Gata3 is Required in Late Proneurosensory Development for Proper Sensory Cell Formation and Organization

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Blinkiewicz, Makayla R. Long, Zachary A. Stoner, Elizabeth M. Ketchum, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2747944/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Aug, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract It has been previously shown that zinc-finger transcription factor Gata3 has dynamic expression within the inner ear throughout embryonic development and is essential for cochlear neurosensory development. However, the temporal window to which Gata3 is required for the formation of the cochlear neurosensory epithelia remains unclear. To investigate the role of Gata3 on cochlear neurosensory development in the late prosensory stages, we used the Sox2-cre ERT2 mouse line to target and conditionally delete Gata3 at E11.5 before the cells have fully committed to a neurosensory fate. While the inner ears of Sox2-cre ERT2 : Gata3 f/f mice appear morphologically normal, the sensory cells in the organ of Corti are partially lost and disorganized in a basal to apical gradient with the apex demonstrating the more severe phenotype. Additionally, spiral ganglion neurons display aberrant peripheral projections, such as increased distances between radial bundles and disorganization upon reaching the organ of Corti. Furthermore, heterozygous Sox2-cre ERT2 : Gata3 f/+ mice show a reduced phenotype in comparison to the homozygous mutant, supporting the concept that Gata3 is not only required for proper formation at the later proneurosensory stage, but also that a specific level of Gata3 is required. Therefore, our studies confirm that Gata3 plays a time-sensitive and dose-dependent role in the development of sensory cells in the late proneurosensory stages. Biological sciences/Cell biology Biological sciences/Developmental biology Biological sciences/Neuroscience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The mammalian inner ear is comprised of six unique sensory organs, but only one of these organs is responsible for the sense of hearing: the cochlea. The cochlea contains the organ of Corti (OC) which is comprised of mechanosensory hair cells (HCs) and their corresponding supporting cells (SCs). HCs transduce sound energy into electrical impulses via innervation by spiral ganglion neurons (SGNs) that project into the hindbrain for further auditory processing. The development of these three sensory cell types has been extensively studied, but there are still gaps in knowledge regarding the transcription factors and gene networks that control the spatial and temporal aspects of this process at later proneurosensory stages. The inner ear is derived from the otic placode, which will invaginate to form the otic cup before developing into the otocyst around embryonic day 9 (E9) 1 , 2 . While several transcription factors are important for neurosensory development in this time frame, the zinc-finger transcription factor Gata3 is particularly interesting due to its dynamic expression throughout development. While Gata3 is initially expressed as early as E8.5 throughout the otocyst, by E10.5 its expression is restricted to the proneurosensory regions 3 – 9 . Gata3 continues to be expressed in SGNs until postnatal day 14 (P14) and remains highly expressed in SCs, with lower levels in HCs, throughout adulthood 10 – 14 .Therefore, it has been postulated that Gata3 plays an important and dynamic role in inner ear development and neurosensory cell formation during this embryonic temporal window. Previous studies have shown that loss of Gata3 in the early proneurosensory region around E8.5 leads to loss of all cochlear neurosensory cells 5 , 15 , while loss of Gata3 one day later around E9.5 leads to a patchy loss of HCs and SCs, and disorganization and patchy loss of SGNs. Other studies have investigated the role of Gata3 postnatally in the maintenance of HCs and SCs 11 , 12 . These studies found that Gata3 is necessary later on to maintain OHCs and to functionally develop IHCs, while loss of Gata3 from postnatal SCs results in an increase in some types of SCs through downregulation of other genes. However, there exists a gap in knowledge about the role of Gata3 later in embryonic development as the proneurosensory cells start to differentiate into HCs, SCs, and SGNs. Specifically, it remains to be seen how long expression is required for proper embryonic development of neurosensory cells before Gata3 switches to its postnatal maintenance role. Additionally, while we know the presence of Gata3 is necessary for proper neurosensory development, the precise level of Gata3 expression is also important for maintenance and function. For example, both Gata3 haploinsufficiency and Gata3 over-expression, as a result of gene duplication, cause human hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome 16 – 21 . While the triad of symptoms of HDR syndrome range in severity, nearly all patients exhibit deafness 16 , 19 , 22 , 23 . Uniquely, deafness is the only symptom of HDR syndrome which can present singularly 16 , 19 , 21 , 23 . This suggests that not only is continued expression of Gata3 required for proper inner ear development, but specific levels of Gata3 are also required. Continued investigation of the dose-dependent requirements of Gata3 would also contribute to the fields’ overall understanding of inner ear gene regulatory networks. Therefore, in this study, we explored the window of developmental plasticity which is governed by Gata3 as a follow up to previous studies showing its loss is detrimental to cochlear neurosensory epithelia 5 – 7 , 10 , 21 , 24 , 25 . Using the Sox2-cre ERT 2 mouse line 26 , we conditionally deleted Gata3 from proneurosensory cells via tamoxifen (TMX) injection at E11.5. Our results show that deletion of Gata3 causes severe loss and disorganization of HCs, SCs, and SGNs in a basal to apical gradient, with a more severe phenotype presenting in the apex. Interestingly, the mutant ears were morphologically normal in that it presented with a full-length cochlea unlike previous Gata3 deletion studies. Overall we show that, while Gata3 is not necessary at E11.5 for overall morphological development and elongation of the cochlea, however, Gata3 is required in the late proneurosensory stages for cochlear sensory epithelia and SGNs both to form and organize properly. Results Gata3 is deleted from HCs, SCs, and SGNs at E11.5 Previous studies have characterized Sox2-cre ERT 2 expression at the placode stage (E8.5), otocyst stage (E10.5), and the late otocyst stage (E12.5) 27 – 32 . At E10.5, Sox2 is present in both the nonsensory cochlear floor and roof 31 . By E12.5, Sox2 is exclusively expressed in OC sensory cells 31 . In order to confirm complete knockout of Gata3 from HCs, SCs, and SGNs, in situ hybridization was performed using a Gata3 riboprobe. While the control shows high expression of Gata3 in all cell types from base to apex, the homozygous mutant shows no expression in the HCs and SCs and greatly reduced expression in the SGN cell bodies (Fig. 1 A-D’), demonstrating that our model is indeed reducing levels of Gata3 in the cell types of interest. Gata3 is required for sustained formation and organization of HCs We first analyzed the effect of deletion of Gata3 on HCs, as previous Gata3 CKOs show either no HC development or only patches of HCs 5 – 7 , 10 . In order to assess the phenotype of the deletion of Gata3 , two different controls were used: Gata3 f/f (Fig. 2 A-A”) and Sox2-cre ERT 2 (Fig. 2 B-B”). Other studies have previously demonstrated that the knock-in Sox2-cre ERT 2 line shows inner hair cell (IHC) duplets, which was confirmed in our study (Fig. 2 B-B’’; white circles). It was important to investigate the IHC duplets in the heterozygous mutant compared to the Sox2-cre ERT 2 control to ensure that the phenotype seen is not the result of using this Cre line (Fig. 2 C-C”). While the base, middle and apex of the heterozygous mutant all contain IHC duplets similar to the Sox2-cre ERT 2 control, it should be noted that the third row of outer hair cells (OHCs) is lost in the middle region of the OC into the apical region (Fig. 2 C-C”). We found that the heterozygous genotype shows a continuous formation of HCs from base to apex, while the homozygous mutant shows some disturbances in the apical region, similar to the previous Gata3 CKO study that showed the presence of patches 6 . The homozygous mutant also shows a worsening phenotype compared to the heterozygous mutant. While the base contains all three rows of OHCs, progressive rows of OHCs are lost (Fig. 2 D-D’). Just two rows of OHCs are present in the middle region and almost no rows of OHCs are present in the apex (Fig. 2 E-F”). Additionally, the entire cochlea contains Myosin VIIa positive cells in the GER with the highest number appearing in the apex, which is similar to a postnatal Gata3 CKO from SCs using this same Cre line 11 . Furthermore, these cells in the GER are associated with SGN endings. Ectopic HCs have been seen in the GER in both CKO and over-expressor models previously 33 – 38 . While ectopic HCs generally are not seen in combination with missing rows of OHCs, previous studies have shown that loss of Gata3 results in missing OHCs postnatally 12 , 21 . The phenotype of both ectopic HCs and missing rows of OHCs as a result of embryonic loss of Gata3 is unique and further supports a role for Gata3 in this specific temporal window in this specific cell type. Gata3 is required for corresponding SC formation and organization Previous studies that have deleted Gata3 have shown either no SC development or only limited patches of SC formation localized to the HC patches 5 , 6 , 10 . However, we still observe SCs in our model throughout the majority of the length of the cochlea. Similar to the HC phenotype in this model, we found that the homozygous mutant shows almost continuous formation of SCs, except for some patches in the apex (Fig. 3 ). The apex also shows disorganization of the SC rows. The homozygous mutant shows a worsening phenotype compared to the heterozygous mutant, similar to that seen in the HCs. The base and middle show disorganized SCs and complete loss of some outer SC rows in the middle region. The apex contains the most severe phenotype in which SC appear to cluster together which is very similar to the SC phenotype seen in other Gata3 CKO studies 10 . Ultimately, the phenotype in the HCs and SCs are consistent in their appearance of progressive loss of OHCs from base to apex, mirroring the loss of SCs from base to apex in the homozygous mutant. While this SC disorganization in our model is also similar to the phenotype seen in other Gata3 CKO studies, it is important to note that the previous study did not also observe ectopic HCs in the GER 10 . Further studies are needed in order to tease apart the specific requirement for Gata3 within this specific time window to determine if Gata3 deletion in one cell population can influence another cell population. Gata3 Is Required For Organization Of Sgn Peripheral Projections Our observation of Myosin VIIa positive cells associated with SGN endings in the homozygous mutant led us to investigate the peripheral projections of SGNs to confirm that they were developing properly. Previous studies examining the effect of Gata3 deletion from the proneurosensory region of the developing otocyst observed a severe reduction in the number of SGNs present in mutant samples, while the SGNs that did form displayed aberrant projection patterns towards the developing OC 6 , 10 . Another study in which Gata3 deletion was restricted to SGNs saw proper formation of SGNs with disorganized peripheral projections 14 , 24 . We first examined peripheral projections in a Sox2-cre ERT 2 mutant sample to establish whether the Cre knock-in displays a SGN phenotype. When compared with control samples (Fig. 4 D-D”), we saw no obvious difference in SGN number or organization (data not shown). We then looked at the peripheral projections in a heterozygous mutant. The number and overall organization of SGNs in the heterozygous mutant largely resemble control samples in the base and middle (Fig. 4 E-E’). However, the radial bundles in the apex of the heterozygous mutant appear to have an increased area separating them relative to the control (F”). The homozygous mutant has a striking phenotype that displays an increase in the distance between radial bundles as well aberrant projections of the radial bundles which becomes progressively more disorganized along the length of the cochlea (Fig. 4 F-F”). The mutant base and middle (Fig. 4 F-F’) reveal irregular distances between radial bundles in addition to extra branches from radial bundles. There are also inconsistencies in their organization, as some gaps are large and others are reduced (Fig. 4 C). This phenotype is even more profound in the apex (Fig. 4 F”). The area between the radial bundles in the apex of control and homozygous mutant samples was measured and quantified (Fig. 4 J). The method for radial bundle distance quantification can be found as Supplementary Fig. S1 online. Analysis of the data showed a statistically significant increase in the distance between radial bundles in the homozygous mutant relative to the control (p < 0.0001). Additionally, the values for the area in mutant samples was highly variable, further supporting that loss of Gata3 results in disorganization of peripheral projections of SGNs. We also examined the peripheral projections where the neurites reach the OC (Fig. 4 G-I”). The basal region of the heterozygous mutant is comparable to the control (Fig. 4 G-H), but peripheral projections are progressively fewer and become disorganized in the heterozygous mutant with progression to the middle and apex (Fig. 4 G’-G”, H’-H”). Peripheral projections in the base of the homozygous mutant appear slightly disorganized upon reaching the OC. Additionally, the density of neurites in the homozygous mutant appears to be less when compared to the base of the control (Fig. 4 I). The disorganization of the neurites and decreased density is even more pronounced in the middle and apex of the homozygous mutant (Fig. 4 I’-I”). Fewer neurites project into the OHC region of the OC in the middle and few-to-no neurites project to the OHC region in the apex. In these regions, not all neurites that are present within the OHC region properly turn towards the base but rather, turn towards the apex. Based upon our results, Gata3 expression is important for the formation of radial bundles with regards to appropriate density and distance between bundles, as well as for proper branching patterns and overall organization. Additionally, Gata3 is needed for peripheral neurites to reach the OC, particularly the OHC region, and to form proper connections with HCs. Importantly, the loss of Gata3 has a phenotype that progressively worsens along the length of the cochlea, with the greatest phenotype observed in the apex. Gata3 Is Required For Proper Central Pathfinding Of Sgns Given that homozygous mutants display aberrant peripheral projections of SGNs, with the phenotype progressively getting more severe in a basal to apical manner (Fig. 4 ), we next investigated whether central projection of SGNs to the cochlear nucleus (CN) was also affected. Previous studies examining the role of Gata3 in spiral ganglion neuron central pathfinding have shown varied results depending on the location and timing of Gata3 deletion 6 , 24 . Early deletion of Gata3 throughout the entire inner ear at E9.5 results in central SGN fibers bifurcating at several branch points, with terminal fibers projecting non-specifically throughout the CN 6 . However, deletion of Gata3 within delaminated SGNs at E9.5 results in normal projection of SGNs within the CN with tonotopy maintained 24 . Taken together these two studies suggest that Gata3 may be affecting SGN neuron central pathfinding in a cell non-autonomous and time-dependent manner. In order to investigate this further, lipophilic dyes were applied to the base (red) and apex (green) of Sox2-cre ERT 2 control, as well as heterozygous and homozygous mutant cochlea (Fig. 5 A) to visualize the projections of SGNs into the CN. Sox2-cre ERT 2 control SGNs entered the hindbrain and bifurcated sending ascending and descending process towards the anteroventral cochlear nucleus (AVCN) and dorsal cochlear nucleus (DCN)/posteroventral cochlear nucleus (PVCN) respectively (Fig. 5 B). Sox2-cre ERT 2 control SGNs remained segregated with basal fibers extending more dorsally and apical fibers more ventrally (Fig. 5 B). This stereotyped central wiring was also maintained in heterozygous (Fig. 5 C) mice. In contrast SGNs in homozygous mice display less segregation between apical and basal fibers. Apical fibers often project more dorsally into spaces occupied by basal fibers. Additionally, some apical fibers upon reaching the hindbrain project outside of cranial nerve VIII into areas outside of the CN (Fig. 5 D). These results provide further evidence for the idea that Gata3 expression plays an important role in the development and wiring of SGNs centrally. Our data along with previous studies 6 , 24 suggest that Gata3 is acting in a cell non-autonomous manner at or before E11.5 to promote proper central wiring of SGNs. Further investigations are needed to elucidate what cell populations require early Gata3 expression in order to promote proper central pathfinding of SGNs. Gata3 deletion at E11.5 results in full morphologic development of the cochlear duct and vestibular system, but shows progressive neurosensory epithelial loss and disorganization Previous Gata3 deletion studies have shown a variety of phenotypes that include morphologic and cochlear neurosensory epithelia defects 5 – 7 , 10 , 21 , 24 , 25 . Gata3 null mice display a severely truncated cochlear and vestibular system which were devoid of sensory epithelia except for a small patch of HCs and SGNs in a portion of the saccule 5 , 7 . Gata3 deletion at E8.5 using the Foxg1-cre mouse line resulted in a truncated cochlea which contained no HCs and abnormal morphologic development of the vestibular system 6 . Gata3 deletion at E9.5 using the Pax2-cre mouse line resulted in similar morphologic defects including a truncated cochlea and abnormal vestibular system. However, deletion at E9.5 resulted in patchy sensory cell development of HCs, SCs, and SGNs 6 , 10 . In studies that have conditionally deleted Gata3 from only SGNs, HCS and SCs form properly 24 , 25 . We contribute results for Gata3 deletion at E11.5, a time in development in which proneurosensory cell differentiation is occurring. Our findings show that Gata3 deletion at E11.5 results in a morphologically sound structure with a full length cochlea and well developed vestibular system (data not shown). Within the cochlea, the sensory cells in the OC are mostly present and have a varying phenotype depending on the cochlear region. In the homozygous mutant cochlear base, HCs and SCs are present with only mild disorganization (Fig. 6 ), while the homozygous mutant basal radial bundles have larger spacing than normal but the neurons are well organized. This contrasts the phenotype seen in the apex since the peripheral projection density of the mutant apex is decreased and those projections which are present appear disorganized (Fig. 6 ). Additionally, the tonotopy of SGN central projections is maintained within the CN in both heterozygous and homozygous mutants (Fig. 5 ). In comparison, the mutant apical HCs are severely reduced to patchy clusters with some ectopic HCs that appear in the GER, while the apical SCs are not organized in rows and instead cluster together (Fig. 6 ). Our data demonstrates a role for Gata3 in all neurosensory cells after their initial specification. Discussion Gata3 was previously shown to be necessary for both proper morphology and cochlear neurosensory epithelia early in development when its expression is high throughout the entire otocyst 5 – 7 , 10 , 21 , 39 . However, the role of Gata3 in HC, SC, and SGN formation after its restriction to the proneurosensory region was unknown. Our study reveals novel findings that Gata3 plays both a dose-dependent and necessary role in the formation and organization of neurosensory cell types, but does not have an impact on the overall morphology of the inner ear at this specific developmental time point. This project contributes new knowledge about the role of Gata3 on proneurosensory epithelia formation in a temporal window that fills a gap between previous studies that investigated Gata3 deletion. From our results we find that deletion of Gata3 from the proneurosensory domain at E11.5 results in a fully formed cochlear duct (data not shown). Regardless of the single or dual loss of Gata3 alleles, both ears formed morphologically normal cochleas. Therefore, Gata3 is not required for morphologic development at E11.5. Given that previous Gata3 deletion studies did not see normal morphology of the cochlea 5 , 6 , it is intriguing that deletion of Gata3 about two days later results in a morphologically sound inner ear with a fully formed cochlea. While HCs, SCs and SGNs do form, they are highly disorganized and this phenotype progressively worsens from base to apex. In Gata3 heterozygous null mice, studies have found that OHC loss occurs without Gata3 12,21,40 . This phenotype is mirrored in our study, despite the difference in timing of which Gata3 is deleted. It is also noteworthy that the heterozygous mutant had a subtler phenotype compared to the homozygous mutant, suggesting that precise levels of GATA3 are needed for proper formation and organization of the proneurosensory epithelia. If precise levels of Gata3 are truly necessary, then increased levels of Gata3 should also have a phenotype in our model. Several other over-expressor studies have been previously studied that demonstrated ectopic HCs in the GER 33 – 36 , 38 . Previous studies have even used the Gata3 over-expressor model in combination with upregulation of other sensory genes in order to increase the efficiency of ectopic HC formation 35 , 36 . Investigating the over-expression of Gata3 in this model would be useful in determining the detrimental effects, if any, of higher levels of Gata3 in the cochlea. While this would further elucidate the specific role of Gata3 in the cochlea, the investigation of Gata3 over-expression is especially pertinent since extra alleles of Gata3 have also been known to cause HDR syndrome 20 . Finally, it should also be noted that we deleted Gata3 from three different cell types: HCs, SCs, and SGNs. While these cell types work together, it is unclear if loss of Gata3 in just one of the cell types is enhancing the overall phenotype we see in our model. Despite the fact that HCs in our model are innervated by SGNs throughout the entire cochlea, we are unable to determine if the HC disorganization is causing the improper SGN peripheral projections when using the Sox2-cre ERT 2 model, or vice versa. Likewise, since the SCs and HCs are connected via tight junctions in the normal OC, our model is unable to determine if a phenotype in one of these cell types is exacerbating the phenotype overall. Therefore, in order to tease apart the role of Gata3 at this specific time point, future studies could use other more cell-specific Cre lines to delete Gata3 . Comparison of our phenotype in this model to Gata3 CKO in HC-specific, SC-specific, or SGN-specific lines could elucidate the exact role of Gata3 in the proneurosensory stage of development. In conclusion, our work demonstrates that Gata3 is essential for proper cochlear neurosensory epithelia development and organization in the late proneurosensory stage at E11.5. Because our study demonstrates a phenotype in the heterozygous mutant in addition to a more severe phenotype in the homozygous mutant, we can confirm that correct levels of Gata3 are also required for proper development. Furthermore, our study performs the latest embryonic Gata3 deletion known in the field and contributes to the understanding that Gata3 is required for proper formation and organization of the cochlea sensory epithelia at E11.5, but not for overall cochlea morphology. Methods Mouse model and genotyping All animal care and procedures were approved by Western Michigan University Institutional Animal Care and Use Committee (IACUC) following the guidelines for use of laboratory animals (IACUC #20-11-01). All experiments were carried out in accordance with the ARRIVE guidelines, and all methods were carried out in compliance with all relevant regulations. The following mouse strains were used: Sox2-cre ERT 2 (Jackson Labs) 26 , tdTomato Ai9 (Jackson Labs) 41 , and Gata3 Flox were provided by Dr. Maxime Bouchard 42 . Sox2-cre ERT 2 males were bred with Gata3 f/f females to produce males that were Sox2-cre ERT 2 : Gata3 f/+ , who were viable. Sox2-cre ERT 2 : Gata3 f/f mice were produced by breeding Sox2-cre ERT 2 : Gata3f/+ or Sox2-cre ERT 2 : Gata3 f/f males with Gata3 f/f or Gata3 f/+ females. Genotyping was performed using the following primers: Cre 5’ CCT GTT TTG CAC GTT CAC CG 3’ and 5’ ATG CTT CTG TCC GTT TGC CG 3’ yield a 280 base pair (bp) mutant and IL2 5’ CTA GGC CAC AGA ATT GAA AGA TCT 3’ and 5’ GTA GGT GGA AAT TCT AGC ATC ATC C 3’ yield a 324 bp control band; Gata3 5’ GAT TCA GTC TCC CTC CTT CTT C 3’ yield a 430 bp mutant band and 5’ GTT CAC ACA CTC CCT GCC TTC TG 3’ yield a 400 bp control band; and tdTomato Ai9 5’ AAG GGA GCT GCA GTG GAG TA 3’and 5’ CCG AAA ATC TGT GGG AAG TC3’ yield a 297 bp WT band; 5’ CTG TTC CTG TAC GGC ATG G 3’ and 5’ GGC ATT AAA GCA GCG TAT CC 3’ yield a 196 bp mutant band. Breedings were performed with E0.5 specified as noon on the day of vaginal plug. Pregnant females received an intraperitoneal injection of 3 mg/40 g tamoxifen (TMX) and 2 mg/40 g progesterone at E11.5 between 9 and 11 am 30 . On the day of collection, the pregnant female was given a lethal intraperitoneal injection of Avertin (500 mg/kg 2.2.2-tribromoethanol). Embryos were dissected from the uterus, perfused with 4% paraformaldehyde (PFA) and stored at 4°C. All images are representative of at least three biological replicates. Whole-mount Immunohistochemistry Whole mount immunohistochemistry was performed on previously fixed tissue 43 . Ears were washed in phosphate buffered saline (PBS), then washed five times five minutes in PBS/0.05% Tween20 followed by blocking for one hour in 5% normal donkey serum, 1% bovine serum albumin, and 0.5% TritonX-100 in PBS. The tissue was incubated in primary antibodies, diluted in blocking buffer, at 4°C for three nights. The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOINVIIA Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 HC Chicken (Aves; 1:200), PROX1 Goat (R & D Systems; 1:200), and SOX2 Rabbit (Sigma; 1:500). Next, the tissue was washed four times thirty minutes, followed by overnight incubation at 4°C in secondary antibody in blocking buffer. Secondary antibodies were conjugated to Alexa flour anti-Mouse 488, anti-Rabbit 488, anti-Chicken 555, anti-Goat 647, or anti-Rabbit 647 (Life Tech; 1:1000). Nuclei were labeled using Hoescht Dye (1:2000), received as a gift from Bernd Fritzsch. Images were taken on either a Nikon C2 confocal microscope or a Leica Stellaris 5 confocal microscope and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017). Spiral Ganglion Neuron Quantification For radial bundle quantification, shown in Fig. 4 and Supplementary Fig. S1 online, cochlea were imaged at the same magnification in the apex for each genotype. Using FIJI imaging software (Version 1.8.0_66), eight spaces between radial bundles were outlined. All area results were recorded in Graph Pad Prism (Version 9.1.2) and a TTEST analysis was performed. Data point plot graphs were constructed, and significance was set at P < 0.05. In situ hybridization Gata3 mRNA labeling was achieved using a previously described in situ hybridization protocol 43 . Mice were fixed in 4% PFA and inner ears were dissected in 0.4% PFA. Control ears and experimental ears were run together throughout the experiment to ensure both ears received the same experimental conditions. Ears were dehydrated overnight in 100% methanol and rehydrated through a graded methanol series. Ears were digested with Proteinase K in PBS (Ambion, Austin, TX, USA). Samples were hybridized overnight at 60°C to the Gata3 riboprobe in hybridization solution consisting of 50% (v/v) formamide, 50% 2X saline sodium citrate (SSC), and 6% (w/v) dextran sulphate. Unbound probe was removed by performing washes with 2X SSC. Samples were then incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) overnight at room temperature. Ears were extensively washed with 1X washing buffer throughout the day, then left overnight in 1X washing buffer at room temperature. Samples were then incubated at room temperature in detection buffer (Roche) before being thoroughly saturated with nitroblue phosphate/5-bromo, 4-chloro, 3-indolil phosphate (BM purple substrate, Roche). Control and mutant samples were developed in BM purple for the same length of time. Ears were mounted in glycerol on a slide and imaged with a Nikon Eclipse E600 microscope and Canon EOS Rebel T7i camera. Images were edited in Corel Draw (version 19.0; 2017). Lipophilic Dye Tracing Neuronal tracing of spiral ganglion neurons was conducted as previously described 43 . Briefly, the lateral half of the inner ear was exposed, and pieces of lipophilic dye-soaked paper was inserted into the base (NeuroVue® Red) and apex (NeuroVue® Maroon) of the cochlea. Heads were then placed into glass vials filled with 4% paraformaldehyde and incubated at 37°C for 3 days to allow for proper dye diffusion. Following incubation, the brains were removed, and the brain stem was flat mounted with the lateral side facing up in glycerol on a slide and imaged within 1 hour. All imaging was performed using a Leica Stellaris 5 confocal microscope with LAS X software and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017). Declarations Acknowledgments Thank you to the Office of the Vice President of Research and the Dean of the College of Arts and Sciences at Western Michigan University for support in this research project. P. Blinkiewicz, M. Long, E. Ketchum, and S. Sheltz-Kempf were supported in part by the College of Arts and Sciences Graduates Teaching Assistantship Awards from Western Michigan University. The use of the Leica Stellaris 5 confocal microscope was made possible by a grant from the WMU ORI. Thank you to Western Michigan University’s Imaging Center for the use of the Nikon C2 confocal microscope. This work was also funded by NIH R21 R21DC017589 (JSD). Author Contributions PVB and MRL contributed to study conception and design; acquisition, analysis,interpretation of data, and drafting and editing of the manuscript. ZAS contributed to acquisition, analysis, interpretation of data, and drafting and editing of the manuscript. EMK, and SNSK contributed to acquisition, analysis, and interpretation of data, and drafting of the manuscript. JSD contributed to editing the manuscript, study conception and design and oversaw the study. Data Availability Data is freely available upon request. Requests for data should be addressed to ZAS (email: [email protected] ). Competing Interests The authors declare no competing interests. References Fritzsch, B., Beisel, K. W. & Hansen, L. A. The molecular basis of neurosensory cell formation in ear development: a blueprint for hair cell and sensory neuron regeneration? Bioessays 28, 1181–1193, doi: 10.1002/bies.20502 (2006). Fekete, D. M. & Wu, D. K. Revisiting cell fate specification in the inner ear. Curr Opin Neurobiol 12, 35–42 (2002). Bouchard, M., Busslinger, M., Xu, P., De Caprona, D. & Fritzsch, B. PAX2 and PAX8 cooperate in mouse inner ear morphogenesis and innervation.. BMC Dev. Biol., 10, 89 (2010). George, K. M. et al. Embryonic expression and cloning of the murine GATA-3 gene. Development 120, 2673–2686 (1994). Duncan, J. S., Lim, K. C., Engel, J. D. & Fritzsch, B. Limited inner ear morphogenesis and neurosensory development are possible in the absence of GATA3. 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SOX2 is required for inner ear growth and cochlear nonsensory formation before sensory development. Development 146, doi: 10.1242/dev.170522 (2019). Kiernan, A. E. et al. Sox2 is required for sensory organ development in the mammalian inner ear. Nature 434, 1031–1035, doi: 10.1038/nature03487 (2005). Liu, Z., Owen, T., Fang, J. & Zuo, J. Overactivation of Notch1 signaling induces ectopic hair cells in the mouse inner ear in an age-dependent manner. PLoS One 7, e34123, doi: 10.1371/journal.pone.0034123 (2012). Liu, Z. et al. Age-dependent in vivo conversion of mouse cochlear pillar and Deiters' cells to immature hair cells by Atoh1 ectopic expression. J Neurosci 32, 6600–6610, doi: 10.1523/JNEUROSCI.0818-12.2012 (2012). Walters, B. J. et al. In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice. Cell reports 19, 307–320, doi: 10.1016/j.celrep.2017.03.044 (2017). Masuda, M., Pak, K., Chavez, E. & Ryan, A. F. TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1. Dev Biol 372, 68–80, doi: 10.1016/j.ydbio.2012.09.002 (2012). Driver, E. C. et al. Hedgehog signaling regulates sensory cell formation and auditory function in mice and humans. J Neurosci 28, 7350–7358, doi: 10.1523/JNEUROSCI.0312-08.2008 (2008). Kelly, M. C., Chang, Q., Pan, A., Lin, X. & Chen, P. Atoh1 directs the formation of sensory mosaics and induces cell proliferation in the postnatal mammalian cochlea in vivo. J Neurosci 32, 6699–6710, doi: 10.1523/JNEUROSCI.5420-11.2012 (2012). Lillevali, K. et al. Gata3 is required for early morphogenesis and Fgf10 expression during otic development. Mech Dev 123, 415–429, doi: 10.1016/j.mod.2006.04.007 (2006). van Looij, M. A. et al. GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice. Neurobiol Dis 20, 890–897, doi: 10.1016/j.nbd.2005.05.025 (2005). Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13, 133–140, doi: 10.1038/nn.2467 (2010). Grote, D. et al. Gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction. PLoS Genet 4, e1000316, doi: 10.1371/journal.pgen.1000316 (2008). Duncan, J., Kersigo, J., Gray, B. & Fritzsch, B. Combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology. J Vis Exp, doi: 10.3791/2451 (2011). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.jpg Cite Share Download PDF Status: Published Journal Publication published 03 Aug, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 05 May, 2023 Reviews received at journal 02 May, 2023 Reviewers agreed at journal 17 Apr, 2023 Reviewers invited by journal 17 Apr, 2023 Editor assigned by journal 13 Apr, 2023 Editor invited by journal 12 Apr, 2023 Submission checks completed at journal 12 Apr, 2023 First submitted to journal 28 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies 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-2747944","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":191163979,"identity":"7817f56f-aadb-40bf-bf20-be4d79a48b2a","order_by":0,"name":"Paige V. Blinkiewicz","email":"","orcid":"","institution":"Western Michigan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paige","middleName":"V.","lastName":"Blinkiewicz","suffix":""},{"id":191163980,"identity":"e706a051-539e-4714-9f40-b374db79430a","order_by":1,"name":"Makayla R. Long","email":"","orcid":"","institution":"Western Michigan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Makayla","middleName":"R.","lastName":"Long","suffix":""},{"id":191163981,"identity":"db55ff6b-a2e8-4e53-90af-043b14cafb62","order_by":2,"name":"Zachary A. Stoner","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYHACNhiD8QGDwQGitRiAGMwGJGthk2BgIEKLfPvhZw8Y/vyRM2fvfVZdUHCHQd69xwCvFsaeNHMDxjYDY8ue42a3Zxg8YzA8cwa/FmaGHDYJxgaDxA030thu8xgcZjCckZaA3yP8b4Be+APUcv8ZWzFRWngkgLYwsIFsYWNjBmmRl0g+gFeLhMQzM4nENmNjgzNpzNJALTwGPIfxa5HvT34m8eGPnJzB8WOMn3n+HJaTb29swKsFDJDdzkNkbKLYS4Qdo2AUjIJRMLIAAMJ9PuNEwKwwAAAAAElFTkSuQmCC","orcid":"","institution":"Western Michigan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zachary","middleName":"A.","lastName":"Stoner","suffix":""},{"id":191163982,"identity":"d7a0fcb2-71ba-4d7c-910e-e97d48be0a79","order_by":3,"name":"Elizabeth M. Ketchum","email":"","orcid":"","institution":"Western Michigan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"M.","lastName":"Ketchum","suffix":""},{"id":191163983,"identity":"a183a84c-93a2-4057-9f8f-1c1797f4da9e","order_by":4,"name":"Sydney N. Sheltz-Kempf","email":"","orcid":"","institution":"Western Michigan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sydney","middleName":"N.","lastName":"Sheltz-Kempf","suffix":""},{"id":191163984,"identity":"23b0f080-e0ba-4a01-ad1c-357378620aab","order_by":5,"name":"Jeremy S. Duncan","email":"","orcid":"","institution":"Western Michigan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"S.","lastName":"Duncan","suffix":""}],"badges":[],"createdAt":"2023-03-28 15:44:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2747944/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2747944/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-39707-0","type":"published","date":"2023-08-03T21:53:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":35787296,"identity":"50f5f404-ccf6-45e6-86f1-5a9600b1fe97","added_by":"auto","created_at":"2023-04-14 21:47:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":400739,"visible":true,"origin":"","legend":"\u003cp\u003eGata3 is conditionally deleted from HCs, SCs, and SGNs at E11.5\u003cstrong\u003e (A-D’)\u003c/strong\u003e Whole mount \u003cem\u003ein-situ\u003c/em\u003e hybridization was performed with a \u003cem\u003eGata3\u003c/em\u003e probe on a \u003cem\u003eGata3 f/f \u003c/em\u003econtrol and a \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: Gata3 f/f\u003c/em\u003e mutant and imaged at the cochlear base and apex. \u003cem\u003eGata3\u003c/em\u003e expression appears in the HCs, SCs, and SGNs of the control and is absent in the HCs and SCs and decreased in the SGNs of the homozygote mutant. Scale bar: 100 µm\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/5c28da30aea163f570194118.jpg"},{"id":35788023,"identity":"8b43db37-40ed-4084-9f19-5e7084bbfbc7","added_by":"auto","created_at":"2023-04-14 21:55:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1201678,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of \u003cem\u003eGata3\u003c/em\u003e results in loss of HCs in a basal to apical gradient\u003cu\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e\u003cstrong\u003e(A-F’)\u003c/strong\u003e Representative images from the basal, middle, and apical regions of the cochlea for HCs indicated by MYOSIN7A+ staining. Two different controls were used, \u003cem\u003eGata3 f/f\u003c/em\u003e and \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e, in order to account for the haploinsufficent phenotype of the Cre line used. Both the heterozygous and homozygous mutant show IHC duplets (white circles) and missing rows of OHCs (white brackets), while the homozygous mutant also shows ectopic HCs in the GER. Scale bar: 50 µm\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/82cd3229f220d43429763f33.jpg"},{"id":35787297,"identity":"8189e0bd-9d9e-4617-9506-f369e99aa1fd","added_by":"auto","created_at":"2023-04-14 21:47:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":589447,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of \u003cem\u003eGata3\u003c/em\u003e results in loss of SCs in a basal to apical gradient\u003cstrong\u003e (A-C’)\u003c/strong\u003e Representative images from the basal, middle, and apical regions of the cochlea showing SCs indicated by SOX2+ staining. The homozygous mutant shows a worsening disorganization of SCs from base to apex, with entire rows of SCs missing in the middle and apex. Scale bar: 25 µm\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/af4ab06fab025f763ab3a852.jpg"},{"id":35787302,"identity":"fe26c146-835a-44fd-be96-143a2ea6dd3d","added_by":"auto","created_at":"2023-04-14 21:47:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2639370,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of \u003cem\u003eGata3\u003c/em\u003e results in fewer of SGNs in a worsening gradient from base to apex\u003cstrong\u003e (A-C)\u003c/strong\u003e Overview of SGNs in the control, heterozygous mutant, and homozygous mutant apex. \u003cstrong\u003e(D, D’, D’’)\u003c/strong\u003e Radial bundles of a control sample. \u003cstrong\u003e(E, E’, E’’)\u003c/strong\u003e Radial bundles of a heterozygous mutant. Slightly increased space between the radial bundles is observed in the apex. \u003cstrong\u003e(F-F”)\u003c/strong\u003e Radial bundles of a homozygous mutant. The distance between radial bundles is increased relative to the control sample. The dotted white outline and white arrowheads in \u003cstrong\u003eF’\u003c/strong\u003e indicate increased branching in the middle. The radial bundles in \u003cstrong\u003eF”\u003c/strong\u003e exhibit even greater amounts of branching as well as an increase and irregular distance between the fibers. \u003cstrong\u003e(F’’)\u003c/strong\u003e \u003cstrong\u003e(G, G’, G’’)\u003c/strong\u003e Peripheral projections of the control as the reach the OC are well organized in the control. \u003cstrong\u003e(H)\u003c/strong\u003e In the heterozygous mutant, all peripheral neurites are present and relatively organized. \u003cstrong\u003e(H’)\u003c/strong\u003e Peripheral neurites in the middle of the heterozygous mutant have some peripheral projections misturn towards the apex instead of the base. Additionally, there are fewer neurites present than in the control. \u003cstrong\u003e(H’’)\u003c/strong\u003e In the apex of the heterozygous mutant, there are fewer peripheral projections and those that are present show disorganization relative to the control sample. \u003cstrong\u003e(I)\u003c/strong\u003e In the base of the homozygous mutant, peripheral projections are present but are fewer in number and show an increased number of neurites turning towards the apex rather than the base. \u003cstrong\u003e(I’)\u003c/strong\u003eThe middle of the homozygous mutant has drastically fewer neurites reaching the OC relative to the control, particularly those neurites that should project to the OHC region. \u003cstrong\u003e(I’’)\u003c/strong\u003e The apex of the homozygous mutant has some peripheral neurites approaching the IHC region of the OC but no peripheral neurites extending to the OHC region. \u003cstrong\u003e(J)\u003c/strong\u003eQuantification of the distance between radial bundles in apex of control samples versus apex in homozygous mutants. The distance between radial bundles is greater in mutant samples than in controls and mutant samples show greater variability in the distance between radial bundles. A TTEST was performed and p \u0026lt; 0.0001. Scale bar: 100 µm\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/1b981a30792fbef1567cc4dc.jpg"},{"id":35788022,"identity":"5002088e-f067-42d9-b963-3cffeb142ec1","added_by":"auto","created_at":"2023-04-14 21:55:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":646141,"visible":true,"origin":"","legend":"\u003cp\u003eSGN central pathfinding unaffected by \u003cem\u003eGata3\u003c/em\u003e deletion\u003cstrong\u003e (A) \u003c/strong\u003eSchematic view of lipophilic dye placement and visualization of SGNs in the CN. \u003cstrong\u003e(B-D) \u003c/strong\u003eLipophilic dye was applied to the base (red) and apex (green) of control and mutant cochlea at E18.5 and their central projections were analyzed. \u003cstrong\u003e(B) \u003c/strong\u003eIn the\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e control, SGNs bifurcate and send processes towards the AVCN and DCN/PVCN. Basal and apical SGN fibers also remain segregated throughout the CN \u003cstrong\u003e(C) \u003c/strong\u003eHeterozygous SGNs bifurcate and maintain tonotopic segregation similar to controls. \u003cstrong\u003e(D)\u003c/strong\u003e Homozygous mutants have aberrant SGN central projections with apical fibers projecting more dorsally and sometimes projecting outside the CN. Additionally, some SGN neurites project outside cranial nerve VIII before reaching the CN.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/a5f7a57a95b41a2d0a1ede95.jpg"},{"id":35787301,"identity":"30c041b5-64aa-4427-8e79-c8f199e8dfe7","added_by":"auto","created_at":"2023-04-14 21:47:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":513140,"visible":true,"origin":"","legend":"\u003cp\u003eLoss of \u003cem\u003eGata3\u003c/em\u003e follows in the timeline of previous studies showing progressive basal to apical loss of sensory cells. Schematic of \u003cem\u003eGata3\u003c/em\u003emutant ear at E18.5, indicating the cochlea (red), vestibular system (green), and endolymphatic duct (purple). Images are representative of the phenotype observed along the length of the cochlea and are taken from figures 2,3, and 4. Our study deleted \u003cem\u003eGata3\u003c/em\u003e later than previous studies at E11.5 and found that the overall morphology of the inner ear is intact unlike previous studies. In addition, there was a basal to apical loss of sensory cells indicating \u003cem\u003eGata3\u003c/em\u003e is still required for their formation and organization.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/cbe54644f49209afb7e0befe.jpg"},{"id":44736800,"identity":"102469a9-a817-4586-bb0a-33fd7351b32f","added_by":"auto","created_at":"2023-10-16 22:31:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1488887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/2bab68ba-fd8a-47c3-b1dc-ac04180627c3.pdf"},{"id":35788021,"identity":"91d76708-135f-4d1d-8581-90f915bd4df1","added_by":"auto","created_at":"2023-04-14 21:55:05","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":747622,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2747944/v1/02ea768b7f38e1172052c91d.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gata3 is Required in Late Proneurosensory Development for Proper Sensory Cell Formation and Organization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mammalian inner ear is comprised of six unique sensory organs, but only one of these organs is responsible for the sense of hearing: the cochlea. The cochlea contains the organ of Corti (OC) which is comprised of mechanosensory hair cells (HCs) and their corresponding supporting cells (SCs). HCs transduce sound energy into electrical impulses via innervation by spiral ganglion neurons (SGNs) that project into the hindbrain for further auditory processing. The development of these three sensory cell types has been extensively studied, but there are still gaps in knowledge regarding the transcription factors and gene networks that control the spatial and temporal aspects of this process at later proneurosensory stages.\u003c/p\u003e \u003cp\u003eThe inner ear is derived from the otic placode, which will invaginate to form the otic cup before developing into the otocyst around embryonic day 9 (E9)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. While several transcription factors are important for neurosensory development in this time frame, the zinc-finger transcription factor \u003cem\u003eGata3\u003c/em\u003e is particularly interesting due to its dynamic expression throughout development. While \u003cem\u003eGata3\u003c/em\u003e is initially expressed as early as E8.5 throughout the otocyst, by E10.5 its expression is restricted to the proneurosensory regions\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eGata3\u003c/em\u003e continues to be expressed in SGNs until postnatal day 14 (P14) and remains highly expressed in SCs, with lower levels in HCs, throughout adulthood\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.Therefore, it has been postulated that \u003cem\u003eGata3\u003c/em\u003e plays an important and dynamic role in inner ear development and neurosensory cell formation during this embryonic temporal window.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that loss of \u003cem\u003eGata3\u003c/em\u003e in the early proneurosensory region around E8.5 leads to loss of all cochlear neurosensory cells\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, while loss of \u003cem\u003eGata3\u003c/em\u003e one day later around E9.5 leads to a patchy loss of HCs and SCs, and disorganization and patchy loss of SGNs. Other studies have investigated the role of \u003cem\u003eGata3\u003c/em\u003e postnatally in the maintenance of HCs and SCs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These studies found that \u003cem\u003eGata3\u003c/em\u003e is necessary later on to maintain OHCs and to functionally develop IHCs, while loss of \u003cem\u003eGata3\u003c/em\u003e from postnatal SCs results in an increase in some types of SCs through downregulation of other genes. However, there exists a gap in knowledge about the role of \u003cem\u003eGata3\u003c/em\u003e later in embryonic development as the proneurosensory cells start to differentiate into HCs, SCs, and SGNs. Specifically, it remains to be seen how long expression is required for proper embryonic development of neurosensory cells before \u003cem\u003eGata3\u003c/em\u003e switches to its postnatal maintenance role. Additionally, while we know the presence of \u003cem\u003eGata3\u003c/em\u003e is necessary for proper neurosensory development, the precise level of \u003cem\u003eGata3\u003c/em\u003e expression is also important for maintenance and function. For example, both \u003cem\u003eGata3\u003c/em\u003e haploinsufficiency and \u003cem\u003eGata3\u003c/em\u003e over-expression, as a result of gene duplication, cause human hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. While the triad of symptoms of HDR syndrome range in severity, nearly all patients exhibit deafness\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Uniquely, deafness is the only symptom of HDR syndrome which can present singularly\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This suggests that not only is continued expression of \u003cem\u003eGata3\u003c/em\u003e required for proper inner ear development, but specific levels of \u003cem\u003eGata3\u003c/em\u003e are also required. Continued investigation of the dose-dependent requirements of \u003cem\u003eGata3\u003c/em\u003e would also contribute to the fields\u0026rsquo; overall understanding of inner ear gene regulatory networks.\u003c/p\u003e \u003cp\u003eTherefore, in this study, we explored the window of developmental plasticity which is governed by \u003cem\u003eGata3\u003c/em\u003e as a follow up to previous studies showing its loss is detrimental to cochlear neurosensory epithelia\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Using the \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e mouse line\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, we conditionally deleted \u003cem\u003eGata3\u003c/em\u003e from proneurosensory cells via tamoxifen (TMX) injection at E11.5. Our results show that deletion of \u003cem\u003eGata3\u003c/em\u003e causes severe loss and disorganization of HCs, SCs, and SGNs in a basal to apical gradient, with a more severe phenotype presenting in the apex. Interestingly, the mutant ears were morphologically normal in that it presented with a full-length cochlea unlike previous \u003cem\u003eGata3\u003c/em\u003e deletion studies. Overall we show that, while \u003cem\u003eGata3\u003c/em\u003e is not necessary at E11.5 for overall morphological development and elongation of the cochlea, however, \u003cem\u003eGata3\u003c/em\u003e is required in the late proneurosensory stages for cochlear sensory epithelia and SGNs both to form and organize properly.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGata3\u003c/span\u003e \u003cb\u003eis deleted from HCs, SCs, and SGNs at E11.5\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have characterized \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e expression at the placode stage (E8.5), otocyst stage (E10.5), and the late otocyst stage (E12.5)\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. At E10.5, \u003cem\u003eSox2\u003c/em\u003e is present in both the nonsensory cochlear floor and roof\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. By E12.5, \u003cem\u003eSox2\u003c/em\u003e is exclusively expressed in OC sensory cells\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In order to confirm complete knockout of \u003cem\u003eGata3\u003c/em\u003e from HCs, SCs, and SGNs, \u003cem\u003ein situ\u003c/em\u003e hybridization was performed using a \u003cem\u003eGata3\u003c/em\u003e riboprobe. While the control shows high expression of \u003cem\u003eGata3\u003c/em\u003e in all cell types from base to apex, the homozygous mutant shows no expression in the HCs and SCs and greatly reduced expression in the SGN cell bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D\u0026rsquo;), demonstrating that our model is indeed reducing levels of \u003cem\u003eGata3\u003c/em\u003e in the cell types of interest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGata3\u003c/span\u003e \u003cb\u003eis required for sustained formation and organization of HCs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe first analyzed the effect of deletion of \u003cem\u003eGata3\u003c/em\u003e on HCs, as previous \u003cem\u003eGata3\u003c/em\u003e CKOs show either no HC development or only patches of HCs\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In order to assess the phenotype of the deletion of \u003cem\u003eGata3\u003c/em\u003e, two different controls were used: \u003cem\u003eGata3 f/f\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-A\u0026rdquo;) and \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-B\u0026rdquo;). Other studies have previously demonstrated that the knock-in \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e line shows inner hair cell (IHC) duplets, which was confirmed in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-B\u0026rsquo;\u0026rsquo;; white circles). It was important to investigate the IHC duplets in the heterozygous mutant compared to the \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e control to ensure that the phenotype seen is not the result of using this Cre line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-C\u0026rdquo;). While the base, middle and apex of the heterozygous mutant all contain IHC duplets similar to the \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e control, it should be noted that the third row of outer hair cells (OHCs) is lost in the middle region of the OC into the apical region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-C\u0026rdquo;). We found that the heterozygous genotype shows a continuous formation of HCs from base to apex, while the homozygous mutant shows some disturbances in the apical region, similar to the previous \u003cem\u003eGata3\u003c/em\u003e CKO study that showed the presence of patches\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The homozygous mutant also shows a worsening phenotype compared to the heterozygous mutant. While the base contains all three rows of OHCs, progressive rows of OHCs are lost (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-D\u0026rsquo;). Just two rows of OHCs are present in the middle region and almost no rows of OHCs are present in the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F\u0026rdquo;). Additionally, the entire cochlea contains Myosin VIIa positive cells in the GER with the highest number appearing in the apex, which is similar to a postnatal \u003cem\u003eGata3\u003c/em\u003e CKO from SCs using this same Cre line\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, these cells in the GER are associated with SGN endings. Ectopic HCs have been seen in the GER in both CKO and over-expressor models previously\u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. While ectopic HCs generally are not seen in combination with missing rows of OHCs, previous studies have shown that loss of \u003cem\u003eGata3\u003c/em\u003e results in missing OHCs postnatally\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The phenotype of both ectopic HCs and missing rows of OHCs as a result of embryonic loss of \u003cem\u003eGata3\u003c/em\u003e is unique and further supports a role for \u003cem\u003eGata3\u003c/em\u003e in this specific temporal window in this specific cell type.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGata3\u003c/span\u003e \u003cb\u003eis required for corresponding SC formation and organization\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies that have deleted \u003cem\u003eGata3\u003c/em\u003e have shown either no SC development or only limited patches of SC formation localized to the HC patches\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, we still observe SCs in our model throughout the majority of the length of the cochlea. Similar to the HC phenotype in this model, we found that the homozygous mutant shows almost continuous formation of SCs, except for some patches in the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The apex also shows disorganization of the SC rows. The homozygous mutant shows a worsening phenotype compared to the heterozygous mutant, similar to that seen in the HCs. The base and middle show disorganized SCs and complete loss of some outer SC rows in the middle region. The apex contains the most severe phenotype in which SC appear to cluster together which is very similar to the SC phenotype seen in other \u003cem\u003eGata3\u003c/em\u003e CKO studies\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Ultimately, the phenotype in the HCs and SCs are consistent in their appearance of progressive loss of OHCs from base to apex, mirroring the loss of SCs from base to apex in the homozygous mutant. While this SC disorganization in our model is also similar to the phenotype seen in other \u003cem\u003eGata3\u003c/em\u003e CKO studies, it is important to note that the previous study did not also observe ectopic HCs in the GER\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Further studies are needed in order to tease apart the specific requirement for \u003cem\u003eGata3\u003c/em\u003e within this specific time window to determine if \u003cem\u003eGata3\u003c/em\u003e deletion in one cell population can influence another cell population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGata3 Is Required For Organization Of Sgn Peripheral Projections\u003c/h3\u003e\n\u003cp\u003eOur observation of Myosin VIIa positive cells associated with SGN endings in the homozygous mutant led us to investigate the peripheral projections of SGNs to confirm that they were developing properly. Previous studies examining the effect of \u003cem\u003eGata3\u003c/em\u003e deletion from the proneurosensory region of the developing otocyst observed a severe reduction in the number of SGNs present in mutant samples, while the SGNs that did form displayed aberrant projection patterns towards the developing OC\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Another study in which \u003cem\u003eGata3\u003c/em\u003e deletion was restricted to SGNs saw proper formation of SGNs with disorganized peripheral projections\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We first examined peripheral projections in a \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e mutant sample to establish whether the Cre knock-in displays a SGN phenotype. When compared with control samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-D\u0026rdquo;), we saw no obvious difference in SGN number or organization (data not shown). We then looked at the peripheral projections in a heterozygous mutant. The number and overall organization of SGNs in the heterozygous mutant largely resemble control samples in the base and middle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-E\u0026rsquo;). However, the radial bundles in the apex of the heterozygous mutant appear to have an increased area separating them relative to the control (F\u0026rdquo;). The homozygous mutant has a striking phenotype that displays an increase in the distance between radial bundles as well aberrant projections of the radial bundles which becomes progressively more disorganized along the length of the cochlea (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-F\u0026rdquo;). The mutant base and middle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-F\u0026rsquo;) reveal irregular distances between radial bundles in addition to extra branches from radial bundles. There are also inconsistencies in their organization, as some gaps are large and others are reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This phenotype is even more profound in the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026rdquo;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe area between the radial bundles in the apex of control and homozygous mutant samples was measured and quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). The method for radial bundle distance quantification can be found as Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online. Analysis of the data showed a statistically significant increase in the distance between radial bundles in the homozygous mutant relative to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Additionally, the values for the area in mutant samples was highly variable, further supporting that loss of \u003cem\u003eGata3\u003c/em\u003e results in disorganization of peripheral projections of SGNs.\u003c/p\u003e \u003cp\u003eWe also examined the peripheral projections where the neurites reach the OC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I\u0026rdquo;). The basal region of the heterozygous mutant is comparable to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H), but peripheral projections are progressively fewer and become disorganized in the heterozygous mutant with progression to the middle and apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u0026rsquo;-G\u0026rdquo;, H\u0026rsquo;-H\u0026rdquo;). Peripheral projections in the base of the homozygous mutant appear slightly disorganized upon reaching the OC. Additionally, the density of neurites in the homozygous mutant appears to be less when compared to the base of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The disorganization of the neurites and decreased density is even more pronounced in the middle and apex of the homozygous mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u0026rsquo;-I\u0026rdquo;). Fewer neurites project into the OHC region of the OC in the middle and few-to-no neurites project to the OHC region in the apex. In these regions, not all neurites that are present within the OHC region properly turn towards the base but rather, turn towards the apex.\u003c/p\u003e \u003cp\u003eBased upon our results, \u003cem\u003eGata3\u003c/em\u003e expression is important for the formation of radial bundles with regards to appropriate density and distance between bundles, as well as for proper branching patterns and overall organization. Additionally, \u003cem\u003eGata3\u003c/em\u003e is needed for peripheral neurites to reach the OC, particularly the OHC region, and to form proper connections with HCs. Importantly, the loss of \u003cem\u003eGata3\u003c/em\u003e has a phenotype that progressively worsens along the length of the cochlea, with the greatest phenotype observed in the apex.\u003c/p\u003e\n\u003ch3\u003eGata3 Is Required For Proper Central Pathfinding Of Sgns\u003c/h3\u003e\n\u003cp\u003eGiven that homozygous mutants display aberrant peripheral projections of SGNs, with the phenotype progressively getting more severe in a basal to apical manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), we next investigated whether central projection of SGNs to the cochlear nucleus (CN) was also affected. Previous studies examining the role of \u003cem\u003eGata3\u003c/em\u003e in spiral ganglion neuron central pathfinding have shown varied results depending on the location and timing of \u003cem\u003eGata3\u003c/em\u003e deletion\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Early deletion of \u003cem\u003eGata3\u003c/em\u003e throughout the entire inner ear at E9.5 results in central SGN fibers bifurcating at several branch points, with terminal fibers projecting non-specifically throughout the CN\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, deletion of \u003cem\u003eGata3\u003c/em\u003e within delaminated SGNs at E9.5 results in normal projection of SGNs within the CN with tonotopy maintained\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Taken together these two studies suggest that \u003cem\u003eGata3\u003c/em\u003e may be affecting SGN neuron central pathfinding in a cell non-autonomous and time-dependent manner. In order to investigate this further, lipophilic dyes were applied to the base (red) and apex (green) of \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e control, as well as heterozygous and homozygous mutant cochlea (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) to visualize the projections of SGNs into the CN. \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e control SGNs entered the hindbrain and bifurcated sending ascending and descending process towards the anteroventral cochlear nucleus (AVCN) and dorsal cochlear nucleus (DCN)/posteroventral cochlear nucleus (PVCN) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e control SGNs remained segregated with basal fibers extending more dorsally and apical fibers more ventrally (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This stereotyped central wiring was also maintained in heterozygous (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) mice. In contrast SGNs in homozygous mice display less segregation between apical and basal fibers. Apical fibers often project more dorsally into spaces occupied by basal fibers. Additionally, some apical fibers upon reaching the hindbrain project outside of cranial nerve VIII into areas outside of the CN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results provide further evidence for the idea that \u003cem\u003eGata3\u003c/em\u003e expression plays an important role in the development and wiring of SGNs centrally. Our data along with previous studies\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e suggest that Gata3 is acting in a cell non-autonomous manner at or before E11.5 to promote proper central wiring of SGNs. Further investigations are needed to elucidate what cell populations require early \u003cem\u003eGata3\u003c/em\u003e expression in order to promote proper central pathfinding of SGNs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGata3\u003c/span\u003e \u003cb\u003edeletion at E11.5 results in full morphologic development of the cochlear duct and vestibular system, but shows progressive neurosensory epithelial loss and disorganization\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious \u003cem\u003eGata3\u003c/em\u003e deletion studies have shown a variety of phenotypes that include morphologic and cochlear neurosensory epithelia defects\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eGata3\u003c/em\u003e null mice display a severely truncated cochlear and vestibular system which were devoid of sensory epithelia except for a small patch of HCs and SGNs in a portion of the saccule\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eGata3\u003c/em\u003e deletion at E8.5 using the \u003cem\u003eFoxg1-cre\u003c/em\u003e mouse line resulted in a truncated cochlea which contained no HCs and abnormal morphologic development of the vestibular system\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eGata3\u003c/em\u003e deletion at E9.5 using the \u003cem\u003ePax2-cre\u003c/em\u003e mouse line resulted in similar morphologic defects including a truncated cochlea and abnormal vestibular system. However, deletion at E9.5 resulted in patchy sensory cell development of HCs, SCs, and SGNs\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In studies that have conditionally deleted \u003cem\u003eGata3\u003c/em\u003e from only SGNs, HCS and SCs form properly\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We contribute results for \u003cem\u003eGata3\u003c/em\u003e deletion at E11.5, a time in development in which proneurosensory cell differentiation is occurring. Our findings show that \u003cem\u003eGata3\u003c/em\u003e deletion at E11.5 results in a morphologically sound structure with a full length cochlea and well developed vestibular system (data not shown). Within the cochlea, the sensory cells in the OC are mostly present and have a varying phenotype depending on the cochlear region. In the homozygous mutant cochlear base, HCs and SCs are present with only mild disorganization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), while the homozygous mutant basal radial bundles have larger spacing than normal but the neurons are well organized. This contrasts the phenotype seen in the apex since the peripheral projection density of the mutant apex is decreased and those projections which are present appear disorganized (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additionally, the tonotopy of SGN central projections is maintained within the CN in both heterozygous and homozygous mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In comparison, the mutant apical HCs are severely reduced to patchy clusters with some ectopic HCs that appear in the GER, while the apical SCs are not organized in rows and instead cluster together (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our data demonstrates a role for \u003cem\u003eGata3\u003c/em\u003e in all neurosensory cells after their initial specification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eGata3\u003c/em\u003e was previously shown to be necessary for both proper morphology and cochlear neurosensory epithelia early in development when its expression is high throughout the entire otocyst\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, the role of \u003cem\u003eGata3\u003c/em\u003e in HC, SC, and SGN formation after its restriction to the proneurosensory region was unknown. Our study reveals novel findings that \u003cem\u003eGata3\u003c/em\u003e plays both a dose-dependent and necessary role in the formation and organization of neurosensory cell types, but does not have an impact on the overall morphology of the inner ear at this specific developmental time point.\u003c/p\u003e \u003cp\u003eThis project contributes new knowledge about the role of \u003cem\u003eGata3\u003c/em\u003e on proneurosensory epithelia formation in a temporal window that fills a gap between previous studies that investigated \u003cem\u003eGata3\u003c/em\u003e deletion. From our results we find that deletion of \u003cem\u003eGata3\u003c/em\u003e from the proneurosensory domain at E11.5 results in a fully formed cochlear duct (data not shown). Regardless of the single or dual loss of \u003cem\u003eGata3\u003c/em\u003e alleles, both ears formed morphologically normal cochleas. Therefore, \u003cem\u003eGata3\u003c/em\u003e is not required for morphologic development at E11.5. Given that previous \u003cem\u003eGata3\u003c/em\u003e deletion studies did not see normal morphology of the cochlea\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, it is intriguing that deletion of \u003cem\u003eGata3\u003c/em\u003e about two days later results in a morphologically sound inner ear with a fully formed cochlea. While HCs, SCs and SGNs do form, they are highly disorganized and this phenotype progressively worsens from base to apex. In \u003cem\u003eGata3\u003c/em\u003e heterozygous null mice, studies have found that OHC loss occurs without \u003cem\u003eGata3\u003c/em\u003e\u003csup\u003e12,21,40\u003c/sup\u003e. This phenotype is mirrored in our study, despite the difference in timing of which \u003cem\u003eGata3\u003c/em\u003e is deleted. It is also noteworthy that the heterozygous mutant had a subtler phenotype compared to the homozygous mutant, suggesting that precise levels of GATA3 are needed for proper formation and organization of the proneurosensory epithelia. If precise levels of \u003cem\u003eGata3\u003c/em\u003e are truly necessary, then increased levels of \u003cem\u003eGata3\u003c/em\u003e should also have a phenotype in our model. Several other over-expressor studies have been previously studied that demonstrated ectopic HCs in the GER\u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Previous studies have even used the \u003cem\u003eGata3\u003c/em\u003e over-expressor model in combination with upregulation of other sensory genes in order to increase the efficiency of ectopic HC formation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Investigating the over-expression of \u003cem\u003eGata3\u003c/em\u003e in this model would be useful in determining the detrimental effects, if any, of higher levels of \u003cem\u003eGata3\u003c/em\u003e in the cochlea. While this would further elucidate the specific role of \u003cem\u003eGata3\u003c/em\u003e in the cochlea, the investigation of \u003cem\u003eGata3\u003c/em\u003e over-expression is especially pertinent since extra alleles of \u003cem\u003eGata3\u003c/em\u003e have also been known to cause HDR syndrome\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, it should also be noted that we deleted \u003cem\u003eGata3\u003c/em\u003e from three different cell types: HCs, SCs, and SGNs. While these cell types work together, it is unclear if loss of \u003cem\u003eGata3\u003c/em\u003e in just one of the cell types is enhancing the overall phenotype we see in our model. Despite the fact that HCs in our model are innervated by SGNs throughout the entire cochlea, we are unable to determine if the HC disorganization is causing the improper SGN peripheral projections when using the \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e model, or vice versa. Likewise, since the SCs and HCs are connected via tight junctions in the normal OC, our model is unable to determine if a phenotype in one of these cell types is exacerbating the phenotype overall. Therefore, in order to tease apart the role of \u003cem\u003eGata3\u003c/em\u003e at this specific time point, future studies could use other more cell-specific Cre lines to delete \u003cem\u003eGata3\u003c/em\u003e. Comparison of our phenotype in this model to \u003cem\u003eGata3\u003c/em\u003e CKO in HC-specific, SC-specific, or SGN-specific lines could elucidate the exact role of \u003cem\u003eGata3\u003c/em\u003e in the proneurosensory stage of development.\u003c/p\u003e \u003cp\u003eIn conclusion, our work demonstrates that \u003cem\u003eGata3\u003c/em\u003e is essential for proper cochlear neurosensory epithelia development and organization in the late proneurosensory stage at E11.5. Because our study demonstrates a phenotype in the heterozygous mutant in addition to a more severe phenotype in the homozygous mutant, we can confirm that correct levels of \u003cem\u003eGata3\u003c/em\u003e are also required for proper development. Furthermore, our study performs the latest embryonic \u003cem\u003eGata3\u003c/em\u003e deletion known in the field and contributes to the understanding that \u003cem\u003eGata3\u003c/em\u003e is required for proper formation and organization of the cochlea sensory epithelia at E11.5, but not for overall cochlea morphology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMouse model and genotyping\u003c/h2\u003e \u003cp\u003e All animal care and procedures were approved by Western Michigan University Institutional Animal Care and Use Committee (IACUC) following the guidelines for use of laboratory animals (IACUC #20-11-01). All experiments were carried out in accordance with the ARRIVE guidelines, and all methods were carried out in compliance with all relevant regulations. The following mouse strains were used: \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e (Jackson Labs)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003etdTomato Ai9\u003c/em\u003e (Jackson Labs)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eGata3 Flox\u003c/em\u003e were provided by Dr. Maxime Bouchard\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e males were bred with \u003cem\u003eGata3 f/f\u003c/em\u003e females to produce males that were \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eGata3 f/+\u003c/em\u003e, who were viable. \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eGata3 f/f\u003c/em\u003e mice were produced by breeding \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eGata3f/+\u003c/em\u003e or \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eGata3 f/f\u003c/em\u003e males with \u003cem\u003eGata3 f/f\u003c/em\u003e or \u003cem\u003eGata3 f/+\u003c/em\u003e females. Genotyping was performed using the following primers: Cre 5\u0026rsquo; CCT GTT TTG CAC GTT CAC CG 3\u0026rsquo; and 5\u0026rsquo; ATG CTT CTG TCC GTT TGC CG 3\u0026rsquo; yield a 280 base pair (bp) mutant and IL2 5\u0026rsquo; CTA GGC CAC AGA ATT GAA AGA TCT 3\u0026rsquo; and 5\u0026rsquo; GTA GGT GGA AAT TCT AGC ATC ATC C 3\u0026rsquo; yield a 324 bp control band; \u003cem\u003eGata3\u003c/em\u003e 5\u0026rsquo; GAT TCA GTC TCC CTC CTT CTT C 3\u0026rsquo; yield a 430 bp mutant band and 5\u0026rsquo; GTT CAC ACA CTC CCT GCC TTC TG 3\u0026rsquo; yield a 400 bp control band; and \u003cem\u003etdTomato Ai9\u003c/em\u003e 5\u0026rsquo; AAG GGA GCT GCA GTG GAG TA 3\u0026rsquo;and 5\u0026rsquo; CCG AAA ATC TGT GGG AAG TC3\u0026rsquo; yield a 297 bp WT band; 5\u0026rsquo; CTG TTC CTG TAC GGC ATG G 3\u0026rsquo; and 5\u0026rsquo; GGC ATT AAA GCA GCG TAT CC 3\u0026rsquo; yield a 196 bp mutant band. Breedings were performed with E0.5 specified as noon on the day of vaginal plug. Pregnant females received an intraperitoneal injection of 3 mg/40 g tamoxifen (TMX) and 2 mg/40 g progesterone at E11.5 between 9 and 11 am\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. On the day of collection, the pregnant female was given a lethal intraperitoneal injection of Avertin (500 mg/kg 2.2.2-tribromoethanol). Embryos were dissected from the uterus, perfused with 4% paraformaldehyde (PFA) and stored at 4\u0026deg;C. All images are representative of at least three biological replicates.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole-mount Immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eWhole mount immunohistochemistry was performed on previously fixed tissue\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Ears were washed in phosphate buffered saline (PBS), then washed five times five minutes in PBS/0.05% Tween20 followed by blocking for one hour in 5% normal donkey serum, 1% bovine serum albumin, and 0.5% TritonX-100 in PBS. The tissue was incubated in primary antibodies, diluted in blocking buffer, at 4\u0026deg;C for three nights. The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOINVIIA Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 HC Chicken (Aves; 1:200), PROX1 Goat (R \u0026amp; D Systems; 1:200), and SOX2 Rabbit (Sigma; 1:500). Next, the tissue was washed four times thirty minutes, followed by overnight incubation at 4\u0026deg;C in secondary antibody in blocking buffer. Secondary antibodies were conjugated to Alexa flour anti-Mouse 488, anti-Rabbit 488, anti-Chicken 555, anti-Goat 647, or anti-Rabbit 647 (Life Tech; 1:1000). Nuclei were labeled using Hoescht Dye (1:2000), received as a gift from Bernd Fritzsch. Images were taken on either a Nikon C2 confocal microscope or a Leica Stellaris 5 confocal microscope and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).\u003c/p\u003e\n\u003ch3\u003eSpiral Ganglion Neuron Quantification\u003c/h3\u003e\n\u003cp\u003eFor radial bundle quantification, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online, cochlea were imaged at the same magnification in the apex for each genotype. Using FIJI imaging software (Version 1.8.0_66), eight spaces between radial bundles were outlined. All area results were recorded in Graph Pad Prism (Version 9.1.2) and a TTEST analysis was performed. Data point plot graphs were constructed, and significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn situ\u003c/span\u003e \u003cb\u003ehybridization\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eGata3\u003c/em\u003e mRNA labeling was achieved using a previously described \u003cem\u003ein situ\u003c/em\u003e hybridization protocol\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Mice were fixed in 4% PFA and inner ears were dissected in 0.4% PFA. Control ears and experimental ears were run together throughout the experiment to ensure both ears received the same experimental conditions. Ears were dehydrated overnight in 100% methanol and rehydrated through a graded methanol series. Ears were digested with Proteinase K in PBS (Ambion, Austin, TX, USA). Samples were hybridized overnight at 60\u0026deg;C to the \u003cem\u003eGata3\u003c/em\u003e riboprobe in hybridization solution consisting of 50% (v/v) formamide, 50% 2X saline sodium citrate (SSC), and 6% (w/v) dextran sulphate. Unbound probe was removed by performing washes with 2X SSC. Samples were then incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) overnight at room temperature. Ears were extensively washed with 1X washing buffer throughout the day, then left overnight in 1X washing buffer at room temperature. Samples were then incubated at room temperature in detection buffer (Roche) before being thoroughly saturated with nitroblue phosphate/5-bromo, 4-chloro, 3-indolil phosphate (BM purple substrate, Roche). Control and mutant samples were developed in BM purple for the same length of time. Ears were mounted in glycerol on a slide and imaged with a Nikon Eclipse E600 microscope and Canon EOS Rebel T7i camera. Images were edited in Corel Draw (version 19.0; 2017).\u003c/p\u003e\n\u003ch3\u003eLipophilic Dye Tracing\u003c/h3\u003e\n\u003cp\u003eNeuronal tracing of spiral ganglion neurons was conducted as previously described\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Briefly, the lateral half of the inner ear was exposed, and pieces of lipophilic dye-soaked paper was inserted into the base (NeuroVue\u0026reg; Red) and apex (NeuroVue\u0026reg; Maroon) of the cochlea. Heads were then placed into glass vials filled with 4% paraformaldehyde and incubated at 37\u0026deg;C for 3 days to allow for proper dye diffusion. Following incubation, the brains were removed, and the brain stem was flat mounted with the lateral side facing up in glycerol on a slide and imaged within 1 hour. All imaging was performed using a Leica Stellaris 5 confocal microscope with LAS X software and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cu\u003eAcknowledgments\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\u003cp\u003eThank you to the Office of the Vice President of Research and the Dean of the College of Arts and Sciences at Western Michigan University for support in this research project. P. Blinkiewicz, M. Long, E. Ketchum, and S. Sheltz-Kempf were supported in part by the College of Arts and Sciences Graduates Teaching Assistantship Awards from Western Michigan University. The use of the Leica Stellaris 5 confocal microscope was made possible by a grant from the WMU ORI. Thank you to Western Michigan University\u0026rsquo;s Imaging Center for the use of the Nikon C2 confocal microscope. This work was also funded by NIH R21 R21DC017589 (JSD).\u003c/p\u003e\u003cp\u003e\u003cu\u003eAuthor Contributions\u003c/u\u003e\u003c/p\u003e\u003cp\u003ePVB and MRL contributed to study conception and design; acquisition, analysis,interpretation of data, and drafting and editing of the manuscript. ZAS contributed to acquisition, analysis, interpretation of data, and drafting and editing of the manuscript. EMK, and SNSK contributed to acquisition, analysis, and interpretation of data, and drafting of the manuscript. JSD contributed to editing the manuscript, study conception and design and oversaw the study.\u003c/p\u003e\u003cp\u003e\u003cu\u003eData Availability\u003c/u\u003e\u003c/p\u003e\u003cp\u003eData is freely available upon request. Requests for data should be addressed to ZAS (email: [email protected]).\u003c/p\u003e\u003cp\u003e\u003cu\u003eCompeting Interests\u003c/u\u003e\u003c/p\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFritzsch, B., Beisel, K. W. \u0026amp; Hansen, L. A. The molecular basis of neurosensory cell formation in ear development: a blueprint for hair cell and sensory neuron regeneration? \u003cem\u003eBioessays\u003c/em\u003e 28, 1181\u0026ndash;1193, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/bies.20502\u003c/span\u003e\u003cspan address=\"10.1002/bies.20502\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFekete, D. M. \u0026amp; Wu, D. K. Revisiting cell fate specification in the inner ear. 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J Vis Exp, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/2451\u003c/span\u003e\u003cspan address=\"10.3791/2451\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2747944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2747944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIt has been previously shown that zinc-finger transcription factor \u003cem\u003eGata3\u003c/em\u003e has dynamic expression within the inner ear throughout embryonic development and is essential for cochlear neurosensory development. However, the temporal window to which \u003cem\u003eGata3\u003c/em\u003e is required for the formation of the cochlear neurosensory epithelia remains unclear. To investigate the role of \u003cem\u003eGata3\u003c/em\u003e on cochlear neurosensory development in the late prosensory stages, we used the \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emouse line to target and conditionally delete \u003cem\u003eGata3\u003c/em\u003e at E11.5 before the cells have fully committed to a neurosensory fate. While the inner ears of \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: Gata3 f/f \u003c/em\u003emice appear morphologically normal, the sensory cells in the organ of Corti are partially lost and disorganized in a basal to apical gradient with the apex demonstrating the more severe phenotype. Additionally, spiral ganglion neurons display aberrant peripheral projections, such as increased distances between radial bundles and disorganization upon reaching the organ of Corti. Furthermore, heterozygous \u003cem\u003eSox2-cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: Gata3 f/+ \u003c/em\u003emice show a reduced phenotype in comparison to the homozygous mutant, supporting the concept that \u003cem\u003eGata3\u003c/em\u003e is not only required for proper formation at the later proneurosensory stage, but also that a specific level of \u003cem\u003eGata3\u003c/em\u003e is required. Therefore, our studies confirm that \u003cem\u003eGata3\u003c/em\u003e plays a time-sensitive and dose-dependent role in the development of sensory cells in the late proneurosensory stages. \u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Gata3 is Required in Late Proneurosensory Development for Proper Sensory Cell Formation and Organization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-14 21:47:00","doi":"10.21203/rs.3.rs-2747944/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-05-05T07:17:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-05-02T18:28:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a6ea0a25-f57c-451a-b268-c02ad121f9c1","date":"2023-04-17T19:12:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-04-17T18:24:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-13T18:17:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-04-12T12:34:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-04-12T12:31:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-03-28T15:38:03+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":"5e749a9e-0018-4c91-9750-3abb2ada188d","owner":[],"postedDate":"April 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":20648808,"name":"Biological sciences/Cell biology"},{"id":20648809,"name":"Biological sciences/Developmental biology"},{"id":20648810,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2023-10-16T22:26:12+00:00","versionOfRecord":{"articleIdentity":"rs-2747944","link":"https://doi.org/10.1038/s41598-023-39707-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-08-03 21:53:05","publishedOnDateReadable":"August 3rd, 2023"},"versionCreatedAt":"2023-04-14 21:47:00","video":"","vorDoi":"10.1038/s41598-023-39707-0","vorDoiUrl":"https://doi.org/10.1038/s41598-023-39707-0","workflowStages":[]},"version":"v1","identity":"rs-2747944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2747944","identity":"rs-2747944","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}