Mammalian Developmental Genetics And Animal Models

哺乳动物发育遗传学和动物模型

• 批准号: 6508734
• 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/6508734
• 关键词: binding proteins; cell growth regulation; developmental genetics; developmental neurobiology; disease /disorder model; ectoderm; embryo /fetus; gene expression; gene targeting; genetic regulation; histogenesis; homeobox genes; laboratory mouse; mammalian embryology; molecular genetics; mutant; neurotrophic factors; secretory protein; site directed mutagenesis; tissue mosaicism; transcription factor

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.

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.