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. Author manuscript; available in PMC: 2011 Apr 7.
Published in final edited form as: Dev Dyn. 2010 Mar;239(3):980–986. doi: 10.1002/dvdy.22217

Dynamic Expression of Groucho-Related Genes Grg1 and Grg3 in Foregut Endoderm and Antagonism of Differentiation

Pilar Santisteban 1,2, Pablo Recacha 1,2, David E Metzger 2, Kenneth S Zaret 2
PMCID: PMC3071952  NIHMSID: NIHMS281662  PMID: 20108349

Abstract

While much is known about Groucho corepressors in Drosophila development, less is known about Grg homologs in mammalian embryogenesis. The transcription factors FoxA1 and FoxA2 are redundantly necessary for liver-inductive competence of the endoderm, and recently we found that FoxA factors bind Grg3, recruit the corepressor to FoxA target genes, and cause transcriptional repression, when Grg3 is ectopically expressed in adult liver cell lines that express little or no endogenous Grg. Unexpectedly, we now find that Grg1 and Grg3 mRNAs are co-expressed with FoxA factors in the foregut endoderm, prior to liver differentiation; though only Grg3 protein is expressed there. Grg3 mRNA and protein are extinguished at the onset of liver differentiation. Lentiviral delivery of Grg3 to explants of foregut endoderm suppresses liver gene induction. We suggest that Grg expression in the endoderm helps suppress the liver program and find that endodermal competence involves a balance between activators and corepressors.

Keywords: Grg, groucho, endoderm, FoxA, competence, corepressor

INTRODUCTION

In mammals, the endoderm germ layer arises during gastrulation and generates diverse tissues, including the thyroid, lung, liver, and pancreas. In the foregut endoderm of the mouse embryo, genes specific to these latter tissues, such as alb1 for liver, become activated by 8.5 days of gestation (E8.5), corresponding to the 7–9 somite pair stage (7–9S) (Cascio and Zaret, 1991; Lazzaro et al., 1991; Jonsson et al., 1994; Gualdi et al., 1996; Zannini et al., 1997). Different inductive signals impinging upon different domains of foregut endoderm activate the tissue programs, leading to the view that endoderm differentiation involves positive regulation (Zaret and Grompe, 2008). Indeed, the FoxA1, FoxA2, GATA4, GATA6, and Sox17 transcriptional activators are expressed in the endoderm and are important for endoderm differentiation (Ang et al., 1993; Sasaki and Hogan, 1993; Bossard and Zaret, 1998; Kanai-Azuma et al., 2002; Zhao and Duncan, 2005; Watt et al., 2007). Chromatin sites for FoxA at alb1 are occupied prior to liver induction (Gualdi et al., 1996), Fox factors open local chromatin structure (Cirillo et al., 2002; Yan et al., 2006; Cuesta et al., 2007; Hatta and Cirillo, 2007), and FoxA1 and FoxA2 are redundantly required for hepatic gene induction (Lee et al., 2005). Together, this has led to the proposal that FoxA proteins are competence or pioneer factors for the endoderm (Zaret, 1999). Yet as described in the present study, a deeper analysis of factors functioning with FoxA in endoderm is needed to understand how their target genes are kept competent for expression.

Groucho in Drosophila, Groucho-related genes (Grgs) in mouse, and Transducin-like Enhancer of Split genes (Tles) in humans encode a family of evolutionary conserved corepressors family that play critical roles in diverse developmental processes (Fisher and Caudy, 1998; Chen and Courey, 2000; Courey and Jia, 2001). Part of the transcriptional repressor activity of the Grgs arises from their recruitment of histone deacetylases (HDACs) (Chen et al., 1999), and hence Grgs can indirectly suppress gene expression (Flores-Saaib and Courey, 2000). However, Grgs also have the intrinsic capacity to compact chromatin structure (Sekiya and Zaret, 2007). Furthermore, Grgs bind to FoxA proteins (Wang et al., 2000) and thereby can become recruited to genes FoxA would otherwise activate, such as the alb1 gene when Grg is ectopically expressed in liver cells (Sekiya and Zaret, 2007). This can lead to local nucleosome structures that exclude other transcription factors from binding, resulting in transcriptional repression.

Based on the interactions of Grgs with FoxA in adult cells, herein we explored the expression profile and function of Grgs during the period when FoxA enables endoderm development and liver gene activation. The results suggest a new model for the FoxA-mediated state of endodermal competence, whereby Grgs restrain FoxA target genes from expression until the developmental time when the target genes are normally activated.

MATERIALS AND METHODS

In situ hybridization

In situ hybridization with 200 ng/ml of DIG-labeled Grg1 and Grg3 cRNA probes was performed as described (Ang and Rossant, 1993; Jung et al., 1999). Grg1 and Grg3 riboprobes, from nucleotides 1091–1617 and 1175–1728, respectively, were cloned from embryonic mRNA into TOPO-TA (Invitrogen).

Foregut endoderm tissue explants, lentivirus infections

Under a dissecting microscope, using electrolytically etched tungsten needles, the presumptive ventral endoderm and its associated cardiogenic mesoderm and septum transversum mesenchyme was separated from the embryo and either used for RNA or cultured in microwells as described (Gualdi et al., 1996).

Grg3-Flag and Grg3-ΔN-Flag lentiviruses were prepared as described (Sekiya and Zaret, 2007). Two hours after starting a foregut explant culture, each explant was infected with 8 × 105 (Grg3-Flag) or 2.4 × 105 (Grg3-ΔN-Flag) viruses. Twenty four hours later, the medium was exchanged for fresh medium without virus. Cultures were maintained for 72 hours, after which RNA was extracted.

RT-PCR

RNA was isolated with the RNeasy Micro Kit (Qiagen), including a DNAase treatment, and eluted in 10 μl of water. For RT-PCR cycle titration analysis, RNA was reverse transcribed with oligo-dT primers and subjected to PCR as described (Gualdi et al., 1996). The primers used were alb1 sense 5′-AGCACACAAGAGTGAGATCGCC-3 ′ , anti sense 5 ′-TGGCATGCTCATCGTATGAGC-3′; β-actin sense, 5 '-AAAGACCTGTACGCCAACACAGTC-3'; antisense, 5'-GTCATACTCCTGCTTGCTGATCCA-3'; Grg 1 sense 5 ′-AAATCGCCAAGAGATTGAAC-3 ′ , anti sense 5 ′-GGGTCCTCGTTAGACACATC-3′; Grg3 sense 5 ′-TGGAGGTCCTGCACCACACTAA-3 ′ , antisense 5 ′-GCTCACAAACCACTTGCCACA-3′; FoxA2 sense 5 ′-TCTCCGTGTCAGGAGCACAA -3 ′ , anti sense 5 ′-TGCTCGGAGGACATGAGGTT -3′. Each point shown in Fig. 3 depicts data from 4–6 separate explants.

Fig. 3. Expression of Grg3 protein in ventral foregut endoderm.

Fig. 3

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A-E. DAPI, Grg3 immunofluorescence (IF), and in situ hybridization (ISH) as shown of sagital sections of 6-7S mouse embryos. The signal in panel E was absent due to the presence of the Grg3 immunogenic peptide as a block. Yellow arrows, ventral foregut endoderm (v. f. e.). F-M, similar DAPI and IF images as above. The boxed regions in H and L are merged with the same domain in G and K and magnified in I and M. The yellow cells co-express FoxA2, a definitive endoderm marker, and Grg3.

For qRT-PCR, approximately 100 ng of RNA was used with the Reverse-ItTM 1st strand Synthesis Kit (Abgene). Quantitative PCR was performed using SYBR® Green PCR Kit (Stratagene). Samples were subjected to 40 cycles of PCR amplification in a Stratagene Mx3000P System for 10 min at 95°C, 30 sec at 95oC, 1 min at 59°C, and 25 sec at 72°C. qPCR were normalized using β-actin as Standard with ROX as Reference dye. The primers were: β-actin sense 5′-ACCCACACTGTGCCCATCTA , antisense 5′-CGCTCAGGAGGAGCAATGAT; alb1 sense 5 ′-AACAAGGAGTGCTGCCATGGT, antisense 5 ′-GCTGGAGATAGTCGCCTGGT; Grg1 sense 5′-AAATCGCCAAGAGATTGAAC′, antisense 5 ′-GGGTCCTCGTTAGACACATC; Grg3 sense 5 ′-TGGAGGTCCTGCACCACACTAA, antisense 5 ′-GCTCACAAACCACTTGCCACA; FoxA2 sense 5 ′-TCTCCGTGTCAGGAGCACAA; antisense 5′-TGCTCGGAGGACATGAGGTT; TTR sense 5′-TTCCATGAATTCGCGGATGT, antisense 5′-AGCCGTGGTGCTGTAGGAGTAT.

Antibodies and Immunofluorescence

Flag-tag antibodies were used as described (Sekiya and Zaret, 2007). The αGrg1 and αGrg3 antibodies were raised in rabbits immunized with peptides containing residues specific to Grg1, LAIKDDKKHHDAERHRDRE, or to Grg3, YSMEAKKRKAEEKDSLSR (ProSci, Inc.; Poway, CA). Immunofluorescence was performed on 8 micron-thick cyrosectioned embryos using αGrg1 (rabbit, 1:2500), αGrg3 (rabbit, 1:3000), and FoxA2 (Santa Cruz Biotech, SC6554, goat, 1:100) primary antibodies. Immunofluorescence development was achieved using tyramide signal amplification (Hunyady et al., 1996) with a TSA Kit (Invitrogen). To assess the specificity of αGrg1 and αGrg3, the antibodies were incubated with immunizing peptide for 1 hour prior to application on sections ("peptide block").

RESULTS AND DISCUSSSION

Grg expression in undifferentiated endoderm becomes repressed at the time of tissue specification

In situ hybridization studies indicated that Grg1 and Grg3 are expressed in various tissues of the mouse embryo at 8.25 days gestation (E8.25; 4–5 somite pair stage (4-5S)) (Fig. 1A, E), corresponding to the time at which the undifferentiated endoderm is receiving patterning signals for organogenesis. Sense strand control probes gave no signal (Fig. 1C, D). Grg1 was expressed prominently in the head folds and dorsal endoderm (Fig. 1A, black and red arrowheads; and data not shown). Shortly later, at E8.75 (12S), Grg1 was expressed in the head and other embryonic tissues, but not the heart and liver/midgut region (Fig. 1B). Grg3 was expressed more pervasively in the E8.25 embryo (Fig. 1E). Detailed section analysis revealed that Grg3 at 4-5S is expressed throughout the foregut endoderm, specifically including the dorsal, ventral-medial, and ventral-lateral domains that contain liver and ventral pancreatic progenitors (Fig. 1G-J, H’, I’) (Tremblay and Zaret, 2005). Several hours later, at 7-8S, the upper part of the dorsal foregut, containing pharyngeal progenitors, still expressed Grg3 (Fig. 1K-M; M’, orange arrowheads), but the ventral foregut began to extinguish expression (Fig. 1K-M; M’, green arrowhead). By 16-18S, Grg3 was not expressed in the heart and liver bud region (Fig. 1N-P, P’, brown arrowhead). In summary, Grgs are expressed in the foregut endoderm in the period preceding tissue specification (4-5S) and their expression diminishes shortly afterward.

Fig. 1. Dynamic expression of Grgs in endoderm development.

Fig. 1

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A-D, E, K, N. Whole mount in situ hybridization with the designated antisense (not denoted) and sense (denoted) probes, at the somite pair stages shown in the Figure. G-J, M, P. Sections as shown in panels F, L, and O, respectively. H', I', M', P', magnifications as shown. Arrowheads indicate tissue domains. Grg1 and Grg3 are expressed in the endoderm at the 4-6S stage but then are suppressed in expression in the nascent liver bud (12-18S).

To more precisely characterize the dynamics in Grg1 and Grg3 expression, we dissected foregut tissues from embryos pooled from carefully timed somite pair stages, isolated RNA, and performed RT-PCR cycle titration analysis (Fig. 2A) and qRT-PCR (Fig. 2B). As seen in Fig. 2, Grg1 and Grg3 mRNAs are present in the foregut until the 6-7S stage, agreeing well with the in situ hybridization data in Fig. 1. However, the abundance of those mRNAs drops strikingly several hours later, by the 9-10S stage, and remains low or absent in the midgut at 18-20S (Fig. 2); agreeing with the in situ data. By contrast, mRNAs for FoxA2, an endoderm marker, and actin, a ubiquitously expressed gene, remain constant. More strikingly, the Grg expression patterns are the exact reciprocal of that for alb1, a definitive marker for the induction of the hepatic program (Gualdi et al., 1996). Notably, the alb1 gene is induced within the hours in which the Grgs are shut off in the endoderm (Fig. 2). This also corresponds to the time when other early liver genes, including Ttr, are induced (Jung et al., 1999) .

Fig. 2. Abrupt down-regulation of Grg1 and Grg3 mRNA at the time of hepatic gene induction in the endoderm.

Fig. 2

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A, left. Diagram of embryonic region dissected for RT-PCR analysis. A, right. Autoradiographs of RT-PCR products at different cycle steps. B. qRT-PCR analysis normalized to fewer cycles of β-actin. Bars depict average of four independent experiments ± S.E.

To assess whether Grg1 and Grg3 proteins follow a similar course of expression, we raised antibodies to several peptides specific to each. We obtained one antibody each that specifically recognizes the expected Grg in Western blots and is of much higher affinity to each protein than a commercially available "pan-Grg" antibody (Supp. Figure 1). The Grg1 antibody gave an immunoflourescence pattern that was spotty throughout much of the embryo at the 3-4S, 7-8S, and 23S stages, but mostly non-nuclear and not blocked by the specific peptide (Supp. Fig. 2 for 7-8S, 23S; data not shown for 3-4S). However, we observed nuclear staining in the head of 7-8S embryos and in the neural tube, dorsal mesenchyme, and lateral endothelium of 23S embryos, and this staining was blocked by the peptide immunogen (Supp. Fig. 2). Notably, no endodermal or liver bud nuclear staining was observed for Grg1 at any stage. We conclude that Grg1 protein expression is much more limited than its mRNA and that it is not expressed in the endoderm, or that the antigen is blocked in endoderm nuclei, in a cell-specific fashion.

The Grg3 antibody, by contrast, gave more interesting results. First, nuclear Grg3 staining was detected in 6-7S (E8.5) ventral foregut endoderm cells in a pattern that is virtually superimposable over that for the in situ hybridization (Fig. 3A-C). Such staining was blocked by peptide immunogen (Fig. 3D, E). The staining was also detected at the earlier 3-4S stage and was superimposable on the staining elicited by FoxA2, a definitive endoderm marker (Ang et al., 1993; Sasaki and Hogan, 1993; Bossard and Zaret, 1998; Kanai-Azuma et al., 2002; Zhao and Duncan, 2005; Watt et al., 2007) (Fig. 3F-I; see yellow, double-labeled cells depicted by arrows in the merged, magnified panel I). By 8-9S, within a few hours of hepatic specification (Gualdi et al., 1996; Jung et al., 1999) Grg3 protein persisted in the ventral foregut endoderm (Fig. 3J-M). These studies show that Grg3 protein persists for several hours after the time its mRNA disappears (Fig. 2)

Notably, several hours later, by 13S, Grg3 protein disappeared from most cells of the nascent liver bud; the latter being marked by FoxA2 expression (Fig. 4A-D; see mostly green, not yellow, cells in the liver bud in the merged, magnified panel D). Interestingly, Grg3 was also expressed with FoxA2 in the neural tube floor plate at 13S (Fig. 4B-D; n.t.f.p.), though Grg3 expression there diminished at later stages (Fig. 4G and data not shown). By 20S and 22-23S, the liver bud cells were completely devoid of Grg3, as seen by the green and not yellow cells in the merged images (Fig. 4E-L). However, Grg3 was robustly expressed in the stromal cells that surround the liver bud and in the midgut epithelium. At E12.5, Grg3 was not expressed in the liver, though the mesothelium (liver capsule) was positive (Fig. 4M, N). The absence of Grg3 protein in the E12.5 liver parenchyma concurs with the absence of Grg3 mRNA there at E14.5, seen in a previous study (Dehni et al., 1995). The presence of Grg3 protein in the liver mesothelium at E12.5 concurs with Grg3 mRNA expression seen there in another previous study (Leon and Lobe, 1997). We conclude that Grg3 protein is markedly down-regulated during the earliest stages of hepatic differentiation, in the cells that express FoxA2. We note that such cells also express FoxA1 (Ang et al., 1993), and that Grg3 interacts with both FoxA1 and FoxA2 (Sekiya and Zaret, 2007).

Fig. 4. Down-regulation of Grg3 protein in the liver bud and fetal liver.

Fig. 4

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A-L. DAPI, immunoflorescence (IF), and merged and magnified images as in Fig. 3. Note absence of red (panels C, G, K) or yellow (panels D, H, L) Grg3-positive cells in the liver bud, but Grg3-positive cells in the nearby midgut and surrounding stroma. M, N. Sagital section of E12.5 embryo, showing the absence of Grg3 in the liver but its presence in the mesothelium surrounding the liver.

Ectopic expression of Grg3 suppresses liver gene induction in endoderm

Given our previous studies showing that FoxA1 recruitment of Grg3 to liver genes suppresses expression (Sekiya and Zaret, 2007), we speculated that Grg expression in the undifferentiated endoderm prevents liver gene induction. To test this hypothesis, we sought to extend the time over which Grg3 is expressed in the endoderm and assess the consequences for alb1 and Ttr. We employed lentiviruses encoding a wild type, Flag-tagged Grg3 protein or Flag-tagged Grg3-ΔN, the latter containing an N-terminal deletion of Grg3 that is not recruited by FoxA1 (Sekiya and Zaret, 2007), as a control. We developed infection conditions whereby Flag antigen could be detected in most epithelial cells of an infected ventral foregut explant (Supp. Fig. 3), when the explant was generated at the 5-6S or 7-8S stages and cultivated for 3 days. Uninfected controls were negative for Flag antigen (Supp. Fig. 3).

As expected from the data in Fig. 2, qRT-PCR analysis showed that the levels of Grg3 mRNA were diminishing in the initial dissected tissues isolated from 7-8S embryos, compared to 5-6S embryos (Fig. 5A, "initial explant"). Endogenous Grg3 mRNA levels declined in cultured explants from both conditions, showing that the down-regulation of Grg3 observed in vivo was recapitulated, at least in part, after three days culture in vitro (Fig. 5A, "cultured explant"). The Grg3-Flag lentivirus infected cultures expressed the Flag sequence (Supp. Fig. 3) and caused the persistence of Grg3 expression in both the 5-6S and 7-8S explants (Fig. 5A). Notably, persistent Grg3 expression caused a failure to induce alb1 and Ttr mRNA expression (Fig. 5B, C), paradigm markers for the induction of the hepatic program (Gualdi et al., 1996; Jung et al., 1999). This effect was not a nonspecific effect of lentiviral infection, because infection with the Grg3-ΔN virus did not prevent alb1 and Ttr induction (Fig. 5B, C). Furthermore, the effect of ectopic expression of full-length Grg3 was specific to the liver genes because wild type Grg3 virus infection had no effect on the expression of FoxA2 (Fig. 5D). This also demonstrated the integrity of the endoderm tissue in the Grg3-infected explant.

Fig. 5. Persistent Grg3 expression suppresses alb1 induction in ventral foregut explants.

Fig. 5

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A-D. qRT-PCR analysis of designated genes in dissected ventral foregut tissues and cultured explants, infected or not with lentivirus constructs encoding Grg3 or Grg3-ΔN. Bars represent expression levels relative to that for β-actin in the same samples as average of 4-6 independent experiments ± S.E. The PCR primers for Grg3 were specific to the N-terminal coding region of the mRNA and did not reveal elevated signals in the Grg3-ΔN-infected cultures; see Supp. Fig. 3 for evidence of Grg3-ΔN expression. E. New model for endoderm competence. The figure depicts liver gene chromatin to which FoxA factors are bound in the endoderm. I. In undifferentiated endoderm, FoxA binding opens the local chromatin, providing entry (green lines) for factors that could bind flanking sequences. II. Grg binding closes the FoxA-bound chromatin and prevents other factors from binding. III. During tissue specification, Grg factors are down-regulated, relieving repression and allowing other factors to enter the chromatin and activate target gene expression.

New model for endodermal competence

Taken together, the data show that Grg3 is expressed in the undifferentiated endoderm, it is rapidly down-regulated during the time of foregut tissue induction, and if Grg3 expression is maintained during this period, it prevents the induction of alb1, a paradigm sentinel of mammalian liver specification. These findings, along with our previous discovery of FoxA factors being able to recruit Grg3 to liver genes and suppress their expression (Sekiya and Zaret, 2007), lead to a revised model for the basis of hepatic competence imparted by FoxA factors. That is, given the potent ability of FoxA factors to enable transcription (see Introduction) (Fig. 5E, panel I), we suggest that FoxA binding of Grg3 may create a closed chromatin structure local to the regulatory sequence (Fig. 5E, panel II), as was seen in our previous study (Sekiya and Zaret, 2007). This would disallow recruitment of other factors or target gene activation until the appropriate time in development, when down-regulation of Grg3 would relieve repression and allow FoxA target genes to be fully induced (Fig. 5E, panel III). Our findings reveal a dynamic balance between activators and repressors during the earliest stages of hepatic programming and add an additional parameter to consider for understanding how endoderm cells can be deliberately programmed to foregut tissues.

Supplementary Material

Supp Figure S1
Supp Figure S2
Supp Figure S3

Acknowledgments

We thank Takashi Sekiya for lentivirus vectors and Eileen Pytko for help with the manuscript. The research was supported by a grant from the Dirección General de Investigación SAF-2007-60614 (MICINN) and Salvador d Madariaga Program (MEC) to P.S., a FPI predoctoral fellowship (MICINN) to P.R., an NIH CA-009035-34 postdoctoral fellowship to D.E.M., and NIH grant R37 GM36477 to K.S.Z.

References

  1. Ang S-L, Rossant J. Anterior mesendoderm induces mouse Engrailed genes in explant cultures. Development. 1993;118:139–149. doi: 10.1242/dev.118.1.139. [DOI] [PubMed] [Google Scholar]
  2. Ang S-L, Wierda A, Wong D, Stevens KA, Cascio S, Rossant J, Zaret KS. The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development. 1993;119:1301–1315. doi: 10.1242/dev.119.4.1301. [DOI] [PubMed] [Google Scholar]
  3. Bossard P, Zaret KS. GATA transcription factors as potentiators of gut endoderm differentiation. Development. 1998;125:4909–4917. doi: 10.1242/dev.125.24.4909. [DOI] [PubMed] [Google Scholar]
  4. Cascio S, Zaret KS. Hepatocyte differentiation initiates during endodermal-mesenchymal interactions prior to liver formation. Development. 1991;113:217–225. doi: 10.1242/dev.113.1.217. [DOI] [PubMed] [Google Scholar]
  5. Chen G, Courey AJ. Groucho/TLE family proteins and transcriptional repression. Gene. 2000;249:1–16. doi: 10.1016/s0378-1119(00)00161-x. [DOI] [PubMed] [Google Scholar]
  6. Chen G, Fernandez J, Mische S, Courey AJ. A functional interaction between the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development. Genes Dev. 1999;13:2218–2230. doi: 10.1101/gad.13.17.2218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cirillo L, Lin FR, Cuesta I, Jarnik M, Friedman D, Zaret K. Opening of compacted chromatin by early developmental transcription factors HNF3 (FOXA) and GATA-4. Molecular Cell. 2002;9:279–289. doi: 10.1016/s1097-2765(02)00459-8. [DOI] [PubMed] [Google Scholar]
  8. Courey AJ, Jia S. Transcriptional repression: the long and the short of it. Genes Dev. 2001;15:2786–2796. doi: 10.1101/gad.939601. [DOI] [PubMed] [Google Scholar]
  9. Cuesta I, Zaret KS, Santisteban P. The forkhead factor FoxE1 binds to the thyroperoxidase promoter during thyroid cell differentiation and modifies compacted chromatin structure. Mol Cell Biol. 2007;27:7302–7314. doi: 10.1128/MCB.00758-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dehni G, Liu Y, Husain J, Stifani S. TLE expression correlates with mouse embryonic segmentation, neurogenesis, and epithelial determination. Mech Dev. 1995;53:369–381. doi: 10.1016/0925-4773(95)00452-1. [DOI] [PubMed] [Google Scholar]
  11. Fisher AL, Caudy M. Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev. 1998;12:1931–1940. doi: 10.1101/gad.12.13.1931. [DOI] [PubMed] [Google Scholar]
  12. Flores-Saaib RD, Courey AJ. Analysis of Groucho-histone interactions suggests mechanistic similarities between Groucho- and Tup1-mediated repression. Nucleic Acids Res. 2000;28:4189–4196. doi: 10.1093/nar/28.21.4189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gualdi R, Bossard P, Zheng M, Hamada Y, Coleman JR, Zaret KS. Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control. Genes Dev. 1996;10:1670–1682. doi: 10.1101/gad.10.13.1670. [DOI] [PubMed] [Google Scholar]
  14. Hatta M, Cirillo LA. Chromatin opening and stable perturbation of core histone:DNA contacts by FoxO1. J Biol Chem. 2007;282:35583–35593. doi: 10.1074/jbc.M704735200. [DOI] [PubMed] [Google Scholar]
  15. Hunyady B, Krempels K, Harta G, Mezey E. Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J Histochem Cytochem. 1996;44:1353–1362. doi: 10.1177/44.12.8985127. [DOI] [PubMed] [Google Scholar]
  16. Jonsson J, Carlsson L, Edlund T, Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature. 1994;371:606–609. doi: 10.1038/371606a0. [DOI] [PubMed] [Google Scholar]
  17. Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver development from endoderm by fibroblasts growth factors. Science. 1999;284:1998–2003. doi: 10.1126/science.284.5422.1998. [DOI] [PubMed] [Google Scholar]
  18. Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, Sanai Y, Yonekawa H, Yazaki K, Tam PPL, Hayashi Y. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 2002;129:2367–2379. doi: 10.1242/dev.129.10.2367. [DOI] [PubMed] [Google Scholar]
  19. Lazzaro D, Price M, de Felice M, Di Lauro R. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development. 1991;113:1093–1104. doi: 10.1242/dev.113.4.1093. [DOI] [PubMed] [Google Scholar]
  20. Lee CS, Friedman JR, Fulmer JT, Kaestner KH. The initiation of liver development is dependent on Foxa transcription factors. Nature. 2005;435:944–947. doi: 10.1038/nature03649. [DOI] [PubMed] [Google Scholar]
  21. Leon C, Lobe CG. Grg3, a murine Groucho-related gene, is expressed in the developing nervous system and in mesenchyme-induced epithelial structures. Dev Dyn. 1997;208:11–24. doi: 10.1002/(SICI)1097-0177(199701)208:1<11::AID-AJA2>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  22. Sasaki H, Hogan BLM. Differential expression of multiple fork head related genes during gastrulation and pattern formation in the mouse embryo. Development. 1993;118:47–59. doi: 10.1242/dev.118.1.47. [DOI] [PubMed] [Google Scholar]
  23. Sekiya T, Zaret KS. Repression by Groucho/TLE/Grg proteins: genomic site recruitment generates compacted chromatin in vitro and impairs activator binding in vivo. Mol Cell. 2007;28:291–303. doi: 10.1016/j.molcel.2007.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tremblay KD, Zaret KS. Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues. Developmental Biology. 2005;280:87–99. doi: 10.1016/j.ydbio.2005.01.003. [DOI] [PubMed] [Google Scholar]
  25. Wang JC, Waltner-Law M, Yamada K, Osawa H, Stifani S, Granner DK. Transducin-like enhancer of split proteins, the human homologs of Drosophila groucho, interact with hepatic nuclear factor 3β. J Biol Chem. 2000;275:18418–18423. doi: 10.1074/jbc.M910211199. [DOI] [PubMed] [Google Scholar]
  26. Watt AJ, Zhao R, Li J, Duncan SA. Development of the mammalian liver and ventral pancreas is dependent on GATA4. BMC Dev Biol. 2007;7:37. doi: 10.1186/1471-213X-7-37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yan J, Xu L, Crawford G, Wang Z, Burgess SM. The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure. Mol Cell Biol. 2006;26:155–168. doi: 10.1128/MCB.26.1.155-168.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zannini M, Avantaggiato V, Biffali E, Arnone MI, Sato K, Pischetola M, Taylor BA, Phillips SJ, Simeone A, Di Lauro R. TTF-2, a new forkhead protein, shows a temporal expression in the developing thyroid which is consistent with a role in controlling the onset of differentiation. Embo J. 1997;16:3185–3197. doi: 10.1093/emboj/16.11.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zaret K. Developmental competence of the gut endoderm: genetic potentiation by GATA and HNF3/fork head proteins. Devel Biol. 1999;209:1–10. doi: 10.1006/dbio.1999.9228. [DOI] [PubMed] [Google Scholar]
  30. Zaret KS, Grompe M. Generation and regeneration of cells of the liver and pancreas. Science. 2008;322:1490–1494. doi: 10.1126/science.1161431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhao R, Duncan SA. Embryonic development of the liver. Hepatology. 2005;41:956–967. doi: 10.1002/hep.20691. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Supp Figure S1
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Supp Figure S3
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