The earliest differentiating neurons in the zebrafish neural plate are generated in well-defined neurogenic domains that are separated by non-neurogenic domains. Analysis of the mechanisms by which these discrete domains are defined in zebrafish provides important clues about how neurogenesis is regulated in the vertebrate nervous system. Previous studies in the frog had suggested a critical role for zic2 in defining non-neurogenic domains, however, analysis in zebrafish has suggested a more complex role in defining both neurogenic and non-neurogenic domains. Our recent studies show how in both contexts the Zic factors regulate neurogenesis by limiting expression of certain Hairy related transcription factors. ? The neural plate eventually undergoes cell movements and rearrangements to form the neural tube. In the hindbrain further patterning and morphogenetic events define segmental compartments called rhombomeres and their boundaries become organizing centers for neurogenesis. Analysis of Mosaic Eyes (Moe), a FERM domain protein, has provided insight into the role of Notch signaling in the morphogentic events leading to the establishment of rhombomere boundaries. Moe was originally identified in a yeast two-hybrid screen with Mind bomb, a critical component of the Notch signaling pathway. Its analysis now provides insight into the poorly understood role of Notch in morphogenesis.? Early neurogenesis occurs in three longitudinal columns within the caudal neural plate in zebrafish and Xenopus embryos. The patterning mechanisms that delineate neurogenic and non-neurogenic domains are poorly understood, but analyses in Xenopus had suggested a role for the Zic zinc-finger transcription factor, Zic2, in defining non-neurogenic domains within the neural plate. To determine if Zic2 related factors have a similar role in zebrafish a loss-of-function approach using morpholino mediated protein knockdown was used to assess the role of zic genes expressed during early neurogenesis. Rather than define non-neurogenic domains, zebrafish zic2a, zic2b and zic3 were found to function together to promote neuronal differentiation in the neural plate. However, consistent with their suggested function in Xenopus, these zic genes also contributed to the suppression of neurogenesis in the adjacent trigeminal placode. The opposing roles of zic2a, zic2b and zic3 genes in the trigeminal placode and neural plate presented a puzzle and we tried to identify potential transcriptional targets to understand how these Zic factors might influence neurogenesis in different ways in these two adjacent neural tissues. ? While these studies were in progress it was reported that a subset of Hairy-Enhancer of split- Related (HER) genes, her3, her5, her9 and her11 have critical roles in defining non-neurogenic territories in the zebrafish neural plate. To determine if some effects of Zic factors on neurogenesis were directly or indirectly related to the function of some of these HER genes we examined changes in the expression of her9, a HER gene whose expression was spatiotemporally most closely related to neurons whose differentiation was altered with zic gene knock-down. Consistent with the combinatorial function of zic2a, zic2b and zic3 in neurogenesis being related, at least in part, to regulation of her9, the expression of her9 was expanded in both the neural plate and adjacent placodal ectoderm in zic2a+zic2b+zic3 morphant embryos. Furthermore, the concurrent knockdown of her9 in the zic2a+zic2b+zic3 morphant embryos """"""""rescued"""""""" neuronal differentiation in both tissues, in spite of their qualitatively opposite phenotypes: neuronal differentiation was restored in the neural plate while the over-production of neurons in the trigeminal placode was suppressed. These observations led to the conclusion that the function of the zic genes in both neural plate and the adjacent trigeminal placode was dependent on their ability to repress her9. Thus, Zic proteins function by limiting her9 expression, thereby either promoting or suppressing neuronal differentiation in a context-dependent manner. Elucidating the mechanism by which HER genes like her9 have opposing effects on neurogenesis in the trigeminal placode and neural placode remains the next question for inquiry.? Delta-Notch signaling limits the differentiation of neural progenitors as early neurons in the zebrafish neural plate. In this context, a broad failure of effective Notch signaling in mind bomb (mib) mutants results in the production of an excess of early neurons. mib encodes an E3 ligase that promotes Delta ubiquitylation and endocytosis, a step that is essential for its function as a Notch ligand. ? In recent studies a FERM domain protein Mosaic Eyes (Moe) was identified as a potential Mib interacting protein. In cell culture Moe is typically associated with the cell surface and in co-transfection experiments it alters Mib?s cellular distribution, reducing Mib in the cytoplasm and stabilizing it at the cell surface. In contrast, co-expression of Mib with a truncated Moe, lacking the N-terminal fragment responsible for its surface localization, fails to stabilize Mib at the cell surface and instead retains it in the cytoplasm. ? Mib ubiquitylates DeltaD and promotes its internalization, dramatically reducing surface DeltaD when the two are co-expressed in COS cells. Co-expression of Moe together with Mib and DeltaD, however, increases surface DeltaD suggesting that either it reduces overall internalization of DeltaD by Mib and/or facilitates recycling to the cell surface. Co-transfection of Mib and DeltaD with N-terminal deleted Moe, also increases surface Delta, though not as effectively as full length Moe. Remarkably, however, co-transfection of DeltaD with the N-terminal deleted Moe reduces DeltaD protein suggesting that retention of Mib in the cytoplasm by N-terminal deleted Moe may facilitate degradation of DeltaD. Destabilization of Delta by the truncated Moe raised the possibility that stabilization of Mib at the cell surface by full length Moe may normally contribute to Delta stability.? To test this prediction endogenous DeltaD expression was examined in zebrafish embryos injected with anti-sense moe morpholinos. Though no change in DeltaD expression was observed during early development, a dramatic loss of DeltaD protein was observed in the hindbrain at 24 hours post fertilization. Knock-down of moe also interfered with morphogenesis of rhombomere boundaries: Genes whose expression is typically restricted to rhombomere boundaries by 24 hours was deregulated and spread throughout the hindbrain in moe morphants. Rhombomere-specific expression of other genes, however, was not affected, suggesting a specific problem with boundary formation not with patterning events that define discrete rhombomere compartments. ? Restriction of Notch activity is thought to be essential for morphogenesis of rhombomere boundaries and cells with relatively high Notch activity segregate to these boundaries. In moe morphants Notch activity is no longer restricted to boundaries and the Notch target gene her4 is expressed throughout some rhombomeres. Interestingly, knock-down of DeltaD with morpholinos also deregulated her4 expression. This supports the idea that DeltaD protein has a critical role in restricting Notch activity to rhombomere boundaries and that loss of DeltaD protein, either due to destabilization in moe morphants or direct knock-down, results in deregulated Notch activity. ? Though it remains unclear how Moe determines DeltaD stability and how Delta limits Notch activity to rhombomere boundaries, these observations identify Moe as a new component of a previously identified patterning mechanism where complementary Notch and Wnt signaling collaborate to restrict Notch signaling and expression of various genes to rhombomere boundaries.
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