Developmental genetics最新文献

The cell-to-cell channels in gap junctions, formed of proteins called connexins (Cxs), provide a direct intercellular pathway for the passage of small signaling molecules (< or = 1 kD) between the cytoplasmic interiors of adjoining cells. It has been proposed that alteration in the expression and function of Cxs may be one of the genetic changes involved in the initiation of neoplasia. To elucidate the role of Cxs in the pathogenesis of human prostate cancer (PCA), the pattern of expression of Cx alpha 1 (Cx43) and Cx beta 1 (Cx32) was studied by immunocytochemical analysis in normal prostate and in prostate tumors of different histological grades. While normal prostate epithelial cells expressed only Cx beta 1, both Cx alpha 1 and Cx beta 1 were detected in PCA cells. The Cxs were localized at the cell-cell contact areas in normal prostate and well-differentiated prostate tumors; however, as prostate tumors progressed to more undifferentiated stages, the Cxs were localized in the cytoplasm, followed by an eventual loss in advanced stages. Thus, epithelial cells from prostate tumors showed subtle and gross alterations with regard to expression of Cx alpha 1 and Cx beta 1 and their assembly into gap junctions during the progression of PCA. Retroviral-mediated transfer of Cx alpha 1 and Cx beta 1 into a Cx-deficient human PCA cell line, LNCaP, inhibited growth, retarded tumorigenicity, and induced differentiation, and these effects were contingent upon the formation of gap junctions. In addition, the capacity to form gap junctions in most Cx-transduced LNCaP cells was lost upon serial passage. Taken together, these findings indicate that the control of proliferation and differentiation of epithelial cells in prostate tumors may depend on the appropriate assembly of Cx beta 1 and Cx alpha 1 into gap junctions and that the development of PCA may involve the positive selection of cells with an impaired ability to form gap junctions.

We report the isolation and characterization of two NK-3-related genes (PwNkx-3.2 and PwNkx-3.3) and their expression patterns during embryonic development, in the adult CNS, and during tail regeneration in the urodele Pleurodeles waltl. PwNkx-3.2 is the ortholog of the mouse and Xenopus genes, Bapx 1 and Xbap, but PwNkx-3.3 has no known homologue in any other vertebrate. We demonstrate that PwNkx-3.2 and PwNkx-3.3 exhibit graded axial expression patterns in adult spinal cord. During tail regeneration, the two genes are expressed in the wound epidermis, the regenerating muscle masses, the regenerating neural tube, the spinal ganglia, and the cartilage rod. The spatial distribution of transcripts in the CNS suggests that these genes could participate in maintaining the position information along the anteroposterior axis and may explain the ability of the adult CNS to regenerate. During tail regeneration, both genes could be implicated in the reformation of the axial skeleton.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

The processes by which undifferentiated cells are assigned to particular fates are far from clear. We review recent work that has examined this problem in the neural crest, a multipotential cell population that gives rise to peripheral neurons in vertebrates. Peripheral neuronal differentiation appears to occur in a series of developmental steps that can be regulated independently by signals in the environment. Furthermore, such steps are reflected by corresponding changes in the pattern of regulatory transcription factor expression in differentiating neural crest cells. The determination of neuronal identity may proceed by a series of parallel regulatory pathways involving transcription factors acting both in cascades and in combinatorial arrays.

The TGF beta family of growth factors has been implicated as playing a significant role in many aspects of embryonic morphogenesis, and also as a mediator of adult tissue repair processes. Unlike the situation in the adult, tissue repair in the embryo does not result in scarring, and it has been suggested that this might be due, in part, to reduced levels of growth factors, particularly TGF beta, at the wound site. We have examined the expression patterns of TGF beta genes following wounding of limb bud lesions in cultured E11.5 mouse embryos. The timetable of wound closure was investigated by standard light and electron microscopy from the time of wounding until the lesion had re-epithelialised 24 hours later. The expression of transcripts for each of the three TGF beta genes was examined at various time points during the healing process using radioactive in situ hybridisation to tissue sections and wholemount non-radioactive in situ hybridisation to embryo pieces. Within 1 to 3 hours of wounding, transcripts encoding TGF beta 1 were rapidly induced within the epithelial cells of the wound margin, particularly those cells at the ventral aspect of the wound. By 3 to 6 hours post-wounding, TGF beta 1 transcripts were detectable in the mesenchyme of the wound bed. No TGF beta 3 induction was observed, and possible TGF beta 2 induction was largely obscured by endogenous expression associated with pre-cartilage mesenchymal condensation. Immunocytochemical analysis of tissue sections of the wound demonstrated a rapid induction of TGF beta 1 protein within 1 hour post-wounding, but also a subsequent rapid clearance of the protein from the wound site such that, by 18 hours post-wounding, TGF beta 1 levels had returned to near background. These data are discussed in terms of the molecular mechanisms underlying embryonic wound healing and the significance of the results to an understanding of scarring following adult tissue repair.

Differentiation inhibiting activity/leukaemia inhibitory factor (DIA/LIF) is a pleiotropic cytokine which has been implicated in a variety of developmental and physiological processes in mammals due to its broad range of biological activities in vitro. A role in very early development is suggested by the requirement for DIA/LIF to support the self-renewal of cultured embryonic stem (ES) cells. Other data point to potential roles in the establishment and maintenance of primordial germ cells, in osteogenesis and in haematopoiesis, and possibly in neuronal specification. DIA/LIF may also act as a mediator of the hepatic acute phase response. In the present study the expression of DIA/LIF transcripts during murine development and in adult mice has been determined using a highly sensitive ribonuclease protection analysis. In contrast to previous reports, it is apparent that DIA/LIF transcripts are present at low levels in many adult mouse tissues. Higher levels of expression are observed in skin, lung, intestine, and uterus. Elevated amounts of mRNA are also found in certain foetal tissue during late gestation and neonatally. In earlier embryogenesis, however, DIA/LIF mRNA is produced primarily in extraembryonic tissues. The alternative transcripts which produce either soluble or matrix-associated DIA/LIF exhibit overlapping but non-identical patterns of expression, consistent with the proposition that the two isoforms may have distinct biological functions. These findings are suggestive of widespread roles for DIA/LIF in vivo and are discussed in the light of available data on the phenotype of homozygous DIA/LIF-deficient mice.

In a search for functions of transforming growth factor-beta during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation proceeds further when the embryoid bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-beta family (TGF-beta 1, TGF-beta 2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 10(-8) M and 10(-7) M induced the formation of neurons but in the absence of RA or at concentrations up to 10(-9) M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10(-9) M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10(-9) M RA. TGF-beta s increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-beta isoform added and the muscle cell type formed. TGF-beta 2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-beta s on the formation of mononucleate flat cells was completely abrogated by RA at 10(-9) M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-beta isoforms may be involved in regulation of differentiation in early myogenesis. In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-beta 2 were injected into the cavity of mouse blastocysts 3.5 days post coîtum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5-9.5 pc.(ABSTRACT TRUNCATED AT 400 WORDS)

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13-20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related alpha, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism.

Two-cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception, the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand-receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation.