In situ expression of 15 kDa interferon alpha responsive gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) and E12.0 to make a profile of the regulator genes for odontogenesis. Fifteen kDa interferon alpha responsive gene (Ifrg15) is one of several highly-expressed genes in the E12.0 mandible. The current study examined the precise expression patterns of Ifrg15 mRNA in the mouse mandibular first molar by in situ hybridization to evaluate the possible functional roles of this gene in odontogenesis. Ifrg15 mRNA was expressed in the epithelial and mesenchymal tissues of the mandible at E10.5 and E12.0. The Ifrg15 in situ signal was detected in the epithelial bud and the surrounding mesenchyme at E14.0, and was present in the enamel organ including the primary enamel knot, and in the underlying mesenchyme at E15.0. The in situ signal was restricted in the inner and outer enamel epithelia and the stratum intermedium at E16.0. The signal of Ifrg15 mRNA was further restricted to the inner enamel epithelium and the adjacent stratum intermedium at E17.0 and E18.0. Consequently, the expression of Ifrg15 mRNA was localized in the ameloblasts and odontoblasts at postnatal days 1.0 to 3.0. However, the in situ signal was markedly weaker than at the embryonic period. The expression of Ifrg15 mRNA was coincidently observed in various craniofacial organs as well as in the tooth germ. These results suggest that Ifrg15 is closely related to odontogenesis, especially the differentiation of the ameloblasts and odontoblasts, and to the morphogenesis of the craniofacial organs.     

In situ expression of the mitochondrial ATPase6 gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0 and E13.0. Strong in situ signals were observed in the epithelium at E14.0, and in the enamel organ excluded the area of the primary enamel knot at E15.0. Atpase6 was strongly expressed in the inner enamel epithelium, the adjacent stratum intermedium, and the outer enamel epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.     

Role of Islet1 in the patterning of murine dentition

It is believed that mouse dentition is determined by a prepatterning of the oral epithelium into molar (proximal) and incisor (distal) regions. The LIM homeodomain protein Islet1 (ISL1) is involved in the regulation of differentiation of many cell types and organs. During odontogenesis, we find Islet1 to be exclusively expressed in epithelial cells of the developing incisors but not during molar development. Early expression of Islet1 in presumptive incisor epithelium is coincident with expression of Bmp4, which acts to induce Msx1 expression in the underlying mesenchyme. To define the role of ISL1 in the acquisition of incisor shape, we have analysed regulation of Islet1 expression in mandibular explants. Local application of bone morphogenetic protein 4 (BMP4) in the epithelium of molar territories either by bead implantation or by electroporation stimulated Islet1 expression. Inhibition of BMP signalling with Noggin resulted in a loss of Islet1 expression. Inhibition of Islet1 in distal epithelium resulted in a loss of Bmp4 expression and a corresponding loss of Msx1 expression, indicating that a positive regulatory loop exists between ISL1 and BMP4 in distal epithelium. Ectopic expression of Islet1 in proximal epithelium produces a loss of Barx1 expression in the mesenchyme and resulted in inhibition of molar tooth development. Using epithelial/mesenchymal recombinations we show that at E10.5 Islet1 expression is independent of the underlying mesenchyme whereas at E12.5 when tooth shape specification has passed to the mesenchyme, Islet1 expression requires distal (presumptive incisor) mesenchyme. Islet1 thus plays an important role in regulating distal gene expression during jaw and tooth development.     

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud,but Dispensable for Kidney Development

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice.     

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.     

The keratocan gene is expressed in both ocular and non-ocular tissues during early chick development.

Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein keratocan gene (Kera) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger-Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that keratocan, or keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.     

Treatment with all-trans-retinoic acid decreases levels of endogenous TGF-beta(1) in the mesenchyme of the developing mouse inner ear

Background: Previous studies have shown that in utero exposure of the mouse embryo to high doses of all-trans-retinoic acid (atRA) produces defects of the developing inner ear and its surrounding cartilaginous capsule, while exposure of cultured periotic mesenchyme plus otic epithelium to high doses of exogenous atRA results in an inhibition of otic capsule chondrogenesis. Methods: In this study, we examine the effects of atRA exposure on the endogenous expression of transforming growth factor-beta(1) (TGF-beta(1)), a signaling molecule that mediates the epithelial-mesenchymal interactions that guide the development of the capsule of the inner ear. Results: Our results demonstrate a marked reduction in immunostaining for TGF-beta(1) in the periotic mesenchyme of atRA-exposed embryos of age E10.5 and E12 days in comparison with control specimens. Consistent with these in vivo findings, high-density cultures of E10.5 periotic mesenchyme plus otic epithelium, treated with doses of atRA that suppress chondrogenesis, showed significantly decreased levels of TGF-beta(1), as compared with TGF-beta(1) levels in untreated control cultures. Furthermore, we demonstrate a rescue of cultured periotic mesenchyme plus otic epithelium from atRA-induced chondrogenic suppression by supplementation of cultures with excess TGF-beta(1). Conclusions: Our results support the hypothesis that TGF-beta(1) plays a role in mechanisms of atRA teratogenicity during inner ear development.     

Eya1 is required for the morphogenesis of mammalian thymus,parathyroid and thyroid

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1(-/-) embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1(-/-) mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1(-/-) embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1(-/-) embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1(-/-) embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.     

Expression of Tcf/Lef and sFrp and localization of beta-catenin in the developing mouse lung.

Recent evidence that Wnts and other genes in the Wnt signaling pathway are expressed in embryonic and adult mouse lung suggests that this pathway is important for cell fate decisions and differentiation of lung cell types. We therefore examined the expression and protein distribution of several Wnt pathway components during prenatal mouse lung development using whole-mount in situ hybridization and immunohistochemistry. Between embryonic days 10.5 and 17.5 (E10.5-E17.5), beta-catenin was localized in the cytoplasm, and often also the nucleus, of the undifferentiated primordial epithelium (PE), differentiating alveolar epithelium (AE; present from E14.5 onward), and adjacent mesenchyme. Tcf1, Lef1, Tcf3, Tcf4, sFrp1, sFrp2 and sFrp4 were also expressed in the PE, AE, and adjacent mesenchyme in specific spatio-temporal patterns.     

In situ expression of ribosomal protein L21 in developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to investigate the key regulator genes in odontogenesis. Ribosomal protein L21 (Rpl21) is one of differentially expressed genes in the E12.0 mandible. This study examined the precise expression pattern of Rpl21 mRNA in the mouse mandibular first molar by in situ hybridization. Rpl21 mRNA was expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0. Strong in situ signals were observed in the epithelial bud at E14.0, and in the enamel organ at E15.0. However, either no (E14.0) or only a weak (E15.0) in situ signal was found in the primary enamel knot at these gestational days. Rpl21 was strongly expressed in the inner enamel epithelium, cervical loop and dental lamina from E16.0 to E18.0. In addition, Rpl21 mRNA was also demonstrated in various developing cranio-facial organs. These results suggest that Rpl21 participates in the synthesis of various polypeptides which might be related to the initiation and the development of such tooth germ, and also in the synthesis of enamel components in the presecretory stage of the ameloblast. Rpl21 for protein synthesis might also be related to the morphogenesis of the developing cranio-facial organs.     

AP-1在腋臭患者发病过程中的作用

目的：腋臭是美容整形外科的常见病,目前发病机制尚不明确,已证实人体大汗腺中的载脂蛋白D（ApoD）在腋臭患者大汗腺中高表达,并且与腋臭的发生密切相关。探明ApoD在大汗腺细胞中的信号转导通路,可以进一步明确其在腋臭发病过程中的作用机制。JNK信号转导通路与多种疾病的发生有关。课题组前期已经做了JNKl对ApoD调控作用的相关研究,证明了在腋臭发病过程中JNK1是通过调控ApoD的转录来上调ApoD的表达。本实验在课题组前期研究基础上,探讨JNKl下游转录因子AP-1是否在JNKl上调ApoD通路中发挥作用。方法：取腋臭志愿者腋区皮肤组织,进行汗腺细胞培养。把汗腺细胞分为5．二氢睾酮处理组、5-二氢睾酮联合姜黄素处理组和空白对照3个组,用姜黄素抑制AP-1的活性,通过Real．timePCR实验方法检测ApoD在姜黄素抑制下的表达变化。结果：在姜黄素的抑制下,ApoD表达明显降低。在体外培养汗腺细胞加入5．二氢睾酮联合姜黄素处理后,ApoD的表达量在mRNA水平低于单独的5-二氢睾酮处理组和正常对照组（P〈0．05）。结论：姜黄素抑制了AP．1的活化导致ApoD的表达降低。在腋臭的发病过程中,JNKl的下游转录因子AP-1对ApoD有明显的上调作用。AP-1可能在JNKl上调ApoD这条通路中扮演了很重要的角色,它可能是JNK1和ApoD的中间转录因子。     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.