Dev Biol. March 15, 2011; 351 (2):

A gene regulatory network controlling hhex transcription in the anterior endoderm of the organizer.

Abstract

Pubmed Id: 21215263

Grant support: DK70858 NIDDK NIH HHS , P30 DK078392 NIDDK NIH HHS , R01 DK070858-01A2 NIDDK NIH HHS , R01 DK070858-02 NIDDK NIH HHS , R01 DK070858-03 NIDDK NIH HHS , R01 DK070858-04 NIDDK NIH HHS , R01 DK070858-05 NIDDK NIH HHS

Genes referenced: cer1 chrd foxh1.2 gsc hhex lhx1 nodal nodal1 nodal3.1 nog otx2 prl.2 sia1 sia2 smad2 tcf3 ventx1.2 ventx2.2 wnt8a

Antibodies referenced:

Paper published

	Fig. 1. A −6Kb:hhex:gfp transgene recapitulates hhex expression. (A) Diagram of the Xenopus laevis hhex genomic locus and −6.0Kb:hhex:gfp transgene. (B) The transgene recapitulates hhex expression in the anterior endoderm from blastula (st9.5) to stages 25. Transgenic embryos assayed by in situ hybridization for hhex or gfp mRNA and for GFP fluorescence with a confocal (st9.5 and 10) or stereomicroscope (st18–35). Embryos from st9.5 to 18 are bisected; in st9.5–11, dorsal is left; and in st18–35, anterior is right. The GFP image at st35 shows both a transgenic (top) and non-transgenic embryos (bottom). Two independent transgenic lines exhibited identical expression patterns. Arrowheads indicate endothelial cells (black), thyroid (white), and liver bud (yellow).
	Fig. 2. Regulation of hhex expression by Nodal and Wnt/β-catenin signaling. (A) Nodal and Wnt signaling are required for hhex expression. In situ hybridization of hhex and gfp in bisected −6.0Kb:hhex:gfp transgenic gastrulae injected with β-catenin-MO (20 ng), ∆Ntcf3 RNA (500 pg), stabilized pt-β-catenin RNA (250 pg), xnr1 RNA (50 pg), and/or cer-s RNA (750 pg). The number of embryos exhibiting the depicted phenotype is indicated. Transgenic F1 males are heterozygous, and approximately 50% of the embryos are expected to express gfp. (B) The schematic indicates the cells injected with the hhex:luc reporter (300 pg) plus pRL-TK:Renilla (25 pg). Co-injection with xnr1 RNA (1–500 pg) and/or β-catenin RNA (20–200 pg) resulted in a dose-dependent activation the hhex:luc reporter in the ectoderm. The histogram shows the average normalized luciferase activity and standard deviation from a single injection experiment performed in biological triplicate. A representative example of 4 independent experiments is shown. (C) hhex is a direct Nodal-target and an indirect Wnt-target. Blastula stage animal cap tissue was cultured either untreated, with Activin or with BIO. Some explants were incubated in CHX for 30 min prior to and during culture to block protein synthesis. At stage 11, the animal caps were assayed by in situ for hhex, gfp, cerb, and xnr3. This experiment was repeated 3 times, with identical results and a total of 12–15 caps per treatment. A representative example is shown.
	Fig. 3. Mapping Nodal and Wnt-responsive cis-elements. (A) Representative examples of GFP fluorescence from hhex:gfp deletion constructs in bisected transgenic gastrulae. Arrowheads show ectopic GFP. (B) Relative luciferase activity of early gastrulae injected with hhex:luc deletion constructs into either dorsal C1 (green), the ventral C4 cells (red) or (C) into the A4 ectoderm cells with xnr1 RNA (50 pg; gray) or stabilized pt-β-catenin RNA (250 pg; black). (B–C) Histograms show the average normalized luciferase activity and standard deviation from injections performed in biological triplicate. Representative examples from 3 independent experiments are shown. *p < 0.05 in Student T-tests when compared to injection of the same reporter alone; ns = no statistical difference. The NRE localized between −0.44 and −0.38 Kb, and the WRE between −0.65 and −0.55 Kb.
	Fig. 5. Nodal-signaling directly activates hhex transcription through Foxh1/Smad2 binding in the proximal −0.44 Kb promoter. (A) Schematic and sequence of the NRE indicating the putative Foxh1 (green) and Smad (red) DNA-binding sites and corresponding ∆F and ∆S mutations. (B–C) Relative luciferase activity in gastrulae injected with the indicated reporter constructs into (B) the C1 dorsal mesendoderm or (C) the A4 ectoderm along with or without xnr1 RNA (50 pg). Histograms show the average normalized luciferase activity and standard deviation from a single injection experiment performed in biological triplicate. A representative from 3 independent experiments is shown. *p < 0.01 in Student T-test in (B) compared to the wt reporter or in (C) compared to injection of the same reporter alone; ns = no statistical difference. (D) Foxh1 and Smad2 regulate endogenous hhex expression. Hhex in situ of gastrulae injected into either dorsal or ventral cells with: foxh1-EnR RNA (500 pg), foxh1-VP16 RNA (200 pg), Smad2a/b -MOs (20 ng each), Smad2-VP16 RNA (200 pg) foxh1 RNA (250 pg), smad2 RNA (250 pg), or foxh1 + smad2 RNAs (125 pg each). (E) Normalized QRT-PCR of hhex and fast3 mRNA levels in gastrulae depleted of maternal Foxh1 and rescued by co-injection of foxH1 RNA (100 pg). (F) Normalized QPCR analysis of chromatin immunoprecipitated (in triplicate) from gastrulae injected dorsally or ventrally with Myc-Foxh1 RNA (50 pg). Primers amplified genomic DNA fragments containing the F1 or F2 Foxh1-sited in the hhex NRE, the mix2 ARE as a positive control and mlc2 promoter as a negative control.
	Fig. 6. Sia/Twn act downstream of Wnt/β-catenin to activate the WRE. (A) In situ hybridization of hhex, twin, and siamois in bisected blastula. (B) Schematic and sequence of the WRE indicating the homeobox sites (HD) and the ∆HD mutations. (C) In situ hybridization of hhex or gfp in bisected −6.0Kb:hhex:gfp transgenic gastrulae injected as follows: a combination of Sia-MO and Twn-MO (20 ng each; dorsal at 4-cell), Sia/Twn-MOs (20 ng each; dorsal at 4-cell) + xnr1 RNA (50 pg; dorsal–vegetal at 8-cell), sia RNA (25 pg; ventral 4-cell), cer-S RNA (500 pg; dorsal 4-cell) to block Nodal signaling, cer-S + sia RNA (500 pg + 25 pg; dorsal 4-cell), β-catenin-MO (20 ng; 2-cell) and β-catenin-MO (20 ng; 2-cell) + sia RNA (50 pg; dorsal–vegetal at 8-cell). (D–E) Sia activates hhex transcription via HD sites in the WRE. Embryos were injected with the indicated hhex:luc constructs into either the C1 dorsal mesendoderm, the C4 ventral mesenoderm or the A4 ectoderm, with or without sia RNA (25 pg). Histograms show the average normalized luciferase activity and standard deviation at stage 10.5 from a single injection experiment performed in biological triplicate. A representative from 3 independent experiments is shown. *p < 0.01 in Student T-test compared to injection of the same reporter alone and ns = no statistical difference.
	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.
	Fig. 8. Sia/Twn promote hhex transcription by inhibiting BMP and Vent. In situ hybridization of −6.0Kb hhex:gfp transgenic embryos injected as follows: Sia/Twn-MOs (20 ng each, 4-cell dorsal), Sia/Twn-MOs (20 ng each, 4-cell dorsal) + tBR mRNA (400 pg, 8-cell dorsal–vegetal), Vent1-MO + Vent2-MO (30 ng each; 4-cell ventral), Sia/Twn-MOs (20 ng each, 4-cell dorsal) + Vent1/2-MOs (30 ng each, 8-cell dorsal–vegetal), Chordin-MO + Noggin-MO (10 ng each, 4-cell dorsal) or sia mRNA (50 pg, 8-cell ventral–vegetal).
	Fig. 9. Vent and Sia directly act on the hhex promoter. (A) Animal cap explants injected with GR-Vent2–VP16 RNA (100 pg) were cultured with or without CHX for 30 min and then treated with DEX. In situ hybridization at stage 11 shows that hhex and otx2, but not chordin, were directly induced by GR-Vent2–VP16 in CHX-treated explants. (B) The indicated hhex:luc reporters were injected into either C1 dorsal (green) or C4 ventral cells (red), with either Vent1/2-MOs (30 ng each) or Sia mRNA (50 pg). The histogram shows the average normalized luciferase activity at stage 10.5 and standard deviation from a single representative injection experiment performed in biological triplicate. *p < 0.05 in Student T-test when compared to injection of reporter alone and ns = no statistical difference. A representative example of 3 independent injection experiments is shown. (C) GR-Sia can directly activate hhex transcription in Vent-depleted animal caps. Animal caps injected with GR-sia RNA (30 pg) with or without Vent1/2-MOs (30 ng each) were cultured with CHX, DEX, or CHX + DEX followed by in situ hybridization at stage 11 for hhex or chordin mRNA. (A, C) Animal cap experiments were repeated twice with identical results and a total of ~ 10 explants/condition. One experiment is shown.
	Fig. 10. A model of the regulatory network controlling hhex transcription. The Nodal and mWnt pathways leading to activation of hhex transcription in the dorsal–anterior endoderm are shown in the green box. Repression of hhex transcription by BMP, zygotic Wnt8, and Vents are shown in the red box. Interactions mediated by secreted factors are indicated by hatched arrows, and interactions not yet known to be direct are indicated by dashed lines. The Nodal- and Wnt-responsive cis-elements are indicated in orange and blue boxes, respectively.
	Figure S1. Efficacy of antisense morpholinos used in this study. Stage 35 embryos injected with the indicated MOs reproduced published phenotypes. Standard control-MO injected embryos have no phenotype. B-catenin-MO results in ventralized embryos (Heasman et al., 2000). Co-injection of Sia-MO + Twn-MOs results in anterior truncation (Ishibashi et al., 2008). Injection of Tcf3-MO results in truncated heads and short axis (Liu et al., 2005). Co-Injection of Vent1-MO + Vent2-MO (Sander et al., 2007) results in loss of ventralosterior structures, while injection of Gsc-MO (Sander et al., 2007), Chordin-MOs (Oelgeschlager et al., 2003) plus Noggin-MO (Kuroda et al., 2004) and the Otx2-MO (Carron et al., 2005) result in loss of dorsalnterior structures. The Lim1-MO causes gastrulation defects (Schambony and Wedlich, 2007). Injection of Smad2a-MO + Smad2b-MO, which target both Xenopus laevis Smad2 pseudo alleles results in a phenotype similar to Nodal inhibition by cerb-S.
	Figure S2. Hhex expression is up regulated in Tcf3-depleted embryos. In situ hybridization to hhex or gfp mRNA in bisected −6.0Kb:hhex:gfp transgenic gastrulae injected with a Tcf3-MO (60 ng). Mating a wild-type female to a heterozygous −6.0Kb:hhex:gfp male approximately 50% of the embryos carry the transgene.
	Figure S6. Gsc-depleted embryos. In situ hybridization of bisected hhex:gfp transgenic embryos injected with Gsc-MO (40 ng). At stage 10.5 Gsc-MO embryos exhibit increased wnt8, vent1, and vent2, mRNA levels, but hhex expression is unchanged. By stage 12.5 Gsc-MO embryos fail to maintain hhex expression.
	Figure S7. Otx2- and Lim1-depleted embryos. In situ hybridization of bisected gastrula embryos injected with Otx2-MO and/or Lim1-MOs (20 ng each).
	sia2 (siamois homeodomain 2)gene expression in bisected Xenopus laevis embryos, NF stage 10.5, as assayed by in situ hybridization. Lateral view. Image extracted from XB-IMG-48873 and originally published in: Rankin SA et al. (2011) Copyright Elsevier , 2011