Molecular and cellular analysis of embryonic avian tongue development

Signalling cascades first described in Drosophila have been found to regulate patterning and outgrowth in a number of structures in higher vertebrates. We sought to determine whether the evolutionarily conserved genes were important during the development of the tongue. In situ hybridisation was used to determine the temporo-spatial expression of a panel of conserved genes. Histological examination and incorporation of BrdU were used to determine the mechanism by which the tongue develops. We show that evolutionarily conserved genes were expressed in distinct dynamic patterns during tongue development. Sonic Hedgehog (Shh) and Patched (Ptc) were found only in the dorsal tongue epithelium. Shh expression was only observed in the suprabasal layers, whereas Ptc was observed in both basal and suprabasal layers. Cell division in the epithelium was concentrated in regions devoid of Shh. Expression of bone morphogenetic protein-7 (BMP) was identical to that of Shh. Shh and Ptc expression were never detected in the mesenchyme. Ectopic expression of Noggin (a potent antagonist of the BMPs) caused severe abnormalities in tongue morphology, including swelling of the mesenchymal component and a thickening of the epithelial layer. Data from this study suggests that the epithelium and mesenchyme express quite different genes during development. However BMP activity acts to inhibit growth in both tissues. Accepted: 22 May 2001     

Sonic Hedgehog及其受体Patched在小鼠视交叉发育过程中的表达

在小鼠胚胎发育过程中,胚胎第13d(E13)至15d(E15)是视交叉发育的主要阶段。在本研究中,我们观察了在E13～E15,Sonic Hedgehog(Shh)及其受体Patched(Ptc)在视觉传导通路的表达。结果发现:在视交叉和视束中,Shh在视神经纤维接近中线时表达上调,越过中线后表达下调,并且主要表达在较深的区域。Ptc在E13～E14的视网膜和E14～E15的视束中有表达,但在视交叉中无表达。Ptc,而不是Shh,表达在体外培养的生长锥中。Shh和Ptc在视觉传导通路发育中的表达提示Shh可能在引导视神经生长方面发挥一定作用。     

Expression and Regulation of Hedgehog-Interacting Protein During Early Tooth Development

Sonic hedgehog ( Shh ) expression is highly localized to the epithelium at the future sites of tooth development. This restricted expression suggests that inhibition of Shh in areas where teeth do not form may be an important mechanism in tooth germ localization. Recently, Hip, a putative vertebrate antagonist of Shh, has been identified. We have investigated the expression of Hip during early tooth development and found it not to be localized in cells immediately adjacent to Shh -expressing cells, but rather at a distance, separated by cells expressing Ptc1 . Hip is also regulated by Shh in the first branchial arch. Shh-soaked agarose beads are able to induce the expression of Hip in odontogenic mesenchyme. A role for Hip might be to prevent the spread of excess Shh ligand beyond an immediate Ptc1-induced zone in odontogenic mesenchyme. This mechanism would therefore restrict Shh signaling specifically to those regions along the oral axis that are destined to form teeth.     

Expression and regulation of hedgehog-interacting protein during early tooth development

Sonic hedgehog (Shh) expression is highly localized to the epithelium at the future sites of tooth development. This restricted expression suggests that inhibition of Shh in areas where teeth do not form may be an important mechanism in tooth germ localization. Recently, Hip, a putative vertebrate antagonist of Shh, has been identified. We have investigated the expression of Hip during early tooth development and found it not to be localized in cells immediately adjacent to Shh-expressing cells, but rather at a distance, separated by cells expressing Ptc1. Hip is also regulated by Shh in the first branchial arch. Shh-soaked agarose beads are able to induce the expression of Hip in odontogenic mesenchyme. A role for Hip might be to prevent the spread of excess Shh ligand beyond an immediate Ptc1-induced zone in odontogenic mesenchyme. This mechanism would therefore restrict Shh signaling specifically to those regions along the oral axis that are destined to form teeth.     

Sonic hedgehog促1型糖尿病小鼠的伤口愈合

目的：研究sonic hedgehog（Shh）及其受体Ptc1在糖尿病小鼠伤口愈合中的作用。方法：分别在正常和链脲佐菌素（STZ）诱导的糖尿病小鼠建立皮肤损伤模型,Western印迹检测Shh和Ptc1的蛋白水平;观察外源性Shh或Ptc1抑制剂cyclopamine对伤口愈合的影响。结果：（1）正常小鼠损伤后皮肤组织中的Shh和Ptcl蛋白质表达明显升高;外源性Shh对伤口愈合无明显促进作用,但cyclopamine可以明显地抑制伤口愈合;（2）STZ诱导的糖尿病小鼠,其皮肤组织中内源性的Shh和Ptcl蛋白水平明显下调;（3）外源性Shh可显著促进糖尿病小鼠伤口的愈合,且呈浓度依赖性;Cyclopamine则明显地抑制糖尿病小鼠的伤口愈合。结论：Shh-Ptc1通路参与了皮肤伤口愈合,糖尿病伤口愈合延迟与Shh-Ptc1表达下调有关。     

音猬因子与胰岛素增强子结合蛋白在小鼠胚胎呼吸系统的早期表达

目的 探讨音猬因子信号通路成员与胰岛素增强子结合蛋白在呼吸系统的早期表达与其发育的联系。方法 胚龄9.0～13d小鼠胚胎呼吸系统各年龄段不小于3个,连续石蜡切片,用抗音猬因子(Shh)、抗patched1(Ptcl)、抗smoothened(Smo)、抗胰岛素增强子结合蛋白(ISL1)和抗α-平滑肌肌动蛋白(α-SMA)抗体进行免疫组织化学染色。结果 胚龄10d,Shh表达在前肠内胚层腹侧壁。胚龄11～12d,Ptc1表达在气管、支气管和肺内分支上皮。胚龄13d,Ptc1只表达在肺内分支上皮。胚龄9d,ISL1表达在前肠内胚层腹侧壁。胚龄10～12d,ISL1表达在前肠（气管）腹侧壁和支气管侧壁上皮及邻近的间充质内。胚龄13d,ISL1表达减弱,始终未见在肺内有阳性表达。胎龄12～13d,与气管后壁、支气管内侧壁上皮Ptc1表达减弱处相邻的间充质出现α-SMA阳性平滑肌细胞,其与对侧间充质ISL1阳性细胞的分布呈背-腹侧或内-外侧模式。气管腹侧及肺芽外侧间充质中可见ISL1与Smo共表达细胞。 结论 ISL1在气管及肺芽的发育中可能与Shh信号系统协同发挥作用。     

Developmentally regulated expression of Shh and Ihh in the developing mouse cranial base: comparison with Sox9 expression

The cranial base, located between the cranial vault and the facial bones, plays an important role in integrated craniofacial development and growth. Transgenic Shh and Sox9-deficient mice show similar defects in cranial base patterning. Therefore, in order to examine potential interactions of Shh, Ihh, another member of the Hh family, and Sox9 during cranial base development and growth, we investigated their cellular mRNA expression domains in the embryonic (E) and early postnatal (PN) cranial base from E10 to PN5 using sectional radioactive 35-S in situ hybridization. Of the Hhs, Shh was observed in the foregut epithelium and the notochord, while Sox9 showed broad expression in the loose mesenchyme of the cranial base area during E10-E11. Subsequently, from E12 onward, all genes were observed in the developing cranial base, and after birth the genes were prominently colocalized in the prehypertrophic chondrocytes of the synchondroses. Collectively, these data suggest that Hh-Sox9 auto- and paracrine signaling interactions may provide a critical mechanism for regulating the patterning of the cranial base as well as for its development and growth.     

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.     

BMP4 modulates fibroblast growth factor-mediated induction of proximal and distal lung differentiation in mouse embryonic tracheal epithelium in mesenchyme-free culture.

Lung morphogenesis and differentiation require interaction between the epithelium and mesenchyme, which is mediated by diffusible molecules such as fibroblast growth factors (FGFs), bone morphogenetic protein 4 (BMP4), and Shh. We have used mesenchyme-free culture to study the effects of these molecules on lung epithelial differentiation. We have tested the individual abilities of FGF1, FGF2, FGF7, FGF9, FGF10, and FGF18, as well as BMP4 and Shh to promote growth and specify distal lung differentiation in mouse tracheal epithelium. The different FGFs exhibited distinct abilities to induce epithelial growth and the expression of the distal lung epithelial marker, surfactant protein C (SP-C), although all FGFs were able to induce expression of BMP4. Tracheal epithelium treated with FGF10 showed little growth and failed to express SP-C as measured by whole-mount in situ hybridization and quantitative real-time polymerase chain reaction. FGF1 treatment resulted in the strongest induction of SP-C. Treatment with BMP4 inhibited epithelial growth and differentiation and antagonized the stimulatory effects of FGF1. In contrast, inhibition of endogenous BMP4 signaling with Noggin protein did not inhibit growth or expression of SP-C but did increase the expression of the proximal lung markers CCSP and HFH4. Expression of Shh was not affected by any of the conditions tested. These results suggest that BMP4 does not signal epithelial cells to adopt a distal fate but may regulate the expansion of proximal epithelial cells in the lung.     

Monoaminergic regulation of Sonic hedgehog signaling cascade expression in the adult rat hippocampus

Monoamines are implicated in the modulation of adult hippocampal neurogenesis in depression models and following chronic antidepressant treatment. Given the key role of Sonic hedgehog (Shh) in adult neurogenesis, we examined whether monoaminergic perturbations regulate the expression of Shh or its co-receptors Smoothened (Smo) and Patched (Ptc). Combined depletion of both serotonin and norepinephrine with para-chlorophenylalanine (PCPA) resulted in a significant decrease in Smo and Ptc mRNA within the dentate gyrus subfield of the hippocampus. However, selective depletion of serotonin, using the serotonergic neurotoxin 5,7-dihyrdroxytryptamine (5,7-DHT), or norepinephrine, using the noradrenergic neurotoxin DSP-4, did not alter expression of Shh and its co-receptors, Smo and Ptc. Acute treatment with the monoamine releasing agent, para-chloroamphetamine (PCA) significantly upregulated Smo mRNA within the dentate gyrus. However, acute or chronic treatment with pharmacological antidepressants that modulate monoaminergic neurotransmission did not regulate Shh cascade expression. These results indicate that robust changes in monoamine levels can regulate the expression of the Shh signaling cascade in the adult rodent brain.     

Telomerase activity detected in oral lichen planus by RNA in situ hybridisation: not a marker for malignant transformation

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory condition. Clinically, it is characterised by the presence of a white lace-like lesion on the buccal mucosa, tongue, and gingivae, with erosions and ulceration. The World Health Organisation considers OLP to be a premalignant condition. AIMS: To investigate expression of the telomerase RNA component (hTR) in OLP compared with normal control buccal mucosa and to assess the possibility of using hTR expression as a marker for malignant transformation in OLP. METHODS: hTR expression was analysed in 40 cases of OLP and 18 normal control buccal mucosa samples using an RNA in situ hybridisation approach. RESULTS: Strong hTR RNA expression was seen in the basal, suprabasal, and to a lesser extent in the upper epithelial layers in 36 of the 40 OLP lesions examined. Infiltrating subepithelial lymphocytes in OLP were also shown to express hTR RNA. Weak hTR RNA expression was seen in seven of the 18 normal control buccal mucosa specimens, with expression confined exclusively to the basal layer of the epithelium and absent in the suprabasal and upper layers. CONCLUSION: The telomerase RNA component hTR is found to be highly expressed in the epithelium of non-dysplastic OLP lesions. It is possible that this high expression is related to the increased cellular proliferation seen in OLP lesions rather than being an indicator of susceptibility to malignancy. Thus, hTR RNA expression may not be a suitable marker for predicting malignant transformation in OLP.     

Spatiotemporal expression of BMP7 in the development of anorectal malformations in fetal rats

The aim of this study was to determine the expression patterns of bone morphogenetic protein 7 (BMP7) during anorectal development in normal rat embryos and in embryos with anorectal malformations (ARM), and to investigate the possible role of BMP7 in the pathogenesis of ARM. ARM was induced by treating rat embryos with ethylenethiourea on the 10th gestational day (GD10). Embryos were harvested by Cesarean delivery and the spatiotemporal expression of BMP7 was evaluated in normal (n=168) and ARM embryos (n=171) from GD13 to GD16 using immunohistochemistry staining and western blot analysis. Immunohistochemical staining in normal embryos revealed that BMP7 was abundantly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13, and BMP7-immunopositive cells were extensively detected in the URS, hindgut, and cloacal membrane by GD14. Increased positive tissue staining was noted on the fused tissue of the URS and the thin anal membrane on GD15. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negatively or only faintly immunostained for BMP7. BMP7 protein expression showed time-dependent changes in the developing hindgut according to western blotting, and reached a peak on GD15 during anus formation. BMP7 expression levels from GD14 to GD15 were significantly lower in the ARM group compared with the normal group (P<0.05). Spatiotemporal expression of BMP7 was disrupted in ARM embryos during anorectal morphogenesis from GD13 to GD16. These results suggest that downregulation of BMP7 at the time of cloacal separation into the primitive rectum and UGS might be related to the development of ARM.     

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development.

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian-specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers.