Mammalian Developmental Genetics And Animal Models Of Hu

哺乳动物分子遗传学部分研究发育中的小鼠胚胎的图案化、轴形成和器官发生的分子遗传学。目前的工作审查了这一过程的关键控制要素。蛋白质-蛋白质相互作用的场景出现了，它控制着身体计划的方方面面。我们利用功能丧失的方法来确定在发育过程中激活靶基因的蛋白质复合体的基本成分。 多年来，或实验室对LIM同源结构域蛋白进行了广泛的研究。这些lhx基因产物是无脊椎动物和脊椎动物胚胎发育的重要转录调控因子。它们参与早期的构图活动、神经系统的发育和器官的发生。它们的作用受到辅因子的促进，这些辅因子被确定为二聚体并与LIM结构域结合，LIM结构域是存在于lhx基因产物和许多其他核蛋白中的一种特殊的锌指结构。在脊椎动物中，这些LIM结合辅助因子被称为LDB、NLI或Clim，在果蝇中被称为Chip，在线虫中被称为Ldb1。涉及LDB/NLI/Clim(以下简称LDB)和ChIP的蛋白质-蛋白质相互作用不仅限于含有LIM结构域的因子，还可以涉及许多其他转录调控因子。有充分的证据支持这一观点，即LDB和CHIP辅因子是由转录调节因子控制的发育程序的重要组成部分。最近，Rlim辅因子被发现，并被证明通过靶向LDB蛋白降解来负面控制转录因子。此外，转录因子与芯片或低密度脂蛋白结合的竞争也可以改变发育细胞的命运。 哺乳动物的LDB1蛋白存在于含有LIM同源结构域、LIM-Only、bHLH、GATA和OTX转录因子的多种蛋白质复合体中。这些蛋白质在胚胎发育过程中发挥着关键作用。我们已经确定，在小鼠中靶向缺失LDB1基因会导致多效性表型，揭示LDB1在关键发育决策中的基本作用。首先，在Ldb1缺失突变胚胎中没有心脏原基。我们的数据表明，在原肠胚中，LDB1基因的功能对于心脏中胚层正确地分配到心域是必不可少的。突变体中心脏中胚层的异常迁移可能会取消该组织与前内胚层的诱导相互作用，从而阻止心脏发育。此外，在突变体中，头部发育严重受阻，头部结构在后脑前面被截断。在大约40%的突变体中，观察到后轴重复。在突变体中，几个Wnt抑制剂的表达受到抑制，这表明Wnt通路可能参与了Ldb1调节的轴向模式事件。组织者基因在原肠发育过程中的异常表达可能是观察到的LDB1突变胚胎中轴缺陷的原因。最后，我们在LDB1缺失突变的胚胎中观察到中胚层来源的胚外结构中的严重缺陷，包括尿囊膜、卵黄囊血岛、原始生殖细胞和羊膜，证明了LDB1在中胚层形成中的关键作用。 为了鉴定含有LDB/Chip的核蛋白复合体的其他成分，我们建立了表达FLAG和HA表位标记的小鼠LDB1的HeLa细胞，利用标记纯化核复合体，并通过胰蛋白酶多肽的质谱学鉴定组成蛋白。我们发现SSDP蛋白(以前被描述为序列特异性的单链DNA结合蛋白)是这些细胞中LDB1相关核复合体的组成部分。SSDP蛋白在多种其他哺乳动物细胞类型中与LDB1相关。这种联系是特定的，不依赖于核酸的存在，并且在功能上具有重要意义。编码SSDP蛋白的基因在从果蝇到人类的进化过程中非常保守。脊椎动物SSDP基因家族有几个密切相关的成员，而果蝇SSDP基因是独一无二的。在非洲爪哇，由果蝇SSDP或小鼠Ssdp1基因编码的SSDP可增强LDB1与LIM-Homeobox基因Xlim1结合的轴向诱导。此外，我们还证实了在果蝇翅膀发育过程中SSDP和ChIP(LDB1的果蝇同源基因)之间的相互作用。这些发现表明，在无脊椎动物和脊椎动物的发育过程中，SSDP作为LDB1的辅助因子在转录调控中具有功能保守性。 该部门已经确定了一些与小鼠胚胎发育有关的新基因。在对野生型和Lhx3缺失突变胚胎的发育中脑垂体差异表达基因的筛选中，检测到其中的前两个基因，THG-1PIT和MPP 2。这些基因在脑下垂体发育的早期阶段是活跃的。第三个基因是MBX，编码一种同源结构域转录因子，最初仅限于小鼠胚胎的中脑区，但后来延伸到发育中的前脑。最后，我们克隆了Frcp1和Frcp2，这两个基因也主要在大脑中表达。这些基因包含一个FNIII基序，具有多种受体和细胞黏附蛋白的特征。

The Section on Mammalian Molecular Genetics studies the molecular genetics of patterning, axis formation and organogenesis in the developing mouse embryo. Current work examines key control elements of this process. A scenario of protein-protein interactions emerges that control every aspect of the body plan. We have utilized a loss-of-function approach to identify basic components of protein complexes that activate target genes during the course of development. Over the years, or laboratory has carried out extensive studies on LIM-homeodomain proteins. These Lhx gene products are important transcriptional regulators of invertebrate and vertebrate embryonic development. They are involved in early patterning events, in the development of the nervous system, and in organogenesis. Their action is facilitated by cofactors that were identified by their ability to dimerize and to bind to the LIM domain, a specialized zinc-finger structure present in Lhx gene products and in a number of other nuclear proteins. In vertebrates, these LIM-binding co-factors are known as Ldb, Nli or Clim, in Drosophila as Chip, and in C. elegans as Ldb1. Protein-protein interactions involving Ldb/Nli/Clim (henceforth referred to as Ldb) and Chip are not restricted to LIM domain-containing factors but can involve a host of other transcriptional regulators as well. There is ample evidence to support the notion that the Ldb and Chip cofactors are essential components of developmental programs controlled by transcriptional regulators. More recently, the Rlim cofactor was identified and shown to negatively control transcription factors by targeting Ldb proteins for degradation. Furthermore, competition of transcription factors for binding to Chip or Ldb can also alter developmental cell fates. The mammalian Ldb1 protein is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. We have established that targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype, revealing fundamental roles of Ldb1 in key developmental decisions. Firstly, there is no heart anlage in the Ldb1 null mutant embryo. Our data suggest that Ldb1 gene function is essential for proper allocation of cardiac mesoderm to the heart field in the gastrulating embryo. Abnormal migration of the heart mesoderm in the mutants may abolish inductive interactions of this tissue with anterior endoderm and thus prevent heart development. Also, head development is severely curtailed in the mutant, and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning events regulated by Ldb1. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in the Ldb1 mutant embryos. Finally, we observed in the Ldb1 null mutant conceptus severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells, and the amnion, attesting to a key role of Ldb1 in mesoderm formation. In an effort to identify additional components of Ldb/Chip-containing nuclear protein complexes, we generated HeLa cells that express FLAG and HA epitope-tagged mouse Ldb1, purified nuclear complexes with the aid of the tags, and identified constituent proteins by mass spectrometry of tryptic peptides. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in these cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely-related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, we were able to demonstrate an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 in transcriptional regulation during invertebrate and vertebrate development. The Section has identified a number of novel genes involved in the development of the mouse embryo. The first two of these, Thg-1pit and MPP 2, were detected in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3 null mutant embryos. The genes are active at early stages of pituitary development. A third gene, Mbx, encodes a homeodomain transcription factor that is initially restricted to the midbrain region of the mouse embryo but later extends to the developing forebrain. Finally, we cloned Frcp1 and Frcp2, two genes that are also primarily expressed in the brain. These genes contain a FNIII motif characteristic for a variety of receptor and cell adhesion proteins.