RGC differentiation is governed by a cascade of gene activation controlling the sequence of distinct developmental stages and promoting axon growth and navigation. By using translation-blocking MOs targeting either of the two Neurolin paralogs, Neurolin-a or Neurolin-b, our study shows that the two gene products have specialized their function because of duplication and each plays a crucial, but distinct role in RGC development. Neurolin-a is necessary for a step in the differentiation of RGCs that correlates with axogenesis (Stuermer,1988; Laessing and Suermer,1996). Loss of Neurolin-a expression inhibits the timely differentiation of RGCs and non-RGC neurons and ultimately leads to cell death. The resulting abnormal retinas lack axons and optic nerves and are probably nonfunctional. Neurolin-b expression in RGCs is delayed relative to Neurolin-a, but is present during axon growth and navigation toward the optic tectum. Accordingly, loss of Neurolin-b expression does not impair RGC differentiation, and eyes develop normally. However, the absence of Neurolin-b causes strikingly aberrant pathways of RGC axons and failure to correctly invade the optic tectum. Together, our results show novel functions of Neurolin/ALCAM in the development of the retina and the formation of tectal connections.
Importance of Neurolin-a during RGC differentiation
In our earlier work, we identified the dynamic spatiotemporal sequence of RGC differentiation in zebrafish by the expression of Neurolin-a (Laessing and Stuermer,1996). Expression proceeds in an arc-like pattern from the nasal via dorsal to temporal and ventral retina and then continues in rings in progressively more peripheral positions (Laessing and Stuermer,1996). In our present experiments, inhibition of Neurolin-a protein synthesis disrupted this pattern of RGC differentiation and blocked the normal order of retina growth, causing reduced eye size. These results suggest that Neurolin-a-based cell-cell communication is necessary for the progression of cell differentiation across the retina. A challenge for future experiments is the analysis of the intracellular signaling cascades triggered by these interactions.
Several other genes involved in zebrafish retinal development are similarly expressed in a wave-like pattern. Both shh and the Iroquois homeobox gene irx1a are required for the propagation of the RGC differentiation wave across the retina (Neumann and Nüsslein-Volhardt,2000; Cheng et al.,2006). Sonic you (syu) mutant zebrafish as well as irx1a morphants show retinal defects that closely resemble MO-Na-affected retinae. Eyes are smaller and development of RGCs is impaired as the spreading waves of shh/irx1a expression are collapsed (Neumann and Nüsslein-Volhardt,2000; Stenkamp et al.,2002; Cheng et al.,2006). Whether this similar phenotype implies any interactions among irx1a, shh, and Neurolin signaling pathways remains to be analyzed. Interestingly, absence of either shh, irx1a, or Neurolin-a also leads to impaired differentiation of neurons in the INL and PRL. It has been shown that shh, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the absence of RGCs (Shkumatava et al.,2004). Therefore, loss of all shh sources explains the retinal phenotype of syu mutants. Irx1a and Neurolin-a, however, are expressed only on RGCs (Laessing and Stuermer,1996; Leppert et al.,1999; Cheng et al.,2006). Although others have claimed Neurolin-a expression in the early eye anlage (Mann et al., 2007) and in amacrine cells (Kay et al.,2001), we and others could not find this by either in situ hybridization or immunostaining (Laessing and Stuermer, 1999; Shkumatava et al.,2004; this paper). RGCs are generally the first retinal neurons to differentiate, and it has been shown that they affect the subsequent differentiation of neurons in a cohort that derives from a common progenitor cell (Harris,1997).
The bHLH transcription factor atoh7 is induced in the first RGCs by pax2a-expressing optic stalk cells in the nasoventral retina prior to Neurolin-a and participates in the induction of RGC cell fate (Masai et al.,2000; Kay et al.,2001). The progression of this induction wave is independent of differentiated RGCs (Kay et al.,2005). An early role for atoh7 in RGC cell fate priming upstream of Neurolin-a function is consistent with our results showing that the wave of atoh7 expression is unaltered in MO-Na-injected zebrafish embryos. Therefore, atoh7 expression is necessary but not sufficient for retinal progenitors to develop into RGCs. Because the optic cup (pax6 expression) and optic stalk (pax2a expression) are normal in MO-Na-treated zebrafish, Neurolin-a functions in RGC differentiation after cell fate determination by atoh7. Surprisingly, an early cell fate error, such as that seen in the atoh7 null mutant lakritz (lak; Kay et al.,2001), seems less detrimental to retinal development than later block of RGC differentiation such as that observed in shh mutants and irx1a and Neurolin-a MO knockdowns (Neumann and Nüsslein-Volhardt,2000, Stenkamp et al.,2002; Cheng et al.,2006; this paper). Although no RGCs differentiate in lak, all other retinal cells develop normally in organized lamina. Rather than losing cells, lak mutants overproduce bipolar, amacrine, and Müller glia cells and misplaced amacrine cells populate the prospective RGCL. Because progenitor cells are not restricted to the RGC fate by atoh7 expression in lak, they can “switch” to a different cell type. The multipotent progenitor cells change their competence to generate different retinal cells in response to tightly controlled position- and stage-dependent environmental cues (Livesey and Cepko,2001).
Analysis of the zebrafish mutant ascending and descending (add) gene revealed that histone deacetylase 1 (hdac1) regulates cell cycle exit as the first step of retinal neurogenesis by suppressing Wnt and Notch signaling pathways (Yamaguchi et al.,2005). Division of retinal progenitors produces two daughters, one of which becomes a postmitotic RGC (Poggi et al.,2005). Based on the atoh7, shh, irx1a, and Neurolin-a knockdown phenotypes, we propose a model in which cells committed to the RGC fate due to atoh7 expression would produce a signaling molecule that would inhibit adjacent progenitor cells to differentiate. Because the differentiation of the different retinal cells is temporally controlled (Ohnuma et al.,2002), this would prevent all progenitor cells from becoming RGCs. One candidate signaling molecule is Notch, which has been shown to maintain proliferation and to inhibit differentiation (Yamaguchi et al.,2005). Once the cells committed to the RGC cell fate actually develop into differentiated RGCs, and hence express shh, irx1a, and Neurolin-a, they then start to express a de-repressor (for example, shh itself) to lift the inhibition of differentiation and to allow progenitors to develop into other cell types like amacrines or photoreceptors.
In accordance with this inhibitor/de-repressor model, loss of atoh7 expression would result in the loss of the differentiation inhibitor, and progenitor cells would be free to develop into all cells except RGCs (lak phenotype). Loss of Neurolin-a (and irx1a), however, would mean persistent block of differentiation of all cell types because the inhibitor cannot be de-repressed, as observed in irx1a and Neurolin-a morphant phenotypes. Because this inhibition/de-repression has to occur very dynamically and be spatiotemporally controlled, it will be challenging to analyze without knowledge of the molecules involved. It would be interesting to know whether Notch expression is at all altered in lak/atoh7 mutants.
Another feature of MO-Na-affected eyes is the presence of fragmented nuclei and cavities indicative of neuronal apoptosis. Again, these are not restricted to the RGCL but are found in all retinal layers and are only detected at later stages of retinal development (4 dpf) well after onset of Neurolin-a expression (32 hpf) and the appearance of a MO-Na phenotype. In syu mutant and irx1a morphant retinas, apoptosis also occurs late and spatially random (Neumann and Nüsslein-Volhardt,2000; Stenkamp et al.,2002; Cheng et al.,2006). Cells that have left the cycle but are not able to complete their differentiation program within a certain time period seem to be condemned to die.
Involvement of Neurolin-b in RGC axon pathfinding
Loss of Neurolin-b function did not produce an overall morphological phenotype. Because Neurolin-b was found to be expressed late (≥48 hpf) in differentiated RGCs, differentiation of retinal cells is unaffected by loss of Neurolin-b function. Instead, Neurolin-b has a role during axon growth and innervation of the optic tectum. In wild-type zebrafish, the first RGC axons enter the tectum at 48 hpf and reach its posterior end within the next 24 hpf (Stuermer,1988). In MO-Nb-injected zebrafish, retinal axons defasciculate in the region between the left and right eye after crossing at the chiasm and stall at the anterior end of the optic tectum or grow to abnormal positions. Therefore, late expression of Neurolin-b in RGCs in comparison with Neurolin-a correlates with a late function in the developing visual system. The observed phenotype suggests a role for Neurolin-b in RGC axon pathfinding and fasciculation not within the retina (Ott et al.,1998; Leppert et al.,1999) but toward the optic tectum.
In the goldfish retina, Neurolin is involved in intraretinal axon pathfinding as the radial growth of axons from newborn RGCs becomes highly abnormal and defasciculated in the presence of Neurolin antibodies (Ott et al., 1997; Leppert et al.,1999). We have not analyzed the pathways of axons within the zebrafish retina of 48 hpf zebrafish because, in our hands, these are too small for such investigations. The avian homologue DM-GRASP has also been associated with axon growth and the establishment of neural connections (Burns et al., 1991; Pourquie et al.,1992; Pollerberg and Mack,1994; DeBernardo and Chang,1996). In the presence of DM-GRASP F(ab) fragments, axons fail to enter the optic nerve and stray away from correctly orientated axons (Avci et al.,2004). Similarly, ALCAM knockout mice show fasciculation and pathfinding defects of RGC and motoraxons (Weiner et al.,2004). The ALCAM knockout retina is normally stratified, but axons fascicles in the optic fiber layer are broader, and aberrant trajectories are observed. Consequently, an axon guidance function, either within the retina (goldfish) or the trajectory to the tectum (mice, zebrafish) seems to be evolutionarily conserved for the neurolin/alcam gene.
In addition to the axon fasciculation and pathfinding defects, the ALCAM knockout retina shows retinal dysplasia indicative of aberrant growth and development. Although this defect was not analyzed in temporal detail or with differentiation markers to elucidate which phase of retinal development was affected, mouse ALCAM also seems to have a basic role in cell differentiation similar to Neurolin-a in zebrafish. However, as mouse ALCAM is still present on neonatal and adult retinal axons (Weiner et al.,2004), it cannot provide a dynamic function that has to be timely and spatially controlled, as is Neurolin-a, which is only expressed in newborn retinal ganglion cells and is downregulated a few hours later (Laessing and Stuermer,1996). Neurolin-a function in the regulation of cell type specification seems therefore to be specific to zebrafish, which would also explain how RGCs properly differentiate and send axons out of the eye in the ALCAM knockout mouse. In contrast, human ALCAM is important for the differentiation of various cell types (hematopoieses: Cortes et al.,1999; Uchida et al.,1997; Ohneda et al.,2001; thymus development: Bowen et al., 1995; bone morphogenesis: Bruder et al., 1998).
Our present data demonstrate that cell differentiation, namely, of retinal cells but also of red blood cells (unpublished result), and axon pathfinding are mediated by two proteins in zebrafish, Neurolin-a and Neurolin-b, respectively. This is in agreement with the duplication-degeneration-complementation (DDC) model that proposes a partitioning of gene functions following gene duplication that renders both duplicates necessary to preserve the function of the single ancestral gene (Force et al., 1999; Meyer and Schartl,1999).