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Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, Texas 77030
Received 29 September 2006/ Returned for modification 17 January 2007/ Accepted 21 May 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Pitx (pituitary homeobox) family of homeobox genes contains three genes, Pitx1, Pitx2, and Pitx3, within the larger paired-related superfamily of homeobox genes (17, 37). The Pitx group, a remarkably important gene subfamily, has been implicated in human development, disease, and evolution (39-41). For example, Pitx2 is the gene mutated in Rieger syndrome I that results in ocular, tooth, and body wall defects (40). A regulatory mutation in Pitx1 has been implicated in the evolution of the pelvis in threespine sticklebacks (41). The last member of the family, Pitx3, is the gene mutated in patients with congenital cataracts and ocular anterior segment defects (39).
Previous studies have shown that Pitx2 is the earliest marker of the oral ectoderm (33); moreover, as the oral ectoderm diversifies into an organ-forming epithelium, such as the dental epithelium or Rathke's pouch (RP), Pitx2 expression becomes restricted to this maturing epithelium. Consistent with the Pitx2 expression pattern, loss-of-function experiments in mice uncovered an important function for Pitx2 in tooth and pituitary development (16, 24, 25, 28, 30); moreover, Pitx2 was critical for left-right asymmetry, a fundamental component of vertebrate organ morphogenesis, in addition to ocular and abdominal wall morphogenesis (26, 27, 30).
Pitx2 played a critical role in pituitary development through a mechanism that involves both proliferation and cell survival (10, 23). Pitx2 has been shown to regulate Pit-1, PLOD-1, and LEF-1 (1, 20, 46). Other work has also indicated that the Hesx1/Rpx1 homeobox gene is a direct target of Pitx genes in the pituitary (11, 16).
Because of its critical role as a late effector of the left-right asymmetry signaling pathways, Pitx2 transcriptional regulation in the left lateral plate mesoderm has been studied. Transgenic analysis of Pitx2 gene regulation uncovered separable mechanisms controlling Pitx2 in the lateral plate and in maturing organs. In the lateral plate, Pitx2 expression was dependent upon a Nodal-FoxH1 pathway that functioned through an intergenic enhancer termed the asymmetric element (ASE). In the organ primordia, Pitx2 maintenance was regulated by an Nkx element also contained within the ASE (42, 43). It is unknown whether similar two-step mechanisms apply to Pitx2 regulation in the oral ectoderm and organ-forming epithelium.
Although Pitx2 transcriptional regulation in oral ectoderm has not been studied directly in vivo, bead implantation experiments indicated that Fgf8 has a positive influence, while Bmp4 restricts Pitx2 in the forming of dental epithelium (44). Whether the influence of Fgf8 and Bmp4 on Pitx2 expression is direct or distant, downstream events are unknown. Other experiments, performed in tissue culture, implicated Pitx2 as a direct target of Wnt signaling in pituitary and skeletal muscle cells (23). A clear picture of the mechanisms regulating Pitx2 expression in the oral ectoderm and organ-forming epithelium within the whole embryo is lacking.
To gain insight into the mechanisms regulating Pitx2 expression in the oral and dental epithelium, we used a series of transgenic reporter genes to define a Pitx2 oral ectoderm enhancer element. We uncovered a 7-kb region 3' of Pitx2 coding sequences that directed LacZ expression in oral ectoderm.
A deletion analysis of this oral ectoderm element uncovered a subregion that directed LacZ expression in RP, an RP enhancer; moreover, in the RP enhancer we found that nuclear factor 1 (NF-1) and T-cell factor (TCF)/LEF binding sites were required for enhancer activity. These findings reveal that NF-1 and TCF/LEF factors regulate Pitx2 in RP and implicate Pitx2 as a direct target of Wnt signaling in early pituitary development.
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MATERIALS AND METHODS |
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Generation and analysis of transgenic mice. All transgenic constructs were digested and gel purified using a spin column kit (QIAGEN) and eluted in 1x microinjection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4). One nanogram per fertilized zygote of purified transgene was injected into the male pronuclei of embryonic day 0.5 fertilized zygotes from FVB mice. Injected zygotes were transferred back to ICR pseudopregnant females, and embryos were harvested at the desired embryonic time points indicated in the text. DNAs of transgenic founders were analyzed by Southern blotting, and DNAs of embryos were analyzed by PCR with either tail snip or yolk sac genomic DNA. LacZ genotyping primers were the 5' primer, 5'-GCA TCG AGC TGG GTA ATA AGG GTT GGC AAT-3', and the 3' primer, 5'-GAC ACC AGA CCA ACT GGT AAT GGT AGC GAC-3', producing a 700-bp LacZ-specific product. Embryos from sacrificed mice were dissected. Yolk sacs were removed for LacZ PCR genotyping. Embryos were fixed in 2% formaldehyde and 0.2% glutaraldehyde in phosphate-buffered saline, washed at room temperature, and then stained overnight in staining buffer [1 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), 5 mM K3Fe(CN)6 and 5 mM K4Fe(CN)6 in phosphate-buffered saline]. For sections, X-gal-stained embryos were dehydrated in a series of 70, 95, and 100% ethanol, embedded in paraffin, sectioned at a thickness of 8 µm, and counterstained with eosin.
Site-directed mutagenesis. Site-directed mutagenesis of the NF-1 and LEF-1 sites was achieved by using the QuikChange II XL site-directed mutagenesis kit (Stratagene). The DNA fragments used for mutagenesis were cloned into the pGEM T-Easy vector (Promega) first, and mutated fragments were then excised and subcloned into the vector with the Hsp68 LacZ cassette to make tg5k-NF-1 M and tg5k-LEF-1 M (see Fig. 6). The sense-strand sequences of the oligonucleotides used for mutation were 5'-CCCTTACTGTCTTCTATCGCACATCGATCGGTTTTACTTTG-3' for NF-1 and 5'-CCCAGCCAAGGTTTTAGCTAGTTTTCTTTTGCTG-3' for LEF-1 (underlined letters indicate the mutant NF-1 and LEF-1 sites). All mutations were confirmed by DNA sequencing. The same mutations were introduced into the NF-1 and LEF-1 sites for the electrophoretic mobility shift assay (EMSA).
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T3-1 cell nuclear extracts, and a TNT-coupled reticulocyte lysate system (Promega) was used to synthesize NF-1X protein in vitro. Purified LEF-1 protein was used for the TCF/LEF gel shift. Briefly, double-stranded oligonucleotides were annealed and end labeled with [
-32P]dATP using T4 polynucleotide kinase (NEB). Labeled probes were purified using a Micro Bio-Spin 6 column (Bio-Rad). Binding reaction mixtures were incubated in ice in 1x binding buffer (100 mM KCl, 20 mM HEPES, 0.5 mM dithiothreitol, 0.2 mM EDTA) containing 10 µg nuclear extract or 2 µg recombinant NF-1X protein or 0.2 µg purified LEF-1 protein, 1 µg of poly(dI-dC) for 5 min and another 5 min after competitor oligonucleotides (100-fold excess) were added. The reaction mixtures were incubated in ice for an additional 15 min after the addition of the probe and another 30 min after the addition of the antibodies. Reaction mixtures were electrophoresed on a 5% nondenaturing polyacrylamide gel. The sense-strand sequences of the oligonucleotides used for EMSAs were 5'-CTTCTTTGTACCCAGCCAAGGTTTTAC-3' for the wild-type NF-1 site, 5'-CTTCTATCGCACATCGATCGGTTTTAC-3' for the mutant NF-1 site, 5'-CCCAGCCAAGGTTTTACTTTGTTTTCTTTTGCTG-3' for the wild-type LEF-1 site, and 5'-CCCAGCCAAGGTTTTAGCTAGTTTTCTTTTGCTG-3' for the mutant LEF-1 site (underlined letters indicate the wild-type and mutated binding sites). ChIP analysis. The chromatin immunoprecipitation (ChIP) analysis was performed using a ChIP assay kit (Upstate) as previously described (14). The two primers for amplifying the LEF-1 and NF-1 binding sites in the Pitx2c promoter were sense, 5'-GCTCCCCAATCACTGTGTGTACGTGT-3', and antisense, 5'-TTGGGGGCTGCAACCAGCTGCTGGCTGAAGG-3'. All the PCR products were evaluated for appropriate size (390 bp) on a 2% agarose gel in 1x Tris-borate-EDTA and were confirmed by sequencing. As controls, the Pitx2c primers were used without chromatin, normal rabbit immunoglobulin G was used as a replacement for the LEF-1 (Upstate) or NF-1 antibody (Santa Cruz) to reveal nonspecific immunoprecipitation of the chromatin, and primers to an unrelated gene were used to demonstrate the specificity of the LEF-1 or NF-1 antibody-immunoprecipitated chromatin.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To scan sequences downstream of Pitx2 for elements that direct Pitx2c expression in oral ectoderm, we added a 7-kb fragment to the 3' end of tg1K, to generate a plasmid referred to as tg3k (Fig. 1A). We also tested a construct that contained the 7-kb downstream sequences in the context of a small (2.8 kb) 5' deletion (referred to as tg2K) (Fig. 1A). Both tg2k and tg3k directed LacZ activity in the oral ectoderm with high efficiency. To better define the oral ectoderm enhancer, we introduced a 4-kb deletion in the 7-kb fragment in the context of the tg3k construct (tg3k D5821-9918). Embryos with tg3k D5821-9918 still expressed LacZ in the oral ectoderm, indicating that the enhancer element was located at either end of the 7-kb fragment (Fig. 1A).
Expression of the Pitx2c oral ectoderm enhancer during development. To determine whether the Pitx2c oral ectoderm enhancer completely recapitulated endogenous Pitx2c expression, we performed a developmental time course using the stable tg3k and tg3k D5821-9918 lines. Two stable transgenic lines were evaluated for both the tg3k and the tg3k D5821-9918 constructs. All stable lines gave comparable results, although the levels of LacZ activity varied and there was more ectopic LacZ activity in the tg3k D5821-9918 lines, suggesting that the deleted region may have a negative regulatory role (not shown).
Pitx2 is expressed in the stomatodeal ectoderm that gives rise to RP, beginning at 8.5 days postcoitum (dpc) (33). At 11.5 dpc, Pitx2c is expressed in the oral ectoderm of the maxillary and mandibular processes, as well as RP that will give rise to the anterior pituitary (Fig. 1B and C). LacZ staining indicated that tg3k directed LacZ activity in oral ectoderm and RP at 11.5 dpc (Fig. 1D, E, and F). Notably, oral ectoderm expression was biased toward the proximal aspect of the branchial arch (Fig. 1D and E). At 13.5 and 14.5 dpc, LacZ activity was detected in the oral ectoderm, rugae of the secondary palate, dental epithelium, and pituitary (Fig. 1G to K). LacZ activity was also detected in the epithelium of the tongue, as has also been shown by immunohistochemistry (Fig. 1H and K) (21). In dental epithelium and the pituitary, LacZ staining was confined to a subpopulation of cells in each organ, indicating that the tg3k transgene lacked elements that direct endogenous Pitx2c expression throughout the dental epithelium and anterior pituitary. Taken together, these data indicate that the tg3k construct contains elements that direct Pitx2c expression in oral ectoderm and RP but lack other elements that are necessary for complete Pitx2c expression in dental epithelium and anterior pituitary.
Identification of a Pitx2c RP enhancer element. We next investigated whether the 7-kb fragment was sufficient to direct LacZ activity in oral ectoderm and RP. The 7-kb fragment was inserted upstream of the Hsp LacZ construct that contained the Hsp heterologous promoter element to generate the tg4k construct. An analysis of founder embryos carrying the tg4k construct indicated that the 7-kb region was sufficient to direct LacZ activity in the RP but not in the oral ectoderm (Fig. 2A to F). Next, we wanted to narrow down the critical region for RP expression. Based on data from the tg3k Del 5821-9918 construct (Fig. 1) that indicated that the Pitx2c enhancer resided at either the 5' or 3' end of the 7-kb fragment, we subcloned the 5' and the 3' regions from the 7-kb fragment upstream of Hsp LacZ and tested for LacZ activity in founder embryos. This analysis indicated that the RP enhancer element was contained in the 1.8-kb sequence at the 5' end of the 7-kb fragment (Fig. 2A, G, and H).
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T3 pituitary cells. We used antibodies specific for NF-1 and LEF-1 in ChIP assays as described previously (14); moreover, T3 cells endogenously express NF-1 and LEF-1, allowing us to investigate transcription factor binding to the Pitx2c enhancer using endogenous levels of protein. ChIP assays revealed that both NF-1 and LEF-1 can specifically bind to the Pitx2c RP enhancer in the native chromatin environment (Fig. 6A to C). TCF-dependent and NF-1-dependent regulation of Pitx2c in RP. To determine whether the NF-1 and TCF/LEF sites were required for transgene expression in vivo, we generated constructs with mutations in the NF-1 (tg5k-NF-1 M) and TCF/LEF (tg5k-TCF/LEFM) sites in the context of the 1.8-kb enhancer element. Of 12 embryos transgenic for the tg5k-NF-1 M construct, 6 failed to show LacZ staining, while another 5 had very weak LacZ activity after 48 h of staining for LacZ (Fig. 7A and B and data not shown). One embryo expressed LacZ in RP at a level similar to that of the wild-type construct (not shown). This embryo also had LacZ expression in other regions of the embryo, such as the limb muscle and eye, suggesting that the integration site was in a region that was permissive for high levels of transcriptional activity. These findings indicate that the NF-1 site is required for optimal transcriptional activity of the Pitx2c RP enhancer in most chromatin environments. However, as revealed by the one embryo that expressed LacZ in RP at a level similar to that of the wild type, the NF-1 site is dispensable for LacZ activity in chromatin environments that are permissive for transcriptional activity.
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NF-1 has also been implicated in regulating the pituitary-specific POU factor, Pit-1, and the growth hormone gene, and NF-1X was expressed in the mouse gonadotrope cell line T3-1 (data not shown) (13, 36). However, it is unknown whether NF-1 genes are expressed in RP. To address this issue, we performed in situ analysis with all four NF-1 genes and found that NF-1B was weakly expressed in the oral ectoderm surrounding RP while NF-1X was weakly expressed in RP (Fig. 7C). This result suggests that NF-1X is the NF-1 gene that regulates the Pitx2c RP enhancer.
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DISCUSSION |
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Regulation of the Pitx2c RP enhancer by NF-1. The NF-1 family contains four homologous members, NF-1A, NF-1B, NF-1C, and NF-1X. In vitro experiments have shown that NF-1 factors act both as transcriptional repressors and activators, depending on the context (19). More recent work has shown that NF-1 can influence transcription by remodeling chromatin (15). Our findings indicate that the requirement for NF-1 binding to the Pitx2c RP enhancer was necessary for optimal transcriptional activity. Some embryos transgenic for the NF-1 mutant construct showed low transcriptional activity; moreover, the requirement for NF-1 binding could rarely be overcome, for example, when the transgene integrated into a region of high transcriptional activity.
The transcriptional regulatory functions of NF-1 have been studied in the context of the mouse mammary tumor virus promoter. In this system, NF-1 is required for full transcriptional activity by stabilizing the chromatin structure and allowing other factors to bind (15). In Xenopus oocytes, NF-1 cooperates with Oct-1 to stabilize an intermediate chromatin structure and likely acts as a platform to recruit other factors (3, 4). Consistent with this notion, NF-1 has been shown to occupy the glial fibrillary acidic protein promoter prior to the differentiation of astrocytes and cooperates with AP1 in astrocytes to regulate gene expression but fails to activate expression on its own (7, 18). Our data indicate that NF-1 may have an analogous function as a potentiator of Pitx2c transcription in RP.
Pitx2c, a target of Wnt signaling in pituitary development. In the presence of Wnt signaling, ß-catenin is stabilized and enters the nucleus, where it interacts with TCF factors, such as LEF-1 or TCF4, to induce downstream gene expression. In the absence of Wnt signaling, ß-catenin is targeted for destruction by the APC, Axin, Gsk3b complex that phosphorylates ß-catenin and directs it to a destruction pathway (22). Canonical Wnt signaling has been shown to be important during pituitary lineage diversification by promoting Pit1 and repressing Hesx1 expression (34). Other experiments indicated that TCF4 had a role in pituitary expansion by influencing anterior pituitary growth while the noncanonical Wnt, Wnt5a, played a role in regulating cell shape (6, 8).
Canonical Wnt signaling is functional in the pituitary during the time window (11.5 to 14.5 dpc) that we focused on in our study, supporting the idea that Pitx2c in RP is regulated by a canonical Wnt signal (34). Previous work showed that TCF3 and TCF4 are expressed in RP at the 11.5 and 12.5 dpc stages that we studied in our analysis of the Pitx2c RP enhancer, suggesting that they are the TCF factors regulating Pitx2c expression. Our data also indicate that LEF-1 binds efficiently to the Pitx2c RP enhancer in gel shift and in ChIP assays. In addition, LEF-1 is expressed transiently in RP, supporting the notion that LEF-1 is involved in the early induction of Pitx2c RP expression (34).
The loss of the TCF/LEF recognition element in the Pitx2c RP enhancer resulted in a complete loss of transcriptional activity, revealing that this is a functional binding site. In our interspecific sequence alignments, we noted that the TCF/LEF binding site was weakly conserved between mouse and human. Phylogenetic sequence conservation is an extremely powerful tool for determining functional transcription factor binding sites (35). However, there is strong evidence that evolutionary genetic drift can result in point mutations or deletions that disrupt functional transcription factor binding sites and also introduce compensatory mutations that maintain normal gene expression (31). The result of such events would be failure to conserve the location of a functional transcription factor binding site.
Previous studies suggested that Pitx2a was regulated in pituitary cells by canonical Wnt signaling (23). Consensus TCF/LEF binding sites identified upstream of Pitx2a were shown to respond to Wnt signals and bind LEF-1 in tissue culture cells (23). Although it is unknown whether this element directs Pitx2a expression in RP, it will be important in the future to determine whether the different TCF/LEF binding elements interact to direct the expression of the Pitx2a and Pitx2c isoforms in RP. Taken together, our data implicate Wnt signaling in the diversification of the oral ectoderm into the organ-forming epithelium of RP.
Implications for mechanisms regulating Pitx2 transcription. Previous studies uncovered a two-step mechanism that functioned through the ASE, regulating Pitx2c in the lateral plate mesoderm and individual organ primordia. The induction of Pitx2c was dependent upon the Nodal-signaling cascade that works through the FoxH1 effector. The second phase of Pitx2c transcription, dependent upon an Nkx binding site in the ASE, was not required for induction but was essential for Pitx2c transcriptional maintenance. Therefore, the mechanism involved in Pitx2c regulation through the ASE likely reflects sequential binding of transcriptional activators, since transgenes with either element still express at the appropriate developmental stage (42, 43).
Although both NF-1 and TCF/LEF factors are expressed in RP, neither NF-1 nor TCF factors are pituitary specific, suggesting the possibility of a cooperative interaction between these two factors in Pitx2c RP regulation. We propose a model in which NF-1 binding to the Pitx2c RP enhancer induces a potentiation of transcription by NF-1, followed by signal-induced transcriptional activation mediated through the TCF/LEF binding site (Fig. 8). TCF/LEF elements have often been shown to function cooperatively with other elements in developmental systems (2, 5); moreover, in other systems, NF-1 is known to function as a transcriptional potentiator in certain contexts (3). Alternative models are also possible. For example, it is possible that the NF-1 and TCF/LEF elements function independently. In this model, there may be other pituitary-specific factors that function cooperatively with the NF-1 or TCF/LEF elements to direct Pitx2c transcription. Further experiments are warranted to investigate this possibility.
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ACKNOWLEDGMENTS |
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T3-1, Richard Gronostajski for the NF-1X expression plasmid, and E. Olson for Hsp LacZ. We thank A. Baldini and W. Klein for comments on the manuscript and discussions. We also thank the TAMHSC-IBT mouse core facility for microinjection. This work was supported by NIH grants R01 DE16329-01 (J.F.M.) and DE13941 (B.A.A.).
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FOOTNOTES |
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Published ahead of print on 11 June 2007.
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