Intestinal secretory lineage development and function

描述(由申请人提供)： 在肠上皮中，Lieberkuhn隐窝内的增殖和凋亡之间的平衡维持绒毛高度和隐窝深度；这种平衡的破坏会导致癌症。在隐窝内，干细胞既产生吸收细胞，又产生分泌细胞。有几个基因参与了这一过程，但缺乏控制肠上皮细胞分化的有序层次结构。这个层次结构中的一个基因Math1是分泌谱系发育所必需的；缺乏Math1的小鼠产生可吸收的肠细胞，但不产生任何分泌谱系。这一提议将检验这样的假设，即肠道内的增殖动态平衡需要局部依赖Math1的分泌谱系，而描绘分泌谱系分化的遗传路径将阐明肠道动态平衡的机制。首先，我将通过基于微阵列的Math1-/-隐窝和Gfi1缺失的隐窝的表达谱来表征肠道分化的遗传途径，Gfi1是一个假定的Math1靶基因，也被证明在肠上皮发育中发挥作用。在转基因细胞中的实验将确定这些转录因子的直接基因靶点。接下来，我将建立一个体内慢病毒-siRNA系统来确定Notch1在肠道中丢失的表型，并确定它在肠道分化层次中的位置。在目标3中，我将评估偶氮甲烷对小鼠肿瘤和异常隐窝的影响，在小鼠的回肠和结肠中含有不产生分泌细胞的Math1/-隐窝。这些实验产生的数据将构成一个独立研究计划的基础，以调查肠道发育和功能。此外，这些目标代表了理解肠道生物学的不同方法，它们将相互信息，而不是相互依赖才能成功。我将得到肠道生物学和发育领域的领军人物苏珊·亨宁博士的指导，并由相关领域的专家组成一个科学咨询委员会。为PI提供了丰富的智力环境和广泛的资源。

DESCRIPTION (provided by applicant): In the intestinal epithelium, the balance between proliferation and apoptosis within the crypts of Lieberkuhn maintains villus height and crypt depth; disruption of this balance leads to cancer. Within the crypts, stem cells produce both absorptive and secretory cells. Several genes have been implicated in this process, but delineation of an ordered hierarchy for control of intestinal epithelial differentiation is lacking. One gene in this hierarchy, Math1, is required for secretory lineage development; mice lacking Math1, produce absorptive enterocytes but none of the secretory lineages. This proposal will test the hypotheses that Math1- dependent secretory lineages are required locally for proliferative homeostasis in the intestine, and that delineation of the genetic pathway for secretory lineage differentiation will clarify the mechanism of intestinal homeostasis. First I will characterize the genetic pathway for intestinal differentiation by microarray-based expression profiling of Math1 -/- crypts and crypts null for Gfi1, a putative Math1 target gene also shown to have a role in intestinal epithelial development. Experiments in transfected cells will define direct gene targets of these transcription factors. Next, I will develop an in vivo lentiviral-siRNA system to ascertain the phenotype of Notch1 loss in the intestine, and ascertain its position in the hierarchy of intestinal differentiation. In Aim 3, I will assess the effect of azoxymethane to produce tumors and aberrant crypt foci in mice in which the ileum and colon contains Math1 -/- crypts that produce no secretory cells. The data generated from these experiments will form the basis of an independent research program to investigate intestinal development and function. In addition, the aims represent distinct approaches to understanding intestinal biology, and they will inform one another while not being interdependent for success. I will be mentored by Dr. Susan Henning, a leader in the field of intestinal biology and development, with a scientific advisory committee composed of experts in relevant fields. A rich intellectual environment and extensive resources are available to the PI.