1 Departments of Internal Medicine and Human Genetics, University of Michigan, Ann Arbor, MI 48109-0650, USA
2 Department of Pediatrics at Childrens Memorial Institute for Education and Research, Northwestern University Medical School, Chicago, IL 60614-3394, USA
*Authors for correspondence (e-mail: n-brown2{at}northwestern.edu and tglaser{at}umich.edu)
Accepted April 13, 2001
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SUMMARY |
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Key words: Math5, Atoh7, Mouse, Retina, Optic nerves
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INTRODUCTION |
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RGCs are the only retinal neurons that extend axons outside the eye. As RGCs differentiate, their axons grow laterally toward the presumptive optic nerve head in response to molecular guidance cues (Birgbauer et al., 2000; Brittis and Silver, 1994; Deiner et al., 1997) and pass outward through the optic stalk (Hinds and Hinds, 1974; Silver and Sidman, 1980). These axons travel along the optic nerve to the chiasm, where they make characteristic pathway choices and project to six specific regions in the brain, including the lateral geniculate nucleus and superior colliculus (Rodieck, 1998). The optic nerve is largely composed of RGC axons, but also contains supporting glia and astrocytes, and the central retinal artery and vein. The physiology and histological characteristics of RGC subtypes are well known (Rodieck, 1998), yet no single intrinsic factor has been demonstrated that specifies RGC fate within the mammalian eye.
The cellular mechanisms of vertebrate and Drosophila retinogenesis are fundamentally different. In vertebrates, the continuous selection of committed neurons from a pool of multipotent progenitors leads to overlapping of neuronal birthdates, stochastic variation in clonal composition (Turner et al., 1990), and random spacing between cell bodies of different neuron types (Rockhill et al., 2000). In Drosophila, a strict spatial and temporal hierarchy of cell-cell inductive interactions creates a highly ordered and invariant array of ommatidia (Brennan and Moses, 2000; Rubin, 1989; Tomlinson, 1988). In addition, all fly photoreceptors synapse directly to neurons within the brain, while vertebrate photoreceptors connect to the brain via specialized interneurons and RGCs, which are the sole projection neurons for the eye.
bHLH transcription factors are central to retinal neurogenesis (Cepko, 1999; Kageyama et al., 1995). In Drosophila photoreceptor development, the proneural gene atonal (ato) specifies the founding R8 neuron in each ommatidium (Dokucu et al., 1996; Jarman et al., 1994; Jarman et al., 1995). In addition to R8 determination, atonal also controls neuronal subtype identity (Chien et al., 1996; Sun et al., 2000) and neurite arborization (Hassan et al., 2000) within the Drosophila peripheral nervous system and brain, respectively. Among murine bHLH genes, Math5 (also known as Atoh7) is the most closely related to atonal within the bHLH domain (Brown et al., 1998; Hassan and Bellen, 2000). This structural homology is consistent with the specific expression of Math5 in the developing mouse retina (Brown et al., 1998), Xath5 in the developing frog retina (Kanekar et al., 1997), Cath5 in the chick eye (Liu et al., 2001; Matter-Sadzinski et al., 2001) and Ath5 in zebrafish retinal progenitors (Masai et al., 2000). Ectopic expression of Xath5 during frog eye development biases progenitors to become RGCs at the expense of later-born neurons such as bipolars and Müller glia (Kanekar et al., 1997). Xath5 is therefore sufficient to specify RGC fate. However, ectopic expression of Math5 in the same assay promotes formation of bipolar cells rather than RGCs (Brown et al., 1998). Thus, despite highly conserved structure and expression patterns, the functional orthology between Math5, Xath5 and atonal is unclear.
In this report, we test the role of Math5 in RGC formation by removing its function in vivo. We show that mice homozygous for a targeted Math5 mutation have grossly normal eyes, but no optic nerves or chiasm. Histological and molecular analyses reveal an almost complete absence of RGCs in postnatal Math5-/- retinae and an increase in cone photoreceptors. We further demonstrate that loss of Math5 alters the early stages of RGC formation and conclude that Math5 acts as a proneural gene for mammalian RGC determination.
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MATERIALS AND METHODS |
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Histology, immunohistochemistry and in situ hybridization
Mice were quickly sacrificed and their eyes, brains, or embryos dissected in cold phosphate-buffered saline (PBS). Tissues were fixed in Bouins or buffered formalin for paraffin embedding, or 4% paraformaldehyde for ß-gal histochemistry, or freshly frozen in OCT (Miles Scientific) for cryosectioning. Paraffin sections (10 µm) were either stained with Hematoxylin and Eosin, counterstained with Neutral Red following ß-gal histochemistry, or processed for immunoperoxidase staining following antigen retrieval (Evers and Uylings, 1997). Cryosections (10 µm) were fixed in 4% paraformaldehyde PBS and used for Math5 digoxigenin in situ hybridization (Brown et al., 1998) or antibody labeling. Immunohistochemical and PNA lectin staining was performed according to published protocols (Rich et al., 1997; Sundin and Eichele, 1990) with the biotin-streptavidin system (ABC, Vector Labs), rabbit Pax6 antiserum (1:2000, Mastick and Andrews, 2001), rabbit S-cone opsin antiserum (1:20,000, Applebury et al., 2000), rabbit recoverin antiserum (1:500, Dizhoor et al., 1991), rabbit caspase-3 antisera (1:200, New England Biolabs), biotin-PNA (5 µg/ml, Vector Labs), and the following monoclonal antibodies: anti-neuron-specific ß-tubulin (TUJ1, 1:1000, Babco), anti-protein kinase C (MC5, 1:400, Sigma), anti-vimentin (LN9, 1:200, Sigma), anti-neurofilament (NN18 for 160 kDa and NE14 for 200 kDa, 1:500, Sigma), anti-tyrosine hydroxylase (TH2, 1:1000, Sigma), anti-syntaxin (HPC1, 1:1000, Sigma), anti-calretinin (mAb1568, 1:500, Chemicon), anti-calbindin (CB955, 1:500, Sigma), VC1.1 (1:200, Sigma), and anti-rhodopsin (RET-P1, 1:1000, Sigma). For ß-gal visualization, tissues were fixed and stained as described previously (Sanes et al., 1986).
Retinal disassociation and cell counts
Cone cell counts were performed on eyes from adult F2 littermates obtained by intercrossing CD-1 Math5+/- mice. At least two mice from each genotype were compared. Neural retinae were dissected free of other ocular tissues in HBSS-CMF (Ca2+/Mg2+-free Hanks buffered saline solution) and gently disassociated as described by Altshuler and Cepko with minor modifications (Altshuler and Cepko, 1992). Dissected retinae were first incubated for 15 minutes at 37°C in normal HBSS with 300 µg/ml hyaluronidase (Sigma, Type IV-S) and 1 mg/ml collagenase (Sigma, Type XI-S), gently washed, and then incubated for 10 minutes at 25°C in HBSS-CMF with 0.1% trypsin (Gibco). After trypsinization, 10% normal donkey serum (NDS) was added and the cells pelleted by low-speed centrifugation. The retinal cells from each mouse were then resuspended in 5 ml HBSS-CMF, 10% NDS, 10 mM Hepes pH 7.4, triturated gently to a single cell suspension, and plated on poly-D-lysine-coated glass slides for 90 minutes at 37°C in a humidified chamber. Slides were fixed in fresh ice-cold PBS with 4% PFA for 10 minutes, and processed for immunohistochemistry in TST (150 mM NaCl, 10 mM Tris pH 7.4, 0.1% Tween20) containing 2.5% NDS. S-cone opsin was detected using a 1:20,000 dilution of polyclonal rabbit antiserum (Applebury et al., 2000), a biotinylated secondary antibody (Jackson Immunoresearch), and streptavidin-DTAF (Molecular Probes). To visualize cell nuclei, RNaseA (200 µg/ml) and propidium iodide (50 µg/ml) were added to the secondary and tertiary reagents, respectively. Processed slides were viewed using a Nikon Eclipse E800 fluorescence microscope and SPOT camera. Two-color images (200x) were captured digitally from random fields selected using a TRITC filter, and the total and S-cone opsin-positive cells in each image counted manually. For each mouse, 10-25 images (800-3000 cells) were analyzed. Differences between wild-type and mutant values were evaluated for statistical significance using the nonparametric Wilcoxon two-sample test for n>10 measurements (Sokal and Rohlf, 1969).
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RESULTS |
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Math5-ßgal expression during retinogenesis
The targeted allele contains a histochemical reporter, Escherichia coli ß-galactosidase, which is transcribed from the endogenous Math5 promoter (Fig. 1A). The ß-gal reporter is an exquisitely sensitive tool for detecting gene expression (Lawson et al., 1999). Although Math5 encodes a nuclear protein, we used a cytoplasmic ß-gal cassette to demarcate Math5 expression domains in the embryo and reveal the processes of differentiated neurons. In heterozygous E11.5 embryos, the pattern of ß-gal expression in retinal progenitors was identical to Math5 mRNA (Fig. 2A,B) and coincides with the onset of histogenesis. After the initiation of RGC differentiation, we noted two layers of ß-gal-expressing cells within the retinal neuroepithelium (Fig. 2C,D). One layer is located at the ventricular (sclerad) surface, closest to the pigmented epithelium (RPE), and corresponds to the zone where retinal progenitors undergo mitosis (Sidman, 1961). These retinal progenitors also express Math5 mRNA (compare Fig. 2D and E). The distribution of Math5 mRNA (Brown et al., 1998) and ß-gal activity within this layer is not uniform, but instead suggests a radial columnar pattern. The second layer is located at the vitreal surface, closest to the lens, where terminally differentiating RGCs accumulate. These cells express ß-gal within their somata and axons (Fig. 2D). We also noted ß-gal expression within the optic nerve at E17.5 (Fig. 2F) and in cone photoreceptors at P21 (Fig. 2G). Consistent with the latter observation, we have noted a low level of Math5 mRNA expression in the photoreceptor cell layer at P0.5 (data not shown). ß-gal expression in these differentiated cell types may reflect perdurance of this stable enzyme (Hall et al., 1983) or persistent, low level transcription of Math5 in some differentiated neurons, combined with the high sensitivity of this reporter system. No other retinal neuron expression domain was observed.
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Specific neuron classes are altered in Math5-/- retinae
To assess the abundance and patterning of the seven major cell types, we examined sections of postnatal Math5+/+, Math5+/- and Math5-/- eyes by light microscopy using a panel of 15 terminal differentiation markers. No significant difference was noted between Math5+/+ and Math5+/- eyes in the expression level or number of positive cells for any marker. Because laminar disruptions in Math5-/- eyes could differentially affect cell populations, we selected regions with relatively normal structure for comparison. Using both 160 kDa and 200 kDa neurofilament markers (Nixon et al., 1989), we discovered a total absence of RGCs and axon bundles in homozygous Math5 mutant retinae (Fig. 4A,B and data not shown). Immunostaining with the neuron-specific ß-tubulin marker TUJ1 (Brittis and Silver, 1994; Macabe et al., 1999; Snow and Robson, 1994; Watanabe et al., 1991) further highlighted a complete absence of RGCs and axon bundles in P21 Math5-/- eyes (Fig. 4C,D).
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Amacrine cells have highly overlapping birthdates with RGCs and cones. This class of cells, originally named because they have no axons (Ramón y Cajal, 1892), is extremely heterogeneous and consists of at least 22 different morphologically and molecularly distinct subtypes (MacNeil and Masland, 1998; Masland, 1988; Strettoi and Masland, 1996). Although the majority of amacrines reside in the INL, a significant proportion are located in the GCL, where they are termed displaced amacrines. We were therefore particularly interested in studying these cells in Math5-/- retinae. The protein syntaxin is present in the dendritic processes and perikarya of all amacrine cells (Barnstable et al., 1985). We observed similar patterns of immunostaining with this pan-amacrine marker in mutant and wild-type eyes (Fig. 5A,B), indicating that amacrine cells as a whole are grossly normal. The transcription factor Pax6 was also similarly expressed by INL and displaced amacrine cells in mutant and wild-type postnatal eyes (Fig. 5C,D). Although Pax6 protein is normally expressed by both amacrines and RGCs in postnatal retinae (Belecky-Adams et al., 1997; Davis and Reed, 1996; Hitchcock et al., 1996; Koroma et al., 1997), ganglion cells are absent in the Math5 mutants (Fig. 4B,D). On the basis of their relative density and expression profile (neurofilament- and TUJ1-negative, syntaxin- and Pax6-positive), we conclude that most, if not all, of cells remaining in the GCL of adult Math5-/- mice (Fig. 3D) are displaced amacrines.
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Loss of Math5 had no apparent effect on the number or arrangement of rods, assessed by rhodopsin immunostaining (data not shown), or horizontal cells, assessed by calbindin (data not shown) and 160 kDa neurofilament immunostaining (Fig. 4AB). However, we did observe a decrease in rod bipolar (Fig. 5G,H) and Müller glial (Fig. 5I,J) cells. The rod bipolars also did not form a defined sublayer in the IPL. A similar decrease in cone bipolars was noted using the marker recoverin (data not shown). This reduction in bipolars and Müller glia may result directly from loss of Math5 activity or indirectly from a depletion of progenitors earlier in development.
RGC development is abnormal in Math5-/- mice
Our initial assessment of the Math5-/- retinal phenotype focused on adult eyes so that all neuron and glial cell classes could be examined simultaneously. However, at birth, mutant retinae appeared thinner and less well laminated (data not shown). This suggested that Math5 is required at earlier, prenatal stages of retinal development. We therefore examined mutant retinae at E15.5, when Math5 mRNA is maximally expressed and at E13.5, which is 1-2 days after the onset of RGC differentiation. At both ages, Math5-/-retinae are noticeably thinner than those of Math5+/+ or Math5+/- littermates (compare Fig. 6A with B, 6E with F, and 6G with H). Hematoxylin and eosin staining at E15.5 showed that RGC axon bundles, normally present at the vitreal surface, are missing or greatly diminished in Math5-/- retinae (arrows in Fig. 6A,B). The reduction in RGC axons was also revealed by comparing the patterns of ß-gal expression in Math5+/- and Math5-/- eyes at E15.5 (arrows in Fig. 6C,D). Intense ß-gal staining of RGC axons was observed at the vitreal margin in Math5+/- but not in Math5-/- eyes. In addition, ß-gal-positive cells, which are confined to two layers in Math5+/- optic cups at E15.5, were distributed across the thickness of the retina (compare Fig. 6C with D). To determine if any differentiating RGCs are present transiently, we examined the E15.5 mutant retinae with the marker TUJ1, which specifically identifies RGCs at this age (Brittis and Silver, 1994; Watanabe et al., 1991). In contrast to the adult eye (Fig. 4C,D), we found that loss of Math5 caused a great reduction, but not a complete absence, of RGCs at E15.5 (Fig. 6E,F).
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Finally, we assessed RGC formation in Math5-/- retinae at E13.5 using the 160 kDa neurofilament marker (Fig. 6G,H). Neurofilament 160 kDa expression in RGCs generally precedes TUJ1 reactivity (Macabe et al., 1999) and can be detected in migrating RGC precursors as well as post-migratory RGCs residing in the GCL. The results were similar to those obtained at E15.5 with TUJ1. In Math5-/- retinae at E13.5, there is a conspicuous deficiency of neurofilament-positive RGCs (Fig. 6G,H). A small number of nascent RGCs can be observed, but these are poorly organized. There is no well formed GCL and the few axons present are not bundled into nerve fibers (Fig. 6H). Normally the optic nerve is easily recognized at E13.5 (arrow in Fig. 6G) and RGC axons have already extended toward the optic chiasm. Serial sectioning of E13.5 and E15.5 mutant eyes (n=8) failed to detect even a rudimentary optic nerve (data not shown). Instead, we consistently saw a concentration of neurofilament staining in the central retina where the optic nerve normally exits (arrow in Fig. 6H). Because some RGCs appear to form in Math5-/- embryos, but do not remain in postnatal eyes, we surveyed mutant retinae for apoptotic cells by activated caspase-3 immunostaining. These experiments provided no evidence of increased programmed cell death in mutant retinae at E13.5, P0.5, P7, P14 and P21 (data not shown). The small number of RGCs that form independently of Math5 function must be eliminated from the eye between the ages of E15.5 and P7, but this may occur gradually.
The increase in cone cell differentiation in Math5-/- mice occurs early
In Math5-/- mice, an increase in cone photoreceptors is correlated with the deficiency of RGCs, suggesting that a shift in progenitor cell fate occurs during retinal histogenesis. To explore this hypothesis further, we sought to test Math5 mutant mice for an increase in cones at the earliest possible age that committed cone photoreceptors can be identified. This is difficult because there is a long delay between cone cell birthdates and overt differentiation. For example, the majority of cones in mice are born between E12 and E15 (Carter-Dawson and LaVail, 1979b), yet cone opsin expression does not begin until P5 (Cepko, 1996; Szel et al., 1993) and focal PNA reactivity is not observed in the outer retina until after P6 (Blanks and Johnson, 1983). To determine whether Math5 deficiency causes an early increase in cones, we therefore tested the developing retinae of newborn mutant and wild-type littermates with antibodies to recoverin, a calcium-binding protein that is expressed by rods, cones and cone bipolar cells (Dizhoor et al., 1991; Milam et al., 1993). Recoverin expression normally begins in the outer margin of the mouse retina between E17.5 and P0.5, and thus significantly precedes the appearance of cone opsin and PNA reactivity. Moreover, at P0.5 recoverin expression is almost exclusively restricted to cone photoreceptors, since few rods and no bipolar cells have differentiated at this stage (Belliveau and Cepko, 1999; Morrow et al., 1998). Our results demonstrate a substantial increase in the density of differentiating cones in Math5-/- mice at P0.5 compared to wild type (Fig. 6I,J). The shift in retinal cell populations is thus likely to be determined early during histogenesis.
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DISCUSSION |
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Although other mutations are known that affect the quantity of RGCs in adult mice (Bonfanti et al., 1996; Burne et al., 1996; Erkman et al., 1996; Gan et al., 1996; Martinou et al., 1994; Rice et al., 1997; Williams et al., 1998), none completely deletes this neuronal cell class. In particular, the less severe phenotype of Brn3b-/- mice (Erkman et al., 1996; Gan et al., 1996), and the dosage-dependent decrease in Brn3b expression in Math5 mutants at E15.5 (Fig. 1D), strongly suggest that Brn3b is a downstream target of Math5 regulation. A comparable relationship has been demonstrated in Caenorhabditis elegans between the Math5 homologue lin32 (Portman and Emmons, 2000) and the POU domain transcription factor unc86 (Baumeister et al., 1996).
Loss of Math5 causes a change in cell fate
In Math5 mutant mice, RGCs are absent and cone photoreceptors increased. This relatively narrow phenotype contrasts sharply with the absence of all photoreceptors in atonal mutant flies (Jarman et al., 1994), which occurs because Drosophila eye development relies upon sequential inductive mechanisms that begin with R8 cells. We believe the cell type shift in Math5-/- mice reflects the continuously available pool of progenitors that underlies vertebrate retinogenesis (Cepko et al., 1996; Turner et al., 1990). A similar binary switch has been observed in developing cochlea of Math1-/- mice, between inner hair cell and supporting cell fates (Bermingham et al., 1999).
We propose that the majority of progenitors that would normally become RGCs switch their primary fate to cones when Math5 function is removed. Indeed, the changes we observed in ganglion and cone cell populations are similar in magnitude, and were detected as early as it is possible to reliably identify these two cell types (Fig. 6). The numerical surplus of cones, compared to the loss of RGCs, could arise because precursor cells undergo one to two additional mitoses before differentiating as cones, or because RGCs are normally overproduced and then selectively culled. A developmental connection between RGCs and cones is supported by independent gain- and loss-of-function experiments in which progenitor cell fate was altered by ectopic expression of Delta1. Retroviral transduction of chick retinae (Henrique et al., 1997) and mRNA injection of frog retinal precursors (Dorsky et al., 1997) with wild-type Delta1 biased progenitor cells to become cones. Conversely, infection of chick retinae with a dominant-negative Delta1 retrovirus produced an excess of RGCs at the expense of photoreceptor cells (Henrique et al., 1997).
Three models can explain the binary cell fate switch in Math5-/- mice (Fig. 7). Each is consistent with the overlapping early birthdates of RGCs and cones (Altshuler et al., 1991), their shared expression of ß-gal in Math5+/- heterozygotes, and retroviral lineage studies (Turner et al., 1990). These models are conceptually distinct, but not mutually exclusive. First, although vertebrate retinogenesis does not utilize a strict lineage mechanism (Cepko et al., 1996), a subpopulation of precursors may emerge with relatively restricted developmental potential, through which particular cell fates are coupled. In the absence of Math5, for example, a bipotential RGC-cone precursor would be expected to differentiate exclusively into cone photoreceptors. Cones would thus represent a specific alternative fate for early progenitors whose RGC development is blocked (Waid and McLoon, 1995). According to this orthodox model, the persistance of ß-gal activity in cones (Fig. 2G) may reflect the legacy of Math5 expression in this bipotential precursor cell. Second, RGCs may act extrinsically to inhibit cone cell determination (Belliveau and Cepko, 1999; Waid and McLoon, 1998), so that loss of Math5 (and RGCs) may indirectly increase the abundance of cones. Third, cones may represent a relatively generic default fate for early progenitors whose exit from the cell cycle is delayed (Henrique et al., 1997). According to this model, the essential and primordial role of Math5 is not to specify ganglion cells per se, but to direct commitment of early retinal neurons to the first appropriate fate, which in vertebrates is an RGC. In the absence of Math5, progenitors are forced to commit to the next available fate, which in mice is a cone photoreceptor. The last model is similar to the first but involves a more fluid and time-dependent view of developmental competence (Cepko et al., 1996). In this context, it is compelling that the lakritz mutation in the zebrafish Ath5 gene causes an equivalent deletion of all RGCs and a concomitant increase in amacrine, bipolar and Müller cells, but no change in cone photoreceptors (Kay et al., 2001). These observations correlate well with the relative birth order of retinal cell types in these two species (Altshuler et al., 1991), since cones and rods are the last-born cell types in the central zebrafish retina (Hu and Easter, 1999).
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RGC development does not depend strictly upon Math5
Because adult Math5-/- retinae are missing virtually all RGCs, our finding that a subset of embryonic RGCs can differentiate was unexpected and suggests that the initial specification and development of some RGCs is Math5-independent. This situation is not unprecedented since mutations in other vertebrate bHLH genes, such as Mash1 and Ngn2, have been shown to cause a marked reduction, but not the complete deletion of particular neuron classes in the central nervous system (Fode et al., 2000). It will be interesting to study this small, transient class of RGCs further, which can initiate development in the absence of Math5. For example, it will be important to determine what becomes of these neurons between ages E15.5 and P21. Since they are so few in number, and unable to organize into an optic nerve or innervate their targets, it is likely that they are cleared from the retina through apoptosis. This occurs during normal postnatal development to a substantial fraction of RGCs whose axons fail to make appropriate synaptic connections in the brain (Bonfanti et al., 1996; Burne et al., 1996; Cowan et al., 1984; Martinou et al., 1994). In Math5 mutant mice, this apoptosis probably occurs in the interval between P0.5 and P7, since we observed no significant increase in programmed cell death outside this window. It will also be important to determine if these short-lived RGCs correspond to a particular functional or anatomical subclass of RGCs. Finally, it is also possible in theory that these cells are not true RGCs, but are a distinct immature cell type that transiently expresses part of the RGC differentiation program, including Brn3b, neural-specific ß-tubulin (TUJ1), and neurofilament 160 kDa. We think this latter possibility is unlikely.
While this paper was under review, a similar Math5 knockout allele was reported, with several notable differences (Wang et al., 2001). First, the ß-gal cassette was targeted to the nucleus, although the insertion site was not detailed. As a result, the descendants of Math5-expressing progenitors and their cellular processes were not histochemically detectable after differentiation. In contrast, the cytoplasmic ß-gal cassette we introduced contains eighteen N-terminal amino acids from the Math5 polypeptide and was readily detected in the optic nerve at E17.5 and in differentiated cones at P21 (Fig. 2F,G). Since the mRNAs transcribed from these two alleles are likely to have similar half-lives, this limited comparison suggests that the ß-galactosidase protein may be stabilized by the Math5 amino terminus or may have significantly greater stability in the cytoplasm of differentiated neurons (Callahan and Thomas, 1994; Schilling et al., 1991). Second, Wang et al. did not evaluate cone photoreceptors in the Math5-/- mice and thus did not observe a major cell fate shift. Third, these authors reported a small number of RGCs in adult Math5-/- mice but none in E13.5 embryos. This discrepancy can be explained if the limited survival of RGCs in Math5-/- mice is somewhat variable and is influenced by genetic background (Williams et al., 1998). This hypothesis is difficult to evaluate at present since detailed information regarding the mouse strains was not provided, and no systematic breeding scheme was described.
Together Math1 and Math5 reconstitute atonal function
Our results significantly advance the concept of functional conservation within the atonal gene family. In Drosophila, atonal controls development of retinal neurons and the chordotonal organs, which are internal mechanosensory structures that act as proprioceptors. In vertebrates, visual system and proprioceptive functions are separated, with Math5 controlling the former and Math1 (Atoh1) regulating the latter. Math1 is expressed in cochlear and vestibular hair cells, vibrissae, the dorsal spinal column, joint capsules, Merkel touch receptors, and the cerebellum (Ben-Arie et al., 1997; Ben-Arie et al., 2000; Bermingham et al., 1999). Each of these structures is associated with mechanosensory perception, locomotory coordination or the integration and processing of three-dimensional spatial information. Math5 and Math1 are the vertebrate bHLH genes most closely related in structure to atonal. This relationship, the absence of significant Math1 expression in the eye, and the findings we present here, strongly suggest that Math5 is the atonal orthologue, and thus the major proneural gene, for the mammalian eye. There are few other examples where two functions identified in the Metazoa are so cleanly partitioned by evolution in the vertebrate lineage. In other gene families, the expression of paralogous genes is largely overlapping so that phenotypes can arise only at the edges where incomplete redundancy is exposed. In contrast, Math1 and Math5 act in independent domains. Taken together they are functionally equivalent to atonal.
Math5 acts during retinal histogenesis, after primary pattern formation in the anterior neural tube, specification of the optic primordia, and the major period of the optic cup growth has occurred. Math5 expression is dependent upon Pax6 (Brown et al., 1998), which has been positioned near the top of a hierarchy for metazoan eye development (Gehring and Ikeo, 1999). Consequently, our results, like those of Neumann and Nüsslein-Volhard (Neumann and Nüsslein-Volhard, 2000) for the hedgehog genes, show that functional homology in visual system development extends more deeply than Pax6. Finally, our results further establish an evolutionary parallel between vertebrate RGCs and fly R8 photoreceptors (Austin et al., 1995; Macabe et al., 1999), the earliest-born neurons of bilaterian visual systems.
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ACKNOWLEDGMENTS |
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