Mammalian Developmental Genetics And Animal Models Of Hu

哺乳动物发育遗传学和胡动物模型

• 批准号: 6991142
• 负责人: HEINER WESTPHAL
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6991142
• 关键词: cell growth regulation; cofactor; developmental genetics; developmental neurobiology; disease /disorder model; functional /structural genomics; gene expression; gene targeting; homeobox genes; laboratory mouse; mammalian embryology; molecular genetics; prosencephalon; protein structure function; transcription factor

The Section on Mammalian Molecular Genetics (SMMG) is focused on the molecular genetics of embryonic development. Transcriptional control is a key element of developmental regulation, involving an elaborate repertoire of cis-regulatory target gene sequences as well as the cooperation and physical interaction of multiple factors that regulate gene expression. Core elements, present in many cell contexts, are thought to form complexes with cell- or tissue-specific factors to establish positive and negative control of gene expression and to bring about cell specification and tissue identity in the developing embryo. We study the molecular genetics of this process. Much of our work deals with the functional evaluation of members of the LIM class of homeobox genes (termed Lhx genes) during mouse development. During the course of these experiments, we identified two novel classes of proteins, encoded by the Ldb and Ssdp gene families, respectively. These are co-factors whose interaction with the Lhx-encoded LIM-homeodomain factors and with other transcriptional regulators is essential for embryonic development. Additional topics of our studies are members of the Dkk family of Wnt inhibitors that form a link between the action of Lhx genes and pattern formation in the developing central nervous system. The closely related Lhx1 and Lhx5 genes are topics of our ongoing work. The two genes are prominently expressed in many regions of the developing central nervous system, including the spinal cord and the cerebellum. Earlier work had shown that Lhx1 null embryos lack anterior head structures and that Lhx5 null mutants are impaired in hippocampal development. Lhx1 null embryos die at an early stage of development, precluding a thorough analysis of possible brain defects. We were also concerned that functional redundancies may exist between Lhx1 and Lhx5. We therefore generated conditional Lhx1 mutants as well as Lhx1/5 double mutants to be able to examine more closely the individual or combined roles of both genes in brain development. The analysis is focused on the developing cerebellum in these mutants. The cerebellar phenotype of embryos that lack either Lhx1 or Lhx5 is not remarkable. However, the examination of Lhx1/5 double mutants revealed that the Purkinje cells of the primordial cerebellum are largely missing, as defined by the absence of the Calbindin marker. Our experiment shows that both Lhx1 and Lhx5 are essential for the generation of Calbindin-positive Purkinje cells and that there is redundancy in this functional aspect. Another aspect of our current work concerns functions of Lhx2 in the context of the development of the pituitary gland. Our previous studies had established a role for this gene in brain and eye development and in hematopoiesis. Null embryos were anophthalmic because of a developmental arrest of the eye anlagen prior to the formation of the optic cup. In addition, deficient cell proliferation in the forebrain resulted in hypoplasia of the neocortex and aplasia of the hippocampal anlagen. The Lhx2-deficient mutants died in utero, possibly because of a cell non-autonomous defect of definitive erythropoiesis that caused severe anemia.Here we describe an additional phenotype. Deletion of Lhx2 impairs formation of the posterior lobe of the pituitary gland. We observe over-proliferation and a lack of proper differentiation of precursor cells in this tissue. Loss of Lhx2 function also leads to a disorganization of the anterior and intermediate lobes of the pituitary gland, possibly secondary to defects in the formation of the posterior pituitary. Our earlier work had identified Lhx3 and Lhx4 as essential regulators of pituitary development. The present work shows that Lhx2 plays an important role in the process as well. Members of the Dkk family of Wnt inhibitors are widely expressed in the developing embryo. We previously reported on Dkk1, a mediator of Lhx and other transcriptional activity during head induction. Severe rostral defects are the result of Dkk1 functional ablation. Our recent work has dealt with the phenotype of Dkk2 knockout mice. These mutants are characterized by a profound defect in cornea cell turnover, indicating that Wnt pathway regulation is essential for the maintenance of ocular surface integrity. The cornea is a very ordered, transparent structure whose epithelium is replaced every three or four weeks by derivatives of stem cells that reside in the limbus, a transitional zone between the corneal and the abutting conjunctival epithelium. The cornea of young adult Dkk2 null mice is opaque and contains cells normally found in skin and conjunctiva, including hair follicles, sebaceous glands and goblet cells. Wnt signals are upregulated in the mutant limbus region. Dkk2 has therefore been identified as a Wnt inhibitor that regulates the turnover of corneal epithelia.

哺乳动物分子遗传学 (SMMG) 部分专注于胚胎发育的分子遗传学。转录控制是发育调节的关键要素，涉及一系列复杂的顺式调节靶基因序列以及调节基因表达的多个因素的合作和物理相互作用。存在于许多细胞环境中的核心元件被认为与细胞或组织特异性因子形成复合物，以建立基因表达的正向和负向控制，并在发育中的胚胎中产生细胞规范和组织特性。我们研究这个过程的分子遗传学。我们的大部分工作涉及小鼠发育过程中 LIM 类同源盒基因（称为 Lhx 基因）成员的功能评估。在这些实验过程中，我们鉴定了两类新的蛋白质，分别由 Ldb 和 Ssdp 基因家族编码。这些辅助因子与 Lhx 编码的 LIM 同源域因子和其他转录调节因子的相互作用对于胚胎发育至关重要。我们研究的其他主题是 Wnt 抑制剂 Dkk 家族的成员，它们在 Lhx 基因的作用和发育中的中枢神经系统中的模式形成之间形成联系。 密切相关的 Lhx1 和 Lhx5 基因是我们正在进行的工作的主题。这两个基因在发育中的中枢神经系统的许多区域显着表达，包括脊髓和小脑。早期的研究表明，Lhx1 无效胚胎缺乏前头部结构，而 Lhx5 无效突变体的海马发育受到损害。 Lhx1 无效胚胎在发育早期死亡，无法对可能的大脑缺陷进行彻底分析。我们还担心 Lhx1 和 Lhx5 之间可能存在功能冗余。因此，我们生成了条件 Lhx1 突变体以及 Lhx1/5 双突变体，以便能够更仔细地检查两个基因在大脑发育中的单独或组合作用。分析的重点是这些突变体中正在发育的小脑。缺乏 Lhx1 或 Lhx5 的胚胎的小脑表型并不显着。然而，对 Lhx1/5 双突变体的检查表明，原始小脑的浦肯野细胞大部分缺失，正如钙结合蛋白标记物缺失所定义的那样。我们的实验表明，Lhx1 和 Lhx5 对于钙结合蛋白阳性浦肯野细胞的生成都是必需的，并且在这个功能方面存在冗余。 我们当前工作的另一个方面涉及 Lhx2 在垂体发育中的功能。我们之前的研究已经确定了该基因在大脑和眼睛发育以及造血中的作用。空胚胎是无眼的，因为在视杯形成之前眼原基发育停滞。此外，前脑细胞增殖缺陷导致新皮质发育不全和海马原基发育不全。 Lhx2 缺陷突变体在子宫内死亡，可能是由于细胞非自主性红细胞生成缺陷导致严重贫血。在这里，我们描述了另一种表型。 Lhx2 的缺失会损害垂体后叶的形成。我们观察到该组织中前体细胞过度增殖且缺乏适当的分化。 Lhx2 功能的丧失还会导致垂体前叶和中叶的紊乱，可能继发于垂体后叶形成的缺陷。我们早期的工作已确定 Lhx3 和 Lhx4 是垂体发育的重要调节因子。目前的工作表明 Lhx2 在此过程中也发挥着重要作用。 Wnt 抑制剂 Dkk 家族的成员在发育中的胚胎中广泛表达。我们之前报道过 Dkk1，它是头部诱导过程中 Lhx 和其他转录活性的介体。严重的吻侧缺陷是 Dkk1 功能性消融的结果。我们最近的工作涉及 Dkk2 敲除小鼠的表型。这些突变体的特点是角膜细胞更新存在严重缺陷，表明 Wnt 通路调节对于维持眼表完整性至关重要。角膜是一种非常有序、透明的结构，其上皮每三到四个星期就会被位于角膜缘（角膜和相邻结膜上皮之间的过渡区）的干细胞衍生物所取代。年轻成年 Dkk2 缺失小鼠的角膜是不透明的，含有通常在皮肤和结膜中发现的细胞，包括毛囊、皮脂腺和杯状细胞。 Wnt 信号在突变角膜缘区域上调。因此，Dkk2 被确定为调节角膜上皮更新的 Wnt 抑制剂。