Ruiz i Altaba A and Melton DA (1989), Bimodal and graded expression of the Xenopus ho...

XB-ART-26796

Development 1989 May 01;1061:173-83.

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Bimodal and graded expression of the Xenopus homeobox gene Xhox3 during embryonic development.

Ruiz i Altaba A , Melton DA .

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A Xenopus laevis homeobox gene, Xhox3, has been isolated using the homeobox of the Drosophila pair-rule gene even skipped as a hybridization probe. Xhox3 is first transcribed at the midblastula transition; RNA levels peak at the early neurula stage and decrease thereafter. During the early period of Xhox3 expression, the gastrula and neurula stages, transcripts are found in a graded fashion along the anteroposterior (A-P) axis in the mesoderm and are most concentrated at the posterior pole. In the late period of expression, the tailbud and tadpole stage, transcripts are concentrated at the two ends of the embryo: in the anterior nervous system and posterior tail bud. Analysis of Xhox3 expression in experimentally perturbed embryos shows that different A-P fates in the mesoderm are correlated with different levels of Xhox3 expression. Based on these results and those with other frog homeobox genes, we propose a role for homeobox genes in the patterning of the A-P embryonic axis.

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Species referenced: Xenopus laevis
Genes referenced: actl6a evx1 ncam1 tbx2

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	Fig. 1. (A) Xhox3 genomic and cDNA plasmid clones. The black box represents the homeobox. The broken lines show the position of the different probes used for hybridization experiments: GP, genomic probe; RP, RNase protection probe. The Eco RI sites shown in parenthesis are derived from the cDNA cloning protocol. (B) Amino acid sequence of the even skipped (Macdonald et al. 1986), Xhox3 and XhoxlA (Harvey et al. 1986) homeoboxes. The brackets represent the putative helix 2 and helix 3 domains of the helix-turn-helix motif (see Laughon & Scott, 1984; Shepherd et al. 1984; Ptashne, 1986). The arrow indicates the position of the splice site deduced from a comparison of the genomic and cDNA clones. The vertical lines depict similar amino acid residues between homeoboxes.

	Fig. 3. Graded distribution of Xhox3 transcripts along the anteroposterior axis during early development. The schematic respresentations of the given stages are shown in the top. Stage 11, mid-gastrula; Stage 13, late gastrula-early neurula; Stage 15, mid-neurula; Stage 19, late neurula. D, dorsal; V, ventral; A, anterior; M, middle; P, posterior. Note the graded distribution of Xhox3 transcripts with the highest levels at the posterior pole. As a control for RNA recovery and quantification, the same RNA samples were used to determine the abundance of EF-Ia mRNA which is a reflection of cell number (Krieg & Melton, 1989). Ten and one embryo equivalents were used for the Xhox3 and EF-Ia probes, respectively. Exposure periods were 4 hours for EF-Ia and 4 days for Xhox3.
	Fig. 4. Xhox3 expression in exogastrulated embryos. Embryos were induced to exogastrulate by incubation in high salt. Neurula stage exogastrulae were carefully dissected into ectodennal, mesodermal and endodermal parts. RNA was extracted and assayed for the presence of Xhox3, muscle-specific actin and EF-la transcripts by RNase protection. Note that muscle-specific (MS)-actin and Xhox3 are expressed only in mesoderm. Ten, one and three embryo equivalents were used for the Xhox3, EF-la and MS-actin probes. Exposure periods were 4 hours for EF-la and MS-actin and 4 days for Xhox3.
	Fig. 5. Localization of Xhox3 •J/ transcripts in late embryos. Tailbud (stage 26) or swimming tadpoles (stage 36) were dissected along the dorsoventral and anteroposterior - axes, and the parts were assayed for the presence of Xhox3 transcripts by RNase protection. As controls, EF-Ia, muscle-specific actin, and N-CAM probes were used with the same RNA samples. Note that the MS-actin reflects the position of the somites (mesoderm) and N-CAM that of the CNS (neural tissue). The N-CAM signal in the most posterior regions is below the level of detection in this assay, probably because of the small size of the spinal cord in this region. D, dorsal; C, central; V, ventral. Numbers refer to the different consecutive dissected parts along the A-P axis. Ten embryo equivalents were used for the Xhox3 and N-CAM probes. One and three embryo equivalents were used for the EF-Ia and MSactin probes, respectively. Exposure periods ranged from 4 hours {EF-Ia and MS-actin) to 4 days (Xhox3 and N-CAM).
	Fig. 6. In situ localization of Xhox3 transcripts in the brain of tadpole stage (stage 36) embryos. A sagittal section hybridized with a 35S-labelled Xhox3 antisense RNA probe is shown after exposure for one month. Xhox3 transcripts are found only in a small area of the brain (large arrow). The white arc around the dorsal half of the eye (small arrow) is due to the presence of pigment cells in the eye of heterozygote albino embryos at this late stage. Anterior is to the left, dorsal is on top. A line drawing of the section (top panel) is shown to orient the hybridization signal in the dark field photograph (bottom panel), eg, cement gland; e, eye; en, endoderm; hb, hind brain; mb, mid brain; n, notochord.
	Fig. 7. Localization of Xhox3 transcripts in feeding larvae. Stage 45 tadpoles were dissected into five parts along the A-P axis. RNA was extracted and assayed for the presence of Xhox3 and EF-la transcripts by RNase protection protocols. The brain of similarly staged tadpoles was dissected and processed in the same way. Ten and one larvae equivalents were used for the Xhox3 and EF-la probes respectively. Exposure times were as in Fig. 4. A, anterior; P, posterior; T, total or whole larvae; B, brain.
	Fig. 8. Xhox3 expression in u.v. and Li treated embryos. Embryos were treated with u.v. or Li and allowed to develop to the neurula stage, u.v.-treated embryos with no head and little trunk and lithium-treated embryos with little or no trunk were selected. RNA from these embryos was assayed by RNase protection assays for the presence of Xhox3 and EF-la transcripts. Ten and one embryos were used for the Xhox3 and EF-la probes respectively. Exposure times were as in Fig. 4.
	in control, u.v. and Li treated embryos during early development. The plotted values were obtained by scanning densitometry of the products of RNase protection assays. Developmental periods and N/F stages are shown below the graph. MBT, midblastula transition; G, gastrulation; N, neurulation; TB, tailbud and TP, tadpole stages. Full bars indicate the periods of Xhox3 expression when transcripts are clearly localized. Broken lines indicate the extent of the expression periods where localization is not well defined.