tbx6 is expressed by ventrolateral mesendodermal precursors in the early zebrafish gastrula. (A) A simplified fate map of the early zebrafish gastrula (Kimmel et al., 1990). At this stage the blastoderm is shaped as an inverted cup covering the animal pole of the yolk cell (gold). Mesoderm and endoderm arise from involuting cells initially present at the blastoderm margin (purple). The remaining cells give rise to ectoderm. Mesodermal tissues are formed from prospective tissue precursors that are organized in the following dorsal (D) to ventral (V) order: notochord (noto.), somites (som.), pronephros (pron.), blood (blo.). (B and B′) Oblique lateral views (animal pole up) of early gastrulae showing the distribution of tbx6 transcripts revealed by whole-mount in situ hybridization. tbx6 is expressed by a continuous arc of cells at the margin of the blastoderm, corresponding to the mesendodermal precursors. The dorsal view in B′ illustrates the absence of the tbx6 expression in dorsal marginal cells. (C) A section through the margin of an embryo in which tbx6 transcripts were detected by in situ hybridization (purple stain) and the No Tail protein was detected by immunohistochemistry (brown nuclear stain) reveals coexpression of the two genes in marginal cells. (D) In situ hybridization to detect transcripts of both tbx6 and the dorsalspecific gene goosecoid indicates that tbx6 is not expressed in the dorsal mesoderm. In this animal pole view of a midgastrula (7 h) embryo, the recently involuted goosecoid-expressing cells (*) appear as a knot of stained cells at the center of the region devoid of tbx6 gene expression. (E) An animal pole view of an early gastrula (5.5 h) embryo showing the distribution of both tbx6 and goosecoid transcripts. At the onset of gastrulation the domain of tbx6 expression (light blue arc) is continuous, and perhaps slightly overlapping, with the domain of goosecoid expression (dark blue staining, *). (F, G) tbx6 expression is down-regulated in embryos dorsalized by exposure to lithium ions. (F) A lateral view of tbx6 transcripts in a control early gastrula. (G) A lateral view of tbx6 transcripts in an early gastrula of an embryo that had been exposed at the 32- to 64-cell stage to 0.3 M LiCl for 10 min.

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Stage: 50%-epiboly

tbx6 and no tail are transcribed in response to mesoderm induction. Animal caps, removed from 2000- to 4000-cell embryos, were incubated for 5 hr in the absence (Control lane) or the presence of activin or bFGF, provided at the indicated concentrations. RNA was harvested from groups of 10 caps and transcribed genes were detected by RT-PCR. The ker8 (cytokeratin 8) gene is specific to the extraembryonic enveloping layer and serves as a control for the viability of the animal caps and the recovery of RNA.

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Stage: High

Northern analysis of the expression of dorsal- and ventral-specific genes at defined developmental time points. Each lane contained RNA harvested from developmentally staged embryos (h, hours of development at 28.5°C). Zygotic gene transcription begins around 3.5 h (Kane and Kimmel, 1993). Transcripts of the panmesodermal gene no tail (2.5-kb transcript) and the organizer-specific gene goosecoid (gsc; predominant transcript 1.4 kb) were first detected at 4.5 h. Although snail1 RNA (1.9-kb transcript) is maternally supplied, a burst of zygotic transcription, corresponding to snail1 gene expression in dorsal mesoderm (Hammerschmidt and Nusslein-Volhard, 1993; Thisse et al., 1993), was detected at 4.5 h. In contrast, transcripts of the ventrally restricted genes eve1 (1.4-kb transcript) and tbx6 (predominant transcript 2.7kb, additional transcript 3.5 kb) were first detected at 5.5 h. eve1, whose domain of expression includes marginal cells of the blastoderm, is maintained in noninvoluting cells of the epiblast. tbx6 is expressed by marginal cells and their involuting derivatives. A photograph of the 18S ribosomal band of the ethidium bromide-stained Northern gel is shown as a loading control.

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Stage: Dome

tbx6 expression is maintained in undifferentiated derivatives of the ventrolateral mesendodermal precursors. (A and A′) Lateral (A, dorsal is right) and dorsal (A′) views of tbx6 expression in late gastrulae (95% epiboly, 9.5 h). During gastrulation, tbx6 continues to be expressed at the margin of the blastoderm and is maintained in recently involuted cells as they move rostrally and converge dorsally toward the developing chordamesoderm (*). (B) A transverse section through the axis (at the level indicated by the bar in A′) of a late gastrula (95% epiboly) embryo indicates that tbx6 is specifically expressed in involuting hypoblast cells. The presumptive notochord (*, dorsal) is to the right. (C) A dorsal view of the midaxial region of a two-somite (11 h) embryo in which tbx6-expressing cells (blue staining) are identified by in situ hybridization and No Tail protein-expressing cells (brown) are identified by immunohistochemistry. The domain of tbx6 expression lies immediately adjacent to the axial mesoderm identified by its expression of the No Tail protein. (D) A ventral view of a 15-h embryo shows that tbx6 is expressed in the region of the early developing tail as an arc of cells whose apex is located at the posterior tip of the tail and whose wings extend anteriorly, bordering the axial mesoderm. A lateral view of a similar embryo (Fig. 6G) shows that tbx6 expression is confined to the ventrolateral aspects of the developing tail at this stage. (E and F) Lateral views of two approximately 24-h embryos show the changing expression of tbx6 in the developing tail and in the blood precursors. The embryo in D is slightly younger than the embryo in E. As tail development proceeds, tbx6 expression becomes progressively restricted to smaller domains at the posterior tip of the tail. In the 24-h embryo, tbx6 is also expressed in blood precursor cells that originate in the intermediate cell mass (arrowhead) and that move rostrally along the ventral midline (arrow) prior to entering the circulation. (G) A dorsal view (anterior to the top) of the midtrunk region of a 26-h embryo shows that blood precursor cells maintain expression of tbx6 as they move laterally over the yolk cell to enter the circulation. Expression of tbx6 among circulating blood cells has not been detected.

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Stage Range: 90%-epiboly to Prim-5

Expression of tbx6 in sibling wild-type and no tail embryos. Staged embryos from matings between no tail/+ heterozygotes were processed to detect tbx6 transcripts (blue–black) by in situ hybridization. 6 and 9 h no tail mutant embryos were identified following immunohistochemical staining (brown) to detect the No Tail protein. At subsequent developmental stages, the no tail embryos could be identified unambiguously by morphological criteria. (A and B) Animal pole views of early gastrulae (6 h; * indicates dorsal) illustrate that tbx6 is initially expressed normally in no tail mutant embryos. (C and D) Dorsal views (animal pole up) of late gastrulae (90% epiboly, 9 h) show that the overall pattern of tbx6 expression in the hypoblast is normal. (E and F) Soon after the tail bud stage (12 h, posterior view), the domain of cells that express tbx6 and the extent of tbx6 expression appear diminished in no tail embryos. (G–J) As tail development proceeds, tbx6 expression is dramatically down-regulated in no tail embryos. Only a small population of cells near the yolk cell of mutant embryos continues to express tbx6 at 15 –16 h. By 18 h, only cells in the region of the intermediate cell mass continue to express the gene in mutant embryos.

Acknowledgments
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Reprinted from Developmental Biology, 183(1), Hug, B., Walter, V., and Grunwald, D.J., tbx6, a brachyury-related gene expressed by ventral mesendodermal precursors in the zebrafish embryo, 61-73, Copyright (1997) with permission from Elsevier. Full text @ Dev. Biol.