摘要:

AbstractAn amputated limb of an adult urodele amphibian is capable of undergoing regeneration. The new structures form from an undifferentiated mass of cells called the regenerative blastema. The cells of the blastema are believed to derive from differentiated tissues of the adult limb. However, the exact source of these cells and the process by which they undergo dedifferentiation are poorly understood. In order to elucidate the molecular and cellular basis for dedifferentiation we isolated a number of genes which are potential regulators of the process. These includeMsx‐1, which is believed to support the undifferentiated and proliferative state of cells in the embryonic limb bud; and two members of the myogenic regulatory gene family,MRF‐4andMyf‐5, which are expressed in differentiated muscle and regulate muscle‐specific gene activity. As anticipated, we find thatMsx‐1is strongly up‐regulated during the initiation of regeneration. It remains expressed throughout regeneration but is not found in the fully regenerated limb. The myogenic geneMRF‐4has the reverse expression pattern. It is expressed in adult limb muscle, is rapidly shut off in early regenerative blastemas, and is only reexpressed at the completion of regeneration. These kinetics are paralleled by those of a musclespecificMyosingene. In contrastMyf‐5, a second member of the myogenic gene family, continues to be expressed throughout the regenerative process. Thus,MRF‐4andMyf‐5are likely to play distinct roles during regeneration.MRF‐4may directly regulate muscle phenotype and as such its repression may be a key event in dedifferentiation.Myf‐5may play a role in maintaining a distinct myogenic lineage during regeneration.

ISSN:1058-8388    

DOI:10.1002/aja.1002020102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractCalvaria from day‐14 calcium‐deficient chick embryos produced by long‐term maintenance in shell‐less culture, exhibit a cartilage‐like phenotype (Jacenko and Tuan [1986] Dev. Biol. 115:215–232), which is restored to an osteogenic phenotype upon calcium repletion to the embryo. The expression of cartilage markers in a typically osteogenic tissue under calcium deficiency implies the presence of chondrogenic cells, and questions the conditions associated with calcium deficiency which may cause their divergent pathway of differentiation. In the present study, by explanting normal and shell‐less embryonic calvarial pairs in organ culture in vitro and experimentally regulating their calcium supply, the calcium status of the calvaria was modulated as a function of medium calcium. Histological and immunoblotting analyses demonstrated for the first time that calvaria possess cells which can form genuine cartilage. This chondrogenic potential is expressed only in a low‐calcium environment, where cartilage forms in both normal and shell‐less calvarial pairs; their calcium‐suplemented counterparts, however, develop as fully osteogenic tissues. Furthermore, chondrogenesis in both normal and shell‐less calvaria indicates that the chondrogenic cells must be endogenous constituents of the calvaria, rather than being derived elsewhere in response to systemic calcium deficiency. Finally, the correlation between matrix under‐calcification and cartilage expression in the embryonic calvarium suggests that calcium, perhaps in the form of matrix mineral, may modulate cell differentiation during skeletogenesis.

ISSN:1058-8388    

DOI:10.1002/aja.1002020103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractChick embryos cultured in the absence of their eggshell are rendered severely calcium‐deficient, and develop a cartilage‐like phenotype in the calvarium, a normally osteogenic tissue. In the preceding paper (Jacenko and Tuan [1995] Dev. Dyn. 202:13–26), experiments using organ cultured calvaria from day‐12 normal and shell‐less embryos showed that depletion of calcium alone may be responsible in promoting chondrogenic differentiation in calvaria. Here these findings were extended using an in vivo calvarial grafting technique, such that the extent of calvarial matrix calcification was a function of the calcium status of both the graft and the host. In these calvarial grafts, undermineralized regions again were shown to support chondrogenesis. To identify possible mechanisms which promote chondrogenesis in the calvaria, cells were enzymatically dissociated from the calvaria and cultured in media with varied levels of soluble calcium, under conditions which should modulate cell‐to‐cell interactions, including monolayer, micromass, agarose gels, and suspension cultures. Soluble calcium had no effect on calvarial cell differentiation, whereas conditions which enhanced cell‐cell interactions, e.g., suspension culture, elicited cartilage expression. Based on these findings, we propose that the calcified matrix of the calvarium is repressive to chondrogenesis during normal development, but that the lack of mineral in a calcium‐deficient calvarium creates a microenvironment permissive for cell‐to‐cell interactions which lead to chondrogenic differentiation.

ISSN:1058-8388    

DOI:10.1002/aja.1002020104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe genes for the α1(IX), α1(IX), α1(II), and α2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promoter usage [for α(IX) and α2(I)] or alternative splicing [for α1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the “housekeeping” enzyme, glycerol‐3‐phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of α1(IX) collagen (termed “long IX”) were predominant at stage 18–20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of α1(IX) was from the downstream promotor (termed “short IX”). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino‐terminal globular domain similar to that found in collagen types I, III, and V. For type I, the “bone/tendon” mRNA isoform of the α2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of α1(II). The downstream transcript of the α2(I) gene (Icart) was found at high levels only i

ISSN:1058-8388    

DOI:10.1002/aja.1002020105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractRetinoids influence both morphogenetic events and differentiation during development of the vertebrate limb. These effects are mediated through nuclear retinoid receptors, which modulate target gene expression. We report here the cloning and characterization of three promoter‐ and splicing‐variants of the retinoic acid receptor‐β (RAR‐β) from chick. These receptor isoforms are independently expressed during limb development. RARβ2 but not RARβ1 transcripts are enriched three‐fold in the posterior limb bud, reflecting the increased RA concentrations in this region. RARβ1 transcripts are initially present throughout the limb bud mesenchyme and ectoderm, then become restricted within perichondrial regions and loose connective tissue of the limb. RARβ1 expression closely overlaps that of NCAM (neural cell adhesion molecule) and tenascin in non‐neuronal tissues. RARβ2 transcripts are present within a subset of those limb tissues which express RARβ1. In the early limb bud RARβ2 transcripts are detected in proximal limb mesenchyme and in the initial mesenchymal condensate. In older limbs RARβ2 mRNAs are abundant in cells lateral to the digit cartilage. Neither RARβ1 nor RARβ2 transcripts are associated specifically with regions of limb cell death. The differential expression and regulation of RARβ isoforms suggests these variants may have different roles in limb development

ISSN:1058-8388    

DOI:10.1002/aja.1002020106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe apical ectodermal ridge (AER) promotes the proliferation and directed outgrowth of the subridge mesodermal cells of the developing limb bud, while suppressing their differentiation. Insulin‐like growth factor‐I (IGF‐I) and its receptor are expressed by the subridge mesodermal cells of the chick limb bud growing out in response to the AER, and specific insulin receptors are present in the limb bud during its outgrowth. To study the possible roles of IGF‐I and insulin in limb outgrowth, we have examined their effects on the morphogenesis of posterior and anterior portions of the distal tip of stage 25 embryonic chick wing buds subjected to organ culture in serum‐free medium in the presence or absence of the AER and limb ectoderm. The distal mesoderm of control posterior explants lacking an AER or all limb ectoderm ceases expressing IGF‐I mRNA, exhibits little or no proliferation, fails to undergo outgrowth, and rapidly differentiates. Exogenous IGF‐I and insulin promote the outgrowth and proliferation and suppress the differentiation of distal mesodermal cells in posterior explants lacking an AER or limb ectoderm, thus mimicking at least to some extent the outgrowth promoting and antidifferentiative effects normally elicited on the subridge mesoderm by the AER. Furthermore, IGF‐I and insulin‐treated posterior explants exhibit high IGF‐I mRNA expression, indicating that IGF‐I and insulin maintain the expression of endogenous IGF‐I by the subridge mesoderm. We have also found IGF‐I and insulin can affect the morphology and activity of the AER. When the posterior portion of the wing bud tip is cultured with the AER intact in control medium, on day 4–5 the AER flattens, ceases expressing high amounts of the AER‐characteristic homeobox‐containing geneMsx2, and concomitantly an elongated cartilaginous element differentiates in the subridge mesoderm. In contrast, in the presence of exogenous IGF‐I or insulin the AER of such explants does not flatten, continues expressing high amounts ofMsx2, and the subridge mesoderm remains undifferentiated and proliferative. Thus, exogenous IGF‐I and insulin maintain the thickness of the AER and sustain its expression ofMsx2, while sustaining the anti‐differentiative effect normally elicited on the subridge mesoderm by a thickned functional AER. Notably, we have also found that exogenous IGF‐I and insulin induce the formation of a thickened ridge‐like structure that expresses high amounts ofMsx2from the normally thin distal anterior ectoderm of the limb bud, while promoting dramatic outgrowth and proliferation of the anterior mesoderm, which normally undergoes little outgrowth or proliferation. These studies provide support for the hypothesis that endogenous IGF‐I and insulin may be involved in promoting the outgrowth and suppressing the differentiation of limb mesoderm in response to the AER, and also in regulating and/or maintaining at least some aspe

ISSN:1058-8388    

DOI:10.1002/aja.1002020107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractIt has been shown that mirrorimage duplications of the zeugopodia and digits are formed when MRC‐5 fibroblasts producing hepatocyte growth factor (HGF) are applied to the anterior region of the chick limb bud (Yonei et al. [1993] Dev. Biol. 160:246–253). To evaluate the role of HGF in limb development, we observed the expression pattern of the HGF gene using in situ hybridization. The HGF gene was expressed in the mesoderm of the limb bud and in the central core region of mandibular arch and maxillary processes at stages 17 to 24. When both wing and leg buds begin to extend distally, the HGF gene is expressed in the mesenchymal cells, but not in the ectodermal cells and somites. Concomitant with establishment of the apical ectodermal ridge, distal mesenchymal cells of the limb bud express the HGF gene intensely with a gradient higher in the distal region. The HGF expression is later confined to the ventral and subapical mesenchyme of the limb bud, although no signal is detectable in the apical and non‐ridge ectoderm. However, signal for thec‐metproto‐oncogene encoding the HGF receptor is not detectable in the limb bud at stages 17 to 24. These results suggest that HGF produced in the limb mesoderm may be involved in initial induction and maintenance of the apical ectoderm during limb development. © 1995 Wiley

ISSN:1058-8388    

DOI:10.1002/aja.1002020108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe expression of matrix‐degrading metalloproteases (MMPs) by human skeletal muscle satellite cells was investigated by zymography of cell culture media and by Northern blot analysis of mRNA prepared from satellite cells. Zymography in gelatin substrate gels revealed that satellite cells constitutively synthesize and secrete 72 kDa gelatinase (MMP‐2). In addition, treatment of satellite cell cultures with phorbol ester resulted in an induction of 92 kDa gelatinase (MMP‐9) activity. On casein substrate gels, little or no proteolytic activity was detectable in control or phorbol ester treated satellite cell cultures, suggesting that compared to fibroblasts, satellite cells secrete little or no interstitial collagenase (MMP‐1) or stromelysin (MMP‐3) activity. Northern blotting, however, revealed that there is detectable expression of mRNA transcripts encoding MMP‐1 in satellite cell cultures, and that increased accumulation of MMP‐1 mRNA transcripts occurs upon treatment of these cells with phorbol ester. In contrast, no constitutive, or induced expression of transcripts encoding MMP‐3 was detectable in satellite cells. These findings show that satellite cells can synthesize and secrete selected members of the MMP family and suggest that skeletal muscle cells may participate directly in remodelling of the extracellular matrix during myogenesis and the regeneration of skeletal muscle. © 1995

ISSN:1058-8388    

DOI:10.1002/aja.1002020109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:1058-8388    

DOI:10.1002/aja.1002020101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY