Determinants of T box protein specificity.

Members of the T box family of transcription factors play important roles in early development. Different members of the family exert different effects and here we show that much of the specificity of the Xenopus T box proteins Xbra, VegT and Eomesodermin resides in the DNA-binding domain, or T box. Binding site selection experiments show that the three proteins bind the same core sequence, but they select paired sites that differ in their orientation and spacing. Lysine 149 of Xbra is conserved in all Brachyury homologues, while the corresponding amino acid in VegT and Eomesodermin is asparagine. Mutation of this amino acid to lysine changes the inductive abilities of VegT and Eomesodermin to resemble that of Xbra.

Xwnt11 and the regulation of gastrulation in Xenopus.

The molecular basis of gastrulation is poorly understood. In this paper we address this problem by taking advantage of the observation that the transcription activator Brachyury is essential for gastrulation movements in Xenopus and mouse embryos. We infer from this observation that amongst the target genes of Brachyury are some that are involved in the regulation of gastrulation. In the course of a screen for Brachyury targets we identified Xwnt11. Use of a dominant-negative Xwntll construct confirms that signalling by this class of Wnts is essential for normal gastrulation movements, and further investigation suggests that Xwntll signals not through the canonical Wnt signalling pathway involving GSK-3 and beta-catenin but through another route, which may require small GTPases such as Rho and Rac. Future work will concentrate on elucidating the Xwnt11 signal transduction pathway and on investigating its influence on cell shape and polarity during Xenopus gastrulation.

Interference with brachyury function inhibits convergent extension, causes apoptosis, and reveals separate requirements in the FGF and activin signalling pathways.

Brachyury plays a key role in mesoderm formation during vertebrate development. Absence of the gene results in loss of posterior mesoderm and failure of the notochord to differentiate, while misexpression of Brachyury in the prospective ectoderm of Xenopus results in ectopic mesoderm formation. Brachyury is therefore both necessary and sufficient for posterior mesoderm formation. Here we present a detailed cellular and molecular analysis of the consequences of inhibiting Brachyury function during Xenopus development. Our results show that Brachyury is required for the convergent extension movements of gastrulation, for mesoderm differentiation in response to FGF, and for the survival of posterior mesodermal cells in both Xenopus and mouse.

The T-box transcription factor Brachyury regulates expression of eFGF through binding to a non-palindromic response element.

Brachyury is a member of the T-box gene family and is required for formation of posterior mesoderm and notochord during vertebrate development. The ability of Brachyury to activate transcription is essential for its biological function, but nothing is known about its target genes. Here we demonstrate that Xenopus Brachyury directly regulates expression of eFGF by binding to an element positioned approximately 1 kb upstream of the eFGF transcription start site. This site comprises half of the palindromic sequence previously identified by binding site selection and is also present in the promoters of the human and mouse homologues of eFGF.

Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm.

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Effects of the TWis mutation on notochord formation and mesodermal patterning.

The mouse T (Brachyury) gene is required for early mesodermal patterning. Mice homozygous for mutations in T die at midgestation and display defects in mesodermal tissues such as the notochord, the allantois and the somitic mesoderm. To examine the role of T in patterning of somitic and posterior mesoderm along the anterior-posterior axis, we have examined the expression of a panel of molecular markers normally localized to the sub-set of cell types affected in TWis mutant mice. Through the use of whole-mount antibody double labelling techniques, we have analysed the spatial relationships of distinct mesodermal populations relative to cells expressing the T protein. We have also examined the consequences of the TWis mutation on mesodermal populations recognised by these markers. We demonstrate that TWis homozygous mutants retain the ability to form notochordal precursor cells, as identified both by the T antibody and the expression of sonic hedgehog/vertebrate homolog of hedgehog 1 (Shh/vhh-1) and goosecoid, however, these cells fail to proliferate or differentiate. These early notochordal defects appear to result in aberrant somitic differentiation as revealed by the distribution of mox-1 protein and twist RNA expression. Moreover, twist expression in paraxial mesoderm appears to be dependent on normal T activity, while Shh/vhh-1, goosecoid, mox-1 and cdx-4 are not T dependent. We propose that T is required for the maintenance of notochordal tissue and subsequent signals required for somite differentiation.

Identification of novel protein kinases expressed in the myocardium of the developing mouse heart.

In Drosophila and Caenorhabditis, signal transduction pathways initiated by the activation of receptor-protein tyrosine kinases can mediate developmental fate decisions. In order to examine whether similar mechanisms are employed during mammalian embryogenesis, we undertook a search for novel protein kinases expressed during heart development in the mouse. The primitive mouse heart is formed between 7.75 and 8.5 days post coitum (dpc) and consists of myocardial and endocardial cells. A reverse transcriptase polymerase chain reaction-based approach was used to amplify protein kinase specific products from cDNAs obtained from 8.5 dpc heart tissue. Twenty independent PCR products corresponding to either protein serine/threonine or tyrosine kinases were identified. In this report, we describe the characterization of two of the genes corresponding to the novel PCR products (designated Hek2 and msk). Hek2 encodes the mouse ortholog of human HEK2, a recently identified member of the eph receptor-protein tyrosine kinase gene family. Prior to and at the time of heart formation (7.5-8.0 dpc), Hek2 is expressed in the cranial (rostral) region of the embryo from which a subpopulation of cells will give rise to the rudimentary heart. Between 8.0 and 9.5 dpc, Hek2 mRNA expression is observed in myocardial cells, head mesenchyme and paraxial mesoderm. Hek2 transcripts are not detected in endocardial cells. After 9.5 dpc, Hek2 expression is downregulated. msk (for myocardial SNF1-like kinase) encodes a putative protein serine/threonine kinase most similar to the yeast gene SNF1. msk mRNA expression is restricted to myocardial cells and their progenitors in the 7.75-8.5 dpc developing heart. Subsequently, msk mRNA expression is rapidly downregulated. The patterns of Hek2 and msk expression suggest that these protein kinases may function during development of the primitive heart.

A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse.

The 413.d insertional mutation arrests mouse development shortly after gastrulation. nodal, a novel TGF beta-related gene, is closely associated with the locus. The present study provides direct evidence that the proviral insertion causes a loss of function mutation. nodal RNA is initially detected at day 5.5 in the primitive ectoderm. Concomitant with gastrulation, expression becomes restricted to the proximal posterior regions of the embryonic ectoderm. nodal RNA is also expressed in the primitive endoderm overlying the primitive streak. A few hours later, expression is strictly confined to the periphery of the mature node. Interestingly 413.d mutant embryos show no morphological evidence for the formation of a primitive streak. Nonetheless, about 25% of mutant embryos do form randomly positioned patches of cells of a posterior mesodermal character. Data presented in this report demonstrate the involvement of a TGF beta-related molecule in axis formation in mammals.

Use of embryonic stem cells to study mutations affecting postimplantation development in the mouse.

The generation and analysis of insertional mutations that perturb early postimplantation development provide a means to identify genes required at this stage of embryogenesis. We have been studying two independently generated insertional mutations termed 413.d and H beta 58 that result in early postimplantation lethality. Each mutation is associated with a distinct phenotype. 413.d mutant embryos become profoundly abnormal around the time of gastrulation: no identifiable embryonic axis or mesodermal structures are formed. H beta 58 mutant embryos proceed further in development, forming a relatively normal anteroposterior axis before developmental arrest occurs. We isolated embryonic stem cell lines homozygous for each of these mutations and assessed their differentiation abilities and developmental potential in vitro and after their introduction into wild-type blastocysts. From these studies we conclude that the 413.d mutation acts in a non-cell-autonomous fashion: mutant cells appear capable of participating, in conjunction with wild-type cells, in the formation of derivatives of all three primary cell lineages of the embryo. H beta 58 mutant embryonic stem cells are clearly pluripotent but they appear to be more restricted in their developmental potential, suggesting that the H beta 58 gene product may be required by specific tissues of the embryo.

A novel retrovirally induced embryonic lethal mutation in the mouse: assessment of the developmental fate of embryonic stem cells homozygous for the 413.d proviral integration.

A genetic screen of transgenic mouse strains, carrying multiple copies of an MPSV neo retroviral vector, has led to the identification of a recessive embryonic lethal mutation, termed 413.d. This mutation is associated with a single proviral insertion and when homozygous, results in the failure of the early postimplantation embryo at the gastrulation stage of development. Embryonic stem cell lines (ES cells) were derived from 413.d intercross embryos. Genotyping, with respect to the 413.d integration site, identified wild-type, heterozygous and homozygous ES cell lines. The differentiation abilities and developmental potential of the ES cell lines were assessed using a number of in vitro and in vivo assays. Results indicate that the ES cell lines, regardless of genotype, are pluripotent and can give rise to tissue and cell types derived from all three germ layers. Furthermore, analysis of midgestation conceptuses (10.5 p.c.) and adult chimeras generated by injecting mutant ES cells into host blastocysts, provides strong evidence that the mutant cells can contribute to all extraembryonic tissues and somatic tissues, as well as to functional germ cells. These results indicate that the homozygous mutant cells can be effectively 'rescued' by the presence of wild-type cells in a carrier embryo.