Knezevic V, De Santo R, Schughart K, Huffstadt U, Chiang C, Mahon K A, Mackem S
Laboratory of Pathology, NCI, NICHD, National Institutes of Health, Bethesda, MD 20892, USA.
Development. 1997 Nov;124(22):4523-36. doi: 10.1242/dev.124.22.4523.
Several 5' members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes has hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 in the limb remains obscure. Using a gain-of-function approach, we find that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of 'true' preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signalling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Furthermore, expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5' Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth.
Hoxd基因簇的几个5'端成员在肢芽的嵌套后远端区域表达，这表明它们在调节骨骼元素的前后模式中发挥作用。虽然功能丧失突变体已证明这些基因在肢体发育中具有调节作用，但不同Hox基因之间广泛的功能重叠阻碍了对单个成员所起作用的阐明。特别是，Hoxd-12在肢体中的功能仍不清楚。通过功能获得方法，我们发现转基因小鼠中Hoxd-12的错误表达会使前指明显转变为后形态并出现指重复，而相关的胫骨半肢畸形和其他变化表明某些骨骼元素的形成/生长受到选择性抑制。如果指弓代表主肢轴的前向弯曲，那么这些结果都与一个模型相符，即Hoxd-12促进从该主轴线分支的轴后软骨生成凝聚物（包括最前端的指）的形成，并选择性地拮抗从该主轴线分支的“真正”轴前凝聚物（如胫骨）的形成。Hoxd-12的错误表达还可诱导异位的音猬因子（Shh）表达，导致肢体出现镜像多指畸形。Hoxd-12在其他侧板衍生物（胸骨、骨盆）中的错误表达同样模拟了几种luxoid/luxate类小鼠突变体，这些突变体都具有异位的Shh信号。这表明Shh表达的反馈激活可能是Hoxd-12的主要功能。Hoxd-12在体外可结合并反式激活Shh启动子。此外，在培养的肢芽细胞中外源表达Hoxd-11或Hoxd-12，再加上成纤维细胞生长因子（FGF），可诱导内源性Shh基因的表达。这些结果共同表明，在肢芽生长过程中，某些5'端Hoxd基因通过增强的正反馈回路直接放大后Shh极化信号。
