摘要:

AbstractWe are interested in the generation of endogenous electric fields associated with ionic currents driven through the vertebrate embryo by the transepithelial potential of its surface ectoderm. Using a non‐invasive vibrating electrode for the measurement of ionic current, we have provided measurements of currents traversing amphibian embryos, and a preliminary report of the internal, extracellular voltage gradient under the neural plate which polarizes the embryo in the rostral/caudal axis (Metcalf et al. [1994] J. Exp. Zool. 268:307–322). Here we complete a description of this gradient in electrical potential (ca. 10 mV/mm, caudally negative), describe a simultaneous gradient organized in the medial/lateral axis (ca. 5–18 mV/mm, negative at the margins of the neural folds), and describe their appearance and disappearance during ontogeny of the axolotl embryo. Both voltage gradients are not expressed until neurulation, and disappear at its climax. This appearance and disappearance correlates with the shunting of current out of the lateral margins of the neural folds in rostral regions of the embryo beginning at stage 15, and is not associated with a more substantial current leak from the blastopore which appears at gastrulation. A steady blastopore current is still present after neural tube formation when intra‐embryonic electric fields have been extinguished. We discuss the direct experimental tests supporting the hypothesis that these extracellular electric fields both polarize the early vertebrate embryo and serve as cues for morphogenesis and pattern. © 1995 Wiley

ISSN:1058-8388    

DOI:10.1002/aja.1002020202

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

年代:1995

数据来源: WILEY

摘要:

AbstractPrevious work in this laboratory has indicated that fibroblast growth factor‐2 (FGF‐2; bFGF) regulates the initial stages of avian heart development in paracrine and autocrine fashion (Parlow et al. [1991] Dev. Biol. 146:139–147; Sugi et al. [1993]Dev. Biol. 157:28–37). Because these findings inferred the presence of a functional receptor for fibroblast growth factor (FGFR), we have immunochemically assessed the appearance of FGFR‐1 (cek‐1;flg) during development. Using a peptide‐generated antibody, Western blots of total embryonic proteins revealed that FGFR‐1 was barely detectable at pre‐heart stages, followed by sequential increases in relative abundance that peaked at stage 24, followed by a decline at days 7–14. Western blots of proteins from isolated embryonic hearts demonstrated a similar developmental pattern, except that FGFR‐1 expression was not decreased at later stages. The presence of FGFR‐1 mRNA was verified by reverse transcription/polymerase chain reaction (RT/PCR) amplification. Immuno‐histochemical examination revealed punctate deposits of FGFR‐1 in the precardiac endoderm at stage 6, followed by detection in the endoderm, foregut, and pre‐cardiac splanchnic mesoderm at stage 8 and in the newly formed myocardium at the heart tube stage (9/10). By stage 13, FGFR‐1 staining was observed only in the myocardium, a pattern which persisted at least until stage 30 (day 7), after which only isolated hearts were examined. After stage 30, staining was diminished in the ventricle, but not in the atrium. Staining of cardiac endothelial cells was not observed at any stage. A functional role for FGFR‐1 was indicated by experiments in which anti‐FGFR‐1, but not pre‐absorbed antiserum, retarded proliferation and multilayering of cardiogenic cells in an in vitro model of c

ISSN:1058-8388    

DOI:10.1002/aja.1002020203

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

年代:1995

数据来源: WILEY

摘要:

AbstractMuscular dysgenesis (mdg)is a recessive lethal mutation in the mouse which drastically affects skeletal muscle development during embryonic life. Physiologically, the disease is characterized by a complete paralysis resulting from a lack of excitation‐contraction coupling. Existing electrophysiological, biochemical, and genetic evidence shows thatmdg/mdgmice express a basic alteration of L‐type voltage‐sensitive Ca2+channels in skeletal muscle. Studies onmdg/mdgmyotubes in primary culture have shown that +/+ fibroblasts or +/+ Schwann cells may fuse with them and correct their functional deficiency by genetic complementation. As the spontaneous formation of heterocaryons is thought to be an exclusive property of myoblasts, we asked whether fibroblasts may have changed their properties before fusion occurred. We used primary cells issued from sciatic nerves dissected from newborn transgenic mice carrying thepHuDes1‐nls‐LacZtransgene (Des‐LacZcells) as non‐muscle cells. These cells were mainly fibroblasts (80%) positive for Thy 1.1 and Schwann cells positive for S100. The cultures were negative for myogenic markers (desmin, troponin T), did not form myotubes long‐term, and did not display significant activation of the muscle reporter gene (pHuDes1‐nls‐LacZ). After a few days in coculture with dysgenic or normal myotubes, the muscle reporter gene (β‐galactosidase) was detected both within dysgenic myotubes, correlating with the restoration of normal contractile activity, and normal myotubes. As well as confirming that fusion takes place, this shows thatDes‐LacZcells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleatedDes‐LacZcells expressing β‐galactosidase were observed, indicating that myogenic genes were being expressed before fusion. This suggests a mechanism of myotube driven myogenic recruitment of cells during the in vitro myogenesis. Analysis of the distribution of the inducedDes‐LacZcells (positive for β‐galactosidase) in compartmentalized muscle cocultures showed that in the presence of dysgenic myotubes, these cells were equally distributed in both myotube free and enriched areas, whereas in the presence of normal myotubes, the positive cells remained in close vicinity of the myotubes. This difference could be explained by the fact that the dysgenic phenotype might include release of the induction process from its normal controls. Our results are consistent with the idea of a transcellular mechanism triggering myogenic differentiation in non‐muscle cells, and that myotubes themselves are able to drive myogenic recruitment of cells during the in vitro myogenesis. This phenomenon could be the result of either a myogenic induction in non‐muscle cells, imposing a phenotypic change, or the activation of pre‐myoblastic quiescent cells by the myo

ISSN:1058-8388    

DOI:10.1002/aja.1002020204

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

年代:1995

数据来源: WILEY

摘要:

AbstractPrevious studies have noted the presence of mesenchymal stem cells located within the connective tissue matrices of avian skeletal muscle, dermis, and heart. In these studies, clonal analysis coupled with dexamethasone treatment revealed the presence of multiple populations of stem cells composed of both lineage‐committed progenitor mesenchymal stem cells and lineage‐uncommitted pluripotent mesenchymal stem cells. The present study was undertaken to assess the distribution of these stem cells in the connective tissues throughout various regions of the body. Day 11 chick embryos were divided into 26 separate regions. Heart, limb skeletal muscle, and limb dermis were included as control tissues. Cells were harvested enzymatically and grown using conditions optimal for the isolation, cryopreservation, and propagation of avian mesenchymal stem cells. Cell aliquots were plated, incubated with various concentrations of dexamethasone, and examined for differentiated phenotypes. Four recurring phenotypes appeared in dexamethasone‐treated stem cells: skeletal muscle myotubes, fat cells, cartilage nodules, and bone nodules. These results suggest that progenitor mesenchymal stem cells and putative pluripotent mesenchymal stem cells with the potential to form at least four tissues of mesodermal origin have a widespread distribution throughout the body, being located within the connective tissue compartments of many organs and organ systems. © 1995 Wiley‐L

ISSN:1058-8388    

DOI:10.1002/aja.1002020205

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe have identified zfh‐4, a new member of a recently recognized zinc finger‐ho‐meodomain (zfh) family of putative transcription factors. Zfh‐4 expression is prominent in developing muscle and brain. In both tissues, zfh‐4 RNA levels are highest embryonically, then decrease gradually to barely detectable levels in adults. In myogenic cell lines, far more zfh‐4 is expressed in proliferating myoblasts than in myotubes, suggesting a cellular basis for the developmental regulation observed in vivo. In contrast, zfh‐4 RNA in brain is more abundant in postmitotic cells of the marginal zone than in proliferating cells of the ventricular zone. Within the brain, zfh‐4 RNA is regionally localized: expression is highest in mid‐brain, readily detectable in hindbrain, and very low in cerebral cortex. Its patterns of expression, and its homology to known DNA binding proteins, support the idea that zfh‐4 may be a regulator of gene expression in developing brain and muscle. ©

ISSN:1058-8388    

DOI:10.1002/aja.1002020206

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

年代:1995

数据来源: WILEY

摘要:

AbstractUsing monoclonal antibodies (mAbs) reactive to newt limb regenerates, we hope to gain insight into the identity and function of regeneration significant molecules. mAb MT4 (matrix 4) identifies an extracellular matrix (ECM) protein that is strongly up‐regulated first in the distal stump and then in the blastema during regeneration. Within the first 24 hr after amputation the MT4 antigen is localized to an acellular space beneath the wound epithelium, and first appears in the basal cells of the wound epithelium between days 5 and 7. At mid‐bud blastema stages, the MT4 antigen is homogeneously distributed as thin fibers in the blastema ECM, and is later largely restricted to the distal tip of the blastema and the areas of cartilage condensation. After extraction and immunoblotting, the MT4 antigen was observed as three reduced species of Mr225, 250, and 260. Taken together, the immunoblot and immunocytochemistry results suggested that mAb MT4 recognized newt fibronectin (FN). Sequence from a cDNA (NvFN.10) obtained by screening a newt blastema cDNA expression library with mAb MT4 conclusively identified the MT4 antigen as FN. To further investigate the expression of FN in regeneration, cDNA NvFN.10 was used to construct a riboprobe and in situ hybridization was done. In the unamputated limb only a few scattered cells expressed the FN gene. Within the first 3 days after amputation strong hybridization signal was observed in the basal cells of the wound epithelium. Most of the stump cells that dedifferentiated and accumulated beneath the wound epithelium at 7 days expressed the FN gene, while the basal cells of the wound epithelium maintained their expression. At mid‐ and late‐bud blastema stages the vast majority of the blastema cells were strongly expressing the FN gene, but the wound epithelial cells now showed only weak FN transcription. Thus initially FN comes from the plasma. Then FN is synthesized by both the wound epithelium and mesenchyme. Finally, at blastema stages FN is produced primarily by the mesenchyme. The expression pattern of FN throughout regeneration suggests that this glycoprotein has roles in wound epithelial and mesenchymal cell migration and mesenchymal cell proliferation and differentiation. © 1995 Wiley

ISSN:1058-8388    

DOI:10.1002/aja.1002020207

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe have studied the angiogenic potential of the unsegmented paraxial mesoderm and epithelial somites of the trunk with homotopical grafts between quail and chick embryos. Quail endothelial cells of the grafts were stained with the QH‐1 antibody after 1–6 days of reincubation. The unsegmented paraxial mesoderm and all parts of the epithelial somite were found to contain angioblasts which develop into QH‐1 positive endothelial cells. These cells are incorporated into the lining of the host's blood vessels such as the perineural vascular plexus and the dorsal branches of the aorta. There is a certain preference as concerns the location of endothelial cells derived from different parts of the somites. Angioblasts from ventral somite halves are mainly found in ventrolateral blood vessels. Those from dorsomedial quadrants form vessels in the dermis of the back, and those from dorsolateral quadrants can be found in the ventrolateral body wall and the wing. With the exception of the dorsal perineural vascular plexus, angioblasts do not cross the median plane of the body. This shows that, although angioblasts migrate extensively, there is bilaterality of the vascular system in the trunk. It remains to be studied whether the notochord plays a role in the establishment of this bilaterality. © 1995 Wiley‐L

ISSN:1058-8388    

DOI:10.1002/aja.1002020208

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

年代:1995

数据来源: WILEY

摘要:

AbstractMolecular mechanisms of endothelial cell differentiation during blood‐brain barrier (BBB) development is not well understood due to the lack of specific molecular markers. Here we describe that expression of the mouse multidrug resistance la/3 (mdrla/3) gene can be detected specifically in subsets of vascular endothelial cells associated with neural tissues at as early as embryonic day 10.5 (E10.5). This onset ofmdrla/3gene expression coincides with the previously described first appearance of morphologically distinct endothelial cells in neural tissues during BBB development. To our knowledge, themdrla/3gene is the earliest endothelial cell differentiation marker gene during BBB development described thus far. In addition, we have found that neither the level nor pattern ofmdrla/3gene expression in BBB endothelial cells is affected by aberrant cortical neuronal layers in mutant mousereeler. © 1995 Wiley‐Liss,

ISSN:1058-8388    

DOI:10.1002/aja.1002020209

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe vasculature of the avian limb bud takes its origin from the intersomitic vessels as can be shown by ink perfusion of the embryo. While the primitive vessels form a central network in the early limb bud, an area of about 100 μm in width from the ectoderm inward remains free from lumenized vessels. However, this subectodermal avascular zone contains isolated angioblasts, which can be demonstrated by confocal laser scanning microscopy in connection with QH‐1‐staining. QH‐1‐positive cells from the avascular zone are capable of giving rise to endothelial cells when grafted ectopically into a “permissive” environment such as the dorso‐lateral paraxial mesoderm. Several grafting sites are compared regarding their permissiveness for capillary formation. In order to investigate the origin of the QH‐1‐positive angioblasts we carried out injections of DiI‐Ac‐LDL, which is specifically taken up by endothelial cells and macrophages, and found the lumenized vessels and a few isolated cells in the peripheral limb mesoderm stained. In double‐labelling studies combining DiI‐Ac‐LDL and QH‐1, it can be shown that there exists a pool of isolated angioblasts that are only QH‐1‐positive, but have not incorporated DiI‐Ac‐LDL. In contrast to the lumenized vessels in the core of the limb bud, we found that angioblasts in the avascular zone do not proliferate, as shown by proliferation studies applying the BrdU‐method to semithin sections in connection with QH‐1‐labelled parallel sections. We conclude that the vascularization of the avian limb bud is achieved by a combination of angiotrophic growth (sprouting of vessels) and angioblastic growth (recruitment of angioblasts fro

ISSN:1058-8388    

DOI:10.1002/aja.1002020210

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

年代:1995

数据来源: WILEY

摘要:

AbstractHomeobox‐containing genes are thought to be involved in regulating pattern formation in a variety of tissues during embryogenesis. We have examined the expression of the homeobox‐related genesMsx‐1andMsx‐2during the development of the chick mandibular arch. Northern blot hybridization indicates that transcripts for bothMsx‐1(1.6 Kb) andMsx‐2(3 Kb) are present in the mandibular arch as early as stage 18. The levels of both transcripts in the whole mandible decrease as cartilage is formed in vivo and in vitro. Using in situ hybridization, transcripts ofMsx‐1were localized in high amounts to the mesenchyme of the mesial tips of the arches.Msx‐2transcripts were localized in high amounts to medial regions of the arches. Little or no hybridization of either probe was detected in the chondrogenic and myogenic regions of the arches. Transcripts of both genes were also excluded from calcified bone and cartilage. Our results further demonstrate that the mesial tip mesenchyme expressingMsx‐1includes areas of highly proliferative cells and has in vitro chondrogenic potential. The region of mesenchymal cells expressing theMsx‐2gene overlap with areas of developmentally programmed cell death which also contain very few proliferative cells and lack chondrogenic potential in vitro. These results are consistent with the possibility thatMsx‐1may be involved in the outgrowth of the mandibular arch andMsx‐2may be involved in both developmentally programmed cell death and delineating the non‐chondrogenic region of the medial part of the mandibular arc

ISSN:1058-8388    

DOI:10.1002/aja.1002020211

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

年代:1995

数据来源: WILEY