GLI基因与肢体发育相关性的研究进展

在肢体发育中,Sonic hedgehog(SHH)蛋白作为极化区(the zone of polarizing actiVity,ZPA)的调节因子,发挥着十分重要的作用。然而SHH是如何沿着肢体的前后轴发挥调控作用的还不是很清楚。最近的报道表明SHH主要是通过阻止转录因子GLI3裂解成抑制形式发挥作用,而后者也能够关闭SHH靶基因的表达。GLI基因家族的成员编码含有锌指结构的转录因子,主要对SHH的靶基因发挥调节作用。现就GLI基因在肢体发育中的表达特点及其临床意义进行综述。     

Neogenin regulates Sonic Hedgehog pathway activity during digit patterning

Background : Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. Results : We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. Conclusions : Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning. Developmental Dynamics 241:627–637, 2012. © 2012 Wiley Periodicals Inc.     

Study of the origin of connective tissue sheaths of peripheral nerves in the limb of avian embryos

Summary Quail-chick and chick-quail chimeras were constructed by grafting, isotopically, the limb bud of quail embryos into a chick of the same developmental stage and vice versa, prior to the entry of nerve fibres into the limb.After 5–14 days reincubation of the embryos, the components of the connective tissue sheaths of the peripheral nerves were observed by using Feulgen-Rossenbeck staining and light microscopy, in order to distinguish quail cells and chick cells.In all the chimeras studied, the connective tissue sheaths of peripheral nerves (the epineurium, perineurium, perineural septa and endoneural fibroblasts) were formed from the mesenchyme of the limb bud, while Schwann cells were of host origin. Also the outer and inner capsule of muscle spindles originated from the limb bud mesenchyme.These experiments suggest that the connective tissue sheaths of peripheral nerves (at least in the limb region of avian embryos) are not of neural crest origin, but are formed from limb bud mesenchyme.     

Patterning the limb before and after SHH signalling

The vertebrate limb is one of the most relevant experimental models for analysing cell-cell signalling during patterning of embryonic fields and organogenesis. Recently, the combination of molecular and genetic studies with experimental manipulation of developing limb buds has significantly advanced our understanding of the complex molecular interactions co-ordinating limb bud outgrowth and patterning. Some of these studies have shown that there is a need to revise some of the textbook views of vertebrate limb development. In this review, we discuss how signalling by the polarizing region is established and how limb bud morphogenesis is controlled by both long-range and signal relay mechanisms. We also discuss recent results showing that differential mesenchymal responsiveness to SHH signalling is established prior to its expression by the polarizing region.     

Endogenous electric current is associated with normal development of the vertebrate limb.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.     

Three Dact gene family members are expressed during embryonic development and in the adult brains of mice.

Members of the Dact protein family initially were identified through binding to Dishevelled (Dvl), a cytoplasmic protein central to Wnt signaling. During mouse development, Dact1 is detected in the presomitic mesoderm and somites during segmentation, in the limb bud mesenchyme and other mesoderm-derived tissues, and in the central nervous system (CNS). Dact2 expression is most prominent during organogenesis of the thymus, kidneys, and salivary glands, with much lower levels in the somites and in the developing CNS. Dact3, not previously described in any organism, is expressed in the ventral region of maturing somites, limb bud and branchial arch mesenchyme, and in the embryonic CNS; of the three paralogs, it is the most highly expressed in the adult cerebral cortex. These data are consistent with studies in other vertebrates showing that Dact paralogs have distinct signaling and developmental roles and suggest they may differentially contribute to postnatal brain physiology.     

在乳腺癌干细胞中Hedgehog信号通路相关基因表达增强

目的 了解Hedgehog信号通路在乳腺癌发生发展中的作用.方法 用免疫磁珠法从无血清培养的乳腺癌悬浮细胞中分选CD44+CD24-细胞和非CD44+CD24-细胞,用real-time RT-PCR法检测Hedgehog信号通路主要分子$HH、PTCH1、SMO和GLI1 mRNA在细胞中的表达,用免疫组织化学法检测上述因子在乳腺癌组织中的表达.结果 分选出的CD44+CIDA-细胞约占乳腺癌悬浮细胞总数的8.25%,分选出的CD44+CD24-细胞表达干细胞标志蛋白ALDHA1和Oct-4;SHH、PTCH1、SMO和GLI1 mRNA在CD44+CD24-细胞中的表达均高于其在非CD44+CD24-细胞中的表达(P＜0.05);SMO和GLI1蛋白在三阴性乳腺癌的表达均高于非三阴性乳腺癌组织(P＜0.05).结论 在乳腺癌干细胞CD44+CD24-细胞中Hedgehog信号通路被激活,抑制癌症干细胞中Hedgehog通路的活化可能会降低或阻止乳腺癌的复发及化疗耐受.     

Retinoic acid is essential for Shh/Hoxd signaling during rat limb outgrowth but not for limb initiation.

Retinoids long have been implicated in limb development and their endogenous contributions to this process are finally being elucidated. Here we use an established model of retinoid depletion during specific gestational windows to investigate the role of endogenous retinoic acid (RA) in supporting limb outgrowth. Rat embryos were deprived of RA starting at days-postcoitum (dpc) 3.0, 5.5, or 7.0 and harvested at the 35-somite stage (dpc 12-12.5). Although embryos from all these windows possessed many characteristics of gestational retinoid deficiency (frontonasal hypoplasia, straight tail, reduced CRBPI and RAR beta), their limb buds emerged with only modest size reductions. Molecular analysis of RA-deficient limb buds revealed enhanced gli-3 and reduced hoxd-12, hoxd-13, shh, and fgf-4, while fgf-8, en-1, and wnt-7a expression remained unaltered. Occasional posterior truncations were observed at low incidence in the longest deficiency window; otherwise, the deficiency window length had no discernable impact on the severity of these changes. At the 45-somite stage, RA-deficient limbs had additional losses of hoxd-13 and fgf-8, accompanied by a flattened AER, suggestive of an ultimate failure in limb bud outgrowth. Results could not confirm a function for endogenous retinoids in limb initiation, but show they are required to maintain the signaling loops between the developing mesenchyme and AER that govern limb outgrowth after the initial emergence of limb bud.     

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.     

Genital abnormalities in Pallister-Hall syndrome: Report of two patients and review of the literature

We describe two patients with Pallister-Hall syndrome (PHS) with genital abnormalities: a female with hydrometrocolpos secondary to vaginal atresia and a male with micropenis, hypoplastic scrotum, and bilateral cryptorchidism. Nonsense mutations in GLI3 were identified in both patients. Clinical and molecular findings of 12 previously reported patients who had GLI3 mutations and genital abnormalities were reviewed. Genital features in the male patients included hypospadias, micropenis, and bifid or hypoplastic scrotum, whereas all the females had hydrometrocolpos and/or vaginal atresia. No hotspot for GLI3 mutations has been found. The urogenital and anorectal abnormalities associated with PHS might be related to dysregulation of SHH signaling caused by GLI3 mutations rather than hormonal aberrations. We recommend that clinical investigations of genital abnormalities are considered in patients with PHS, even those without hypopituitarism.     

Differential response of Shh expression between chick forelimb and hindlimb buds by FGF-4.

The interactions of Sonic hedgehog (Shh) and fibroblast growth factor (FGF) play important roles in vertebrate limb pattern formation. In the posterior region of the chick limb bud, Shh and FGF-4 each maintain expression in a positive feedback loop. In the anterior region, Shh can also induce Fgf-4 expression in the anterior apical ectodermal ridge. However, the possibility of Shh induction by FGF protein is unclear. Because many experiments to analyze gene expression have been carried out by using the forelimb bud of the chick embryo, we investigated gene expression of the cells in the anterior region of the chick hindlimb bud after FGF-4 application and compared the results with those for the forelimb bud. When an FGF-4-containing bead was implanted into the anterior region of a stage 20 hindlimb bud, ectopic expression of Shh was induced in the mesenchyme beneath the anterior end of the apical ectodermal ridge at 36 hr after implantation. Subsequent to Shh activation, Hoxd13 was also observed in the anterior-distal region of the limb bud. Furthermore, FGF-4 implantation to the hindlimb bud caused additional digit formation accompanying respecification of positional value in the anterior tissue. Ectopic Shh was induced in cells located distal to the FGF-4 bead, and the cells of the flank region did not contribute to ectopic Shh induction. On the other hand, no ectopic Shh and Hoxd13 expression was detected by grafting an FGF-4 bead into the forelimb bud. Although FGF-4 implantation to the forelimb bud occasionally induced extra digit 2 formation, no embryos had an extra digit 3 or digit 4, and many specimens exhibited normal skeletal pattern. These results demonstrate the difference between the fore- and hindlimb buds in the cell competence of Shh induction in response to FGF-4, suggesting the possibility that the responsiveness of mesenchymal cells in signaling molecules is not the same in the fore- and hindlimb buds.     

Gene expression analysis of the hedgehog signaling cascade in the chick midbrain and spinal cord.

The signaling molecule Sonic Hedgehog (SHH) plays a critical role in patterning the ventral midbrain of vertebrates. Our recent studies have established that the requirement for Hedgehog (HH) signaling in the chick midbrain is modulated spatially and temporally in a complex manner across the midbrain anlage. Unfortunately, the patterns of expression of downstream regulators that might modulate the HH signal in the midbrain are not currently known. To fill this gap, we have examined across time, the expression pattern of 14 genes that function in the HH signaling cascade in the midbrain and spinal cord. Our results suggest that SHH expression in the axial mesendoderm begins before the expression of known HH receptors/HH-binding proteins (e.g., PTC1, PTC2, HHIP, BOC, MEGALIN). In the midbrain, PTC and GLI genes are expressed and then eliminated very early from the ventral midline. However, they exhibit high and persistent expression in the midbrain region circumscribing the SHH source. Intriguingly, multiple HH-binding proteins (BOC, MEGALIN) and HH effectors (GLI1-3, SMO, SUFU, DZIP) are expressed in the dorsal midbrain and the midbrain-hindbrain boundary. Finally, we report for the first time that IHH is expressed in intermediate regions of the spinal cord, where its expression does not overlap with that of SHH.     

A role for epithelial-mesenchymal interactions in tail growth/morphogenesis and chondrogenesis in embryonic mice

Neurulation involves development from primary germ layers before any differentiation of embryonic mesenchyme. Subsequently, secondary organogenesis is via epithelial-mesenchymal interaction. It is unclear whether formation of the caudal body axis and tail bud in vertebrate embryos is by temporal and causal extension of primary neurulation, by secondary neurulation, or by secondary induction (epithelial-mesenchymal interactions) as seen in organogenesis of the limb buds, kidneys, heart and other embryonic regions. Reports of a ventral ectodermal ridge (VER) associated with tail bud development in rodent embryos imply that tail bud development may share features with limb bud development, in which the apical ectodermal ridge (AER) directs limb bud outgrowth and skeletal patterning. Organ culture or grafting to the chorioallantoic membranes of host chick embryos, of tail bud mesenchyme with or without tail epithelium, demonstrates that both survival and growth of tail mesenchyme depend on the presence of tail epithelium. Initiation of chondrogenesis of tail mesenchyme was similarly dependent on tail epithelium until 10.5 days of gestation, which is when the VER is at its maximal extent. Initiation of myogenesis was independent of the presence of tail epithelium. These results are discussed in relation to the similarity of tail bud to limb bud developed, and to the different mechanisms employed in differentiation of the cranial and caudal ends of vertebrate embryos. Secondary induction of the caudal body region is argued to be fundamental in vertebrate embryogenesis.     

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod

Retinoic acid, when administered to pregnant mice on d 11·0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4 , a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8 , which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh , nor of its receptor and downstream target gene Ptc , suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4-induced skeletogenic mesenchymal cell proliferation.     

Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles

We investigated the roles of bare morphogenetic protein (BMP), sonic hedgehog (SHH) and fibroblast growth factor (FGF)-expressing signaling centers in regulating the patterned outgrowth of the telencephalic and optic vesicles. Implantation of BMP4 beads in the anterior neuropore of stage 10 chicken embryos repressed FGF8 and SHH expression. Similarly, loss of SHH expression in Shh mutant mice leads to increased BMP signaling and loss of Fgf8 expression in the prosencephalon. Increased BMP signaling and loss of FGF and SHH expression was correlated with decreased proliferation, increased cell death, and hypoplasia of the telencephalic and optic vesicles. However, decreased BMP signaling, through ectopic expression of Noggin, a BMP-binding protein, also caused decreased proliferation and hypoplasia of the telencephalic and optic vesicles, but with maintenance of Fgf8 and Shh expression, and no detectable increase in cell death. These results suggest that optimal growth requires a balance of BMP, FGF8 and SHH signaling. We suggest that the juxtaposition of Fgf8, Bmp4 and Shh expression domains generate patterning centers that coordinate the growth of the telencephalic and optic vesicles, similar to how Fgf8, Bmp4 and Shh regulate growth of the limb bud. Furthermore, these patterning centers regulate regional specification within the forebrain and eye, as exemplified by the regulation of Emx2 expression by different levels of BMP signaling.In summary, we present evidence that there is cross-regulation between BMP-, FGF- and SHH-expressing signaling centers in the prosencephalon which regulate morphogenesis of, and regional specification within, the telencephalic and optic vesicles.     

Sonic hedgehog: restricted expression and limb dysmorphologies

Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre-axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long-range cis-acting regulator of Shh expression. By inference, human pre-axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre-axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies.     

Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition

Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin βA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin βA mutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin βA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFβ family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.