Our earlier studies on the regulation of dorsal axis formation by the E3 ubiquitin ligase Lnx-2b were continued by analyzing the biological function of Lnx-2b in caudal body development. Formation of the caudal domain and tissue differentiation in this region, such as hematopoiesis, requires Wnt signaling, which leads to the expression of transcription factors in the Cdx family. Several Cdx factors occur in vertebrates;in zebrafish, Cdx4 is the key factor responsible for caudal tissue formation and for hematopoiesis. We found that the transcription factor E4f1 enhances the expression of Cdx4. The mechanism underlying this effect is the interaction of E4f1 with the Wnt effector molecule Tcf3. In the absence of Wnt signaling, Tcf3 is associated with co-repressors such a Groucho/TLE and HDAC, forming a repressor complex that keeps the cdx4 gene silent. E4f1 can de-repress the cdx4 gene by dissociating co-repressors from Tcf3, without interfering with the binding of Tcf3 to its cognate sites in the DNA. Lnx-2b counteracts this effect by stabilizing the co-repressor complex. These studies have revealed a novel mechanism that modulates Wnt signaling during embryogenesis, and is likely to contribute to a robust read-out of the Wnt gradient that determines formation of the caudal body axis. 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. We have continued the study of the biological function and molecular mechanism of Kctd15 action. For this purpose we pursue the generation of transgenic zebrafish lines that can express Kctd15 under controlled conditions. Specifically, we are using a dual system based on the Gal4 DNA binding domain (DBD) and its UAS target sequence. The Gal4 DBD is fused to the ecdysone receptor which keeps the molecule inactive until an ecdysone agonist is added. This allows controlled expression of the UAS-driven transgene. We further are in the process of attempting to generate a targeted mutation in the kctd15 gene with the use of TALENs. The application of TALENs to gene manipulation is discussed in the report from our laboratory entitled Gene Expression During Embryonic Development of Xenopus laevis. Among genes identified 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. The biological function of Unc119c is being investigated by morpholino antisense oligonucleotide injection to inhibit its expression in the zebrafish embryo. Med12 is a component of the Mediator, a large complex involved in the transcriptional activation of many genes. With some variations, the Mediator complex and its components are conserved from yeast to humans. We have previously characterized a mutation, named kohtalo, that affects Med 12. Multiple tissues in the embryo develop abnormally in kohtalo embryos. Recently we have studied the nature of hindbrain patterning in this mutant. The hindbrain is segmented into units named rhombomeres, which are characterized by the expression of specific marker genes. Rhombomeres also have substructure that can be visualized by marker gene expression. We found that segmentation takes place in kohtalo mutants, but rhombomere boundary cells do not form. This finding confirms other results suggesting that segmentation per se does not require boundary cell differentiation, and further illustrates the specific effects that may arise in development as a result of loss of a widely expressed transcriptional regulatory molecule. 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
6
Fiscal Year
2012
Total Cost
$1,198,110
Indirect Cost
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Won, Minho; Ro, Hyunju; Dawid, Igor B (2015) Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas. Proc Natl Acad Sci U S A 112:12426-31
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) The BTB-containing protein Kctd15 is SUMOylated in vivo. PLoS One 8:e75016
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
Coffey, CaitrĂ­n M; Solleveld, Patricia A; Fang, Joyce et al. (2013) Novel oxytocin gene expression in the hindbrain is induced by alcohol exposure: transgenic zebrafish enable visualization of sensitive neurons. PLoS One 8:e53991
Kawahara, Atsuo; Endo, Sumie; Dawid, Igor B (2012) Vap (Vascular Associated Protein): a novel factor involved in erythropoiesis and angiogenesis. Biochem Biophys Res Commun 421:367-74
Zhao, Hui; Han, Dandan; Dawid, Igor B et al. (2012) Homeoprotein hhex-induced conversion of intestinal to ventral pancreatic precursors results in the formation of giant pancreata in Xenopus embryos. Proc Natl Acad Sci U S A 109:8594-9
Lei, Yong; Guo, Xiaogang; Liu, Yun et al. (2012) Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 109:17484-9
Takesono, Aya; Moger, Julian; Farooq, Sumera et al. (2012) Solute carrier family 3 member 2 (Slc3a2) controls yolk syncytial layer (YSL) formation by regulating microtubule networks in the zebrafish embryo. Proc Natl Acad Sci U S A 109:3371-6

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