Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

Analysis of limb patterning in BMP-7-deficient mice

Bone morphogenetic proteins `BMPs' are polypeptide signaling molecules, belonging to the TGF-β superfamily. They were originally identified by their ability to induce ectopic bone formation, but their expression patterns in embryos suggest multiple functions. BMP-7-deficient mice show among other mesodermal and skeletal patterning defects, polydactyly in the hindlimbs `Luo G, Hofmann C, Bronckers ALJJ, Sohocki M, Bradley A, Karsenty G `1995': Genes Dev 9:2808-2820; Dudley AT, Lyons KM, Robertson EJ `1995': Genes Dev 9:2795-2807'. Here we report a more detailed analysis of the limb phenotype in BMP-7-deficient mice using in situ hybridization to monitor expression of molecules implicated in patterning processes of the developing vertebrate limb. In previous studies we showed that Sonic hedgehog (Shh) was expressed normally, but Hoxd-13 expression in limb mesenchyme was lower in BMP-7 mutant limbs. Here we show that Hoxd-11 expression domains are also contracted and decreased in intensity in mutant limbs, suggesting that 5′ genes of the Hoxd cluster are coordinately downregulated, while another Bmp, Bmp-2, which can be activated by Shh, is similarly expressed. The mutant limb buds are broader than normal buds, and fibroblast growth factor Fgf-8 is expressed throughout the extended ridge. However, expression of the homeobox gene Msx-1, which has been shown to be involved in epithelial-mesenchymal interactions during limb development, was decreased in the mesenchyme of BMP-7 mutant limbs. Taken together, our data suggest that BMP-7 is involved in regulating proliferation and/or epithelial-mesenchymal interactions in the developing limb. © 1996 Wiley-Liss Inc.     

Immunohistochemical study on a macrophage calcium-type lectin in mouse embryos: transient expression in chondroblasts during endochondral ossification

We investigated expression of mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin (MMGL) in mouse embryos using a rat monoclonal antibody (mAb) LOM-14 that we previously developed. Immunoblot analysis revealed that a significant expression of MMGL was first detected in detergent extracts of whole embryos of 11 days post coitus (dpc) and the level of its expression increased during further fetal development (examined up to 18-dpc embryos). Tissue sections of 12, 14, 16, and 18-dpc embryos, newborn and adult mice were investigated by immunohistochemical staining. In embryos of 12-dpc and later stages, mesenchymal cells (typically distributed in the embryonic skin) exhibited positive signals for MMGL. Interestingly, a conspicuous staining was observed during endochondral ossification in temporary cartilage tissue, in which chondroblasts were transiently positive for MMGL. The staining intensity for the chondroblasts peaked in 14-dpc embryos and then gradually decreased. The staining was diminished while hypertrophy and maturation of chondrocytes proceeded, and was eliminated in areas with calcification. Immunoelectron microscopic study demonstrated the presence of MMGL in rough endoplasmic reticulum in the chondroblasts in the temporary cartilage tissue in 14-dpc embryos. These results provide first evidence showing the expression of MMGL in cells other than macrophages. © 1998 Rapid Science Ltd     

Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting-out experiment in vitro

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting-out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo-distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting-out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated 'positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.     

Retinoic acid enhances and depresses in vitro development of cartilaginous bone anlagen in embryonic mouse limbs

Summary Forelimbs of Day 11 and Day 12 embryonic mice were excised and cultured for 3 d in the presence of either 0.25 μg (8×10⁻⁷ M), 0.5 μg(1.7×10⁻⁶ M), or 1.0 μg (3.3×10⁻⁶ M) of all-rans retinoic acid (RA) per milliliter of culture medium. Cultured limbs were fixed, stained, and mounted whole on glass slides and evaluated with computerized optical image analysis for RA-induced effects on the area and shape of the total limb and individual bone anlagen. Relative effects of RA on total bone, soft tissue, long bone, and paw regions were also examined. With Day 11 forelimbs total bone area was increased by 10.5% by the low dose of RA. The increase was mostly in long bones and at the expense of soft tissue. Total bone area was increased 9.3% with Day 12 forelimbs. This increase was primarily in the paw. The high dose of RA decreased Day 11 forelimb area, primarily affecting long bones. Day 12 forelimbs were not significantly affected by the high dose of RA. Effects of the imtermediate dose were primarily limited to reduction in soft tissue area. Long bone:paw and soft tissue: bone ratios reflected these effects. The high dose produced a consistent rounding or shortening of Day 11 forelimb bones. On Day 12 0.5 μg/ml RA produced an inconsistent pattern of rounding of bone anlagen. Treatment with the high dose on Day 12 produced angular rather than rounded contours in many cases, as indicated by shape factor values closer to zero than obtained with controls. These data show that direct exposure to RA can affect both the size and shape of bone anlagen of the developing limb; the low dose enhances and the high dose depresses development. The results support previous studies which suggest that RA may play a critical role in the control of cell activities such as cell migration, proliferation, and cytodifferentiation in the development of the cartilaginous bone anlagen.     

Differential Expression of c-myc and N-myc during Oral Organogenesis of the Mouse Embryo

The expressions of the c- and N- myc proto-oncogenes during oral development of midgestational mouse embryos were examined by in situ hybridization in order to analyze their roles. In the mandibular rudiment, c- myc RNA was strongly expressed in the mesenchymal condensation around the ossification center in which high-level expression of 2 ar (osteopontin) was detected. In tooth germs, c- myc was strongly expressed in the epithelia at the bud stage, and its expression gradually became restricted to the inner enamel epithelia from the cap to bell stages. In contrast, N- myc expression was detected in the undifferentiated mesenchymal cells of the dental papilla. Incorporation of BrdU was examined immunohistochemically to study the relationship between the expressions of c- and N- myc and cell proliferation. Unexpectedly, the distribution of BrdU labelled regions was not coincident with the expressions of c- and N- myc . These results suggest that the level of myc expression is not necessarily related to cell proliferation.     

Ypel3基因在小鼠胚胎发育中的表达与调控

目的:探讨小鼠胚胎发育过程中Ypel3基因的时空特异性表达与调控,为后续功能研究奠定基础。方法:选取胎龄(E)10.5、12.5、14.5、16.5和18.5 d的小鼠胚胎,利用荧光定量RT-PCR技术研究Ypel3基因mRNA的时序性动态表达谱;采用原位杂交技术观察Ypel3基因mRNA在胚胎发育E11.5和E15.5的空间表达谱;应用定量RT-PCR技术检测表观遗传学修饰对Ypel3基因mRNA表达丰度的影响。结果:定量RT-PCR表明该基因从胚胎发育的早中期开始表达,到出生前表达量呈逐渐升高趋势;原位杂交显示E11.5信号出现在脑和心脏中,E15.5信号在脑、舌、心、肺、胸腺、肝、肾等主要脏器中均有表达;甲基化转移酶抑制剂5-氮胞苷(5-Aza)处理的Neuro-2a(N2a)细胞中,Ypel3的表达水平未产生显著变化,而去乙酰化酶抑制剂4-苯丁酸(4-PBA)处理后该基因表达显著升高,5-Aza和4-PBA联合处理后表达水平进一步升高。结论:Ypel3基因在小鼠胚胎发育各阶段有广泛的表达,提示其具有重要作用,且该基因的表达可能受到组蛋白乙酰化的调控。     

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.     

Cloning,expression, and localization of a new member of a Paracentrotus lividus cell surface multigene family

We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16-cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso-macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed. © 1996 Wiley-Liss, Inc.     

Structure and site of expression of a murine type II hair keratin

We present here a 1770 bp-long cDNA which encodes a murine type II keratin. Sequence comparisons of the keratin with those of various type II keratins expressed in mouse epidermis and internal stratified epithelia reveal that the new keratin is unrelated to epithelial keratins. Rather the structural organization of its amino- and carboxyterminal domains and the high content of cysteine and proline residues in these regions suggest that the keratin represents a murine type II hair keratin. This assumption was confirmed by in situ hybridization which localized the mRNA of the keratin in upper cells of the hair cortex and in suprabasal cells of the central core unit of filiform papillae of the tongue. Hybrid selection analyses revealed that the keratin has a molecular weight of 58 kD. It remains to be seen whether the keratin corresponds to MHb 3 or MHb 4.     

Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis

Spermatogenesis is a complex process. Duringspermatogenesis, the production of sperm occurs withinthe testicular seminiferous tubules through three separatedphases. First of all, diploid germ cells, primitivespermatogonia, will self renew to amplify and producetypes A and B spermatogonia. Type B spermatogonia willdifferentiate into primary spermatocytes. Then, meioticdivisions of spermatocytes will produce round spermatids.Finally, after a series of biochemical and morphologicalchanges, sper…     

红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致     

斑马鱼整体原位杂交法研究MMP15a的发育相关表达的初步观察

克隆斑马鱼基质金属蛋白酶15a（MMP15a）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP15a RNA探针,采用全胚胎原位杂交方法研究MMP15a基因在胚胎斑马鱼的表达。结果MMP15a基因在胚胎受精后一个细胞时期就开始表达,从受精后24h起,在眼睛处表达明显,从受精后48h MMP15a在胸鳍和耳囊有特异性表达至到受精后96h。MMP15a在斑马鱼胚胎发育不同时期表达明显,且在胸鳍和耳囊处有持续表达。