Mitsugu Maéno

Expression and Function of Xmsx-2B in Dorso-Ventral Axis Formation in Gastrula Embryos

Abstract Msx is a homeodomain-containing transcriptional factor that plays an essential role in pattern formation in vertebrata and invertebrata embryos. In Xenopus laevis, two msx genes have been identified (Xmsx-1 and Xmsx-2). In the present study, we attempted to elucidate the expression and function of Xmsx-2B (formerly designated as Xhox7.1′) in early embryogenesis. Whole mount in situ hybridization analyses showed that the expression pattern of Xmsx-2B at gastrula and neurula stages was very similar to that of Xmsx-1: the transcript of Xmsx-2B was observed in ventral and lateral sides of the embryo. At the tailbud stage, however, the expression pattern of Xmsx-2B in neural tissues was distinct from that of Xmsx-1. An RNA injection experiment revealed that, like BMP-4, Xmsx-2B has a strong ventralizing activity. In the Xmsx-2B -injected embryos, differentiation of axial structures such as the notochord, muscle, and neural tissue was completely suppressed, whereas α-globin mRNA, a blood cell marker, was highly expressed. Simultaneous injection of Xmsx-1 and Xmsx-2B RNAs showed that they function in an additive manner. It was also shown that coinjection of Xmsx-2B with a dominant-negative BMP-4 receptor (tBR), which can induce formation of secondary axis when injected alone in ventral blastomeres, suppressed secondary axis formation. Furthermore, Xmsx-2B also suppressed secondary axis formation, which was induced by a dominant-negative form of Xmsx-1 (VP16/msx-1). Therefore, like Xmsx-1, Xmsx-2B is a downstream nuclear factor of the BMP-4-derived ventralizing signal, and these two factors probably share the same target molecules. In conclusion, Xmsx-1 and Xmsx-2B function in dorso-ventral axis formation in early Xenopus laevis development.

Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryo

The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)‐4 signal and a key regulator for neural tissue differentiation. Xmsx‐1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription–polymerase chain reaction assays. In animal cap explants, Xmsx‐1 and BMP‐4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss‐of‐function study using the Xmsx‐1/VP‐16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N‐CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP‐4 and Xmsx‐1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell‐lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP‐4 or Xmsx‐1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP‐4‐injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant‐negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N‐CAM and hoxB‐9) if co‐injected with BMP‐4 RNA, although XFD alone at the same dose did not shut down the expression of N‐CAM in the combination explants. Therefore, it is proposed that an FGF‐related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.

XBMP-1B (Xtld), a Xenopus homolog of dorso-ventral polarity gene in Drosophila, modifies tissue phenotypes of ventral explants

Previously we have isolated a Xenopus cDNA homolog of bone morphogenetic protein‐1 (XBMP‐1A). In the present report we describe a new cDNA clone called XBMP‐1B (or Xtld) from a Xenopus embryonic library. Sequence analysis indicates that these two clones share an identical N‐terminal sequence, including a region of metalloprotease domain, three copies of a repeat first found in complement proteins C1r/s and an epidermal growth factor (EGF)‐like sequence. XBMP‐1B protein has an additional copy of an EGF‐like sequence followed by two copies of complement 1 r/s repeat in the C‐terminus. The overall protein structure predicted from the XBMP‐1B sequence reveals that it encodes a protein homologous to Drosophila tolloid. Three XBMP‐1 transcripts (2.9, 5.2 and 6.6 kb) were detected by northern blot analysis. However, the 2.9 kb transcript hybridized specifically with XBMP‐1A and the 5.2 and 6.6 kb transcripts hybridized with XBMP‐1B. In Drosophila, a major function of tolloid is to augment the activity of the decapentaplegic gene product, a close relative of tumor growth factor (TGF)‐β superfamily members, BMP‐2/4. Although XBMP‐1 and XBMP‐4 are detected in various adult tissues of Xenopus, the expression pattern of these two genes was not tightly correlated. In the embryo, the expression of XBMP‐1 increased gradually from the morula to the swimming tadpole stages. Injection of XBMP‐1B RNA into the ventral blastomeres at the 4‐cell stage caused an elongation of the ventral marginal zone explants and converted globin‐positive blood cells to mesenchymal and muscle tissues at later stages. It was shown that XBMP‐1A was less active and a 1A mutant lacking the signal sequence was inactive. Further studies revealed that injection of XBMP‐1B RNA into the ventral marginal zone induced up‐regulation of dorsal marginal zone markers, such as goosecoid and chordin, at the gastrulation stage. These data indicate that XBMP‐1 may have a role in determining dorso–ventral patterning in Xenopus, but in a different way from the dpp/tolloid system demonstrated in Drosophila.

Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos

In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie‐2, a Xenopus homolog of the tie‐2 receptor tyrosine kinase, was examined. Whole‐mount in situ hybridization analysis revealed that Xtie‐2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie‐2‐positive cells are predominantly present just outside the boundary of α‐globin‐positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie‐2 and α‐globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a ‘dorsal determinant’ from the vegetal cytoplasm at the 1‐cell stage. Second, expression of both Xtie‐2 and α‐globin were markedly induced in ectodermal explants (animal caps) from embryos co‐injected with activin and bone morphogenetic protein (BMP)‐4 RNA. Furthermore, both Xtie‐2 and α‐globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP‐4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP‐4‐dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.

The keratin-related Ouroboros proteins function as immune antigens mediating tail regression in Xenopus metamorphosis

Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.

Regulatory signals and tissue interactions in the early hematopoietic cell differentiation in Xenopus laevis embryo.

Abstract Bone morphogenetic protein-4 (BMP-4) has been shown as an essential factor in differentiation of the primitive blood cells in Xenopus laevis embryo. Organizer factors, in contrast, function as a negative regulator for the blood cell differentiation. Differentiation of both blood cells and muscle tissue are negatively regulated by the organizer activity. However, blood cells but not muscle tissue can differentiate in the organizer-depleted embryos produced by removal of vegetal cortex cytoplasm at the one-cell stage. Thus the blood cells are totally independent cells from the organizer activity. The down-stream target molecules of the BMP-4 signaling, such as vent-1/2 and msx-1/2 are the positive regulators for muscle tissue differentiation, whereas these factors do not promote blood cell formation. It has not yet been elucidated how the BMP-4 signaling promotes the differentiation of blood cells, but it is likely that transcription factors such as GATA-2, SCL, LMO-2, biklf and GATA-1 are at least involved in the initial blood program. Experiments using combined germ layer explants suggest that interaction between ectoderm and mesoderm at the gastrula stage is important for the blood cell formation in mesoderm. BMP-4 produced in the ectodermal cells is essential for this interaction. For understanding the whole program in the embryonic blood cell differentiation, it is important to elucidate the molecular mechanisms underlying the tissue-tissue interaction, in addition to the analysis of the regulatory cascade that takes place in the mesodermal cells.

Positive and Negative Regulation of the Differentiation of Ventral Mesoderm for Erythrocytes in Xenopus laevis

To elucidate the mechanism of determination and regulation of hemopoiesis in the early Xenopus embryo, explants of dorsal and ventral mesoderm from various stage embryos were cultured alone or combined with various tissues derived from the same stage embryo. Western blot analysis of larvae‐specific globin expression using monoclonal antibody L5.41 revealed that extensive erythropoiesis occurred in the explants of ventral mesoderm from st. 22 tailbud embryo, but not in those of dorsal mesoderm. Experiments using combined explants at this stage demonstrated that the in vitro differentiation of erythrocytes in the ventral mesoderm could be completely inhibited by the dorsal tissue, including neural tube, notochord, and somite mesoderm, but not by other mesoderms, gut endoderm, or forebrain. Subsequent explant studies showed that the notochord alone is sufficient for this inhibition. Furthermore, the ventral mesoderm explant from the st. 10+ early gastrula embryo was not able to differentiate into erythroid cells. However, small amounts of globin were expressed if ventral mesoderm of this stage was combined with animal pole cells which were mainly differentiated to epidermis. This stimulation was enhanced when both tissues were excised together without separation, while none of the other parts of st. 10+ embryo had this stimulatory effect. These observations found in the combined explants suggest that in vivo interactions between the ventral mesoderm and adjacent tissues are important for normal development of erythroid precursor cells.

Characterization of myeloid cells derived from the anterior ventral mesoderm in the Xenopus laevis embryo

A recent study revealed the presence of a unique population of myeloid cells in the anterior ventral (AV) mesoderm of Xenopus laevis embryo, as characterized by the expression of peroxidase 2 (POX2), which encodes for a leukocyte‐specific enzyme. The current report further characterized the POX2‐positive cells in terms of their contribution to hematopoiesis in tadpole and regulatory mechanism in differentiation. Grafting experiments with cytogenetically labeled tissues revealed that AV‐derived mesoderm supplies a transient population of migrating leukocytes in the mesenchyme of early tadpole. These cells were rarely found in blood vessels at any stages. Using a ventral marginal zone explant system, we demonstrated that dkk1, shown as a heart inducer in this system, has a strong ability to induce the expression of POX2. Injection of a high dose dkk1 RNA induced a heart marker while a low dose of dkk1 preferentially induced the expression of POX2, suggesting that dkk1 works as a morphogen to determine the different lineages. Overall results indicate that wnt signal inhibitors induce leukocytes at the early neurula stage and that these cells spread to the entire body and exist until the ventral blood island‐derived leukocytes appear in the body.

Xenopus msx-1 regulates dorso-ventral axis formation by suppressing the expression of organizer genes☆

We demonstrated previously that Xmsx-1 is involved in mesoderm patterning along the dorso-ventral axis, under the regulation of BMP-4 signaling. When Xmsx-1 RNA was injected into the dorsal blastomeres, a mass of muscle tissue formed instead of notochord. This activity was similar to that of Xwnt-8 reported previously. In this study, we investigated whether the activity of Xmsx-1 is related to the ventralizing signal and myogenesis promoting factor, Xwnt-8. Whole-mount in situ hybridization showed that Xmsx-1, Xwnt-8, and XmyoD were expressed in overlapping areas, including the ventro-lateral marginal zone at mid-gastrula stage. The expression of XmyoD was induced by the ectopic expression of either Xmsx-1 or Xwnt-8 in dorsal blastomeres, and Xwnt-8 was induced by the ectopic expression of Xmsx-1. On the other hand, the expression of Xmsx-1 was not affected by the loading of pCSKA-Xwnt-8 or dominant-negative Xwnt-8 (DN-Xwnt-8) RNA. In addition, Xmsx-1 RNA did not abrogate the formation of notochord if coinjected with DN-Xwnt-8 RNA. These results suggest that Xmsx-1 functions upstream of the Xwnt-8 signal. Furthermore, the antagonistic function of Xmsx-1 to the expression of organizer genes, such as Xlim-1 and goosecoid, was shown by in situ hybridization analysis and luciferase reporter assay using the goosecoid promoter construct. Finally if Xmsx-1/VP-16 fusion RNA, which was expected to function as a dominant-negative Xmsx-1, was injected into ventral blastomeres, a partial secondary axis formed in a significant number of embryos. In such embryos, the activity of luciferase, under the control of goosecoid promoter sequence, was significantly elevated at gastrula stage. These results led us to conclude that Xmsx-1 plays a central role in establishing dorso-ventral axis in gastrulating embryo, by suppressing the expression of organizer genes.

Distinct mechanisms control the timing of differentiation of two myeloid populations in Xenopus ventral blood islands.

Previous study has suggested that distinct populations of myeloid cells exist in the anterior ventral blood islands (aVBI) and posterior ventral blood islands (pVBI) in Xenopus neurula embryo. However, details for differentiation programs of these two populations have not been elucidated. In the present study, we examined the role of Wnt, vascular endothelial growth factor (VEGF) and fibroblast growth factor signals in the regulation of myeloid cell differentiation in the dorsal marginal zone and ventral marginal zone explants that are the sources of myeloid cells in the aVBI and pVBI. We found that regulation of Wnt activity is essential for the differentiation of myeloid cells in the aVBI but is not required for the differentiation of myeloid cells in the pVBI. Endogenous activity of the VEGF signal is necessary for differentiation of myeloid cells in the pVBI but is not involved in the differentiation of myeloid cells in the aVBI. Overall results reveal that distinct mechanisms are involved in the myeloid, erythroid and endothelial cell differentiation in the aVBI and pVBI.