-183C in sensory nerves results in decreased basal tear production It is known that corneal sensory innervation plays important role in regulating tear production 27,99 . Since inactivation of miR-183C in sensory neurons resulted in decreased sensory nerve density and sensitivity to mechanical stimuli (Fig. 3 – 5 ), we further hypothesize that miR-183C may play a role in tear production through its regulation of corneal sensory innervation. To test this hypothesis, we measured tear volumes using the PRT test. Our result showed that in both the miR-183C KO and SNS-CKO mice, tear volume was significantly decreased when compared to their WT littermates (Fig. 6 A&B). In male mice, the tear volume was decreased by ~ 81% in the miR-183C KO and ~ 71% in the SNS-CKO mice; while in female, it was decreased by ~ 55% and 59% in the conventional KO and SNS-CKO, respectively (Fig. 6 A&B). However, tear volume of MS-CKO mice showed no difference compared to their WT littermate controls (Fig. 6 C). These data suggest that miR-183C modulates basal tear production through regulation of corneal sensory innervation. In addition, our data showed that the tear volume in female vs male WT mice was consistently reduced in all three strains, by ~ 77%, 28% and 43% in the conventional KO, SNS-CKO and MS-CKO mice, respectively, suggesting a sex-dependent difference of tear production (Fig. 6 A-C). miR-183C modulates the functions of sensory neurons and myeloid cells through its regulation of cell type-specific target genes To start to understand the molecular mechanisms underlying miR-183C’s regulation of corneal functions, we performed 3’ mRNA sequencing 88,100 in the TG and corneas of young adult, naïve SNS-CKO, MS-CKO and miR-183C KO mice and their corresponding WT littermate controls. Comparison of the transcriptomes identified a series of DEGs in the TG and corneas of SNS-CKO, MS-CKO and miR-183C KO mice vs their WT controls ( Fig.S3; Tables S1-6 ). Since miRNAs negatively regulate gene expression 35 , to identify tissue-specific targets of miR-183C, we searched predicted target genes of miR-183C among the upregulated genes in KO or CKO vs their WT controls in both TG and cornea. This analysis yielded a series of tissue-specific target genes of miR-183C ( Tables S7-12 ). Statistical analysis showed that miR-183C predicted target genes are significantly enriched among the upregulated genes in the TG and cornea of SNS-CKO or KO mice vs their corresponding WT littermate controls. However, no enrichment of miR-183C predicted target genes was detected in the upregulated genes of either TG or cornea of miR-183C MS-CKO mice, possibly a result of the rarity of the CRMCs in the cornea 4,7 . Functional annotation analyses of the tissue-specific target genes identified in the TG and cornea of SNS-CKO and KO mice showed similar overlapping results ( Tables S13-20 ). Since miR-183C is predominantly expressed in the TG sensory neurons when compared to myeloid cells (> 50 folds. Fig.S4 ) and that CRMCs are a rare population in the cornea (1–3% of total corneal cells) 4,7 , we reasoned that gene expression changes and miR-183C targets identified in the TG and cornea of SNS-CKO and KO mice represents TG sensory neuron-specific changes and miR-183C targets. Gene ontology (GO) analysis of the tissue-specific target genes identified in the TG of SNS-CKO and KO mice ( Tables S13,S17,S21 ) showed significant enrichment in neuronal axon projection-, immune response- and epithelial cell proliferation and migration-related biological processes (Table 1 ). Consistent with the GO analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in axon guidance (Fig. 7 A), synaptic function pathways (Table 2 ; Tables S14,18,22 ). In the cornea, miR-183C target genes identified in the SNS-CKO and miR-183C KO mice ( Table S15,16,19,20,23,24 ) are prominently enriched in regulation of synaptic functions, in addition to axonogenesis and axon projection (Tables 3 & 4 ). These data collectively suggest that miR-183C regulates sensory neuronal projection and synaptic function through directly targeting genes involved in these biological processes. Besides neuronal related functions, miR-183C targets in TG sensory neurons are also enriched in epithelial cell proliferation and migration-, cell-cell adhesion-, fibroblast and endothelial cell migration-related biological processes, suggesting indirect regulation of miR-183C on other cell types in the cornea through its modulation of corneal sensory innervation (Table 1 – 4 ). Furthermore, genes involved in chemokine signaling pathway, e.g. CX3CL1, GNG5, GNG12, GNAI3, ADCY6, SHC1, BRAF, ELMO1, RAC1, MAP2K1, FOXO3 ( Fig. 7 B ) , leukocyte trans-endothelial migration, e.g. GNAI3, MSN, PRKCA, RAC1, and FcγR-mediated phagocytosis pathways, e.g. MAP2K1, PRKCE, PRKCA, RAC1, are also enriched in miR-183C target genes of TG sensory neurons (Tables 2 , 4 ), suggesting potential roles in neuropeptide production and neuroimmune interactions. Table 1 Neuronal, immune and epithelial-related GO terms enriched in miR-183C targets in TG sensory neurons GO ID Terms P Genes Fold Enrichment Neuronal related 0010976 positive regulation of neuron projection development 0.0023 EHD1, RGS2, ANKRD27, ITGA3, STMN2, FYN, CX3CL1 5.17 1902667 regulation of axon guidance 0.0428 NOVA2, MYCBP2 45.59 0031175 neuron projection development 0.0431 EHD1, GDNF, SHC1, STMN2, FYN 3.78 0007411 axon guidance 0.0508 KLF7, GAP43, MYCBP2, PAX6, ANK3 3.58 Immune related 0050729 positive regulation of inflammatory response 0.0355 CEBPA, CD47, LDLR, CX3CL1 5.53 0071560 cellular response to transforming growth factor beta stimulus 0.0003 ZEB1, NOX4, FYN, WNT2, TGFBR1, WNT4 10.39 0036120 cellular response to platelet-derived growth factor stimulus 0.0152 PRKCE, RASA1, FYN 15.78 Epithelial related 0010634 positive regulation of epithelial cell migration 0.0373 ITGA3, PRKCE, PPM1F 9.77 0050680 negative regulation of epithelial cell proliferation 0.0279 ZEB1, CELF1, PAX6, MTSS1 6.08 Table 2 Neuronal and immune-related KEGG pathways enriched in miR-183C targets in TG sensory neurons KEGG ID Term P Genes Fold Enrichment Neuronal related mmu04725 Cholinergic synapse 0.0124 GNA11, KCNJ14, FYN, GNG12, ADCY6 5.48 mmu04724 Glutamatergic synapse 0.0132 GRIA1, PPP3R1, SLC1A1, GNG12, ADCY6 5.39 mmu04722 Neurotrophin signaling pathway 0.0161 MAP3K3, SHC1, PSEN2, BRAF, FOXO3 5.08 mmu04360 Axon guidance 0.0574 PPP3R1, RASA1, NCK2, FYN, WNT4 3.39 Immune related mmu04062 Chemokine signaling pathway 0.0189 SHC1, BRAF, FOXO3, GNG12, CX3CL1, ADCY6 3.84 Table 3 Neuronal, cellular adhesion and migration-related GO terms enriched in miR-183C targets in the cornea of SNS-CKO and KO mice GO ID Term P Genes Fold Enrichment Neuronal function related 0010976 positive regulation of neuron projection development 3.21E-04 ANKRD27, FUT9, AVIL, DAB2IP, CNTN1, FYN, PRKD1, CX3CL1 6.16 2000300 regulation of synaptic vesicle exocytosis 0.002954 NRN1, SV2C, PRKCE, PRKCA, CACNA1E 8.38 0001764 neuron migration 0.004744 MEF2C, TRIM46, FYN, FAT3, RAC1, CELSR3 5.48 0048813 dendrite morphogenesis 0.005398 KLF7, ELAVL4, FYN, RAC1 11.18 0030030 cell projection organization 0.014212 UBXN10, RAB34, GPR22, TSC1, FAM149B, TAPT1 4.19 0030517 negative regulation of axon extension 0.0173 TRIM46, AATK, RTN4 14.75 0048812 neuron projection morphogenesis 0.022254 MAP2K1, ANKRD27, DAB2IP, RAC1 6.63 0010977 negative regulation of neuron projection development 0.024328 TSC1, HES1, RTN4, PTPRG 6.41 0048870 cell motility 0.030182 MAP2K1, ELMO1, RAC1 10.96 0050772 positive regulation of axonogenesis 0.033101 ROBO2, ZEB2, MAP2K1 10.43 0007411 axon guidance 0.044784 ROBO2, KLF7, CNTN1, UNC5D, RAC1 3.73 Cellular adhesion and migration related 0007155 cell adhesion 0.001191 CD164, PRKCE, FIBCD1, PRKCA, CX3CL1, PCDH17, CELSR3, CNTN1, HES1, FAT3, TGFBI, RAC1, DGCR2 3.03 0010595 positive regulation of endothelial cell migration 9.78E-04 ANXA3, PDCD6, PRKCA, PRKD1, RAC1 11.31 0010764 negative regulation of fibroblast migration 0.002543 ZEB2, RAC1, CYGB 38.87 0022409 positive regulation of cell-cell adhesion 0.01618 SOX2, MAGI1, PDE4D 15.27 Table 4 Neuronal and immune-related KEGG pathways enriched in miR-183C targets in the cornea of SNS-CKO and KO mice KEGG ID Term P Genes Fold Enrichment Neuronal and synaptic function related mmu04360 Axon guidance 0.0011 ROBO2, GNAI3, FYN, PRKCA, UNC5D, RAC1, SSH2 5.84 mmu04725 Cholinergic synapse 0.0060 MAP2K1, GNG5, GNAI3, FYN, PRKCA 6.74 mmu04726 Serotonergic synapse 0.0103 MAP2K1, GNG5, DUSP1, GNAI3, PRKCA 5.76 mmu04921 Oxytocin signaling pathway 0.0174 MAP2K1, MEF2C, CACNA2D1, GNAI3, PRKCA 4.94 mmu04727 GABAergic synapse 0.0201 GABARAPL2, GNG5, GNAI3, PRKCA 6.79 mmu04713 Circadian entrainment 0.0258 GNG5, GNAI3, PRKCA, GRIA3 6.16 mmu04724 Glutamatergic synapse 0.0380 GNG5, GNAI3, PRKCA, GRIA3 5.30 Immune function related mmu04062 Chemokine signaling pathway 0.0081 MAP2K1, GNG5, ELMO1, GNAI3, RAC1, CX3CL1 4.72 mmu04666 Fc gamma R-mediated phagocytosis 0.0225 MAP2K1, PRKCE, PRKCA, RAC1 6.50 mmu04670 Leukocyte transendothelial migration 0.0414 GNAI3, MSN, PRKCA, RAC1 5.12 To further identify myeloid cell-specific target genes of miR-183C, we isolated the Csf1r-EGFP + CRMCs from young adult miR-183C MS-CKO and conventional KO mice and their age- and sex-matched WT control mice. KEGG pathway analyses of miR-183C targets identified in CRMCs of both MS-CKO and conventional KO ( Tables S25&S26 ) are consistently enriched in multiple immune/inflammation-related pathways (Table 5 ; Table S27 ), including FcγR-mediated phagocytosis (Fig. 8 A), chemokine signaling (Fig. 8 B), FcεRI signaling, TGFβ signaling pathway, TNF signaling pathways, and several microbial infection pathways (Table 5 ; Table S27 ). These data suggest that miR-183C regulates the functions of myeloid cells through simultaneously targeting multiple genes in different pathways. Intriguingly, miR-183C targets are also enriched in neuronal function, especially synaptic function-related pathways ( Table S28 ), and cell-cell interaction pathways ( Table S29 ), suggesting potential indirect modulation of neuronal innervation and other cell types of the cornea. Table 5 Immune/infection-related KEGG pathways enriched in miR-183C targets of CRMCs of KO and MS-CKO mice KEGG ID Term P Genes Fold Enrichment mmu05100 Bacterial invasion of epithelial cells 2.98E-04 ITGB1, ACTR2, CTTN, SHC1, CAV1, ARPC1B, RAC1, WASL, CRK, WASF2, CD2AP 4.12 mmu04666 Fc gamma R-mediated phagocytosis 3.87E-04 ACTR2, MAP2K1, MARCKS, PAK1, PRKCE, ARPC1B, MAPK1, ASAP1, GAB2, RAC1, CRK, WASF2 3.67 mmu04664 Fc epsilon RI signaling pathway 0.002041 MAP2K3, MAPK9, MAP2K1, MAPK1, GRB2, FYN, KRAS, GAB2, RAC1 3.88 mmu05135 Yersinia infection 0.002728 ITGB1, MAP2K3, ACTR2, GIT2, MAP2K1, ARHGEF12, ARPC1B, WASL, MAPK9, MAPK1, RAC1, CRK, WASF2 2.74 mmu04062 Chemokine signaling pathway 0.003045 MAP2K1, SHC1, GNAI3, BRAF, GNG12, PRKCZ, ADCY6, TIAM1, PAK1, GNG5, MAPK1, GRB2, KRAS, RAC1, CRK, PRKACB 2.37 mmu05166 Human T-cell leukemia virus 1 infection 0.006333 ATF2, MAP3K3, EGR1, MAP2K1, CRTC1, XIAP, TGFBR1, ADCY6, PPP3CA, MAPK9, ZFP36, CCND2, CREB3L1, CREB3L2, MAPK1, EP300, KRAS, PRKACB 2.06 mmu05132 Salmonella infection 0.007681 MAP2K3, ACTR2, RALA, MAP2K1, ARPC1B, WASL, PIK3C2A, RHOB, MAPK9, SNX18, PAK1, BCL2, EXOC4, MAPK1, RAC1, PFN1, KPNA3, PFN2 2.02 mmu05163 Human cytomegalovirus infection 0.007979 ATF2, MAP2K1, ARHGEF12, GNAI3, TSC1, GNG12, ADCY6, PPP3CA, GNG5, GNA11, CREB3L1, CREB3L2, MAPK1, GRB2, KRAS, RAC1, CRK, PRKACB 2.02 mmu05165 Human papillomavirus infection 0.015215 MAGI1, ITGB1, PRKCI, MAP2K1, ITGA3, TSC1, PPP2R5C, LAMC1, PRKCZ, PPP2CA, PPP2CB, CCND2, CREB3L1, CREB3L2, MAPK1, EP300, GRB2, HES1, KRAS, WNT2, PRKACB, WNT4 1.74 mmu04650 Natural killer cell mediated cytotoxicity 0.020466 PPP3CA, MAP2K1, PAK1, SHC1, MAPK1, GRB2, FYN, BRAF, KRAS, RAC1 2.45 mmu05145 Toxoplasmosis 0.037313 ITGB1, MAP2K3, MAPK9, GNAI3, BCL2, XIAP, MAPK1, LAMC1, LDLR 2.35 mmu04350 TGF-beta signaling pathway 0.039069 ACVR1, PPP2CA, SMAD1, PPP2CB, EP300, MAPK1, SKIL, TGFBR1, SMAD7 2.33 mmu05170 Human immunodeficiency virus 1 infection 0.040389 MAP2K3, MAP2K1, FBXW11, GNAI3, GNG12, PPP3CA, MAPK9, PAK1, GNG5, GNA11, BCL2, MAPK1, KRAS, RAC1, CRK 1.79 mmu04668 TNF signaling pathway 0.048672 MAP2K3, CYLD, MAPK9, ATF2, MAP2K1, CREB3L1, CREB3L2, XIAP, MAPK1 2.23 Discussion To delineate the cell type-specific functions of miR-183C in corneal sensory nerves and innate immune cells, we created and characterized a SNS- and a MS-CKO mouse models, in comparison to the conventional KO mice. Our data showed that inactivation of miR-183C in corneal nerves, but not in innate immune cells, resulted in decreased corneal sensory nerve density and reduced sensitivity to mechanical stimuli, suggesting intrinsic regulation of miR-183C on corneal sensory innervation. Our data showed that, in the SNS-CKO mice, the reduction of sensory nerve density occurs specifically in the fine, terminal neurites in the epithelial layer, including the subbasal plexus, while the large-diameter nerves in the stromal layer remain unaffected, suggesting a critical role of miR-183C in the terminal differentiation and projection of corneal sensory nerves. This is consistent with its functions in the other sensory organs, e.g. the photoreceptors of the retina 51,54 and the inner ear hair cells 101,102 , as well as myeloid cells 59 , suggesting a common feature of miR-183C regulating terminal functional differentiation of primary sensory neurons and innate immune cells. Consistent with this observation, our RNA seq data showed that target genes of miR-183C in the TG were significantly enriched with genes involved in axonal guidance and neuronal projection regulation as well as synaptic functions, suggesting that miR-183C regulates corneal sensory nerve functions by directly targeting neuronal projection and synaptic release-related genes. Multiple genes in the same pathway are simultaneously targeted. This exemplifies the mode of action of miRNAs – quantitatively regulating multiple genes in the same pathway. Their effect on each individual gene may not cause a major phenotypic change; however, working in concert, they impose significant functional consequence 7,103–106 . In this case, the simultaneous regulation of multiple genes in the neuronal projection and synaptic functional pathways contributes to the decreased sensory nerve density and levels of neuropeptides in the cornea of the SNS-CKO and miR-183C KO mice, which affects the neuroimmune response of the cornea to bacterial infection 60 . MS-CKO of miR-183C resulted in increased CRMCs, suggesting intrinsic regulation of miR-183C in CRMCs. Our comparative study of the transcriptomes of CRMCs of miR-183C KO and MS-CKO mice vs their WT controls revealed myeloid-specific target genes of miR-183C, which are significantly enriched in a range of immune/infection related pathways, including chemokine signaling and phagocytosis pathways. These data provide new insight into the mechanisms of intrinsic regulation of miR-183C on the functions of innate myeloid cells, e.g. cytokine production and phagocytosis and intracellular killing capacity that we reported previously 7,60,61 . Intriguingly, SNS-CKO of miR-183C also led to increased number of CRMCs in naïve mouse cornea, suggesting that normal function of miR-183C in sensory nerves has an extrinsic regulation on the establishment of CRMCs, indicative of neuroimmune interaction in the maintenance of the homeostasis of the cornea. KEGG pathway analysis of miR-183C target genes in TG sensory neurons showed significant enrichment of genes involved in chemokine signaling pathway, including Cx3cl1. Neuron-produced chemokine Cx3cl1, also called fractalkine, is known to promote chemotactic migration of microglia in the central nervous system 107,108 , mediate the homing of resident myeloid cells in the cornea 109 and recruitment of macrophages in other tissues 110 . Meanwhile, Cx3cl1 has also been shown to enhances neuron adhesion to extracellular matrix and reduce neuronal migration 111 . These observations support a hypothesis that inactivation of miR-183C disinhibits Cx3cl1 in the TG sensory neurons and resulted in increased expression of Cx3cl1 in the cornea of SNS-CKO mice; then elevated level of Cx3cl1 may enhance the recruitment of myeloid cells to the cornea and result in increased number of the corneal resident myeloid cells. Additional studies are warranted to further test this hypothesis. Furthermore, our RNA seq data showed that miR-183C target genes in TG sensory neurons are also enriched in pathways regulating other corneal cell types, e.g. epithelial, endothelial cell and fibroblast migration; while target genes in CRMCs are also enriched in neuronal synaptic functions and cell-cell interaction and migration pathway. These data suggest that, in addition to the intrinsic regulation of sensory innervation and CRMCs, miR-183C exerts extrinsic regulation on neuro-immune-epithelial-stromal-endothelial interactions. This is consistent with our recent report of a single-cell transcriptome study that miR-183C serves as a checkpoint of corneal resident immune cells and shapes the cellular landscape of the cornea 4 . Different cell types form the microenvironment or niche of the cornea and work in concert to endow the cornea with its unique architecture and functionalities and maintain homeostasis 1–4 . Perturbation of the functions of one cell type imposes global impact on other cell types and the overall function of the cornea in both physiological and various pathological conditions 4 . For the first time, here we discovered that both naïve miR-183C KO and SNS-CKO, but not the MS-CKO mice, had significantly decreased tear volume, when compared to their age- and sex-matched WT controls. These data suggest miR-183C enhances basal tear production through its regulation of corneal sensory innervation. It is known that the primary afferent sensory neurons innervating the cornea play important roles in the basal tear production and secretion evoked by noxious stimulation of the cornea through the brainstem tear reflex arc 27 . Corneal sensory neurons sense dry condition and other environmental stressors, send primary afferent projections to activate the neurons in the spinal trigeminal nucleus (Vsp) and regulates lacrimation through the efferent autonomic arm of the reflex arc, which include the preganglionic parasympathetic neurons in/around the superior and inferior salivatory nuclei (SSN and ISN) and postganglionic parasympathetic neurons in the pterygopalatine ganglion (PG) 27 . The autonomic reflex promotes the production of the watery component of the tears by the lacrimal glands; mucin component of the tears by the conjunctival goblet cells, and lipid content by the meibomian glands 27 . We speculate that decreased sensory innervation caused by loss of miR-183C in sensory neurons imposes a negative impact on proper lacrimation, and therefore, miR-183C may play a role in dry eye disease (DED). Further studies will be needed to confirm this hypothesis. To our surprise, we observed significant decrease of basal tear volume in female vs male mice in all three strains used in this study, which are in a mixed genetic background of C57BL/6 and129S 51 . It is well known that DED is more prevalent in women compared to men 112–122 , especially in the autoimmune DED, Sjögren’s syndrome 123 . Over 90% of all patients with Sjögren’s syndrome are women 123 . The female gender is considered a risk factor for the development of DED 112 . Increased inflammation in lacrimal glands has been reported in several mouse models of Sjögren’s syndrome 124 . Data from rats suggest that gender has significant influence on lacrimal gland structure during development and aging 125 . The acinar area of the lacrimal glands of males are reported to be larger than that of females in multiple species including human 126 . Sex hormones modulates lacrimal gland morphology, tear volume and secretory components and protein contents of the tears 120–122,126−128 . However, sex-related difference in basal tear volumes has not been reported in either humans or animals 120,125,128 . Our observation of decreased tear volume in female mice appears to be in-line with the fact that female gender has been considered a risk factor for the development of DED 112 . The discrepancy between our observation and others 125,129 could come from different species and/or strains of animals as well as different methodology of measurement 130 . Nevertheless, our data suggest a sex-dependent difference in basal tear volume and further studies are warranted. To our knowledge, this is the first example that a miRNA cluster plays significant, functional roles in the corneal homeostasis through direct regulations of both sensory innervation and CRMCs and modulation of neuroimmune interaction. The SNS-CKO and MS-CKO mouse models developed in this report provide critical tools to fully uncover the roles of miR-183C in sensory nerves or myeloid cells under different pathological conditions, e.g., bacterial keratitis and DED. Such knowledge will be essential to develop cell type-specific strategies targeting miRNAs for the treatment of corneal diseases. Declarations Acknowledgement This work is supported by grants from the National Eye Institute, National Institutes of Health (R01 EY026059 to SX; R01 EY016058, R01 EY035231,and P30 EY004068 to LDH); a Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology, Visual and Anatomical Science, Wayne State University School of Medicine; and the Bridge fund from the Office of Vice President for Research (OVPR) of the Wayne State University. Author contributions N.G., M.S., K.G., G.L., H.Z., S.M., A.P., M.S., M.F.A.S. conducted experiments and data collection. N.G., G.L. S.X. performed data analyses. L.D.H participated in experimental design and manuscript revision. S.X. conceived, designed and directed the entire study, conducted experiments, data collection, wrote, revised and submitted the manuscript. Competing Interests The authors declare no conflict of interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3678621","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":264307091,"identity":"0bd7f9ef-f576-4b92-b41b-9ce25620c17f","order_by":0,"name":"Naman Gupta","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Naman","middleName":"","lastName":"Gupta","suffix":""},{"id":264307092,"identity":"1804bb08-bcb9-417a-b47a-090d15e497b4","order_by":1,"name":"Mallika Somayajulu","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Mallika","middleName":"","lastName":"Somayajulu","suffix":""},{"id":264307093,"identity":"c6fa992d-ae3b-4524-a874-06d60c7895d3","order_by":2,"name":"Katherine Gurdziel","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Katherine","middleName":"","lastName":"Gurdziel","suffix":""},{"id":264307094,"identity":"f3903c96-c2a9-4539-8611-a038904ce5d8","order_by":3,"name":"Giovanni LoGrasso","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"LoGrasso","suffix":""},{"id":264307095,"identity":"03d1b00d-c7c6-4b33-a0aa-c604065eaf3d","order_by":4,"name":"Haidy Aziz","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Haidy","middleName":"","lastName":"Aziz","suffix":""},{"id":264307096,"identity":"775458d6-9be7-42d9-b237-99cb18f57ada","order_by":5,"name":"Sharon McClellan","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Sharon","middleName":"","lastName":"McClellan","suffix":""},{"id":264307097,"identity":"fb5fbaf1-fde5-4b51-bb46-33de5538198f","order_by":6,"name":"Ahalya Pitchaikannu","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Ahalya","middleName":"","lastName":"Pitchaikannu","suffix":""},{"id":264307098,"identity":"54ec9ed0-21b7-4ed4-8a4d-8922ef8ed484","order_by":7,"name":"Manoranjan Santra","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Manoranjan","middleName":"","lastName":"Santra","suffix":""},{"id":264307099,"identity":"a24ec7ee-e216-4dcc-bb7a-179f964039a7","order_by":8,"name":"Muhammed Shukkur","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Muhammed","middleName":"","lastName":"Shukkur","suffix":""},{"id":264307100,"identity":"2c957db6-b4b5-4241-b3f9-d24e526a266c","order_by":9,"name":"Linda Hazlett","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Hazlett","suffix":""},{"id":264307102,"identity":"9fa89c85-5f96-415f-82fa-61b7d266eadc","order_by":10,"name":"Shunbin Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYHACxgcMDMwIXgMRWpgNSNbCJkGaFvkZOWZVNyqsGQyOnzF78IHBRnbDAQJaDM6cMbudcyYdyMgxN5zBkGZMWAt7j9nt3LbDDGYHcsykeRgOJxLUIt/MY1ac+w+o5fwbM+k/DP8Ja2E43mPGnNsA1HIDaAsDwwHCWgzOHCuWzjmWzmN/41mZZI9BsvFMgg6bkbzxc06NtZxkf/I2iR8VdrJ9BB0GBTwMDBzAKDUgUjkUsD8gTf0oGAWjYBSMGAAAoO5BGD2tZVMAAAAASUVORK5CYII=","orcid":"","institution":"Wayne State University","correspondingAuthor":true,"prefix":"","firstName":"Shunbin","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2023-11-28 22:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3678621/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3678621/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-58403-1","type":"published","date":"2024-04-01T15:00:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49482704,"identity":"4413f44d-7c66-48b8-9856-2f6e17fabbdf","added_by":"auto","created_at":"2024-01-11 15:31:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C has no significant impact on the gross histological architecture of the cornea\u003c/strong\u003e. A. H\u0026amp;E staining of cross-sections of the corneas of MS-CKO, SNS-CKO and miR-183C conventional KO and age- and sex-matched WT control littermates. B. Measurement of the thickness of the corneas. Epi: epithelium; Endo: endothelium.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/8c0d830ce567457658686349.png"},{"id":49482710,"identity":"6fd26524-9b84-4d21-99a2-1dcb715c542e","added_by":"auto","created_at":"2024-01-11 15:31:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3130065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C in either sensory neurons or myeloid cells results in increased number of corneal resident myeloid cells (CRMCs). \u003c/strong\u003eCompressed confocal images of flatmount cornea of young adult MS-CKO (A) and SNS-CKO sand their age- and sex-matched WT control littermates (B). **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/0eeecb36ca69f5077f472835.png"},{"id":49483145,"identity":"5844ee0a-e329-4469-a3b5-2abc1966602e","added_by":"auto","created_at":"2024-01-11 15:39:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4386475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C in sensory neurons, but not in the CRMCs, results in decreased sensory nerve density. \u003c/strong\u003eCompressed confocal images of flatmount corneas of young adult MS-CKO (A) and SNS-CKO sand their age- and sex-matched WT control littermates (B). *: p\u0026lt;0.05; **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/22c13af33f95dd7204e675ac.png"},{"id":49482708,"identity":"5931692a-bfef-43a9-a475-d5a258f4969c","added_by":"auto","created_at":"2024-01-11 15:31:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2771031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduction of corneal sensory nerve density in the SNS-CKO is caused by decreased sensory nerve density in the epithelial layer but not the stromal layer.\u003c/strong\u003e Compressed confocal images of corneal sensory nerves of the epithelial layers and stromal layers of the whorl center areas (A) and peripheral regions (B) of flatmount corneas of young adult, male SNS-CKO sand their age- and sex-matched WT control littermates. *: p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/f58107743920cb917ae8cbc6.png"},{"id":49483146,"identity":"97a0b682-fef2-4ebb-ada7-dc4e488a7395","added_by":"auto","created_at":"2024-01-11 15:39:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":168234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C in sensory nerves, but not in the myeloid cells, results in decreased corneal sensitivity to mechanical stimuli. \u003c/strong\u003eSensitivity test using a Cochet and Bonnet aesthesiometer in young adult (8-12 weeks old) miR-183C conventional KO (A), SNS-CKO (B) and MS-CKO mice and their age- and sex-matched WT control littermates (C). **: p\u0026lt;0.01; ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/37e7a075981061f14bfc430b.png"},{"id":49482707,"identity":"57382481-a6fd-417c-8c56-6e95e95f98a6","added_by":"auto","created_at":"2024-01-11 15:31:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":188019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C in sensory nerves, but not in myeloid-cells, results in decreased basal tear volume. \u003c/strong\u003ePhenol-red thread assays in young adult (8-12 weeks old) miR-183C conventional KO (A), SNS-CKO (B) and MS-CKO mice and their age- and sex-matched WT control littermates (C). *: p\u0026lt;0.05; **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/194c3e7812b4586535422ca2.png"},{"id":49482706,"identity":"fd92a54e-1773-47af-843f-2eda928fad43","added_by":"auto","created_at":"2024-01-11 15:31:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":625160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-183C target genes in TG sensory neurons are enriched in axon guidance (A) chemokine signaling pathways (B).\u003c/strong\u003e Modified from www.genome.jp/kegg/pathway.html. Star-labeled molecules or complexes are or contain target genes of miR-183C.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/18ff6932f2af90622fc453a8.png"},{"id":49482711,"identity":"14fc8e96-349f-4c8c-b3d0-ffa8883b976c","added_by":"auto","created_at":"2024-01-11 15:31:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":635283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-183C target genes in CRMCs are enriched in FcgR-mediated phagocytosis (A) and chemokine signaling pathways (B).\u003c/strong\u003e Modified from www.genome.jp/kegg/pathway.html. Star-labeled molecules or complexes are or contain target genes of miR-183C.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/7e3cf4415788a2d86b5f40e5.png"},{"id":54303773,"identity":"07660d86-7cbf-4eac-997f-cfa80f918672","added_by":"auto","created_at":"2024-04-08 15:11:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5177188,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/0bde5b09-70a0-4dbc-8dd3-5e58b8371607.pdf"},{"id":49482712,"identity":"fcd4a41f-f449-4624-a02f-4b90c9c8de3f","added_by":"auto","created_at":"2024-01-11 15:31:50","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":4960793,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationrev.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3678621/v1/5dfa9b33b4943b5536847146.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell-type-specfic target genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cornea is an avascular, transparent tissue between the eye and the external environment. It provides visual clarity and two thirds of the refractive power of the eye\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. It also serves as first-line defense against microbial infection and other insults\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Multiple cell types of different origin, including corneal epithelium, stromal keratocytes, endothelial cells, resident immune cells (CRICs) and nerves\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e, works in sync to confer the unique architecture and functionalities of the cornea\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. Although rare (1.2\u0026ndash;5% in mouse\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e), CRICs are a diverse population and have essential roles in almost every aspect of the development and functions of the cornea under both physiological and pathological conditions\u003csup\u003e8\u0026ndash;16\u003c/sup\u003e. The cornea is the most densely sensory-innerved tissue in the body\u003csup\u003e17\u0026ndash;19\u003c/sup\u003e. Sensory innervation of the cornea not only provides bodily sensation to various stimuli but also plays important roles in the homeostasis of the cornea as well as the pathogenesis of corneal diseases through neuroimmune interactions\u003csup\u003e18\u0026ndash;33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003emicroRNAs (miRNAs) are small, non-coding RNAs and are post-transcriptional regulators of gene expression\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e. They play an important role in human diseases\u003csup\u003e38\u0026ndash;45\u003c/sup\u003e and are viable therapeutic targets\u003csup\u003e46\u0026ndash;49\u003c/sup\u003e. However, their roles in the neuroimmune interaction in health and diseases of the cornea are still largely unknown. The evolutionarily-conserved, paralogous miRNA cluster, miR-183/96/182 cluster (referred to as the miR-183C from here on) is highly, specifically expressed and required for the normal development and functions of sensory neurons\u003csup\u003e50\u0026ndash;52\u003c/sup\u003e. Inactivation of miR-183C in conventional knockout (KO) mouse models results in multisensory defects\u003csup\u003e51,53\u0026ndash;55\u003c/sup\u003e. Point mutations in the seed sequence of miR-96 results in non-syndromic hearing loss in both mouse\u003csup\u003e56\u003c/sup\u003e and human\u003csup\u003e57\u003c/sup\u003e. In addition, this miRNA cluster is also expressed and plays important roles in both innate\u003csup\u003e4,7,58\u0026ndash;62\u003c/sup\u003e and adaptive immune cells \u003csup\u003e63\u0026ndash;70\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the cornea, we demonstrated that inactivation of miR-183C in a conventional KO mouse model results in decreased nerve density and reduced expression of capsaicin receptor TRPV1 and pro-inflammatory neuropeptide substance P (sP) precursor gene Tac1\u003csup\u003e60\u003c/sup\u003e. Inactivation or knockdown of miR-183C in innate immune cells, e.g. Mφ and neutrophils, reduces their production of pro-inflammatory cytokines, however, enhances their phagocytosis and intracellular bacterial killing capacity\u003csup\u003e60,61\u003c/sup\u003e. These result in a reduced inflammatory response to bacterial infection, e.g. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PA) and contribute to a decreased severity of PA keratitis \u003csup\u003e60,61,71\u003c/sup\u003e. In addition to its effect on peripheral Mφ and neutrophils, we showed that inactivation of the miR-183C results in increased number of steady-state CRMCs in na\u0026iuml;ve mice \u003csup\u003e4,7\u003c/sup\u003e and myeloid cell infiltration into the cornea in the event of PA infection\u003csup\u003e7\u003c/sup\u003e. Collectively, these findings suggest a pivotal regulatory role for miR-183C in fine-tuning neuroimmune interaction in the cornea.\u003c/p\u003e \u003cp\u003eHowever, since in the conventional KO mice, miR-183C is simultaneously inactivated in all miR-183C-expressing cells, including both CSN and CRMCs\u003csup\u003e7,60\u003c/sup\u003e, the phenotype observed reflects a composite effect of loss-of-function of miR-183C in both cell types. This precludes distinguishing whether the changes observed in CSN and CRMCs are intrinsic functions of miR-183C in sensory nerves and myeloid cells, respectively, or an extrinsic effect through neuroimmune interaction. To resolve this conundrum, we produced sensory neuron-specific (SNS) and myeloid cell-specific (MS) conditional knockout mice (CKO). Here we report the first characterization of these CKO mice in comparison to their corresponding wild type (WT) littermate control mice at their steady state. Our results reveal sensory neuron- and myeloid cell-specific functions of miR-183C and their impact on the homeostasis of the cornea.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eMice.\u003c/b\u003e All experiments and procedures involving animals and their care were pre-reviewed and approved by the Wayne State University Institutional Animal Care and Use Committee and carried out in accordance with National Institute of Health and Association for Research in Vision and Ophthalmology (ARVO) guidelines (Approved protocol number: IACUC-22-05-4618). The study is reported in accordance with ARRIVE guidelines. Euthanasia was performed by cervical dislocation under anesthesia with isoflurane followed by thoracotomy.\u003c/p\u003e \u003cp\u003eThe miR-183C KO \u0026ndash; the miR-183C\u003csup\u003eGT/GT\u003c/sup\u003e mice, are on a 129S2/BL6-mixed background \u003csup\u003e51\u003c/sup\u003e and were originally derived from a gene-trap (GT) embryonic stem cell clone \u003csup\u003e51,72,73\u003c/sup\u003e. Csf1r-EGFP or MacGreen mice \u003csup\u003e74\u003c/sup\u003e were purchased from the Jackson lab (Stock number. 018549). In this strain, the EGFP transgene is under the control of the 7.2-kb mouse colony stimulating factor 1 receptor (Csf1r\u003cem\u003e)\u003c/em\u003e promoter, allowing specific expression of EGFP in the mononuclear phagocyte system (MPS) myeloid cells, including monocytes, macrophages and dendritic cells\u003csup\u003e74,75\u003c/sup\u003e. The Csf1r-EGFP mice were bred with miR-183C\u003csup\u003eGT/+\u003c/sup\u003e to produce miR-183C KO [Csf1r-EGFP(+);miR-183C\u003csup\u003eGT/GT\u003c/sup\u003e] and WT mice [Csf1r-EGFP(+);miR-183C\u003csup\u003e+/+\u003c/sup\u003e] on the background of Csf1r-EGFP as described previously\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMice with miR-183C CKO allele, the miR-183C\u003csup\u003ef/f\u003c/sup\u003e, were provided by Dr. Patrick Ernfors, Karolinska Institutet, Sweden through the European Mouse Mutant Archive (EMMA. ID: EM12387). The miR183C\u003csup\u003ef\u003c/sup\u003e allele has two loxP sites flanking the 5\u0026rsquo; and 3\u0026rsquo; ends of the miR-183C for robust Cre-mediated miR-183C CKO\u003csup\u003e76\u003c/sup\u003e. The myeloid-specific, LysM-Cre mice\u003csup\u003e77\u003c/sup\u003e were purchased from The Jackson Laboratory (Stock number: 004781). The LysM-Cre knock-in/knock-out allele has a nuclear-localized (NLS)-Cre recombinase inserted into the first coding ATG of the lysozyme 2 gene (\u003cem\u003eLyz2\u003c/em\u003e), both abolishing endogenous \u003cem\u003eLyz2\u003c/em\u003e gene function and placing NLS-Cre expression under the control of the endogenous \u003cem\u003eLyz2\u003c/em\u003e promoter/enhancer elements. Therefore, the LysM-Cre allows myeloid cell-specific expression of Cre recombinase. The sensory nerve specific Nav1.8-Cre mice \u003csup\u003e78\u003c/sup\u003e were kindly provided by Dr. John N. Wood, University College London, through Dr. Theodore J. Price, University of Texas Dallas. This strain is now available at the Jackson Laboratory (Stock Number: 036564). The voltage-gated sodium channel Na\u003csub\u003ev\u003c/sub\u003e1.8 (encoded by the Scn10a gene) is one of the signature genes of the majority of nociceptive sensory neurons in the TG and dorsal root ganglia (DRG)\u003csup\u003e33,79\u0026ndash;81\u003c/sup\u003e. Na\u003csub\u003ev\u003c/sub\u003e1.8 promoter-driven Cre recombinase (Na\u003csub\u003ev\u003c/sub\u003e1.8-Cre) is expressed in nearly all corneal nociceptive sensory nerves\u003csup\u003e33,78,82\u003c/sup\u003e. The reporter strain, R26\u003csup\u003eLSL\u0026thinsp;\u0026minus;\u0026thinsp;RFP(+/+)\u003c/sup\u003e mice\u003csup\u003e83\u003c/sup\u003e, also known as Ai14 (Stock number: 007914, the Jackson Laboratory) has a loxP-flanked STOP cassette (LSL) in front of a tdTomato RFP cassette, all of which are inserted into the ROSA26 locus\u003csup\u003e83\u003c/sup\u003e. The LSL prevents the transcription of tdTomato RFP; however, when Cre recombinase is present, the LSL cassette will be excised to allow the expression of tdTomato RFP.\u003c/p\u003e \u003cp\u003emiR-183C MS-CKO mice [LysM-Cre(+/-);miR-183C\u003csup\u003ef/f\u003c/sup\u003e;Csf1r-EGFP(+/+)], in which MPS myeloid cells are labeled with EGFP, were produced by breeding of the LysM-Cre(+/-), miR-183C\u003csup\u003ef/f\u003c/sup\u003e and Csf1r-EGF(+/-) mice. Their littermates [LysM-Cre(-/-);miR-183C\u003csup\u003ef/f\u003c/sup\u003e;Csf1r-EGFP(+/+)] are used as WT mice. SNS-CKO mice [Nav1.8-Cre(+/-);miR-183C\u003csup\u003ef/f\u003c/sup\u003e;R26\u003csup\u003eLSL\u0026thinsp;\u0026minus;\u0026thinsp;RFP(+/+)\u003c/sup\u003e;Csf1r-EGFP(+/+)] and their WT littermates [Nav1.8-Cre(+/-);miR-183C\u003csup\u003e+/+\u003c/sup\u003e;R26\u003csup\u003eLSL\u0026thinsp;\u0026minus;\u0026thinsp;RFP(+/+)\u003c/sup\u003e;Csf1r-EGFP(+/+)] were produced by breeding of Nav1.8-Cre (+/-), miR-183C\u003csup\u003ef/f\u003c/sup\u003e, R26\u003csup\u003eLSL\u0026thinsp;\u0026minus;\u0026thinsp;RFP\u003c/sup\u003e and Csf1r-EGF(+/-) mice. All breeding showed a normal mendelian inheritance pattern. Sex is considered as a biological variance in all studies. 8\u0026ndash;12 weeks old, male and female mice were used as separate groups in all experiments. The age, sex and number of the mice in each experiment are specified in the figure legends and/or the text.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHematoxylin and eosin (H\u0026amp;E) staining of paraffin sections of mouse cornea\u003c/b\u003e. Mouse eyes were enucleated and fixed in 4% formalin. The embedding, sectioning and H\u0026amp;E staining were performed by Excalibur Pathology Inc (Oklahoma City, OK). Comparable sagittal sections through the optic nerves were imaged under a DM4000b brightfield microscope (Leica).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence-activated cell sorting (FACS).\u003c/b\u003e 8\u0026ndash;12 weeks old, na\u0026iuml;ve MS-CKO and KO mice and age- and sex matched WT littermate controls were used to isolate Csf1r-EGFP\u0026thinsp;+\u0026thinsp;myeloid cells from the cornea and spleen for DNA and RNA preparations (see below). For corneal samples, corneas anterior to the limbi from 6\u0026ndash;7 mice/genotype were carefully dissected and pooled for single cell preparation as described before \u003csup\u003e4,7\u003c/sup\u003e. For spleen cells, mononuclear cells were isolated following a standard protocol\u003csup\u003e84\u003c/sup\u003e. FACS was conducted on a Sony SY3200 cell sorter at the Microscopy, Imaging and Cytometry Resources Core (MICR), Wayne State University (WSU) to isolate Csf1r-EGFP\u0026thinsp;+\u0026thinsp;myeloid cells as we described previously\u003csup\u003e4,7\u003c/sup\u003e. EGFP- corneal and spleen mononuclear cells were used as negative controls to optimize the gating.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA and genotyping PCR.\u003c/b\u003e Genomic DNA was prepared from mouse tail, cornea, TG, FACS-sorted Csf1-EGFP\u0026thinsp;+\u0026thinsp;myeloid cells using the gMax DNA mini kit (IBI Scientific) following manufacturer\u0026rsquo;s instruction as described previously\u003csup\u003e85\u003c/sup\u003e. Subsequently, DNA concentration and quality were assayed on the Nanodrop 2000 (ThermoFisher Scientific). 100 ng (from mouse cornea, TG and tails) or 4 ng (from FACS-sorted myeloid cells of the cornea and spleen) genomic DNA was used for genotyping PCR to detect tissue and/or cell type-specific recombination at genomic DNA level as we described previously\u003csup\u003e51,85\u003c/sup\u003e. PCR of 18s rRNA was amplified and used as a loading control. The genomic structure and primers are illustrated in Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The sequences of the primers are: 7Fintron1: 5\u0026rsquo;-TACCCTGAGTGTGTCTCAATC-3\u0026rsquo;; CKO-R: 5\u0026rsquo;-GCAGAGTCACAAACATGTGTAGC-3\u0026rsquo;; 18s rRNA Forward: 5\u0026rsquo;-GTAACCCGTTGAACCCCATT-3\u0026rsquo;; 18s rRNA Reverse: 5\u0026rsquo;-CCATCCAATCGGTAGTAG CG-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA preparation and qRT-PCR.\u003c/b\u003e Total RNA was prepared using the miRVana miRNA isolation kit (Life Technologies, Foster City, CA, USA) or the RNeasy (Qiagen, Frederick, MD, USA) for miRNA or mRNA studies, respectively, as described previously\u003csup\u003e50,86,87\u003c/sup\u003e. qRT-PCR for miRNAs was performed using Taqman miRNA primers and RT-PCR kit (Life Technologies) with snRNA U6 as an endogenous control as described before\u003csup\u003e50,87\u003c/sup\u003e. For protein-coding genes, qRT-PCR was performed using a QuantiFast SYBR Green RT-PCR kit and QuantiTect primers (Qiagen) with 18s rRNA as endogenous controls\u003csup\u003e50,51,87\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTear volume measurement.\u003c/b\u003e Tear volume was measured by the Zone-Quick Phenol red thread (PRT) test (Yokota Co. Ltd). Manufacturer\u0026rsquo;s instruction was followed with modifications. Briefly, under light anesthesia by isoflurane, the lower eyelid was pulled down slightly; the PRT is placed between the palpebral conjunctiva of the lower eyelid and the eyeball at a point approximately 1/3 of the distance from the lateral canthus for 15 seconds. The length of the thread which turned red was measured.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorneal sensitivity test\u003c/b\u003e. Corneal sensitivity to mechanical stimuli was measured by a blink threshold test using a Cochet and Bonnet aesthesiometer (Western Ophthalmics). Briefly, the tip of a nylon filament, starting from 6 cm, was applied perpendicularly to the central cornea. The length at which the mouse blinks was registered as the blink threshold.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorneal flatmount, immunofluorescence (IF), confocal microscopy, and quantification of Csf1r-EGFP\u0026thinsp;+\u0026thinsp;corneal resident MPS cells and corneal nerve density.\u003c/b\u003e Corneal flatmount and IF was performed as described previously\u003csup\u003e7,71\u003c/sup\u003e. Briefly, mice were euthanized; eyes were enucleated and transferred to cold phosphate buffered saline (PBS). Under a dissecting scope (VWR International, Radnor, PA), the cornea anterior to the limbus was carefully dissected out. The corneas were transferred to cold 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4 for 1 h at 4\u0026deg;C. For direct confocal microscopy, the cornea was flattened by six evenly spaced cuts from the periphery toward the center and mounted in Vectashield media with DAPI (Vector Laboratories, Burlingame, CA) on Superfrost Plus slides (Fisherbrand). For IF, after fixation, the corneas were incubated in a blocking buffer with 2% normal goat serum (NGS) (Vector labs) in PBS for 30 min at room temperature (RT); then, permeabilized with 0.1% Triton X-100 in the blocking buffer for 30 min at RT. Subsequently, corneal tissues were incubated with mouse anti-\u003cem\u003eβ\u003c/em\u003e-Tubulin III (1/600 dilution. Cat. No. 801201, BioLegend, San Diego, CA, USA) antibodies in the blocking buffer for 72 hours at 4\u0026deg;C. After washes with PBS, the corneas were incubated with AF546\u0026ndash;conjugated goat anti-mouse IgG (1/1000 dilution. Cat. No. A-11003, Thermo Fisher Scientific) for overnight at 4\u0026deg;C. After washing, the corneas were flattened and mounted on slides. Negative controls were treated similarly with omission of the primary antibody. All slides were imaged using a TCS SP8 laser confocal microscope (Leica Microsystems Inc. Buffalo Grove, IL). To capture the images of the entire cornea, a series of Z-stacked images under 10x objective were taken across the entire cornea and stitched together. These stitched Z-stacked images were merged/flattened for cell counting or nerve density quantification using Adobe Photoshop CS6 (64 bit) and ImageJ 1.52p software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imagej.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://imagej.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. NIH, Bethesda, MD, USA) as described previously\u003csup\u003e7\u003c/sup\u003e. Briefly, the raw image was first converted to the 16-bit grayscale; then the black \u0026amp; white binary image is optimized for the threshold to faithfully represent the original image and the cell density. The EGFP\u0026thinsp;+\u0026thinsp;cells in the entire cornea were counted. To quantify nerve density, the mean value of pixels in a 500-\u0026micro;m\u003csup\u003e2\u003c/sup\u003e square covering the center of the whorl-like subbasal plexus was quantified as the nerve density of the center; the ones in three 500-\u0026micro;m\u003csup\u003e2\u003c/sup\u003e squares randomly placed in the periphery of the cornea were measured, the average of which was recorded as nerve density of the peripheral cornea. Corneas from at least n\u0026thinsp;=\u0026thinsp;3 mice/sex/genotype were quantified.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3\u0026rsquo; RNA sequencing and data analysis\u003c/b\u003e: mRNAs were isolated from TG, corneas or FACS-sorted Csf1r-EGFP\u0026thinsp;+\u0026thinsp;myeloid cells of young adult (8\u0026ndash;12 weeks old) male mice. For miR-183C KO strain, n\u0026thinsp;=\u0026thinsp;3 for both KO and WT controls; for SNS-CKO strain, n\u0026thinsp;=\u0026thinsp;3 for SNS-CKO mice, n\u0026thinsp;=\u0026thinsp;4 for WT controls; for MS-CKO strain, n\u0026thinsp;=\u0026thinsp;5 for both MS-CKO and WT controls were used. To isolate Csf1r-EGFP\u0026thinsp;+\u0026thinsp;myeloid cells, 12 corneas of 6 KO and 14 corneas of 7 WT control mice or 20 corneas of 10 MS-CKO or 10 WT control mice were pooled for FACS isolation. Subsequently, 3\u0026rsquo; mRNA-Seq libraries were prepared by the Genome Sciences Core (GSC), WSU using the QuantSeq 3\u0026rsquo; mRNA-Seq Library Prep kit FWD (Lexogen, Greenland, NH)\u003csup\u003e88\u003c/sup\u003e. High sensitivity D1000 ScreenTape peak range between 100\u0026ndash;700 bp were in accordance with Lexogen guidelines. The libraries were sequenced on NovaSeq sequencer (Illumina). Reads were aligned to the mouse genome (Build mm10) using a public available software \u003cem\u003eSTAR\u003c/em\u003e \u003csup\u003e89\u003c/sup\u003e and tabulated for each gene region using \u003cem\u003eHTSeq\u003c/em\u003e \u003csup\u003e90\u003c/sup\u003e. Differential expressed genes (DEGs) between KO or CKO vs WT controls were identified by \u003cem\u003eedgeR\u003c/em\u003e \u003csup\u003e91\u003c/sup\u003e. miR-183C predicted target genes were identified by TargetScan algorithm (Targetscan.org)\u003csup\u003e92\u0026ndash;96\u003c/sup\u003e in the upregulated genes in CKO or KO vs WT control and are recognized as miR-183C target genes in these tissues. Statistical significance of enrichment of miR-183C targets in upregulated genes was analyzed by Chi-square with Yates\u0026rsquo; correction using the Analyze a 2x2 Contingency Table (GraphPad. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad.com/quickcalcs/contingency1/\u003c/span\u003e\u003cspan address=\"https://www.graphpad.com/quickcalcs/contingency1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Functional annotation analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID)\u003csup\u003e97\u003c/sup\u003e as described before\u003csup\u003e98\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e When the comparison was made among more than 2 conditions, one-way ANOVA with Bonferroni\u0026rsquo;s multiple comparison test was employed (GraphPad Prism); adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. Otherwise, a two-tailed Student\u0026rsquo;s t test was used to determine the significance; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. Each experiment was repeated at least once to ensure reproducibility and data from a representative experiment are shown. Quantitative data is expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSensory nerve- and myeloid cell-specific knockout of miR-183C results in no major morphological changes of the cornea.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo uncover tissue-specific functions of the miR-183C, we produced sensory neuron- and myeloid cell-specific miR-183C CKO mice (\u003cstrong\u003eFig.\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). To detect whether inactivation of miR-183C in sensory nerves and/or myeloid cells causes major histological and morphological changes in the cornea, we performed H\u0026amp;E staining in cross-sections of the corneas of SNS-CKO, MS-CKO and KO mice and their WT control littermates. No gross morphological changes were observed in the cornea of miR-183C SNS-CKO, MS-CKO and conventional KO mice, when compared to their WT controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The total thickness (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and thickness of the epithelial and stromal layers (not shown) had no significant differences between the CKOs and KO vs their age- and sex-matched WT controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C in either sensory neurons or myeloid cells results in increased number of CRMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of Csf1r-GFP\u0026thinsp;+\u0026thinsp;CRMCs of young adult, na\u0026iuml;ve mice showed that the numbers of Csf1r-EGFP\u0026thinsp;+\u0026thinsp;resident myeloid cells in MS-miR-183C CKO mice [3278\u0026thinsp;\u0026plusmn;\u0026thinsp;268/cornea (n\u0026thinsp;=\u0026thinsp;3) in male; 2234\u0026thinsp;\u0026plusmn;\u0026thinsp;80/cornea (n\u0026thinsp;=\u0026thinsp;3) in female] were significantly increased in both male and female mice, when compared to age-matched WT controls [1697\u0026thinsp;\u0026plusmn;\u0026thinsp;225/cornea (n\u0026thinsp;=\u0026thinsp;3) in male, 1507\u0026thinsp;\u0026plusmn;\u0026thinsp;107/cornea (n\u0026thinsp;=\u0026thinsp;3) in female] (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting miR-183C intrinsically regulates the number of CRMCs under homeostatic condition. In WT controls, male and female mice showed no difference in the number of CRMCs, however, in MS-CKO, the male vs female mice showed increased number of CRMCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting a potential sex-related modulation.\u003c/p\u003e\n\u003cp\u003eTo test whether miR-183C in CSN has an extrinsic regulation on the CRMC population, we quantified the number of CRMCs in the SNS-CKO mice. Intriguingly, sensory nerve-specific inactivation of miR-183C also resulted in an increased number of CRMCs when compared to age- and sex-matched WT controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). In male, it was increased by ~\u0026thinsp;35% [3197\u0026thinsp;\u0026plusmn;\u0026thinsp;151/cornea in SNS-CKO (n\u0026thinsp;=\u0026thinsp;3) vs 2335\u0026thinsp;\u0026plusmn;\u0026thinsp;121/cornea in WT controls (n\u0026thinsp;=\u0026thinsp;4)]; while in the female by ~\u0026thinsp;56% in female [3197\u0026thinsp;\u0026plusmn;\u0026thinsp;151/cornea in SNS-CKO (n\u0026thinsp;=\u0026thinsp;3) vs 2772\u0026thinsp;\u0026plusmn;\u0026thinsp;298/cornea in WT controls (n\u0026thinsp;=\u0026thinsp;4)](Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The numbers of CRMCs showed no significant differences between male and female in both WT or SNS-CKO mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInactivation of miR-183C in sensory neurons, but not in CRMCs, results in decreased sensory nerve density in the epithelial layer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreviously, we showed that corneal nerve density is significantly decreased in the miR-183C KO mice\u003csup\u003e60\u003c/sup\u003e. To dissect the contribution of miR-183C in corneal sensory nerves or resident myeloid cells to this phenotype, we quantified corneal nerve density in the SNS-CKO and MS-CKO mice. \u0026beta;-III tubulin IF of flatmount cornea showed no difference in corneal nerve density of the MS-CKO vs their WT littermate control mice in both male and female (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggesting loss of miR-183C in CRMCs had no contribution to the decreased corneal nerve density in the miR-183C KO mice\u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, the corneal nerve density was significantly decreased in the SNS-CKO vs WT littermate control mice in both male and female (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), in both the whorl center and peripheral regions of the cornea, suggesting that decreased nerve density in the miR-183C conventional KO mice\u003csup\u003e60\u003c/sup\u003e is a result of loss of miR-183C in corneal sensory nerves; miR-183C imposes an intrinsic regulation of corneal sensory innervation.\u003c/p\u003e\n\u003cp\u003eTo further probe into the reduction of sensory nerve density, we quantified the sensory nerve density in the epithelial and stromal layers separately (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The layer-specific analyses showed that, in both the whorl center and peripheral regions of male mice, it is the density of the fine terminal nerves in the epithelial layer that is significantly decreased, but not the large nerves in the stroma layer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that miR-183C regulates the terminal differentiation and/or extension of sensory nerve in the epithelial layer of the cornea, but has no significant impact on the establishment of large sensory nerves in the stromal layer. Similar effect was observed in female mice (\u003cstrong\u003eFig.S2\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eInactivation of miR-183C in sensory nerves results in decreased corneal sensitivity to mechanical stimuli\u003c/h2\u003e\n\u003cp\u003eSince inactivation of miR-183C in sensory neurons resulted in decreased sensory nerve density (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026amp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), we hypothesize that the functions of the sensory nerve are compromised in the cornea. To test this hypothesis, we first tested corneal sensitivity to mechanical stimuli using a Cochet and Bonnet aesthesiometer. Our result showed in both miR-183C KO (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) and SNS-CKO mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), corneal sensitivity was significantly decreased, when compared to their WT littermate. In the conventional KO, corneal sensitivity was decreased by ~\u0026thinsp;23% and 33% in male and female mice, respectively. In the SNS-CKO, corneal sensitivity was significantly reduced by ~\u0026thinsp;10 and 11% in male and female mice, respectively. However, in MS-CKO mice, inactivation of miR-183C showed no impact on corneal sensitivity. These data suggest that miR-183C in corneal sensory nerves intrinsically regulate its function. Decreased corneal sensitivity in miR-183C KO is a result of loss of miR-183C in the sensory nerve.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eInactivation of miR-183C in sensory nerves results in decreased basal tear production\u003c/h2\u003e\n\u003cp\u003eIt is known that corneal sensory innervation plays important role in regulating tear production \u003csup\u003e27,99\u003c/sup\u003e. Since inactivation of miR-183C in sensory neurons resulted in decreased sensory nerve density and sensitivity to mechanical stimuli (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), we further hypothesize that miR-183C may play a role in tear production through its regulation of corneal sensory innervation. To test this hypothesis, we measured tear volumes using the PRT test. Our result showed that in both the miR-183C KO and SNS-CKO mice, tear volume was significantly decreased when compared to their WT littermates (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026amp;B). In male mice, the tear volume was decreased by ~\u0026thinsp;81% in the miR-183C KO and ~\u0026thinsp;71% in the SNS-CKO mice; while in female, it was decreased by ~\u0026thinsp;55% and 59% in the conventional KO and SNS-CKO, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026amp;B). However, tear volume of MS-CKO mice showed no difference compared to their WT littermate controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). These data suggest that miR-183C modulates basal tear production through regulation of corneal sensory innervation.\u003c/p\u003e\n\u003cp\u003eIn addition, our data showed that the tear volume in female vs male WT mice was consistently reduced in all three strains, by ~\u0026thinsp;77%, 28% and 43% in the conventional KO, SNS-CKO and MS-CKO mice, respectively, suggesting a sex-dependent difference of tear production (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-183C modulates the functions of sensory neurons and myeloid cells through its regulation of cell type-specific target genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo start to understand the molecular mechanisms underlying miR-183C\u0026rsquo;s regulation of corneal functions, we performed 3\u0026rsquo; mRNA sequencing\u003csup\u003e88,100\u003c/sup\u003e in the TG and corneas of young adult, na\u0026iuml;ve SNS-CKO, MS-CKO and miR-183C KO mice and their corresponding WT littermate controls. Comparison of the transcriptomes identified a series of DEGs in the TG and corneas of SNS-CKO, MS-CKO and miR-183C KO mice vs their WT controls (\u003cstrong\u003eFig.S3; Tables S1-6\u003c/strong\u003e). Since miRNAs negatively regulate gene expression\u003csup\u003e35\u003c/sup\u003e, to identify tissue-specific targets of miR-183C, we searched predicted target genes of miR-183C among the upregulated genes in KO or CKO vs their WT controls in both TG and cornea. This analysis yielded a series of tissue-specific target genes of miR-183C (\u003cstrong\u003eTables S7-12\u003c/strong\u003e). Statistical analysis showed that miR-183C predicted target genes are significantly enriched among the upregulated genes in the TG and cornea of SNS-CKO or KO mice vs their corresponding WT littermate controls. However, no enrichment of\u0026nbsp;miR-183C predicted target genes was detected in the upregulated genes of either TG or cornea\u0026nbsp;of miR-183C MS-CKO mice, possibly a result of the rarity of the CRMCs in the cornea\u003csup\u003e4,7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFunctional annotation analyses of the tissue-specific target genes identified in the TG and cornea of SNS-CKO and KO mice showed similar overlapping results (\u003cstrong\u003eTables S13-20\u003c/strong\u003e). Since miR-183C is predominantly expressed in the TG sensory neurons when compared to myeloid cells (\u0026gt;\u0026thinsp;50 folds. \u003cstrong\u003eFig.S4\u003c/strong\u003e) and that CRMCs are a rare population in the cornea (1\u0026ndash;3% of total corneal cells)\u003csup\u003e4,7\u003c/sup\u003e, we reasoned that gene expression changes and miR-183C targets identified in the TG and cornea of SNS-CKO and KO mice represents TG sensory neuron-specific changes and miR-183C targets. Gene ontology (GO) analysis of the tissue-specific target genes identified in the TG of SNS-CKO and KO mice (\u003cstrong\u003eTables S13,S17,S21\u003c/strong\u003e) showed significant enrichment in neuronal axon projection-, immune response- and epithelial cell proliferation and migration-related biological processes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Consistent with the GO analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in axon guidance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), synaptic function pathways (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e; \u003cstrong\u003eTables S14,18,22\u003c/strong\u003e). In the cornea, miR-183C target genes identified in the SNS-CKO and miR-183C KO mice (\u003cstrong\u003eTable S15,16,19,20,23,24\u003c/strong\u003e) are prominently enriched in regulation of synaptic functions, in addition to axonogenesis and axon projection (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). These data collectively suggest that miR-183C regulates sensory neuronal projection and synaptic function through directly targeting genes involved in these biological processes. Besides neuronal related functions, miR-183C targets in TG sensory neurons are also enriched in epithelial cell proliferation and migration-, cell-cell adhesion-, fibroblast and endothelial cell migration-related biological processes, suggesting indirect regulation of miR-183C on other cell types in the cornea through its modulation of corneal sensory innervation (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, genes involved in\u0026nbsp;chemokine signaling pathway, e.g. CX3CL1, GNG5, GNG12, GNAI3, ADCY6, SHC1, BRAF, ELMO1, RAC1, MAP2K1, FOXO3 \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e, leukocyte trans-endothelial migration, e.g. GNAI3, MSN, PRKCA, RAC1, and Fc\u0026gamma;R-mediated phagocytosis pathways, e.g. MAP2K1, PRKCE, PRKCA, RAC1, are also enriched in miR-183C target genes of TG sensory neurons (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting potential roles in neuropeptide production and neuroimmune interactions.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eNeuronal, immune and epithelial-related GO terms enriched in miR-183C targets in TG sensory neurons\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGO ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerms\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold Enrichment\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNeuronal related\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0010976\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of neuron projection development\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0023\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEHD1, RGS2, ANKRD27, ITGA3, STMN2, FYN, CX3CL1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.17\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1902667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eregulation of axon guidance\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0428\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNOVA2, MYCBP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45.59\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0031175\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eneuron projection development\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0431\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEHD1, GDNF, SHC1, STMN2, FYN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.78\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0007411\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eaxon guidance\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0508\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKLF7, GAP43, MYCBP2, PAX6, ANK3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.58\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eImmune related\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0050729\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of inflammatory response\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0355\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCEBPA, CD47, LDLR, CX3CL1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.53\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0071560\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecellular response to transforming growth factor beta stimulus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZEB1, NOX4, FYN, WNT2, TGFBR1, WNT4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.39\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0036120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecellular response to platelet-derived growth factor stimulus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0152\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRKCE, RASA1, FYN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.78\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEpithelial related\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0010634\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of epithelial cell migration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0373\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eITGA3, PRKCE, PPM1F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.77\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0050680\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enegative regulation of epithelial cell proliferation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0279\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZEB1, CELF1, PAX6, MTSS1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.08\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eNeuronal and immune-related KEGG pathways enriched in miR-183C targets in TG sensory neurons\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKEGG ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerm\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold Enrichment\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNeuronal related\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04725\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCholinergic synapse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0124\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGNA11, KCNJ14, FYN, GNG12, ADCY6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04724\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlutamatergic synapse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0132\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGRIA1, PPP3R1, SLC1A1, GNG12, ADCY6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.39\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04722\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNeurotrophin signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP3K3, SHC1, PSEN2, BRAF, FOXO3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.08\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04360\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAxon guidance\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0574\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePPP3R1, RASA1, NCK2, FYN, WNT4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.39\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eImmune related\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChemokine signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0189\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSHC1, BRAF, FOXO3, GNG12, CX3CL1, ADCY6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eNeuronal, cellular adhesion and migration-related GO terms enriched in miR-183C targets in the cornea of SNS-CKO and KO mice\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGO ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerm\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold Enrichment\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNeuronal function related\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0010976\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of neuron projection development\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.21E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eANKRD27, FUT9, AVIL, DAB2IP, CNTN1, FYN, PRKD1, CX3CL1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2000300\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eregulation of synaptic vesicle exocytosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002954\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNRN1, SV2C, PRKCE, PRKCA, CACNA1E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.38\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0001764\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eneuron migration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.004744\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMEF2C, TRIM46, FYN, FAT3, RAC1, CELSR3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0048813\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003edendrite morphogenesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.005398\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKLF7, ELAVL4, FYN, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0030030\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecell projection organization\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.014212\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUBXN10, RAB34, GPR22, TSC1, FAM149B, TAPT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.19\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0030517\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enegative regulation of axon extension\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0173\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTRIM46, AATK, RTN4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.75\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0048812\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eneuron projection morphogenesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.022254\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, ANKRD27, DAB2IP, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.63\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0010977\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enegative regulation of neuron projection development\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.024328\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTSC1, HES1, RTN4, PTPRG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.41\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0048870\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecell motility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.030182\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, ELMO1, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.96\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0050772\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of axonogenesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.033101\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eROBO2, ZEB2, MAP2K1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.43\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0007411\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eaxon guidance\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.044784\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eROBO2, KLF7, CNTN1, UNC5D, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.73\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCellular adhesion and migration related\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0007155\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecell adhesion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.001191\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCD164, PRKCE, FIBCD1, PRKCA, CX3CL1, PCDH17, CELSR3, CNTN1, HES1, FAT3, TGFBI, RAC1, DGCR2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0010595\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of endothelial cell migration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.78E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eANXA3, PDCD6, PRKCA, PRKD1, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.31\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0010764\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enegative regulation of fibroblast migration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002543\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZEB2, RAC1, CYGB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38.87\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0022409\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epositive regulation of cell-cell adhesion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.01618\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOX2, MAGI1, PDE4D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.27\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eNeuronal and immune-related KEGG pathways enriched in miR-183C targets in the cornea of SNS-CKO and KO mice\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKEGG ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerm\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold Enrichment\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eNeuronal and synaptic function related\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04360\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAxon guidance\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0011\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eROBO2, GNAI3, FYN, PRKCA, UNC5D, RAC1, SSH2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04725\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCholinergic synapse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0060\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, GNG5, GNAI3, FYN, PRKCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.74\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04726\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSerotonergic synapse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0103\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, GNG5, DUSP1, GNAI3, PRKCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.76\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04921\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOxytocin signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0174\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, MEF2C, CACNA2D1, GNAI3, PRKCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.94\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04727\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGABAergic synapse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0201\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGABARAPL2, GNG5, GNAI3, PRKCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.79\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04713\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCircadian entrainment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0258\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGNG5, GNAI3, PRKCA, GRIA3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04724\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlutamatergic synapse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0380\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGNG5, GNAI3, PRKCA, GRIA3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.30\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eImmune function related\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChemokine signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0081\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, GNG5, ELMO1, GNAI3, RAC1, CX3CL1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.72\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04666\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFc gamma R-mediated phagocytosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0225\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, PRKCE, PRKCA, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04670\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLeukocyte transendothelial migration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0414\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGNAI3, MSN, PRKCA, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.12\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTo further identify myeloid cell-specific target genes of miR-183C, we isolated the Csf1r-EGFP\u0026thinsp;+\u0026thinsp;CRMCs from young adult miR-183C MS-CKO and conventional KO mice and their age- and sex-matched WT control mice. KEGG pathway analyses of miR-183C targets identified in CRMCs of both MS-CKO and conventional KO (\u003cstrong\u003eTables S25\u0026amp;S26\u003c/strong\u003e) are consistently enriched in multiple immune/inflammation-related pathways (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; \u003cstrong\u003eTable S27\u003c/strong\u003e), including Fc\u0026gamma;R-mediated phagocytosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA), chemokine signaling (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB), Fc\u0026epsilon;RI signaling, TGF\u0026beta; signaling pathway,\u0026nbsp;TNF signaling pathways, and several microbial infection pathways (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; \u003cstrong\u003eTable S27\u003c/strong\u003e). These data suggest that miR-183C regulates the functions of myeloid cells through simultaneously targeting multiple genes in different pathways. Intriguingly, miR-183C targets are also enriched in neuronal function, especially synaptic function-related pathways (\u003cstrong\u003eTable S28\u003c/strong\u003e), and cell-cell interaction pathways (\u003cstrong\u003eTable S29\u003c/strong\u003e), suggesting potential indirect modulation of neuronal innervation and other cell types of the cornea.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eImmune/infection-related KEGG pathways enriched in miR-183C targets of CRMCs of KO and MS-CKO mice\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKEGG ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerm\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold Enrichment\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBacterial invasion of epithelial cells\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.98E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eITGB1, ACTR2, CTTN, SHC1, CAV1, ARPC1B, RAC1, WASL, CRK, WASF2, CD2AP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.12\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04666\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFc gamma R-mediated phagocytosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.87E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eACTR2, MAP2K1, MARCKS, PAK1, PRKCE, ARPC1B, MAPK1, ASAP1, GAB2, RAC1, CRK, WASF2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.67\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04664\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFc epsilon RI signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002041\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K3, MAPK9, MAP2K1, MAPK1, GRB2, FYN, KRAS, GAB2, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.88\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05135\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYersinia infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002728\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eITGB1, MAP2K3, ACTR2, GIT2, MAP2K1, ARHGEF12, ARPC1B, WASL, MAPK9, MAPK1, RAC1, CRK, WASF2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.74\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChemokine signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.003045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K1, SHC1, GNAI3, BRAF, GNG12, PRKCZ, ADCY6, TIAM1, PAK1, GNG5, MAPK1, GRB2, KRAS, RAC1, CRK, PRKACB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.37\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05166\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman T-cell leukemia virus 1 infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.006333\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eATF2, MAP3K3, EGR1, MAP2K1, CRTC1, XIAP, TGFBR1, ADCY6, PPP3CA, MAPK9, ZFP36, CCND2, CREB3L1, CREB3L2, MAPK1, EP300, KRAS, PRKACB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05132\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSalmonella infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.007681\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K3, ACTR2, RALA, MAP2K1, ARPC1B, WASL, PIK3C2A, RHOB, MAPK9, SNX18, PAK1, BCL2, EXOC4, MAPK1, RAC1, PFN1, KPNA3, PFN2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.02\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05163\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman cytomegalovirus infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.007979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eATF2, MAP2K1, ARHGEF12, GNAI3, TSC1, GNG12, ADCY6, PPP3CA, GNG5, GNA11, CREB3L1, CREB3L2, MAPK1, GRB2, KRAS, RAC1, CRK, PRKACB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.02\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05165\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman papillomavirus infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.015215\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAGI1, ITGB1, PRKCI, MAP2K1, ITGA3, TSC1, PPP2R5C, LAMC1, PRKCZ, PPP2CA, PPP2CB, CCND2, CREB3L1, CREB3L2, MAPK1, EP300, GRB2, HES1, KRAS, WNT2, PRKACB, WNT4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.74\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04650\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNatural killer cell mediated cytotoxicity\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.020466\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePPP3CA, MAP2K1, PAK1, SHC1, MAPK1, GRB2, FYN, BRAF, KRAS, RAC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.45\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05145\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eToxoplasmosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.037313\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eITGB1, MAP2K3, MAPK9, GNAI3, BCL2, XIAP, MAPK1, LAMC1, LDLR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.35\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04350\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTGF-beta signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.039069\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eACVR1, PPP2CA, SMAD1, PPP2CB, EP300, MAPK1, SKIL, TGFBR1, SMAD7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu05170\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman immunodeficiency virus 1 infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.040389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K3, MAP2K1, FBXW11, GNAI3, GNG12, PPP3CA, MAPK9, PAK1, GNG5, GNA11, BCL2, MAPK1, KRAS, RAC1, CRK\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.79\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emmu04668\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTNF signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.048672\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMAP2K3, CYLD, MAPK9, ATF2, MAP2K1, CREB3L1, CREB3L2, XIAP, MAPK1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.23\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo delineate the cell type-specific functions of miR-183C in corneal sensory nerves and innate immune cells, we created and characterized a SNS- and a MS-CKO mouse models, in comparison to the conventional KO mice. Our data showed that inactivation of miR-183C in corneal nerves, but not in innate immune cells, resulted in decreased corneal sensory nerve density and reduced sensitivity to mechanical stimuli, suggesting intrinsic regulation of miR-183C on corneal sensory innervation. Our data showed that, in the SNS-CKO mice, the reduction of sensory nerve density occurs specifically in the fine, terminal neurites in the epithelial layer, including the subbasal plexus, while the large-diameter nerves in the stromal layer remain unaffected, suggesting a critical role of miR-183C in the terminal differentiation and projection of corneal sensory nerves. This is consistent with its functions in the other sensory organs, e.g. the photoreceptors of the retina\u003csup\u003e51,54\u003c/sup\u003e and the inner ear hair cells\u003csup\u003e101,102\u003c/sup\u003e, as well as myeloid cells\u003csup\u003e59\u003c/sup\u003e, suggesting a common feature of miR-183C regulating terminal functional differentiation of primary sensory neurons and innate immune cells. Consistent with this observation, our RNA seq data showed that target genes of miR-183C in the TG were significantly enriched with genes involved in axonal guidance and neuronal projection regulation as well as synaptic functions, suggesting that miR-183C regulates corneal sensory nerve functions by directly targeting neuronal projection and synaptic release-related genes. Multiple genes in the same pathway are simultaneously targeted. This exemplifies the mode of action of miRNAs \u0026ndash; quantitatively regulating multiple genes in the same pathway. Their effect on each individual gene may not cause a major phenotypic change; however, working in concert, they impose significant functional consequence\u003csup\u003e7,103\u0026ndash;106\u003c/sup\u003e. In this case, the simultaneous regulation of multiple genes in the neuronal projection and synaptic functional pathways contributes to the decreased sensory nerve density and levels of neuropeptides in the cornea of the SNS-CKO and miR-183C KO mice, which affects the neuroimmune response of the cornea to bacterial infection\u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMS-CKO of miR-183C resulted in increased CRMCs, suggesting intrinsic regulation of miR-183C in CRMCs. Our comparative study of the transcriptomes of CRMCs of miR-183C KO and MS-CKO mice vs their WT controls revealed myeloid-specific target genes of miR-183C, which are significantly enriched in a range of immune/infection related pathways, including chemokine signaling and phagocytosis pathways. These data provide new insight into the mechanisms of intrinsic regulation of miR-183C on the functions of innate myeloid cells, e.g. cytokine production and phagocytosis and intracellular killing capacity that we reported previously\u003csup\u003e7,60,61\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIntriguingly, SNS-CKO of miR-183C also led to increased number of CRMCs in na\u0026iuml;ve mouse cornea, suggesting that normal function of miR-183C in sensory nerves has an extrinsic regulation on the establishment of CRMCs, indicative of neuroimmune interaction in the maintenance of the homeostasis of the cornea. KEGG pathway analysis of miR-183C target genes in TG sensory neurons showed significant enrichment of genes involved in chemokine signaling pathway, including Cx3cl1. Neuron-produced chemokine Cx3cl1, also called fractalkine, is known to promote chemotactic migration of microglia in the central nervous system\u003csup\u003e107,108\u003c/sup\u003e, mediate the homing of resident myeloid cells in the cornea\u003csup\u003e109\u003c/sup\u003e and recruitment of macrophages in other tissues\u003csup\u003e110\u003c/sup\u003e. Meanwhile, Cx3cl1 has also been shown to enhances neuron adhesion to extracellular matrix and reduce neuronal migration \u003csup\u003e111\u003c/sup\u003e. These observations support a hypothesis that inactivation of miR-183C disinhibits Cx3cl1 in the TG sensory neurons and resulted in increased expression of Cx3cl1 in the cornea of SNS-CKO mice; then elevated level of Cx3cl1 may enhance the recruitment of myeloid cells to the cornea and result in increased number of the corneal resident myeloid cells. Additional studies are warranted to further test this hypothesis.\u003c/p\u003e \u003cp\u003eFurthermore, our RNA seq data showed that miR-183C target genes in TG sensory neurons are also enriched in pathways regulating other corneal cell types, e.g. epithelial, endothelial cell and fibroblast migration; while target genes in CRMCs are also enriched in neuronal synaptic functions and cell-cell interaction and migration pathway. These data suggest that, in addition to the intrinsic regulation of sensory innervation and CRMCs, miR-183C exerts extrinsic regulation on neuro-immune-epithelial-stromal-endothelial interactions. This is consistent with our recent report of a single-cell transcriptome study that miR-183C serves as a checkpoint of corneal resident immune cells and shapes the cellular landscape of the cornea\u003csup\u003e4\u003c/sup\u003e. Different cell types form the microenvironment or niche of the cornea and work in concert to endow the cornea with its unique architecture and functionalities and maintain homeostasis\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. Perturbation of the functions of one cell type imposes global impact on other cell types and the overall function of the cornea in both physiological and various pathological conditions\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor the first time, here we discovered that both na\u0026iuml;ve miR-183C KO and SNS-CKO, but not the MS-CKO mice, had significantly decreased tear volume, when compared to their age- and sex-matched WT controls. These data suggest miR-183C enhances basal tear production through its regulation of corneal sensory innervation. It is known that the primary afferent sensory neurons innervating the cornea play important roles in the basal tear production and secretion evoked by noxious stimulation of the cornea through the brainstem tear reflex arc \u003csup\u003e27\u003c/sup\u003e. Corneal sensory neurons sense dry condition and other environmental stressors, send primary afferent projections to activate the neurons in the spinal trigeminal nucleus (Vsp) and regulates lacrimation through the efferent autonomic arm of the reflex arc, which include the preganglionic parasympathetic neurons in/around the superior and inferior salivatory nuclei (SSN and ISN) and postganglionic parasympathetic neurons in the pterygopalatine ganglion (PG) \u003csup\u003e27\u003c/sup\u003e. The autonomic reflex promotes the production of the watery component of the tears by the lacrimal glands; mucin component of the tears by the conjunctival goblet cells, and lipid content by the meibomian glands\u003csup\u003e27\u003c/sup\u003e. We speculate that decreased sensory innervation caused by loss of miR-183C in sensory neurons imposes a negative impact on proper lacrimation, and therefore, miR-183C may play a role in dry eye disease (DED). Further studies will be needed to confirm this hypothesis.\u003c/p\u003e \u003cp\u003eTo our surprise, we observed significant decrease of basal tear volume in female vs male mice in all three strains used in this study, which are in a mixed genetic background of C57BL/6 and129S\u003csup\u003e51\u003c/sup\u003e. It is well known that DED is more prevalent in women compared to men\u003csup\u003e112\u0026ndash;122\u003c/sup\u003e, especially in the autoimmune DED, Sj\u0026ouml;gren\u0026rsquo;s syndrome\u003csup\u003e123\u003c/sup\u003e. Over 90% of all patients with Sj\u0026ouml;gren\u0026rsquo;s syndrome are women\u003csup\u003e123\u003c/sup\u003e. The female gender is considered a risk factor for the development of DED\u003csup\u003e112\u003c/sup\u003e. Increased inflammation in lacrimal glands has been reported in several mouse models of Sj\u0026ouml;gren\u0026rsquo;s syndrome\u003csup\u003e124\u003c/sup\u003e. Data from rats suggest that gender has significant influence on lacrimal gland structure during development and aging\u003csup\u003e125\u003c/sup\u003e. The acinar area of the lacrimal glands of males are reported to be larger than that of females in multiple species including human\u003csup\u003e126\u003c/sup\u003e. Sex hormones modulates lacrimal gland morphology, tear volume and secretory components and protein contents of the tears\u003csup\u003e120\u0026ndash;122,126\u0026minus;128\u003c/sup\u003e. However, sex-related difference in basal tear volumes has not been reported in either humans or animals \u003csup\u003e120,125,128\u003c/sup\u003e. Our observation of decreased tear volume in female mice appears to be in-line with the fact that female gender has been considered a risk factor for the development of DED\u003csup\u003e112\u003c/sup\u003e. The discrepancy between our observation and others \u003csup\u003e125,129\u003c/sup\u003e could come from different species and/or strains of animals as well as different methodology of measurement\u003csup\u003e130\u003c/sup\u003e. Nevertheless, our data suggest a sex-dependent difference in basal tear volume and further studies are warranted.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first example that a miRNA cluster plays significant, functional roles in the corneal homeostasis through direct regulations of both sensory innervation and CRMCs and modulation of neuroimmune interaction. The SNS-CKO and MS-CKO mouse models developed in this report provide critical tools to fully uncover the roles of miR-183C in sensory nerves or myeloid cells under different pathological conditions, e.g., bacterial keratitis and DED. Such knowledge will be essential to develop cell type-specific strategies targeting miRNAs for the treatment of corneal diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by grants from the National Eye Institute, National Institutes of Health (R01 EY026059 to SX; R01 EY016058, R01 EY035231,and P30 EY004068 to LDH); a Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology, Visual and Anatomical Science, Wayne State University School of Medicine; and the Bridge fund from the Office of Vice President for Research (OVPR) of the Wayne State University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.G., M.S., K.G., G.L., H.Z., S.M., A.P., M.S., M.F.A.S. conducted experiments and data collection. N.G., G.L. S.X. performed data analyses. L.D.H participated in experimental design and manuscript revision. S.X. conceived, designed and directed the entire study, conducted experiments, data collection, wrote, revised and submitted the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets of 3\u0026rsquo; RNA sequencing generated during the current study are available in the Gene Expression Omnibus (GEO) repository (accession number: GSE250213). All data and protocols generated in this study are available upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLwigale, P. Y. 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To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contribute to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult na\u0026iuml;ve mice; 3\u0026rsquo;RNA sequencing (Seq) in the trigeminal ganglion (TG), cornea and CRMCs.\u003c/p\u003e \u003cp\u003eOur results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.\u003c/p\u003e","manuscriptTitle":"The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell-type-specfic target genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-11 15:31:44","doi":"10.21203/rs.3.rs-3678621/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvited","content":"","date":"2023-12-22T10:15:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-22T09:48:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-11-28T22:49:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fabef7a4-65a6-4d1e-9ceb-e6fede6f5b68","owner":[],"postedDate":"January 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27864071,"name":"Biological sciences/Cell biology"},{"id":27864072,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":27864073,"name":"Biological sciences/Immunology"},{"id":27864074,"name":"Biological sciences/Molecular biology"},{"id":27864075,"name":"Biological sciences/Neuroscience"},{"id":27864076,"name":"Biological sciences/Systems biology"},{"id":27864077,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":27864078,"name":"Health sciences/Diseases/Eye diseases/Corneal diseases"}],"tags":[],"updatedAt":"2024-04-08T15:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-3678621","link":"https://doi.org/10.1038/s41598-024-58403-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-04-01 15:00:59","publishedOnDateReadable":"April 1st, 2024"},"versionCreatedAt":"2024-01-11 15:31:44","video":"","vorDoi":"10.1038/s41598-024-58403-1","vorDoiUrl":"https://doi.org/10.1038/s41598-024-58403-1","workflowStages":[]},"version":"v1","identity":"rs-3678621","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3678621","identity":"rs-3678621","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}