Pinhead signaling regulates mesoderm heterogeneity via the FGF receptor-dependent pathway

ABSTRACTAmong the three embryonic germ layers, the mesoderm plays a central role in the establishment of the vertebrate body plan. The mesoderm is specified by secreted signaling proteins from the FGF, Nodal, BMP and Wnt families. No new classes of extracellular mesoderm-inducing factors have been identified in more than two decades. Here, we show that the pinhead (pnhd) gene encodes a secreted protein that is essential for the activation of a subset of mesodermal markers in the Xenopus embryo. RNA sequencing revealed that many transcriptional targets of Pnhd are shared with those of the FGF pathway. Pnhd activity was accompanied by Erk phosphorylation and required FGF and Nodal but not Wnt signaling. We propose that during gastrulation Pnhd acts in the marginal zone to contribute to mesoderm heterogeneity via an FGF receptor-dependent positive feedback mechanism.

Basic fibroblast growth factor positively regulates hematopoietic development

Recently identified BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, we show that BL-CFC expresses the receptor tyrosine kinase, Flk1, and thus we took advantage of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1(+) cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, we have analyzed ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, for their potential to generate BL-CFCs and Flk1(+) cells, to further define events leading to hemangioblast development. Our data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast.

Hemangioblast development and regulation

Hematopoietic and endothelial cell lineages are the first to mature from mesoderm in the developing embryo. However, little is known about the molecular and (or) cellular events leading to hematopoietic commitment. The recent applications of technology utilizing gene targeted mice and the employment of many available in vitro systems have facilitated our understanding of hematopoietic establishment in the developing embryo. It is becoming clear that embryonic hematopoiesis occurs both in the extra-embryonic yolk sac and within the embryo proper in the mouse. The existence of the long pursued hemangioblast, a common progenitor of hematopoietic and endothelial cells, is now formally demonstrated. Based on this new information, many studies are being conducted to understand hematopoietic commitment events from mesoderm. In this review, we will first discuss the establishment of the hematopoietic system with special emphasis on the most primitive hematopoietic committed cells, the hemangioblast. We will then discuss mesoderm-inducing factors and their possible role in hematopoietic lineage commitment.Key words: hematopoietic commitment, hemangioblast, in vitro embryonic stem cell differentiation.

Bix1, a direct target of Xenopus T-box genes, causes formation of ventral mesoderm and endoderm

Brachyury, a member of the T-box gene family, is required for posterior mesoderm and notochord differentiation in vertebrate development, and mis-expression of Xenopus Brachyury causes ectopic mesoderm formation. Brachyury is a transcription activator, and its ability to activate transcription is essential for its biological function, but Brachyury target genes have proved difficult to identify. Here we employ a hormone-inducible Brachyury construct and subtractive hybridization to search for such targets. Using this approach we have isolated Bix1, a homeobox gene expressed both in the marginal zone of Xenopus and in the vegetal hemisphere. Expression of Bix1 is induced in an immediate-early fashion by mesoderm-inducing factors such as activin as well as by the products of the T-box genes Xbra and VegT (also known as Antipodean, Brat and Xombi). Activation of Bix1 in response to Xbra is direct in the sense that it does not require protein synthesis, and both Xbra and VegT activate expression of a reporter gene driven by the Bix 5′ regulatory region, which contains an Xbra/VegT binding site. Mis-expression of low levels of Bix1 causes formation of ventral mesoderm, while high levels induce endodermal differentiation. These results suggest that Bix1 acts downstream of both VegT and Xbra to induce formation of mesoderm and endoderm.

Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra

Analysis of gene function in Xenopus development frequently involves over-expression experiments, in which RNA encoding the protein of interest is microinjected into the early embryo. By taking advantage of the fate map of Xenopus, it is possible to direct expression of the protein to particular regions of the embryo, but it has not been possible to exert control over the timing of expression; the protein is translated immediately after injection. To overcome this problem in our analysis of the role of Brachyury in Xenopus development, we have, like Kolm and Sive (1995; Dev. Biol. 171, 267–272), explored the use of hormone-inducible constructs. Animal pole regions derived from embryos expressing a fusion protein (Xbra-GR) in which the Xbra open reading frame is fused to the ligand-binding domain of the human glucocorticoid receptor develop as atypical epidermis, presumably because Xbra is sequestered by the heat-shock apparatus of the cell. Addition of dexamethasone, which binds to the glucocorticoid receptor and releases Xbra, causes formation of mesoderm. We have used this approach to investigate the competence of animal pole explants to respond to Xbra-GR, and have found that competence persists until late gastrula stages, even though by this time animal caps have lost the ability to respond to mesoderm-inducing factors such as activin and FGF. In a second series of experiments, we demonstrate that Xbra is capable of inducing its own expression, but that this auto-induction requires intercellular signals and FGF signalling. Finally, we suggest that the use of inducible constructs may assist in the search for target genes of Brachyury.

FGF is a prospective competence factor for early activin-type signals in Xenopus mesoderm induction

Normal pattern formation during embryonic development requires the regulation of cellular competence to respond to inductive signals. In the Xenopus blastula, vegetal cells release mesoderm-inducing factors but themselves become endoderm, suggesting that vegetal cells may be prevented from expressing mesodermal genes in response to the signals that they secrete. We show here that addition of low levels of basic fibroblast growth factor (bFGF) induces the ectopic expression of the mesodermal markers Xbra, MyoD and muscle actin in vegetal explants, even though vegetal cells express low levels of the FGF receptor. Activin, a potent mesoderm-inducing agent in explanted ectoderm (animal explants), does not induce ectopic expression of these markers in vegetal explants. However, activin-type signaling is present in vegetal cells, since the vegetal expression of Mix.1 and goosecoid is inhibited by the truncated activin receptor. These results, together with the observation that FGF is required for mesoderm induction by activin, support our proposal that a maternal FGF acts at the equator as a competence factor, permitting equatorial cells to express mesoderm in response to an activin-type signal. The overlap of FGF and activin-type signaling is proposed to restrict mesoderm to the equatorial region.

Expression of inhibin subunits and follistatin during postimplantation mouse development: decidual expression of activin and expression of follistatin in primitive streak, somites and hindbrain

Members of the activin family are believed to act as mesoderm-inducing factors during early amphibian development. Little is known, however, about mesoderm formation in the mammalian embryo, and as one approach to investigating this we have studied activin and follistatin expression during early mouse development. Activins are homo- or heterodimers of the beta A or beta B subunits of inhibin, itself a heterodimer consisting of one of the beta subunits together with an alpha subunit. Follistatin is a single-chain polypeptide which inhibits activin function. Expression of the inhibin alpha chain could not be detected in embryonic or extraembryonic tissues at any of the stages studied (5.5 to 8.5 days) and expression of the beta A and beta B subunits could only be observed in the deciduum in cells surrounding the embryo. Expression of follistatin could also be detected in the deciduum, but in a pattern complementary to that of the beta subunits. Embryonic expression of follistatin first occurred in the primitive streak, and at later stages transcripts were detectable in the somites and in rhombomeres 2, 4 and 6 of the hindbrain. These results are consistent with a role for activin in mesoderm formation in the mouse embryo, and suggest functions for follistatin in addition to its role as an inhibitor of activin.