摘要:

AbstractThe most important portion of the mammary gland development occurs postnatally, with distinct periods of intensive morphogenesis taking place btween birth and puberty and during pregnancy and lactation. To characterize the differentiation process of mammary myopithlial cells, we have studied the expression patterns of several smooth musle phenotypic markers, including contractile proteins, α‐smooth muscle‐actin (α‐SM‐actin), smooth muscle myosin heavy chains (SM‐MHC), and calponin; components of cell‐extracellular matrix aderens junctions, phosphoglucomutase‐related protein (PGM), vinculin variants, integrin subunits; and laminin variant chains in the developing rat mammary gland using immunofluorescence microscopy. α‐SM‐actin‐ and SM‐MHC‐positive cells were found first in newborn animals, while calponin, PGM, and α1integrin subunit began to be expressd in prepubertal animals (1.5 weeks). Vinculin, β1and α3integrin subunit were largely confined to the basal cell layer at all developmental stages examined with greater staining starting at 1.5 weeks. Meta‐vinculin was identifed only in myoepithelial cells of the lactating gland. γ1 laminin chain was present in the mammary gland basement membrane throughout development, while the β2 chain was revealed in 3‐week‐old animals and accumulated later in pospubertal animals (7 weeks). Similarly, β2 laminin chain was absent from the forming alveoli basement membrane in pregnant rats and started to accumulate in the lactating gland. In addition to temporal changes, we have observed spatial differences in the distribution of the phenotypic markers. Both in pre‐ and in postpubertal animals, α‐SM‐actinand SM‐MHC‐positive cells of the growing ductal ends contained low amounts if any of calponin, PGM, and β2 laminin chain. We conclude that during postnatal development, mammary myoepithelial cells progressively acquire a differentiated phenotype as revealed by the expression of various smooth muscle markers. Maturation of the myoepithelial cells is accompanied by upregulation of the smooth muscle integrin expression followed by accumulation of β2‐containing laminin variant. Thus, changes in adhesion system parallel with the myoep

ISSN:1058-8388    

DOI:10.1002/aja.1002040202

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe major mechanism for proteloysis in eucaryotes involves an ATP‐dependent pathway for which the covalent attachment of ubiquitin targets proteins for degradation. The involvement of ubiquitin conjugation in early embryonic vertebrate development was investigated by examinining the amounts and localization of ubiquitin conjugates at different stages of development in the chicken using an affinity‐purified antibody specific for conjugated ubiquitin. Solid phase immunochemical assays measuring whole embryo pools of free and conjugated ubiquitin demonstrated a progressive increase increase in conjugate pools to stage 18, followed by a decline to stage 24. In contrast, levels of free polypeptide showed a dramatic increase after stage 5, indicating a change in the dynamics of the two pools during develoment. Immunohistochemistry revealed that the distribution of ubiquitin adducts between stages 3 and 22 was pronounced in regions undergoing extensive cellular remodeling. Ubiquitin conjugates were detected in the primitive streak where cells ingress during gastrulastion. The presence of these degradative intermediate in both neuroectodermal cells of the neural folds and subsquent neural crest cells migrating from the dorsum of the neural tube is consistent with an involvement in key morphogenetic events. The localization of ubiquitin conjugates at other selected tissue interfaces including limb bud ectoderm/mesoderm, and cardiac atrioventricular myocardium/endothelium suggests an active role for ubiquitin‐mediated protein modification in similar developmental interactions. Conjugates were distributed first between somites, then in myotomes with a pattern spatially identical to that of the ubiquitin conjugating enzyme, E214K, the major cognate isozyme for isopeptide ligase (E3)‐dependent degradation. The potential involvement of ubiquitin conjugation at sites of epithelialmesenchymal associations was further analyzed ion culture using atrioventricular canal (AV) endothelium. Immunoreactivity was abundant in cells immediately prior to and during their transformation into mesenchyme. Collectively, the specific temporal and, spatial changes in ubiquitin conjugates during early vertebrate development suggest a regulatory role for this degradative pathway in the cellular remodeling accompanying embryonic growth and differentiation. © 1995 wiley

ISSN:1058-8388    

DOI:10.1002/aja.1002040203

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor with a basic region/helix‐loop‐helix (bHLH) motif. AhR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, although these areas continue to be investigated. AhR mediates many biological effects such as developmental toxicity, including induction of cleft palate and hydronephrosis. This regulatory protein is expressed in embryonic liver and has been immunohistochemically localized in cells of human and mouse secondary palate. The expression of AhR in embryonic tissues and its ability to disrupt development suggests a significant role for this protein in development. The present study examines the pattern of AhR expression in the C57BL/6N mouse embryo from gestation days (GD) 10–16, using in situ hybridization and immunohistochemical analysis. AhR mRNA was localized with35S‐RNA antisense riboprobe (cAh1 probe, 1.8 Kb amino terminal DNA). AhR protein was localized with purified monoclonal antibody (RPT‐9) raised against the N‐terminal peptide sequence. AhR mRNA and protein were expressed in GD 10–13 neuroepithelium, and as development progressed the levels in brain decreased. GD 10–12 embryos also showed AhR in branchial arches, heart, somites, and liver. AhR protein and mRNA in heart were highest at GD 10–11 and decreased with age. In liver, AhR mRNA and protein levels increased and nuclear localization became more pronounced with gestational age. In GD 14–16 embryos levels in liver and adrenal were highest, but AhR was present in ectoderm, bone, and muscle. AhR expression was specific for both cell type, organ/tissue, and developmental stage, suggesting that this novel ligand‐activated transcriptional regulator may be important in normal embryonic development. © 1995 wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in th

ISSN:1058-8388    

DOI:10.1002/aja.1002040204

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic‐helix‐loop‐helix (bHLH) proteins involved in transcriptional regulation. The AhR is a ligand‐activated partner of the ARNT protein. Both proteins are required to transcriptionally regulate gene expression. ARNT must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR‐ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and ARNT are expressed in human embryonic palatal cells and AhR was recently shown to have a specific developmental pattern of expression in the mouse embryo. In the present study, expression of ARNT is characterized in C57BL/6N mouse embryos from gestation day (GD) 10–16 using immunohistochemistry and in situ hybridization. An affinity purified antibody against human ARNT (1.1 μg/ml) was detected with an avidinbiotin‐peroxidase complex. ARNT mRNA was localized with a35S‐RNA probe from pBM5/NEOM1–1. Specific spatial and temporal patterns of ANRT expression emerged and mRNA and protein expression correlated. The GD 10–11 embryos showed highest levels of ARNT in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also had significant expression of ARNT with strong nuclear localization. After GD11, expression in heart and brain declined. In GD 12–13 embryos expression was highest in the liver where expression increased from GD 12–16. At GD 15–16 the highest levels of ARNT occurred in adrenal gland and liver, although ARNT was also detected in submandibular gland, ectoderm, tongue, bone, and muscle. In all of these tissues ARNT was cytoplasmic as well as nuclear, except in some of the cortical adrenal cells in which ARNT was strongly cytoplasmic with little or no nuclear localization. These specific patterns of ARNT expression, which differ in certain tissues from the expression of AhR, suggest that ARNT may have additional roles in normal embryonic developme

ISSN:1058-8388    

DOI:10.1002/aja.1002040205

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe nuclear Limb deformity (Ld) proteins (formins) are expressed during the avian primitive streak stages. Initially, they are detected predominantly in cells of the forming notochord, scattered mesodermal precursors and the induced neural plate. No expression is detected in endodermal cells. The subsequent graded distribution of Ld positive cells along the anterior‐posterior axis of the neural tube follows the antero‐posterior progression of its differentiation. The Ld proteins are also differentially expressed during induction and development of both the inner ear and eye. An unequal distribution of Ld proteins along the dorso‐ventral axis of the otic vesicle is observed during its initial patterning. In the eye, the Ld proteins are expressed by the optic vesicle during secondary induction of the lens placode. Following induction, the proteins are also expressed by the newly formed lens placode, a process which is reminiscent of homeogenetic induction. During differentiation of the retina and lens, the Ld domains seem to demarcate territories, giving rise to specific eye structures. A comparative analysis of the Ld distribution and BrdU incorporation in the two sense organs indicates that the proteins are predominantly expressed by committed and/or differentiating (post‐mitotic) cells. In general, expression of Ld proteins is induced during determination and remains during differentiation of particular cell‐types. This study implies that the nuclear Ld rproteins are involved in morphogenesis of both neuro‐ectodermal and mesodermal structures. © 1995 wil

ISSN:1058-8388    

DOI:10.1002/aja.1002040206

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

年代:1995

数据来源: WILEY

摘要:

AbstractDeveloping muscles contain at least two types of myoblasts. Early myoblasts are the first myoblast to form and are the only myoblasts present during primary myotube formation. By the time secondary myotube formation begins, early myoblasts are rare and late myoblasts are common. The late myoblasts have been postulated to give rise to secondary myotubes. While this is generally accepted, it is unclear whether late myoblasts also contribute to the growth of primary myotubes. One study has produced evidence that myoblasts present during secondary myogenesis selectively fuse with each other or with secondary myotubes, but not with primary myotubes (Harris et al. [1989a] Development 107:771–784). However, the sizes of primary myotubes increase during secondary myotube formation. We have therefore re‐examined the question of whether primary myotubes absorb new nuclei during secondary myotube formation. Pregnant rats were given a single intraperitoneal injection of 5 mg of 5‐bromodeoxyuridine (BrdU) on one embryonic day (from E13 to E19) and their embryos removed on E20. The brominated‐nuclei were labelled with an antibody to BrdU and the myotubes were marked with anti‐myosin antibodies. Double labelled sections from the soleus, tibialis anterior, and extensor digitorum longus muscles were examined with a confocal microscope. The numbers and locations of labelled nuclear profiles in primary and secondary myotubes were counted and recorded. The results show: (1) that primary myotubes absorb nuclei at all stages of development, including the period of secondary myotube formation; (2) that in the early stages of secondary myotube formation, more myoblasts fuse with primary than secondary myotubes whereas this situation is reversed by the end of secondary myotube formation; and (3) that the nuclei added to primary and secondary myotubes during the early stages of their formation are located within the middle of E20 muscles. The nuclei added to growing myotubes are preferentially located at the ends of the muscles. © 1995 wiley

ISSN:1058-8388    

DOI:10.1002/aja.1002040207

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

年代:1995

数据来源: WILEY

摘要:

Abstractα dystroglycan (156 kDa DAG) and β dystroglycan (43 kDa DAG) are encoded by the same gene and are components of the dystrophin‐associated membrane glycoprotein complex. The dystroglycans together with dystrophin form a link between the extracellular matrix and the intracellular cytoskeleton of the muscle fibre. Using in situ hybridisation to mRNA in embryo sections we have examined the expression of the mouse dystroglycan gene. Dystroglycan transcripts are ubiquitously expressed throughout developmnet but are most abundant in cardiac, skeletal and smooth muscle and in ependymal cells lining the developing neural tube and brain. The expression patterns of dystroglycan and dystrophin ovrlap in the major muscle systems during development, suggesting that the dystrophin‐dystroglycan complex plays an important role during myogenesis. In contrast, the major sites of utrophin expression do not co‐localize with those of dystroglycan suggesting that utrophin may interact with a distinct membrane‐associated complex in these non‐muscle sites. Inmdxembryos the pattern of distribution of dystroglycan mRNA remains unchanged, as do those of utrophin and apo‐dystrophin mRNAs. This observation implies that the observed changes in the relative abundance of DAGs and utrophin in dystrophin‐deficient muscle occur post‐transcriptionally. © 19

ISSN:1058-8388    

DOI:10.1002/aja.1002040208

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

年代:1995

数据来源: WILEY

摘要:

AbstractInteraction of ectoderm and underlying mesoderm is essential for normal vertebrate limb morphogenesis. One of the functions of limb bud ectoderm is its influence on the composition of extracellular matrix in subectodermal mesoderm, which in turn participates in morphogenesis of this region of the limb. This matrix is highly enriched in hyaluronan, even at the time when the level of hyaluronan in the chondrogenic and myogenic regions of the limb decreases, due to secretion of a stimulatory factor by the ectoderm. In this study we show that limb bud ectoderm not only stimulates hyaluronan synthesis but induces formation of large hyaluronan‐dependent, pericellular matrices around cultured limb bud mesodermal cells. The ectodermal activity is mimicked in great part by fibroblast growth factor‐2 and transforming growth factor‐β, and antibodies to these proteins inhibit induction of mesodermal pericellular matrix by the ectodermal factor. It has been shown by other investigators that fibroblast growth factor‐2 is produced by limb ectoderm whereas transforming growth factor‐β is present in limb mesodermal tissues. Thus we conclude that the unique properties of mesodermally produced matrix underlying limb bud ectoderm are regulated, at least in part, by ectodermal fibroblast growth factor‐2, probably in concert with mesodermal transforming growth factor‐β. © 199

ISSN:1058-8388    

DOI:10.1002/aja.1002040209

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe enteric nervous system is formed from neural crest‐derived cells. These cells enter the gut, migrate, proliferate, and ultimately differentiate into neurons and glia. We have used a human anti‐neuronal autoantibody (ANNA‐1), which recognizes neuron‐specific RNA‐binding proteins of the Hu family as an early marker of neuronal phenotype, to study the appearance of enteric neurons in the developing chicken gut. Immunoreactive cells appear first in the gizzard primordium at E3.5 and are found at progressively more caudal locations in the gut as development proceeds. Nascent neurons are present at the yolk stalk at E4.5, at the ileocecal junction at E6.5, and within the rectum at E7.5–8.5. Neurons appear slightly later in the esophagus. Aggregates of cells resembling developing ganglia were first observed at E6.5 in the distal esophagus and at E8.5 in the proximal esophagus. A small number of cells appeared in the vagus nerve trunks at E4.5 and that number increased at E7.5–8.5. Immunoreactive cells were also found in the sympatho‐aortic plexus between the mesonephri and in the dorsal mesentery. These cells appeared to coalesce and form the ganglionated Nerve of Remak which contained positive cells at E3.5. This Nerve extended to the yolk stalk at E4.5 and to duodendum at E6.5. We conclude that the appearance of nascent neurons occurs first in the gizzard and proceeds more rapidly in a distal than proximal direction along the gut. Furthermore, cells that appear to be nascent neurons are found in the vagus and in the dorsal mesentery. © 1995

ISSN:1058-8388    

DOI:10.1002/aja.1002040210

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe correct temporal and spatial expression of the type II collagen gene is believed to be important for normal development and growth of the skeleton and the eye, i.e., tissues where the protein product is predominantly found. To study transcriptinal activation of type II collagen gene in skeletal and nonskeletal tissues we produced transgenic mice carrying murine proα1(II) collagen/β‐galactosidase fusion gene constructs. The expression of the fusion gene was found to depend on the presence of intron 1 sequences: constructs with most of intron 1 deleted failed to reveal any β‐galactosidase activity confirming the important role of regulatory sequences within intron 1 of the gene. High‐level expression of the functional construct was clearly confined to cartilaginous tissues but transient low‐level expression was also observed in extraskeletal locations, such as the developing brain and the notochord. The results demonstrate that the regulatory elements in the proα1(II) collagen/β‐galactosidase fusion gene construct confer both temporal and spatial specificity indistinguishable from that of the endogenous proα1(II) collagen gene as determined by the presence of the corresponding mRNA by in situ hybridization. Furthermore the β‐galactosidase activity correlated well with the progression of chondrogenesis as seen by staining of whole mouse embryos with Alizarin red S and Alcian blue in the hybrid mouse strain used for microinjections. The transgenic mouse line produced should prove useful for studies on various aspects of chondrogenesis. Furthermore, the data shows that the regulatory elements present in the construct are sufficient for targetting the expression of other genes in cartilage. © 1

ISSN:1058-8388    

DOI:10.1002/aja.1002040211

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

年代:1995

数据来源: WILEY