THE EVOLUTION AND DYNAMICS OF GENETIC REGULATORY NETWORKS IN ZEBRAFISH NEUROGENESIS
, Head, Section on Neural Developmental Dynamics
Michael Keller, PhD, Postdoctoral Fellow
Kinneret Rand, PhD, Visiting Fellow
Miho Matsuda, PhD, Visiting Fellow
Gali Prag, PhD, Visiting Fellow
Min Jung Kim, PhD, Visiting Fellow
Seok-Yong Choi, PhD, Visiting Fellow
Gregory Palardy, BS, Research Technician
Chongmin Wang, MS, Research Technician
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 provide insight into 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 the fish-specific gene Zic6, which has been lost in frogs, birds, and mammals. Its analysis provides a basis for understanding the evolution of the Zic genes and the significance of the paired arrangement of Zic genes in the genome. As the nervous system develops, compartment boundaries in the hindbrain become Wnt signaling centers and induce new neurogenic zones in adjacent domains. Analysis of a Mind bomb–interacting protein, Mosaic Eyes, showed that the protein is essential for the function of the genetic regulatory network that both establishes Notch-dependent Wnt signaling centers and maintains the boundaries. We have used computer simulations to visualize the dynamics of this genetic network. The simulations have helped us 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.
Insights into the evolutionary history of the vertebrate zic3 locus from a teleost-specific zic6
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 fates within the developing embryo. The Zic genes are typically expressed in ectodermal tissues contributing to the nervous system, neural crest, and somatic mesoderm. Strong experimental evidence supports a combined role of the Zic genes in neurulation, neurogenesis, neural crest specification, and establishment of left-right 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 the genes’ broadly overlapping expression with the potential for competition for DNA-binding sites as well as by cross-regulatory 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.
Despite significant conservation in the structure of the Zic protein DNA-binding domain, which consists of five zinc fingers, considerable divergence occurs in other parts of the protein and may be correlated with altered post-translational regulation, protein-protein 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. With the exception of zic3, four known vertebrate homologues occur as zic1/zic4 and zic2/zic5 bigenes; zic3 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 the description to infer the evolutionary relationships of the Zic family members in vertebrates that include fish, frogs, birds, and mammals. We found the zic6 gene 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 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 that the gene plays a different regulatory role during early embryonic development of fish.
Keller MJ, Chitnis AB. Insights into the evolutionary history of the vertebrate zic3 locus from a teleost-specific zic6 gene in the zebrafish, Danio rerio. Dev Genes Evol 2007;217:541-7.
Mosaic eyes and the dynamics of the genetic circuit that maintains Wnt signaling centers at rhombomere boundaries
Mind bomb (Mib) ubiquitylates the transmembrane protein Delta at the cell surface, and ubiquitylated Delta recruits the endocytic machinery, resulting in internalization of Delta from the surface. This step is essential for Delta’s effective activation of 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 plays a role in establishing 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 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. These interactions play 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.
Chitnis AB. Notch signaling: a versatile tool for fine patterning of cell fate in development. In: Moody S, ed. Principles of Developmental Genetics. Elsevier, 2007;316-40.
For further information, contactchitnisa@mail.nih.gov.
