Biomedical subjects

R S Beddington

Publications and source records attributed to R S Beddington.

At least 19 recordsLinked to original sources

PubMed1996

Genetic mapping and embryonic expression of a novel, maternally transcribed gene Mem3.

To study the molecular function of genes expressed during preimplantation development, we isolated a novel maternal transcript SSEC (Stage Specific Embryonic cDNA)-26 from a partial subtraction library of mouse unfertilized eggs and preimplantation embryos. The SSEC-26 transcript is abundant in the unfertilized egg and also actively transcribed from the newly formed zygotic genome. On the basis of its expression in eggs and embryos, this new mouse gene is named Mem (maternal-embryonic) 3. The genomic locus of Mem3 has been mapped to Chromosome (Chr) 8 near the D8Mit78 marker and the glutaryl CoA dehydrogenase (Gcdh) locus. The deduced amino acid sequence of MEM3 resembles that of the yeast VPS (Vacuolar Protein Sorting) 35 in two separate domains. A cDNA sequence of the potential human homolog of Mem3 has been assembled with partial clones from the EST database and assigned to human Chr 16.

Amino Acid Sequence↗

PubMed1996

Cell fate and morphogenetic movement in the late mouse primitive streak.

A prospective fate map of the late gastrulation mouse primitive streak has been charted in 8.5 dpc mouse embryos developed in culture, using the lineage marker DiI to label groups of cells. As at earlier stages, the fate of cells in the 8.5 dpc primitive streak is regionalised such that successively more caudal regions of the streak give rise to more lateral mesoderm. While most labelled cells over a 24 or 48 h culture period exit from the primitive streak, some are consistently found to remain within it. The most conspicuous resident population is present in the node. To determine when ingression of ectoderm through the streak ceases, ectoderm cells of the streak and posterior neuropore of 8.5-10.0 dpc embryos were labelled. Involution of surface cells to form mesoderm continues until closure of the posterior neuropore but is not seen thereafter.

Affinity Labels↗

PubMed1995

Capturing genes encoding membrane and secreted proteins important for mouse development.

A strategy based on the gene trap was developed to prescreen mouse embryonic stem cells for insertional mutations in genes encoding secreted and membrane-spanning proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to generate an active beta-galactosidase fusion protein. Insertions were found in a cadherin gene, an unc6-related laminin (netrin) gene, the sek receptor tyrosine kinase gene, and genes encoding two receptor-linked protein-tyrosine phosphatases, LAR and PTP kappa. Analysis of homozygous mice carrying insertions in LAR and PTP kappa showed that both genes were effectively disrupted, but neither was essential for normal embryonic development.

Animals↗

PubMed1995

The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation.

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. T/T embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells (Wilson, V., Rashbass, P. and Beddington, R. S. P. (1992) Development 117, 1321-1331). To determine the origin of this abnormal tissue distribution, we have isolated T/T and control T/+ ES cell clones which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. We conclude that prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed. Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak.

Animals↗

PubMed1995

Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo.

A total of 5 conventional, directionally cloned plasmid cDNA libraries have been constructed from the entire embryonic region of the mid-gastrulation mouse embryo and from its four principal tissue constituents (ectoderm, mesoderm, endoderm and primitive streak). These libraries have been validated with respect to the number of independent clones, insert-size and appropriate representation of diagnostic marker genes. Subtractive hybridisation has been used to remove clones common to the Endoderm and Mesoderm cDNA libraries resulting in an Endoderm minus Mesoderm subtracted library. Probe prepared from this subtracted library has been hybridised to a grid containing approximately 18,500 Embryonic Region library clones. Three novel clones have been recovered as well as expected genes already known to be highly expressed in the primitive endoderm lineage at this stage of development. In situ hybridisation to early postimplantation embryos has revealed the expression patterns of these novel genes. One is highly expressed exclusively in visceral endoderm, one is expressed in ectodermal and endodermal tissues, and the third proves to be an early marker of prospective and differentiated surface ectoderm as well as being expressed in endoderm and its derivatives.

Animals↗

PubMed1994

Induction of a second neural axis by the mouse node.

The anterior aspect of the mouse primitive streak resembles the organizer of Xenopus and chick in terms of its developmental fate, ability to alter pattern in the chick limb bud and with respect to the repertoire of genes that its constituent cells express. However, until now there has been no direct evidence that the mouse node organizes pattern during gastrulation, nor that the exceptionally small mouse embryonic egg cylinder can be induced to form a second axis. Grafts of transgenically marked midgastrulation mouse node, or node labelled with DiI, to a posterolateral location in a host embryo of the same developmental stage results in the induction of a second neural axis and the formation of ectopic somites. The graft gives rise predominantly to notochord and endoderm tissue whereas the neurectoderm and somites are mainly of host origin. The ectopic notochord formed is derived solely from the donor node which suggests that the node can serve as a 'stem cell' source of axial mesoderm. This is corroborated by the observation that labelling in situ the population of cells on the outer surface of the mid-gastrulation node with DiI results in continuous labelling of the notochord. DiI-labelled cells are present throughout the notochord from a rostral boundary in the cranial region to its most caudal extreme and the node itself always remains labelled.

Animals↗

PubMed1994

Expression of inhibin subunits and follistatin during postimplantation mouse development: decidual expression of activin and expression of follistatin in primitive streak, somites and hindbrain.

Members of the activin family are believed to act as mesoderm-inducing factors during early amphibian development. Little is known, however, about mesoderm formation in the mammalian embryo, and as one approach to investigating this we have studied activin and follistatin expression during early mouse development. Activins are homo- or heterodimers of the beta A or beta B subunits of inhibin, itself a heterodimer consisting of one of the beta subunits together with an alpha subunit. Follistatin is a single-chain polypeptide which inhibits activin function. Expression of the inhibin alpha chain could not be detected in embryonic or extraembryonic tissues at any of the stages studied (5.5 to 8.5 days) and expression of the beta A and beta B subunits could only be observed in the deciduum in cells surrounding the embryo. Expression of follistatin could also be detected in the deciduum, but in a pattern complementary to that of the beta subunits. Embryonic expression of follistatin first occurred in the primitive streak, and at later stages transcripts were detectable in the somites and in rhombomeres 2, 4 and 6 of the hindbrain. These results are consistent with a role for activin in mesoderm formation in the mouse embryo, and suggest functions for follistatin in addition to its role as an inhibitor of activin.

Activins↗

PubMed1994

Alterations in gene expression during mesoderm formation and axial patterning in Brachyury (T) embryos.

The mouse T (Brachyury) deletion causes defective mesoderm formation and notochord morphogenesis, and abnormalities in the caudal neural tube and somites. To investigate the effect of the wild type T gene on concurrently expressed genes, we have compared expression of a panel of such genes in homozygous T mutants with that in wild type and heterozygous T/+ control embryos. Two classes of genes were used in this study: those implicated in primitive streak or mesoderm formation, and those which are differentially expressed in regions of the neural tube and somites. Results of wholemount in situ analysis show that the mRNA levels of Evx-1, Wnt-3a and Wnt-5a decrease in T/T embryos late in gastrulation, although earlier expression patterns are similar to control embryos. In contrast, BMP-4 and Msx-1 expression patterns remain similar throughout the period studied. Pax-3 and Pax-6, which are expressed in specific dorsoventral domains of the neural tube, both have ventrally extended expression domains in caudal T/T neural tube. This is consistent with a missing ventral signal provided by the notochord. However, the expression of Msx-1 in the most dorsal domain of the neural tube is unaltered in T/T embryos. Pax-1 and Pax-3, which are expressed in the sclerotome and dermamyotome respectively, are expressed correctly in anterior T/T somites, although the Pax-3 expression domain is widened ventromedially. This extension into ventromedial somite domains is more pronounced caudally, supporting a function for the notochord in ventralizing somites.

Abnormalities, Multiple↗

PubMed1993

Quantitative analysis of protein synthesis in mouse embryos. II: Differentiation of endoderm, mesoderm, and ectoderm.

The changes in protein synthesis that occur during differentiation of the primitive germ layers were examined by high-resolution, two-dimensional gel electrophoresis of proteins synthesized in 6.5 and 7.5 days postcoitum (d.p.c.) mouse embryos. For 6.5 d.p.c. embryos, protein synthesis patterns were compared between whole extraembryonic and embryonic regions and between embryonic visceral endoderm and embryonic ectoderm. For 7.5 d.p.c. embryos, comparisons were made between extraembryonic and embryonic regions and between isolated embryonic endoderm, mesoderm, and ectoderm. Each of the isolated 7.5 d.p.c. germ layers was divided into anterior and posterior fragments in order to evaluate possible regional differences in gene expression along the anterior-posterior axis. Comparisons of protein synthesis patterns revealed the greatest difference between isolated endoderm and ectoderm, indicating that by as early as 6.5 d.p.c. patterns of gene expression differ significantly between these tissues. The greatest similarities were found between ectoderm and whole embryonic regions and between endoderm and whole extraembryonic regions, which most likely reflects the overall cellular compositions of the embryonic and extraembryonic regions. Based on their patterns of synthesis, four groups of proteins were identified that were preferentially synthesized in either endoderm or ectoderm. These provide useful markers for studying differentiation in these tissues. One other protein, migrating at the position expected for vimentin, was synthesized at an elevated rate in isolated mesoderm. We also observed differences in rates of synthesis of alpha-tubulin and tropomyosin-5 indicative of potential differences in cytoskeletal composition among the germ layers beyond those previously described. The difference in overall protein synthesis patterns between anterior and posterior regions was greatest in the embryonic endoderm, indicating that differentiation along the anterior-posterior axis may be initiated sooner or may proceed more rapidly in the endoderm than in the other germ layers. These data provide the first quantitative evaluation of the degree to which differentiation of the three primitive germ layers affects protein synthesis patterns and reveal potentially useful markers of endoderm and ectoderm differentiation.

Animals↗

PubMed1993

Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions.

Non-isotopic whole-mount in situ hybridization of mRNA is a novel technique that has greatly facilitated the precise three-dimensional localization of transcripts from genes whose expression is important during development. This methodology has recently been applied to the study of the mouse embryo and offers particular advantages over conventional procedures.

Animals↗

PubMed1993

Control of vertebrate gastrulation: inducing signals and responding genes.

Recently, genes with similar expression patterns in the early gastrulae of several different vertebrate species have been identified. The remarkable conservation of these expression patterns suggests that fundamental similarities exist within the vertebrates at remarkably early stages. It has yet to be established exactly how these genes are activated in the correct spatial patterns and what their functions might be.

Animals↗

PubMed1993

Chimeric analysis of T (Brachyury) gene function.

To investigate T(Brachyury) gene function, a chimeric analysis of midgestation (9.5-11.5 days post coitum) embryos has been performed. Embryonic stem (ES) cell lines homozygous or heterozygous for the T gene have been introduced into wild-type host embryos by blastocyst injection, and the resulting chimeras scored for morphological abnormality and extent of colonization by T/T cells. As observed previously in earlier stage chimeras (Rashbass, P., Cooke, L. A., Herrmann, B. G. and Beddington, R. S. P. (1991) Nature 353, 348-350), 9.5-11.5 dpc T/T<==>+/+ chimeras exhibit many morphological features of intact T/T mutants. In addition, a dramatic bias of T/T cells towards caudal regions (such as tail and allantois) was observed in all chimeras tested. This is likely to result from accumulation of nascent T/T mesoderm cells with time near the primitive streak, possibly because of altered migration or adhesion properties. T/+ cells colonized rostral regions efficiently, but a slight bias towards the distal end of the tail was still evident. No such bias was observed in control chimeras. The presence of T/T cells in the allantois resulted in its failure to form a correct placental connection and thus arrested later development. In contrast, chimeras in which T/T cells were present predominantly in the tail developed normally but exhibited severe tail abnormalities such as foreshortening, branching and haemorrhagic cavities. Moreover, in these embryos, much higher levels of chimerism were present in the distal end of the tail than in younger (9.5 dpc) embryos. Later in gestation, such abnormal tails probably degenerated, giving rise to neonates with absent or severely abnormal tails but no evidence of chimerism. In situ analysis of T expression in the tail reveals that normally T is expressed highly in the tailbud (the growing portion of the tail) during its elongation between 9.5 and 11.5 dpc. Thus, evidence both from chimeras and from T expression in the tail suggest that T may play a role in the correct deployment of cells emerging from the tailbud.

Allantois↗

PubMed1992

Establishment and organization of germ layers in the gastrulating mouse embryo.

By following the distribution of wheat germ agglutinin-gold-labelled cells in primitive streak stage embryos, we obtained direct evidence for a continuous recruitment of the embryonic ectoderm cells to the definitive endoderm and to the embryonic and extraembryonic mesoderm during gastrulation. The majority of the definitive endodermal cells ingressed through the anterior end of the primitive streak and were incorporated initially into the midline endoderm at the archenteron, but a small population of endodermal cells may be recruited by direct delamination from the embryonic ectoderm. The pre-existing visceral embryonic endoderm was progressively replaced, but not totally, by the newly recruited population which colonized the embryonic foregut and the notochord. The developmental fate of the recruited endoderm and that of cells in the embryonic ectoderm and the mesoderm of late primitive streak stage embryos indicate that concomitant with the establishment of the germ layers, an orderly allocation of prospective fetal tissues to specific parts of the body occurs simultaneously in all three germ layers.

Animals↗

PubMed1992

Use of chimeras to study gene function in mesodermal tissues during gastrulation and early organogenesis.

The origin of different mesodermal tissues during gastrulation and the developmental lability of mesodermal precursors can be mapped by transplanting marked epiblast cells to the same or a different position in a host egg cylinder, and assessing the subsequent fate of transplanted tissue. This information provides the context for assessing the role of particular patterns of gene expression during mesoderm formation and differentiation. For example, the stability of Hox gene expression can be examined by transplanting transgenically marked somites that express a particular Hox gene to a position in the somite file where it is not normally expressed. Such experiments can reveal not only the cues required for Hox gene expression but also the relevance of a circumscribed pattern of Hox gene expression to a specific developmental fate. A different approach to resolving gene function is to mix mutant cells known to affect mesoderm formation with normal cells and to determine the cell autonomy of mutant cells in a normal environment. Homozygous Brachyury (T/T) embryonic stem cell lines have been isolated and injected into normal blastocysts. The presence of T/T cells in chimeras results in mesodermal defects similar to those seen in the intact mutant.

Animals↗

PubMed1992

Brachyury--a gene affecting mouse gastrulation and early organogenesis.

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+<-->T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+<-->T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

A cell autonomous function of Brachyury in T/T embryonic stem cell chimaeras.

Developmental genetics has shown that the Brachyury (T) gene has a key role in mesoderm formation during gastrulation in the mouse. Homozygous embryos have a defective allantois, degenerate or absent notochord and disrupted primitive streak and node. The neural tube is kinked and somite formation interrupted. The T gene has been cloned and is expressed during the early stages of gastrulation, being restricted to the primitive streak region, nascent mesoderm and notochord. Neither the sequence of the gene nor its expression pattern define its developmental function. To study the cell autonomy of the T mutation we have isolated and genetically characterized embryonic stem cell lines and studied their behaviour in chimaeras. T/+ embryonic stem cells form normal chimaeras, whereas T/T in equilibrium with +/+ chimaeras mimic the T/T mutant phenotype. The results indicate that the T gene acts cell autonomously in the primitive streak and notochord but may activate a signalling pathway involved in the specification of other mesodermal tissues.

Animals↗

PubMed1990

Permissiveness to murine leukemia, virus expression during preimplantation and early postimplantation mouse development.

Permissiveness to Moloney Murine Leukemia Virus (MoMuLV) expression was examined during preimplantation and early postimplantation development of the mouse embryo. Blastocysts and 8th, 9th and 10th day postimplantation embryos were infected in vitro with a MoMuLV-based retroviral vector expressing the lacZ gene driven off an internal rat beta-actin promoter. Beta-galactosidase-positive cells were identified in all embryonic tissues including inner cell mass, epiblast, mesoderm, endoderm and definitive ectoderm. In contrast, embryos infected with a MoMuLV-based vector expressing the lacZ gene driven off the viral LTR showed beta-galactosidase-positive cells only in mesoderm and definitive ectoderm. We conclude that permissiveness to transcriptional activity of the LTR is acquired immediately upon differentiation of epiblast during gastrulation of the mouse embryo.

Animals↗

PubMed1989

An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo.

Embryonic stem cells (ES) cells were injected into host blastocysts either in groups of 10-15 cells or as single cells in order to test their developmental potential in the developing embryo. The analysis of midgestation chimaeras, by electrophoretic separation of glucose phosphate isomerase (GPI) isozymes, showed that ES cells were capable of colonizing trophectoderm and primitive endoderm derivatives at a low frequency, as well as producing a high rate of chimaerism in tissues of the fetus and extraembryonic mesoderm.

Animals↗