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

基本信息

批准号：
10926136
负责人：
Susan Mackem
金额：
$ 44.31万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926136
关键词：
Adult Affect Anterior Apoptosis Apoptotic Autocrine Communication Basal Cell Nevus Syndrome Biological Assay Brain Bypass Cell Death Cell Proliferation Cell Survival Cell physiology Cells Cessation of life Child Code Communities Complement component C5 Complementary DNA Complex Coupled Development Developmental Biology Digit structure ERG gene Elements Embryo Ensure Enterobacteria phage P1 Cre recombinase Exons Family member Feedback Fibroblast Growth Factor Fingers Gene Deletion Gene Expression Profile Gene Expression Regulation Genes Genetic Genetic Diseases Genetic Transcription Genomics Goals Growth Holoprosencephaly Hour Human Infant Intercept Joints Learning Length Ligands Limb Bud Limb Development Limb structure Link Maintenance Malignant Childhood Neoplasm Malignant Neoplasms Mesoderm Mission Mitogens Modeling Morbidity - disease rate Morphogenesis Mus Mutant Strains Mice Mutate Mutation Neoplasm Metastasis Neoplasms Neural Crest Normal tissue morphology Nucleic Acid Regulatory Sequences Organ Pancreas Pathologic Pathology Pathway interactions Pattern Phalanx Phase Physiological Processes Play Population Process Production Proliferating Prostate Proteomics Refractory 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 Therapeutic Intervention Thumb structure Time Tissues Tumor Biology body system cancer type cell behavior cell motility cellular targeting design developmental genetics genome-wide insight model building morphogens mortality mutant neoplastic cell novel paracrine progenitor programs response selective expression smoothened signaling pathway stem cell population tissue culture tissue regeneration tool transcription factor transcriptome transcriptomic profiling transcriptomics tumor tumorigenesis

项目摘要

Sonic hedgehog (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. 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 novel 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. Our results, deleting Shh function at different time points, are most consistent with a model in which Shh activity is required only very transiently (several hours) to specify the complete complement of 5 different digit types, but is required for a prolonged time (about 2 days) to maintain cell survival and proliferation, enabling 5 normal digits to form. To further test this model for Shh function, we have assessed whether restoring cell survival can rescue normal digit formation in mutant embryos after a transient period of Shh activity that is terminated by Shh gene deletion. 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 results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. In addition, genetic lineage tracing of cells in the limb that have responded to Shh signals at different time points indicates that Shh only signals directly to the very posterior part of the limb bud at the time when all 5 digit progenitors have been specified. This result indicates that Shh acts indirectly in the early limb bud, via a system of relay signals, to specify digits. We developed a genetic assay to test for relay signaling by artificially enforcing Shh-response in the posterior limb bud in embryos lacking all Shh function (Shh KO). This partly rescues anterior digit formation, and strongly suggests the presence of relay signals acting downstream of Shh. Our results are incompatible with Shh acting as a classic morphogen in the limb and suggest that Shh acts as a trigger to activate a relay mechanism. 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 (see also Project : Genome-wide target analysis of Shh-activated transcription network in limb bud; ZIA BC 0111120). ......................................................................................................................................................................... Our results also indicate that both Shh-expression and response are highly dynamic and we are characterizing these features using several approaches. Our lineage tracing results of Shh production and response indicate that Shh producing cells arise from a renewing progenitor pool in the proximal limb bud margin, providing a high level of robustness to Shh signaling in the limb. In parallel, we performed single cell transcriptome profiling from normal limb buds to identify expression signatures in the transient Shh signaling phase and characterize immediate-early response genes. These studies have revealed a potential renewing progenitor population by trajectory analysis and identified progenitor-specific markers. We are following up on these observations using a conditional Cre driven by marker regulatory regions to genetically mark and further characterize the progenitor lineage. Understanding the basis for robustness and renewal capacity in Shh-expressing progenitors may be valuable in devising therapeutic intervention for Shh-driven cancers. ......................................................................................................................................................................... The lineage analysis of Shh-production and -response also suggest that Shh-producing cells are completely refractory to autocrine signaling response, which is a feature shared by many Shh-driven tumors in humans (signaling is often paracrine and regulates a supportive tumor niche). Our genetic and transcriptomic studies suggest that non-responsiveness to Shh signaling is tightly coupled to functional Shh-ligand production, but occurs in a cell autonomous manner in the producing cells. We are dissecting the requirements for ligand production in a tissue culture model using comparison of the endogenous Shh locus vs Shh cDNA with only coding exon sequences to assess the basis for this autonomous non-responsiveness. Understanding the underlying mechanisms for this strict inhibition of autonomous response will also provide insights on how this inhibition is bypassed in some Shh-driven cancers.

在正常组织更新和许多类型的癌症中，Sonic刺猬（SHH）在许多成年过程中充当有丝分裂原和细胞存活因子，但在几种发育环境中充当了形态学。 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）。为了执行此分析，我们生成并表征了一种新型的条件性CRE重组酶系，在早期肢体中胚层，神经rest，肠和尾巴中有选择地表达。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。我们的结果是在不同时间点上删除SHH函数，与仅需要非常短的（几个小时）需要SHH活性的模型来指定5种5种不同数字类型的完整补体，但需要长时间（约2天）来维持细胞存活和增殖，从而形成5个正常数字。为了进一步测试该模型的SHH功能，我们已经评估了恢复细胞存活是否可以在SHH基因缺失终止的SHH活性后，是否可以挽救突变体胚胎中的正常数字形成。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。我们的结果表明，可以通过简单地恢复SHH突变体胚胎中的细胞存活和增殖来挽救正常数字和模式（形态发生）。此外，在不同时间点对SHH信号响应的肢体中细胞的遗传谱系追踪表明，在指定所有5个数字祖细胞时，SHH仅直接向肢体芽的后部直接信号。该结果表明，SHH通过继电器信号系统间接起作用在早期的肢体芽中，以指定数字。我们开发了一种遗传测定，通过缺乏所有SHH功能（SHH KO）的胚胎后肢体中的人为实施SHH反应来测试继电器信号传导。这部分挽救了前数字的形成，并强烈暗示存在SHH下游作用的继电器信号。我们的结果与SHH充当肢体中经典的形态学不相容，并表明SHH充当激活继电器机制的触发因素。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。我们比较了SHH突变体和救出肢体芽的转录组，以表征这两个差异调节的目标类别中的基因类型（另请参见项目：全基因组范围的目标分析，对肢体芽中SHH激活的转录网络； Zia BC 0111120）。 ......................................................................................................................................................................... Our results also indicate that both Shh-expression and response are highly dynamic and we are characterizing these features using several approaches.我们的SHH产生和反应的谱系追踪结果表明，SHH产生细胞是由近端肢体芽缘的更新祖细胞池引起的，为肢体中的SHH信号传导提供了高度的鲁棒性。同时，我们从正常的肢体芽中进行了单细胞转录组分析，以识别瞬态SHH信号阶段中的表达特征，并表征了即时响应基因。这些研究揭示了通过轨迹分析的潜在更新祖细胞种群，并确定了祖细胞特异性标记。我们正在使用由标记调节区域驱动的条件CRE来跟进这些观察结果，以遗传标记并进一步表征祖细胞谱系。了解表达SHH祖细胞中鲁棒性和更新能力的基础可能对于设计用于SHH驱动的癌症的治疗干预可能是有价值的。 ......................................................................................................................................................................... The lineage analysis of Shh-production and -response also suggest that Shh-producing cells are completely refractory to autocrine signaling response, which is a feature shared by many Shh-driven tumors in humans (signaling is often paracrine and regulates a supportive tumor利基）。我们的遗传和转录组研究表明，对SHH信号传导的无反应性与功能性SHH配体的产生紧密耦合，但在生产细胞中以细胞自主方式发生。我们使用仅使用编码外显子序列的内源性SHH基因座与SHH cDNA进行比较，在组织培养模型中解剖了配体产生的需求，以评估这种自主性无反应性的基础。了解严格抑制自主反应的基本机制也将提供有关在某些SHH驱动的癌症中如何绕过这种抑制的见解。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Tracing the evolution of avian wing digits.

DOI：
10.1016/j.cub.2013.04.071
发表时间：
2013-06-17
期刊：
Current biology : CB
影响因子：
0
作者：
Xu X;Mackem S
通讯作者：
Mackem S

Tamoxifen-dependent, inducible Hoxb6CreERT recombinase function in lateral plate and limb mesoderm, CNS isthmic organizer, posterior trunk neural crest, hindgut, and tailbud.

DOI：
10.1002/dvdy.21846
发表时间：
2009-02
期刊：
DEVELOPMENTAL DYNAMICS
影响因子：
2.5
作者：
Nguyen, Minh-Thanh;Zhu, Jianjian;Nakamura, Eiichiro;Bao, Xiaozhong;Macken, Susan
通讯作者：
Macken, Susan

Sonic hedgehog is not a limb morphogen but acts as a trigger to specify all digits in mice.