低氧诱导因子-1在小鼠胚胎神经胚形成阶段的发育学表达

为研究低氧诱导因子-1(HIF-1)在小鼠胚胎神经胚形成阶段的时空表达规律,探讨HIF-1在胚胎神经系统发生和发育过程中的作用。本研究以地高辛标记的HIF-1αcRNA为探针,采用全胚胎原位杂交技术,观察HIF-1α在神经胚形成阶段的表达规律。结果显示在E8.5d,整个鼠胚神经管的头端和尾端均可观察到HIF-1αmRNA的表达,此时表达较弱。在E9.5d,随着神经管的逐渐关闭,HIF-1αmRNA在前脑泡、第一鳃弓、第二鳃弓、后脑泡处急剧增多,并在发育中的眼部有较强的表达;此外在中脑泡以及心脏原基有较弱的表达。到E10.5d,阳性信号除在E9.5d检测到的部位继续富集外,在端脑、间脑、发育中的肢芽以及尾部也出现较强的HIF-1αmRNA的表达,且在心脏原基的表达亦进一步增强。E11.5d时在连合板、喙区、第一鳃弓、第二鳃弓、后脑、延脑、发育中的肢芽以及尾部末端可检测到HIF-1αmRNA的明显表达。以上结果提示HIF-1可能参与了小鼠神经胚形成的发育过程。     

FGF10及其受体FGFR2 Ⅲb在小鼠肾脏发育中的表达

 摘要：目的 观察FGF10及其受体FGFR2 Ⅲb在小鼠肾脏发生发育中的表达和分布,探讨它们在肾脏发生发育中的作用。方法 选取胚龄12、14、16、18 d和生后1、7、14、21、42 d小鼠肾组织,用免疫组织化学和蛋白印迹技术（Western blot）,检测FGF10和FGFR2 Ⅲb的表达。结果 E12 d FGF10及FGFR2Ⅲb开始在输尿管芽微弱表达;随着肾脏发育成熟,FGF10及FGFR2Ⅲb主要表达于远端小管和集合管;FGF10在近曲小管表达,近直小管无表达;FGFR2Ⅲb在近端小管无阳性表达;生后肾组织及发育各阶段肾小体未见FGF10及FGFR2Ⅲb表达。Western blot显示随着胚（日）龄的增加,FGF10及FGFR2Ⅲb在肾脏的表达量先增后减。结论 FGF10和FGFR2Ⅲb在肾脏发生发育过程中发挥重要作用。     

移行上皮反应基因在小鼠主要器官发育过程中的表达

目的:研究移行上皮反应基因(TERE1)在小鼠胚胎着床后至新生儿断乳期主要器官中的正常表达规律.方法:于E11 d～E18 d、P0 d、P7 d、P14 d、P21 d,取得小鼠胚胎脑、眼、心、肺、胃、肝、肢、肾(肾自E13 d开始),利用实时荧光定量PCR方法检测TERE1基因在各器官的表达丰度.结果:除仅在P21 d肢未检出外,TERE1基因在小鼠主要器官的发育过程中均有表达,且在脑、眼、心、肺、胃、肝、肾的表达水平较高;该基因的表达除在小鼠肾发育过程中呈升高趋势外,在脑、眼、心、肺、胃、肝、肢的表达均呈波动性.结论:TERE1基因在小鼠器官发育过程中呈广泛表达,且表达丰度不同,表达模式也不同.     

Expression of FGF10 and FGFR2Ⅲb in development of mouse kindey

目的 观察FGF10及其受体FGFR2Ⅲb在小鼠肾脏发生发育中的表达和分布,探讨其在肾脏发生发育中的作用.方法 选取胚龄12、14、16及18 d和出生后1、7、14、21及42 d小鼠肾组织,用免疫组织化学和Western blot,检测FGF10和FGFR2Ⅲb的表达.结果 胚龄12 d组FGF10及FGFR2Ⅲb开始在输尿管芽微弱表达;随着肾脏发育成熟,FGF10及FGFR2Ⅲb主要表达于远端小管和集合管;FGF10在近曲小管表达,近直小管无表达;FGFR 2Ⅲb在近端小管无阳性表达;生后肾组织及发育各阶段肾小体未见FGF10及FGFR2Ⅲb表达.Western blot显示随着胚(日)龄的增加,FGF10及FGFR2Ⅲb在肾脏的表达量先增后减.结论 FGF10和FGFR2Ⅲb在肾脏发生发育过程中发挥重要作用.     

软骨发育不全的产前基因诊断

目的 通过产前成纤维细胞生长因子受体3(fibroblast growth factor receptor 3,FGFR3)基因检测确诊胎儿软骨发育不全.方法 经脐带穿刺获取78例短肢发育异常胎儿脐血,常规行核型分析,并提取基因组DNA,扩增FGFR3基因第10外显子,应用限制酶Bfm I进行限制性片段长度多态性分析,并对其行DNA双向测序.同法分析阳性胎儿双亲FGFR3基因第10外显子.结果 短肢发育异常78例胎儿中,8例为G1138A杂合突变,诊断为软骨发育不全,其核型分析正常.余70例短肢发育异常胎儿,FGFR3基因第10外显子第1138位核苷酸检测结果正常,排除软骨发育不全.8例患病胎儿双亲中,1例父亲同为G1138A杂合突变,余检测结果正常.结论 对超声诊断的胎儿短肢发育异常,通过脐血FGFR3基因第10外显子的PCR-限制性片段长度多态分析和DNA双向测序,产前能明确诊断软骨发育不全.     

内皮型一氧化氮合酶和神经型一氧化氮合酶在胚胎小鼠肾发育过程中的表达

目的:观察内皮型一氧化氮合酶(eNOS)和神经型一氧化氮合酶(nNOS)在胚胎小鼠肾发育过程中的时空性表达,探讨它们在小鼠发育早期肾组织中的作用.方法:选取胚龄(E)13、14、15、16、18d及新生组(P)0d小鼠,应用免疫组织化学方法显色进行图像分析;用免疫印迹法测定各组小鼠肾组织中的eNOS以及nNOS的含量.结果:免疫组织化学显色显示,eNOS阳性表达最早出现于E14d的生肾区;E16d时在生肾区表达减弱,近端小管呈强阳性;P0d时远端小管可见弱表达,集合管也有一定程度表达,致密斑无表达.nNOS在E13d时即表达于皮质输尿管芽;E16d时近端小管弱表达,远端小管强表达;P0d时在远端小管和致密斑强表达,近端小管弱表达,集合管无表达.图像分析和免疫印迹法结果显示,eNOS和nNOS在E14d含量较少,随后逐渐增多,至P0d达到高峰.结论:在胚胎小鼠肾发育过程中,eNOS和nNOS的表达有一定的时空顺序,它们对肾组织的发育及形成起一定的作用.     

小鼠胚胎心脏发育期T型钙离子通道的表达

目的 检测哺乳动物心脏中T型钙离子通道α1亚基在小鼠胚胎心脏发育过程中表达部位和时间的分布特征。 方法 对小鼠胚胎心脏发育关键时期（Ｅ9.5至胚胎E18.5共9d）的胚胎进行连续切片,用RT-PCR和免疫组织化学的方法对α1G和α1H蛋白进行阳性检测。 结果 α1H从胚胎期10.5d左右开始表达并一直延续到发育晚期（E18.5）,而α1G则从E11.5d延续到E18.5;免疫组织化学的结果显示,T型钙离子通道主要在心肌层以及左右传导束支稳定表达,而在心内膜垫以及心室肌小梁的心内膜内皮和间充质细胞中不表达。 结论 T型钙离子通道对于心肌细胞以及心脏传导组织的形成起到了一定的作用。     

NDRG2在小鼠胚胎脑组织的表达特点

目的:观察NDRG2 mRNA在小鼠胚胎脑组织的表达与分布。方法:采用胚胎期小鼠E10.5 d、E12.5 d、E16.5d和E18.5 d进行脑切片的原位杂交。结果:NDRG2 m RNA在小鼠胚胎发育各期均有持续性表达,主要定位于端脑、中脑、菱脑、间脑、眼原基、脊髓、小脑原基、海马原基、丘脑、下丘脑和许多神经核团,神经细胞定位主要集中在细胞质中。结论:脑内NDRG2作用贯穿了整个脑的发育阶段,提示该基因在小鼠胚胎组织发育过程中有重要作用。     

肝细胞核因子6在小鼠肝内胆管发育过程中的表达

目的 探讨肝细胞核因子6(HNF6)在肝内胆管发育过程中的作用. 方法 用RT-PCR及免疫组织化学等方法 检测HNF6在小鼠胚胎发育的各个阶段以及成年肝中的表达. 结果 RT-PCR结果 显示,HNF6 mRNA的表达在E9d开始出现,与肝芽形成的时间吻合,E13d HNF6 mRNA的表达消失,E15d又重新出现,并一直维持到出生.免疫组织化学显示,CK19免疫反应在E13d开始出现,此时免疫反应阳性细胞在肝索内散在分布.E15d时在近肝门处的门管区丌始出现由单层的、CK19阳性细胞组成的胆管板,之后,阳性细胞反应主要分布于胆管板和小叶间胆管.E9～11d,多数肝索细胞呈HNF6阳性反应,E13d的肝索中未观察剑HNF6的表达.E15～17d,HNF6阳性反应的分布与CK19相似,成年小鼠肝的胆管上皮细胞仍呈HNF6阳性反应. 结论 HNF6可能与肝干细胞的特化关系不大,而与肝发育的启动、肝干细胞向胆管上皮细胞的分化及其分化状态的维持有关.     

组蛋白乙酰化酶p300和CREB结合蛋白在小鼠胚胎心发育中的时序表达

目的:探讨p300和CREB结合蛋白(CBP)在小鼠胚胎心发育过程中的时序表达规律.方法:选取胎龄7.5～18 d、出生1 d、3月小鼠正常心,采用免疫组织化学法观察p300和CBP在小鼠胎心发育中的表达分布及变化规律.结果:p300在胚胎心发育各个时期及出生1 d和3月成年鼠的心各部分均有很强表达.CBP在E7.5不表达;在E8.5～E9.5的心管中较强表达;E10.5～E16.5及E18和生后1 d心肌较强表达,房室瓣和小梁网较弱表达,室间隔肌部弱表达而膜部几乎不表达;3月成年鼠心肌和小梁较弱表达,室间隔极弱表达.结论:p300对发育早期的生心细胞诱导特化和中期的室间隔形成作用较CBP更大.     

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation.     

FGFs,their receptors,and human limb malformations: clinical and molecular correlations

Fibroblast growth factors (FGFs) comprise a family of 22 distinct proteins with pleiotropic signaling functions in development and homeostasis. These functions are mediated principally by four fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase family, with heparin glycosaminoglycan as an important cofactor. Developmental studies in chick and mouse highlight the critical role of FGF-receptor signaling in multiple phases of limb development, including the positioning of the limb buds, the maintenance of limb bud outgrowth, the detailed patterning of the limb elements, and the growth of the long bones. Corroborating these important roles, mutations of two members of the FGFR family (FGFR1 and FGFR2) are associated with human disorders of limb patterning; in addition, mutations of FGFR3 and FGF23 affect growth of the limb bones. Analysis of FGFR2 mutations in particular reveals a complex pattern of genotype/phenotype correlation, which will be reviewed in detail. Circumstantial evidence suggests that the more severe patterning abnormalities are mediated by illegitimate paracrine signaling in the mesoderm, mediated by FGF10 or by a related FGF, and this is beginning to gain some experimental support. A further test of this hypothesis is provided by a unique family segregating two FGFR2 mutations in cis (S252L; A315S), in which severe syndactyly occurs in the absence of the craniosynostosis that typically accompanies FGFR2 mutations.     

Distribution of type I collagen, type II collagen and PNA binding glycoconjugates during chondrogenesis of three distinct embryonic cartilages

Summary Previous studies of chondrogenesis have been focused on limb bud cartilage, whereas little is known about chondrogenic processes of other cartilages with different developmental fates. We hypothesize that cartilages with various developmental fates might show identical characteristics of chondrogenesis. The chondrogenic processes in the nasal septum, the mandible, and the limb bud of the mouse were examined by means of PNA-binding glycoconjugate, and types I and II collagen expression. Swiss-Webster mouse embryos of 11 days (E11) to 14 days (E14) gestation were fixed and processed for imniuno- and lectin histochemistry. The blastema of mesenchymal cell aggregates stained positively with anti-type I collagen, but very weakly with anti-type II collagen in all three models at E12, whereas PNA bound to the blastema in the limb bud but not in nasal septum or mandible. Types I and II collagens coexisted in cartilages at E13. Type II collagen was predominant in E14; type I collagen was confined to the peripheral region. The synchronized transitional expression of the collagen phenotypes in all three embryonic cartilages may be systemically regulated. The presence or absence of the PNA-binding glycoconjugates may be involved in characterizing the nature of the cartilages.     

FGFs,their receptors,and human limb malformations: Clinical and molecular correlations

Fibroblast growth factors (FGFs) comprise a family of 22 distinct proteins with pleiotropic signaling functions in development and homeostasis. These functions are mediated principally by four fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase family, with heparin glycosaminoglycan as an important cofactor. Developmental studies in chick and mouse highlight the critical role of FGF‐receptor signaling in multiple phases of limb development, including the positioning of the limb buds, the maintenance of limb bud outgrowth, the detailed patterning of the limb elements, and the growth of the long bones. Corroborating these important roles, mutations of two members of the FGFR family (FGFR1 and FGFR2) are associated with human disorders of limb patterning; in addition, mutations of FGFR3 and FGF23 affect growth of the limb bones. Analysis of FGFR2 mutations in particular reveals a complex pattern of genotype/phenotype correlation, which will be reviewed in detail. Circumstantial evidence suggests that the more severe patterning abnormalities are mediated by illegitimate paracrine signaling in the mesoderm, mediated by FGF10 or by a related FGF, and this is beginning to gain some experimental support. A further test of this hypothesis is provided by a unique family segregating two FGFR2 mutations in cis (S252L; A315S), in which severe syndactyly occurs in the absence of the craniosynostosis that typically accompanies FGFR2 mutations. © 2002 Wiley‐Liss, Inc.     

In situ hybridization and immunohistochemistry of versican, aggrecan and link protein, and histochemistry of hyaluronan in the developing mouse limb bud cartilage

We investigated the expression pattern of versican, aggrecan, link protein and hyaluronan in the developing limb bud cartilage of the fetal mouse using in situ hybridization and/or immunohistochemistry. Versican mRNA and immunostaining were detected in the mesenchymal cell condensation of the future digital bone at E13. Versican mRNA expression rapidly disappeared from the tibial cartilage, as cartilage formation progressed during E13-15, but the immunostaining was gradually replaced by aggrecan immunostaining from the diaphysis. Immunostaining for both molecules thus had a 'nega-posi' pattern and consequently versican immunostaining was still detected at the epiphyseal end at E15. This result indicated that versican functions as a temporary framework in newly formed cartilage matrix. An aggrecan-positive region within the cartilage invariably had intense hyaluronan staining, whereas a versican-positive region also had affinity for hyaluronan within the cartilage, but not in the mesenchymal cell condensation. Therefore, the presence of versican aggregates was not confirmed in the developing limb bud cartilage. Furthermore, although link protein was more closely related with aggrecan than versican during limb bud cartilage formation, there was a discrepancy between the expression of aggrecan and link protein in tibial cartilage at E15. In particular, only a link protein-positive region was present in the marginal area of the metaphysis and the epiphysis at this stage. This finding may indicate a novel role for link protein.     

A point mutation in the pre-ZRS disrupts sonic hedgehog expression in the limb bud and results in triphalangeal thumb–polysyndactyly syndrome

PurposeThe zone of polarizing activity regulatory sequence (ZRS) is an enhancer that regulates sonic hedgehog during embryonic limb development. Recently, mutations in a noncoding evolutionary conserved sequence 500 bp upstream of the ZRS, termed the pre-ZRS (pZRS), have been associated with polydactyly in dogs and humans. Here, we report the first case of triphalangeal thumb–polysyndactyly syndrome (TPT-PS) to be associated with mutations in this region and show via mouse enhancer assays how this mutation leads to ectopic expression throughout the developing limb bud.MethodsWe used linkage analysis, whole-exome sequencing, Sanger sequencing, fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, single-nucleotide polymorphism array, and a mouse transgenic enhancer assay.ResultsTen members of a TPT-PS family were included in this study. The mutation was linked to chromosome 7q36 (LOD score 3.0). No aberrations in the ZRS could be identified. A point mutation in the pZRS (chr7:156585476G>C; GRCh37/hg19) was detected in all affected family members. Functional characterization using a mouse transgenic enhancer essay showed extended ectopic expression dispersed throughout the entire limb bud (E11.5).ConclusionOur work describes the first mutation in the pZRS to be associated with TPT-PS and provides functional evidence that this mutation leads to ectopic expression of this enhancer within the developing limb.