Atoh8, a bHLH Transcription Factor, Is Required for the Development of Retina and Skeletal Muscle in Zebrafish

Math6/atoh8, a bHLH transcription factor, is thought to be indispensable for early embryonic development and likely has important roles in vertebrate tissue-specific differentiation. However, the function of Atoh8 during early development is not clear because homozygous knockout causes embryonic lethality in mice. We have examined the effects of the atoh8 gene on the differentiation of retina and skeletal muscle during early development in zebrafish.We isolated a Math6 homologue in zebrafish, designated as zebrafish atoh8. Whole -mount in situ hybridization analysis showed that zebrafish atoh8 is dynamically expressed mainly in developing retina and skeletal muscle. Atoh8-MO knock-down resulted in reduced eye size with disorganization of retinal lamination. The reduction of atoh8 function also affected the arrangement of paraxial cells and differentiated muscle fibers during somite morphogenesis.Our results show that Atoh8 is an important regulator for the development of both the retina and skeletal muscles necessary for neural retinal cell and myogenic differentiation during zebrafish embryogenesis

Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

Background: The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6(Sey/Sey)) and are abnormally small in Pax6(Sey/+) mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results: Eyes form in PAX77(+/+) embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77(+/+) retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77(+/+) embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6(Sey/+), Pax6(+/+), PAX77(+) and PAX77(+/+)) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection. Conclusion: Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6(+/-) heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm

Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis-acting regulatory elements within the 5'-flanking region of the alpha 7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which alpha 7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that alpha 7 regulatory elements acquire their neuronal specificity in the course of embryogenesis

Neurog2 controls the leading edge of neurogenesis in the mammalian retina

AbstractIn the mammalian retina, neuronal differentiation begins in the dorso-central optic cup and sweeps peripherally and ventrally. While certain extrinsic factors have been implicated, little is known about the intrinsic factors that direct this process. In this study, we evaluate the expression and function of proneural bHLH transcription factors during the onset of mouse retinal neurogenesis. Dorso-central retinal progenitor cells that give rise to the first postmitotic neurons express Neurog2/Ngn2 and Atoh7/Math5. In the absence of Neurog2, the spread of neurogenesis stalls, along with Atoh7 expression and RGC differentiation. However, neurogenesis is eventually restored, and at birth Neurog2 mutant retinas are reduced in size, with only a slight increase in the retinal ganglion cell population. We find that the re-establishment of neurogenesis coincides with the onset of Ascl1 expression, and that Ascl1 can rescue the early arrest of neural development in the absence of Neurog2. Together, this study supports the hypothesis that the intrinsic factors Neurog2 and Ascl1 regulate the temporal progression of retinal neurogenesis by directing overlapping waves of neuron formation