Functional characterization of isl2/nr2f1b activation target stap2b in vascular development
2016
Hochschulschrift
Zugriff:
104
Genetic and signal interaction that regulating the vein identity, intersegmental vessels (ISV) and cardinal vein pluses (CVP) patterning is still not fully understood. We previously identified the transcription factors Islet2 (Isl2) and nr2f1b required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish. To understand the molecular mechanisms behind the isl2/ nr2f1b regulation, we performed the microarray experiments to identify the potential downstream targets. In this study, we focus on the potential activation targets and we confirmed several genes expressed in vessels during early development indicating those potential genes function in vascular development likely downstream of isl2/ nr2f1b. We selectively assay the functional relevance of one potential target, signal-transducing adaptor protein 2b (stap2b). Stap2 is identified as an adaptor protein that has been showed functions in a variety of cellular signal pathways in immune system, however, there is no description of stap2b functioning in vascular development so far. Our in-situ hybridization results showed stap2b mRNA is expressed is expressed in developing vessels from 18S to 48 hpf stages, suggesting its roles in vasculization. Knockdown of stap2b by morpholino injection causes vascular defects, suggesting the role of stap2b in controlling ISV and CVP growth. We confirmed the specificity of stap2b MO knockdown by using 2nd MO interfere block splicing site, examining knockdown efficiency, and expressing stap2b mRNA to rescue stap2b morphants. AO staining and TUNEL assay showed that vascular defects do not caused by cell death, but due to the impairment of migration and/or proliferation by examining ISV integrity and ISV cell numbers in Tg(kdrl:mCherry; fli:neGFPy7) fish. To test molecular mechanisms of vascular defects in stap2b morphants, we examined the expression of vascular markers. We found that stap2b morphants showed a decreased expression of vascular markers flk and stabilin, consistent with the role of stap2b in vascular development. While loss of stap2b cause vascular defects, we expect overexpression of stap2b will enhance vascular growth. Overexpression of stap2b enhanced the fluorescent signals in vessels and the growth of ISV. These data suggest that stap2b is necessary and sufficient to promote vascular development. We further examine the interaction between stap2b and Notch signals, we showed stap2b is regulated by Notch signals to control ISV growth. In addition, stap2b may interact with BMP signaling to function in CVP formation. Together, we conclude that our microarray results identify many vascular genes acting downstream of isl2/ nr2f1b pathway. We also showed that stap2b plays an important role for vascular development in zebrafish.
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Functional characterization of isl2/nr2f1b activation target stap2b in vascular development
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Autor/in / Beteiligte Person: | Wang, Yi-shan ; 王乙珊 |
Link: | |
Veröffentlichung: | 2016 |
Medientyp: | Hochschulschrift |
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