Development. February 1, 2008; 135 (3):

The Gata5 target, TGIF2, defines the pancreatic region by modulating BMP signals within the endoderm.

Abstract

Pubmed Id: 18094028

Genes referenced: a2m bmp4 c19orf25 chrd fam21a fzd7 gata4 gata5 gata6 hdgf hhex ins odc1 pdx1 rcc2 smad1 smad2 t tgif2 vegt ventx2.2

Antibodies referenced:

Paper published

	Fig. 2. Whole-mount in situ hybridization of selected array clones. (A) Xenopus embryos at early gastrula (st. 10), tailbud (st. 30) stage and dissected whole gut (st. 43) were hybridized with antisense probe for Fz7/clone 10B11. Arrowheads indicate expression in the endoderm; the dotted line depicts the dorsal blastopore lip. Staining in endodermal derivatives, such as the hepatic and pancreatic regions, is indicated by a bracket in the middle panel. (B) Embryos at early gastrula (st. 10), tadpole (st. 35) stage and dissected whole gut (st. 43) were hybridized with HDGF/clone 7C5. Arrowheads point to expression in the endoderm; and the bracket in the middle panel indicates endodermal derivatives, such as the hepatic and pancreatic regions. (C) Embryos at gastrula (st. 10.5), tadpole (st. 37) stage and dissected whole gut (st. 42) were hybridized with clone 7G9 [EST similar to hypothetical protein (BG410109)] indicating expression in the ectoderm and hepatic rudiment (bracket in the middle panel). (D) Embryos at gastrula (st. 10), tadpole (st. 36) stage and dissected whole gut (st. 42) were hybridized with clone 8C11 [EST similar to hypothetical protein KIAA0592 (BG410148)] showing expression in the dorsal ectoderm, brain, pronephros, liver and duodenum. (E) Embryos at gastrula (st. 10), early tailbud (st. 27) stage and dissected whole gut (st. 42) were hybridized with clone 7B7 [EST similar to hypothetical protein (BC094159)] indicating expression in the ectoderm, brain, branchial arches and pancreas. (F-J) In situ hybridizations on serial sections of gastrula and neurula stage embryos using Xenopus TGIF2 (xTGIF2) and Gata5 probes show expression of both genes in a subset of endodermal cells. xTGIF2 is also strongly expressed in the ectoderm and mesoderm. (F,F′) In situ hybridization on section of stage 10 embryo using antisense probe for TGIF2/clone 8B1 (* indicates the dorsal side). Red dashed box outlines the TGIF2-positive endodermal region, which is magnified in F′. Arrowheads point to endodermal cells. (G) Gata5 in situ hybridization on section of stage 10 embryo. Arrowheads point to endodermal cells. (H,H′) In situ hybridization on section of stage 10.5 embryo shows enriched staining in dorsal endodermal cells (* indicates the dorsal side). Red dashed box outlines the TGIF2-positive endodermal region, which is magnified in H'. Arrowheads point to endodermal cells. (I,I′) In situ hybridization on neurula stage whole (I) and sectioned (I′) embryos shows TGIF2 staining in anterior endodermal cells (red dashed box in I′). (J) Gata5 in situ hybridization on section of stage 17 embryo. (K) Dissected whole gut (st. 43) stained with TGIF2 probe shows expression in the pancreatic rudiment. a, anterior; d, duodenum; lv, liver; pa, pancreas; p, posterior.
	Fig. 3. xTGIF2 behaves as a modifier of the endoderm. (A) Xenopus eight-cell stage embryos were injected into both ventral (VV) or dorsal vegetal (VD) blastomeres with xTGIF2 (1 ng) mRNA. Vegetal explants were dissected at early gastrula stage (stage 10). Uninjected VV or VD pole halves were used as control for their regional differences in the expression of endodermal markers. All explants were collected at tailbud stage and assayed for expression of the indicated markers by RT-PCR analysis. (B) Animal caps injected with VegT (60 pg) mRNA and/or xTGIF2 mRNA, as indicated. The explants were cultured until stage 30 (tailbud) and analyzed for expression of the indicated markers. (C) Eight-cell stage embryos were injected into ventral vegetal (VV) blastomeres with xTGIF2 (1 ng) mRNA. Uninjected VV or VD pole halves were used as control. All explants assayed at tailbud stage for expression of the indicated mesodermal markers by RT-PCR analysis. (D-E″) Lineage-tracing analysis of endodermal cells injected with xTGIF2 mRNA. (D) Ventral vegetal explants isolated from uninjected or xTGIF2+lacZ-co-injected embryos were stained for β-gal and assayed for Pdx1 expression by in situ hybridization at tailbud stage. The β-gal staining (blue) and ectopic Pdx1 (red) expression co-localize in endodermal explants (see arrowheads). (E) Whole-mount in situ hybridization analysis of Pdx1 expression in xTGIF2+lacZ-co-injected embryos. White dashed outline demarcates the region of the embryo magnified in E′. Arrowheads indicate purple cells stained for β-gal (blue) and positive for Pdx1 (red). (E″) Transverse section through the stained endodermal region shown in E′.
	Fig. 4. xTGIF2 is required in vivo for establishing the pancreatic region within the endoderm. (A) Both dorsal vegetal (VD) blastomeres of eight-cell stage embryos were injected with a combination (5 ng each) of two antisense morpholino oligonucleotides (TGIF2-Mo) targeting both Xenopus laevis TGIF2 pseudoalleles (see Fig. S3 in the supplementary material). TGIF2-Mo-injected VD, uninjected VD and ventral vegetal (VV) explants were dissected at stage 10 and assayed for expression of the indicated markers by RT-PCR analysis. (B) Whole-mount in situ hybridization using Pdx1 probe. Embryos injected with TGIF2-Mo showed reduction of Pdx1 expression domain (80%; n=40). Pdx1 staining was rescued in embryos injected with TGIF2-Mo and mouse Tgif2 (mTgif2; 500 pg) mRNA (70%; n=25). Embryos left untreated show normal Pdx1 expression domain in the pancreatic/duodenum endoderm, as indicated by the white bracket. (C) Whole-mount in situ hybridization with Hex. Embryos injected with TGIF2-Mo or left untreated were stained at stage 34. Hex expression domain in the hepatic endoderm is indicated by the yellow bracket. (D) Analysis of the expression of the pancreatic differentiation markers, insulin and amylase, by whole-mount in situ hybridization. Arrowheads indicate normal domains of expression of insulin and amylase in the pancreatic bud of tadpole gut tubes.
	Fig. 1. Schematic of experimental strategy. (A) Xenopus embryos were injected into both animal blastomeres at the two-cell stage with Gata5 (500 pg) mRNA. Ectodermal explants were isolated at stage 9 from both Gata5-injected and uninjected embryos and cultured until stage 28 (tailbud). Transcriptional differences were analyzed on 5000-clone gastrula-stage cDNA microarray (Munoz-Sanjuan et al., 2002). An aliquot of each RNA sample was assayed for expression of the indicated Gata5 targets by RT-PCR. ODC was used as loading control. (B) Pie chart of the classification of the genes upregulated by Gata5 based on the GO molecular function categories. The majority of the upregulated clones in the Gata5 array fall into four main categories: (1) catalytic activity (26%); (2) binding activity (26%), including the large group of nucleic-acid-binding as well as protein-binding factors; (3) hypothetical/unknown function (13%), including full-length sequences conserved in the mouse and human databases the function of which is unknown; (4) no database match (13%), including a number of clones with no hits in database searches, which might be either genes unique to the frog or partial cDNAs. Edd, endodermin; -RT, minus reverse transcriptase; ODC, ornithine decarboxylase.
	Fig. 5. Inhibitory effects of xTGIF2 on TGFβ and BMP signalings in Xenopus. (A) Partial secondary axis (indicated by *) was observed in tadpole stage embryos injected with xTGIF2 (1 ng) mRNA into one ventral vegetal cell. (B) xTGIF2 (1 ng) mRNA and activin (100 pg) mRNA were injected separetely or together into the animal pole of two-cell stage embryos. Animal caps were analyzed at gastrula stage (stage 11) for the expression of indicated markers by RT-PCR. (C) Animal caps injected with xTGIF2 (1 ng) mRNA were analyzed at gastrula stage (stage 11) for the expression of indicated markers by RT-PCR. (D) Luciferase assay with BMP inducible VENT2-luciferase (VENT2-Luc.) reporter construct. Two-cell stage embryos were injected with VENT2-Luc. alone or in combinations with BMP4 (200 pg) and/or xTGIF2 (1 ng) mRNAs, as indicated. Embryos were harvested at the onset of gastrulation and assayed for luciferase activity. (E) Immunoprecipitation (IP) of flag-xTGIF2 and endogenous Smad1 or Smad2. Four-cell stage embryos were injected into the the vegetal pole (ventrally for Smad1 IP; dorsally for Smad2 IP) with flag-xTGIF2 (1 ng), lysates were prepared at gastrula stage (stage 11) and immunoprecipitated with anti-flag antibody and analyzed by immunoblot (IB) with anti-Smad1 and anti-Smad2 antibodies. The expression of flag-xTGIF2, Smad1 and Smad2 was checked by immunoblotting on the crude extracts used for the IP reaction. As loading control, the membranes were stripped and reprobed with anti-α-tubulin. Chd, chordin; EK, epidermal keratin; xbra, brachyury.