Hoxd and Gli3-Hoxd interaction roles in Hedgehog regulated digit morphogenesis

5‘HOXD基因在肢体发育过程中发挥多种作用，可能控制后期的形态发生效应。HOXD基因如何引导手指形态发生及其下游靶标仍然是个谜。利用小鼠的遗传学方法，我们发现，除了在启动Sonic hedgehog(Shh)表达方面发挥作用外，5‘Hoxd基因还决定了初级肢轴的极性，并在手指缩合形成后的后期调节手指模式和形态发生，包括关节形成和定位；这是Hoxd基因调节手指识别的主要机制。我们以前发现了5Hoxd和Gli3之间的遗传和物理相互作用，改变了Gli3R(Gli3R)的功能(因此Shh输出)，拮抗Gli3R，并有可能将其转化为激活剂。我们发现，Gli3-Hox相互作用既调节肢体轴形成的极性，又调节手指软骨与关节形成的起搏，这可能与骨骼动态平衡和疾病以及骨骼出生缺陷有关。................................................................................................................................................................................................................................................5‘HOXD基因和HOXD-Gli3相互作用在决定初级肢体轴形成极性中的....................................................................Role作用：在大多数脊椎动物中，四肢主轴穿过后肢，尺骨/指骨(D4)首先凝聚。在尾目两栖动物中，如Axolotl，它们在成年后仍保持再生肢体的能力，前肢轴占主导地位(首先出现的是半径/D2)。根据表达模式的改变，有人认为尾足的轴移动是由于5‘Hoxd基因在肢体远端的表达不能扩展所致。我们对5‘Hoxd突变体(Hoxd11-13缺失)的肢体轴形成进行了分析，发现前轴的形成先于尾骨突变体。此外，我们还发现，在化合物5‘Hoxd；Gli3突变体中，恢复了后轴优势。5‘Hoxd同源框转录因子在复制许可和细胞黏附中发挥作用。Gli3R在前面表达，也调节增殖和凝集，并拮抗5‘Hoxd功能。我们正在分析这些突变体中肢芽不同区域的相对增殖时间和速度以及细胞聚集/凝聚的变化是如何改变的，以及它们是否与前轴和后轴优势相关。我们认为，拮抗的5‘Hoxd-Gli3功能之间的平衡控制着初级肢轴形成的极性，并正在研究改变的轴极性与再生能力之间的潜在关系。................................................................................................................................................................................................................................................Hoxd基因的....................................................................Role和Hoxd-Gli3在软骨分化和关节形成中的相互作用以确定不同的数字身份：即使在数字原始软骨形成凝结形成后，数字身份仍然是可塑性的，并受指间区带调节，它们也是5‘Hoxd和Gli3表达的晚期位点。我们发现，几个Hoxd基因(d11-d13)的遗传缺失会导致关节形成异常，指关节丢失和/或关节位置异常，以及短的双指指。与玛丽安·罗斯(大学)合作我们还在研究Hoxa13在数字形成中的作用。Hoxa13作用于Hoxd13表达的上游，并诱导Hoxd13表达的晚期，可能在调节正常拇指的形成过程中起着明显的作用。典型的Wnt途径在关节形成中起着重要的作用，我们发现在5‘Hoxd突变的手指中，激活的β-连环蛋白可以恢复正常的关节形成。但令人惊讶的是，拯救需要选择性地激活指间组织中稳定的β-连环素，这表明至少在关节形成中，β-连环素和5‘Hoxd功能的某些方面是通过指间信号间接发生的。Gli3(Shh和Hoxd蛋白相互作用的转录效应因子)也在数字上对软骨分化和关节形成有显着的影响。在指骨前体的关节形成过程中，Gli3突变体形成异常的节段，过度的关节形成延伸到软骨元素。在遗传学上，5‘Hoxd和Gli3基因总剂量之间的平衡调节正常关节和哺乳动物手指典型的正常3个骨段的周期性形成。我们的遗传证据表明，Hoxd-Gli3平衡从指间间质间接地作用于调节BMP的活动，从而调节指尖祖细胞池中指尖元素(指骨)和关节的周期性出现。我们正在扩展我们的分析以确定：1)受Gli3-Hoxd相互作用调节的靶标；2)调节指尖祖细胞池以确定指骨数量和大小的其他信号输入。与Steve Vokes(德克萨斯大学奥斯汀分校)合作，我们将研究染色质修饰物PRMT5在维持这个祖细胞库中的作用，它选择性地表达在指尖祖细胞中。即使在哺乳动物中，远端指尖也保持着有限的再生能力，并了解这个远端指祖细胞库的调节，它的维持将为与骨骼再生潜力相关的研究提供新的见解。

5'Hoxd genes play many roles during limb development and may control the effectors of morphogenesis at late stages. How Hoxd genes guide digit morphogenesis and their downstream targets remain enigmatic. Using genetic approaches in mice we find that, in addition to a role in initiating Sonic hedgehog (Shh) expression, 5'Hoxd genes determine the polarity of the primary limb axis early, and regulate digit pattern and morphogenesis at late stages, after digit condensations have already formed, including joint formation and positioning; a major mechanism by which Hoxd genes regulate digit identity. We previously discovered genetic and physical interactions between 5Hoxd and Gli3 that modify Gli3 repressor (Gli3R) function (and hence Shh output), antagonizing Gli3R and potentially converting it to an activator. We find that Gli3-Hox interactions both modulate the polarity of limb axis formation and regulate the pacing of cartilage vs joint formation in digits, which may have relevance for skeletal homeostasis and disease, as well as skeletal birth defects. ....................................................................................................................................................................................................................................................................................................................Role Role of 5'Hoxd genes and Hoxd-Gli3 interaction in determining polarity of primary limb axis formation: In most vertebrates, the primary limb axis runs through the posterior limb with the ulna/digit4 (d4) condensing first. In urodele amphibians such as axolotl, which retain the ability to regenerate limbs as adults, the anterior limb axis is dominant (radius/d2 appear first). Based on altered expression patterns, it has been proposed that the axis shift in Urodeles results from a failure to expand 5'Hoxd gene expression in the late distal limb. We have analyzed limb axis formation in the 5'Hoxd mutant (Hoxd11-13 deleted) and found that the anterior axis forms first as in urodeles. Furthermore, we find that in compound 5'Hoxd;Gli3 mutants, posterior axial dominance is restored. The 5'Hoxd homeobox transcription factors play roles in replication licensing and cell adhesion. Gli3R, expressed anteriorly, also regulates proliferation and condensation, and antagonizes 5'Hoxd function. We are analyzing how changes in the relative timing and rate of proliferation and of cell aggregation/condensation in different zones of the limb bud are altered in these mutants, and if they correlate with anterior vs posterior axial dominance. We propose that the balance between antagonistic 5'Hoxd-Gli3 functions governs the polarity of primary limb axis formation and are investigating the potential relation between altered axis polarity and regenerative capacity. ....................................................................................................................................................................................................................................................................................................................Role of Hoxd genes and Hoxd-Gli3 interaction in cartilage differentiation and joint formation to determine distinct digit identities: Digit identity remains plastic even after the formation of the digit primordial chondrogenic condensations and is regulated by interdigit zones, which are also late sites of 5'Hoxd and Gli3 expression. We found that genetic removal of several Hoxd genes (d11-d13) results in abnormal joint formation, both loss of digit joints and/or abnormal joint position, as well as short, biphalangeal digits. Collaborating with Marian Ros (Univ. Cantabria) we are also examining the role of Hoxa13 in digit formation. Hoxa13 acts upstream of and induces the late phase of Hoxd13 expression and may have a distinct role in regulating the formation of a normal thumb. The canonical Wnt pathway plays an essential role in joint formation and we find that activated beta-catenin restores normal joint formation in the 5'Hoxd mutant digits. But surprisingly, selective activation of stabilized beta-catenin in the interdigital tissues is required for rescue, suggesting that at least some aspects of beta-catenin and 5'Hoxd function in joint formation occur indirectly, via interdigit signaling. Gli3 (the transcriptional effector of Shh and Hoxd protein interactor) also has striking effects on cartilage differentiation and joint formation in digits. During joint formation in digit precursors, Gli3 mutants form abnormal segments with excessive joint formation extending into the cartilage elements. Genetically, the balance between total 5'Hoxd and Gli3 gene dosage regulates the periodic formation of normal joints and the normal 3 bony segments typical of mammalian digits. Our genetic evidence indicates that the Hoxd-Gli3 balance acts indirectly, from interdigital mesenchyme, to modulate Bmp activity and thereby regulate the periodic appearance of digit elements (phalanges) and joints from a digit tip progenitor pool. We are extending our analysis to determine: 1) targets regulated by Gli3-Hoxd interaction and 2) other signaling inputs that regulate the digit tip progenitor pool to determine phalanx number and size. Collaborating with Steve Vokes (UT-Austin) we will examine the role of the chromatin modifier Prmt5, which is selectively expressed in the digit tip progenitors, in maintaining this progenitor pool. Even in mammals, distal digit tips retain a limited capacity for regeneration and understanding the regulation of this distal digit progenitor pool and it's maintenance will provide new insights relevant to skeletal regeneration potential.