Larger Image Fig. 7. Otx2 and Lim1 promote hhex transcription downstream of Sia/Twn. (A) GR-sia RNA (30 pg) + gfp RNA (200 pg) was injected into ventral–vegetal cells and explants were isolated at blastula stage based on GFP fluorescence. Explants were cultured with or without CHX for 30 min, and then DEX was added to activate the GR-Sia as indicated. In situ hybridization at stage 11 shows that chordin, otx2, and lim1, but not hhex are directly induced by GR-Sia in CHX treated explants. This experiment was repeated 3 independent times with identical results and > 10 explants for each condition. (B) A model showing two mechanisms by which Sia/Twn could promote hhex transcription. (C) Induction of hhex by Sia/Twn is partially mediated by Otx2 and Lim1. In situ hybridization of hhex, gsc, otx2, and lim1 in gastrulae injected as follows: Sia/Twn-MOs (20 ng each, dorsal 4-cell), sia RNA (50 pg, ventral–vegetal 8-cell), Sia/Twn-MOs (20 ng each, dorsal 4-cell) + otx2 and lim1 RNA (50 pg each dorsal–vegetal 8-cell), otx2 and lim1 RNA (50 pg each ventral–vegetal 8-cell), Otx2-MO + Lim-MO1 (20 ng each, dorsal 4-cell) or Gsc-MO (60 ng, dorsal 4-cell). (D) WT or ∆HD mutant −0.65 Kb hhex:luc reporters were injected into either C1 dorsal (green) or C4 ventral cells (red), with or without the indicated mRNAs (pg). Histograms show the average normalized luciferase activity and standard deviation from a single injection experiment performed in biological triplicate. A representative example from 3 independent experiments is shown. *p < 0.05 in Student T-test when compared to injection of reporter alone and ns = no statistical difference. Image published in: Rankin SA et al. (2011) Copyright © 2011. Image reproduced with permission of the Publisher, Elsevier B. V. Gene Synonyms Species Stage(s) Tissue hhex.L hex, tHex, XHex X. laevis Throughout NF stage 10.5 endoderm endomesoderm gsc.L goosecoid, gsc-a, gsc-b, Xgsc X. laevis Throughout NF stage 10.5 endomesoderm prechordal plate involuted dorsal mesoderm otx2.S otx-2, otx2-a, otx2-b, otxA, Xotx-2, Xotx2 X. laevis Throughout NF stage 10.5 involuting marginal zone mesoderm prechordal plate lhx1.L lim-1, Lim1, lim1, Xlim-1, Xlim1 X. laevis Throughout NF stage 10.5 involuted ventral mesoderm involuted dorsal mesoderm Image source: Published Experiment + Assay Source Phenotypes and Disease Xla Wt + sia1-GR + CHX + animal cap explant + NF11 (in situ hybridization) fig.7.a Expression Phenotype decreased amount chrd.1.L expression in animal cap decreased amount hhex.L expression in animal cap decreased amount lhx1.L expression in animal cap decreased amount otx2.L expression in animal cap increased amount ventx1.2 expression in animal cap Xla Wt + sia1-GR + CHX + DEX + animal cap explant + NF11 (in situ hybridization) fig.7.a Expression Phenotype increased amount chrd.1.L expression in animal cap decreased amount hhex.L expression in animal cap increased amount lhx1.L expression in animal cap increased amount otx2.L expression in animal cap increased amount ventx1.2 expression in animal cap Xla Wt + sia1-GR + DEX + animal cap explant + NF11 (in situ hybridization) fig.7.a Expression Phenotype increased amount chrd.1.L expression in animal cap increased amount hhex.L expression in animal cap increased amount lhx1.L expression in animal cap increased amount otx2.L expression in animal cap decreased amount ventx1.2 expression in animal cap Xla Wt + lhx1 + otx2 + NF11 (in situ hybridization) fig.7.c Expression Phenotype increased amount gsc.L expression in dorsal endomesoderm increased amount gsc.L expression in ventral endoderm mislocalised gsc.L expression in ventral endoderm increased amount hhex.L expression in dorsal endoderm mislocalised hhex.L expression in ventral endoderm increased amount hhex.L expression in ventral endoderm increased amount otx2.L expression in ventral endoderm Xla Wt + lhx1 + otx2 + sia1 MO + sia2 MO + NF11 (in situ hybridization) fig.7.c Expression Phenotype increased amount gsc.L expression in dorsal endomesoderm decreased amount hhex.L expression in dorsal endoderm Xla Wt + lhx1 MO + otx2 MO + NF11 (in situ hybridization) fig.7.c Expression Phenotype decreased amount gsc.L expression in dorsal endomesoderm decreased amount hhex.L expression in dorsal endoderm Xla Wt + sia1 + NF11 (in situ hybridization) fig.7.c Expression Phenotype increased amount gsc.L expression in dorsal endomesoderm mislocalised gsc.L expression in ventral endoderm increased amount hhex.L expression in dorsal endoderm mislocalised hhex.L expression in ventral endoderm increased amount lhx1.L expression in dorsal endoderm increased amount lhx1.L expression in ventral endoderm mislocalised otx2.L expression in ventral endoderm increased amount otx2.L expression in ventral endoderm Xla Wt + sia1 MO + sia2 MO + NF11 (in situ hybridization) fig.7.c Expression Phenotype decreased amount gsc.L expression in dorsal endomesoderm decreased amount hhex.L expression in dorsal endoderm decreased amount lhx1.L expression in dorsal endoderm decreased amount lhx1.L expression in ventral endoderm decreased amount otx2.L expression in dorsal endoderm Permanent Image Page Printer Friendly View XB-IMG-48874