摘要:

AbstractWe 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

ISSN:1058-8388    

DOI:10.1002/aja.1002010402

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

年代:1994

数据来源: WILEY

摘要:

AbstractRetinoic acid (RA) has been shown to affect skeletal patterning in vivo in both developing and regenerating limbs. Regional differences in RA concentrations alone cannot account for the region‐specific cell behaviors involved in limb‐skeletal morphogenesis. The present study explores a role for regional differences in signal interpretation in RA's effects along the anteroposterior and proximodistal axes of stage 21–22 and 23–24 chick wing‐buds. Mesenchymal cells isolated from specific limb regions were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high‐density microtiter cultures. Previous studies showed that RA's effects on chondrogenesis and growth in such cultures differed depending on the position along the limb's proximodistal axis from which the cells were isolated. The present study is the first to show that such differences in RA‐responsiveness also exist along the limb's anteroposterior axis, especially in the distal subridge mesenchyme. The region‐dependent relationships between RA's effects on growth and chondrogenesis suggest that RA affects these two behaviors through different mechanisms. The regional differences in the responsiveness of these cells to exogenous RA are discussed with respect to their correspondence to the in vivo patterns of expression of RA‐binding proteins, RA‐receptors, and other patterning‐related genes.

ISSN:1058-8388    

DOI:10.1002/aja.1002010403

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

年代:1994

数据来源: WILEY

摘要:

AbstractPlacental growth is largely determined by the proliferation of cytotrophoblast cells. However, the distribution of cytotrophoblast cells engaged in the cell cycle during placental development is poorly understood. Recently, antibodies have been developed that identify two proteins directly involved with DNA synthesis: Ki‐67 protein and proliferating cell nuclear antigen (PCNA). Immunolocalization of Ki‐67 and PCNA provides a measure of the proliferating cells in tissues. We examined, in macaque placentas, the spatiotemporal pattern of expression of these proteins during gestation. Tissues from 24 macaque placentas collected from 22–153 days of pregnancy were prepared for paraffin sections. Standard immunoperoxidase techniques were used to identify Ki‐67 and PCNA. The proteins generally co‐localized, although PCNA was usually represented in more cells than Ki‐67. Early in gestation the cell columns contained many labeled cells. The cytotrophoblastic shell was occupied by numerous cells with PCNA positive nuclei, but few were reactive for Ki‐67. By 45 days of pregnancy the immunolabeled cells in the cell columns were concentrated in the proximal regions, adjacent to the anchoring villus tips. The number of positive cells descreased by 100 days when the cell columns were diminished, leaving the anchoring villus tips buried in the shell. Labeled cells were rarely present in the shell at late pregnancy. The single layer of cytotrophoblast cells in the chorionic plate contained numerous reactive cells throughout early and mid‐gestation. After approximately 100 days the cytotrophoblast layer of the chorionic plate was stratified over large areas. Soon thereafter few cells of the chorionic plate were labeled. The chorionic villi contained reactive cytotrophoblastic cells throughout gestation. Extravillous cytotrophoblast cells invading spiral arteries were sometimes labeled for PCNA but not Ki‐67. We conclude that compartments of the placenta are distinguished by specific patterns of cytotrophoblast cell proliferation. Moreover, these patterns correspond to macroscopic growth parameters of the placenta. Evidence suggests that the macaque placenta slows its rate of diametrical growth at approximately 100 days of gestation. It is at about this time that the cell columns are absorbed into the trophoblastic shell and this pool of proliferating cells is diminished. The growth in diameter of the chorionic plate matches that of the shell. In this compartment also the architecture changes at about 100 days as the cytotrophoblast layer stratifies. This stratification may result from continued proliferation of cytotrophoblast cells when the diametrical rate of growth is decreasing. Soon thereafter, proliferation decreases in this compartment also. By contrast, labeled cells were found in chorionic villi throughout gestation. Continued villous growth and branching probably accounts for most of the increases in placental thickness and weight that occurs during late gestation. © 19

ISSN:1058-8388    

DOI:10.1002/aja.1002010404

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

年代:1994

数据来源: WILEY

摘要:

AbstractRetinoic acid receptors α, β and γ (RARα, β and γ) are ligand‐inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the threeRARgenes are expressed in stage‐ and tissue‐specific patterns during embryonic development. In order to understand the role of the different RARs in chick,RARγ2cDNAs were isolated from an 8.5‐day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RARγ2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino‐terminal region (domains A2 and B), and a truncated acidic carboxy‐terminal region (F domain). However, the pattern ofRARγ expression in chick embryo resembles that reported in mouse, particularly in skin whereRARγ expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RARγ isoforms could be successively required during chick development.

ISSN:1058-8388    

DOI:10.1002/aja.1002010405

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

年代:1994

数据来源: WILEY

摘要:

AbstractF9 embryonal carcinoma cells resemble epithelial cells when in monolayer culture. After treatment with retinoic acid these cells differentiate into fibroblastic‐like cells in a sequence that has been modeled as the mammalian equivalent of the differentiation from stem cells of the inner cell mass to parietal endoderm. This study examined the changes in integrin subtypes that accompany retinoic acid‐induced differentiation of F9 cells. Although several integrins were found to be present on the surface of F9 cells and retinoic acid‐induced (RA) cells, the two dominant integrins were α3β1 and α5β1. Differentiation of F9 cells resulted in about 10‐ to 25‐fold increase in the amount of α3β1 integrin protein as measured by immunoprecipitation of cell surface labeled material. There was a corresponding several‐fold reduction of α5β1 protein. The concentration of α3 mRNA was about the same in F9 and RA cells while the concentration of α5 mRNA dropped several‐fold after retinoic acid treatment. Thus α3 regulation appeared to be largely posttranscriptional while the drop in α5 protein may have been a result of transcriptional down‐regulation. Quantitative measurement of adhesion suggested that most of the F9 and RA cell‐substrate adhesion to fibronectin or laminin is mediated by these integrins. They are the dominant integrins present, and antibodies to either these integrins or to the substrate blocked the adhesion.Despite the large switch in integrin subtype protein expression there was little difference between the two cell types in initial cell interactions when adhesive affinities were measured quantitatively. Also there was no difference between the two phenotypes in rate of initial adhesive strengthening. The phenotypic difference was first observed with later events in the attachment and spreading of the RA‐treated cells to the substrate. These results show that retinoic acid treatment alters the amounts of α5 and α3 integrin subunits during the F9 to RA phenotypic switch. The data show that these integrins are important in the cell‐substrate adhesion to fibronectin and laminin. They show, however, that the phenotypic changes observed with differentiation are not associated with the initial preferential adhesions to the substrate, but rather with consequences that alter the cytoskeletal architecture

ISSN:1058-8388    

DOI:10.1002/aja.1002010406

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe cellular protooncogene, c‐ski, is expressed in all cells of the developing mouse at low but detectable levels. In situ hybridization and Northern blot analyses reveal that some cells and tissues express this gene at higher levels at certain stages of embryonic and postnatal development. RT‐PCR results indicate that alternative splicing of exon 2, known to occur in chickens (Sutrave and Hughes [1989] Mol. Cell. Biol. 9:4046–4051; Grimes et al. [1993]Oncogene 8:2863–2868) does not occur in adult mouse tissues. In the embryo, neural crest cells express the c‐skigene during migration at 8.5 to 9.5 days post coitum (p.c.). Neural crest derivatives such as dorsal root ganglia and melanocytes stain positively with an antibody to theskiprotein. At 9 days p.c., the entire neural tube has high levels of c‐skigene expression. By 12–13.5 days only the ependymal layer expresses c‐skiabove background levels. At 14–16 days p.c., c‐skimRNAs are detected at high levels in the cortical layers of the brain and in the olfactory bulb. In 2 week and 6 week postnatal brains, c‐skigene transcripts are also detected in the hippocampus and in the granule cell layer of the cerebellum. The allantois and placenta exhibit high levels of c‐skimRNAs. Neonatal lung tissue increases c‐skigene expression approximately two‐fold compared to prenatal levels. These results suggest thatskiplays a role in both the proliferation and differentiation of specific cell populations of the central and peripheral nervous systems and of other tiss

ISSN:1058-8388    

DOI:10.1002/aja.1002010407

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

年代:1994

数据来源: WILEY

摘要:

AbstractA neomycin resistance (neo) gene driven by the phosphoglycerokinase (PGK) promoter was inserted into theHoxd‐10homeobox 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 describedHoxgene gain‐of‐function phenotypes. The spatial expression patterns of variousHoxdgene transcripts were analysed in chimeric mutant embryos by in situ hybridization. Two main observations were made: (1) a wide ectopic expression domain of theHoxd‐9gene was found in the spinal cord of these embryos, and (2) theneogene exhibited a specificHox‐like expression domain which extended far more rostrally than that of theHoxd‐10gene, showing that, in the context of this mutation, the PGK promoter could be regulated as aHoxpromoter. These results provide the first evidence that a targeted insertion into aHoxgene coding sequence, in the context of its own cluster, could result in misexpression of a neighbour gene of the complex. © 1994 W

ISSN:1058-8388    

DOI:10.1002/aja.1002010408

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

年代:1994

数据来源: WILEY

摘要:

AbstractRemarkably, 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 Wil

ISSN:1058-8388    

DOI:10.1002/aja.1002010409

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.1002010401

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

年代:1994

数据来源: WILEY