Kumano G and Smith WC (2000), FGF signaling restricts the primary blood islan...

XB-ART-9860

Dev Biol 2000 Dec 15;2282:304-14. doi: 10.1006/dbio.2000.9937.

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FGF signaling restricts the primary blood islands to ventral mesoderm.

Kumano G , Smith WC .

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According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. It is proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. We present evidence here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. We further observed that inhibition of FGF signaling with a dominant negative receptor resulted in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories was observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. On the other hand, we found no evidence that manipulation of BMP signaling, either positively or negatively, altered the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a bmp4 cd4 eomes fgf4 igf2bp3 mix1 myod1 nodal2 nodal3.1 nog tbx2 tbxt ventx1.2

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	FIG. 1. Xenopus fate map and 32-cell stage designations. The revised Xenopus fate map for the blastula marginal zone (Lane and Smith, 1999). In this fate map, the VBIs are shown to arise not only from the “ventral marginal zone,” but also from mesoderm situated near the Spemann organizer. Furthermore, blastomeres in the “ventral marginal zone” are shown to contribute to both dorsal (somite) and ventral (VBI) posterior mesoderm. Somites (pink) map to the animal region of all sectors of the marginal zone (blastomeres C1–C4, B1–B4, and A1–A4). Codistribution of somites (pink) with notochord (blastomeres C1, C2, B1, B2, B3, A1, A2, and A3) is shown in green. Blood arises from the leading edge mesoderm (orange), situated in the vegetal region of the marginal zone (blastomeres C1–C4 and D1–D4). The approximate cleavage plains for the 32-cell stage embryo are shown, and labeling of blastomeres is indicated according to Nakamura and Kishiyama (1971). Only notochord, somites, and the VBIs are shown for mesoderm. Lateral plate and other mesodermal territories are omitted for simplicity, and each territory labeled may also include other fates.
	FIG. 2. FGF signaling is essential for dorsoventral mesoderm patterning. (A) Control marginal zone explants containing B4 and C4 descendants. Explants excised at stage 9 were cultured to stage 32 for detection of globin expression by in situ hybridization. Note that globin expression is localized to the vegetal poles of the explants as shown previously (Kumano et al., 1999). (B) Marginal zone explants as in (A) with the exception that both the B4 and the C4 blastomeres were injected with 100 pg of XFD RNA. Globin expression is expanded toward the animal pole of the explants. The center regions of the explants without globin expression are probably endodermal derivatives. (C) Marginal zone explants as in (A) with the exception that the B4 blastomeres were injected with 100 pg of XFD RNA. globin expression was detected at the animal, as well as at the vegetal, pole. (D) Marginal zone explants which had been injected with 500 pg of XeFGF RNA into both the CD4 blastomeres, the mother cells of the C4 and the D4. No globin expression was detected by in situ hybridization at stage 32. All explants (A–D) are oriented with animal poles (pigmented) to the top. (E) Posterior region of a stage 38 Xenopus embryo injected at 32-cell stage with 200 pg of lacZ RNA in the B4 and C4 blastomeres. LacZ staining was observed in the somites (segments, arrowheads), the ventral region, and the epidermis. (F) Posterior region of an embryo treated the same as (E) with the exception that 100 pg of XFD RNA was co-injected with the lacZ RNA. This embryo showed abnormal tail formation and no LacZ staining in somites.
	FIG. 3. Noggin does not induce muscle actin in XFD RNA-injected explants. (A) Control marginal zone explants containing B4 and C4 derivatives from embryos injected with 50 pg of GFP RNA in both the B4 and the C4 blastomeres at the 32-cell stage. Shown is staining for muscle actin expression by in situ hybridization. No expression is observed under these conditions. (B) Marginal zone explants as in (A) with the exception that 25 pg of pCSKA-noggin plasmid was co-injected with the GFP RNA. All explants express muscle actin. (C) Marginal zone explants as in (B) with the exception that XFD RNA was co-injected with the GFP RNA and the pCSKA-noggin plasmid. Almost no muscle actin expression was observed when explants were stained as in (A) and (B).
	FIG. 4. FGF signaling establishes unique expression domains along animal/vegetal axis of midgastrula embryos. (A) Control embryo at stage 10.5 showing Xbra expression by in situ hybridization. The expression is not seen in the region just above the blastopore lip (arrowhead). (B) Embryo injected in B4 and C4 with XFD and lacZ RNAs. In situ hybridization for Xbra at stage 10.5 is shown. No Xbra expression in the region of LacZ staining was observed. (C) Control embryo at stage 11 stained for myoD expression by in situ hybridization. Expression is observed widely around the marginal zone, excluding the Spemann organizer. (D) Embryo injected at 32-cell stage in both B4 and C4 with 100 pg of XFD and 200 pg of lacZ RNAs. Shown is staining at stage 11 for myoD expression by in situ hybridization and for LacZ by X-gal staining. Note the gap in myoD expression localizing with the LacZ staining. (E) Double in situ hybridization at stage 10.5 for Xbra (red, arrow) and Xnr-2 (black/purple, arrowhead pointing down). Duplicates are shown. The expression domains of those genes were exclusive along the animal–vegetal axis. The bottom picture is an enlargement of the marginal zone in the top embryo. (F) Double in situ hybridization at stage 10.5 for Xbra (red, arrow) and Xnr-2 (black/purple, arrowhead pointing down) in embryos that had been injected with XFD RNA in the marginal zone (B4 and C4 blastomeres). Duplicates are shown. The marginal zones (bottom in each embryo) on the injected sides no longer express Xbra and show expansion of Xnr-2 toward the animal pole (arrowhead). The bottom picture is again an enlargement of the marginal zone in the top embryo. The blastopore lips of the embryos in (E) and (F) are indicated by arrowheads pointing up. Embryos in (A–F) are vegetal views, orientated with the Spemann organizer at the top. (G) Lateral view of representative embryo as shown in (F). (H) Lateral view of control embryo at stage 10 stained for eomesodermin expression by in situ hybridization. (I) Lateral view of embryo which had been injected with XFD and lacZ RNA in the B4 and C4 blastomeres. eomesodermin expression by in situ hybridization and LacZ staining are shown. XFD did not effect eomesodermin expression. (J) Double in situ hybridization at stage 10.5 for Xbra (red, arrow) and Mix.1 (black/purple, arrowhead). (K) The same as in (J) with the exception that the embryo had been injected with XFD RNA in the B4 and C4 blastomeres. Note that only Mix.1 expression persists on the injected side (arrowhead), whereas Xbra expression is eliminated. Embryos in (J) and (K) are vegetal views, oriented the same as (A–F).
	FIG. 5. dnBMP-R or dnXTcf-3 injection does not alter the early mesoderm patterning along the animal–vegetal axis. (A) Stage 10.5 embryo which had been injected with 250 pg of dnBMP-R RNA in both B4 and C4 blastomeres at the 32-cell stage. Representative embryo is shown with double in situ hybridization staining for Xbra (red, arrow) and Xnr-2 (black/purple, arrowhead). Note that the Xbra and Xnr-2 expression pattern is the same as that in control embryos (Fig. 4E). (B) Representative embryo treated the same as in (A) but subjected to in situ hybridization for Xvent-1. Note that Xvent-1 expression is eliminated from the injected side (arrowhead). (C) Control stage 10.5 embryo showing Xnr-3 expression by in situ hybridization. In (D) and (E) embryos were injected at the 4-cell stage with 500 pg of dnXTcf-3 RNA in the equatorial region of the two blastomeres containing the presumptive Spemann organizer. (D) dnXTcf-3 RNA-injected embryo at stage 10.5 embryo showing Xnr-3 expression by in situ hybridization. Note that Xnr-3 expression is eliminated, indicating that the wnt pathway had been inhibited. (E) Representative stage 10.5 dnXTcf-3 RNA-injected embryo subjected to double in situ hybridization for Xbra (red, white arrow) and Xnr-2 (black/purple, white arrowhead). The normal pattern of expression of these two genes is maintained on the uninjected side despite the absence of the Spemann organizer. Why Xbra and Xnr2 are interrupted on the injected side is not known.
	FIG. 6. globin expression in dnXTcf-3- and BMP-4-injected embryos is restricted to the leading edge mesoderm. (A) Suggested gastrulation movements of the leading edge mesoderm (yellow band). Normal and ventralized embryos are shown at the end of gastrulation. In normal embryos the leading edge mesoderm converges ventrally and ends up the ventral midline of the tadpole. Anterior is to the left. In “ventralized” embryos all sectors of the leading edge mesoderm migrate straight up toward the animal pole and come to lie under the pole. Animal pole is to the left. Blastopores are shown in black spots at the right of the embryos. (B) Stage 41 “ventralized” embryos produced by injection of dnXTcf-3 RNA in the presumptive organizer region at the 4 cell stage. The descendents of the C4 blastomeres were marked by lacZ RNA injection at the 32-cell stage. The location of the C4 descendants is shown by X-gal staining (green), and globin expression is indicated by black/purple in situ hybridization signal. (C) Stage 41 embryos treated the same as in (B) with the exception that embryos were injected with BMP-4 RNA in all the cells at the 4-cell stage instead of dnXTcf-3 in the presumptive organizer region. Note that in (B) and (C) globin expression is restricted to the leading edge mesoderm and that the C4 descendants show a small portion of overlap with the domain of globin-expressing cells. Blastopores of the embryos in (B) and (C) are shown by “bp.” (D, E) Close-up of section through globin-expressing region of BMP-4 RNA-injected (D) and dnXTcf-3 RNA-injected (E) embryos. The BMP-4 RNA injection results in globin expression in the surface cells, unlike in the dnXTcf-3 RNA-injected embryos.