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.     

Shh反义核酸对体外培养小鼠牙胚发育的影响

目的 用Shh反义寡脱氧核苷酸（ODN）抑制体外培养小鼠牙胚中Shh的翻译，观察该抑制反应对牙胚生长及矿化的影响。方法 培养牙胚取自胎龄为E17．5d昆明小鼠下颌第一磨牙，利用简易Trowell支架，建立小鼠牙胚体外培养模型，实验共分为三组：反义ODN组；正义ODN组及空白对照组，核酸浓度为30μmol/L。体外分别培养10d后，进行HE染色、von Kossa染色。结果 蛋白印迹结果显示所应用的反义核酸阻断了牙胚中Shh的表达。HE、von Kossa染色结果显示：培养10d，S-ODN组与空白对照牙胚进入钟状晚期，细胞分化，并可见分泌的基质带；钙磷酸盐在成牙本质细胞与成釉细胞间的基质中沉积。AS-ODN组牙胚培养10d，牙胚进入钟状期，但牙胚体积明显变小，牙尖形态发育不良，成牙本质细胞排列紊乱，但仍可见高柱状分泌期细胞；von Kossa染色未见明显钙磷酸盐沉积。结论 Shh反义核酸减缓体外培养牙胚的发育，可能通过对成牙本质细胞分泌功能的影响而影响牙胚的矿化。     

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.     

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。     

Shh、Ptch1及Ptch2基因在小鼠磨牙发育过程中的表达

目的通过原位杂交的方法研究Sonic Hedgehog（Shh）、Patched1（Ptch1）及Patched2（Ptch2）在小鼠下颌磨牙发育过程中的表达，以探讨该信号通路在牙胚发育中的作用。方法制备小鼠下颌第一磨牙发育各期标本，原位杂交检测Shh、Ptch1及Ptch2的表达部位及强度。结果Shh在帽状期表达于釉结，钟状早期表达于内釉上皮，钟状晚期表达于成釉细胞；Ptch1在帽状期表达于牙囊、外釉上皮及星网状层，钟状早期表达于牙囊、外釉上皮及牙乳头，钟状晚期表达于成牙本质细胞及牙乳头；Ptch2在帽状期表达于釉结，钟状早期表达于内釉上皮，钟状晚期无表达。结论Shh信号系统在牙胚发育各期有各自的表达特点，提示可能在牙胚形态的调控，成釉细胞、成牙本质细胞分化的诱导中起重要作用。     

Sonic hedgehog regulates epithelial proliferation and cell survival in the developing tooth germ.

Shh expression is highly restricted to the future sites of tooth development during the initiation of odontogenesis. This suggests a role for Shh as a proliferative factor, as localized epithelial thickenings invaginate to form a tooth bud. We have investigated this role by blocking Shh signaling between E10.5 and E12.5 in murine mandibular processes using a 5E1 blocking antibody and the PKA activator Forskolin. This results in down-regulation of Ptc, a principle target of Shh signaling. The effects of inhibition varied with developmental time. At E10.5, tooth development was arrested as epithelial thickenings and the numbers of teeth developing were considerably reduced. Inhibition at E12.5 produced localized apoptosis in the epithelium at the tip of the tooth buds, although some teeth were able to develop. Thus, Shh has dual roles in early odontogenesis, first in bud formation by stimulating epithelial proliferation, and second in the development of cap-stage tooth germs by increasing epithelial cell survival.     

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.     

牙齿形状调控相关基因Wnt6的克隆

目的 :获得新生猪牙胚的差异表达基因。方法 :利用消减杂交方法获得新生猪前后恒牙胚的差异表达基因 ,并通过反向点杂交排除假阳性 ,尔后进行测序定性分析。结果 :通过消减杂交获得Wnt6基因。结论 :Wnt6可能在决定牙齿形状发育的分子调控网络中起重要作用     

Tooth and jaw: molecular mechanisms of patterning in the first branchial arch

The mammalian jaw apparatus is ultimately derived from the first branchial arch derivatives, the maxillary and mandibular processes, and composed of a highly specialised group of structures. Principle amongst these are the skeletal components of the mandible and maxilla and the teeth of the mature dentition. Integral to the development of these structures are signalling interactions between the stomodeal ectoderm and underlying neural crest-derived ectomesenchymal cells that populate this region. Recent evidence suggests that in the early mouse embryo, regionally restricted expression of homeobox-containing genes, such as members of the Dlx, Lhx and Gsc classes, are responsible for generating early polarity in the first branchial arch and establishing the molecular foundations for patterning of the skeletal elements. Teeth also develop on the first branchial arch and are derived from both ectoderm and the underlying ectomesenchyme. Reciprocal signalling interactions between these cell populations also control the odontogenic developmental programme, from early patterning of the future dental axis to the initiation of tooth development at specific sites within the ectoderm. In particular, members of the Fibroblast growth factor (Fgf), Bmp, Hedgehog and Wnt families of signalling molecules induce regionally restricted expression of downstream target genes in the odontogenic ectomesenchyme. Finally, the processes of morphogenesis and cellular differentiation ultimately generate a tooth of specific class. Many of the same genetic interactions that are involved in early tooth development mediate these effects through the activity of localised signalling centres within the developing tooth germ.     

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     

Patterning of Molar Tooth Roots in Mammals

Tooth morphogenesis is regulated by reciprocal interactions between the dental epithelium and odontogenic mesenchyme. As tooth roots are fundamental structures of the tooth support system, the morphology and functions of the roots are very important. However, basic information on the morphology of tooth root patterning and the molecular mechanism of root morphogenesis is largely unavailable. Following tooth crown formation, the dental epithelium forms a double-layered Hertwig's epithelial root sheath (HERS) derived from inner and outer enamel epithelium. Previous studies have reported that HERS plays an important role in tooth root development. Here, we report the correlation between the number of major cusps of the tooth crown and number of tooth roots of first molar and last premolar teeth in several extant mammals. We also discuss the molecular mechanism of tooth root patterning by introducing studies of mouse mutants and human syndromes associated with an abnormal molar morphology.     

Analysis of dental anomalies in the Siberian mole, Talpa altaica (Insectivora, Talpidae)

We re-examined tooth variation in specimens of the Siberian mole, Talpa altaica, from the collection of the Siberian Zoological Museum and discuss the mechanisms of dental evolution. The number of teeth counted in 1789 specimens ranged from 34 to 47, and supernumerary, absent, and connate teeth were observed. The most frequent tooth anomaly was an absent tooth in the premolar region (200 maxillary first premolars and 190 mandibular third premolars), which does not support Fujita and Kirino's terminal reduction hypothesis in the mandible [Fujita T, Kirino T. Ha No Kaibougaku. 21st ed. Tokyo: Kanehara Publishers Inc.; 1976 (in Japanese)]. Supernumerary teeth were found in premolar rows and in the incisor and molar regions. An maxillary fourth molar, positioned distal to the normal third molar, was thought to result from a genetically programmed atavistic event during the natal stages. Connate teeth were observed only in the premolar rows and were thought to have developed with the fusion of two independent tooth germs. Connate premolars appeared to result from an expression of an incomplete division of tooth germ at an early developmental stage or a reunion of independent tooth germs, based on the morphological similarity of the normal and supernumerary premolars. These extraordinarily frequent tooth anomalies of T. altaica are of much interest both in terms of tooth development and classification.     

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.     

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。     

A new, accurate and fast digital method to predict unerupted tooth size

The sum of the lower incisor tooth width has been proposed as the best predictor for calculating unerupted canine and premolar mesiodistal tooth sizes. The aims of this study were to develop a new, fast, and accurate computerized method to predict unerupted mesiodistal tooth sizes and to determine which reference tooth or combination of reference teeth was the best predictor for canines and premolars in a Spanish sample. The dental casts of 100 Spanish adolescents with permanent dentition were measured to the nearest 0.05 mm with a two-dimensional computerized system. The goal was to predict unerupted canine and premolar mesiodistal tooth sizes using the sizes of the upper central incisor, upper and lower first molar, or a combination of these as a reference and using a specific mesiodistal tooth-size table. The results showed that the Digital Method proposed was very accurate in predicting unerupted canine and premolar tooth size. The combination of the sums of the permanent upper central incisor and the lower first molar was the best predictor for canines and premolars in this sample. Upper arch teeth were better predicted than lower arch teeth. The upper lateral incisor provided the worst predictions.     

Pax6基因纯合突变胎鼠上前牙区额外牙发生的初步研究

研究Pax6基因纯合突变胎鼠上前牙区额外牙的发生情况,为研究Pax6在上前牙发生、发育中的作用及额外牙的发生机制提供依据.方法收集E18.5 DEBA Pax6基因纯合突变胎鼠20只,制作胎鼠头部组织冠状面石蜡连续切片,HE染色,观察上前牙区额外牙的发生情况;E18.5 C57BL/6NCrlBR Pax6基因野生型胎鼠18只作为对照.结果 20只Pax6基因纯合突变胎鼠中,4只上前牙区显示有额外牙牙胚;18只Pax6基因野生型胎鼠上前牙区均无额外牙牙胚.Pax6基因纯合突变胎鼠和Pax6基因野生型胎鼠的下前牙区和上下颌第一、第二磨牙区牙胚的数目相同,形态结构未见明显差别.结论本实验结果提示Pax6基因在胎鼠上前牙发生过程中有非常重要的作用.     

An experimental study of timing and topography of early tooth development in the mouse embryo with an analysis of the role of innervation

The putative involvement of the innervation in determining the sites of dental development was investigated by intra-ocular homografting and organ culture methods. Mandibular arches were dissected from 9 and 10-day-old (E9-E10) mouse embryos and grafted to the anterior eye chambers of homologous adult mice for 12-14 days. There was no significant difference in the incidence of tooth formation between grafts in which the trigeminal ganglion was included (n = 82, 68 per cent) or excluded (n = 72, 65 per cent). A parallel in vitro study in which E9 and E10 mouse embryo mandibular arches were cultured in the absence of trigeminal innervation showed that definitive tooth germs were formed during the 7-9 day culture period. It is concluded that innervation plays no part in determination of tooth development. In further experiments with E9 and E10 material, the complete mandibular arch, the hemimandibular arch and the ventral midline region of the mandibular arch (including the median epithelial isthmus) were each grafted for a period of 21 days. Alizarin-red whole-mount staining of recovered grafts revealed that bone had been deposited and mineralized in the majority of grafts of all types. Incisor and molar teeth with near normal crown shapes developed in grafts of complete mandibular arches. Hemimandibular arches gave rise almost exclusively to molars. Grafts of the E10 ventral midline region gave rise exclusively to incisors, but the same graft performed at E9 did not produce teeth. It is concluded that incisor primordia are localized in or near the median epithelial isthmus (and are thus destroyed or damaged when the mandibular arch is hemisected) and that incisors are not determined until E10. At E9 the odontogenic neural crest has yet to complete its ventrad migration into proximity with presumptive incisor epithelium. By this time, however, odontogenic crest and presumptive molar epithelium have already reached juxtaposition and molar primordia are fully competent. In grafts of the frontonasal and maxillary processes made at E10, prior to merging of their respective mesenchymes, frontonasal processes gave rise exclusively to incisors, whereas maxillary processes gave rise exclusively to molars.     

82对双生子牙齿发育和萌出指标的遗传学分析

目的 对牙齿发育指标和牙齿萌出指标进行遗传学分析。方法 收集６－１２岁女性双生子８２对,通过ＤＮＡ指纹图进行卵型诊断。通过口腔检查和全口曲面断层片记录第三磨牙存在数、先天缺牙、上颌侧切牙畸形和萌出恒牙数、估算其遗传度。结果 所测指标依 由大到小依次为第三磨牙存在数、上颌侧切牙畸形、先天缺牙、萌出恒牙数。结论牙齿发育指标受遗传控制强于牙齿萌出指标。