Mammalian Developmental Genetics And Animal Models

我们所有实验的共同目标是了解哺乳动物基因调控的细节，并将这些知识应用于人类状况。主要研究课题涉及胚胎发育的分子遗传学。对控制发育中的小鼠胚胎早期轴和器官形成事件的因素的功能分析构成了我们目前研究的重点。在过去的12个月里，我们主要致力于研究Dickkopf分泌蛋白家族成员Dkk1的功能。此前，Dkk1基因产物被描述为非洲爪哇分泌的Wnt抑制物，被认为参与诱导信号。我们对携带Dkk1基因零突变的小鼠胚胎进行了表型分析。突变胚胎缺乏位于中脑前面的头部结构。对嵌合小鼠的分析表明，Dkk1在前中轴中胚层中是必需的，但在前内脏内胚层中不需要，以促进头部的形成。Dkk1在几种与Spemann组织器功能相关的生长因子拮抗剂(如noggin、Chordin、Follistatin、Cerberus、frzb)中是独一无二的，因为它是第一个消融导致组织器相关表型的药物，即轴性缺陷。这意味着，在早期胚胎发育的关键时期，该配体的功能是不可替代的。因此，Dkk1缺失突变体代表了脊椎动物头部诱导研究的一个重要模型。此外，Dkk1缺失突变的胚胎显示出前肢指头的复制和融合。这种表型的分子特征和对雏鸡肢体错误表达的分析表明，Dkk1在顶端外胚脊和程序性细胞死亡中发挥了作用。我们的结果揭示了在小鼠轴形成和肢体形态发生过程中抑制Wnt信号的必要性。观察到的表型表明，Dkk1是Wnt活性的调节器，参与调节肢体生长过程中细胞增殖和细胞死亡之间的平衡。据我们所知，这是基因产物第一次被直接牵连到这一过程中，但人们对此知之甚少。我们发现，Dkk1活性的缺乏(因此，Wnt活性的增加)与顶端外胚层脊中成纤维细胞生长因子活性的增加有关。这说明了在肢体发育过程中成纤维细胞生长因子和Wnt信号之间的重要关系。该实验室的一个长期项目涉及LIM-Homeobox(LHx)基因的功能。Lhx基因编码转录因子，在无脊椎动物和脊椎动物的发育过程中发挥关键的控制功能。我们使用功能缺失的方法分析了发育中的小鼠胚胎中各种LIM-Homeobox(LHx)基因的功能。从我们对lhx基因在发育中的大脑、脑下垂体、脊索、性腺和其他领域的发育中的作用的观察中，出现了共同的主题。随着细胞开始表达传达特定身份的决定因素，lhx基因变得活跃起来。在不同类型的前体细胞中，lhx基因家族各个成员的单独或联合活动表明了导致细胞增殖和初始分化的共同机制，这是发育中的胚胎中新生组织正确安排的先决条件。本研究探讨了两个密切相关的LHX基因LHX2和LHX9在脑形成过程中的单独和联合功能。这两个基因在发育中的前脑的不同区域显示出不同的和部分重叠的表达模式。为了区分它们在神经元前体细胞中的单独和冗余功能，我们产生了携带这两个基因的零等位基因的突变。从对这些突变胚胎的分析中，我们有望获得有关控制新生前脑细胞分化和迁移模式的途径的详细知识。虽然通过突变分析我们对lhx基因的功能有了很大的了解，但它们的作用机制仍然是个谜。LHX蛋白可以与LIM结合蛋白家族以及其他蛋白形成复合体。这种复杂的结构在不同的细胞环境中似乎是必要的功能要求。因此，我们希望关于这种复合体的组成及其在特定细胞和组织中组装的干扰的知识将使我们更接近于建立一个框架来理解LHX的作用机制。对LDB的果蝇同源物CHIP的研究表明，LIM结合蛋白在多肽复合体的组装中发挥着核心作用。ChIP是母体提供给卵子的，可以与不同的同源结构域蛋白相互作用，介导关键的发育调节事件。如果同样适用于LDB蛋白，那么当同源结构域因子开始发挥其构型活性时，它们的消融应该会在很早的时候对脊椎动物的发育产生负面影响。事实的确如此。我们已经通过定向突变LIM结合蛋白LDB1和LDB2的各自的基因来去除它们的功能。虽然在Ldb2 KO突变体中没有观察到表型，但我们的初步发现表明，Ldb1基因敲除会导致早期和致命的胚胎表型。前部结构的严重截断，原始条纹的频繁重复，心脏的缺乏，以及胚胎外结构的缺陷表明多个调节回路的崩溃，这表明在不同的发育关键调控因子的背景下，LDB1的功能需要同时进行。

The goal common to all of our experiments is to learn details of mammalian gene regulation and control, and to apply this knowledge to the human condition. Major research topics address the molecular genetics of embryonic development. The functional analysis of factors that control early axis and organ forming events in the developing mouse embryo constitutes the current focus of our studies. During the past 12 months, our main efforts have been directed toward studying the function of Dkk1, a member of the Dickkopf family of secreted proteins. The Dkk1 gene product has previously been described as a secreted Wnt inhibitor of Xenopus, thought to be involved in inductive signaling. We performed a phenotypic analysis of mouse embryos that carry a null mutation in the Dkk1 gene. The mutant embryos lack head structures anterior to the midbrain. An analysis of chimeric mice showed that Dkk1 is required in anterior axial mesendoderm but not in anterior visceral endoderm to promote head formation. Dkk1 is unique among several growth factor antagonists linked to Spemann organizer function (e.g. noggin, chordin, follistatin, cerberus, frzb) because it is the first one whose ablation results in an organizer-related phenotype, i.e., an axial defect. This implies that the function of this ligand is irreplaceable during a critical time of early embryonic development. As such, Dkk1 null mutants represent an important model for studies of head induction in vertebrates. In addition, Dkk1 null mutant embryos display duplications and fusions of forelimb digits. Molecular characterization of this phenotype together with misexpression analysis in chick limbs indicates a role for Dkk1 in the apical ectodermal ridge and in programmed cell death. Our results reveal a requirement for inhibition of Wnt signaling during mouse axis formation and limb morphogenesis. The observed phenotype identifies Dkk1 as a modulator of Wnt activity involved in regulating the balance between cell proliferation and cell death during limb outgrowth. This is, to the best of our knowledge, the first instance in which a gene product has been directly implicated in this as yet poorly understood process. We find that lack of Dkk1 activity (and hence, an increase of Wnt acticity ) correlates with an increase of FGF activity in the apical ectodermal ridge. This illuminates an important relationship between FGF and Wnt signaling during limb outgrowth. A long-term project of the laboratory concerns the function of LIM-homeobox (Lhx) genes. The Lhx genes encode transcription factors that exert crucial control functions during the development of invertebrate and vertebrate organisms. We have used a loss-of-function approach to analyze functions of various LIM-homeobox (Lhx) genes in the developing mouse embryo. From our observation of Lhx gene action in the prospective brain, pituitary gland, spinal chord, gonad and other fields of the developing embryo, common themes have emerged. The Lhx genes become active as cells begin to express determinants that convey specific identities. The individual or combined activities of individual members of the Lhx gene family in different types of precursor cells suggest common mechanisms leading to cell proliferation and initial differentiation, a prerequisite for correct arrangement of nascent tissues in the developing embryo. A present study addresses the individual and combined functions of two closely related Lhx genes, Lhx2 and Lhx9, during brain formation. These two genes display distinct and partially overlapping expression patterns in different regions of the developing forebrain. In an effort to distinguish between their individual and redundant functions in neuronal precursor cells, we have generated mutants that carry null alleles of both genes. From the analysis of these mutant embryos, we expect to derive detailed knowledge concerning pathways that control cell differentiation and migration patterns in the nascent forebrain. While our knowledge of Lhx gene function has been considerably advanced through mutant analysis, their mechanism of action has remained enigmatic. Lhx proteins can engage in complex formation with the family of LIM binding proteins, as well as with other proteins. This complex formation appears to be a necessary functional requirement in different cellular contexts. Therefore, we hope that knowledge with regard to the composition of such complexes and interference with their assembly in specific cells and tissues will bring us closer to establishing a framework for understanding the mechanism of Lhx action. Studies with CHIP, the Drosophila homolog of Ldb, suggest that the LIM binding proteins play a central role in the assembly of polypeptide complexes. CHIP is maternally supplied to the egg and can interact with a diverse array of homeodomain proteins to mediate key developmental regulatory events. If the same holds for Ldb proteins as well, their ablation should have negative effects on vetrebrate development very early on when homeodomain factors begin to exert their patterning activities. This is indeed the case. We have ablated the functions of the LIM binding proteins Ldb1 and Ldb2 by targeted mutagenesis of the respective genes. While no phenotype was observed in Ldb2 KO mutants, our preliminary findings indicate that knockout of Ldb1 results in an early and lethal embryonic phenotype. Severe truncation of anterior structures, frequent duplication of the primitive streak, lack of a heart, and defective extraembryonic structures indicate a breakdown of multiple regulatory circuits, suggesting a simultaneous functional requirement of Ldb1 in the context of different key regulators of development.