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: 9556462
• 负责人: Susan Mackem
• 金额: $ 42.47万
• 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/9556462
• 关键词: Adult; Affect; Anterior; Apoptosis; Apoptotic; Basal Cell Nevus Syndrome; 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; Digit structure; Embryo; Ensure; Enterobacteria phage P1 Cre recombinase; Family member; Feedback; 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 Childhood Neoplasm; Malignant Neoplasms; 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; Pathologic; Pathology; Pathway interactions; Pattern; Phalanx; Phase; Physical condensation; Physiological Processes; Play; Process; Prostate; Proteomics; Recruitment Activity; Regulation; Research; Role; SHH gene; Shapes; Signal Pathway; Signal Transduction; Skeleton; Skin; Sonic Hedgehog Pathway; Specific qualifier value; Stomach; Structure; Study models; System; Systems Biology; Tamoxifen; Testing; Thumb structure; Time; Tissues; Tumor Biology; Vertebrates; Work; body system; cancer type; cell behavior; cell motility; cellular targeting; design; developmental genetics; insight; morphogens; mortality; mutant; neoplastic cell; programs; response; selective expression; smoothened signaling pathway; stem cell population; tool; transcription factor; transcriptome; 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. 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. In parallel, single cell transcriptome analysis from normal limb buds will be performed to identify expression signatures in the transient Shh signaling phase and characterize immediate-early response zones. 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 downstream of hedgehog signaling to alter digit patterns at 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-posterior order of digit condensations 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.

Shh 在正常组织更新期间的许多成体过程和许多类型的癌症中充当有丝分裂原和细胞存活因子，但在多种发育环境中充当形态发生素。在发育过程中，Shh 的促有丝分裂作用是否以及如何与形态发生作用相结合仍知之甚少。在肢体中，Shh 调节数字数量和不同数字的身份（A 到 P，拇指到小指）。据认为，Shh 充当形态发生素，沿肢体 AP 轴形成梯度，浓度越高，指定的后指类型越多。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在小鼠的不同时间去除Shh）。为了进行该分析，我们生成并表征了在早期肢体中胚层、神经嵴、肠道和尾芽中选择性表达的条件性 Cre 重组酶系。该品系为科学界提供了一个极好的工具，可用于阐明关键发育调节因子在使用小鼠突变体的几个重要发育模型中的不同时间作用，以及用于小鼠遗传谱系追踪研究。作为该模型的一项测试，我们表明不同类别的突变体中数字数量减少的 Shh 活性谱支持该模型。特别是，中央数字首先丢失的突变体的Shh活动持续时间缩短，但早期短暂的模式阶段（其中后数字被指定）保持完整。相反，选择性丢失数字 5 的突变体延迟了 Shh 表达的开始，并在早期模式形成阶段改变了 Shh 活性。为了进一步测试该模型的Shh功能，我们评估了在后期Shh删除后恢复突变胚胎的存活和/或增殖是否可以挽救数字形成。为了挽救细胞存活，引入了促凋亡 Bcl2 家族成员 Bax/Bak（在正常指间凋亡中发挥作用）的复合突变体来灭活内在死亡途径。我们的初步结果表明，通过简单地恢复Shh突变胚胎中的细胞存活和增殖，可以挽救正常的数字数量和模式（形态发生）。迄今为止，我们的结果挑战了Shh作为经典形态发生素的观点，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Shh响应性靶基因，一类响应瞬时信号并稳定表达，另一类需要连续信号传导以维持表达。我们正在比较 Shh 突变体和拯救的肢芽的转录组，以表征这两个差异调节目标类别中的基因类型。同时，将对正常肢芽进行单细胞转录组分析，以识别瞬时 Shh 信号阶段的表达特征并表征立即早期反应区。在这些差异调节靶标类别的背景下了解Shh的增殖和抗凋亡作用，将为破译和拦截Shh在癌症中的作用提供参考。为了了解间充质细胞的增殖和募集与凝聚形成的交替顺序有何关系，并将我们的结果与其他有关Shh功能的研究相结合，我们正在开发工具来对非常早期的凝聚进行成像，并从遗传上追踪细胞谱系（与Dr.史蒂芬·洛克特，NCI）。例如，凝聚是通过前体在交替序列中的空间受限增殖形成的，还是通过细胞粘附的交替焦点形成的？我们还使用遗传工具，包括作用于刺猬信号下游的突变体，在早期改变数字模式，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数字压缩的前后交替顺序是如何调节的，以及该序列在不同脊椎动物中是否在进化上保守。数字形成交替模式的可能性也可能导致对数字数量适应性变化的物种进化数字丧失机制的新见解。