Discovery Pipeline for Genetic Defects in Hypothalamic-pituitary Development Using International Mouse Phenotyping Consortium Mice

摘要 先天性垂体功能减退症(CH)是一种常见的出生缺陷，常与综合征有关 中枢神经系统、眼睛结构、面部和性腺的异常。最严重的疾病 有中线发育异常，包括无前脑畸形，这通常是胚胎致命的，或者 隔-视神经发育不良。不太严重的出生缺陷只会扰乱下丘脑或脑下垂体的发育，导致 影响生存、生长、生育、新陈代谢和应激反应的激素缺乏，并可能 需要终生护理。已知有60多个基因导致CH，其中许多是首先在小鼠身上发现的。 尽管如此，81%的CH患者仍然缺乏分子诊断，这对于计划是非常有价值的 治疗和预测未来风险。我们提出了现有胚胎致死基因敲除的详细表型。 具有已知基因缺陷并导致下丘脑和/或垂体畸形的小鼠。这将会扩大 人类CH和相关的中线缺陷的分子诊断和增加我们的 了解这些关键组织的器官发生。我们确定了一组优先考虑的18个胚胎 在下丘脑和/或脑下垂体有明显畸形的致死或亚存活的小鼠品系。这 一组基因根据功能进行分组，包括表观遗传调节因子、纤毛成分、蛋白质 修饰等新的功能类别。我们组建了一支调查团队，他们的专长是 下丘脑-垂体发育、小鼠表型和生物信息学。我们将深入开展 对选定的IMPC小鼠的下丘脑和脑下垂体进行彻底的表型分析。我们的管道已经 三个步骤，每个阶段适当地重新确定优先顺序。第一，时间和空间的表达 选定的基因将在野生型小鼠中确定，IMPC菌株将被进口， 畸形的组织学特征是从妊娠中期到出生。其次，这些菌株将是 评估血管形成缺陷以及下丘脑神经元和垂体激素的规格 使用scRNA-seq和组织学方法相结合的方法生产细胞。第三，机械主义者 对发育缺陷的病理生理学的了解将通过组织学、细胞学 培养分析和/或建立组织特异性cre驱动程序以测试从scRNA-seq开发的假说 和荟萃分析。这项工作将建立新基因的基因型/表型关系 未确诊的CH患者的候选对象。数据传播将是及时和开放的，建立在我们 现有小鼠脑垂体分析数据库(MUSEPAD)和BLOG，以及IMPC网站。未来的扩张 将增加额外的致死或亚存活的IMPC菌株，这些菌株的基因突变与 下丘脑-垂体发育，但尚未研究。我们已做好准备，将对 在小鼠和人中发现了影响下丘脑-垂体发育的基因缺陷。

Abstract Congenital hypopituitarism (CH) is a common birth defect frequently associated with syndromic abnormalities in the central nervous system, ocular structures, face, and gonads. The most severe disorders have midline developmental anomalies and include holoprosencephaly, which is usually embryonic lethal, or septo-optic dysplasia. Less severe birth defects disrupt only hypothalamic or pituitary development, causing hormone deficiencies that affect viability, growth, fertility, metabolism, and the stress response, and may require life-long care. Over 60 genes are known to cause CH, many of which were first discovered in mice. Nonetheless, 81% of CH patients still lack a molecular diagnosis, which would be invaluable for planning treatment and predicting future risk. We propose detailed phenotyping of existing embryonic lethal knockout mice with known genetic defects that result in hypothalamic and/or pituitary malformations. This will expand the molecular diagnoses for CH and associated midline deficiencies in humans and increase our understanding of organogenesis of these critical tissues. We identified a prioritized set of 18 embryonic lethal or sub-viable mouse lines with obvious malformations in the hypothalamus and/or pituitary gland. This set of genes are grouped based on function including epigenetic regulators, components of cilia, protein modification, and other novel functional categories. We assembled a team of investigators with expertise in hypothalamic-pituitary development, mouse phenotyping, and bioinformatics. We will conduct deep and thorough phenotyping of the hypothalamus and pituitary gland in the selected IMPC mice. Our pipeline has three steps with appropriate re-prioritization at each stage. First, temporal and spatial expression of the selected genes will be determined in wild type mice, the IMPC strains will be imported, and the dysmorphology will be characterized histologically from mid- gestation to birth. Second, the strains will be assessed for defects in vascularization and specification of hypothalamic neurons and pituitary hormone producing cells using a combination of scRNA-seq and histological methods. Third, a mechanistic understanding of the pathophysiology of the developmental defects will be determined using histology, cell culture assays and/or established tissue-specific cre drivers to test hypotheses developed from scRNA-seq and meta-analyses. This work will establish genotype/phenotype relationships for novel genes that are candidates for the undiagnosed CH patients. Data dissemination will be timely and open, building on our existing mouse pituitary analysis database (mousePAD) and blog, and the IMPC website. Future expansion of our pipeline will add additional lethal or sub-viable IMPC strains with mutations in genes implicated in hypothalamic-pituitary development, but not yet studied. We are poised to make a significant impact on the discovery of genetic defects that affect hypothalamic-pituitary development in mouse and man.