Ajay B. Chitnis - US grants

zebrafish; developmental neurobiology; neurogenesis; mutant; developmental genetics; nervous system transplantation; Xenopus; single strand conformation polymorphism;

Early differentiating neurons in the zebrafish are generated in discrete neurogenic domains of the neural plate. The mechanisms by which these discrete domains are defined in zebrafish provide insight about how neurogenesis is regulated in the vertebrate nervous system. Previously we described the role of Zic genes in defining boundaries in the neural plate adjacent to which neurons differentiate. Now we have characterized a fish-specific gene, Zic6, which has been lost in frogs, bird and mammals. Its analysis serves as a basis for understanding the evolution of the Zic genes and the significance of paired arrangement of Zic genes in the genome. [unreadable] The Zic gene family encodes a group of C2H2 zinc-finger transcription factors that are important regulators of early vertebrate development. They are part of a larger Gli/Zic/NKL gene superfamily and, together with the Gli genes, are thought to help define tissue compartments with specific fate within the developing embryo. The Zic genes are typically expressed in ectodermal tissues contributing to the nervous system and neural crest, as well as somatic mesoderm. There is strong experimental support for a combined role of the Zic genes in neurulation, neurogenesis, neural crest specification, and establishment of leftright asymmetry. Deficits in Zic gene family members have been linked to developmental defects such as spina-bifida, holoprosencephaly, and X-linked heterotaxia. Understanding the significance of Zic gene function during embryonic development is confounded by their broadly overlapping expression with the potential for competition for DNA-binding, sites as well as crossregulatory and physical interactions among orthologues. It is therefore essential to define the combined expression of the Zic gene family members and understand their evolutionary relationships. Although there is significant conservation in the structure of the Zic protein DNA-binding domain, consisting of five zinc-fingers, there is also considerable divergence in other parts of the protein that may be correlated with altered post-translational regulation, proteinprotein interactions and repressor/activator activities. The evolutionary diversification among family members may, however, be constrained by their physical arrangement as paired genes (bigenes) that share a limited amount of upstream DNA. [unreadable] Four known vertebrate homologues occur as zic1/zic4 and zic2/zic5 bigenes, with the exception being zic3, which is a single-gene locus, located on the X chromosome in mammals. We have described the structure, genomic context, and embryonic expression of zebrafish zic6 and use this analysis to infer the evolutionary relationships of the Zic family members in vertebrates that include fish, frogs, birds and mammals. The zic6 gene was found to be teleost-specific, occurring among a broad range of fishes, but absent from the genomes of frogs, birds, and mammals. Genomic analysis established that zic6 is paired with zic3, in opposite orientation, as is the case with the zic1/zic4 and zic2a/zic5 gene pairs. Synteny of flanking genes confirmed that the zic3 loci of fish and the other vertebrate taxa are true homologues, supporting the conclusion that zic6 was the product of a chromosomal duplication before the divergence of fishes and tetrapods and was subsequently lost in the tetrapod lineage. The expression of zic6 in the neural plate lacked the lateral and rostral domains typical of the other Zic gene orthologues, indicating it has a different regulatory role during early embryonic development of fish. We are currently examining if interactions between bigenes influences the spatiotemporal pattern of their expression.[unreadable] [unreadable] As the nervous system develops, compartment boundaries in the hindbrain become Wnt signaling centers and they induce new neurogenic zones in adjacent domains. Analysis of a Mind bomb interacting protein, Mosaic Eyes, showed that it is essential for the function of the genetic regulatory network that establishes and restricts Notch-dependent Wnt signaling centers to the boundaries. We have used computer simulations to visualize the dynamics of this genetic network. The simulations have helped understand how the signaling centers are established and how establishment of boundary-associated signaling centers could fail with dysfunction of specific components of the genetic network.[unreadable] Mib is an essential component of the Delta-Notch signaling system, which plays a critical role in determining how many progenitors are allowed to become neurons within a neurogenic domain. Mib ubiquitylates the transmembrane protein Delta at the cell surface and the ubiquitylated Delta is internalized from the surface. This step is essential for Delta to effectively activate its receptor, Notch, in neighboring cells. Mosaic Eyes (Moe) is a Mib-interacting protein that stabilizes Mib at the lateral surface of epithelial cells. While Moe is not essential during early neurogenesis, it is essential for regulating Notch function in the hindbrain, where Notch has a role in establishment of Wnt signaling centers at rhombomere boundaries. Reduction of Moe function results in failure of the mechanism that normally restricts Wnt signaling centers to boundaries and it allows spreading of the Wnt signaling center to adjacent non-boundary cells. In this context, Delta is expressed in para-boundary cells, where its interactions with Notch allow it to maintain Notch activation in adjacent boundary cells. At the same time, interactions of Delta with Notch within the para-boundary cells prevent Notch activation, and these interactions have a critical role in preventing Notch-mediated Wnt expression in non-boundary cells. Computer simulations of the genetic regulatory circuit that maintains Notch-dependent Wnt signaling at rhombomere boundaries reveal how loss of Delta-mediated inhibition of Notch signaling can account for failure to restrict Wnt signaling centers to rhombomere boundaries. Furthermore, the simulations illustrate how asymmetric signaling properties established in even and odd-numbered rhombomeres by early patterning mechanisms could set up the conditions for emergence of Wnt signaling centers at rhombomere boundaries later in development.

BACKGROUND Formation of the Posterior Lateral Line system in zebrafish is pioneered by the posterior Lateral Line (pLL) primordium, a group of about 150 cells that forms near the ear. While leading cells in the pLL primordium have a relatively mesenchymal morphology, trailing cells are more epithelial; they have distinct apical basal polarity and they reorganize to sequentially form nascent neuromasts or protoneuromasts. The pLL primordium begins migration toward the tip of the tail at about 22 hours post fertilization (hpf). Proliferation adds to the growth of the primordium, nevertheless, as the primordium migrates, the length of the column of cells undergoing collective migration progressively shrinks as cells stop migrating are deposited from the trailing end: cells that were incorporated into protoneuromasts are deposited as neuromasts, while cells that were not, are deposited between neuromasts as interneuromast cells. Eventually, the primordium ends its migration a day later after depositing 5-6 neuromasts and by resolving into 2-3 terminal neuromasts. Establishment of polarized Wnt and FGF signaling systems coordinates morphogenesis and migration of the primordium: Wnt signaling dominates at the leading end and is thought to determine the relatively mesenchymal morphology of leading cells, while FGF signaling dominates in the trailing end. There, FGF determines reorganization of groups of trailing cells to form rosettes as they constrict at their apical ends. Furthermore, FGF signaling determines the specification of a central cell in each rosette as a sensory hair cell progenitor and it helps determine collective migration of the pLL primordium cells. Wnt signaling promotes its own activity and at the same time drives expression of fgf3 and fgf10. However, leading cells do not respond to these FGF ligands because Wnt signaling simultaneously promotes expression of intracellular inhibitors of the FGF receptor. Instead, the FGFs activate FGF receptors and initiate FGF signaling at the trailing end of the primordium, where Wnt signaling is weakest. There, FGF signaling determines expression of the diffusible Wnt antagonist Dkk1b, which counteracts Wnt signaling to help establish stable FGF responsive centers. Once established, the trailing FGF signaling system coordinates morphogenesis of nascent neuromasts by simultaneously promoting the reorganization of cells into epithelial rosettes and by initiating expression of factors that help specify a sensory hair cell progenitor at the center of each forming neuromast. Over time, the leading domain with active Wnt signaling shrinks closer to the leading edge and additional FGF signaling centers form sequentially in its wake, each associated with formation of additional protoneuromasts. QUESTIONS The interactions between the leading Wnt system and the trailing FGF system provide a useful framework for understanding the self-organization of neuromast formation and deposition by the migrating pLL primordium, however, many questions remain unanswered. The Wnt and FGF signaling systems act simply as a means of communication between cells and it remains unclear what molecular mechanisms are being regulated by them to specifically determine morphogenesis of epithelial rosettes and the collective migration of primordium cells. Furthermore, the mechanics of collective migration in the primordium remains poorly understood, specifically, how the pull of leading cells, which migrate in response to chemokine cues in their path, determines the FGF-dependent collective migration of trailing cells in the primordium. Finally, the summary above suggests that morphogenesis of epithelial rosettes during the assembly of nascent neuromasts is entirely dependent on FGF signaling. However, it has been seen that in the absence of collective migration mediated by chemokines in the leading cells, the trailing cells in the primordium come together to form one or two large rosettes. These and other observations related to the changes in the number and size of epithelial rosettes in the presence and absence of collective migration suggest that primordium cells have an inherent potential to form epithelial rosettes and their potential to form epithelial rosettes can be influenced by a variety of signaling systems and/or by migratory behavior of leading cells. Our attention has now shifted to answering some of the questions outlined here. Below we summarize the observations of a recently published study that describes the role of chemokine signaling in determining expression of the transcription factor Snail1b in leading cells, the role of Snail1b in kickstarting collective migration of the pLL primordium, and the surprising role of collective migration in determining sequential formation of protoneuromasts in the migrating primordium. CXCL12a INDUCES SNAIL1B EXPRESSION TO INITIATE COLLECTIVE MIGRATION AND SEQUENTIAL FGF-DEPENDENT NEUROMAST FORMATION IN THE ZEBRAFISH POSTERIOR LATERAL LINE PRIMORDIUM Our discovery that snail1b is expressed in leading cells led us to speculate that its expression might be determined by Wnt signaling and that, as a factor known for its role in promoting EMT, it might determine mesenchymal morphology of leading cells. However, our analysis revealed that Wnt signaling does not determine snail1b expression in the leading zone. Instead, its expression in the leading zone is promoted by chemokine signals first encountered by leading cells of the primordium, while Fgf signaling prevents snail1b expression in trailing cells. The effect of knocking down snail1b function was also not what we expected. It did not compromise the ability of leading cells to adopt a mesenchymal morphology. Instead, sequential morphogenesis of epithelial rosettes associated with formation of protoneuromasts in the trailing domain was delayed in snail1b morphants. Our analysis revealed, however, that the delay in protoneuromast formation is not related to a specific role of Snail1b in morphogenesis of epithelial rosettes. Instead, it is indirectly related to the role of Snail1b in helping to initiate effective collective migration of the primordium, a role consistent with its previously described role in promoting cell movement. Interestingly, we found that other manipulations that prevent effective primordium migration also cause a similar delay in sequential formation of Fgf signaling centers, associated protoneuromasts and shrinking of the leading Wnt system. These observations, together, reveal an unexpected role for collective migration of the primordium in kick-starting sequential formation of additional protoneuromasts. Finally, we showed that problems in initiating collective migration in the primordium may, at least in part, be related to aberrant expansion of the cell adhesion molecule epcam into the leading zone and/or the loss of cdh2 expression from the leading zone of the primordium following knock-down of snail1b function.