Role of Shh in developmental patterning and growth of digit skeleton

Shh 在发育模式和数字骨骼生长中的作用

• 批准号: 9153785
• 负责人: Susan Mackem
• 金额: $ 36.44万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/9153785
• 关键词: Accounting; Adult; Affect; Anterior; Apoptosis; Apoptotic; Brain; Cancer Biology; Cell Adhesion; Cell Death; Cell Lineage; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cessation of life; Child; Communities; Complex; Development; Developmental Biology; Differentiation and Growth; Digit structure; Early identification; Embryo; Ensure; Erinaceidae; Family member; Fibroblast Growth Factor; Fingers; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genomics; Goals; Growth; Hereditary Disease; Holoprosencephaly; Human; Image; Infant; Intercept; Joints; Lead; Learning; Length; Limb Bud; Limb Development; Limb structure; Link; Maintenance; Malignant Neoplasms; Mediator of activation protein; Mesenchymal; Mesoderm; Mission; Mitogens; Modeling; Morbidity - disease rate; Morphogenesis; Mus; Mutant Strains Mice; Mutate; Mutation; Neoplasm Metastasis; Neoplasms; Neural Crest; Normal tissue morphology; Organ; Pallister-Hall syndrome; Pancreas; Pathology; Pathway interactions; Pattern; Phalanx; Phase; Physical condensation; Physiological Processes; Play; Process; Prostate; Proteomics; Regulation; Research; Role; Shapes; Signal Pathway; Signal Transduction; Skeleton; Skin; Sonic Hedgehog Pathway; Specific qualifier value; Staging; Stomach; Structure; Study models; System; Systems Biology; Tamoxifen; Testing; Thumb structure; Time; Tissues; Transgenes; Tumor Biology; Vertebrates; Work; body system; cancer type; cell behavior; cell motility; cellular targeting; design; developmental genetics; genome-wide analysis; insight; model building; morphogens; mortality; mutant; neoplastic cell; programs; promoter; recombinase; selective expression; smoothened signaling pathway; stem cell population; tool; transcription factor; tumorigenesis; Accounting; Adult; Affect; Anterior; Apoptosis; Apoptotic; Brain; Cancer Biology; Cell Adhesion; Cell Death; Cell Lineage; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cessation of life; Child; Communities; Complex; Development; Developmental Biology; Differentiation and Growth; Digit structure; Early identification; Embryo; Ensure; Erinaceidae; Family member; Fibroblast Growth Factor; Fingers; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genomics; Goals; Growth; Hereditary Disease; Holoprosencephaly; Human; Image; Infant; Intercept; Joints; Lead; Learning; Length; Limb Bud; Limb Development; Limb structure; Link; Maintenance; Malignant Neoplasms; Mediator of activation protein; Mesenchymal; Mesoderm; Mission; Mitogens; Modeling; Morbidity - disease rate; Morphogenesis; Mus; Mutant Strains Mice; Mutate; Mutation; Neoplasm Metastasis; Neoplasms; Neural Crest; Normal tissue morphology; Organ; Pallister-Hall syndrome; Pancreas; Pathology; Pathway interactions; Pattern; Phalanx; Phase; Physical condensation; Physiological Processes; Play; Process; Prostate; Proteomics; Regulation; Research; Role; Shapes; Signal Pathway; Signal Transduction; Skeleton; Skin; Sonic Hedgehog Pathway; Specific qualifier value; Staging; Stomach; Structure; Study models; System; Systems Biology; Tamoxifen; Testing; Thumb structure; Time; Tissues; Transgenes; Tumor Biology; Vertebrates; Work; body system; cancer type; cell behavior; cell motility; cellular targeting; design; developmental genetics; genome-wide analysis; insight; model building; morphogens; mortality; mutant; neoplastic cell; programs; promoter; recombinase; selective expression; smoothened signaling pathway; stem cell population; tool; transcription factor; tumorigenesis

Shh acts as a mitogen and cell survival factor in many adult processes during normal tissue renewal and in many types of cancer, but acts as a morphogen in several developmental contexts. Whether and how the mitogenic role of Shh is integrated with the morphogenetic role in developmental contexts is still poorly understood. In the limb, Shh regulates both digit number and identity of different digits (A-to-P, thumb to pinky). Shh is thought to act as a morphogen forming a gradient along the limb AP axis, with higher concentrations specifying more posterior digit types. We have determined the time-requirements for Shh function in limb (using a tamoxifen-regulated Cre to remove Shh at different times in mice). To perform this analysis, we generated and characterized a conditional Cre recombinase line selectively expressed in early limb mesoderm, neural crest, gut and tailbud. This line provides an excellent tool available to the scientific community to illuminate different temporal roles of key developmental regulators in several important developmental models using mouse mutants, as well as for genetic lineage tracing studies in mice. As one test of this model, we showed that the Shh activity profiles in different classes of mutants with reduced digit number support the model. In particular, mutants in which central digits are lost first have a reduced duration of Shh activity, but the early transient patterning phase, in which posterior digits are specified, remains intact. Conversely, mutants with selective loss of digit 5 have delayed onset of Shh expression and altered Shh activity during the early patterning phase. To further test this model for Shh function, we have assessed whether restoring survival and/or proliferation in mutant embryos after later-stage Shh deletion can rescue digit formation. To rescue cell survival, the compound mutant for the pro-apoptotic Bcl2 family members Bax/Bak (which play roles in normal interdigital apoptosis) has been introduced to inactivate the intrinsic death pathway. Myc has been identified as a direct target of Hedgehog signaling that regulates proliferation in several systems. To attempt rescue of proliferation, we are employing a RosaMycER transgene (from Dr. Gerard Evan) to provide a proliferative impulse whose timing, duration and level of action can be closely regulated by tamoxifen. Our preliminary results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. Our results thus far challenge the view that Shh behaves as a classic morphogen and suggest that the roles of Shh during organ morphogenesis and during tumorigenesis may be very similar. Furthermore, our results indicate that there are 2 classes of Shh responsive target genes, those that respond to a transient signal and become stably expressed, and those that require continuous signaling to maintain expression. We are comparing the transcriptomes of Shh mutant and rescued limb buds to characterize the types of genes in these two differentially regulated target classes. Understanding the proliferative and anti-apoptotic actions of Shh in the context of these differentially regulated target classes, will provide a reference for deciphering and intercepting Shh roles in cancer. To learn how proliferation and recruitment of mesenchymal cells relate to the alternating order in which condensations form and integrate our results with other work on Shh function, we are developing tools to image very early condensations, and to trace cell lineage genetically (collaboratively with Dr. Stephen Lockett, NCI). For example, do condensations form via spatially restricted proliferation of precursors in an alternating sequence, or by alternating foci of cell adhesion? We are also using genetic tools including mutants that act to alter digit patterns at very early times to learn when and how the alternating sequence of digit formation is determined. These tools will help unravel how the observed alternating anterior-to-posterior order of condensation is regulated, and whether this sequence is evolutionarily conserved among different vertebrates. The possibility of alternating patterns of digit formation may also lead to new insights on the mechanisms of evolutionary digit loss in species with adaptive changes in digit number.

在正常组织更新和许多类型的癌症中，SHH充当许多成年过程中的有丝分裂原和细胞存活因子，但在几种发育环境中起到了形态学。 SHH的有丝分裂作用与在发育环境中形态发生的作用是否整合在一起的方式仍然很少了解。在肢体中，SHH调节了不同数字的数字数量和身份（A-to-P，拇指至小指）。 SHH被认为是沿肢体AP轴形成梯度的形态学，其浓度更高，指定了更多的后数类型。我们已经确定了肢体中SHH功能的时间要求（使用他莫昔芬调节的CRE在小鼠的不同时间删除SHH）。为了进行此分析，我们生成并表征了有条件的CRE重组酶系在早期肢体中胚层，神经rest，肠道和尾巴中选择性表达。这条线为科学界提供了一种出色的工具，可以在几种重要的开发模型中使用小鼠突变体以及小鼠的遗传谱系跟踪研究来阐明关键发育调节剂的不同时间作用。作为该模型的一项测试，我们表明，不同类别的突变体中的SHH活性曲线支持该模型。特别是，首先丢失了中央数字的突变体的SHH活性持续时间降低，但是指定后数字的早期瞬态图案阶段仍然完好无损。相反，具有选择性丧失数字5的突变体在早期构图阶段延迟了SHH表达的发作和SHH活性改变。为了进一步测试该模型的SHH功能，我们评估了后期缺失后突变胚胎中的生存和/或增殖是否可以挽救数字形成。为了挽救细胞存活，已经引入了促凋亡Bcl2家族成员Bax/Bak的复合突变体（在正常跨层凋亡中起着作用），以使固有的死亡途径失活。 MYC已被确定为刺猬信号的直接目标，可调节多种系统中的增殖。为了尝试营救增殖，我们正在采用玫瑰色的转基因（来自Gerard Evan博士）提供了增生性冲动，其时间，持续时间和作用水平可以受到他莫昔芬的严格调节。我们的初步结果表明，可以通过简单地恢复细胞存活和SHH突变胚胎的增殖来挽救正常的数字和模式（形态发生）。迄今为止，我们的结果挑战了SHH表现为经典形态学的观点，并表明SHH在器官形态发生和肿瘤发生过程中的作用可能非常相似。此外，我们的结果表明，有两类的SHH响应靶基因，那些对瞬态信号响应并稳定表达的靶基因，以及需要连续信号传导以保持表达的那些。我们正在比较SHH突变体的转录组和拯救肢体的芽，以表征这两个差异调节的靶类别类别中的基因类型。在这些差异调节的目标类别的背景下，了解SHH的增生和抗凋亡作用，将为解密和拦截癌症中的SHH角色提供参考。要了解间充质细胞的增殖和募集与凝结形成的交替顺序如何相关，并将我们的结果与SHH功能的其他工作相结合，我们正在开发图像非常早期冷凝的工具，并在遗传上（与NCI的Stephen Lockett博士协作）。例如，是通过在交替序列中的前体的空间限制前体的增殖或通过细胞粘附的交替灶来形成冷凝的？我们还使用遗传工具，包括在很早的时间内改变数字模式的突变体，以了解何时以及如何确定数字形成的交替序列。这些工具将有助于揭示观察到的交替的凝结前至后方级的调节，以及该序列在不同脊椎动物之间是否在进化上保守。数字形成模式交替模式的可能性也可能导致对数字自适应变化物种进化数字损失机制的新见解。