Northrop JL and Kimelman D (1994), Dorsal-ventral differences in Xcad-3 expression...

XB-ART-21651

Dev Biol 1994 Feb 01;1612:490-503. doi: 10.1006/dbio.1994.1047.

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Dorsal-ventral differences in Xcad-3 expression in response to FGF-mediated induction in Xenopus.

Northrop JL , Kimelman D .

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We have identified a dorsal-ventral difference in the specification of mesoderm in vivo by examining the effect of the dominant-negative FGF receptor on a new member of the Xenopus caudal gene family, Xcad-3. Xcad-3 is expressed throughout the marginal zone during the gastrula stages and serves as a useful marker for events occurring within the mesoderm. Disruption of the FGF signaling pathway by the dominant-negative FGF receptor, disrupts the Xcad-3 expression pattern, eliminating expression preferentially from the dorsal regions of the embryo. We also find that the expression of the Xenopus brachyury homolog, Xbra, is more readily eliminated from the dorsal than the ventral region of the embryo by the dominant-negative FGF receptor, indicating that the observed dorsal-ventral differences are not unique to Xcad-3. These results demonstrate the importance of regional effects on FGF-mediated induction in vivo and suggest that FGF-dependent expression of mesodermal genes depends upon the localization of other factors which establish dorsal-ventral differences within the embryo.

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Species referenced: Xenopus
Genes referenced: cad cdx4 fgf2 fgfr1 mif sprn tbx2 tbxt tlr1

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	FIG. 1. (A) Amino acid comparison of the caudal homeobox sequence from several species. The amino acid sequence of the homeobox is shown for thr eeXen&Jl'US caudal genes (Blumberg et al, 1991; this work), the mouse Cdx gene (Duprey et aL, 1988), the chicken CHvx-cad gene (Frumkin et aL , 1991). zebrafish ZF'·oodl {Joly et a.l., 1992), and the Drosophila caudal gene (Mlodzik etal, 1985). (B) eDNA sequence and predicted amino acid sequence for Xcad-!1. The XC<Ul-9 sequence is sho....-n in uppercase letters with the predicted amino acid sequence shown underneath in three-letter code. The homeobox sequence is underlined. (C) Schematic of the eDNA clone of Xca4·3. The open reading frame is boxed and the homeobox region is shaded. Antisense RNA probes synthesized for RNase protections (probe 11) and for in 8it·u hybridizations (probes b and c) are indicated.
	FIG. 2. Temporal ex pression of Xcarl -3. Twenty micrograms of RNA was extracted from em hryos at t he indicated stages and analy-.:ed by RNase protection using 112P-Iabeled RNA synthesized from the 3' end of Xcad-S (Fig. 1C, proben). Expression of Xcad-S was direetly compared to tlle expression profile o£ EF'- Ia by including the antisense probe for EF-la in the s>.lmc reactions. Note that the EF-1a gene is first expressed at the onset of zygotic transcription and increases in expression un til early gastrulation when it. is CXJ>ressed at a constant rat~ (Krieg et aL, 1989).
	F1c. 3. Spatial distribution of Xcad-3 transcripts in the early Xenopus embryo. Localization of Xcad-3 transcripts was determined by wholemount in situ hybridization using a digoxygenin·labeled antisense RNA probe (Fig. 1C, probe b). Embryos inA-Care albino embryos and those shown in D-F are pigmented embryos which have been cleared in benzyl alcohol:benzyl benzoate. (A) St.nge 8 embryo. A faint stain of Xccul-3, barely above backgrouud levels, is detected. (B) Stage 9 embryos. At stage 9, the animal cap (An) distinctly stains for Xcad-S expression while there is no visible stain in the vegetal hemisphere (Vg). The unlabeled embryos are posi tioned with their vegetal side up. (C) Stage 10 embryos. Xcad-3 is expressed in a band at the mnrginal zone. The uppermost embryo is tilted such that the ma..ginal zone is perpendicular to the page and a distinct band of stain tan be seen encircling the embryo. The embryo at the lower left is shown in a vegetal view and the dorsal blastopore lip is indicated. The embryo to the riiht is viewed from the animal hemisphere. (D) Stage 12 embryo. The band of Xcad-a expression encircles the blastopore lip. The embryo is oriented with the dorsal side up. (E) Stage 15 embryo. Xcad-3 is expressed in the dorsal and ventral posterior mesoderm and in the neural fold region. The embryo is oriented with anterior to the right and dorsal to the top of the page. (F) Stage 22 embryos. Xcad-3 is expressed primarily in the posterior neural tube. The embryos are oriented with anterior to the right and dorsal to the top of the page.
	FIG. 4. Induction of Xcad-3 expression by activin and bFGF. Animal caps were dissected at stage 8-9 and incubated in the presence or absence of either recombinant Xenopus bFGF at 100 ng/ml or activin in the form of a 1:10 dilution of XTC- MIF. Animal caps were harvested at stage 12 and the RNA was analyzed by RNase protection.

	FIG. 6. Effect of localized injection of the dominant-negative FGF receptor on Xcad-3 expression. Uninjected embryos (A, E) or embryos injected twice laterally (B, F), injecced twice dorsally (C, G), or injected twice ventrally (D, H) into the marginal zone with 1-2 ng of dominant negative FGF receptor RNA per injection are shown. The embryos were allowed to develop until stage 12 and then fixed and analy~ed by whole-mount in llit1t hybridization fc>r XtXUl-:J expression. The view is vegetal, looking down onto the blastopore, and dorsal is oriented toward the top of the page.
	FIG. 7. Titer of dominant-negative FGF receptor RNA injected into the dorsal and ventral regions- effect on Xcad-3 expression. Embryos were injected both twice dorsally and twice ventrally (a total of four injections per embryo) with increasing amounts of RNA encoding the dominant-negative FGF receptor. These embryos were allowed to develop until stage 12 and then stained for Xcad-3 expression by in situ hybridization. Embryos were injected with 0 ng (A), 0.2 ng (B), 0.4 ng (C), 0.8 ng (D), 1.2 ng (E), or 1.8 ng (F) of RNA per injection. All embryos were cleared and photographed looking down on the blastopore lip with the dorsal region oriented toward the top of the page.
	FIG. 8. Titer of dominant-negative FGF receptor RNA injected into the dorsal and ventral regions- effect on Xbra expression. Embryos were injected both twice dorsally and twice ventrally (a total of {our injections per embryo) wi th increasing amounts of RNA encoding the dominant· negative FGF receptor. These embryos were allowed to develop until stage 12 and then stained for Xbra expression by in situ hybridization. Embryos were injected with 0 ni (A), 0.04 ng (B), 0.08 ng (C), 0.16 ng (D), 0.24 ng (E), or 0.36 ng (F) of RNA per injection. All embryos were cleared and photographed looking down on the blastopore with the dorsal region oriented toward the top of the page.