A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as the pharyngeal arches. We found that the BTB-domain containing protein Kctd15 that is first expressed in the embryo in the neural plate border, is an important factor in regulating the domain in which neural crest forms. Overexpression of Kctd15 strongly inhibits neural crest specification, while attenuation of Kctd15 expression leads to expansion of the neural crest precursor domain. In contrast, we have shown that anterior placodal domains are expanded after Kctd15 overexpression, supporting the idea that Kctd15 limits the extent of the neural crest domain and prevents incursion of the placodal domain by neural crest precursor cells. Recently we have found that Kctd15 inhibits the activity of transcription factor AP-2, a factor well known to be required at several stages of neural crest formation. Mechanistic studies on the interaction between AP-2 and Kctd15 have been carried out. Ap-2 is a major regulator of neural crest formation, being involved in the initial specification of neural crest cells as well as in later stages such as their migration and differentiation into multiple derivatives. Kctd15 is capable of binding AP-2 in co-immunoprecipitation experiments, and inhibits the activation of an AP-2 reporter both in cultured cells and in zebrafish embryos. In studying the mechanism of this inhibition we found that Kctd15 does not affect the level of AP-2 in the cell, does not inhibit dimerization of AP-2 which is required for activity, and does not affect nuclear localization of AP-2. Also, AP-2 remains bound to its cognate sites in chromatin in the presence of Kctd15. To probe the mechanism further we studied the activation domain of AP-2. Kctd15 binds to the activation domain, and specifically requires proline 59 for this interaction. An AP-2 mutant in which this proline is changed to alanine (P59A) is active in the reporter assay but cannot bind Kctd15, and its activity is not inhibited by Kctd15. We conclude that Kctd15 inhibits AP-2 activity by specific binding to its activation domain which precludes its function. Among genes identified in our DNA microarray studies as differentially expressed in the pineal gland we noted a gene encoding a homolog of the Unc119 protein family. Whereas two Unc119 homologs are known in humans, the gene we isolated is the third family member noted in zebrafish;accordingly we named this protein Unc119c. The unc119c gene is specifically expressed in the pineal, with a low level of expression in the retina. In fish the pineal is a photosensitive organ and shows many similarities in gene expression to the retina. Unc119 proteins interact with small GTPases of the Arl3 family, and we have shown that Unc119c binds to Arl3l2 when both are coexpressed in heterologous cells. Using morpholino antisense oligonucleotide (MO) mediated knock-down of Unc119c expression we found that this protein is required for the formation of the habenular commissure (HC) in the zebrafish. The HC crosses the midline in close proximity to the pineal gland. Knock-down of the Unc119c binding partners Arl3l1 or Arl3l2 also affect HC formation. We hypothesized that Unc119c might be involved in protein trafficking or secretion of a guidance factor involved in HC formation. Based on the literature and expression pattern we picked Wnt4a as a candidate target gene;Wnt4a has been reported by others to be required for HC formation, a fact confirmed in our experiments. We found that Wnt4a accumulation and secretion from cultured HEK293T cells is stimulated by Unc119c and Arl3l1/2. Thus we propose that Unc119c is required in the pineal gland to stimulate Wnt4a secretion, while Wnt4a in turn acts as a guidance cue for the HC as it traverses the zebrafish forebrain. The yolk syncytial layer (YSL) of the zebrafish embryo arises during early stages of development. It is a multinucleated syncytium that is generated by the collapse of membranes between cells adjoining the yolk layer. The YSL is essential for multiple steps in embryo development, for example mesoderm induction and the establishment of the dorsoventral axis. In spite of its importance the molecular mechanisms underlying YSL formation have not been fully elucidated at this time. We have shown that the zebrafish homolog of the protein named solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL, and is required for its normal development. When the expression of Slc3a2 is reduced by morpholino oligonucleotide-mediated knockdown, YSL development becomes abnormal, showing clustering of the yolk syncytial nuclei, an enhanced level of cell fusion, and the disruption of microtubule networks that normally have a highly ordered structure in the YSL. The artificial introduction of a constitutively active version of the small GTPase RhoA leads to a similar YSL phenotype as that generated by Slc3a2 knockdown. Conversely, the reduction of RhoA activity rescues the Slc3a2 knockdown phenotype. Likewise, inhibition of Rock, a downstream effector of RhoA, also rescues the Slc3a2 phenotype. In addition, Slc3a2 knockdown strongly inhibits tyrosine phosphorylation of c-Src. Consistent with this observation, overexpression of a constitutively active Src rescues the phenotype generated by the reduction in Slc3a2 expression. These observations suggest that the signaling pathway that regulates YSL formation involves the inhibition of RhoA/Rock by Slc3a2 as a consequence of the phosphorylation of c-Src. We suggest that this signal transduction pathway modulates microtubule dynamics in the developing YSL. Loss of microtubule structure then causes the abnormalities in the arrangement of yolk syncytial nuclei and in the regulation of cell fusion that are observed in Slc3a2 knockdown embryos. These studied shed light on more general aspects of the regulation of cell fusion, a process that has many roles in different biological circumstances.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$1,064,966
Indirect Cost
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Kim, Mi Ha; Rebbert, Martha L; Ro, Hyunju et al. (2014) Cell adhesion in zebrafish embryos is modulated by March 8. PLoS One 9:e94873
Zarelli, Valeria E; Dawid, Igor B (2013) Inhibition of neural crest formation by Kctd15 involves regulation of transcription factor AP-2. Proc Natl Acad Sci U S A 110:2870-5
Toyama, Reiko; Kim, Mi Ha; Rebbert, Martha L et al. (2013) Habenular commissure formation in zebrafish is regulated by the pineal gland-specific gene unc119c. Dev Dyn 242:1033-42
Ro, Hyunju; Dawid, Igor B (2009) Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like. Nat Cell Biol 11:1121-7
Brown, Jacob D; Dutta, Sunit; Bharti, Kapil et al. (2009) Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure. Proc Natl Acad Sci U S A 106:1462-7
Hong, Sung-Kook; Dawid, Igor B (2009) FGF-dependent left-right asymmetry patterning in zebrafish is mediated by Ier2 and Fibp1. Proc Natl Acad Sci U S A 106:2230-5
Kansara, Maya; Tsang, Michael; Kodjabachian, Laurent et al. (2009) Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice. J Clin Invest 119:837-51
Toyama, Reiko; Chen, Xiongfong; Jhawar, Nupur et al. (2009) Transcriptome analysis of the zebrafish pineal gland. Dev Dyn 238:1813-26
Toyama, Reiko; Rebbert, Martha L; Dey, Anup et al. (2008) Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis. Dev Dyn 237:1636-44
Hong, Sung-Kook; Dawid, Igor B (2008) Alpha2 macroglobulin-like is essential for liver development in zebrafish. PLoS One 3:e3736

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