bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of M‐cadherin protein in myogenic cells during prenatal mouse development and differentiation of embryonic stem cells in culture
Authors: Rose Olaf , Rohwedel Jürgen , Reinhardt Sigrid , Bachmann Michael , Cramer Matthias , Rotter Max , Wobus Anna , Starzinski‐Powitz Anna
Published: February 2005

Molecules regulating morphogenesis by cell‐cell interactions are the cadherins, a class of calcium‐dependent adhesion molecules. One of its members, M‐cadherin, has been isolated from a myoblast cell line (Donalies et al. [1991] Proc. Natl. Acad. Sci. U.S.A. 88:8024—8028). In mouse development, expression of M‐cadherin mRNA first appears at day 8.5 of gestation (E8.5) in somites and has been postulated to be down‐regulated in developing muscle masses (Moore and Walsh [1993] Development 117:1409—1420). Affinity‐purified polyclonal M‐cadherin antibodies, detecting a protein of approximately 120 kDa, were used to study the cell expression pattern of M‐cadherin protein. It was first visualized in somites at E10 1/3 and could be confined to desmin positive, myotomal cells. At all subsequent prenatal stages, M‐cadherin was only found in myogenic cells of somitic origin. The detection of the protein at E10 1/3 suggests a translational delay of M‐cadherin mRNA of 1 to 2 days (E8.5 vs. E10 1/3). This was further supported by the finding that during differentiation of ES cell line BLC6 into skeletal muscle cells in culture, expression of M‐cadherin mRNA can be detected 2 days prior to M‐cadherin protein. During prenatal development, the pattern of M‐cadherin expression changes: In E10 1/3 embryos and also in myotomal cells of later stages, M‐cadherin is evenly distributed on the cell surface. In developing muscle masses (tested at E16 to E18), however, M‐cadherin protein becomes clustered most likely at sites of cell‐cell contact as indicated by double‐labelling experiments: M‐cadherin‐staining is the positive image of laminin negative areas excluding the presence of a basal lamina at M‐cadherin positive sites. Furthermore, M‐cadherin is coexpressed with the neuronal cell adhesion molecule N‐CAM which has been shown to mediate cell‐cell contact in myogenic cells. In summary, our results are in line with the idea that M‐cadherin might play a central role in myogenic morphogenesis. © 1994 Wiley‐Liss, Inc.
Prospective fate map of the mouse primitive streak at 7.5 days of gestation
Authors: Smith Jodi L. , Gesteland Katherine M. , Schoenwolf Gary C.
Published: February 2005

Fluorescent dyes were used in the present study to map three rostrocaudal levels of the primitive streak of mouse embryos at 7.5 days of gestation. Injections at the most rostral level, the 1/4 streak level, principally labeled cells of the paraxial mesoderm, including mesenchyme in the head, and somites and segmental plate mesoderm in the trunk. Injections at the intermediate level, the 1/2 streak level, principally labeled cells of the lateral plate mesoderm of the trunk. Injections at the most caudal level, the 3/4 streak level, principally labeled cells of the extraembryonic mesoderm at the periphery (i.e., caudolateral portion) of the embryo adjacent to the developing amnion, yolk sac, and allantois. However, injections placed at all three primitive‐streak levels provided some labeled cells to the areas of the ingressed mesoderm derived from immediately adjacent levels, suggesting that prospective mesodermal areas are not fully spatially segregated from one another at this stage. These data combined with those from a companion study in which the structure of the node was defined and its derivatives were mapped (Sulik et al. [1994] Dev. Dyn. 201:260—278) allowed us to construct a prospective fate map of the mouse primitive streak at 7.5 days of gestation. This map revealed that mediolateral subdivisions of the ingressed mesoderm were arrayed in rostrocaudal sequence within the primitive streak, such that more rostral levels of the streak contained prospective medial cells (e.g., notochord and paraxial mesoderm), intermediate levels contained prospective intermediate cells (e.g., intermediate mesoderm or nephrotome and lateral plate mesoderm), and more caudal levels contained prospective lateromost cells (e.g., extraembryonic mesoderm). Prospective mesodermal cells, with the exception of those of the notochord, ingressed bilaterally from the primitive streak, contributing to the mesoderm on both the right and left sides of the axis. Our prospective fate map of the mouse primitive streak at 7.5 days of gestation, when compared to a map of the avian primitive streak at a comparable stage of development (Schoenwolf et al. [1992] Dev. Dyn. 193:235—248), demonstrates that considerable congruity exists between the locations of mesoderm precursor cells in birds and mammals. Thus, similar morphogenetic movements underlie mesoderm formation during avian and mammalian gastrulation. Moreover, prospective mesodermal locations within the mouse primitive streak correspond to recently described patterns of early gene expression (Sasaki and Hogan [1993] Development 118:47—59; Takada et al. [1994] Genes Dev. 8:174—189; Rossant, personal communication). © 1994 Wiley‐Liss, Inc.
Temporally and spatially restricted expression of apolipoprotein J in the developing heart defines discrete stages of valve morphogenesis
Authors: Witte David P. , Aronow Bruce J. , Dry Jane K. , Harmony Judith A. K.
Published: February 2005

During cardiac valve morphogenesis, a series of interactions between the mesodermal‐derived myocardium and the overlying endothelium lead to condensed leaflet structure formation. At the atrioventricular (AV) canal, endocardial cells are transformed by specialized underlying myocardial cells into endocardial cushions, and then remodeled into mitral and tricuspid valves. Aortic and pulmonary valves develop by a similar mechanism in the primitive outflow tract. Few genes exhibit restricted spatiotemporal expression in these critical embryonic structures, thus limiting the clues to the sequence of molecular events necessary for valvulogenesis. Apolipoprotein J (ApoJ), a secreted glycoprotein expressed in a variety of cell types at tissue interfaces, exhibits a highly restricted and dynamic expression pattern in the developing heart. ApoJ transcripts were detected in mice at day 9.0 of gestation in the wall of the developing truncus arteriosus. By day 10, intense signal occurred in a thin layer of myocardial cells adjacent to developing endocardial cushions of both atrioventricular canal and truncus arteriosus. No apoJ mRNA was present in the overlying endocardial cushions until day 13.5 when prevalvular condensation begins. Intense expression occurred in the stromal connective tissue throughout leaflet formation. The highly restricted spatiotemporal expression pattern of apoJ in the developing heart implicates its role in the morphogenesis of the AV canal and outflow tract into cardiac valves. © 1994 Wiley‐Liss, Inc.
Influence of digits, ectoderm, and retinoic acid on chondrogenesis by mouse interdigital mesoderm in culture
Authors: Lee Kenneth K. H. , Li Felix C. H. , Young W. T. , Kung Jean L. S. , Ng Jim L. , Cheah Kathryn S. E.
Published: February 2005

We have cultured tissues isolated from the interdigital zones (IDZ) of the mouse footplate in the presence of the digits, ectoderm, and all‐trans retionic acid. The objective was to understand how these various factors influence the developmental fate of the interdigital tissues. Neutral red staining showed that these tissues normally differentiate by dying between day 12.5–14.5. However, if they were isolated from the footplate between day 12.5–13.5 (when cell death is not overtly obvious in the IDZ) and maintained in organ culture, these tissues would develop into cartilage and soft connective tissues. In culture, chondrogenesis is initiated very rapidly in the interdigital explants as revealed by in situ hybridization with riboprobes specific for type IIA and IIB procollagen mRNAs. The ability of interdigital tissues to form cartilage is not attributed to factors present in the serum of the culture medium as this phenomenon is also observed in serumless cultures. We have found that if all‐trans retinoic acid, at concentrations of 10–50 ng/ml culture medium, were added to the explants it could inhibit chondrogenesis and promote cell death. Moreover, in some of the cultures, a single digit was left attached to the interdigital tissue. This also dramatically reduced the incidence of chondrogenesis. We have tried to determine whether the digits and ectoderm can produce a diffusible factor that can prevent cartilage from developing by culturing day 12.5 interdigital tissues in ectoderm and digit conditioned media. The ectoderm conditioned medium had no effects on interdigital growth or chondrogenesis. In contrast, the size of interdigital explants cultured in the presence of digit conditioned medium was shown to be significantly smaller than the control. These explants also produced a smaller quantity of cartilage as revealed by Alcian blue binding assay. In sum, our results showed that the fate of the interdigital tissues are not fully determined until after day 13.5. These tissues have the potentials to form cartilage and soft connective tissues. We tentatively propose that these interdigital tissues do not normally realize their histogenetic potentials because of the antichondrogenic influence of the digits and retinoic acid. © 1994 Wiley‐Liss, Inc.
Insertion of a targeting construct in a Hoxd‐10 allele can influence the control of Hoxd‐9 expression
Authors: Rijli Filippo M. , Dollé Pascal , Fraulob Valérie , LeMeur Marianne , Chambon Pierre
Published: February 2005

A neomycin resistance (neo) gene driven by the phosphoglycerokinase (PGK) promoter was inserted into the Hoxd‐10 homeobox by homologous recombination in embryonic stem (ES) cells. Chimeric mice derived from ES cell‐injected blastocysts died shortly after birth. Craniofacial and axial abnormalities were found in the skeleton of these chimeras, resembling some of the previously described Hox gene gain‐of‐function phenotypes. The spatial expression patterns of various Hoxd gene transcripts were analysed in chimeric mutant embryos by in situ hybridization. Two main observations were made: (1) a wide ectopic expression domain of the Hoxd‐9 gene was found in the spinal cord of these embryos, and (2) the neo gene exhibited a specific Hox‐like expression domain which extended far more rostrally than that of the Hoxd‐10 gene, showing that, in the context of this mutation, the PGK promoter could be regulated as a Hox promoter. These results provide the first evidence that a targeted insertion into a Hox gene coding sequence, in the context of its own cluster, could result in misexpression of a neighbour gene of the complex. © 1994 Wiley‐Liss, Inc.
Expression of β1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels
Authors: Zuk Anna , Hay Elizabeth D.
Published: February 2005

Remarkably, a number of definitive epithelia, such as that of the anterior lens, give rise when suspended within 3D gels of type I collagen, to elongate, bipolar shaped cells that exhibit the ultrastructure, polarity, and migratory ability of mesenchymal cells. They begin producing type I collagen and stop producing crystallins, type IV collagen, and laminin. Here, we investigated changes in β1 integrins and their extracellular matrix (ECM) ligands during this transdifferentiation. The former free surface of the lens epithelium that is now in contact with collagen begins within a day to stain intensely for β1 and it is this surface rather than the surface facing the basement membrane that gives rise to mesenchymal cells. Immunoprecipitation experiments reveal a large increase in the β1 integrin subunit on mesenchymal cells as compared to the epithelium of origin. The α5 integrin subunit, which is barely detectable in the lens, increases in the mesenchymal cells and α3 continues to be expressed at about the same level as in the epithelium. α6, the epithelial integrin subunit, and laminin, its ECM ligand, are not detected immunohistochemically or biochemically in the mesenchyme. Rather, the mesenchymal cells secrete abundant fibronectin, the major ECM ligand for α5β1. RGD peptides do not inhibit the transformation but antibodies to β1 do perturb the emigration of mesenchymal cells from the lens apical surface. We conclude that the β1 integrins newly expressed on the apical epithelial surface interact with the surrounding 3D collagen gel to help bring about this unusual epithelial‐mesenchymal transition. © 1994 Wiley‐Liss, Inc.
Chondrogenic potential of chick embryonic calvaria: I. Low calcium permits cartilage differentiation
Authors: Jacenko Olena , Tuan Rocky S.
Published: February 2005

Calvaria 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. © 1995 Wiley‐Liss, Inc.
Chondrogenic potential of chick embryonic calvaria: II. Matrix calcium may repress cartilage differentiation
Authors: Jacenko Olena , Antonio James D. San , Tuan Rocky S.
Published: February 2005

Chick 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. © 1995 Wiley‐Liss, Inc.
Differential expression of retinoic acid receptor‐β isoforms during chick limb ontogeny
Authors: Smith Susan M. , Kirstein Isaac J. , Wang Zhong‐Shan , Fallon John F. , Kelley John , Bradshaw‐Rouse Judith
Published: February 2005

Retinoids 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. © 1995 Wiley‐Liss, Inc.
Expression of the hepatocyte growth factor gene during chick limb development
Authors: Myokai Fumio , Washio Norifumi , Asahara Yoji , Yamaai Tomoichiro , Tanda Nobuya , Ishikawa Tetsuya , Aoki Shunsuke , Kurihara Hidemi , Murayama Yoji , Saito Taiichi , Matsumoto Kunio , Nakamura Toshikazu , Noji Sumihare , Nohno Tsutomu
Published: February 2005

It 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 the c‐met proto‐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‐Liss, Inc.
Synthesis and secretion of matrix‐degrading metalloproteases by human skeletal muscle satellite cells
Authors: Guérin Claude W. , Holland Paul C.
Published: February 2005

The 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 Wiley‐Liss, Inc.
Three‐dimensional gradients of voltage during development of the nervous system as invisible coordinates for the establishment of embryonic pattern
Authors: Shi Riyi , Borgens Richard B.
Published: February 2005

We 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‐Liss, Inc.
Developmental expression of fibroblast growth factor receptor‐1 (cek‐1; flg) during heart development
Authors: Sugi Yukiko , Sasse Joachim , Barron Matthew , Lough John
Published: February 2005

Previous 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 cardiac morphogenesis. © Wiley‐Liss, Inc.
Myotube driven myogenic recruitment of cells during in vitro myogenesis
Authors: Breton Muriel , Li Zhen‐Lin , Paulin Denise , Harris John A. , Rieger François , Pinçon‐Raymond Martine , Garcia Luis
Published: February 2005

Muscular 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 that mdg/mdg mice express a basic alteration of L‐type voltage‐sensitive Ca2+ channels in skeletal muscle. Studies on mdg/mdg myotubes 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 the pHuDes1‐nls‐LacZ transgene (Des‐LacZ cells) 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 that Des‐LacZ cells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleated Des‐LacZ cells 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 induced Des‐LacZ cells (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 myotubes themselves. © 1995 Wiley‐Liss, Inc.
Expression of zfh‐4, a new member of the zinc finger‐homeodomain family, in developing brain and muscle
Authors: Kostich Walter A. , Sanes Joshua R.
Published: February 2005

We 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. © 1995 Wiley‐Liss, Inc.
Mouse multidrug resistance 1a/3 gene is the earliest known endothelial cell differentiation marker during blood‐brain barrier development
Authors: Qin Ying , Sato Thomas N.
Published: February 2005

Molecular 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 of mdrla/3 gene expression coincides with the previously described first appearance of morphologically distinct endothelial cells in neural tissues during BBB development. To our knowledge, the mdrla/3 gene 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 of mdrla/3 gene expression in BBB endothelial cells is affected by aberrant cortical neuronal layers in mutant mouse reeler. © 1995 Wiley‐Liss, Inc.
Blood vessel formation in the avian limb bud involves angioblastic and angiotrophic growth
Authors: Brand‐Saberi Beate , Seifert Roswitha , Grim Milos , Wilting Jörg , Köhlewein Martin , Christ Bodo
Published: February 2005

The 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 from the limb mesoderm). © 1995 Wiley‐Liss, Inc.
Collagen fibrillogenesis in situ: Fibril segments undergo post‐depositional modifications resulting in linear and lateral growth during matrix development
Authors: Birk David E. , Nurminskaya Maria V. , Zycband Emanuel I.
Published: February 2005

Elucidating how collagen fibril growth is regulated is important in determining how tissues are assembled. Fibrils are deposited as segments. The growth of these segments is an important determinant of tissue architecture, stability, and mechanical attributes. Fibril segments were isolated from developing tendons and their structure characterized. The post‐depositional changes leading to linear and lateral growth of fibrils also were examined. Segments extracted from 14‐day chicken embryo tendons had a mean length of 29 μm. The segments were asymmetric, having a short and a long tapered end. Most of the segments were centrosymmetric with respect to molecular packing. Segments extracted from 12‐to 16‐day tendons had the same structure, but mean segment length increased incrementally due to the addition of an increasingly large population of longer segments. At 17 days of development there was a precipitous increase in segment length. The morphological data indicate that the increase in length was the result of lateral associations among adjacent segments. Analysis demonstrated that this fibril growth was associated with a significant decrease in fibril associated decorin. Using immunoelectron microscopy, decorin was seen to decrease significantly at 18 days of development. When decorin content was biochemically determined, a decrease also was observed. Decorin mRNA also decreased relative to fibrillar collagen mRNA during the same period. These data support the hypothesis that a decrease in fibril‐associated decorin is necessary for fibril growth associated with tissue maturation. Growth through post‐depositional fusion allows for appositional and intercalary growth and would be essential for normal development, growth, and repair. © 1995 Wiley‐Liss, Inc.
Cyclopamine, a steroidal alkaloid, disrupts development of cranial neural crest cells in Xenopus
Authors: Dunn Michael K. , Mercola Mark , Moore David D.
Published: February 2005

Cyclopamine is a steroidal alkaloid which causes limb and craniofacial defects in many vertebrate species. We have used Xenopus laevis as a model system to characterize the defects caused by cyclopamine at the cellular level. The most dramatic consequence of cyclopamine treatment in the Xenopus embryo is a defect in formation of craniofacial cartilage. Much of this cartilage is absent in treated animals. As in avian and mammalian species, Xenopus craniofacial cartilage is derived primarily from cells of the cranial neural crest. Grafting experiments show that development of the cartilaginous derivatives of the cranial neural crest is impaired after cyclopamine treatment, and this is at least partially due to a direct effect on presumptive crest cells. A culture system was used to determine the cellular response to the drug. Cyclopamine did not block the initial emigration of cells from a neural plate explant. However, cell death is seen in treated cultures after 4 days. Trunk neural crest cells and transformed cell lines are resistant to cyclopamine. We therefore conclude that cyclopamine specifically causes death of cranial neural crest cells and that lethality is likely to account for the teratogenic effects of this compound. © 1995 Wiley‐Liss, Inc.
Retinoic acid‐induced cell death in the wound epidermis of regenerating zebrafish fins
Authors: Ferretti Patrizia , Géraudie Jacqueline
Published: February 2005

Previous work has shown that treatment with retinoic acid (RA) can induce malformations in regenerating pectoral and caudal fins. RA‐treated regenerates are narrower than unamputated and regenerated control fins because of a decrease in the distance between rays, and either partial or total fusion of some of them. In order to tackle the issue of how RA induces its teratogenic effects on regenerating fins, and which cell types may be specifically affected by RA, we have examined the cellular changes occurring in early regenerates following treatment with retinoids. The work presented here shows for the first time that RA induces significant apoptosis in the wound epidermis, but not in the mesenchyme, of a regenerating appendage, besides inhibiting blastema development as reported in other species. We also show that RA does not retard regeneration by inhibiting accumulation of blastemal cells, but probably by impairing their ability to migrate distal to the amputation plane. This effect is rapidly reversed by discontinuing the treatment, and within 24 hr of removing the drug, blastema development is well advanced. By this time the teratogenic effects induced by RA are already apparent. A correlation between the length of the apical ectodermal ridge (AER) and the number of digits formed has been demonstrated in developing limb buds. We therefore suggest that RA‐induced patterning abnormalities in regenerating fins are the consequence of a reduction in the size of the wound epidermis, due to increased cell death, which would affect patterning of the underlying mesenchyme. © 1995 Wiley‐Liss, Inc.
Expression of two new protein isoforms of the neurofibromatosis type 1 gene product, neurofibromin, in muscle tissues
Authors: Gutmann David H. , Geist Robert T. , Rose Kamala , Wright Douglas E.
Published: February 2005

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, termed neurofibromin, which is expressed predominantly in neurons, Schwann cells, oligodendrocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alternative use of exons 23a and 48a. Previously we described the identification of an NF1 mRNA isoform containing an additional 54 nucleotides from exon 48a (type 3 NF1 ) in human skeletal, cardiac and smooth muscle tissues by reverse‐transcribed (RT)‐PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recognize this muscle‐specific neurofibromin isoform. An NF1 cDNA was generated containing human exon 48a sequences and expressed as a fusion protein in bacteria. The muscle‐specific neurofibromin antibodies detected this exon 48a fusion protein by Western immunoblotting. Immunoprecipitation using these type 3 neurofibromin antibodies also specifically detected a 250 kDa protein in human and rat muscle tissues. Type 3 neurofibromin was found in rat heart and muscle, but not in live, brain, kidney or spleen with levels of expression declining after postnatal day 7. Expression of total NF1 RNA during rat embryonic development was detected at high levels in E15 heart, tongue, and limb bud. In addition, using type 2 neurofibromin‐specific antibodies, the existence of a fourth isoform of neurofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demonstrated in rat heart muscle tissues. The identification of two muscle‐specific isoforms of neurofibromin expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation. © 1995 Wiley‐Liss, Inc.
Endogenous distribution of retinoids during normal development and teratogenesis in the mouse embryo
Authors: Horton Claire , Maden Malcolm
Published: February 2005

We have analysed the endogenous retinoids present in whole mouse embryos from day 9 to day 14 of development and in individual components of the embryo at two stages, day 10.5 and day 13, by HPLC. We can only detect two retinoids, all‐trans ‐RA (tRA) and all‐trans ‐retinol (t‐retinol), and t‐retinol is 5‐10‐fold in excess over tRA. We cannot detect 9‐cis ‐RA or any didehydroretinoids; thus mammalian embryos seem to differ in their retinoid content from other embryos such as chick, Xenopus, and fish. The levels of tRA do not change significantly over the 6 days of development analysed, whereas t‐retinol rises sharply as the liver develops. Within the embryo, tRA is present at high levels in the developing spinal cord and at very low levels in the forebrain; indeed there is a gradient of endogenous tRA from the forebrain to the spinal cord. Other parts of the embryo had intermediate levels of tRA. When a teratogenic dose of RA was administered to day 10.5 embryos, the levels of tRA present in individual tissues of the embryo rose dramatically—from 175‐fold to 1,400‐fold—and the levels rose in all tissues not in any exclusive areas. We then determined which areas of the embryo were malformed by such a teratogenic dose. The lower jaw, palate, vertebrae, tail, and limbs were consistently abnormal, and since these areas received a dose of tRA no higher than any other it was concluded that cell‐specific factors must determine the teratogenic response of these tissues. We then considered whether cellular retinoic acid‐binding protein I or II (CRABP I or II) played any role in this response by determining their relative levels in each of the tissues analysed. There was no correlation between the presence of CRABP I and II and the distribution of administered RA. Neither was there a clear correlation in detail between the presence of CRABP I and II and the sites of teratogenesis. We therefore conclude that other factors, for example, nuclear factors, must be responsible for the teratogenic response to RA. © 1995 Wiley‐Liss, Inc.
Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier
Authors: Hallmann R. , Mayer D. N. , Berg E. L. , Broermann R. , Butcher E. C.
Published: February 2005

Few markers specific for mouse endothelium exist. We describe here one such marker, MECA‐32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA‐32 antigen has a Mr of 50−55 − 103 under reducing conditions and Mr of 100−120 − 103 under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA‐32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA‐32 antigen expression. Between E12 and E16 MECA‐32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA‐32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA‐32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier. © 1995 Wiley‐Liss, Inc.
Posterior extension of the chick nephric (Wolffian) duct: The role of fibronectin and NCAM polysialic acid
Authors: Bellairs Ruth , Lear Pamela , Yamada Kenneth M. , Rutishauser Urs , Lash James W.
Published: February 2005

The nephric duct of the chick embryo starts to form at about stage 10 of Hamburger and Hamilton ([1951] J. Morphol. 88:49–92) and extends posteriorly, fusing with the cloaca at about the end of the third day of incubation (HH stage 17). Evidence from the literature suggests that the extension involves active migration of the posterior tip. This investigation concerned some molecules that might control this migration: fibronectin, vitronectin, the β1 integrin receptor, and NCAM polysialic acid. The concentration of fibronectin in the extracellular matrix was found by immunocytochemistry to be negligible at the posterior end of the duct; treatment of the living embryo with GRGDS failed to halt further extension of the duct; SEM examination of embryos treated with the synthetic peptides of fibronectin GRGDS, GRDGS, SDGR, and GRGES, or with vitronectin, revealed negligible morphological effects on the duct. It is concluded that there is yet no evidence that fibronectin is an important factor in duct migration. NCAM polysialic acid had a similar distribution to fibronectin, but treatment of the living embryo with Endo‐N caused cessation of extension of the duct. Endo‐N is an enzyme that specifically degrades PSA without affecting the NCAM polypeptide itself. It is suggested therefore that PSA may play an important role in duct extension. The synthetic peptides of fibronectin each produced distinctive patterns of blebbing on the surfaces of cells in trunk mesoderm, but the duct cells were unaffected. GRGES and SDGR caused blebbing on cells in the somites and the anterior segmental plate, though not on cells in the posterior segmental plate. This suggests that integrin receptors change in the anterior segmental plate as the mesoderm forms somites from somitomeres. © 1995 Wiley‐Liss, Inc.
Muscle cell death during the development of head and neck muscles in the chick embryo
Authors: McClearn Deedra , Medville Richard , Noden Drew
Published: February 2005

Degenerating myofibers have been reported in the embryos and neonates of a number of birds and mammals, but neither the pervasiveness of the phenomenon nor the spatiotemporal patterns of degeneration has been examined in detail. Using transmission electron microscopy, we determined the patterns of muscle cell death in the chick biventer cervicis, a head extensor muscle. Cell death is most pronounced at incubation days 10 through 15, and occurs throughout the muscle. This is the period during which many myofiber clusters segregate into individual fibers, each with a separate basal lamina, and secondary myofibers become demarcated. Cells of largest diameter, presumably the primary myofibers, are preferentially affected. Degenerating cells exhibit a cohort of cytological features consistent with apoptosis, including the presence of dense, darkly‐staining, hypercontracted myofibrils, misshapen nuclei with irregular chromatin condensations along the nuclear envelope, and scores of cytoplasmic vesicles and vacuoles. In cross section some large diameter muscle cells are characterized by sparse, flocculent cytoplasm that is devoid of myofibrils and organelles. Some show disintegrating cell membranes. In longitudinal section 200–300 μm long regions of hypercontracted myofibrils alternate with areas devoid of fibrils; this arrangement suggests that the myofibrils break into segments that are in register along one part of a muscle fiber and entirely absent from the adjacent length of fiber. We have observed similar patterns of muscle cell degeneration in the complexus, splenius cervicis, depressor mandibulae, and branchiomandibularis muscles. By day 18 of incubation most signs of degeneration are absent and by hatching (day 21) the muscle fibers all appear healthy. Many of these cytological changes in embryonic head muscle cells are characteristic of programmed cell death. We hypothesize that large‐scale death of myocytes is a normal part of avian myogenesis and an important mechanism for affecting the transformation from embryonic to hatching muscle patterning. © 1995 Wiley‐Liss, Inc.