Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

Immunohistochemical localization of cyclic AMP during normal and abnormal chick and mouse limb development

This paper describes the immunohistochemical localization of cAMP during limb chondrogenesis in talpid3 chick, brachypod mouse, and normal embryos. Comparisons were made between chick wing buds at Stages 22, 25, and 30, and mouse hind limb buds at Days 11, 12.5 and 14. At Stage 22, the normal mesenchyme in the chick displayed areas of bright fluorescence compared to a lesser intense and more evenly distributed fluorescence in talpid3. Sections of the central region from normal Stage 25 limb buds exhibited an intense fluorescence that was uniformly distributed, whereas, in talpid3 staining was more mosaic with some areas fluorescing brightly and others showing little fluorescence. At Stage 30 the staining pattern was similar between normal and talpid3, with the fluorescence being brighter in the cartilage tissue than in the surrounding soft tissue. Difference in the staining patterns of normal and brachypod limb tissue were not detectable. At Days 11 and 12.5, tissue from both genotypes displayed a very bright, uniform fluorescence. In the 14-day hind limb buds, the staining patterns were comparable to those observed in Stage 30 chick wing buds. However, under in vitro conditions conducive for the expression of the chondrogenic phenotype, differences in the intensity and extensiveness of fluorescent staining were detectable in cultures derived from 12-day normal and brachypod hind limb mesenchyme. Compared to the control, the uneven distribution of immunofluorescence in the talpid3 limb buds and the differences in intensity and extensiveness of fluorescence in the brachypod cultures support the hypothesis that cAMP is involved in limb cartilage differentiation.     

An immunofluorescence study of chondrogenesis in murine mandibular ectomesenchyme

The temporal and spatial distribution of type I collagen, type II collagen, cartilage-specific proteoglycan (CSPG) and fibronectin in mouse mandible is described. CD-1 mouse embryos of 12-, 15-, and 18-day gestation were used, and matrix molecules were localized using indirect immunofluorescence. On day 12, accumulation of type II collagen, CSPG, and fibronectin within regions of condensed mesenchyme was noted. On day 15, intense staining for type II collagen and CSPG occurred. Fibronectin was less brilliant with its greatest concentration near the perichondrium. On day 18, the cartilage matrix was undergoing osseous replacement concurrent with loss of type II collagen and CSPG. Type I collagen was seen in the perichondrium, membranous bone and sub-basement membrane region in specimens of all ages. Synthesis and expression of extracellular matrix molecules reflect patterns of differentiation in mandibular mesenchyme.     

Affinity of intramitochondrial granules for ruthenium red accompanying induced cell death in chick embryos.

To test the hypothesis that ruthenium red binding of intramitochondrial granules might reflect an altered or pathological state of membranes associated with degeneration, embryos were treated with 6-AN to induce cell death in cartilaginous skeletons of chick embryos. Cervical cartilage from normal, 6-AN-treated and nicotinamide-alleviated 6-AN embryos was examined ultrastructurally for presence of IM RR-positive granules. Mitochondria of normal cervical chondroblasts which undergo normal phenotypic expression acquire RR-positive granules, although few mature cells are observed in young embryos. Necrotic chondroblasts, chondroblasts in various stages of degeneration, and proliferating chondrogenic cells of 6-AN-treated embryos all demonstrated induced RR-positive IM granules. Foci of degenerating chondroblasts, with mitochondria demonstrating RR granules, were observed infrequently in teratogen-alleviated tissue. The cytological features induced by 6-AN confirm its lethal effect and the degenerative effect on membranes presumably "unmasks" mitochondrial Ca-affinity sites which then become RR-positive. Cytochemical observations correspond with the biochemical and structural changes induced by 6-AN and confirm the hypothesis that RR-positive sites are the result of pathological changes.     

Cwc15家族同源基因mED1在小鼠早期胚胎发育中的表达模式(英文)

RNA选择性剪接机制涉及基因表达模式的多样性、胚胎发育的调控和疾病的发展与转归.Cwf15/Cwc15蛋白家族与RNA剪接体的功能相关,其基因序列在很多物种之间是十分保守的.然而,在哺乳类,Cwf15/Cwc15基因的表达模式和生物学功能研究至今未见实验性研究报道.本文首次报道了Cwc15家族同源基因mED1在小鼠早期胚胎发育过程中的表达规律.RT-PCR结果表明,mED1基因的转录水平从小鼠桑葚胚到器官形成期呈逐渐上升趋势;整体原位杂交结果显示mED1基因主要在小鼠6.5-dpc胚胎的ICM、8.5-dpc胚胎的神经褶衍生物和10.5-dpc的头部、鳃弓和体节中表达.说明mED1基因参与了小鼠胚胎的早期发育.此外,GFP-融合蛋白的亚细胞定位实验表明,mED1蛋白具有核定位的功能(剪接体蛋白的必要特性),验证了其核定位序列(NLS)的预测.本文是关于Cwc15家族基因的首次实验研究报道.     

增强子驱动的Cre转基因小鼠的构建及Cre基因的表达鉴定

目的:探索将增强子应用于构建Cre转基因小鼠品系,为以条件基因敲除为基础的基因功能研究提供更多的工具。方法:通过PCR方法从小鼠的细菌人工染色体扩增UH增强子片段,构建含有Hsp68基础启动子、增强子UH、Cre重组酶基因和SV40 polyA的转基因载体pLW400,将3.3 kb的转基因片段通过显微注射导入小鼠受精卵;为了检测Cre在转基因小鼠中的表达,将转基因一代小鼠与纯合子ROSA26报告小鼠(R/R)交配,收集第14 d胚胎期(E14)的舌组织进行LacZ染色检测鉴定。结果:经鉴定,31只子代小鼠中有6只携带外源基因,整合率为19.4%;与R/+对照相比,E14期的双基因型Cre,R/+舌组织为阳性结果(蓝色)。这表明Cre基因在转基因小鼠舌组织内得到表达,并在体内介导ROSA26基因座loxP位点间的重组,且有效删除了2个loxP之间的片段,从而启动了LacZ基因的表达。结论:构建了UH增强子-Hsp68Cre的转基因小鼠,在舌肌中特异表达Cre基因,提示增强子可以被选择应用于Cre转基因小鼠的构建;为舌肌的发育和再生研究奠定了基础。     

Antifungal triazole derivative triadimefon induces ectopic maxillary cartilage by altering the morphogenesis of the first branchial arch

BACKGROUND: The triazole derivative, triadimefon (FON), induces branchial arch abnormalities in post-implantation rat embryos cultured in vitro, and cranio-facial malformations in mouse fetuses. Ectopic maxillary cartilage has been also described as a typical FON-related malformation. This work studies the morphogenesis of the ectopic cartilage in rat embryos and fetuses exposed in vivo to FON during the early postimplantation period. METHODS: Pregnant rats were treated with 0, 250, and 500 mg/kg FON on Day 9.5 of pregnancy (D9.5) and sacrificed at term (D20), during the early fetal period (D17) or at different embryogenetic periods (D10, D11, D12). The skeleton was examined after stain of bone and cartilage or of cartilage alone respectively at term or at D17. The neural crest cell (NCC) migration and compaction was investigated at D10 and D11 and the cranial nerve organization described at D12. RESULTS: Triadimefon is teratogenic in rats under the chosen experimental conditions. The malformations were at the level of the cranio-facial and axial skeleton at term and of the hindbrain nerves in embryos. A NCC abnormal migration and compaction was observed at the level of the first branchial arch: in FON-exposed embryos NCC were detected at the level of both maxillary and mandibular processes, whereas control embryos showed the immunostained tissue only at the level of the mandibular bud. CONCLUSIONS: The pathogenic pathway, proposed to explain the ectopic cartilage, is the displacement of part of the NCC-derived tissues at the maxillary region of the first branchial arch.     

Visualization of whole-mount skeletal expression patterns of LacZ reporters using a tissue clearing protocol.

Gene targeting or trapping constructs that utilize the lacZ gene encoding beta-galactosidase activity to trap promoter expression have become an increasingly important way to disrupt gene function and monitor gene expression. A number of genes targeted in this way have revealed both expected and unexpected developmental abnormalities of the skeleton. The use of X-gal staining to monitor gene expression in developing skeletal structures is hampered in these mutants because, during the critical latter stages of mouse embryonic development, visualization is hindered by the opacity of overlying soft tissue. Here, we report the development of a reliable method to clear exogenous tissue in late-stage embryos and neonates that still preserves skeletal X-gal staining patterns. This protocol reveals (i) specific cell staining in localized regions of developing bone and cartilage in two different genetic models and (ii) that the intensity of X-gal staining is consistent with the level of expression of lacZ. We conclude that this protocol accurately reflects both the specificity and intensity of expression and will facilitate the analysis of mouse skeletal development.     

Members of the ErbB receptor tyrosine kinases are involved in germ cell development in fetal mouse gonads.

To isolate the genes involved in mouse primordial germ cell (PGC) development, we carried out subtraction cDNA cloning between PGC-derived embryonic germ (EG) cells and inner cell mass-derived embryonic stem cells. Among the genes preferentially expressed in EG cells, we found a gene encoding a receptor tyrosine kinase ErbB3. By in situ hybridization and immunohistochemical staining, the expression of ErbB3 as well as that of ErbB2, a coreceptor for ErbB3, was detected in PGCs in genital ridges at 12.5 dpc (days postcoitum). The expression was, however, downregulated at 14.5 dpc when the PGCs underwent growth cessation. Neuregulin-beta, a ligand for ErbB2 and ErbB3, was also expressed in genital ridges. In addition, a recombinant Neuregulin-beta enhanced the number of PGCs in 12.5-dpc embryos in culture. Taken together, these observations suggest that ErbB signaling controls the growth or survival of PGCs in genital ridges.     

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast- specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole- like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.     

The development of the conduction system in the mouse embryo heart: III. The development of sinus muscle and sinoatrial node

The origin and development of the sinus musculature, and the sinoatrial node (SAN), were studied in mouse embryo heart from the 8th day postcoitum (dpc) to the neonate. In the medial wall of the right common cardinal vein (RCCV), the muscle cells clearly derive from the splanchnic epithelium, whereas in the dorsolateral wall of the sinus horns, the loose mesenchymal cells appear to transform into the early sinus muscle. The early sinus muscle is particularly voluminous around the right venous valve (RVV). The 9-dpc heart shows regular contractions, but a morphologically definable SAN is not seen until 11 dpc, located in the medioanterior wall of the RCCV. There is indication that the loose mesenchymal cells play a role in the development of the nodal fibers. The SAN and the atrioventricular conduction system (AVCS) develop simultaneously in the 11-to 12-dpc mouse embryo heart. In the medioanterior wall of the left common cardinal vein (LCCV), a transient node-like structure was found. This, however, integrates into the left atrial wall in the 13-dpc and older embryos. Growth and early differentiation of the sinus muscle proceed distally during embryonic life to the point where it is indistinguishable from the atrial musculature.     

The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells

MicroRNAs (miRNA) are short RNA molecules regulating the expression of specific mRNAs. We investigated the expression pattern and potential targets of mouse miR-140 and found that miR-140 is specifically expressed in cartilage tissues of mouse embryos during both long and flat bone development. MiR-140 expression was detected in the limbs of E11.5 embryos in the primorida of future bones both in the fore and hindlimb and across autopod, zeugopod and stylopod. All digits of E14.5 fore- and hindlimbs showed accumulation of miR-140, except the first digit of the hindlimb. MiR-140 expression was also detected in the cartilagenous base of E17.5 skulls and in the sternum, the proximal rib heads and the developing vertebral column of E15.5 embryos. A potential target of miR-140, histone deacetylase 4, was validated experimentally and the possible role of miR-140 in long bone development is discussed.     

Nerve growth factor in skeletal tissues of the embryonic chick

Summary This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6–9 of incubation at 12 h intervals, and on days 10–18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24–36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.     

Immunohistochemical evidence of Muc1 expression during rat embryonic development

During embryonic development, studies on mouse and human embryos have established that Muc1/MUC1 expression coincides with the onset of epithelial sheet and glandular formation. This study aimed therefore at evaluating the temporal and spatial expression of Muc1 at different stages of rat development. In this experiment, 80 animals were included: 64 rat foetuses at 13, 14, 15, 16, 17, 18, 19 and 20 days of gestation from pregnant females (WKAH/Hok), 8 embryos each stage. Standard immunohistochemistry was performed using anti-MUC1 cytoplasmic tail polyclonal antibody (CT33). The reaction was considered positive when more than 5% of the cells were stained; reaction patterns were: L = linear, membrane, C = cytoplasmic and M = mixed; nuclear staining was also recorded. Intensity was graded as negative (-), low (+), moderate (++) and strong (+++). Muc1 expression was observed with a low intensity on 13th day (13 d) in the stomach, lung and kidney; at 14 d, small intestine and pancreas were also reactive; at 16 d, liver and esophagus and at 18 d, trachea and salivary glands. During the development, intensity increased while the pattern of expression changed: at the first days of gestation, it was predominantly linear and apical while during further development an increase in cytoplasmic expression was observed. Trachea, stomach, kidney and lung epithelia were the more reactive tissues. In specimens belonging to neonates and adults, all tissues analyzed showed similar Muc1 expression. The findings of this study assess that Muc1 is highly expressed in the epithelial rat embryonic development.     

Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone

We examined immunohistochemically the fracture repair process in rat tibial bone using antibodies to PCNA, BMP2, TGF-beta 1,-2,-3, TGF-beta R1,-R2, bFGF, bFGFR, PDGF, VEGF, and S-100. The peak level of cell proliferation as revealed by PCNA labelling appeared first in primitive mesenchymal cells and inflammatory cells at the fracture edges and neighboring periosteum at 2-days after fracture, followed by the peaks of periosteal primitive fibroblasts and chondroblasts, which appeared at fracture edges at 3- and 4-days after fracture, respectively. BMP2 was weakly positive in primitive mesenchymal cells, osteoblasts and chondroblasts. At 3-days post-fracture, periosteal osteoblasts produced osteoid tissue and callus with marrow spaces lined by osteoblasts and osteoclasts, and all primitive mesenchymal cells and osteoblasts were positive for TGF-beta 1,-2,-3, and TGF-beta R1,-R2. They were also positive for vascular growth factors bFGF, FGFR and PDGF, but negative for VEGF, and the peak of PCNA labelling of vascular endothelial cells in the marrow space was delayed to 4-days after fracture. Chondroblasts at fracture edges produced hypertrophic chondrocytes at 5-days after fracture and they were positive for TGF-beta 1,-2,-3, and TGF-beta R1,-R2. Primitive chondroblasts were positive for vascular growth factors VEGF as well as bFGF, FGFR, and the peak of PCNA labelling of vascular endothelial cells in the cartilage was at 5-days after fracture. Hypertrophic chondrocytes were also positive for these growth factors but negative for bFGF and bFGFR. S-100 protein-induced calcification was only positive on chondroblasts and hypertrophic chondrocytes. At 7-days after fracture, bone began to be formed from the cartilage at fracture edges, by a process similar to bone formation in the growth plate. Enchondral ossification established a bridge between both fracture edges and periosteal membranous ossification encompassed the fracture site like a sheath at 14 day after fracture. Our study of fracture repair of bone indicates that this process is complex and occurs through various steps involving various growth factors.     

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.