Regulation of geminin expression in embryonic ectoderm

描述(申请人提供)：我们的长期目标是剖析脊椎动物早期胚胎发育过程中神经外胚层决定细胞命运的分子电路。从这些研究中获得的信息最终可能会影响人类的多种健康问题，包括我们操纵胚胎干细胞用于神经系统人类治疗的能力，以及我们对出生缺陷(包括全前脑畸形和脊柱裂)的分子基础的理解。这些出生缺陷是人类最常见的先天性畸形之一。我们的总体策略是专注于一组基因，这些基因在早期胚胎发育过程中充当神经命运的主要效应者。这些基因在未来神经板的表达代表了外胚层对来自相邻细胞的神经诱导信号的最早转录反应。这一表达将胚胎外胚层划分为神经性和非神经性领域。目前尚不清楚这种转录是如何发生的，也不知道哪些分子决定因素和信号通路是直接调节因子。在这里，我们使用了一个可操作的转基因非洲爪哇胚胎系统来重建这种基因gminin的早期神经表达的顺式调控控制。我们发现，Gminin的5‘调控序列概括了它的表达，并以与内源基因相同的方式对诱导信号做出反应。我们建议使用几种方法来识别离散的顺式元件和蛋白质复合体，这些方法直接控制Ginin最初的神经特异性转录程序的启动，包括体内注射和转基因方法以及体外的各种分子方法。这些研究应该会填补我们对神经细胞命运决定的分子基础的理解的一个关键空白。

DESCRIPTION (provided by applicant): Our long-term objective is to dissect the molecular circuitry that underlies cell fate determination in the neurectoderm during early vertebrate embryogenesis. Information obtained in these studies may ultimately impact multiple human health issues, including our ability to manipulate embryonic stem cells for human therapies in the nervous system, and our understanding of the molecular basis of birth defects, including holoprosencephaly and spina bifida. These birth defects are among the most common congenital malformations in humans. Our general strategy is to focus upon a group of genes that act as primary effectors of neural fate during early embryogenesis. Expression of these genes in the future neural plate represents the earliest transcriptional response of ectoderm to neural-inducing signals from adjacent cells. This expression demarcates the embryonic ectoderm into neural versus non-neural territories. It is not known how this transcription arises or which molecular determinants and signaling pathways are direct regulators. Here, we have used a manipulatable transgenic Xenopus embryo system to reconstitute cis-regulatory controls underlying the early neural expression of one such gene, geminin. We show that 5' regulatory sequences from geminin recapitulate its expression and respond to inductive signals in the same manner as the endogenous gene. We propose use of several approaches to identify discrete cis-elements and protein complexes that directly control the onset of geminin's initial neural-specific transcriptional program, including injection and transgenic methodologies in vivo and various molecular methodologies in vitro. These studies should fill a critical gap in our understanding of the molecular basis of neural cell fate determination.