Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter.

The homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation.

Opposite effects of FGF and BMP-4 on embryonic blood formation: roles of PV.1 and GATA-2.

In adult vertebrates, fibroblast growth factor (FGF) synergizes with many hematopoietic cytokines to stimulate the proliferation of hematopoietic progenitors. In vertebrate development, the FGF signaling pathway is important in the formation of some derivatives of ventroposterior mesoderm. However, the function of FGF in the specification of the embryonic erythropoietic lineage has remained unclear. Here we address the role of FGF in the specification of the erythropoietic lineage in the Xenopus embryo. We report that ventral injection of embryonic FGF (eFGF) mRNA at as little as 10 pg at the four-cell stage suppresses ventral blood island (VBI) formation, whereas expression of the dominant negative form of the FGF receptor in the lateral mesoderm, where physiologically no blood tissue is formed, results in a dramatic expansion of the VBI. Similar results were observed in isolated ventral marginal zones and animal caps. Bone morphogenetic protein-4 (BMP-4) is known to induce erythropoiesis in the Xenopus embryo. Therefore, we examined how the BMP-4 and FGF signaling pathways might interact in the decision of ventral mesoderm to form blood. We observed that eFGF inhibits BMP-4-induced erythropoiesis by differentially regulating expression of the BMP-4 downstream effectors GATA-2 and PV.1. GATA-2, which stimulates erythropoiesis, is suppressed by FGF. PV.1, which we demonstrate to inhibit blood development, is enhanced by FGF. Additionally, PV.1 and GATA-2 negatively regulate transcription of each other. Thus, BMP-4 induces two transcription factors which have opposing effects on blood development. The FGF and BMP-4 signaling pathways interact to regulate the specification of the erythropoietic lineage.

Transcriptional regulation of BMP-4 in the Xenopus embryo: analysis of genomic BMP-4 and its promoter.

Recent experiments in the Xenopus embryo suggest that proper regulation of BMP-4 signaling is critical to the dorsal ventral specification of both mesoderm and ectoderm. Regulation of BMP-4 signaling is known to occur extracellularly by direct binding with chordin, noggin, and follistatin, and intracellularly through the antagonistic signal interaction with dorsalizing TGF-beta family member activin. However, tight repressional regulation of BMP transcription may also be required to sustain the dorsal and neural status of the induced cells. Here we demonstrate that the dominant negative mutant of the BMP receptor (DN-BR) or the BMP-4 antagonizers, chordin and noggin, negatively regulate BMP-4 transcription in animal cap explants. We suggest that repression of BMP-4 transcription is important in the maintenance of dorsal fate and that continuous input of BMP-4 signaling is required to sustain the expression of BMP-4 transcription in the maintenance of epidermal/ventral fate. Consistent with this postulation, we found that the promoter region of the isolated BMP-4 genomic DNA includes several consensus binding sites for transcriptional regulators functioning under BMP-4 signaling such as GATA binding and ventralizing homeobox genes. In a functional assay we found that the GATA binding and ventral homeobox proteins can positively modulate BMP-4 promoter activity. We also observed that DN-BR decreases BMP-4 promoter activity. This was likely due to a repression of the above-mentioned transcription factors. The significance of these observations to embryonic patterning is discussed.

The homeobox gene PV.1 mediates specification of the prospective neural ectoderm in Xenopus embryos.

Bone morphogenetic protein 4 (BMP4), a member of the TGF beta superfamily, has been implicated in the dorsoventral specification of both mesoderm and ectoderm. High levels of BMP4 signaling appear to specify ventral lineages, while lower levels are causally associated with the development of dorsal lineages. We have previously identified a homeobox-containing transcription factor (PV. 1) which is a likely mediator of the ventralizing effects of BMP4 in the mesoderm. Here we provide evidence that PV.1 also functions downstream of BMP4 in the patterning of ectoderm, specifying epidermal and suppressing neural gene expression. PV.1 is expressed in the prospective neuroectoderm at the time of ectodermal fate determination. BMP4 and xSmad1 (a downstream effector of BMP4) induce PV.1 in uncommitted ectoderm and the dominant negative form of the BMP4 receptor (DN-BR) blocks PV.1 expression. In animal pole explants PV.1 counteracts the neuralizing effects of chordin and the DN-BR and restores them to their original epidermal fate. To address the physiological significance of these observations we employed an animal cap transplantation system and demonstrated that overexpression of PV.1 in the prospective neuroectoderm specifically blocks neurogenesis in intact embryos. Thus, PV.1 plays an important role in the ventralization of both mesoderm and ectoderm. We have previously shown that PV.1 is also preferentially expressed in the ventral endoderm, suggesting that this transcription factor may be involved in the ventralization of all three germ layers.

Modulation of Xenopus embryo mesoderm-specific gene expression and dorsoanterior patterning by receptors that activate the phosphatidylinositol cycle signal transduction pathway.

A role for the phosphatidylinositol (PI) cycle signal transduction pathway in Xenopus mesoderm induction has been revealed by observations of PI cycle activation coincident with this process, combined with the demonstration that Li+ (a PI cycle inhibitor) blocks this response and hyperdorsalizes mesoderm induction in intact embryos or augments growth factor-mediated induction in animal caps. It has been suggested that spatially restricted PI cycle activity in the marginal zone might modulate (but not, itself, activate) mesoderm induction. To better characterize the ability of PI cycle activity to modulate the pattern of mesoderm-specific gene expression elicited by mesoderm-inducing growth factors we have expressed in the embryo exogenous 5-hydroxytryptamine receptors that activate the PI cycle. In embryos, ventral expression and activation of these receptors during mesoderm induction are without obvious effect, whereas dorsal expression and activation yield dorsoanterior-deficient tadpoles. In animal caps induced with activin, simultaneous activation of exogenous 5-hydroxytryptamine receptors inhibits both convergent extension movements associated with dorsal mesoderm induction and the expression of goosecoid, a dorsal-specific gene, but is without effect on expression of a 149 generic mesodermal marker, Xbra. All of these effects of a 149 PI cycle-stimulating receptor are the opposites of those previously reported for the PI cycle inhibitor, Li+. PI cycle activity thus proves able to modulate the dorsal/ventral character of early mesodermal gene expression elicited by growth factor, suggesting a model for mesodermal patterning.

A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.

Scavenging of singlet molecular oxygen by imidazole compounds: high and sustained activities of carboxy terminal histidine dipeptides and exceptional activity of imidazole-4-acetic acid.

Singlet molecular oxygen was generated by illumination of phenosafranin in phosphate buffer at pH 7.5. Relative efficiencies of various imidazole compounds to form endoperoxides were assayed by following at 25 degrees C the rate of light- and imidazole-dependent bleaching of N,N-dimethyl-4-nitrosoaniline. Of over 30 imidazole compounds tested, imidazole-4-acetic acid, a major catabolite of histamine in mammals, exhibited the highest activity. L-Carnosine (beta-alanyl-L-histidine), a natural dipeptide prevalent in striated muscle of mammals, possessed several properties important for a physiologically significant scavenger of singlet oxygen. On a molar basis, this readily water-soluble C-terminal histidine dipeptide reacted with singlet oxygen two- to four-fold faster than free L-histidine and approximately two-fold faster than the N-terminal L-histidine dipeptides tested. Furthermore scavenging ability of L-carnosine did not appreciably increase or decrease with time of reaction, in contrast to behaviors exhibited by a number of other imidazole compounds that included some other C-terminal L-histidine dipeptides. The fungal metabolite, ergothioneine, blocked singlet oxygen generation by illuminated phenosafranin.