WINGED HELIX TRANSCRIPT FACTOR C-KIT IN NEURAL CREST DEV & EVOLUTION (PROJ CHNG)

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 在过去的一年里，我的实验室一直专注于具体目标#1和具体目标#3，我的资助实验2。 具体目标#1是鉴定驱动FoxD 3（cFoxd）的鸡同源物的神经嵴特异性表达的调控元件。 为此，我们克隆了cFoxD 3 5'端约3 kb的基因组DNA片段，将其插入到pBGZA报告载体中，并用于卵电穿孔以将构建体转染到11期鸡胚的背神经管中。 这个构建体不能驱动报告基因在神经管或神经嵴细胞中表达，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涉及确定来自AmphifoxD的调节元件是否能够驱动脊椎动物神经管和/或神经嵴细胞中的报告基因表达。 迄今为止，我们的数据表明，它们不是，这表明文昌鱼和脊椎动物胚胎之间的foxD 3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。 除了继续我们在具体目标#3上的工作之外，我们将开始具体目标#2（产生cFoxD 3的组成型激活物和阻遏物形式）和具体目标#3的工作，实验1（测试AmphifoxD在脊椎动物神经管中表达时诱导异位神经嵴细胞的能力）。 我将和雷切尔·帕特森一起做这项工作，她是生物学专业的大二学生，也是我的第一个INBRE学者。

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.