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Experiment details for hhex

Homeoprotein hhex-induced conversion of intestinal to ventral pancreatic precursors results in the formation of giant pancrea...

Homeoprotein hhex-induced conversion of intestinal to ventral pancreatic precursors results in the formation of giant pancreata in Xenopus embryos.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 11 involuted dorsal mesoderm , endomesoderm
hhex.L laevis NF stage 12 gastrocoel roof plate , endomesoderm , anterior
hhex.L laevis NF stage 13 to NF stage 18 prechordal plate , gastrocoel roof plate , anterior
hhex.L laevis NF stage 24
hhex.L laevis NF stage 28
hhex.L laevis NF stage 33 and 34

  Fig. 1. vpp1 and hhex display partially overlapping expression in gas- trulating dorsal endoderm and later demarcate ventral pancreatic buds and liver anlagen, respectively. (A) Comparison of vpp1 and hhex expression as revealed by whole mount in situ hybridization analysis with bisected (stages 118) or whole (stages 243) embryos. (1, 2, 5, 6, 9, 10, 13, and 14) Dorsal is toward the right; (172) lateral view, head toward the left. Images 3, 7, 11, and 15 are cartoons drawn representing both the single staining (images 1, 2, 5, 6, 9, 10, 13, and 14) and double staining whole mount in situ hybrid- ization data (Fig. S3B), illustrating vpp1 (purple) and hhex (turquoise) ex- pression. Boxed regions are magnified in 4, 8, 12, and 16, respectively. (B) RT-PCR analysis compares the temporal expression profile of vpp1 and hhex during Xenopus embryogenesis. Ornithine decarboxylase (odc) was used as the RNA loading control.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 13 endoderm , dorsal , anterior
hhex.L laevis NF stage 24 endoderm , foregut , liver primordium
hhex.L laevis NF stage 32 endoderm , alimentary system , liver , pancreas , foregut
hhex.L laevis NF stage 42 alimentary system , liver , pancreas , foregut

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  Fig. 5. Liver and ventral pancreas defects in hhex morphants. Probes or methods for whole mount staining are indicated on the left. (A and B) Bisected embryos, dorsal toward top. (C) Lateral view, head toward the left. (I and J) Bisected embryos, anterior toward the left. (K) Lateral view, head toward the left. The white arrowhead (N) indicates the missing expression of fibrinogen in the liver anlage. (S) Bisected embryos, anterior toward the left. Phenotype is as follows: B, 19/20; D, 20/22; F, 24/25; H, 34/41; J, 25/27; L, 38/44; N, 19/20; P, 36/42; R, 16/72 (for detailed phenotype analysis, see Fig. S7); T, 22/23; V, 41/41. (W) Statistical analysis of phospho-histone H3-positive cells in the outlined areas in U and V. Thirty-four samples from 17 bisected embryos were counted from either control MO or hhex MO-injected embryos. P value (0.309) was determined by the paired, two-tailed t test.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 18 endoderm , anterior

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  Fig. 3. Ectopic hhex converts intestine-forming cells into vpp1-expressing cells. Whole mount staining was performed with probes indicated on the left. (A) Bisected embryos, anterior toward the left. Phenotype is as fol- lows: B, 28/32; E, 34/40; H, 21/21; K, 42/42. (L) Statistic analysis of phospho- histone H3-positive cells on the whole surface of bisected vegetal endoderm, as demarcated by the red dashed lines in J and K. As the distribution of phospho-histone H3-positive cells across the whole vegetal endoderm sur- face did not show obvious bias, for simplicity, we counted the whole area rather than only putative vpp1-positive area. Forty-two bisected samples were counted from either control or hhex-injected embryos. P value (0.156) was determined by the paired, two-tailed t test.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 18 endoderm , liver diverticulum , ventral , anterior
hhex.L laevis NF stage 24 endoderm , foregut , ventral , anterior , liver primordium

  Fig. S3. vpp1 is undetectable in blastulae, and a small number of cells coexpresses vpp1 and hhex at neurula and tail bud stages. (A) No vpp1 expression was detected in stage 7 and stage 9 embryos, and weak expression was detected in the subblastoporal endodermal cells at stage 10 and stage 11 by whole mount in situ hybridization. Red arrowheads in 3 and 4 point to the dorsal blastopore lips. (B and C) Embryos were subjected to double label whole mount in situ hybridization by using hhex (turquoise) and vpp1 (purple) specific probes. (B) Stage 18. (1) Ventral view, anterior toward the left. (2) Serial vibratome sections obtained from the area between the dashed lines in 1 sequentially from top to bottom. Red arrowheads in 3 point to the cells that coexpress vpp1 and hhex. The red arrowhead highlighted areas in 5 are magnified in 92, respectively. A, anterior; P, posterior. (C) Stage 24. (1) Lateral view, head toward the left. (214) Serial vibratome sections obtained from the area between the dashed lines in 1 in the order from left to right. Red arrowheads in 83 highlight the cells that coexpress vpp1 and hhex. Boxed region in 7a is magnified in 7b.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 37 and 38 endoderm , alimentary system , liver , pancreas , foregut

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  Fig. S2. Injection of vpp1 mRNA (A, D, and G) or morpholino (C, F, and I) had no obvious effects on the expression of pancreas and liver marker genes compared with the uninjected controls (B, E, and H). vpp1 mRNA (2 ng) and vpp1 splice blocking MO (5 picomoles) were injected into four vegetal blastomeres of eight-cell stage embryos. Whole mount staining was performed with probes indicated on the left. (A) All images show lateral view, head toward the left.