转录调节因子LMO4在牙胚发育中的基因表达

目的观察转录调节因子LMO4在鼠磨牙牙胚形态发生中的基因表达，并与Shh信号分子的基因表达进行比较。方法制备昆明小鼠磨牙形态发育各期标本（E11．5～P1．5），wholemount原位杂交分析LM04 mRNA在鼠胚中的表达与分布。切片原位杂交分析LMO4及Shh mRNA在牙胚中的表达与分布。用免疫组化SP法对增殖细胞核抗原（PCNA）进行定位研究。结果wholemount原位杂交发现，在E11．5，LMO4 mRNA在上下颌突、肢芽、脑、表皮和体节中呈阳性表达。切片原位杂交发现，E13．5～E16．5，LMO4 mRNA分别在牙蕾上皮、成釉器两侧尖部和颈环处呈阳性表达，Shh mRNA表达于釉结；E18．5～P1．5，LMO4 mRNA在成釉细胞层和中间层呈阳性表达。免疫组化染色发现，E13．5～E16．5，部分牙蕾上皮及颈环处细胞PCNA阳性，恰与LMO4基因表达部位重合。结论LMO4在牙胚形态发生中呈时空特异性表达并且局限表达于上皮来源的组织。LMO4与Shh信号分子表达范围邻近，在牙胚发育早期可能调控细胞增殖，晚期可能参与成釉细胞的分化。     

Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.     

Noggin is required for early development of murine upper incisors

BMP signaling plays crucial roles in the development of many organs, including the tooth. Equally important is BMP signaling homeostasis, as demonstrated by multiple organ defects in mice lacking the extracellular BMP antagonist Noggin. Here, we show that Noggin is initially expressed in the maxillary mesenchyme adjunct to the upper incisor at the initiation stage, and then in the developing teeth, including incisors and molars, from the bud stage. Noggin mutants develop normal molars and mandibular incisors, but form a single, medially located upper incisor that is arrested at the late bud stage. Histological and molecular marker analyses demonstrated that two distinct upper incisor placodes initiate independently at E11.5, but begin to fuse at E12.5, coupling with elevated cell proliferation rates in the developing tooth germs. We further found that Chordin and Gremlin, two other BMP antagonists, are co-expressed with Noggin in the developing lower incisor and molar teeth. These observations indicate the importance of BMP signaling homeostasis, and suggest a functional redundancy between BMP antagonists during tooth development.     

Expression of the Hedgehog antagonists Rab23 and Slimb/betaTrCP during mouse tooth development

The sonic hedgehog signalling peptide has been demonstrated to play an important role in the growth and patterning of several organs including the tooth. Inappropriate activation of Shh signalling in the embryo causes various patterning defects and complex regulation of this pathway is important during normal development. A growing list of diverse antagonists have been identified that restrict Shh signalling in the embryo, however, only Ptc1, Gas1 and Hip1 have been studied during tooth development. We have examined the expression pattern of the putative antagonists Rab23 and Slimb/betaTrCP during early murine odontogenesis and find that these molecules are expressed in the developing tooth. Interestingly, Rab23 demonstrates contrasting expression domains in the incisor and molar dentition during the cap stage, being restricted to the mesenchymal compartment of molar teeth and the epithelium of the enamel knot in incisor teeth. These findings provide the first evidence of distinct regulatory pathways for Shh in teeth of different classes.     

Shh及其受体Ptc1、Ptc2在鼠帽状期磨牙的基因表达

目的 观察信号分子Sonic Hedgehog(Shh)及其受体Ptcl、Ptc2在小鼠下颌第一磨牙帽状期的基因表达，以探讨Shh信号路径在牙胚形态发育早期的作用。方法 制备昆明小鼠磨牙形态发育早期标本(E10．5～E15．5)，常规HE染色观察组织形态。免疫组化SP法对增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)进行定位研究。原位杂交法分析Shh、Ptcl、Ptc2 mRNA在牙胚中的表达与分布。结果 E14．5，内、外釉上皮，星网状层及牙乳头细胞PCNA阳性，大部分釉结细胞PCNA阴性，少量釉结细胞PCNA阳性。Shh、Ptcl、Ptc2 mRNA在内、外釉上皮、星网状层及釉结中均有表达。结论 在帽状期，信号分子Shh可能经自分泌或旁分泌途径作用于釉结以外的上皮和间充质，促进其增殖。     

Tissue engineering of teeth using adult stem cells

Tooth development, a process which occurs in the developing embryo, involves the reciprocal and sequential signalling between epithelial and mesenchymal tissue of the developing first branchial arch. The oral epithelium produces the first inductive signals for odontogenesis at around E10.0, which trigger off a cascade of events that result in the formation of a tooth. We have engineered a tooth in vitro by harnessing the basic principles of odontogenesis and the inductive capability of the oral epithelium of the developing embryo. We replaced the mesenchymal portion of the developing mandibular primordium with aggregates of stem cells from embryos as well as stem cells taken from adult mice. The cell aggregates were covered with embryonic epithelium from E10.0 mouse embryos to form recombinant explants. In vitro culture of these recombinant explants resulted in the induction of early tooth marker genes in the cell aggregates, indicating that the cells were able to respond to the odontogenic signals produced by the oral epithelium. In vivo culture of explants resulted in the induction of Dspp within the cell aggregates indicating that tooth tissue was present. Three recombinant explants, where the cell aggregates consisted of adult bone marrow cells, produced teeth. To determine whether the oral cavity would be able to sustain the growth of an implanted tooth germ, E14.5 molar rudiments were implanted into the diastema region of the maxilla of adult mice. The resulting teeth appeared to be normal in size and were connected to the underlying bone. These experiments are an indication that it is possible to induce odontogenesis and engineer a tooth using adult cells of non-dental origin. They also indicate that developing tooth germs could be successfully implanted into the gingiva of patients.     

Regulation of tooth development by the novel type I TGFbeta family member receptor Alk8.

We have recently identified, in zebrafish, a novel type I receptor of the TGFbeta family, alk8, that participates in Bmp signaling pathways to mediate early dorsoventral patterning of neurectodermal and mesendodermal tissues. Since Bmps play significant roles in tooth specification, initiation, and differentiation, we hypothesized that alk8 may play a role in directing the Bmp-mediated epithelial mesenchymal cell interactions regulating tooth development. Immunohistochemical analysis demonstrates that Alk8 is expressed in developing zebrafish and mouse teeth. Examination of tooth development in zebrafish with disrupted alk8 signaling revealed specific defects in tooth development. Ectopic expression of constitutively active Alk8 results in the formation of elongated tooth structures, while expression of dominant-negative Alk8 results in arrested tooth development at the bud stage. These results are consistent with the established requirements for Bmp signaling in tooth development and demonstrate that Alk8 is a key regulator of tooth development.     

初级纤毛在牙发育中的分布及相关信号通路的研究进展

初级纤毛是位于大多数哺乳动物细胞表面,感受外环境刺激并传导信息的一种细胞器,在组织发育过程中参与调控各种信号通路。本文就初级纤毛在牙发育中的分布及相关信号通路的研究进展作一综述。文献复习结果表明,在牙发育过程中,初级纤毛在上皮与间充质的相互诱导中发挥重要作用,且随细胞不断增殖分化,初级纤毛的分布呈现出时间和空间依赖性。尽管此分布特征的原因尚不明确,但部分实验证据表明其与初级纤毛所分布的细胞与组织的功能相适应。初级纤毛在牙发育过程中主要参与调控Hedgehog和Wnt两种重要的信号通路,编码纤毛蛋白的基因(如Kif3a、Evc/Evc2和Ift等)可通过对这两种信号通路的调控来影响牙齿的发育,并且两种信号通路之间存在交互作用。相关基因(如Ofd1,Bbs等)的缺失也可通过损害纤毛的结构或功能破坏上下游信号的传导,引起多种类型的牙齿发育不良,包括小牙、釉质发育不全、牙齿缺失或颅面部畸形。     

Fibroblast growth factor signaling in mammalian tooth development

In this review, we discuss the central role of fibroblast growth factor (FGF) signaling in mammalian tooth development. The FGF family consists of 22 members, most of which bind to four different receptor tyrosine kinases, which in turn signal through a cascade of intracellular proteins. This signaling regulates a number of cellular processes, including proliferation, differentiation, cell adhesion and cell mobility. FGF signaling first becomes important in the presumptive dental epithelium at the initiation stage of tooth development, and subsequently, it controls the invagination of the dental epithelium into the underlying mesenchyme. Later, FGFs are critical in tooth shape formation and differentiation of ameloblasts and odontoblasts, as well as in the development and homeostasis of the stem cell niche that fuels the continuously growing mouse incisor. In addition, FGF signaling is critical in human teeth, as mutations in genes encoding FGF ligands or receptors result in several congenital syndromes characterized by alterations in tooth number, morphology or enamel structure. The parallel roles of FGF signaling in mouse and human tooth development demonstrate the conserved importance of FGF signaling in mammalian odontogenesis.     

阻遏蛋白β-TrCP在小鼠牙胚不同发育时期的表达与意义

目的观察Sonic hedgehog（shh）信号转导通路的阻遏蛋白β- TrCP在小鼠牙胚发育不同时期的表达情况，探讨β- TrCP在晚期牙胚发育中的作用与意义。方法取不同发育时期的鼠胚、乳鼠鼠头标本，用标记生物素链亲和素LsAB法观察β- TrCP蛋白在不同发育时期牙胚组织的表达情况。结果β- TrCP在胚胎10.5、13.5、14.5、16.5、18.5 d的小鼠牙胚上皮层与间充质层，以及出生后0、3、6 d的小鼠成釉细胞与成牙本质细胞胞浆呈特征性表达。结论β- TrCP在正常发育小鼠牙胚及成釉细胞与成牙本质细胞特征性表达，提示β- TrCP在牙胚发育中维持/限定shh信号通路的正常信号转导具有重要意义。     

Wnt signaling in the murine diastema

The correct number and shape of teeth are critical factors for an aesthetic and functional dentition. Understanding the molecular mechanisms regulating tooth number and shape are therefore important in orthodontics. Mice have only one incisor and three molars in each jaw quadrant that are divided by a tooth-less region, the diastema. Although mice lost teeth in the diastema during evolution, the remnants of the evolutionary lost teeth are observed as transient epithelial buds in the wild-type diastema during early stages of development. Shh and Fgf signaling pathways that are essential for tooth development have been shown to be repressed in the diastema. It remains unclear however how Wnt signaling, that is also required for tooth development, is regulated in the diastema. In this study we found that in the embryonic diastema, Wnt5a expression was observed in mesenchyme, whereas Wnt4 and Wnt10b were expressed in epithelium. The expression of Wnt6 and Wnt11 was found in both tissues. The Wnt co-receptor, Lrp6, was weakly expressed in the diastema overlapping with weak Lrp4 expression, a co-receptor that inhibits Wnt signaling. Secreted Wnt inihibitors Dkk1, Dkk2, and Dkk3 were also expressed in the diastema. Lrp4 mutant mice develop supernumerary teeth in the diastema that is accompanied by upregulation of Wnt signaling and Lrp6 expression. Wnt signaling is thus usually attenuated in the diastema by these secreted and membrane bound Wnt inhibitors.     

Opg, Rank, and Rankl in tooth development: co-ordination of odontogenesis and osteogenesis

Osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB (RANK), and RANK ligand (RANKL) are mediators of various cellular interactions, including bone metabolism. We analyzed expression of these three genes during murine odontogenesis from epithelial thickening to cytodifferentiation stages. Opg showed expression in the thickening and bud epithelium. Expression of Opg and Rank was observed in both the internal and the external enamel epithelium as well as in the dental papilla mesenchyme. Although Rankl expression was not detected in tooth epithelium or mesenchyme, it was expressed in pre-osteogenic mesenchymal cells close to developing tooth germs. All three genes were detected in developing dentary bone at P0. The addition of exogenous OPG to explant cultures of tooth primordia produced a delay in tooth development that resulted in reduced mineralization. We propose that the spatiotemporal expression of these molecules in early tooth and bone primordia cells has a role in co-ordinating bone and tooth development.     

Shh在大鼠牙胚发育过程中的表达

目的 :通过检测Shh(SonicHedgehog)在大鼠牙胚发育不同时期的表达 ,探讨其在牙齿发育过程中的作用。方法 :采用免疫组化方法 ,观察大鼠牙齿发育各阶段标本中Shh的表达。结果 :大鼠牙齿发育早期 ,Shh表达于口腔上皮和牙上皮 ;帽状期表达于成釉器 ;钟状期 ,成釉细胞和成牙本质细胞表达阳性。结论 :Shh在牙齿发育过程中的表达有时空特异性 ,提示它参与牙釉质和牙本质的形成     

Early stages of tooth morphogenesis in mouse analyzed by 3D reconstructions

Computer-aided 3D reconstructions were used to investigate early odontogenesis in the ICR mouse, from the dental lamina to the cap stage. The diastemal region of the maxilla was not an empty zone: five transient epithelial rudiments (D1–D5) were found between ED 12.5–13.5. Two further rudiments (R1 and R2) were observed between D5 and the maxillary first molar primordium, whose bud emerged at ED 13.5. These rudiments might be related to vestiges of ancestral teeth. During this period, only an epithelial lamina was observed in front of the bud-shaped molar epithelium in the cheek region of the mandible. Apoptosis plays an important role in the reduction of antemolar rudiments in the maxilla and in the remodeling of the epithelium anterior to the M1 bud and cap in both jaws: two successive waves of apoptosis were detected in the mandible and in the maxilla. Computer-aided 3D reconstructions clearly demonstrated that morphologically different developmental stages coexist along the anteroposterior axis of M1 in both jaws.     

The eternal tooth germ is formed at the apical end of continuously growing teeth

Rodent incisors are known to be continuously growing teeth that are maintained by both the cell-proliferation at the apical end and the attrition of the incisal edge. This type of tooth had a special epithelial structure for the maintenance of stem cells, showing the bulbous epithelial protrusion at the apical end. The morphological transition of the epithelial-mesenchymal compartment by serial transverse sections of the apical end toward the incisal direction is likely to reflect the development of the tooth germ in the prenatal stage. Based on the present histological and previous molecular biological studies, the special structure at the apical end is obviously different from the cervical loop giving rise to Hertwig's epithelial root sheath (HERS), in human, mouse and rat molar tooth germs. Hence, we propose a new concept that the eternal tooth bud producing various dental progeny is formed at the apical end of continuously growing teeth, and a new term "apical bud" for indicating this specialized epithelial structure. Furthermore, BrdU labelling analysis suggested that the guinea-pig molars, which were continuously growing teeth, also possessed plural specific proliferative regions and "apical bud" at the apical end.     

大鼠第一腮弓肾被膜下种植的成牙能力研究

目的:观察E12.5d大鼠第一腮弓肾被膜下的成牙能力。方法:切取E12.5d大鼠第一腮弓(下颌突),种植于肾被膜下,4周后收获种植的组织块,组织学观察种植的第一腮弓的发育情况。结果:种植4周,第一腮弓在肾被膜下继续发育,形成牙齿样结构,包含牙釉质、牙本质、牙髓、成牙本质细胞、上皮根鞘样结构及骨样组织。形成的组织结构与正常发育的牙齿形态结构相似。结论:E12.5d大鼠第一腮弓在肾被膜下可以模拟体内牙齿的发育过程,继续发育形成牙齿样结构。     

Development of heterodont dentition in house shrew (Suncus murinus)

Mammalian heterodont dentition comprises incisors, canines, premolars, and molars. Although there has been intensive research, the patterning of these specific tooth types has not yet been elucidated. In order for the gene expression data to be linked with tooth type determination, it is first necessary to determine precisely the incisor-, canine-, premolar-, and molar-forming regions in the jaw primordia. To accomplish this, we studied dentition development in the house shrew (Suncus murinus), which has retained all the tooth types, using three-dimensional reconstructions from serial histological sections and the Sonic hedgehog (Shh) expression patterns. Before the appearance of morphological signs of odontogenesis, Shh expression localized to the presumptive tooth-forming regions, in which the mesial and distal expression domains corresponded to the incisor- and premolar-forming regions, respectively. The upper incisor region was found to extend across the boundary between the frontonasal and the maxillary processes. The canine-forming regions later appeared in the intermediate portions of the maxillary and the mandibular processes. The molar-forming regions later appeared distal to the initially demarcated tooth-forming regions by secondary extension of the distal ends. The demarcation visualized by the Shh expression pattern in the jaw primordia of the house shrew probably represents the basic developmental pattern of mammalian heterodont dentition.