A SHH-independent regulation of Gli3 is a significant determinant of anteroposterior patterning of the limb bud

The family of GLI proteins (GLI1-3) comprises the intracellular mediators of the hedgehog pathway, which regulates a myriad of developmental processes, one of which is limb development. Whereas GLI1 and GLI2 seem to be dispensable during limb development, GLI3 is especially crucial since all GLI3-associated human congenital diseases comprise limb malformations. Furthermore, Gli3^−/− mouse embryos exhibit pronounced polydactyly in conjunction with a loss of digit identities.Here we examined how the quantity of GLI3 contributes to its function by using different Gli3 mutants in order to vary overall GLI3 levels. In addition, we made use of the Gli3^Δ699 allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3^Δ699 mutant made it feasible to analyze isoform-specific contributions of GLI3 within the context of anteroposterior patterning of the limb bud. We revealed a so far unappreciated variation in the quantitative demand for GLI3 within different phases and aspects of distal limb formation. In addition, our analyses provide evidence that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud. Instead, digit identities are most likely defined by GLI3 repressor activity alone. Furthermore, we present evidence that the anteroposterior grading of GLI3 activity by the action of SHH is supported by a prototype patterning, which regulates Gli3 independently from SHH.     

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Gα_i proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Gα_i protein activity appears to significantly reduce Hh pathway activity in Ptc^−/− mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Gα_i protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Gα_i protein, Gα_i2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Gα_i activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.     

Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh(-/-), Gli2(-/-) and Gli3(-/-) embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2(-/-) embryos displays ventral patterning defects as severe as those observed in Shh(-/-) embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain.     

胶质瘤相关癌基因蛋白在肿瘤发生中的作用

胶质瘤相关癌基因蛋白(glioma-associated oncogene1,Gli)是Hedgehog(Hh)信号通路的转录因子,定位于细胞核和细胞浆,将信号传送至核内。脊椎动物中已鉴定出3个成员,分别为Gli 1、Gli2和Gli3,该蛋白家族成员只有在维持全长时才具有转录激活子的功能,羧基端被蛋白酶体水解后,就形成了转录抑制子。近年来,Gli与肿瘤的关系日益受到人们的重视,以前普遍认为的Gli目的基因的调控和Gli蛋白的转录后修饰是通过Hh通路实现受到挑战,越来越多的研究证明有许多非经典机制可以不通过Hh通路来调节Gli目的基因的表达。Gli研究将有助于我们对肿瘤的认知和治疗。     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.