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: 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
• 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

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.

Sonic hedgehog(Shh)在正常组织更新的许多成体过程中和在许多类型的癌症中作为有丝分裂原和细胞生存因子，但在一些发育背景下作为形态原。Shh的有丝分裂作用如何与发育环境中的形态发生作用相结合仍然知之甚少。在肢体中，Shh同时调节数字和不同手指的身份(A-P、拇指-小指)。Shh被认为是一种沿着肢体AP轴形成梯度的形态生成物，浓度越高，指定的后部手指类型越多。我们已经确定了肢体Shh功能的时间要求(使用他莫昔芬调节的Cre在不同时间在小鼠体内清除Shh)。为了进行这一分析，我们构建并鉴定了一个新的条件Cre重组酶系，该重组酶系选择性地在早期肢体中胚层、神经脊、肠道和尾芽中表达。这条线为科学界提供了一个极好的工具，可以用来阐明关键发育调节因子在几个使用小鼠突变的重要发育模型中的不同时间角色，以及用于小鼠的遗传谱系追踪研究。我们的结果，在不同的时间点删除Shh功能，与一个模型最一致，在该模型中，只需要非常短暂的(几个小时)Shh活性来确定5个不同的数字类型的完整补充，但需要较长的时间(约2天)来维持细胞的存活和增殖，从而形成5个正常的数字。为了进一步测试Shh功能的这一模型，我们评估了在Shh活性被Shh基因缺失终止的短暂时期后，恢复细胞存活是否可以挽救突变胚胎中正常的手指形成。为了挽救细胞的存活，已经引入了促凋亡的Bcl2家族成员Bax/Bak的复合突变体(它们在正常的指间凋亡中发挥作用)来灭活固有的死亡途径。我们的结果表明，通过简单地恢复Shh突变胚胎的细胞存活和增殖，可以挽救正常的位数和模式(形态发生)。此外，对肢体中在不同时间点对Shh信号做出反应的细胞的遗传谱系追踪表明，当所有5位数的祖细胞都已被指定时，Shh仅直接向肢体芽的最后面部分发出信号。这一结果表明，Shh在早期肢芽中通过一系列中继信号间接作用于指定的数字。我们开发了一种遗传测试方法，通过在缺乏Shh功能的胚胎(Shh KO)的后肢芽中人为地强制Shh反应来测试中继信号。这在一定程度上挽救了前指的形成，并强烈表明Shh下游存在继电信号。我们的结果与Shh在肢体中作为经典形态生成物的作用是不相容的，并表明Shh作为触发器激活了一种继电机制。此外，我们的结果表明，Shh反应靶基因有两类，一类是对瞬时信号做出反应并稳定表达的基因，另一类是需要持续信号来维持表达的基因。我们正在比较Shh突变体和挽救的肢芽的转录本，以表征这两个不同调控靶类中的基因类型(另见项目：肢芽中Shh激活的转录网络的全基因组靶标分析；ZIA BC 0111120)。.........................................................................................................................................................................我们的结果还表明Shh表达和反应都是高度动态的，我们正在使用几种方法来表征这些特征。我们对Shh产生和反应的谱系追踪结果表明，Shh产生细胞来自近端肢体芽缘的更新的祖细胞池，为肢体中的Shh信号提供了高度的稳健性。同时，我们对正常肢芽进行了单细胞转录组谱分析，以确定瞬时Shh信号阶段的表达特征，并表征即刻-早期反应基因。这些研究通过轨迹分析和识别的祖细胞特异性标记揭示了一个潜在的更新的祖细胞群体。我们正在使用由标记调节区驱动的条件CRE来跟踪这些观察结果，以遗传标记并进一步表征祖细胞谱系。了解Shh表达的祖细胞的健壮性和更新能力的基础，对于设计Shh驱动的癌症的治疗干预措施可能是有价值的。.........................................................................................................................................................................Shh产生和反应的谱系分析还表明，Shh产生细胞对自分泌信号反应完全不敏感，这是许多Shh驱动的人类肿瘤的共同特征(信号通常是旁分泌的，并调节一个支持性的肿瘤生态位)。我们的遗传学和转录学研究表明，对Shh信号的无反应与功能Shh配体的产生密切相关，但在产生Shh信号的细胞中以细胞自主的方式发生。我们正在通过比较内源性Shh基因座和仅编码外显子序列的Shh基因来分析组织培养模型中对配体生产的要求，以评估这种自主无反应的基础。理解这种对自主反应的严格抑制的潜在机制也将为在一些Shh驱动的癌症中如何绕过这种抑制提供见解。

项目成果

Tracing the evolution of avian wing digits.

追踪鸟翼数字的演变。

• DOI: 10.1016/j.cub.2013.04.071
• 发表时间: 2013-06-17
• 期刊: Current biology : CB
• 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.

• DOI: 10.1016/j.devcel.2022.07.016
• 发表时间: 2022-09-12
• 期刊: DEVELOPMENTAL CELL
• 影响因子: 11.8
• 作者: Zhu, Jianjian;Patel, Rashmi;Trofka, Anna;Harfe, Brian D.;Mackem, Susan
• 通讯作者: Mackem, Susan

Evolutionary developmental biology: Use it or lose it.

进化发育生物学：使用它或失去它。

• DOI: 10.1038/nature13509
• 发表时间: 2014
• 期刊: Nature
• 影响因子: 64.8
• 作者: Huang,Bau-Lin;Mackem,Susan
• 通讯作者: Mackem,Susan

Analysis of mutants with altered shh activity and posterior digit loss supports a biphasic model for shh function as a morphogen and mitogen.

对 shh 活​​性改变和后指缺失的突变体的分析支持 shh 作为形态发生素和有丝分裂原发挥作用的双相模型。

• DOI: 10.1002/dvdy.22637
• 发表时间: 2011
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Zhu,Jianjian;Mackem,Susan
• 通讯作者: Mackem,Susan