A post-mid-blastula transition requirement for TGFbeta signaling in early endodermal specification.
(49/1708)
In Xenopus, endodermal cell fate is determined gradually from late blastula to early gastrula stages; cell-cell interaction plays an important role in this process. Here we use a cell dissociation assay to show that extracellular signaling is required continuously before endoderm determination. Activin and Vg1, but not BMP2 or basic FGF, rescue the expression of endodermal markers in dissociated cells when provided at the mid-blastula transition (MBT, the time in which zygotic transcription begins). Removal of exogenously added activin or Vg1 before MBT results in reduction of endodermal gene expression in dissociated vegetal cells. In vivo, endogenous endodermal markers are reduced in vegetal explants when activin-like signaling is blocked with dominant negative receptors. VegT, a maternal transcription factor shown to be critical for endoderm specification, relies on an active TGFbeta pathway to induce endoderm in animal caps. These results indicate that TGFbeta signaling may be activated by the maternally expressed VegT to participate in endoderm determination. In addition, VegT function seems to be required in parallel with the TGFbeta pathway, as overexpression of activin does not relieve endoderm repression by a dominant negative VegT mutant in vegetal cells. Our data suggest that maternal VegT first activates a zygotic TGFbeta signal, then cooperates with this signal to determine the endodermal cell fate. (+info)
Developmental regulation of Tbx5 in zebrafish embryogenesis.
(50/1708)
T-box (tbx) genes constitute a large family of transcriptional regulators involved in developmental patterning processes. In tetrapods, tbx5 has been implicated in specifying forelimb type identity. Here, we report the cloning of the zebrafish tbx5.1 gene and characterise its expression during zebrafish embryogenesis and early larval development of wild type and mutant embryos that affect pectoral fin patterning. tbx5.1 is expressed during development of the heart, the pectoral fins and the eye. Notably, its expression in the lateral plate mesoderm defines a single and continuous region of heart and pectoral fin precursor cells, and constitutes the earliest specific marker for pectoral fin development in the zebrafish. (+info)
Transient depletion of xDnmt1 leads to premature gene activation in Xenopus embryos.
(51/1708)
In Xenopus laevis zygotic transcription begins at the midblastula transition (MBT). Prior to this the genome is organized into chromatin that facilitates rapid cycles of DNA replication but not transcription. Here we demonstrate that DNA methylation contributes to the overall transcriptional silencing before MBT. Transient depletion of the maternal DNA methyltransferase (xDnmt1) by anti sense RNA during cleavage stages is associated with a decrease in the genomic 5-methyl-cytosine content and leads to the activation of zygotic transcription approximately two cell cycles earlier than normal. Hypomethylation allows the early expression of mesodermal marker genes such as Xbra, Cerberus, and Otx2, which are subsequently down-regulated during gastrulation of the xDnmt1-depleted embryos. The temporal switch in gene expression may account for the appearance of body plan defects that we observe. Loss of xDnmt1 can be rescued by the coinjection of mouse or human Dnmt1 protein. These results demonstrate that DNA methylation has a role in the regulation of immediately early genes in Xenopus at MBT. (+info)
Regulation of the early expression of the Xenopus nodal-related 1 gene, Xnr1.
(52/1708)
The Xenopus nodal related-1 (Xnr1) gene has a complex expression pattern in embryos, with two temporal phases. In the first phase, transcripts are first detected in perinuclear sites in the vegetal region of the blastula. During gastrulation, this expression disappears and transcripts become localised to the dorsal marginal zone. Expression stops and then restarts in a second phase at neurula and tailbud stages, firstly in two symmetric patches near the posterior end of the notochord, and then asymmetrically in a large domain in the left lateral plate mesoderm. In this study, we have investigated the regulation of the early phase of expression of Xnr1. We show that the T-box transcription factor VegT can induce Xnr1. It had previously been shown that Xnr1 can induce VegT in ectoderm cells and we show that the early expression of Xnr1 is regulated by an autoregulatory loop. By inspection of the Xnr1 promoter sequence, we have identified two non-palindromic T-box-binding sites, which are 10 bp apart. Using mutational analysis, we have shown that these elements are required for the VegT induction of Xnr1. The Xnr1 promoter shows striking homologies with the Xnr3 promoter. In particular, two elements that are required for Wnt signaling are conserved between these two promoters, but the two T-box sites are not conserved, and Xnr3 is not induced by VegT. A region of the promoter containing the T-box sites and the Wnt sites is sufficient to drive expression of a reporter gene in a dorsal domain in transgenic Xenopus at the gastrula stage. We show that this pattern of expression of the transgene in gastrulae is not dependent on the T-box sites. (+info)
Xenopus Xenf: an early endodermal nuclear factor that is regulated in a pathway distinct from Sox17 and Mix-related gene pathways.
(53/1708)
We report a novel zygotic gene encoding a Xenopus endodermal nuclear factor, Xenf. Expression of Xenf starts at the late blastula stages and is decreased after gastrulation. Xenf shows no structural homology to any known proteins. When GFP-tagged Xenf is overexpressed in Xenopus cells, Xenf protein is localized to the nucleus, associating closely with the chromosomes. In animal cap assays, Xenf expression is strongly activated by mRNA injection of Vg1 and VegT, maternal vegetal genes that can induce endodermal differentiation. In contrast, Xenf is not induced by endoderm-inducing zygotic transcription factors such as Sox17 and Mix-related genes. In turn, Xenf does not activate expression of Sox17, Mixer or Milk. Thus, Xenf is regulated by maternal vegetal positional information in a parallel manner to Sox17 and Mix-related gene pathways. (+info)
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.
(54/1708)
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. (+info)
Brachyury is a target gene of the Wnt/beta-catenin signaling pathway.
(55/1708)
To identify target genes of the Wnt/beta-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/beta-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/beta-catenin complex and respond specifically to beta-catenin-dependent transactivation. From these results we conclude that Brachyury is a target gene for Wnt/beta-catenin signaling. (+info)
Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus.
(56/1708)
We report here the identification of four members of T-box family genes, Xltbx2-Xltbx5, in Xenopus. Two of them are probable pseudovariant genes of XTbx5 and ET, a putative Xenopus ortholog of Tbx3. We compared their expression patterns in both embryos and limbs. In embryos, expression of Xltbx2 and Xltbx3 showed novel diversities, such as Xltbx2 in the neural crest cells and Xltbx3 in the ventral spinal cord, together with mutual similarities in the following regions: dorsal retina, proctoderm, lateral line organ, cement gland and cranial ganglia. The patterns in limbs were highly conserved with mouse and chick orthologs, including the limb-type specific expression of Xltbx4 and Xltbx5. In addition, RT-PCR analysis showed that they are expressed weakly even in adult limbs as previously reported in the newt. (+info)