Hh signaling in the zebrafish forebrain and pituitary

描述（由申请人提供）：脑垂体被称为内分泌主腺，控制多种激素反应，包括调节生殖、体内平衡和应激反应。垂体的激素分泌叶，或腺垂体，通过神经和表皮组织之间的诱导相互作用在发育中的胚胎的前端形成。Hedgehog （Hh）信号分子有助于介导这些诱导事件，从鱼类到哺乳动物，这一作用在脊椎动物物种中一直是保守的。人类Hh信号突变可导致多种影响垂体发育的综合征，包括前脑畸形和帕利斯特-霍尔综合征。事实上，人类先天性垂体缺陷是相当普遍的，范围从所有内分泌功能的丧失（全垂体功能低下）到单一激素功能的丧失。生长激素缺失是人类最常见的单一内分泌缺陷，每4000个胚胎中就有1个发生。我们之前的研究表明，这一范围的垂体缺陷也发生在斑马鱼Hh通路突变体中，为垂体发育的研究提供了独特的资源。其中，未表征的uml突变消除了一些细胞类型（如GH），并独特地影响垂体细胞的命运决定。我们还发现Gli介导的Hh信号是垂体诱导和内分泌细胞分化所必需的。在这里，我们建议继续使用斑马鱼作为模型系统来研究Hh调节脑垂体细胞分化的分子和细胞机制。我们将首先测试Hh在垂体发育中是否作为形态因子或有丝分裂原，并确定哪些Gli转录因子介导垂体Hh反应。利用新开发的技术暂时和细胞自主地干扰Hh信号，我们将测试垂体前体细胞和内分泌细胞系对Hh信号的直接需求，并确定这些细胞分化事件何时需要直接Hh信号。最后，我们将确定斑马鱼umleitung （uml）突变的分子基础，作为Hh参与内分泌细胞谱系测定的遗传研究的一部分。这项工作将提供关于Hh信号在指导脊椎动物垂体细胞规范中的作用的基础知识。我们的研究计划利用斑马鱼，将基因、细胞和分子分析结合起来，达到其他脊椎动物无法达到的水平。我们的新基因工具将有助于研究人员在任何胚胎组织中研究Hh。对Hh信号的潜在新调控因子（uml位点）的表征也可能为整个发育中的胚胎中Hh信号的调控提供新的见解。由于Hh信号通路在进化过程中高度保守，这项工作将直接应用于我们对高等脊椎动物Hh信号的理解，并将影响我们指导干细胞分化的治疗目的的能力。最终，这项斑马鱼研究有望有助于我们理解影响垂体的人类出生缺陷，并可能阐明由出生后Hh信号调节不当引起的肿瘤发生。

DESCRIPTION (provided by applicant): The pituitary gland is known as the master endocrine gland and controls multiple hormonal responses including those regulating reproduction, homeostasis, and responses to stress. The hormone-secreting lobe of the pituitary gland, or adenohypophysis, forms at the anterior end of the developing embryo through inductive interactions between neurally and epidermally derived tissues. Hedgehog (Hh) signaling molecules help mediate these inductive events, a role that has been conserved across vertebrate species from fish to mammals. Human mutations in Hh signaling lead to a variety of syndromes that affect pituitary development, including Holoprosencephaly and Pallister-Hall syndrome. In fact, human congenital pituitary defects are quite common and range from the loss of all endocrine function (panhypopituitarism) to the loss of single hormone function. The loss of GH is the most common single endocrine deficiency in humans, occurring in 1 in 4000 embryos. We previously showed that this range of pituitary defects also occurs in zebrafish Hh pathway mutants, providing a unique resource for the study of pituitary development. Among these, the uncharacterized uml mutation eliminates some cell types (e.g. GH) and uniquely affects cell fate decisions in the pituitary. We also showed that Gli mediated Hh signaling is needed for pituitary induction and endocrine cell differentiation. Here we propose to continue our use of the zebrafish as a model system to investigate the molecular and cellular mechanisms of Hh regulated cell differentiation in the vertebrate pituitary gland. We will first test whether Hh acts as a morphogen or mitogen in pituitary development and determine which Gli transcription factors mediate the pituitary Hh response. Using newly developed techniques to temporally and cell-autonomously disrupt Hh signaling, we will then test the direct requirement for Hh signaling in pituitary precursor cells and endocrine cell lineages and determine when direct Hh signaling is needed for these cell differentiation events. Finally, we will determine the molecular basis of the zebrafish umleitung (uml) mutation as part of a genetic investigation of Hh involvement in endocrine cell lineage determination. This work will provide fundamental knowledge about the role of Hh signaling in guiding cell specification in the vertebrate pituitary. Our research plan takes advantage of zebrafish to combine genetic, cellular, and molecular analyses at a level not possible in other vertebrates. Our new genetic tools will be useful to researchers investigating Hh in any embryonic tissue. The characterization of a potentially novel regulator of Hh signaling (the uml locus) is also likely to provide new insights into the regulation of Hh signaling throughout the developing embryo. Because the Hh signaling pathway has been highly conserved through evolution, this work will apply directly to our understanding of Hh signaling in higher vertebrates and will impact on our ability to direct stem cell differentiation for therapeutic purposes. Ultimately, this zebrafish research promises to contribute to our understanding of human birth defects affecting the pituitary and may shed light on tumorigenesis caused by mis-regulation of Hh signaling postnatally.