Role of Drosophila Bunched Protein Isoforms in Regulating Notch Signaling at Cell Fate Boundaries

在多细胞动物的发育过程中，细胞增殖必须与细胞分化协调，才能形成适当的组织。细胞通过分离到不同的区域或区室来协调它们的活动，并在它们的边界建立减少细胞混合的区域。细胞命运边界组织了多种组织的模式和形态发生，包括果蝇的眼赤道和翅膀边缘，以及脊椎动物的顶端外胚层脊、体和后脑菱形体。关于产生细胞命运边界和将室边界转化为成熟组织结构特征的机制知之甚少。果蝇束状基因是高度保守的TSC-22/GILZ转录因子家族的一员，在卵发生过程中建立上皮细胞命运边界。束状蛋白编码三种不同的蛋白同型，其结构特征与小鼠TSC-22基因的三种同型相对应。束状蛋白受远程形态因子的调控，以确定细胞命运边界的位置。Bunched1同种异构体调节细胞亲和性和细胞命运边界上的短距离Notch信号，这与苍蝇蛋壳的一个关键结构特征——卵盖的颈圈相吻合。使用一个简单的模型组织并应用强大的分子和遗传学方法，本提案的目标是：(1)确定远程形态因子信号如何通过Bunched1被细化为短程Notch激活；(2)描述三个束状异构体之间的相互作用如何影响细胞命运。这项建议的长期目标是阐明细胞命运边界建立的复杂、保守的机制。更广泛的影响发育中的一个基本问题是组织的细分是如何通过基因发生的，这些基因最初在广泛的重叠结构域中表达，这些结构域调节下游基因的表达以建立一个确定的模式。TSC-22蛋白家族在动物中广泛保守，但大量的哺乳动物组织培养研究尚未揭示其发育功能。提出的实验将提供基本的见解，如何束蛋白异构体相互作用，以调节基因转录在形成细胞命运边界和阐明类似的调控过程在四肢，神经系统和其他脊椎和无脊椎动物组织。提出的实验旨在将研究与教学和培养本科生和研究生的计划结合起来。果蝇基因类似于人类TSC-22（一种与几种人类癌症和糖尿病性神经病变相关的基因），将向学生传达模式生物的潜力，以阐明作为理解人类疾病基础的发育机制。这些目标中详细介绍的逻辑系列实验可分为适合研究生的大项目和适合本科生的小项目。这里概述的工作将补充本科研究，包括扩展研究和夏季研究的学者项目（SEARCH和Sapere Vedere奖学金）和博士培训项目。该项目将加强教学：(1)本科生发育生物学和高级细胞生物学，这是一门研究生水平的课程，强调使用动物模型系统，如果蝇、秀丽隐杆线虫和酵母作为基因组工具。这项工作将进一步发展发育生物学的实验课程，部分基于这里计划的基因实验。

B. PROJECT SUMMARYRole of Drosophila Bunched protein isoforms in regulating Notch signaling at cell fate boundaries Intellectual meritDuring development of a multicellular animal, cell proliferation must be orchestrated with cell differentiation for proper tissue formation. Cells coordinate their activities by segregating into separate domains - or compartments - and establishing regions of reduced cell mixing at their boundaries. Cell fate boundaries organize patterning and morphogenesis of a wide range of tissues, including the eye equator and wing margin in Drosophila, and the apical ectodermal ridge, somite and hindbrain rhombomeres in vertebrates. Little is known about the mechanisms that generate cell fate boundaries and translate compartment boundaries into the structural features of mature tissue.The Drosophila bunched gene, a member of the highly conserved TSC-22/GILZ family of transcription factors, establishes an epithelial cell fate boundary during oogenesis. bunched, which encodes three distinct protein isoforms whose structural features correspond to three isoforms of the mouse TSC-22 gene, is regulated by long-range morphogens to set the position of a cell fate boundary. The Bunched1 isoform regulates cell affinities and short-range Notch signaling at a cell fate boundary that coincides with a key structural feature of the fly eggshell, the collar of the operculum.Using a simple model tissue and applying powerful molecular and genetic approaches, the goals of this proposal are to: (1) determine how long-range morphogen signaling is refined to short-range Notch activation via Bunched1; and (2) characterize how interactions among the three Bunched isoforms pattern cell fates. The long term goal of this proposal is to elucidate the complex, conserved mechanism by which cell fate boundaries are established.Broader ImpactA fundamental question in development is how tissue subdivision occurs by genes, initially expressed in broad, overlapping domains, which regulate the expression of downstream genes to establish a defined pattern. The TSC-22 family of proteins is widely conserved in animals but extensive mammalian tissue culture studies have shed no light on their developmental function. Experiments proposed will give basic insights into how Bunched protein isoforms interact to regulate gene transcription at forming cell fate boundaries and illuminate similar regulatory processes in limbs, nervous system and other vertebrate and invertebrate tissues. Experiments proposed are designed to integrate research with a program of teaching and training undergraduate and graduate students. The focus on a Drosophila gene similar to human TSC-22, a gene associated with several human cancers and diabetic neuropathy, will convey to students the potential of model organisms to elucidate developmental mechanisms as a basis to understand human disease. The logical series of experiments detailed in these Aims are divisible into larger projects suitable for graduate students and smaller projects appropriate for undergraduates. Work outlined here will complement undergraduate research, including scholars programs for extended research and summer research (SEARCH and Sapere Vedere scholarships) and the Ph.D. training program. This project will enhance teaching: (1) Developmental Biology for undergraduates and Advanced Cell Biology, a graduate level course emphasizing the use of animal model systems like Drosophila, C. elegans, and yeast as genomic tools. This work will further development of a lab course for Developmental Biology, based in part on the genetic experiments planned here.