Sonic hedgehog信号通路在脑损伤中的作用

Sonic hedgehog(Shh)是神经系统发育和肿瘤发生发展中的重要调节因子。近年的研究显示,Shh在脑损伤疾病中发挥重要的作用。在脑损伤后,Shh的表达上调,通过其信号通路促进脑损伤后的修复过程。研究Shh信号通路有助于深入地了解脑损伤的病理过程,同时也为脑损伤的临床治疗提供新的途径。     

腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响

主要嗅觉表皮(main olfactory epithelium, MOE)是哺乳动物感知气味分子的主要嗅觉器官。在MOE组织内,大多数嗅觉神经元通过cAMP信号传导通路感知气味信息。作为嗅觉cAMP信号通路的主要成员之一,腺苷酸环化酶3（adenylyl cyclase 3, ac3）基因敲除小鼠嗅觉探测功能丧失。除cAMP信号传导通路外,MOE内AC3相关因子AC2和AC4,以及肌醇1,4,5-三磷酸（inositol 1,4,5-trisphosphate,IP3）信号通路和Sonic Hedgehog（Shh）信号通路均有表达。然而,敲除ac3是否会对ac2和ac4以及IP3和Shh信号通路成员产生影响,尚不清楚。本文以AC3缺失（AC3-/-）及其野生型小鼠（AC3+/+）MOE为材料,采用实时荧光定量PCR（qRT-PCR）和免疫荧光组织化学方法,发现AC3缺失后,MOE内的ac2和ac4,以及IP3信号通路中的IP3受体ip3r1及钙调蛋白calm1和calm2表达水平均明显降低。Shh信号通路中的受体patched(ptch)与smoothened(smo)、以及核转录因子gli1与gli2的表达也受到了影响。总之,AC3基因缺失不但导致小鼠MOE组织中cAMP信号通路受损,同时AC3相关因子,IP3信号通路和Shh信号通路的传导也受到抑制。本文对于阐明AC3基因敲除小鼠嗅觉丧失的原因及其嗅觉探测机制具有重要启示作用。     

Wnt信号在胚胎干细胞自我更新和分化中的作用

胚胎干细胞分离自胚泡内细胞团,具有无限自我更新和多向分化潜能,有很大的医学应用前景。Wnt家族是一类分泌型的细胞信号传导蛋白,可以通过复杂的信号传递通路调控胚胎的早期发育,对细胞的分化、增殖及生长具有重要的调节作用。该文就Wnt信号通路调节胚胎干细胞的自我更新和分化作一综述。     

Shh信号参与调控BMP9诱导的间充质干细胞成骨分化

目的：观察sonic hedgehog（Shh）信号通路在骨形态发生蛋白9（BMP9）诱导的小鼠间充质干细胞（MSCs）C3H10T1/2和C2C12成骨分化中的作用,并初步探讨其作用机制。方法：Shh信号通路抑制剂Cyclopamine和激活剂Purmorphamine以及过表达Shh腺病毒分别作用于BMP9处理的C3H10T1/2和C2C12细胞,碱性磷酸酶（ALP）检测早期成骨指标ALP,茜素红S染色检测晚期成骨指标钙盐沉积,RT-PCR检测Shh信号相关基因以及成骨关键转录因子的表达,Western blot检测Shh的表达,荧光素酶报告基因检测Smad1/5/8的转录调控活性。结果：BMP9促进Shh信号相关基因的表达,激活Shh信号可增强BMP9诱导的C3H10T1/2和C2C12细胞早晚期成骨分化并促进了BMP9诱导的Smad荧光素酶活性,抑制Shh信号后作用相反。结论：激活Shh信号通路可促进BMP9诱导的小鼠MSCs成骨分化,抑制其活性后作用相反。     

Wnt信号通路在视网膜色素上皮发育中的作用

Wnt（wingless-type MMTV integration site family members）信号通路与细胞的发育分化密切相关,尤其对动物胚胎期中枢神经系统的发育至关重要。在眼的早期发育中,视泡背部视网膜色素上皮细胞（RPE）Wnt/βcatenin信号通路高度活跃,对神经视网膜及RPE的发育调控起重要作用。本文结合目前该领域研究进展,综合评述Wnt信号通路、Wnt蛋白家族以及Wnt信号通路与RPE发育的关系。     

鸟类仍保留牙齿发生定位的分子机制

众所周知现代鸟类不长牙齿,而其侏罗纪和白垩纪的祖先则长有牙齿,然而,在发育中鸡胚口腔中却残留着牙齿发生的原基,在形态上与哺乳动物臼牙牙原基极为相似,现代鸟类的胚胎组织是否具有牙齿发育的潜能,目前已有不少研究者对这一问题进行了探讨,Kollar和Fisher等人将鸡胚胎下颌靠近口腔面的上皮与小鼠的牙间充质进行组织重组实验,并植入小鼠眼球中作intraocular grafting培养,他们的实验结果表明重组后的组织块可以发育形成牙齿的结构,包括形成成釉细胞（ameloblast),并能分泌釉质,Kollar等认为在进化进程中鸟类牙齿的消失并非由于口腔上皮中有关釉质合成的遗传信息的丢失,而是牙齿发育过程中的组织之间所必须的相互作用（次级诱导）受阻而造成的。Lemus和Fuenzalida等人的实验结果进一步证实了这一结论。他们用鹌鹑胚胎躯体的上皮组织与蜥蚁或兔子的牙间充质重组后,用鸡胚绒毛进行培养,得到了发育很好的牙齿结构,发现鹌鹑的上皮细胞也可以分形成釉质细胞,并分泌牙釉质,Cummings将鹌鹑胚胎的牙上皮组织与小鼠胚胎的牙间充质组织重组后也得到类似的结果。根据小白鼠牙齿发育中已知的调控分子信号通路,我们曾对鸟类不长牙齿的分子机制进行了研究,我们的研究发现鸟类牙组织仍保留与哺乳动物早期牙齿发生相类似的信号通路,能表达相关的基因并产生相应的信号分子,鸟类牙齿发育停滞在牙原基时期的可能原因是Bmp4不在预定牙上皮组织中表达,导致发育信号的传递受阻,因此,鸟类胚胎的牙原基组织是一个很好的实验模型,用于研究上皮与间充质组织之间的相互作用,导致器官发育的分子机制。在本研究中,我们又进一步对鸡胚发育中与牙原基定位的有关分子信号通路进行了研究,研究证实了,与小白鼠的发育相似,在鸡胚发育中,在任何可见的牙齿发生了形态变化出现之前,Pax9作为预定牙间充质的标记基因（Fig.1),利用体外器官培养,组织重组和原位杂交等方法,证实了Pax9在下颚间充质中的定位表达是由其上方上皮中的两种信号分子所决定（Fig.2),其中FGF8诱导Pax9的表达（Fig.3).而BMP4则抑制该表达（Fig.4)。通过这两种信号之间的诘抗作用决定了间充质中Pax9的表达部位,亦即牙原基的发生部位,因此,与小白鼠相似,在鸟类中牙原基的发生部位是由两类具有诘抗作用的信号分子所决定的,本研究结果进一步证实了在鸟类胚胎发育过程中仍保留与牙齿发育有关的早期信号通路。     

舌的发育及其调控机制

舌是参与咀嚼、吸允、吞咽、发音等功能的重要器官。舌发育异常会导致无舌、小舌、舌裂、结舌、巨舌症、腭裂等严重的先天舌发育缺陷疾病。发育完全的舌由肌肉组织、结缔组织、黏膜组织及血管组成。调控舌发育的基因有Pax3/Pax7、Dlx基因家族、TGF-β家族和FGF等,同时还受Shh与Wnt等信号通路调控。舌发育分子调控机制的研究对相关舌发育畸形疾病的诊断与治疗有重要意义。     

鳜Shh基因分子特征和时空表达规律及其对肌肉生长的调控

研究从鳜(Siniperca chuatsi)基因组中获得Shh(Sonic hedgehog)基因序列,对该基因编码的蛋白质和同源进化特征进行了分析。鳜Shh基因的开放阅读框(ORF)为1242 bp,编码413个氨基酸,分子量为46.01 kD,等电点(pI)为6.57,脂溶系数为82.83,亲水性平均系数为-0.292,拥有一个跨膜区,被推定为亲水性膜结合蛋白。Shh蛋白具有两个结构域,即Hh-N和Hh-C结构域。鳜Shh蛋白与自身所属的鲈形目鱼类Shh蛋白同源性较高。通过实时荧光定量PCR技术检测了鳜Shh基因的时空表达水平。结果显示:Shh的表达量在神经胚期表达显著上调,并在胚胎发育中后阶段保持较高水平。Shh在鳜不同组织中存在一定的表达差异,在脑、肠道中表达量较高,而在红肌、白肌等组织中表达量相对较低。通过环巴胺(Cyclopamine)处理鳜胚胎来抑制Shh信号通路的实验表明, Shh信号通路被抑制后, Pax3、Pax7、Myomaker、生肌调节因子及肌球蛋白重、轻链等基因的表达均显著降低,推测Shh参与调控鳜肌细胞的早期分化和发育过程。研究将有助于从分子水平了解Sh...     

FGF8在脊椎动物早期胚胎发育中的调控作用

成纤维细胞生长因子8 (fibroblast growth factor 8,FGF8)是成纤维细胞生长因子家族的成员之一,是一种组织发育过程中的重要分泌性调控信号分子,参与脊椎动物的多种组织器官的发生与发育.早期胚胎细胞通过表达FGF8在组织和器官发育、血管发生、血细胞生成、附肢发生和伤口愈合等方面发挥着重要作用.FGF8不但可以在细胞外通过胞内信号通路,而且也可以进入细胞内部发挥生物学功能.本文就FGF8在脊椎动物神经系统、内脏器官、肢体发育及不对称发育等组织、器官发育中的调控作用予以阐述.     

生长激素/催乳素家族配体和受体成员在斑马鱼早期胚胎中的表达比较和交叉活性分析

为了进一步研究GH/PRL家族信号通路在鱼类早期胚胎发育中的作用, 研究以斑马鱼为模型, 通过Real-time PCR技术和原位杂交技术刻画并比较了GH/PRL家族成员及其受体家族成员在胚胎发育早期的表达模式。结果发现, 在配体家族成员中, 生长激素（Growth hormone, GH）和生长催乳素（Somatolactin, smtl）存在母源表达, 在受体家族成员中, ghra、ghrb存在母源表达。利用荧光素酶分析spi2.1启动子活性的结果初步证明, 在斑马鱼早期胚胎发育中, 各配体家族成员与GHRa之间可以发生广泛的互作。这一系列结果对于我们认识GH/PRL家族信号通路在斑马鱼早期发育中的作用具有重要的指导意义。       

Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata)

Here we study the role of Shh signaling in tooth morphogenesis and successional tooth initiation in snakes and lizards (Squamata). By characterizing the expression of Shh pathway receptor Ptc1 in the developing dentitions of three species (Eublepharis macularius, Python regius, and Pogona vitticeps) and by performing gain- and loss-of-function experiments, we demonstrate that Shh signaling is active in the squamate tooth bud and is required for its normal morphogenesis. Shh apparently mediates tooth morphogenesis by separate paracrine- and autocrine-mediated functions. According to this model, paracrine Shh signaling induces cell proliferation in the cervical loop, outer enamel epithelium, and dental papilla. Autocrine signaling within the stellate reticulum instead appears to regulate cell survival. By treating squamate dental explants with Hh antagonist cyclopamine, we induced tooth phenotypes that closely resemble the morphological and differentiation defects of vestigial, first-generation teeth in the bearded dragon P. vitticeps. Our finding that these vestigial teeth are deficient in epithelial Shh signaling further corroborates that Shh is needed for the normal development of teeth in snakes and lizards. Finally, in this study, we definitively refute a role for Shh signaling in successional dental lamina formation and conclude that other pathways regulate tooth replacement in squamates.     

Shh signaling within the dental epithelium is necessary for cell proliferation,growth and polarization

Sonic hedgehog (Shh), a member of the mammalian Hedgehog (Hh) family, plays a key role during embryogenesis and organogenesis. Tooth development, odontogenesis, is governed by sequential and reciprocal epithelial-mesenchymal interactions. Genetic removal of Shh activity from the dental epithelium, the sole source of Shh during tooth development, alters tooth growth and cytological organization within both the dental epithelium and mesenchyme of the tooth. In this model it is not clear which aspects of the phenotype are the result of the direct action of Shh on a target tissue and which are indirect effects due to deficiencies in reciprocal signalings between the epithelial and mesenchymal components. To distinguish between these two alternatives and extend our understanding of Shh's actions in odontogenesis, we have used the Cre-loxP system to remove Smoothened (Smo) activity in the dental epithelium. Smo, a seven-pass membrane protein is essential for the transduction of all Hh signals. Hence, removal of Smo activity from the dental epithelium should block Shh signaling within dental epithelial derivatives while preserving normal mesenchymal signaling. Here we show that Shh-dependent interactions occur within the dental epithelium itself. The dental mesenchyme develops normally up until birth. In contrast, dental epithelial derivatives show altered proliferation, growth, differentiation and polarization. Our approach uncovers roles for Shh in controlling epithelial cell size, organelle development and polarization. Furthermore, we provide evidence that Shh signaling between ameloblasts and the overlying stratum intermedium may involve subcellular localization of Patched 2 and Gli1 mRNAs, both of which are targets of Shh signaling in these cells.     

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

The Shh signalling pathway in early tooth development.

The Sonic Hedgehog (Shh) signalling pathway has been proposed to play an important role in mammalian tooth development. We describe the spatial and temporal expression of genes in this pathway during early tooth development and interpret these patterns in terms of the likely roles of Shh signalling. We show that the two putative receptors of the Shh ligand, Ptc and Ptch-2, localise in different cells, suggesting Shh may function in different ways as an epithelial and mesenchymal signal. Shh signalling has previously been shown, in other organs, to stimulate cell proliferation. In this paper we analyse the Fgf signalling pathway in Gli-2 mutants and propose a mechanism as to how Gli-2 may regulate cell proliferation in tooth development.     

Sonic hedgehog is required early in pancreatic islet development

Pancreatic organogenesis relies on a complex interplay of cell-autonomous and extracellular signals. We demonstrate that the morphogen sonic hedgehog (Shh) is required for pancreatic development in zebrafish. Genetic mutants of Shh and its signaling pathway establish this dependence as specific to endocrine, but not exocrine, pancreas. Using cyclopamine to inhibit hedgehog signaling, we show that transient Shh signaling is necessary during gastrulation for subsequent differentiation of endoderm into islet tissue. A second hedgehog-dependent activity occurring later in development was also identified and may be analogous to the known action of Shh in gut endoderm to direct localization of pancreatic development. The early action of Shh may be part of a more general process allowing neuroendocrine cells to originate in nonneuroectodermally derived tissues.     

Epiprofin/Sp6: a new player in the regulation of tooth development

Odontogenesis is governed by a complex network of intercellular signaling events between the dental epithelium and mesenchyme. This network leads to the progressive determination of tooth shape, and to the differentiation of these tissues into enamel-producing ameloblasts and dentin-producing odontoblasts respectively. Among the main signaling pathways involved in the regulation of tooth development, Bone Morphogenetic Protein (BMP), Sonic hedgehog (Shh) and Wingless-type MMTV integration site (Wnt) pathways have been reported to play significant roles. Recently, the phenotype of mice deficient in Epiprofin/Sp6 (Epfn) has been found to present striking dental abnormalities, including a complete lack of differentiated ameloblasts and consequently no enamel, highly altered molar cusp patterns and the formation of multiple supernumerary teeth. In this article, we review the interaction of Epfn with the BMP, Shh and Wnt pathways in the regulation of tooth development, based on the data obtained from the study of several genetically modified mice.     

Sonic hedgehog expression during early tooth development in Suncus murinus

Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.     

The epidemiology of supernumerary teeth and the associated molecular mechanism

Supernumerary teeth are common clinical dental anomalies. Although various studies have provided abundant information regarding genes and signaling pathways involved in tooth morphogenesis, which include Wnt, FGF, BMP, and Shh, the molecular mechanism of tooth formation, especially for supernumerary teeth, is still unclear. In the population, some cases of supernumerary teeth are sporadic, while others are syndrome-related with familial hereditary. The prompt and accurate diagnosis of syndrome related supernumerary teeth is quite important for some distinctive disorders. Mice are the most commonly used model system for investigating supernumerary teeth. The upregulation of Wnt and Shh signaling in the dental epithelium results in the formation of multiple supernumerary teeth in mice. Understanding the molecular mechanism of supernumerary teeth is also a component of understanding tooth formation in general and provides clinical guidance for early diagnosis and treatment in the future.