摘要:

AbstractThe mouse cellular retinoic acid binding protein‐I (CRABP‐I) gene is specifically up‐regulated by retinoic acid (RA) in P19 mouse embryonal carcinoma cells, and its expression in animals is spatially and temporally restricted to RA‐sensitive tissues during embryonic development. This study demonstrates that, in adult mouse tissues and P19 cells where the expression of CRABP‐I is detected at the basal level, the 5′‐ flanking region of the CRABP‐I gene is hypermethylated at the C residues of all theHpaII sites. Conversely, in mouse embryos during early stages of development when the expression of CRABP‐I gene is detected at a much higher level, this region is demethylated at theseHpaII sites. In P19, enhancement on the RA‐induced up‐regulation of CRABP‐I can be observed in cells treated with 5‐azacytidine (5‐AzaC) in conjunction with RA, where partial demethylation in the 5′‐flanking region of CRABP‐I gene is observed. Nuclear run‐on experiments indicate that increased message levels of CRABP‐I in P19 cells can be accounted for, at least partially, by increases in its transcription rates. The induction of retinoic acid receptor (RAR) β by RA can also be enhanced by 5‐AzaC, but to a much lesser degree. In contrast, all theHpaII sites in the structural gene portion, at least in the first two exons, are fully demethylated a

ISSN:1058-8388    

DOI:10.1002/aja.1002010102

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

年代:1994

数据来源: WILEY

摘要:

AbstractParthenogetically activated, diploid mouse oocytes can develop to midgestation stages in utero. However, even these advanced parthenogenones appear to die because of much reduced trophoblast and yolk sac development. Previous studies have compared the general features of parthenogenetic and androgenetic development and determined the fate of uniparental cells in chimeras with normal embryos. These studies led to the concept of genomic imprinting as the cause for developmental failure when either the maternal or the paternal genome is duplicated, with the corresponding deficiency of the other. Genomic imprinting appears to arise during gametogenesis and to act through dosage effects in a set of imprinted genes, whose expression depends on their parental origin. In this study we undertook a more detailed morphological analysis of parthenogenetic development in the mouse and established a classification system to quantify the developmental extent of parthenogenones. We found that the failure of parthenogenones occurred at different times during early postimplantation development, generating a spectrum of concepti which had developed to different extents, with only a small fraction of the embryos reaching advanced somite stages. In all parthenogenones differentiation and proliferation of the trophectoderm and primitive endoderm lineages (both extraembryonic) was abnormal, and in all, even the best‐developed parthenogenones, we observed similar deficiencies in the embryonic lineages, especially the mesoderm. Common to all abnormally developed lineages was that the proportion of undifferentiated precursor cells was much reduced, while their differentiated descendants were relatively abundant. We propose, therefore, that the failure of parthenogenones to develop to term is due to abnormal regulation of differentiation and proliferation in both embryonic and extraembryonic lineages. In this hypothesis, the apparent tissue specific defects observed in parthenogenones arise as a consequence of the functional importance of certain tissues (like the trophoblast) early in development. The spectrum of parthenogenones thus appears to reflect critical events in early development, whose regulation are affected by genomic imprinting. © 1994 Wiley‐Liss,

ISSN:1058-8388    

DOI:10.1002/aja.1002010103

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

年代:1994

数据来源: WILEY

摘要:

AbstractTo study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time‐lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine α‐bungarotoxin (R‐BT) continuously present in the medium. Since TIRF illuminates only cell‐substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the cell culture during imaging procedures is much enhanced relative to standard epi‐ (or trans‐) illumination. During the experiment, cells were kept alive on the microscope stage at 37°C in an atmosphere of 10% CO2·Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week‐long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest‐lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell‐cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour. ©

ISSN:1058-8388    

DOI:10.1002/aja.1002010104

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe have studied the effect of adding extra satellite cells or soluble factors from crushed muscle on regeneration of minced fragments from rat tibialis muscle. The muscle mince was wrapped in an artificial epimysium to prevent adhesions and cell immigration from adjacent muscles. Regeneration was quantitatively assessed by electrophoretic determination of the muscle‐specific form of creatine kinase. Control minces exhibited three periods of change in creatine kinase activity during a 7‐week regeneration period. Activity fell rapidly during the first week, then rose gradually from 1–3 weeks and increased more rapidly from 3–7 weeks. To augment the original complement of myogenic cells, satellite cells were isolated from the contralateral muscle, purified by density gradient centrifugation, and expanded in culture for 3 days before adding to the muscle mince. The added cells resulted in a 3‐fold enhancement of creatine kinase activity throughout the regeneration period. Soluble muscle extract incorporated into a collagen matrix also stimulated regeneration when added to muscle mince. The extract accelerated the rate of creatine kinase increase during the 1–3 week period beyond that observed in the control or cell augmented mince, suggesting that factors in the extract may facilitate revascularization or reinnervation. The specific activity of creatine kinase was increased in regenerates augmented with both cells and extract, indicating that the effects enhance primarily myogenic processes. © 1994 Wil

ISSN:1058-8388    

DOI:10.1002/aja.1002010105

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe mechanism(s) which control patterning in the face remain elusive, due in large part to the absence of morphologically identifiable controlling regions such as the AER of the limb bud. In order to identify the controlling region(s) and timing of patterning in the face, an investigation was launched to determine the spatial organization of tissues within this region, beginning with the chondrogenic zones of the avian (chick and quail) mandible. The mandibles from HH stage 23/24 chick and equivalent stage quail embryos were initially bisected in three planes giving rostral or caudal, proximal or distal, and medial or lateral halves. The mesenchyme from these various regions was isolated, plated out in high density micromass cultures, and grown for 4 days. Additionally, further cultures were grown, consisting of mandibular mesenchyme subdivided into quarters along the long axis of the mandible (e.g., rostro‐proximal quarter) as well as the bisecting of medial or lateral halves (e.g., medialrostral quarter). Nodule number and area were determined by morphometric analysis for each culture as well as whole mandible controls. The demarcation between chondrogenic and non‐chondrogenic regions was dramatic. Of the bisected halves, proximal and lateral were the most chondrogenic with the lateral subdivision displaying much more cartilage than whole mandible. The nodules of the lateral cultures fused into a sheet of cartilage. In constrast mesenchyme from the medial half was virtually non‐chondrogenic. When ranked by the amount of chondrogenesis, the order was, lateral>proximal = whole = core>distal>caudal>rostral>periphery ≫ medial. Interestingly, when subdivided further an altered pattern appeared. For example, the rostromedial quarter displayed a sheet of cartilage; more than the disto‐lateral or even the proximolateral which, based on the bisected‐mandible data, should have yielded the most cartilage. One possible explanation for the variance in the cartilage produced by the quartered mandibles from the amount predicted by the bisected‐mandible data is that further isolating portions of the mesenchyme changed the ratios of other cell types in the cultures and that cell‐cell interactions affect chondrogenic differentiation as suggested for limb mesenchyme. © 1994

ISSN:1058-8388    

DOI:10.1002/aja.1002010106

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe enteroendocrine cell system of the mammalian gastrointestinal tract is comprised of at least 16 different subpopulations. Each subpopulation shows a characteristic distribution along both the crypt‐villus and cephalocaudal axes. In both the small intestine and colon of adult mice, multilabel immunohistochemistry has demonstrated that two or more neuroendocrine products can be coexpressed in various combinations in single cells along the crypt‐villus axis, suggesting that enteroendocrine phenotypes may be actively regulated. Using bromodeoxyuridine (BrdU) incorporation and multilabel immunohistochemistry, we have previously demonstrated an enteroendocrine cell differentiation pathway consisting of two subpopulations of cells in the mouse proximal small intestine—one involving the sequential expression of substance P, serotonin, and secretin in cells migrating out of the crypts into the villi, and a second involving the expression of substance P and serotonin in cells which remain in the crypts. In this report, we use double label immunohistochemistry and BrdU incorporation to define the temporal and spatial interrelationships between gastrin, cholecystokinin (CCK), glucagon‐like peptide‐1 (GLP‐1), and gastric inhibitory peptide (GIP) immunoreactive cells in the mouse proximal small intestine. The expression of these products was compared with that of substance P, serotonin, and secretin. Minimal overlap of expression was found in cells immunoreactive for substance P or serotonin with gastrin, CCK, GLP‐1, or GIP; however, secretin was found colocalized in villus‐associated gastrin, CCK, and GLP‐1 containing cells. We demonstrate that, similar to the bidirectionally migrating substance P and serotonin expressing cells, gastrin, CCK, GLP‐1, and secretin are expressed in upwardly migrating cells, and gastrin, CCK, and GLP‐1 are expressed in downwardly migrating cells that fail to express secretin. GIP containing cells only rarely coexpressed any of the products examined, but were found both in the villi and the crypts, suggesting both upwardly and downwardly migrating populations. These findings demonstrate several novel enteroendocrine cell differentiation pathways. In addition, the expression of secretin in the villi, but not in the crypts, by two otherwise distinct differentiation pathways, and the lack of secretin expression by villus‐associated GIP expressing cells, suggests that local factors present in the crypts and/or on the villi are necessary, but not sufficient, for secretin expression.

ISSN:1058-8388    

DOI:10.1002/aja.1002010107

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe intestinal tissue is characterized by important morphogenetic movements during development as well as by a continuous dynamic crypt to villus epithelial cell migration leading to differentiation of specialized cells. In this study, we have examined the spatio‐temporal distribution of laminin A and M chains as well as of α6 and β4 integrin subunits in adult and developing human and mouse intestine by indirect immunofluorescence. Selective expression of the constituent polypeptides of laminin isoforms (A and M chains) was demonstrated. In the mature human intestine, A and M chains were found to be complementary, the M chain being restricted to the base of crypts and the A chain lining the villus basement membrane. In the developing human intestine, M chain expression was delayed as compared to that of A chain; as soon as the M chain was visualized, it exhibited the typical localization in the crypt basement membrane. A somewhat different situation was found in the adult mouse intestine, since both M and A chains were found in the crypts. During mouse intestinal development the delayed expression of the M chain as compared to that of the A chain was also obvious. The absence of M chain expression in mutantdymouse did not impair intestinal morphogenesis nor cell differentiation. The expression of α6 and β4 subunits was not coordinated. In both species the α6 expression preceded that of β4. Furthermore, while β4 staining in adult mouse intestine was detected at the basal surface of all cells lining the cryptvillus, that of α6 was mainly confined to the crypt cell compartment. An overall similarity of location between α6 integrin subunit and laminin A chain at the epithelial/stromal interface was noted. These data indicate that the spatial and temporal distribution of laminin variants in the developing intestine may be characteristic for each species and that interactions of laminin variants with particular receptors may be important for induction and/or maintenance of differentiated cells. © 1994 Wile

ISSN:1058-8388    

DOI:10.1002/aja.1002010108

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

年代:1994

数据来源: WILEY

摘要:

Abstractsqt‐3mutants ofCaenorhabditis elegansform dumpy larvae and adults and display allele‐specific defects in locomotion, fertility, and viability. We have determined that thesqt‐3locus encodes COL‐1 collagen. We physically mapped thecol‐1gene to a cosmid on chromosome V whose position is consistent with the location of thesqt‐3gene. We also observed morphological defects insqt‐3mutants at stages that correlate with the mRNA expression patterns ofcol‐1.Sequence analysis of thecol‐1gene in the three temperature‐sensitive mutants revealed that each allele ofsqt‐3has a unique missense mutation causing arginine or glutamic acid to replace glycine in a Gly‐X‐Y triple helical domain. These glycine substitutions may result in longer non‐collagenous domains, which may decrease the thermal stability or impart additional flexibility to mutant trimers. In addition, we describe four corrections to the published sequence ofcol‐1, including one fifteen nucleotide addition that completes a conserved domain in the amino terminal coding r

ISSN:1058-8388    

DOI:10.1002/aja.1002010109

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

年代:1994

数据来源: WILEY

ISSN:1058-8388    

DOI:10.1002/aja.1002010101

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

年代:1994

数据来源: WILEY