Brachyury Expression Research Articles

Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression.

Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with beta-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in Lef1(-/-)Tcf1(-/-) mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the beta-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation.

FGF Signaling Regulates Mesoderm Cell Fate Specification and Morphogenetic Movement at the Primitive Streak

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. Here, we demonstrate that FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, we show that FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, we provide evidence that the attenuation of Wnt3a signaling observed in Fgfr1 −/− embryos can be rescued by lowering E-cadherin levels. We propose that modulation of cytoplasmic β-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak.

Characterisation of Wnt gene expression during the differentiation of murine embryonic stem cells in vitro: role of Wnt3 in enhancing haematopoietic differentiation.

The first haematopoietic stem cells in mammalian and non-mammalian vertebrates are derived from mesoderm, therefore genes that are important in mesoderm patterning and formation might also play an essential role in haematopoietic stem cell commitment and differentiation. Several members of the Wnt gene family are expressed in very specific patterns in embryonic mesoderm and have previously been shown to act as haematopoietic growth factors. In order to investigate in detail the role that such secreted proteins play in the biology of early haematopoietic commitment we have used in vitro differentiation of murine embryonal stem (ES) as a model system. Using reverse-transcriptase polymerase chain reaction analysis we identified several candidate Wnt genes whose expression pattern was consistent with a role in generation, maintenance and/or differentiation of early haematopoietic progenitor cells including three genes previously shown to have a role in haematopoiesis (Wnt5a, Wnt2b and Wnt10b). The most interesting candidate was Wnt3, because of its strong and regulated expression during in vitro differentiation of murine ES cells as well as its early embryonic expression in mesoderm. Overexpression of Wnt3 was sufficient to cause a consistent increase in the number of embryoid bodies committing to haematopoiesis further strengthening the evidence that this protein can enhance haematopoietic commitment during in vitro differentiation of ES cells. In addition, overexpression of Wnt3 caused a marked upregulation of Brachyury expression, thus providing some evidence that Brachyury may be one of the target genes for the Wnt3 signalling pathway.

Evolution of the bilaterian larval foregut.

Bilateria are subdivided into Protostomia and Deuterostomia. Indirect development through primary, ciliary larvae occurs in both of these branches; however, the closing blastopore develops into mouth and anus in Protostomia and into anus only in Deuterostomia. Because of this important difference in larval gut ontogeny, the tube-shaped guts in protostome and deuterostome primary larvae are thought to have evolved independently. To test this hypothesis, we have analysed the expression of brachyury, otx and goosecoid homologues in the polychaete Platynereis dumerilii, which develops by means of a trochophora larva-the primary, ciliary larva prototypic for Protostomia. Here we show that brachyury expression in the ventral portion of the developing foregut in Platynereis and also otx expression along ciliated bands in the mouth region of the trochophora larva parallels expression in primary larvae in Deuterostomia. In addition, goosecoid expression in the foregut of Platynereis mirrors the function in higher Deuterostomia. We present molecular evidence for the evolutionary conservation of larval foreguts and mouth regions of Protostomia and Deuterostomia. Our data indicate that Urbilateria, the common bilaterian ancestors, developed through a primary, ciliary larva that already possessed a tripartite tube-shaped gut.

MesP1 and MesP2 are essential for the development of cardiac mesoderm.

The transcription factors, MesP1 and MesP2, sharing an almost identical bHLH motif, have an overlapping expression pattern during gastrulation and somitogenesis. Inactivation of the Mesp1 gene results in abnormal heart morphogenesis due to defective migration of heart precursor cells, but somitogenesis is not disrupted because of normal expression of the Mesp2 gene. To understand the cooperative functions of MesP1 and MesP2, either a deletion or sequential gene targeting strategy was employed to inactivate both genes. The double-knockout (dKO) embryos died around 9.5 days postcoitum (dpc) without developing any posterior structures such as heart, somites or gut. The major defect in this double-knockout embryo was the apparent lack of any mesodermal layer between the endoderm and ectoderm. The abnormal accumulation of cells in the primitive streak indicates a defect in the migratory activity of mesodermal cells. Molecular markers employed to characterize the phenotype revealed a lack of the cranio-cardiac and paraxial mesoderm. However, the axial mesoderm, as indicated by brachyury (T) expression, was initially generated but anterior extension was halted after 8.5 dpc. Interestingly, a headfold-like structure developed with right anterior-posterior polarity; however, the embryos lacked any posterior neural properties. The persistent and widely distributed expression of Cerberus-like-1(Cer1), Lim1 and Otx2 in the anterior endoderm might be responsible for the maintenance of anterior neural marker expression. We also performed a chimera analysis to further study the functions of MesP1 and MesP2 in the development of mesodermal derivatives. In the chimeric embryos, dKO cells were scarcely observed in the anterior-cephalic and heart mesoderm, but they did contribute to the formation of the somites, notochord and gut. These results strongly indicate that the defect in the cranial-cardiac mesoderm is cell-autonomous, whereas the defect in the paraxial mesoderm is a non-cell-autonomous secondary consequence.

A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1

In this report, we describe the identification and characterization of an early embryoid body-derived colony, termed the transitional colony, which contains cell populations undergoing the commitment of mesoderm to the hematopoietic and endothelial lineages. Analysis of individual transitional colonies indicated that they express Brachyury as well as flk-1, SCL/tal-1, GATA-1, (beta)H1 and (beta)major reflecting the combination of mesodermal, hematopoietic and endothelial populations. This pattern differs from that found in the previously described hemangioblast-derived blast cell colonies in that they typically lacked Brachyury expression, consistent with their post-mesodermal stage of development (Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N. and Keller, G. (1997) Nature 386, 488-493). Replating studies demonstrated that transitional colonies contain low numbers of primitive erythroid precursors as well as a subset of precursors associated with early stage definitive hematopoiesis. Blast cell colonies contain higher numbers and a broader spectrum of definitive precursors than found in the transitional colonies. ES cells homozygous null for the SCL/tal-1 gene, a transcription factor known to be essential for development of the primitive and definitive hematopoietic systems, were not able to form blast colonies but did form transitional colonies. Together these findings suggest that the transitional colony represents a stage of development earlier than the blast cell colony and one that uniquely defines the requirement for a functional SCL/tal-1 gene for the progression to hematopoietic commitment.

Exogenous FGF-4 Can Suppress Anterior Development in the Mouse Embryo during Neurulation and Early Organogenesis

Members of the fibroblast growth factor (FGF) family of peptide growth factors are widely expressed in the germ layer derivatives during gastrulation and early organogenesis of the mouse. We have investigated the effect of administering recombinant FGF-4 in the late-primitive streak stage embryo to test if the patterning of the body plan may be influenced by this growth factor. Shortly after FGF treatment the embryonic tissues up-regulated the expression of Brachyury and the RTK signaling regulator Spry2, suggesting that FGF signaling was activated as an immediate response to exogenous FGF. Concomitantly, Hesx1 expression was suppressed in the prospective anterior region of the embryo. After 24 h of in vitro development, embryos displayed a dosage-related suppression of forebrain morphogenesis, disruption of the midbrain–hindbrain partition, and inhibition of the differentiation of the embryonic mesoderm. Overall, development of the anterior–posterior axis in the late gastrula is sensitive to the delivery of exogenous FGF-4. The early response associated with the expression of Spry2 suggests that the later phenotype observed could be primarily related to an inhibition of the FGF signaling pathway.

XTIF2, a Xenopus homologue of the human transcription intermediary factor, is required for a nuclear receptor pathway that also interacts with CBP to suppress Brachyury and XMyoD

Ligand-bound nuclear receptors (NRs) recruit cofactors such as members of the p160 family and CREB-binding protein (CBP) to activate transcription. We have cloned the Xenopus homologue of the human transcription intermediary factor 2 (TIF2), a member of the p160 family of cofactors. Xenopus TIF2 ( XTIF2) mRNA is expressed homogeneously during late blastula–early gastrula stages and later becomes highly expressed in the notochord. To study the function of XTIF2 during development, we have used two dominant negative constructs, one encompassing the NR-binding domain and the other the CBP interacting region of XTIF2. Overexpression of the XTIF2 dominant negative mRNAs causes ectopic expression of Xenopus Brachyury ( Xbra) and MyoD in all tissue layers. Moreover, ectopic expression of the dominant negative construct that contains the CBP-binding region produces strong phenotypes at hatching stage such as loss of head structures, shortened trunks and open blastopores, which can be rescued by XTIF2 coexpression. These observed defects are due, at least in part, to repression of dorsal mesoderm and endoderm genes. Our data suggest the existence of a NR pathway that requires XTIF2 and CBP to repress Xbra and XMyoD.

The VT+ and VT− Isoforms of the Fibroblast Growth Factor Receptor Type 1 Are Differentially Expressed in the Presumptive Mesoderm of Xenopus Embryos and Differ in Their Ability to Mediate Mesoderm Formation

Previously, we cloned a variant form of the type 1 fibroblast growth factor receptor (FGFR1), FGFR-VT-, from Xenopus embryos (Gillespie, L. L., Chen, G., and Paterno, G. D. (1995) J. Biol. Chem. 270, 22758-22763). This isoform differed from the reported FGFR1 sequence (FGFR-VT+) by a 2-amino acid deletion, Val(423)-Thr(424), in the juxtamembrane region. This deletion arises from the use of an alternate 5' splice donor site, and the activity of the VT+ and VT- forms of the FGFR1 was regulated by phosphorylation at this site. We have now investigated the expression pattern and function of these two isoforms in mesoderm formation in Xenopus embryos. Cells within the marginal zone are induced to form mesoderm during blastula stages. RNase protection analysis of blastula stage embryos revealed that the VT+ isoform was expressed throughout the embryo but that the VT- isoform was expressed almost exclusively in the marginal zone. The ratio of VT+:VT- transcripts in the marginal zone indicated that the VT+ form was predominant throughout blastula stages except for a brief interval, coinciding with the start of zygotic transcription, when a dramatic increase in VT- expression levels was detected. This increase could be mimicked in part by treatment of animal cap explants with FGF-2. Overexpression of the VT+ isoform in Xenopus embryos resulted in development of tadpoles with severe reductions in trunk and tail structures, while embryos overexpressing the VT- isoform developed normally. A standard mesoderm induction assay revealed that a 10-fold higher concentration of FGF-2 was required to reach 50% induction in VT+-overexpressing animal cap explants compared with those overexpressing the VT- isoform. Furthermore, little or no expression of the panmesodermal marker Brachyury (Xbra) was detected in VT+-overexpressing embryos, while VT--overexpressing embryos showed normal staining. This demonstrates that VT+ overexpression had a negative effect on mesoderm formation in vivo. These data are consistent with a model in which mesoderm formation in vivo is regulated, at least in part, by the relative expression levels of the VT+ and VT- isoforms.

Brachyury is a target gene of the Wnt/β-catenin signaling pathway

To identify target genes of the Wnt/β-catenin signaling pathway in early mouse embryonic development we have established a co-culture system consisting of NIH3T3 fibroblasts expressing different Wnts as feeder layer cells and embryonic stem (ES) cells expressing a green fluorescent protein (GFP) reporter gene transcriptionally regulated by the TCF/β-catenin complex. ES cells specifically respond to Wnt signal as monitored by GFP expression. In GFP-positive ES cells we observe expression of Brachyury. Two TCF binding sites located in a 500 bp Brachyury promoter fragment bind the LEF-1/β-catenin complex and respond specifically to β-catenin-dependent transactivation. From these results we conclude that Brachyury is a target gene for Wnt/β-catenin signaling.

BMP-4 inhibits neural differentiation of murine embryonic stem cells

Members of the transforming growth factor-beta superfamily, including bone morphogenetic protein 4 (BMP-4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP-4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron-specific antibody TuJ1, aggregates maintained for 8 days in serum-free medium containing BMP-4 generated 5- to 10-fold fewer neurons. The action of BMP-4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP-4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK-1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP-4-treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP-4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP-4 antagonist noggin counteracted the effect of exogenous BMP-4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP-4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells.

Brachyury (T) expression in embryonal carcinoma P19 cells resembles its expression in primitive streak and tail-bud but not that in notochord.

Brachyury (T) is involved in mesoderm induction during early mouse development. T expression in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro, was studied. Endogenous T expression in P19 cells was transiently induced when the cells were allowed to form aggregates. This expression was enhanced by dimethyl sulfoxide. In situ hybridization showed that T expressing cells formed clusters on the aggregates. Transfection of plasmids encoding reporter genes under the control of the upstream region of T showed that the sequence up to -351 bp can resemble the differentiation-dependent expression of T in P19 cells. To define the promoter region regulating T expression, transgenic mice carrying LacZ under the control of the upstream region were prepared. The region up to -351 bp is sufficient to direct the expression in the primitive streak and tail-bud. The upstream region up to -2400 bp does not support expression in the notochord. The sequence between -987 and -585 bp enhances expression in the primitive streak and tail-bud. In the tail-bud where new cells for elongation of the anteroposterior axis were continuously supplied, the sequence up to -987 bp drove lacZ expression in gut endoderm and prospective neuroectoderm as well as in mesoderm derivatives.

Embryonic Stem Cell Development in a Chemically Defined Medium

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cellin vitrodifferentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported inXenopus,due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possiblyin vivo.

A molecular basis for retinoic acid-induced axial truncation.

Dietary deprivation and gene disruption studies clearly demonstrate that biologically active retinoids, such as retinoic acid (RA), are essential for numerous developmental programs. Similar ontogenic processes are also affected by retinoic acid excess, suggesting that the effects of retinoid administration reflect normal retinoid-dependent events. In the mouse, exogenous retinoic acid can induce both anterior (anencephaly, exencephaly) and posterior (spina bifida) neural tube defects depending on the developmental stage of treatment. Retinoic acid receptor gamma (RARgamma) mediates these effects on the caudal neural tube at 8.5 days postcoitum, as RARgamma-/- mice are completely resistant to spina bifida induced by retinoic acid at this stage. We therefore used this null mouse as a model to examine the molecular nature of retinoid-induced caudal neural tube defects by using a panel of informative markers and comparing their expression between retinoic acid-treated wild-type and RARgamma-/- embryos. Our findings indicate that treatment of wild-type embryos led to a rapid and significant decrease in the caudal expression of all mesodermal markers examined (e.g., brachyury, wnt-3a, cdx-4), whereas somite, neuroepithelial, notochord, floorplate, and hindgut markers were unaffected. RARgamma-/- mutants exhibited normal expression patterns for all markers examined, consistent with the notion that mesodermal defects underlie the etiology of retinoid-induced spina bifida. We also found that posterior somitic, but not caudal presomitic, embryonic tissues contained detectable bioactive retinoids, an observation which correlated with the ability of caudal explants to rapidly clear exogenous RA. Interestingly, transcripts encoding mP450RAI, a cytochrome P450, the product of which is believed to catabolize retinoic acid, were abundant in the retinoid-poor region of the caudal embryo. mP450RAI was rapidly induced by retinoic acid treatment in vivo, consistent with previous studies suggesting that it plays a critical role in retinoid signaling. These data suggest that nascent mesoderm is highly sensitive to retinoic acid and that mP450RAI serves to tightly regulate retinoid levels in the caudal embryo. These findings also raise the possibility that RA may play a role in the generation of posterior mesoderm derivatives in part by affecting brachyury expression.

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo.

Oct-1, a member of the POU family of transcription factors, is expressed at relatively high levels in ectodermal and mesodermal cell lineages during early Xenopus embryogenesis (Veenstra et al, 1995). Here we show that overexpression of Oct-1 induces programmed cell death concomitant with the loss of the posterior part of the body axis. Truncated Oct-1 variants, missing either the C-terminal or N-terminal trans-activation domain, exhibit a different capacity to cause such developmental defects. Oct-1-induced cell death is rescued in unilaterally injected embryos by non-injected cells, indicative of the non-cell autonomous character of the developmental effects of Oct-1. This was confirmed by marker gene analysis, which showed a significant decrease in brachyury expression, suggesting that Oct-1 interferes with an FGF-type signalling pathway.

FGF-2 alters the fate of mouse epiblast from ectoderm to mesoderm in vitro

We have developed an in vitro differentiation assay to characterize the ability of peptide growth factors to induce differentiation in mouse epiblast. We report that culturing explants of mouse anterior epiblast, a tissue normally fated to give rise to neuroectoderm and surface ectoderm, in a serum-free, chemically defined medium with 10–50 ng/ml of FGF-2 induced gross changes in cell morphology. Treated cells adopted an elongated, flattened morphology but did not migrate from the explant. Instead, FGF-2-treated cells condensed into multicellular mounds or ridges. Immunocytochemistry showed that cells in treated explants expressed vimentin and in situ hybridization demonstrated that FGF-2 induced the expression of brachyury, goosecoid, and myo-D in regions of treated explants displaying morphological differentiation. Control explants cultured with platelet-derived growth factor AA (PDGF AA), transforming growth factor-β1 (TGF-β1), or in defined medium alone showed no morphological or biochemical differentiation. These results indicate that FGF-2 altered the fate of mouse anterior epiblast from ectoderm to mesoderm in vitro. Cell migration, which is characteristic of primitive streak mesoderm in vivo, was not induced by FGF-2 in these assays. However, the changes in morphology and the expression of mesodermal genes in vitro do support an early role for FGF signaling in the induction of mouse primitive streak mesoderm, as well as in later patterning events during embryogenesis.

Abnormalities of floor plate, notochord and somite differentiation in the loop-tail ( Lp) mouse: a model of severe neural tube defects

Mouse embryos homozygous for the loop-tail ( Lp) mutation fail to initiate neural tube closure at E8.5, leading to a severe malformation in which the neural tube remains open from midbrain to tail. During initiation of closure, the normal mouse neural plate bends sharply in the midline, at the site of the future floor plate. In contrast, Lp/Lp embryos exhibit a broad region of flat neural plate in the midline, displacing the sites of neuroepithelial bending to more lateral positions. Sonic hedgehog ( Shh) and Netrin1 are expressed in abnormally broad domains in the ventral midline of the E9.5 Lp/Lp neural tube, suggesting over-abundant differentiation of the floor plate. The notochord is also abnormally broad in Lp/Lp embryos with enlarged domains of Shh and Brachyury expression. The paraxial mesoderm shows evidence of ventralisation, with increased expression of the sclerotomal marker Pax1, and diminished expression of the dermomyotomal marker Pax3. While the expression domain of Pax3 does not differ markedly from wild-type, there is a dorsal shift in the domain of Pax6 expression in the neural tube at caudal levels of Lp/Lp embryos. We suggest that the Lp mutation causes excessive differentiation of floor-plate and notochord, with over-production of Shh from these midline structures causing ventralisation of the paraxial mesoderm and, to a lesser extent, the neural tube. Comparison with other mouse mutants suggests that the enlarged floor plate may be responsible for the failure of neural tube closure in Lp/Lp embryos.

Pre-MBT patterning of early gene regulation in Xenopus: the role of the cortical rotation and mesoderm induction

Patterning events that occur before the mid-blastula transition (MBT) and that organize the spatial pattern of gene expression in the animal hemisphere have been analyzed in Xenopus embryos. We present evidence that genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16–128-cell stage) cultured until gastrula stages, we demonstrate by RT-PCR analysis that the expression of goosecoid ( gsc), wnt-8 and brachyury ( bra) is dependent on mesoderm induction. In contrast, nodal-related 3 ( nr3) and siamois ( sia) are expressed in a manner that is independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. Our data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo.

Genetic patterning of the developing mouse tail at the time of posterior neuropore closure

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. We report a detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridisation, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, we report continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organised at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation.

Temporal alteration of dose-dependent response to activin in newt animal-cap explants.

In this study, we examined the dose-dependent responses of animal-pole cells of the Japanese newt, Cynops pyrrhogaster, to activin. Cynops has a slower developmental rate and a simpler animal cap structure than Xenopus. These features enable temporal differences in animal-cap competence to be identified more easily and relatively sharp dose-response profiles can be obtained without cell dissociation. When Cynops caps were excised at the mid-blastula stage and transcript levels of marker genes were examined at the early gastrula stage, the strongest induction of brachyury occurred at a low activin dose, suggesting that cells can recognize changing concentrations of an inducing signal in the embryo. Furthermore, the time course of brachyury expression revealed that caps from the mid-blastula stage exposed to a high dose of activin maintained a low expression level after induction. This suggests that Cynops animal-pole cells can assess activin concentration in a simple and direct manner. In addition, we found that animal-cap competence significantly changes during the blastula stage. The data presented here suggest that this change does not autonomously occur within animal-pole cells but requires signals that emanate from other germ layers.