A single Musashi gene allele is sufficient to maintain mouse photoreceptor cells

A single Musashi gene allele is sufficient to maintain mouse photoreceptor cells | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A single Musashi gene allele is sufficient to maintain mouse photoreceptor cells Bohye Jeong , View ORCID Profile Peter Stoilov doi: https://doi.org/10.1101/2025.11.26.690869 Bohye Jeong 1 Department of Biochemistry and Molecular Medicine, West Virginia University (WVU) , Morgantown, WV, 26506, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peter Stoilov 1 Department of Biochemistry and Molecular Medicine, West Virginia University (WVU) , Morgantown, WV, 26506, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peter Stoilov For correspondence: pstoilov{at}hsc.wvu.edu Abstract Full Text Info/History Metrics Preview PDF Abstract In vertebrates, two genes, Musashi1 (Msi1) and Musashi2 (Msi2) , encode for highly similar Musashi protein paralogs. The Musashi proteins are known to bind to 3’-UTRs and control translation. In photoreceptor cells, the Musashi proteins promote the inclusion of photoreceptor-specific alternative exons by binding to the proximal downstream of their introns. While the Musashi proteins are expressed in various cell types, their role in regulating splicing appears to be confined to photoreceptor cells, where the two proteins have exceptionally high expression levels. To test if the photoreceptor-specific role of MSI1 and MSI2 in splicing is due to their expression levels in photoreceptor cells, we generated combined Msi1 and Msi2 knockouts that progressively reduced the number of Musashi alleles in photoreceptor cells. We analyzed the splicing of photoreceptor-specific exons in the Cc2d2a , Cep290 , Prom1 , and Ttc8 genes and the function of photoreceptor cells in the knockouts. We found that a single allele from either Msi1 or Msi2 is sufficient to maintain photoreceptor function and support high inclusion levels of the photoreceptor-specific exons. Introduction The Musashi proteins are a family of RNA binding proteins conserved in metazoans. First discovered in Drosophila, Musashi ( dMsi ) was implicated in the maintenance of neural and germ stem cells [ 1 – 4 ]. Drosophila genome contains a paralog dMsi , Rbp6, but the two proteins appear to perform nonredundant functions in the fly [ 5 ]. Vertebrate genomes contain two Musashi paralogs, Msi1 and Msi2, that are more closely related to Rbp6 than dMsi [ 5 ]. The vertebrate Musashi genes were shown to support multiple stem cell populations and stem cell fate transition in mammals, and control the expression of maternally produced transcripts in Xenopus oocytes [ 6 – 10 ]. The canonical role of the Musashi proteins is to bind to the 3’-UTRs of their mRNA targets to regulate translation [ 11 – 13 ]. The Musashi proteins regulate translation through distinct mechanisms that involve interactions with the cytoplasmic poly-A polymerase, poly-A binding proteins, and LSM14B [ 14 – 17 ]. In vertebrates, MSI1 and MSI2 are expressed across multiple cell types, including terminally differentiated neurons. However, their functions outside of stem cell populations are less well understood. Recently, the Musashi proteins were shown to regulate the plasticity of differentiated gonadotrope cells in the pituitary by controlling protein translation [ 18 ]. We have previously demonstrated that MSI1 and MSI2 are particularly abundant in the vertebrate retina, where they are required for the development and maintenance of photoreceptor cells [ 19 , 20 ]. In photoreceptor cells, the Musashi proteins promote the expression of a broad range of proteins [ 19 ]. Unique to photoreceptor cells, the Musashi proteins promote the inclusion of alternative exons when bound to the downstream proximal intron [ 21 ]. We were interested in understanding why the Musashi proteins play such a prominent role in regulating alternative splicing specifically in photoreceptor cells, despite being expressed in many mammalian cell types. The binding of MSI1 to RNA has been shown to inhibit its translocation to the nucleus, as its nuclear localization signal (NLS) is also part of the RNA-binding surface [ 22 ]. Thus, we reasoned that the stoichiometry between Musashi proteins and their cytoplasmic RNA targets may dictate the amount of Musashi protein in the nucleus and, consequently, its effect on pre-mRNA splicing. Such a mechanism will be consistent with exceptionally high expression of Musashi proteins in the retina and the activation of photoreceptor-specific exons in cultured cells by overexpression of MSI1. To test this hypothesis, we manipulated the Musashi gene dosage in mature photoreceptor cells by generating a series of Msi1 and Msi2 knockouts that eliminated one, two, three, or all four combined Msi1 and Msi2 alleles. We observed a complete redundancy between Msi1 and Msi2 in photoreceptor cells, where a single allele from either gene was sufficient to support the splicing of photoreceptor-specific exons and photoreceptor function. Materials and Methods Animal Models The procedures on animal experiments in this study were carried out upon the approval of the Institutional Animal Care and Use Committee (IACUC) at West Virginia University (WVU). Animal lines are routinely backcrossed to C57BL/6J (Jackson Laboratory, Strain # 000664) in order to avoid the effects of inbreeding. Our animal lines are routinely outcrossed to avoid effects of inbreeding and genotyped for rd1 and rd8 mutations that are known to affect vision in mice. The mice were genotyped at weaning using primers listed in S1 Table. For Msi1 genotyping PCR reactions were supplemented with Betain (1M) and DMSO (5%) to minimize mispriming associated with the GC-rich template. Experimental animals were produced by crossing male mice carrying floxed alleles for either or both Msi1 and Msi2 , with Pde6g CreERT2 with female mice carrying only floxed alleles. The floxed Msi1 and Msi1 alleles and the Pde6g CreERT2 mice were described previously [ 19 , 23 , 24 ]. All the experiments in this study were performed in both male and female mice under C57BL/6J background. Tamoxifen-induced knockouts The intraperitoneal injection of tamoxifen (Sigma-Aldrich, Catalog # T5648-1G) was conducted on the animals at postnatal day 30 unless otherwise specified, as we have done previously [ 19 ]. Briefly, tamoxifen was thoroughly dissolved in ethanol (Sigma-Aldrich, Catalog # E7023) (100mg/ml) using a thermal mixer (ThermoFischer, Catalog# 13687717) at 40 °C. After that, it was diluted in corn oil (10mg/ml). Following vacuum centrifugation at 30 °C for 10 minutes to remove the ethanol, tamoxifen (100mg/kg) is administered via intraperitoneal injection for 3 consecutive days. Western Blot Mouse retina were collected 14 days after the first tamoxifen injection and lysed with RIPA buffer (50 mM Tris HCl-pH 8.0, 150 mM NaCl, 1.0% TritonX-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and protease inhibitor cocktail (Sigma-Aldrich, Catalog# P8340). The protein concentration was measured using a BCA protein assay kit (ThermoFischer, Catalog# 23225) and Synergy H4 hybrid reader (BioTek). Protein samples (30 µg) were resolved in 4–20% gradient polyacrylamide SDS–PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) transfer membrane (ThermoFischer, Catalog# 88518). Membranes were blocked with BSA (5%) in 1X TBST (10 mM Tris HCl-pH7.5, 150 mM NaCl, 5 mM EDTA, and 0.1% Tween) for 1 hour at room temperature and incubated with primary antibodies diluted in BSA (3%) in 1X TBST overnight at 4 °C. The next day, secondary antibodies diluted in BSA (3%) in 1X TBST were used to incubate membranes for 1 hour at room temperature. The membranes were then imaged on Amersham Typhoon (Cytiva). Primary and secondary antibodies used for western blot are listed in S2 Table. Immunofluorescence Mouse eyeballs were enucleated 143 days after the first tamoxifen injection and a small incision was made along the cornea. Following fixation of eyeballs in paraformaldehyde fixative solution (4% PFA in 1X PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 ) (Electron Microscopy Sciences, Cat# 15710) for 2 hours at room temperature on a rotator, the eyeballs were dehydrated in sucrose (20% in 1X PBS) (Fischer Scientific, Cat# S5-3) overnight at 4 °C. The next day, the eyeballs were embedded and frozen in optimal cutting temperature (OCT) compound (Sakura, Cat# 4583) in Tissue-Tek cryomold (Sakura, Cat# 4565). The frozen eyeballs were sectioned with 16 µm thickness using CyoStar NX50 cryostat (Epredia) and then mounted onto Superfrost Plus™ microscope slides (Fischer Scientific, Cat# 1255015). The mounted sections were washed three times with 1X PBS for 5 minutes each and then blocked with a blocking buffer (10% goat sera, 0.5% Triton X-100, and 0.05% sodium azide in 1X PBS) for 1 hour at room temperature. After that, the sections were incubated with primary antibodies diluted (1:500) in antibody dilution buffer (5% goat sera, 0.5% Triton X-100, and 0.05% sodium azide in 1X PBS) overnight at 4 °C. The following day, the sections were washed three times with 1X PBS for 10 minutes each and then incubated with secondary antibodies diluted (1:1,000) in antibody dilution buffer for 1 hour at room temperature. After the sections were washed three times with 1X PBS for 10 minutes each, the sections were mounted with ProLong™ Glass Antifade Mountant with NucBlue™ (ThermoFischer, Catalog# P36981) and covered with Microscope Cover Glass (Fischer Scientific, Cat# 12544B). Following securing the coverslip with clear nail polish, the sections were imaged using a Nikon AX inverted confocal microscope. Throughout the scanning process, the imaging settings remained the same for all slides. Primary and secondary antibodies used for immunofluorescence are listed in S2 Table. Image analysis Image J was used to quantify the signal intensities across the retinal layers. First the background was subtracted using the rolling ball algorithm with a ball radius of 500 pixels (74 µm). For each retinal section the fluorescence signal profile was collected along the length of three 300 pixel (44 µm) rectangular sections (technical replicates) perpendicular to the retinal layers. Subsequent data analysis was performed in R. The intensity profile was separated in segments corresponding to the inner segment (IS), outer nuclear layer (OS), and the combined inner neuronal layers ( Fig. 4 ). The signal intensities for the photoreceptor cells (inner segment and outer nuclear layer segments) were integrated and normalized to the integrated intensity of the inner neurons segment in each section). The three technical replicates for each retina were averaged to generate one biological replicate. Two way ANOVA and TukeyHSD tests were used to determine statistical significance. The full set of images used in the analysis is shown on S4 Fig. Raw data and data analysis code in R are provided in Supplementary data and code 2. RNA extraction and RT-PCR Mouse retina were collected 14 days after the first tamoxifen injection. The RNA was extracted using TRIzol Reagent (ThermoFischer, Catalog# 15596026), chloroform (Fisherscientific, Catalog# C298-500), and isopropanol (Sigma-Aldrich, Catalog # 650447). The RNA was then treated with Dnase I recombinant (Sigma-Aldrich, Catalog# 4716728001) for 20 minutes at 37 °C. The reactions were extracted with chloroform and the RNA was precipitated with ethanol and sodium acetate (3 M, pH 5.2). The RNAs (200 ng) were reverse-transcribed to cDNA using random hexamers (50 µM) and oligo-dT (10 µM), which was then amplified with fluorescently labeled primer sets spanning the alternatively spliced region listed in S1B Table. The PCR products were resolved on Urea (7.5 M)/ denaturing polyacrylamide gel (4% of 19:1 acrylamide/bis-acrylamide ratio) electrophoresis and scanned on Amersham Typhoon imager (Cytiva). Image Quant software (Cytiva) was utilized to quantify the band intensities on the gels. The individual band intensities and the quantification of the exon inclusion ratio are listed in S3 Table. Electroretinography (ERG) Mice were dark-adapted overnight prior to recording ERGs. After that, testing was done under the red light. The mice were anesthetized with isoflurane (5%) in oxygen (2.5%) in the induction chamber and placed onto a heated platform (37 °C) equipped with a nose cone with a constant flow of isoflurane (1.5%) in oxygen (2.5%). The eyes were dilated using a drop from a 1:1 mixture of tropicamide (1%, Sandoz) and phenylephrine-Hydrochloride (2.5%, Paragon) for 10 minutes. A reference electrode was placed subcutaneously between the ears, and a ground electrode was placed in the mouse’s thigh. The mice eyes were lubricated with GenTeal gel (0.3% Hypromellose, Alcon) before positioning silver wire electrodes near the center of the cornea surface. ERG recordings for both scotopic and photopic responses were collected using UTAS Visual Diagnostic System with UBA-4200 amplifier and interface, Big-Shot Ganzfeld device, and EMWIN 9.0.0. software (LKC Technologies, Gaithersburg, MD, USA). Scotopic ERG was recorded using LED white light flashes at increasing flash intensities (−40, −24, −12, and −4 dB). To record photopic responses, the rod photoreceptors were saturated by light-adapting the mice for 5 minutes using 30 cd-s/m 2 white background light. After light-adaptation, photopic ERG was recorded using flash intensity (3 dB). ERG data was analyzed in R (Supplementary data and code 1) and the results for scotopic (−12 dB) and photopic (3 dB) stimulation are presented on Fig. 3 . Statistics analysis Three replicates were used for western blot and RT-PCR experiments. ERG recordings were performed on four to five animals per group and each eye was treated as a separate replicate in the analysis. Statistical significance was determined by two-way ANOVA. Tukey HSD or pairwise T-test was used for pairwise comparisons as indicated. All data were presented as the mean ± standard error of the mean (SEM), unless otherwise noted. Results Combinatorial ablation of Musashi 1 ( Msi1) and/or Musashi 2 ( Msi2 ) alleles in mature photoreceptor cells To manipulate the total Musashi protein levels in mature photoreceptor cells, we crossed mice carrying floxed Msi1 and/or Msi2 alleles to Pde6g CreERT2 knock-in mice [ 19 , 20 ]. To account for potential differences in the expression of Msi1 and Msi2 , we generated eight combinations of floxed Msi1 and Msi2 alleles covering all possible single, double, triple, and quadruple allele knockout combinations ( Fig. 1A and S1 Fig.). To delete the alleles from the mature photoreceptor cells, the animals were injected with tamoxifen at postnatal day 30. Animals with matching floxed Musashi alleles but lacking the Cre allele were used as controls. Western blot was used to confirm that the selected genotypes produced the expected changes in MSI1 and MSI2 protein levels ( Fig. 1B and S2 Fig.). Download figure Open in new tab Figure 1. Confirmation of four different allelic knockout of Musashi 1 (Msi1) and/or Musashi 2 (Msi2) (A) Agarose gels showing the genotypes of the animals used in this study (only one replicate is shown here). Msi1/Msi2 genotypes are listed at the top. Below, “+” indicates Cre-positive and “−” indicates Cre-negative. Water (no template negative control), wild-type, and Flox/Cre-positive controls were used. “Flox” and “Wild type” denote floxed allele and wild-type allele, respectively. The Flox/Cre-positive control represents Msi1 flox/flox , Msi2 flox/flox , and Pde6g CreERT2 . The diagram at the bottom indicates the knockout allele status in photoreceptor cells. Filled circles indicate the presence of the allele, while empty circles represent the absence of the allele. (B) Western blot showing the expression of MSI1, MSI2, and TUBB (as a loading control) from retinal tissues of different genotypes (labeled at the top). The retina were collected 14 days after the first tamoxifen injection at postnatal day 30. A single allele from either Msi1 or Msi2 is sufficient to support splicing of photoreceptor-specific exons We first sought to understand the effect of Musashi gene dosage on alternative splicing. We analyzed the splicing of four Musashi -dependent photoreceptor-specific alternative exons in the Cc2d2a, Cep290, Prom1, and Ttc8 genes [ 20 , 21 ]. RT-PCR was conducted on eight knockout allele combinations and matching controls ( Fig. 2 and S3 Fig.). Splicing of the photoreceptor-specific exons in the Cc2d2a, Cep290, Prom1, and Ttc8 genes was not significantly affected unless all four Msi1 and Msi2 alleles were deleted ( Fig. 2 ). The photoreceptor-specific exon in the Prom1 gene showed a minor 12% reduction in inclusion rates when only a single Musashi allele was expressed. The same effect on Prom1 splicing was observed, regardless if the expressed allele was from the Msi1 or Msi2 gene ( Fig. 2 ). Complete deletion of all Musashi alleles resulted in significant downregulation of all four exons. These findings indicated that a single Musashi allele is sufficient to support high levels of inclusion of photoreceptor-specific exons. Download figure Open in new tab Figure 2. Alternative exon splicing on photoreceptor-specific compound Musashi ( Msi ) allelic knockouts. (A) RT-PCR analysis of four photoreceptor-specific alternative exons ( Cc2d2a exon 32 , Cep290 exon 8 , Prom1 exon 19 , and Ttc8 exon 2a ) that were previously shown to be dependent on MSI1 [ 21 ]. The retina were collected 14 days after the first tamoxifen injection at postnatal day 30. Genotypes for Msi1/Msi2/Cre are listed at the top of the gel images. The schematic at the bottom of each image indicates the status of the knockout alleles in photoreceptor cells. (B) Quantification of the exon inclusion levels, grouped by the number of Musashi alleles that were knocked out. Data are presented as mean ± SEM. Statistical significance relative to the control was determined using Tukey HSD. Significance level is indicated as: *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. A single allele of Musashi is sufficient to maintain photoreceptor cell function Previously, we have shown that the Musashi genes are required for photoreceptor development and for the maintenance of the mature photoreceptor cells. Deletion of both Msi1 and Msi2 in mature photoreceptors resulted in loss of the retina response to light within 100 days after the knockout was induced [ 19 ]. As a single Musashi allele was sufficient to support splicing, we asked whether a single allele from either Msi1 or Msi2 could also support vision. We monitored the retina response to light by Electroretinogram (ERG) in mice expressing a single Msi1 or Msi2 allele and matched controls ( Fig. 3 ). We did not observe a significant difference between the knockout animals and the controls for over 100 days after the knockout was induced. Both the A-wave corresponding to photoreceptor cell hyperpolarization and the B-wave corresponding to ON bipolar cell depolarization were normal, indicating that the phototransduction cascade and synaptic function in photoreceptor cells were unaffected. Thus, the physiological role for Musashi in vision can be fulfilled by a single Msi1 or Msi2 allele. Download figure Open in new tab Figure 3. Electroretinography (ERG) responses in photoreceptor-specific triple Musashi (Msi) knockouts Data were obtained from triple allelic knockouts from Msi 1 and Msi 2 (purple and red) along with matched controls (blue, orange). (A) Scotopic A-wave (top) and photopic B-wave (bottom) responses from the triple allelic knockout animals over time after the first tamoxifen injection. Scotopic ERGs were performed after overnight dark adaptation using 0.151 cd*s/m 2 flashes, and photopic ERGs were recorded after light adaptation using 4.88 cd*s/m 2 flashes. (B) Plots of A-wave intensity from both scotopic (top) and photopic (bottom). (C) Plots of B-wave intensity from both scotopic (top) and photopic (bottom). A feedback loop compensates for the loss of Musashi alleles Neither alternative splicing nor light sensing were significantly affected in photoreceptors expressing a single Msi1 or Msi2 allele. Thus, we used immunofluorescence to determine if the Msi1 and Msi2 allele knockouts produced the expected decrease in protein levels and characterize the subcellular distribution of the two proteins. We probed sections from retinas expressing a single Msi1 or Msi2 allele with antibodies to MSI1 and MSI2. The immunofluorescence signal intensities in the photoreceptor inner segment and outer nuclear layer were quantified and normalized to the signal intensity of the inner neurons. We observed clear loss of MSI1 and MSI2 protein when both alleles for each gene were targeted ( Fig. 4A ). However, we did not observe a significant change in MSI1 or MSI2 protein levels in photoreceptors (the sum of the inner segment and outer nuclear layer signal) relative to the controls when either protein was expressed from a single allele of the respective gene ( Fig. 4A and 4B ). This data indicates that a feedback mechanism exists that increases expression from the intact allele. Download figure Open in new tab Figure 4. Immunofluorescence analysis of photoreceptors expressing a single Msi1 or Msi2 allele. Retinal sections were collected 143 days after the first tamoxifen injection from triple allelic knockouts and their genotype-matched controls. (A) Retinal cross-section stained against MSI1 (green), MSI2 (magenta), and DAPI for nuclei (blue) and scanned using laser lines, 488 nm (MSI1), 561 nm (MSI2), and 405 nm (DAPI) with 40X objective. The scale bar is 50 µm. Retinal layers are labeled as IS for inner segment, ONL for outer nuclear layer, photoreceptors (IS+ONL), and inner neurons. (B) Quantification of MSI1 and MSI2 protein expression in photoreceptor cells in controls and animals expressing a single Musashi allele. The signal intensities are normalized to the signal in inner neurons where all Musashi alleles are intact. (C) Quantification of the MSI1 and MSI2 protein levels in the inner segment and outer nuclear layer of photoreceptor cells expressing a single Musashi allele and matched controls. We also examined the distribution of the MSI1 and MSI2 proteins between the cytoplasm and the nuclei of the photoreceptor cells by comparing the immunofluorescence signal intensities in the inner segment (photoreceptor cytoplasms) and the outer nuclear layer (photoreceptor nuclei). Consistent with the preserved function in splicing, we did not observe a decrease in the MSI1 or MSI2 protein levels in the outer nuclear layer when only a single Musashi allele was expressed ( Fig. 4A and 4C ). In fact, there was a trend for elevated Musashi protein levels in the outer nuclear layer in the knockout animals. However this trend did not reach statistical significance. Discussion Msi1 and Msi2 were partially redundant in developing photoreceptors, but were fully redundant in the mature photoreceptor [ 19 , 20 ]. This difference in the requirement for Msi1 and Msi2 was attributed to variations in the expression levels of the two proteins in developing photoreceptor cells. In the developing mouse retina, MSI1 protein levels sharply increase perinatally and peak one week after birth. In contrast, MSI2 protein levels were significantly upregulated after postnatal day 8 and reached its peak approximately thirty days after birth. Consequently, deletion of Msi1 but not Msi2 in retinal progenitors significantly reduced total Musashi protein levels, causing early vision defect and minor effect on alternative pre-mRNA splicing. The knockout of Msi2 in retinal progenitors had little effect at the time of eye opening, but the response of the retina to light progressively decreased with age. RNA binding by MSI1 is reported to interfere with nuclear import of the protein [ 22 ]. Thus, it seemed plausible that the photoreceptor-specific role of Musashi proteins in regulating alternative splicing can be achieved by increasing Musashi proteins levels so that they saturate their cytoplasmic targets. Indeed, the retina expresses exceptionally high levels of both MSI1 and MSI2 [ 20 ]. We used progressive reduction of Musashi gene dosage to test this hypothesis. We find that reducing gene dosage of Msi1 and Msi2 , short of complete knockout of all alleles from both genes, has little effect on splicing of photoreceptor-specific alternative exons. Similarly, the ability of photoreceptor cells to sense light remained unaffected when only a single Msi1 or Msi2 allele was present. The remarkable redundancy between Msi1 and Msi2 appears at least in part to be due to a robust feedback loop that upregulates protein expression from the intact Musashi allele in our knockout photoreceptors. Consequently, in our experiments, the Musashi protein levels do not decrease proportionally to the reduction in Musashi gene dosage. For this reason, we cannot conclusively rule out the stoichiometry between Musashi proteins and their cytoplasmic targets as a mechanism controlling the Musashi protein nuclear localization and function in pre-mRNA processing. Nevertheless, in the light of our results, such mechanisms appear less likely. Acknowledgments This work is supported by NIH/NEI grant R01EY025536 to P.S. References 1. ↵ Sakakibara S , Imai T , Hamaguchi K , Okabe M , Aruga J , Nakajima K , et al. Mouse-Musashi-1, a Neural RNA-Binding Protein Highly Enriched in the Mammalian CNS Stem Cell . Developmental Biology . 1996 ; 176 : 230 – 242 . doi: 10.1006/dbio.1996.0130 OpenUrl CrossRef PubMed Web of Science 2. Nakamura M , Okano H , Blendy JA , Montell C . Musashi, a neural RNA-binding protein required for drosophila adult external sensory organ development . 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Share A single Musashi gene allele is sufficient to maintain mouse photoreceptor cells Bohye Jeong , Peter Stoilov bioRxiv 2025.11.26.690869; doi: https://doi.org/10.1101/2025.11.26.690869 Share This Article: Copy Citation Tools A single Musashi gene allele is sufficient to maintain mouse photoreceptor cells Bohye Jeong , Peter Stoilov bioRxiv 2025.11.26.690869; doi: https://doi.org/10.1101/2025.11.26.690869 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41953) Biophysics (21456) Cancer Biology (18595) Cell Biology (25521) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22511) Immunology (17738) Microbiology (40401) Molecular Biology (17184) Neuroscience (88623) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)