D'Souza A et al. (2003), Molecular components of the endoderm specificat...

XB-ART-5958

Dev Dyn 2003 Jan 01;2261:118-27. doi: 10.1002/dvdy.10201.

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Molecular components of the endoderm specification pathway in Xenopus tropicalis.

D'Souza A , Lee M , Taverner N , Mason J , Carruthers S , Smith JC , Amaya E , Papalopulu N , Zorn AM .

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Xenopus laevis has been instrumental in elucidating a conserved molecular pathway that regulates vertebrate endoderm specification. However, loss-of-function analysis is required to resolve the precise function of the genes involved. For such analysis, antisense oligos and possibly forward genetics are likely to be more effective in the diploid species Xenopus tropicalis than in the pseudotetraploid Xenopus laevis. Here we have isolated most of the tropicalis genes in the endoderm specification pathway, specifically, tVegT, tMixer, tMix, tBix, tGata6, tSox17alpha, tSox17beta, tFoxA1, tHex, and tCerberus, which lack the redundant copies that are found in laevis. In situ hybridization analysis has revealed identical expression patterns between the orthologous tropicalis and laevis endoderm genes, thus suggesting conserved genetic functions. Furthermore, we noted that the smaller tropicalis embryos gave better probe penetration than in laevis whole-mount in situ hybridizations-allowing us to visualize transcripts in the deep endoderm in tropicalis, which is difficult in laevis. This study illustrates how an entire genetic pathway can be quickly transferred from laevis to tropicalis due to high sequence conservation between the sister species and the large number of tropicalis-expressed sequence tags that are now available.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: bix1.1 cer1 foxa1 gata6 hhex mix1 mixer sox17a sox17b.1 vegt

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	Figure 1. In situ hybridization. A comparison of the whole-mount in situ hybridization procedure between Xenopus tropicalis and Xenopus laevis embryos. A: Whole-mount in situ hybridization with species-specific Sox17 alpha probes to laevis (left) and tropicalis (right) gastrula. A ventral view with dorsal up is shown. Probes penetrate more efficiently in tropicalis whole-mount hybridizations than they do in laevis embryos. Compare a midsagittal, Vibratome section of a laevis gastrula previously hybridized by whole-mount with a Sox17 alpha probe (B) with a laevis embryo for which the Sox17 alpha in situ hybridization was preformed on a paraffin section (C). Notice the lack of staining in the central deep endoderm of the sectioned laevis whole-mount. D: In contrast, a midsagittal, Vibratome section of a tropicalis gastrula, previously stained by tSox17 alpha , whole-mount shows complete penetration of the probe into the deep endoderm. E: A schematic of a midsagittal gastrula embryo. As in all the sections above, dorsal is to the left. Yellow indicates the deep and superficial endoderm, orange the mesoderm, and white the ectoderm.
	Figure 2. Xenopus tropicalis VegT. Whole-mount in situ hybridization analysis of tVegT mRNA expression in tropicalis oocyte and embryos. A: tVegT mRNA is localized to the vegetal half of a stage VI oocyte. The bleached animal pole is up. A lateral view of a cleared whole-mount in situ hybridization of early gastrula embryo (B) and a midsagittal section of the same whole-mount embryo(C) shows tVegT expression throughout the endoderm with stronger expression in the mesoderm. Dorsal is to the left. The brown color in the animal cap is pigment. st., embryonic stage.
	Figure 4. Xenopus tropicalis Gata6. Whole-mount in situ hybridization analysis of tGata6 expression in tropicalis embryos. A vegetal view of stage (st.) 10 (A) and stage 11 (B) gastrula embryos show tGata6 transcripts localized to the deep endoderm but absent from the superficial endoderm (red arrow). C: A midsagittal hemisection of a stage 10.5 gastrula previously stained by whole-mount (dorsal lip to the left) shows tGata6 transcripts abundant in the leading edge endoderm and confirms the lack of expression in the superficial endoderm (red arrow). A tGata6 whole-mount of a stage 22, early tail bud embryo(D) and a midsagittal section of the same embryo (E) is shown with anterior to the right. ld, liver diverticulum; cm, cardiac mesoderm; lpm, lateral plate mesoderm. F: A section of a stage 26, whole-mount embryos shows tGata6 expression in the endoderm and the cardiac mesoderm. G: By stage 32, tGata6 is strongly expressed in the mid-gut endoderm as well as the cardiac and lateral plate mesoderm.
	Figure 5. Xenopus tropicalis Sox17 alpha / beta . In situ hybridization analysis of tropicalis embryos with Sox17 alpha (A-E) and Sox17 beta (F-J) antisense probes. Whole-mount (A,C,F,H) and sectioned (B,G) gastrula show that tSox17 alpha and tSox17 beta are identically expressed in throughout the deep and superficial endoderm (red arrows). D: At early tail bud stage (anterior to the left), tSox17 alpha is strongly expressed in the posterior endoderm but absent in the anterior endoderm, except behind the cement gland. I: By comparison at stage 22 (anterior to the left), tSox17 beta transcripts are almost absent from the entire endoderm, except behind the cement gland. E: At stage 32, tSox17 alpha expression persists in the extreme posterior endoderm, the gall bladder (g) precursors and in endothelial cells. J: In late tail bud stages, tSox17 beta is only expressed in a small patch of cells in the head. st, embryonic stage.
	Figure 6. Xenopus tropicalis FoxA1. Whole-mount in situ hybridization analysis of tFoxA1 mRNA expression in tropicalis embryos. A: A vegetal view of a midgastrula embryo shows tFoxA1 transcripts in the endoderm. B: A midsagittal section of the same embryo (dorsal to the left) shows strong tFoxA1 expression in the chordomesoderm and weaker expression throughout the endoderm. The dark staining in the animal cap is pigment. C: A dorsal view (anterior up) of a stage 18 embryo shows tFoxA1 expression in the notochord and also in ciliated cells of the epithilium. A lateral view of a tail bud embryo (D) and a cross-section of the same embryo (E) shows tFoxA1 transcripts expressed throughout the endoderm, as well as in the notochord and floor plate. F: Analysis of stage 40 embryos shows that tFoxA1 transcripts mark the endoderm late in development. st., embryonic stage.
	Figure 7. Xenopus tropicalis Hex and Cerberus. In situ hybridization analysis of tropicalis embryos with tHex (A-D) and tCerberus (E-G) antisense probes. The top row shows vegetal views (dorsal up), and the second row shows lateral views (dorsal left) of tHex (A,B) and tCerberus (E,F) expression in the anterior endoderm of the early gastrula. Note tCerberus is more broadly expressed than tHex. C,G: Midsagittal sections (dorsal left) of gastrula embryos, hybridized in whole-mount with tHex and tCerberus probes. D: A cleared tail bud embryo (anterior right) shows tHex transcripts localized to the presumptive liver region. st., embryonic stage.
	foxa1 (forkhead box A1) gene expression in a Xenopus tropicalis embryo, NF stage 26/27, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	Figure 3. Xenopus tropicalis Mix-like genes. Whole-mount in situ hybridization analysis of tropicalis gastrula embryos with tMixer (A), tMix (D), and tBix (G) antisense probes. The top row (A,D,G) shows stage (st.) 10.5 early gastrula embryos. The middle row (B,E,H) shows later stage 11 gastrula. In each case, a vegetal view is shown with dorsal up. The bottom row (C,F,I) shows midsagittal sections of whole-mount, stage 10.5 embryos. Dorsal is to the left.
	hhex (hematopoietically expressed homeobox ) expression in X. tropicalis embryo, NF stage 22, lateral view, anterior right, dorsal up, indicating the ventral position of the liver diverticulum.
	sox17b.1 (SRY (sex determining region Y)-box 17 beta, gene 1) gene expression in a X. tropicalis embryo, NF stage 10, assayed via in situ hybridization, blastoporal view, dorsal up.
	sox17b.1 (SRY (sex determining region Y)-box 17 beta, gene 1) gene expression in a X. tropicalis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10, dorsal right, animal hemisphere up.
	sox17b.1 (SRY (sex determining region Y)-box 17 beta, gene 1) gene expression in a X. tropicalis embryo, assayed via in situ hybridization, NF stage 22, later view, anterior left, dorsal up.
	sox17b.1 (SRY (sex determining region Y)-box 17 beta, gene 1) gene expression in a X. tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, dorsal up.
	Gene expression of sox17a (SRY-box 17 alpha) (D, left panel) compared with sox17b.1 (SRY (sex determining region Y)-box 17 beta, gene 1) (I, right panel) in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up. Both genes are expressed in the anterior endoderm, directly underlying the cement gland, but only sox17a is also extensively expressed in the entire endoderm.