Co-ordinating retinal histogenesis: early cell cycle exit enhances early cell fate determination in the Xenopus retina

The laminar arrays of distinct cell types in the vertebrate retina are built by a histogenic process in which cell fate is correlated with birth order. To explore this co-ordination mechanistically, we altered the relative timing of cell cycle exit in the developing Xenopus retina and asked whether this affected the activity of neural determinants. We found that Xath5, a bHLH proneural gene that promotes retinal ganglion cell (RGC) fate, ( Kanekar, S., Perron, M., Dorsky, R., Harris, W. A., Jan, L. Y., Jan, Y. N. and Vetter, M. L. (1997) Neuron 19, 981-994), does not cause these cells to be born prematurely. To drive cells out of the cell cycle early, therefore, we misexpressed the cyclin kinase inhibitor, p27Xic1. We found that early cell cycle exit potentiates the ability of Xath5 to promote RGC fate. Conversely, the cell cycle activator, cyclin E1, which inhibits cell cycle exit, biases Xath5-expressing cells toward later neuronal fates. We found that Notch activation in this system caused cells to exit the cell cycle prematurely, and when it is misexpressed with Xath5, it also potentiates the induction of RGCs. The potentiation is counteracted by co-expression of cyclin E1. These results suggest a model of histogenesis in which the activity of factors that promote early cell cycle exit enhances the activity of factors that promote early cellular fates.     

Pushing the envelope of retinal ganglion cell genesis: context dependent function of Math5 (Atoh7)

The basic-helix-loop helix factor Math5 (Atoh7) is required for retinal ganglion cell (RGC) development. However, only 10% of Math5-expressing cells adopt the RGC fate, and most become photoreceptors. In principle, Math5 may actively bias progenitors towards RGC fate or passively confer competence to respond to instructive factors. To distinguish these mechanisms, we misexpressed Math5 in a wide population of precursors using a Crx BAC or 2.4 kb promoter, and followed cell fates with Cre recombinase. In mice, the Crx cone-rod homeobox gene and Math5 are expressed shortly after cell cycle exit, in temporally distinct, but overlapping populations of neurogenic cells that give rise to 85% and 3% of the adult retina, respectively. The Crx>Math5 transgenes did not stimulate RGC fate or alter the timing of RGC births. Likewise, retroviral Math5 overexpression in retinal explants did not bias progenitors towards the RGC fate or induce cell cycle exit. The Crx>Math5 transgene did reduce the abundance of early-born (E15.5) photoreceptors two-fold, suggesting a limited cell fate shift. Nonetheless, retinal histology was grossly normal, despite widespread persistent Math5 expression. In an RGC-deficient (Math5 knockout) environment, Crx>Math5 partially rescued RGC and optic nerve development, but the temporal envelope of RGC births was not extended. The number of early-born RGCs (before E13) remained very low, and this was correlated with axon pathfinding defects and cell death. Together, these results suggest that Math5 is not sufficient to stimulate RGC fate. Our findings highlight the robust homeostatic mechanisms, and role of pioneering neurons in RGC development.     

Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors

We have addressed the role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells. We show that mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a premature and excessive generation of astrocytic precursors. An analysis of wild-type and mutant cortical progenitors in culture showed that a large fraction of Ngn2; Mash1 double-mutant progenitors failed to adopt a neuronal fate, instead remaining pluripotent or entering an astrocytic differentiation pathway. Together, these results demonstrate that proneural genes are involved in lineage restriction of cortical progenitors, promoting the acquisition of the neuronal fate and inhibiting the astrocytic fate.     

Overexpression of FGF-2 alters cell fate specification in the developing retina of Xenopus laevis

The developing vertebrate retina produces appropriate ratios of seven phenotypically and functionally distinct cell types. Retinal progenitors remain multipotent up until the last cell division, favoring the idea that extrinsic cues direct cell fate. We demonstrated previously that fibroblast growth factor (FGF) receptors are necessary for transduction of signals in the developing Xenopus retina that bias cell fate decisions (S. McFarlane et al., 1998, Development 125, 3967-3975). However, the precise identity of the signal remains unknown. To test whether an FGF signal is sufficient to influence cell fate choices in the developing retina, FGF-2 was overexpressed in Xenopus retinal precursors by injecting, at the embryonic 16-cell stage, a cDNA plasmid encoding FGF-2 into cells fated to form the retina. We found that FGF-2 overexpression in retinal precursors altered the relative numbers of transgene-expressing retinal ganglion cells (RGC) and Müller glia; RGCs were increased by 35% and Müller glia decreased by 50%. In contrast, the proportion of retinal precursors that became photoreceptors was unchanged. Within the photoreceptor population, however, we found a twofold increase in rod photoreceptors at the expense of cone photoreceptors. These data are consistent with an endogenous FGF signal influencing cell fate decisions in the developing vertebrate retina.     

Roles of homeobox and bHLH genes in specification of a retinal cell type

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.