ABSTRACT
Musashi family of RNA-binding proteins are known for their role in stem-cell renewal and are negative regulators of cell differentiation. Interestingly, in the retina, Musashi proteins, MSI1 and MSI2 are differentially expressed throughout the cycle of retinal development including robust expression in the adult retinal tissue. To study the role of Musashi proteins in the retina, we generated a pan-retinal and rod photoreceptor neuron specific conditional knockout mouse lacking MSI1 and MSI2. Independent of sex, photoreceptor neurons with simultaneous deletion of Msi1 and Msi2 were unable to respond to light and displayed severely disrupted OS morphology and ciliary defects. The retina lacking Musashi exhibited neuronal degeneration with complete loss of photoreceptors by six months. In concordance with our earlier studies that proposed a role for Musashi in regulating alternative splicing, the loss of Musashi prevented the use of photoreceptor-specific exons in transcripts critical for OS morphogenesis, ciliogenesis and synaptic transmission. Overall, we demonstrate a critical role for Musashi in the morphogenesis of terminally differentiated photoreceptor neurons. This role is in stark contrast with the canonical function of these two proteins in maintenance and renewal of stem cells.
INTRODUCTION
In eukaryotes, alternative splicing of pre-mRNA increases protein diversity and controls gene expression. Diversification of proteomes through alternative splicing is a defining characteristic of metazoans and was expanded dramatically in bilaterians (1). Alternative splicing is prevalent in vertebrate neurons and is critical for the development and function of vertebrate nervous systems (2–7).
We previously showed that photoreceptor neurons exploit a unique splicing program (8). Motif enrichment analysis suggested that Musashi-1 (MSI1) and Musashi-2 (MSI2) promote the use of photoreceptor specific exons (8). We further showed that MSI1 is critical for utilization of photoreceptor specific exon in the Ttc8 gene (8). In addition, Musashi promotes the splicing of several photoreceptor specific exons when over-expressed in cultured cells (8). Recently, analysis of a comprehensive gene expression data set demonstrated that photoreceptors utilize a unique set of alternative exons that are primarily regulated by MSI1 and MSI2 (9).
The MSI1 and MSI2 proteins have two highly conserved RNA binding domains (RBDs) in the N-terminal region which show close to 90% sequence identity and recognize a similar UAG motif in RNA (10). The two RBDs of MSI1 and MSI2 are followed by a less conserved C-terminal region which shows approximately 70% sequence identity (11). The high degree of sequence identity between the MSI1 and MSI2 results in functional redundancy between the two proteins (12, 13).
Vertebrate photoreceptors are neurons specialized in detecting and transducing light stimuli. Photoreceptors are characterized by segmented morphology which compartmentalizes phototransduction, core cellular functions, and synaptic transmission. The light sensing machinery is confined to the outer segment, a stack of membranes that is elaborated by cell’s modified primary cilium. The outer segment is dynamic structure that is remade every 7 to 10 days. Consequently, maintenance of the outer segment requires high rate of transport of membranes and proteins through the connecting cilium (14).
The predicted splicing targets of Musashi in photoreceptors include pre-mRNAs from ciliary (Ttc8, Cep290, Cc2d2a, Prom1) and synaptic-associated genes (Cacna2d4, Slc17a7) (15–21). These genes are crucial for photoreceptor development and function (15–21). We proposed that production of photoreceptor specific splicing isoforms that is promoted by Musashi is necessary for the development and maintenance of photoreceptor cells in vivo (8).
To test if Musashi drives photoreceptor development and function, we removed Msi1 and Msi2 in the developing retina and rod photoreceptor cells. We find that Musashi proteins are essential for photoreceptor function, morphogenesis, and survival but not their specification. Specifically, the Musashi proteins are crucial for outer segment (OS) and axoneme development. As expected, disruption of the Musashi genes led to loss of expression of photoreceptor specific splicing isoforms.
MATERIALS AND METHODS
Generation of mice and genotyping
Mice carrying floxed alleles for Msi1 and Msi2 were provided by Dr. Christopher Lengner from the University of Pennsylvania. Six3-Cre transgene or Nrl-Cre transgenes were used to delete the floxed alleles in the developing retina or rod photoreceptors (Stock Nos. 019755, 028941, Jax labs). All mouse lines in this study are in C57 Black6/J background (https://www.jax.org/strain/000664) and were devoid of naturally occurring rd1 and rd8 alleles (22, 23). Males hemizygous for the Six3-Cre transgene or Nrl-Cre transgene and floxed for either Msi1, Msi2, or both Msi1 and Msi2 were mated with females floxed for either Msi1, Msi2, or both Msi1 and Msi2 to obtain experimental knockout mice and littermate control. The offspring of breeding pairs were genotyped using PCR from ear biopsies. The Msi1 wildtype and floxed alleles were identified using following primers: (5’-CGG ACT GGG AGA GGT TTC TT-3’ and 5’-AGC TCC CCT GAT TCC TGG T-3’). The Msi2 wildtype and floxed alleles were identified by using following primers: (5’-GCT CGG CTG ACA AAG AAA GT-3’ and 5’-TCT CCT TGT TGC GCT CAG TA-3’). The presence of the Six3 Cre, Nrl Cre transgene and Cre recombinase were determined using following primers respectively: (5’-CCC AAA TGT TGC TGG ATA GT-3’ and 5’-CCC TCT CCT CTC CCT CCT-3’), (5’-TTT CAC TGG CTT CTG AGT CC-3’ and 5’-CTT CAG GTT CTG CGG GAA AC-3’) and (5’-CCT GGA AAA TGC TTC TGT CCG-3’ and 5’-CAG GGT GTT ATA AGC AAT CCC-3’).
All experiments were conducted with the approval of the Institutional Animal Care and Use Committee at West Virginia University. All experiments were carried out with adherence to the principles set forth in the ARVO Statement for the Ethical Use of Animals in Ophthalmic and Vision Research which advocates the use of the minimum number of animals per study needed to obtain statistical significance.
Electroretinography, Immunoblotting, and Reverse Transcriptase PCR
Electroretinography, immunoblotting, and reverse transcriptase PCR were conducted using previously described protocol from our laboratory (8, 24, 25).
Immunofluorescence Microscopy
Immunofluorescence microscopy was carried out using a modified procedure in our laboratory(24, 25). Briefly, eyes were enucleated, and the cornea and lens were discarded. After dissection, eyes were fixed by immersion in 4% paraformaldehyde in PBS for one hour. After washing the eyes in PBS three times for ten minutes each, they were dehydrated by overnight incubation in 30% sucrose in PBS. Eyes were then incubated in a 1:1 solution of OCT:30% sucrose in PBS for one hour and frozen in OCT (VWR). The frozen tissues were sectioned using a Leica CM1850 cryostat for collecting serial retinal sections of 16μm thickness. The retinal cross-sections were then mounted onto Superfrost Plus microscope slides (Fisher Scientific). Slide sections were then washed and permeabilized with PBS supplemented with 0.1% Triton X-100 (PBST) and incubated for one hour in a blocking buffer containing 10% goat serum, 0.3% Triton X-100, and 0.02% sodium azide in PBS. Retinal sections were then incubated with primary antibody in a dilution buffer containing 5% goat serum, 0.3% Triton X-100, 0.02% sodium azide, and primary antibody at 1:500 dilution in PBS overnight at 4°C followed by three 5-minute washes using PBST. Sections were then incubated in the same dilution buffer containing secondary antibody and DAPI at 1:1000 for one hour. Slides were washed with PBST three times for five minutes each before treating with Prolong Gold Antifade reagent (ThermoFisher) and securing the coverslip. The images were collected using a Nikon C2 Confocal Microscope.
Retinal histology of the mouse models
Following euthanasia, eyes were enucleated using C-shaped forceps after marking the superior pole and incubated in Z-fixative for >48 hours before shipment and tissue processing by Excalibur Pathology Inc. (Norman, OK). The embedding, serial sectioning, mounting, and hematoxylin/eosin (H&E) staining were performed by Excalibur Pathology. A Nikon C2 Microscope equipped with Elements software was used to image the slides.
Transmission Electron Microscopy
After euthanasia, a C-shaped forceps was used to enucleate the eye, and the cornea was discarded (24, 25). Eyes were then incubated in a fixative solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 100mM sodium cacodylate buffer at pH 7.5 for 45 minutes before removal of the lens. After lensectomy, eyes were placed back into fixative for 72 hours before shipment, tissue processing, and imaging at the Robert P. Apkarian Integrated Electron Microscopy Core at Emory University.
Antibodies and stains
The following primary antibodies were used throughout our studies: rat anti-MSI1 (1:1000; MBL International Cat# D270-3, RRID:AB_1953023), rabbit anti-MSI2 (1:2000; Abcam Cat# ab76148, RRID:AB_1523981), mouse anti-α-tubulin (1:10 000; Sigma-Aldrich Cat# T8328, RRID:AB_1844090), rhodamine peanut agglutinin (1:1000; Vector Laboratories Cat# RL-1072, RRID:AB_2336642), rabbit anti-peripherin-2 (1:2000) was a kind gift by Dr. Andrew Goldberg from Oakland University, rabbit anti-PDE6β (1:2000; Thermo Fisher Scientific Cat# PA1-722, RRID:AB_2161443), mouse anti-acetylated α-tubulin (1:1000; Santa Cruz Biotechnology Cat# sc-23950, RRID:AB_628409), guinea pig anti-MAK (1:500; Wako, Cat# 012-26441, RRID:AB_2827389), mouse anti-glutamylated tubulin (1:500; AdipoGen Cat# AG-20B-0020B, RRID:AB_2490211), mouse anti-Ttc8 (1:1000; Santa Cruz Biotechnology Cat# sc-271009, RRID:AB_10609492), rabbit anti-Ttc8 Exon 2A (1:1000; Peter Stoilov, West Virginia University, Cat# Anti-Bbs8 exon 2A, RRID:AB_2827390), mouse anti-GAPDH (1:10,000; Fitzgerald Industries International Cat# 10R-G109a, RRID:AB_1285808), and 4′,6-diamidino-2-phenylindole (DAPI: nuclear counterstain; 1:1000; ThermoFisher, Waltham, MA).
Statistical analysis
Unless otherwise stated, the data is presented as mean of at least three biological replicates with error bars representing the standard error of the mean. Statistical significance was determined by homoscedastic, two-tailed unpaired T-test.
RESULTS
Validation of the conditional knockout mouse models
We analyzed the expression of Musashi proteins in various tissues from adult mice. Out of all the tissues we tested, retina showed the highest expression of MSI1 and MSI2 proteins (Figure 1A), in line with the previously reported high transcript levels for Msi1 and Msi2 in rod photoreceptors (9). To test the biological significance of Musashi protein expression in the murine retina, we used Cre-LoxP conditional recombination to remove either Msi1, Msi2, or both the Msi1 and Msi2 genes throughout the entire retina and ventral forebrain using the Six3 Cre transgene (26). Throughout this work, we refer to Musashi floxed mice which are hemizygous for the Six3 Cre transgene as ret-Msi-/- mice. The conditional recombination results in the deletion of Msi1’s transcription start site, exon 1, and exon 2 (13). For Msi2, the transcription start site and the first four exons are removed after cre-mediated recombination (13). The ablation of MSI1 and MSI2 was confirmed by immunoblotting retinal lysates from knockout mice at postnatal day 10 (PN10) (Figure 1B). Immunofluorescence microscopy of retinal cross sections obtained from the knockout mice affirmed the absence of MSI1 and MSI2 expression in the retina (Figure 1C).
The Musashi proteins are crucial for photoreceptor function
To determine if the Musashi proteins are required for photoreceptor function, we performed electroretinographic (ERG) recordings of the Musashi conditional knockout mice at PN16 and monitored for changes in retinal function up to PN180. Figure 2A shows the representative scotopic and photopic ERG waveforms of the ret-Msi1-/-, ret-Msi2-/-, and ret-Msi1-/-:Msi2-/- mice at PN16 immediately after mice open their eyes (27). When both Musashi genes are removed, no scotopic or photopic response remains as shown by absence of conspicuous “a”-waves and “b”-waves (Figure 2A). However, significant photoreceptor function remains in the ret-Msi1-/- and ret-Msi2-/- single knockout mice. We characterized the photoreceptor function of the ret-Msi1-/- and ret-Msi2-/- mice further to see if there was a progressive loss of vision as the mice aged (Figure 2B–E). In ret-Msi1-/- mice, there was a statistically significant reduction in photoreceptor “a”-wave amplitudes at almost all light intensities (Figure 2B). This reduction in the photoreceptor “a”-wave amplitude was stationary and persisted in ret-Msi1-/- mice up to PN180 (Figure 2C). On the other hand, ret-Msi2-/- mice at PN16 had normal photoreceptor function at all the light intensities we tested (Figure 2D). The “a”-wave amplitude began to decrease progressively in ret-Msi2-/- mice as they aged, and this became significant at PN120 (Figure 2E).
The two Musashi protein share high degree of sequence similarity and are proposed to be functionally redundant, yet the progression of vision loss in the single Msi1 and Msi2 knockouts was significantly different. We tested if changes in expression levels of the two proteins after birth may account for this discrepancy. Western blot analysis of the Musashi protein expression levels in the retina between postnatal days 0 and 110, showed a distinct pattern of expression (Figure 3A and B). MSI1 levels spike by postnatal day 4 and remain high until P13-P16, a time frame that includes the period of photoreceptor outer segment morphogenesis (Figure 3A and B). After eye opening MSI1 protein expression declines (Figure 3A and B). MSI2 shows inverse pattern of protein expression to that of MSI1: relatively low levels after birth that gradually increase and peak after postnatal day 16 as the MSI1 protein levels decline (Figure 3A and B). Overall, our data shows that the Musashi proteins essential for photoreceptor function. The two proteins are functionally redundant, but appear to act at different time points of the retinal development.
Intrinsic expression of Musashi in photoreceptors is crucial for photoreceptor function
We next sought to determine if the phenotype of the ret-Msi-/- mice was due to the absence of Musashi protein expression in photoreceptors or if deletion of Musashi in other retinal cell types or retinal progenitors were contributing to the loss of vision. To this end, we generated rod-specific Musashi conditional knockouts by crossing Musashi floxed mice with mice hemizygous for the Nrl Cre transgene where the Nrl promoter activates Cre expression in rod photoreceptors (28). Throughout this work, we refer to the Musashi floxed mice that are hemizygous for the Nrl Cre transgene as rod-Msi-/- mice. We used ERG to analyze the retinal function of the knockout mice after ablation of the Musashi genes in rods (Figure 4 A–E). Figure 4A shows the scotopic and photopic ERG waveforms of the rod-Msi1-/-, rod-Msi2-/-, and rod-Msi1-/-:Msi2-/- mice at PN16. As observed in the ret-Msi1-/-:Msi2-/- mice, no significant rod function was observed in the rod-Msi1-/-:Msi2-/- mice at PN16 which is demonstrated by absence of conspicuous “a”-wave under scotopic testing conditions (Figure 4A). We examined the rod-Msi1-/- and rod-Msi2-/- single knockout mice to see if the photoresponse phenotype was comparable to that obtained from the ret-Msi1-/- and ret-Msi2-/- mice. In rod-Msi1-/- mice at PN16, there was a reduction in photoreceptor “a”-wave amplitudes at multiple light intensities (Figure 4B). This reduction in “a”-wave amplitude persisted as these mice aged up to PN180 (Figure 4C). Contrarily, PN16 rod-Msi2-/- mice had no changes in photoreceptor function at all the light intensities examined (Figure 4D). As observed in the ret-Msi2-/- mice, the “a”-wave amplitude began to decrease progressively as these mice aged, and this decrease became statistically significant at PN90 (Figure 4E). The similar phenotypes of the ret-Msi and rod-Msi knockout mice shows that the intrinsic expression of Musashi proteins in photoreceptors is crucial for their function and that deletion of Musashi proteins in other cell types likely does not contribute significantly to the phenotype observed in the ret-Msi-/- mice. Therefore, throughout the rest of our studies, we focus on the ret-Msi1-/-:Msi2-/- mouse model for our experiments since there is a compensation in function occurring between MSI1 and MSI2 in the single knockout mice and to avoid confounding results that might be obtained when Msi1 and Msi2 are deleted only in rod but not cone photoreceptors.
Progressive neuronal degeneration in the absence of the Musashi proteins
We next wanted to examine the mechanism behind the photoreceptor dysfunction seen in the ret-Msi1-/-:Msi2-/- mouse model. One of the common causes of a reduced ERG is photoreceptor cell death. Therefore, we performed histological analysis of the ret-Msi1-/-:Msi2-/- mice at PN5, PN10, PN16, and PN180 (Figure 5A–D). In ret-Msi1-/-:Msi2-/- mice at PN5, even before the neural retina has completely differentiated, there is a reduction in the neuroblast layer (NBL) thickness which was quantified across the superior-inferior axis (Figure 5A, left and right panels). There is also a more disordered arrangement of NBL nuclei in ret-Msi1-/-:Msi2-/- mice with cells more tightly packed together compared to its littermate control (Figure 5A, left panel). At PN10, the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the retina all form in ret-Msi1-/-:Msi2-/- mice but there is a reduction in the number of layers of photoreceptor nuclei (Figure 5B, left and right panels). At PN16, the number of layers of ONL nuclei continue to decrease suggesting that photoreceptor cell death is occurring (Figure 5C, left and middle panels). However, at this age, there are no statistically significant changes in the number of layers of INL nuclei (Figure 5C, left and right panels). By 6 months of age, the retina of ret-Msi1-/-:Msi2-/- mice was severely degenerated with a complete loss of ONL nuclei in addition to a significant reduction in the number of layers of INL nuclei (Figure 5D, left, middle, and right panels).
The Musashi proteins are crucial for photoreceptor outer segment and axoneme development
Photoreceptor cells are present in the ret-Msi1-/-:Msi2-/- as indicated by the well-defined ONL (Figure 1C). We therefore examined the structure of the OS in ret-Msi1-/-:Msi2-/- mice at PN16 by immunofluorescence microscopy using three different OS markers, anti-Peripherin-2 (PRPH2: OS marker), anti-Phosphodiesterase-6β (PDE6β: rod OS marker), and peanut agglutinin (PNA: cone OS marker). After staining retinal cross sections from ret-Msi1-/-:Msi2-/- mice with PRPH2 and PNA, we observed a severe shortening of the photoreceptor outer segment (Figure 6A). This result was not limited to PRPH2, as staining with the rod OS marker PDE6β demonstrated the same phenotype (Figure 6B). The outer segment of cone photoreceptors also appears to be severely shortened as shown by the abnormal PNA staining (Figure 6A–B). Lastly, no mislocalization of PDE6β or PRPH2 is found in the ONL or inner segment of ret-Msi1-/-:Msi2-/- mice suggesting that while the Musashi proteins are required for outer segment formation they are not regulating trafficking or localization of OS-resident proteins (Figure 6B).
Using transmission electron microscopy, we imaged ultrathin retinal sections from ret-Msi1-/-:Msi2-/- mice at PN10 when the OS begins to elaborate. When examining the OS/IS boundary in ret-Msi1-/-:Msi2-/- mice by electron microscopy, we observed very little, if any, conspicuous OS (Figure 6C). Instead, the IS of the ret-Msi1-/-:Msi2-/- mice appears to come in direct contact with the RPE (Figure 6C–D). At higher magnification, the photoreceptors of ret-Msi1-/-:Msi2-/- mice displayed either no OS or aberrant and undersized OS (Figure 6D left, middle, and right panels).
To examine the structure of the connecting cilium and the axoneme, we stained retinal cross sections from ret-Msi1-/-:Msi2-/- mice at PN10 using antibodies directed against the established markers of murine connecting cilium (glutamylated and acetylated tubulin) and axoneme (MAK) (29–32). Probing with glutamylated and acetylated α-tubulin antibodies showed that there were no changes in the length of the CC (Figure 7A, C-D). Contrarily, staining with the anti-MAK antibody showed a substantial reduction in the length of the axoneme accompanied with punctate staining suggesting a severe structural defect of the axoneme (Figure 7A–B).
The Musashi proteins promote splicing of photoreceptor specific exons
Our previous studies suggested that the Musashi proteins are regulating alternative splicing of their target pre-mRNAs in vertebrate photoreceptors (8). To test if the Musashi proteins are responsible for the inclusion of photoreceptor specific exon, we analyzed the splicing in ret-Msi1-/-:Msi2-/- mice of pre-mRNAs from cilia-and OS-related genes that we previously showed to express photoreceptor specific isoforms (Figure 8). We witnessed a drastic reduction in alternative exon inclusion in ret-Msi1-/-:Msi2-/- mice for all tested transcripts (Figure 8A). We also analyzed isoform expression at the protein level for TTC8 (Tetratricopeptide repeat domain 8) since we had an antibody that specifically recognizes the photoreceptor-specific isoform. TTC8 also referred as Bardet-Biedl Syndrome Protein (BBS8) is part of the BBSome complex that is known play an important role in photoreceptor outer segment morphogenesis (33, 34). We used two different antibodies, a pan-antibody that recognizes all TTC8 protein isoforms (Pan-TTC8) and the other that recognizes the photoreceptor-specific isoform of Ttc8 by binding the epitope encoded by Exon 2A (the photoreceptor-specific exon of Ttc8) (Figure 8B). After probing retinal lysates from the ret-Msi1-/-:Msi2-/- mice with the pan-TTC8 antibody, we observed faster migration of the TTC8 protein compared to the littermate control suggesting that the Exon 2A was not included (Figure 8B). Concordantly, when probing for the photoreceptor-specific isoform of TTC8 using the Ttc8 Exon 2A antibody, we saw the absence of this isoform in ret-Msi1-/-:Msi2-/- mice (Figure 8B). Taken together, these results demonstrate that the Musashi proteins are required for the inclusion of photoreceptor specific alternative exons.
DISCUSSION
MSI1 and MSI2 are required for photoreceptor morphogenesis but not specification
Our data shows the requirement for MSI1 and MSI2 in photoreceptor cells. Double knockout of Msi1 and Msi2 in retinal progenitors results in complete loss of vision. Two lines of evidence demonstrate that this loss of vision is due to a defect in photoreceptor morphogenesis, rather than early developmental defects. First, the specification of retinal progenitors to photoreceptor cells was not affected by loss of Musashi. The retina of the knockout mice had laminated nuclear layers indicating normal development of the retina. The photoreceptor cells retained their characteristic morphology and expressed cell type specific proteins such as peripherin and PDE6. Importantly, removal of Msi1 and Msi2 in rod photoreceptors driven by Nrl-Cre caused loss of scotopic photoresponse. Thus, the vision phenotype is not due to impairment of the early stages of retinal development and is caused by a defect specific to photoreceptor cells.
Morphological examination by electron microscopy and immunofluorescence showed that the outer segment of the photoreceptors lacking Musashi is either missing or is stunted and disorganized. In addition, axoneme was shortened. In contrast, the connecting cilium has normal length and did not have obvious defects. Trafficking of PDE6 and peripherin through the connecting cilium also appears to be unaffected and the two proteins localize to the stunted outer segment wherever one is present. Taken together our findings demonstrates a requirement for Musashi in the morphogenesis and function of the photoreceptor outer segment that appears not to affect protein trafficking.
Musashi is needed for inclusion of photoreceptor-specific exons
RT-PCR analysis of alternative splicing in the retina of Msi1 and Msi2 knockout mice showed that inclusion of photoreceptor specific exons in the mature transcripts is dependent on the Musashi proteins. We confirmed this finding using immunoblotting with antibody that recognizes photoreceptor-specific isoform of TTC8. The effect of Msi1 knockout on splicing is stronger compared to Msi2 knockdown. It remains to be determined if this observation reflects a dominant role for MSI1 in splicing control, or derives from to the timing of the embryonic knockout of the two genes relative to the postnatal developmental switch from Msi1 to Msi2 expression in the retina. Our data demonstrates for the first time that Musashi regulates splicing in vivo and impacts dramatically the inclusion levels of the exons it controls. This is a novel role for Musashi that is distinct from it known function in controlling translation in the cytosol.
Functional redundancy and developmental switch within the Musashi protein family
In vertebrates, the Musashi protein family consists of two paralogues, MSI1 and MSI2, which have high degree of sequence identity, and have arisen from a gene duplication event (35, 36). The RNA binding domains of MSI1 and MSI2 have approximately 90% sequence identity and recognize the same UAG sequence motif in vitro and in vivo (37–40). The high degree of similarity suggest that the two proteins are likely to be functionally redundant when co-expressed in the same cells. Indeed, we observed only minor reductions in visual function after the loss of either MSI1 or MSI2 alone whereas the combined loss of MSI1 and MSI2 resulted in a complete loss of visual function (Figure 2). Similarly, inclusion of photoreceptor specific exons is promoted by both proteins, and the double knockout produces stronger effect on splicing than the knockouts of either Msi1 or Msi2. The functional redundancy in photoreceptor cells that we observe is in agreement with previous reports of redundancy between MSI1 and MSI2 in other cell types (12, 13).
Despite the proposed functional redundancy between the two Musashi proteins the phenotype of the single Msi1 and Msi2 knockouts show distinct progression of vision loss. Msi1 knockouts have reduced vision at birth, followed by minor decline as the animals age. This decline is unlikely to be associated with the lack of Musashi, as it tracks the normal reduction in visual response observed in the wild type controls. In contrast, Msi2 knockouts do not show significant visual defect at the time of eye opening (P16), but their vision progressively deteriorates with age. This difference in the phenotypes can be explained by the developmental timing of the MSI1 and MSI2 protein expression. A burst in MSI1 protein expression precedes the critical period for rod photoreceptor outer segment morphogenesis between birth and eye opening and MSI1 levels remain high until the eyes open at P16. The MSI1 expression begins a gradual decline at P13 and the MSI1 protein is replaced by increase in MSI2 levels. This data shows distinct roles for MSI1 and MSI2 in photoreceptor morphogenesis and photoreceptor maintenance, respectively. The developmental switch we observe raises the question of potential functional differences in the two Musashi protein, that require MSI1 expression during photoreceptor morphogenesis and MSI2 for photoreceptor maintenance.
Our work highlights roles for MSI1 and MSI2 in photoreceptor morphogenesis and survival. An interesting aspect of the function of the Musashi proteins in the retina is their apparently mutually exclusive roles at different stages of development. At early stages of development, MSI1 and MSI2 support the renewal and proliferation of retinal precursor cells. At late stages of retinal development and in the adult retina MSI1 and MSI2 are required for morphogenesis of the differentiated photoreceptor cells and survival of mature neurons. Our studies point to the need for MSI in controlling the alternative splicing in photoreceptor cells. It is important to note that the canonical function of the Musashi proteins is to control mRNA translation in the cytosol (41, 42), where they can either block or enhance translation of mRNA depending on cellular context (43–48). Future studies will be aimed at determining the mechanism(s) for the need for Musashi in vision and the regulation of the developmental switch between MSI1 and MSI2.
Conflict of interest statement
The authors declare no conflicts of interest.
FUNDING
This work was supported by the National Institutes of Health [grant numbers RO1 EY028035, R01 EY025536, and R21 EY027707]; the West Virginia Lions Club Foundation; and Lions Club International Foundation.
AUTHOR CONTRIBUTIONS
P.S. and V.R jointly conceived and supervised this study and edited the manuscript. J.S. designed and performed experiments and wrote the manuscript. F.M and B.J. designed and performed experiments.
Acknowledgments
The authors thank Maxim Sokolov, John Hollander, Ronald Gross for their feedback on the work. We also thank Dr. Christopher Lengner for the generous donation of the Msi1fl/fl Msi2fl/fl mice and Dr. Andrew Goldberg for PRPH2 antibody.
Footnotes
Figure 1: Panel quantifying Msi1 and Msi2 protein expression in single Msi2 or Msi1 knockouts was removed. Figure 2 and new Figure 3. Panel showing developmental regulation of Msi1 an Msi2 protein was moved to a new Figure 3 and now includes quantitative data on the protein levels. Figure 4 (former Figure 3). Immunofluorescnece panel was removed. Figures 4 and 5 are now Figures 5 and 6. Figure 6 showing expression of apoptosis and cell proliferation markers was removed and the data is no longer discussed in the manuscript.