WINGED HELIX TRANSCRIPTION FACTOR FOXD3 IN NEURAL CREST DEV AND EVOLUTION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在过去的一年里，我的实验室一直专注于我资助的具体目标#1和具体目标#3，实验2。具体目标#1是确定驱动FoxD3(CFoxd)鸡同源基因神经脊特异性表达的调控元件。为此，我们克隆了一个约3kb的基因组DNA片段cFoxD3，将其插入到pBGZA报告载体中，并将其用于卵子电穿孔，将其导入第11期鸡胚胎的背神经管。这种结构不能驱动报告基因在神经管或神经脊细胞中的表达，所以今年夏天我们将开始克隆更大的5‘片段和一些3’片段进行测试。具体目标#3，实验2涉及确定AmphifoxD的调控元件是否能够驱动报告基因在脊椎动物神经管和/或神经脊细胞中的表达。到目前为止，我们的数据表明它们不是，这表明文昌鱼和脊椎动物胚胎foxD3表达的差异主要是由于顺式调节因子而不是反式调节因子。除了继续我们在特定目标#3上的工作外，我们还将开始特定目标#2(产生cFoxD3的结构性激活和抑制形式)和特定目标#3，实验1(测试AmphifoxD在脊椎动物神经管中表达时诱导异位神经脊细胞的能力)的工作。我将和瑞秋·帕特森一起做这项工作，她是生物学专业的大二学生，也是我的第一位INBRE奖学金获得者。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the past year my lab has focused on specific aim #1 and specific aim #3, experiment 2 of my grant. Specific aim #1 is to identify the regulatory elements driving neural crest-specific expression of the chicken homology of FoxD3 (cFoxd). To this end we cloned an approximately 3kb genomic DNA fragement 5' to cFoxD3, inserted it into the pBGZA reporter vector, and used in ovo electroporation to transfect the construct into the dorsal neural tube of stage 11 chicken embryos. This construct did not drive reporter gene expression the neural tube or neural crest cells, so this summer we will begin cloning larger 5' fragments and some 3' fragments to test. Specific aim #3, experiment 2 involves determining whether the regulatory elements from AmphifoxD are capable of driving reporter gene expression in the vertebrate neural tube and/or neural crest cells. Our data thus far indicate that they are not, suggesting that that the difference in expression of foxD3 between amphioxus and vertebrate embryos is due primarily to cis-regulatory factors rather than trans-regulatory factors. In addition to continuing our work on specific aim #3, we will begin work on specific aim #2 (generating constitutive activator and repressor forms of cFoxD3) and specific aim #3, experiment 1 (testing the ability of AmphifoxD to induce ectopic neural crest cells when expressed in the vertebrate neural tube). I will be doing this work with Rachel Patterson, a sophomore Biology major and my first INBRE scholar.