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Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development
Developmental Biology 300 (2006) 748 – 757 www.elsevier.com/locate/ydbio
Genomes & Developmental Control
Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development Xin Zhang a , Sheldon Rowan b , Yingzi Yue b , Shaun Heaney b , Yi Pan a , Andrea Brendolan c , Licia Selleri c , Richard L. Maas b,⁎ a
Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 W. Walnut St., IB244, Indianapolis, IN 46202, USA b Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA c Department of Cell and Developmental Biology, Cornell University, Weill Medical School, New York, NY 10021, USA Received for publication 6 February 2006; revised 13 June 2006; accepted 21 June 2006 Available online 27 June 2006
Abstract Pancreatic development depends on the transcription factor Pax6, which controls islet cell differentiation and hormone production. To understand the regulation of Pax6 pancreatic expression, we have identified a minimal Pax6 pancreatic enhancer and show that it contains a composite binding site for Meis and Pbx homeoproteins. We further show that Meis proteins are expressed during pancreatic development, and together with Pbx, are able to form a synergistic binding complex on the Pax6 pancreatic enhancer. When tested in transgenic mice, both the Meis and Pbx sites are essential for Pax6 pancreatic enhancer activity, and the composite site can be functionally replaced by a consensus Meis–Pbx sequence. In addition, analysis of Pbx1 and Pbx2 knockout mice demonstrates that, during pancreatic islet formation, Pax6 expression becomes dependent upon Pbx1 and Pbx2 function. As Meis homeoproteins have been previously demonstrated to regulate Pax6 expression during lens development, these results suggest a conserved mechanism of Pax6 regulation by Meis homeoproteins in two different organs. © 2006 Elsevier Inc. All rights reserved. Keywords: Pax6; Meis; Prep1; Pbx1; Pbx2; Transcription factors; Pancreas; Development enhancer; DNA binding; Mouse
Introduction Vertebrate pancreatic cells derive from a common pool of pluripotent progenitors. A complex network of interacting transcription factors, including the paired domain and homeodomain containing transcription factor Pax6, underlies endocrine cell differentiation during pancreatic development. Several lines of evidence support the functional role of Pax6 in pancreatic development. In the Pax6 mutant, Sey1Neu, all endocrine cell types are reduced in number (Sander et al., 1997). In the Pax6−/− knockout, glucagon-producing α cells are absent and islet structure is disrupted (St-Onge et al., 1997). Interestingly, this is compensated for by a significant increase in the number of ghrelin expressing ε cells (Heller et al., 2004; Prado et al., 2004). Glucose intolerance and diabetes have also been observed in patients with PAX6 mutations (Yasuda et al., ⁎ Corresponding author. E-mail address: [email protected] (R.L. Maas). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.06.030
2002). The sensitivity of pancreatic development to Pax6 gene dosage is further demonstrated by transgenic mice overexpressing Pax6, which develop diabetes and pancreatic adenomas (Yamaoka et al., 2000). Pax6 is a key regulator of islet hormone gene expression. The promoter regions of the genes encoding glucagon, insulin and somatostatin all contain Pax6 binding sites, and Pax6 can activate transcription of these genes in cell culture (Andersen et al., 1999; Ritz-Laser et al., 1999; Sander et al., 1997). As a consequence, hormone production is substantially reduced in the Pax6 mutant pancreas, and mice with a pancreas specific knockout of Pax6 die soon after birth from overt diabetes (Ashery-Padan et al., 2004; Sander et al., 1997). Pax6 can also interact with other transcription factors in the pancreas. For example, the transactivation ability of Pax6 on the glucagon promoter is significantly enhanced by its direct interaction with Cdx-2/3 and Maf (Hussain and Habener, 1999; Planque et al., 2001; Ritz-Laser et al., 1999). More recently, Pax6 binding to Pax4 and Pdx1 control sequences has been demonstrated, and
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reduction of Pdx1 expression was observed in Pax6 mutant mice (Ritz-Laser et al., 2002; Samaras et al., 2002; Wang et al., 2004). Thus, Pax6 also plays a direct role in the regulation of key pancreatic regulators. During pancreatic development, a number of transcription factors are known to act earlier than Pax6. These include Hb9 and Pdx1 (also known as Ipf1, Idx-1 and Stf-1), two homeodomain proteins expressed in the pre-pancreatic endoderm (Ahlgren et al., 1996; Guz et al., 1995; Harrison et al., 1999; Li et al., 1999; Miller et al., 1994; Offield et al., 1996). Loss of Hb9 expression blocks dorsal but not ventral pancreatic bud formation from the endoderm (Harrison et al., 1999; Li et al., 1999). Meanwhile, abrogation of Pdx1 results in an arrest of pancreatic development at the bud stage (Ahlgren et al., 1996; Jonsson et al., 1994; Offield et al., 1996). A conditional gene inactivation experiment reveals that Pdx1 is required for the differentiation of both islet and acinar cells in later development (Ahlgren et al., 1998). Another pancreatic bud specific transcription factor is P48 (also known as Ptf1a), which can bias gut epithelium toward pancreatic instead of duodenal fates (Kawaguchi et al., 2002; Krapp et al., 1998). The TALE (three amino acid loop extension) family homeoprotein Pbx1 is a known binding partner of Pdx1, and both exocrine and endocrine cell differentiation are severely disrupted in the Pbx1 mutant (Dutta et al., 2001; Kim et al., 2002; Peers et al., 1995; Swift et al., 1998). The first endocrine lineage specific gene is Neurogenin3 (Ngn3), which is only transiently expressed in the pancreatic epithelium (Apelqvist et al., 1999; Jensen et al., 2000; Schwitzgebel et al., 2000). Nevertheless, inactivation of Ngn3 results in a complete loss of endocrine cells (Gradwohl et al., 2000). Conversely, ubiquitous expression of Ngn3 converts the entire pancreas to an endocrine fate (Apelqvist et al., 1999; Schwitzgebel et al., 2000). Expressed slightly later than Ngn3, the LIM homeodomain protein Islet-1 is expressed in newly differentiated endocrine cells, and its function is clearly demonstrated by its mutant phenotype, which includes a failure of all endocrine cells to differentiate (Ahlgren et al., 1997). At the onset of Pax6 expression, other endocrine-cell-specific transcription factors also appear in the pancreas. These include NeuroD1, Nkx2.2, Pax4, Nkx6.1 and Brn4, all of which affect endocrine cell subtype specification (Hussain et al., 2002; Naya et al., 1997; Sander et al., 2000; Sosa-Pineda et al., 1997; Sussel et al., 1998). Many of these genes are candidates to encode direct regulators of Pax6 during pancreatic development. In a recent study of vertebrate eye development, we uncovered a role for the Meis family homeoproteins Meis1 and Meis2 in controlling Pax6 lens placode expression (Zhang et al., 2002). In this role, Meis recognizes a conserved sequence in the Pax6 lens placode enhancer and forms a multi-protein complex with currently unknown co-factor(s) to directly regulate Pax6 lens placodal expression. To search for direct upstream regulators of Pax6 in the pancreas, we previously identified a Pax6 pancreatic enhancer which resides on a 450 bp element 1.9 kb upstream of the Pax6 P0 promoter (Zhang et al., 2003). In transgenic mice, this enhancer is specifically active in the embryonic pancreas where its
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expression recapitulates endogenous Pax6 expression. In the current study, we have further refined the Pax6 pancreatic enhancer to a minimal 237 bp element. Biochemical studies show that this enhancer contains an evolutionarily conserved composite Meis–Pbx binding site, which exhibits distinct binding activities for different Meis family members. By transgenic analysis, we demonstrate that this Pax6 regulatory sequence that binds Meis and Pbx is required for Pax6 pancreatic enhancer activity in vivo and that it can be replaced by a consensus Meis–Pbx site. Finally, genetic deletion of Pbx1 and Pbx2 results in the down-regulation of Pax6 expression during pancreatic development. Therefore, Pbx and Meis homeoproteins collectively regulate Pax6 expression in the pancreas. Materials and methods Transgenic constructs All transgenic constructs were derived from the plasmid P0–3.9LacZ, which contains a 4.3 kb Pax6 genomic sequence that includes the Pax6 lens ectoderm enhancer, the Pax6 embryonic pancreatic enhancer, the Pax6 P0 promoter and a LacZ reporter gene (Zhang et al., 2002). For mapping the Pax6 pancreatic enhancer, a 1782 bp NsiI–XhoI fragment of the P0–3.9LacZ vector was shortened to 284 bp (PCR amplified using primers: 5′-TAAGATGCATGGCTACCGGGAGAACCTGACTCAGGC-3′ and 5′-AGCCCTCGAGCTTGTGCGGATCCCTGCAACCCAGC-3′); 237 bp (PCR primers: 5′-CTTAATGCATGGTGACCCCAACGCCCGCTCCCTCTCGCC3′ and 5′-AGCCCTCGAGCTTGTGCGGATCCCTGCAACCCAGC-3′); 167 bp (PCR primers: 5′-GAGCATGCATAAGCGTTGGCCTGGAGCTCGGGAGGGC-3′ and 5′-AGCCCTCGAGCTTGTGCGGATCCCTGCAACCCAGC-3′); or 246 bp (PCR primers: 5′-TAAGATGCATGGCTACCGGGAGAACCTGACTCAGGC-3′ and 5′-TGCTCTCGAGCGAAGCTGGAAACGCCACAGCC-3′). For transgenic constructs m5, mC, mR6 and Tg-con, the respective nucleotides were modified using a QuickChange site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing.
Mice Transgenic mice were generated as described previously (Zhang et al., 2002). Pbx1 and Pbx2 knockout mice are maintained in C57BL/6 and Black Swiss background respectively (Capellini et al., 2006; Kim et al., 2002). We also analyzed Pbx1 embryos from the original colony maintained by Dr. Michael Cleary (Stanford University) and consistently observed a similar pancreatic phenotype.
RT-PCR Pancreatic tissue was dissected in ice-cold PBS and immediately placed in liquid nitrogen. RNA was isolated from tissue extracts using a kit (RNA isolation kit, Qiagen), and reverse transcription was carried out according to the manufacturer's instructions (Invitrogen). The primers used for PCR are, Meis1 (forward: 5′AACCTCAACGCATCCGCAGGC-3′; reverse: 5′-GATTGACCAGTCAAATCGAGC-3′), Meis2 (forward: 5′-GGCATCCACTCGTTCAGGAGGA-3′, reverse: 5′-GATAGACCAGTCCAACCGAGC-3′), Meis3 (forward: 5′-CAGTGGTCTGTACATCTGGGG-3′, reverse: 5′-GATTGACCAGTCTAATCGCAC-3′), Prep1 (forward: 5′-CAGACTCAGCCCTACAACAGGG-3′, reverse: 5′-GCTTGATGCCAGCAACCCAGA-3′), Prep2 (forward: 5′-CAGCCTCAGCACTCCAGCAGGG-3′, reverse: 5′-GCTTGATGCCAGCAACCCAGA-3′).
EMSA EMSA was performed as described (Zhang et al., 2002). The probe for the region containing the Meis–Pbx site in the wild-type Pax6 pancreatic element is 5′-GCCCTTTATTGACAGACAGATAGATAAGCTGG-3′. The consensus
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Meis–Pbx binding site probe is 5′-CGAAGCCGGACCTGTCAATCATAGAA3′. The probes were made by annealing oligonucleotides of complementary sequence. Antibodies for DNA binding interference assays were anti-Pbx1, anti-Prep1 and anti-TGIF (all from Santa Cruz Biotechnology, Santa Cruz, CA). Meis1, Meis2, Pbx1, Prep1 and Prep2 proteins were made by in vitro transcription and translation using a kit (Promega). Expression vectors were kindly provided by Dr. Neal Copeland (Meis1, Meis2) (NCI, Frederick), Dr. Galvin Swift (Pbx1) (UT Southwestern, Dallas), Dr. Kenneth Gross (Prep1) (Roswell Park Cancer Institute, Buffalo) and Dr. Mark Featherstone (Prep2) (McGill Cancer Center, Montreal).
Histology, in situ hybridization and immunohistochemistry X-gal staining, in situ hybridization and immunohistochemistry were performed as previously described (Zhang et al., 2003). The following antibodies were used: mouse anti-Pax6 and mouse anti-Islet-1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-Pax6 (Covance, Berkeley, CA), mouse anti-insulin and rabbit antiglucagon (BioGenex, San Ramon, CA), guinea pig anti-Glucagon (Linco, St. Charles, MO), rabbit anti-Meis1 and anti-Meis2 (kind gift from Dr. Arthur Buchburg, Jefferson Medical College), rabbit anti-Pbx1/2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Pdx1 (kind gift from Dr. Chris Wright, Vanderbilt University), rabbit anti-IDX-1 (kind gift from Dr. Joel Habener, MGH, Harvard Medical School) and rabbit anti-Prep2 (kind gift from Dr. Mark Featherstone, McGill Cancer Center). Rabbit and guinea pig Prep1 antibodies were raised against the C-terminal 102 amino acids of mouse Prep1 fused to GST using the expression vector pGEX-6P-3 (Amersham Bioscience). Both rabbit and guinea pig polyclonal antibodies were raised by Covance and affinity-purified. The resulting anti-Prep1 antibodies were tested by Western blot and immunostaining and specifically recognized Prep1 in transfected but not control cells. To determine the Pax6/Pdx1 ratio, mouse embryos were sectioned serially at 10 μm increments and stained with Pax6 and Pdx1 antibodies. The total number of Pax6 and Pdx1 positive cells was counted on every 6th section for E14.5 embryos or on at least 5 sections for E10.5 embryos. At least 3 embryos of each genotype were analyzed. The statistical significance was calculated using Student's t test.
Results The minimal Pax6 embryonic pancreatic enhancer contains a composite Meis–Pbx binding site To study the transcriptional regulation of Pax6, we first determined the minimal sequence required for proper Pax6 pancreatic enhancer activity in transgenic mice. To do this, a LacZ reporter gene was fused downstream of 4.3 kb of murine Pax6 genomic sequence, containing the endogenous Pax6 promoter (P0), the embryonic pancreatic enhancer (Pan) and the lens ectoderm enhancer (Lens) (Zhang et al., 2002). After two consecutive deletions to the previously determined 450 bp Pax6 pancreatic enhancer element, the resulting 237 bp sequence fully retained pancreatic expression in all 5 E10.5 transgenic embryos analyzed (Figs. 1A, B). Further deletion of 70 bp from the 5′ end or of 38 bp from the 3′ end of the 237 bp sequence, however, abolished all pancreatic enhancer activity. This was not due to transgene integration effects since lens expression was not affected. Therefore, this 237 bp region is both necessary and sufficient for Pax6 pancreatic enhancer activity. Comparison with the genomes of mouse, human and Fugu fish revealed that, although the 96 bp at the 3′ end of the enhancer is conserved
Fig. 1. The minimal Pax6 pancreatic enhancer contains a conserved Meis–Pbx site. (A) The Pax6 lens ectoderm enhancer (Lens), Pax6 embryonic pancreatic enhancer (Pan) and the Pax6 P0 promoter are shown in the 3.9 kb Pax6 genomic sequence. The Meis–Pbx site lies at the beginning of the human/ mouse/fugu fish homologous region (the 5′ boundary denoted by a vertical line). For transgenic analysis, the number of β-galactosidase expressing lines is shown together with the total number of independent transgenic embryos. B: BamHI, E: EcoRI, H: HindIII. (B) Transient transgenic embryos were collected at E10.5, and the reporter gene activities in pancreas detected by X-gal staining (arrow). Note that lens expression is preserved in each transgenic line, while truncation of the minimal 237 bp element by 70 bp at the 5′-end (167 bp construct) or by 38 bp at the 3′-end (246 bp construct) results in loss of pancreatic expression.
between all three species, the 5′ portion of the 237 bp element is homologous only between human and mouse. This suggests the existence of mammalian specific regulatory elements that control embryonic Pax6 pancreatic expression.
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Analysis of the Pax6 pancreatic enhancer sequence revealed a perfect consensus Meis binding site (5′-TGACAG-3′) at the 5′ end of homologous region between human, mouse and Fugu fish (Chang et al., 1997; Shen et al., 1997) (Fig. 1A). Consistent with evolutionary significance, this Meis binding site is perfectly conserved between human, mouse and fugu fish (Miles et al., 1998). This observation is particularly intriguing in light of our previous finding that the Pax6 embryonic lens enhancer also contains a Meis binding site (5′-TGACAA-3′) and is regulated by Meis family members (Zhang et al., 2002). In the developing lens, the Meis protein forms a nuclear protein complex with additional unknown co-factors to regulate Pax6 expression. Further sequence analysis failed to identify any additional homology between the lens and pancreatic Pax6 enhancers. However, immediately downstream of the murine pancreatic Meis site is a region (5′-AGATAGAT-3′) which closely resembles the consensus Pbx binding site (5′-TGATTGAT-3′) (Van Dijk et al., 1993) (Fig. 1A). In addition, previous studies have established that Meis and Pbx can form functional heterodimers and bind DNA synergistically (Berthelsen et al., 1998; Chang et al., 1997), suggesting a model in which Meis and Pbx cooperatively bind the Pax6 pancreatic enhancer to regulate Pax6 expression. Meis and Pbx family members are expressed throughout pancreatic development The vertebrate Meis family has five members, Meis1, Meis2, Meis3, Prep1 and Prep2 (Berthelsen et al., 1998; Fognani et al., 2002; Haller et al., 2002; Moskow et al., 1995; Nakamura et al., 1996). These proteins all contain an N-terminal Meis domain, which mediates the interaction with Pbx, and a C-terminal homeodomain that can bind DNA. To determine whether Meis family members are expressed in the pancreas, we first performed RT-PCR to examine Meis transcripts in the whole pancreas. In both postnatal day P3 and adult pancreata, Meis1, Meis2 and Prep1 were clearly expressed (Fig. 2A). In embryonic E14.5 pancreata, transcripts of all five Meis family genes were detected (Fig. 2A). Consistent with this finding, we also observed Meis1, Meis2, Prep1 and Prep2 protein expression in the developing E10.5 pancreas by immunostaining (Figs. 2B–E). The antibodies used were specific to each of the four proteins, as demonstrated by their unique staining patterns in the spinal cord (data not shown). At E10.5, Meis1 and Meis2 were strongly expressed in pancreatic mesenchyme and only weakly co-localized with scattered Pax6 positive pancreatic epithelial cells (Figs. 2B, C). In contrast, Prep1 protein was ubiquitously expressed in pancreatic epithelial cells, thus overlapping with Pax6 positive cells (Fig. 2D). Prep2 showed strong pancreatic mesenchymal expression, but also colocalized with Pax6 positive epithelial cells (Fig. 2E). These results were further confirmed by RNA in situ hybridization (data not shown). We next examined Pbx gene expression in the E10.5 pancreas. Adjacent pancreatic sections from E10.5 embryos were analyzed for Pax6, Pbx1, Pbx2 and Pbx3 transcripts by
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RNA in situ hybridization and for total Pbx protein by immunofluorescence with a pan-Pbx reactive antibody (Figs. 2F–I). While Pax6 was detected only in clusters of islet cells at this stage (Fig. 2F), Pbx1 and Pbx2 were widely expressed in the pancreatic region (Figs. 2G, H). Pbx1 expression was detected at high levels in pancreatic mesenchyme and apparent only at lower levels in pancreatic epithelium (Fig. 2G). Pbx2 was expressed in both the epithelial and mesenchymal compartments (Fig. 2H), while Pbx3 pancreatic expression was not detected at this stage by this method (data not shown). Consistent with these results, a pan-Pbx antibody detected expression of Pbx proteins in both pancreatic epithelial and mesenchymal cells, and a subset of Pbx positive epithelial cells co-expressed Pax6 (Fig. 2I). Prior studies have also detected Pbx1 and Pbx3 expression in developing islet cells (Gu et al., 2004; Kim et al., 2002). Taken together, we conclude that Pax6, Meis and Pbx family members exhibit overlapping expression in the developing endocrine pancreas. Prep1 and Pbx1 bind the Pax6 pancreatic enhancer synergistically Our previous studies indicated that both Meis1 and Meis2 have the potential to regulate Pax6 expression in the lens (Zhang et al., 2002). We thus performed DNA electrophoretic mobility shift assays (EMSAs) to test whether these two proteins could also bind the Pax6 pancreatic enhancer element. Interestingly, neither Meis1, Meis2 nor Pbx1 (lane 4) alone bound a 26 bp oligonucleotide containing the putative Meis– Pbx site (Fig. 3 and data not shown). However, inclusion of both Meis1 and Pbx1 (lane 2) or Meis2 and Pbx1 (lane 3) proteins in the EMSA resulted in a moderately abundant shift in DNA mobility, indicating the formation of binary protein complexes on the DNA (Fig. 3). Moreover, the synergistic binding between Meis1 or 2 and Pbx1 was abolished by an anti-Pbx antibody but not by a control anti-Prep1 antibody (data not shown). Thus, Meis1 or 2 and Pbx1 cooperatively bind the Pax6 pancreatic enhancer in vitro. We next tested the DNA binding activity of Prep1. While both Meis1 and Meis2 bound the Pax6 enhancer element in conjunction with Pbx1, a markedly stronger binding was observed when Prep1 and Pbx1 were tested together (Fig. 3, lane 6 vs. lanes 4 and 5). The Prep1–Pbx1 complex was sensitive to antibodies against Prep1 or Pbx (lanes 7 and 8), but not to a control anti-TGIF antibody (lane 9; TGIF is another closely related TALE family homeoprotein). Similarly, we also observed strong cooperative binding of Prep2 and Pbx1 to the 26 bp probe (data not shown). The differential binding affinities exhibited by Meis1, Meis2, Prep1 and Prep2 are surprising because PCR-based binding site selection experiments suggest that Meis1 and Prep1 prefer identical consensus sequences (Berthelsen et al., 1998; Chang et al., 1997). Indeed, Meis1, Meis2 and Prep1 also bound equally strongly with Pbx1 protein on a probe containing a Meis–Pbx composite site (5′TGATTAGCAG-3′) taken from the literature (Fig. 3; lanes 11, 12 and 15) (Chang et al., 1997). Therefore, the preferential binding of Prep1 and Pbx1 or of Prep2 and Pbx1 to the Pax6
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Fig. 2. Expression analysis of Meis and Pbx family members during pancreatic development. (A) RT-PCR analysis was carried out for adult, day P3 and E14.5 mouse pancreas. In the adult and P3 pancreas, Meis1, Meis2 and Prep1 are expressed. In E14.5 pancreata, all 5 Meis family genes were detected. Pax6, β-actin and ribosomal L19 transcripts were present in all three samples. The nominally equivalent efficiencies of the PCR primers for the different Meis family genes were established by testing equal amounts of plasmid DNA for each gene as template (data not shown). (B–E) At E10.5, immunohistochemistry detected Meis1 and Meis2 proteins mainly in pancreatic mesenchyme, while Prep1 and Prep2 proteins are present throughout the pancreas and co-localize with Pax6 in islet cells (yellow nuclei). (F–G) Pax6, Pbx1 and Pbx2 transcripts and protein detected by RNA in situ hybridization and immunohistochemistry. Circled regions depict the boundary of the pancreatic epithelium containing prospective islet cells. Pbx1 expressed is detected at higher levels in pancreatic mesenchyme relative to epithelium, whereas Pbx2 is expressed in both compartments. The pan-Pbx antibody detects co-expression of Pax6 (red fluorophore) and Pbx (green fluorophore) in islet cells (yellow nuclei).
pancreatic enhancer is due to the unique sequence of this element, and not to a difference in protein concentrations or activities. Especially when taken with the results from preceding expression experiments, these results strongly suggest that Prep1 and Prep2 are the most likely Meis family candidates to be involved in regulation of the Pax6 embryonic pancreatic enhancer. Mutational analysis of the Meis–Pbx site We next undertook an in vitro mutational analysis of the Prep1 and Pbx1 composite binding site in the Pax6 pancreatic enhancer sequence (Fig. 4A). In EMSAs, whereas mutation of 2 bases 5′ to the Meis site did not affect Prep1–Pbx1 binding, replacement of 5 of the 6 bases of the Meis binding site (mutant C) completely abolished complex formation in vitro (Fig. 4B and data not shown). Moreover, as further evidence that the Meis site is required for Prep1–Pbx1 binding, point mutations
(mutant m5 and mutant m1) within the Meis site significantly inhibited protein–DNA complex formation (Fig. 4B). In the 8 bp Pbx site, mutation of 2 bases (mutant R2), 4 bases (mutant R4) or 6 bases (mutant mR6) progressively reduced the binding affinity of the Prep1–Pbx1 complex to DNA (Fig. 4C). However, deletion from the probe of all 8 bases comprising the Pbx1 site (mutant RΔ) did not abolish complex binding, which is thus relatively independent of the sequence of the Pbx site (Fig. 4C). This finding is surprising considering that Pbx1 protein is critically required for Prep1 to bind to the Pax6 pancreatic enhancer (see Fig. 3) and suggests that Pbx1 contributes to the stability of the binding complex by inducing a conformational change in Prep1. Lastly, we replaced the Pbx site in the Pax6 pancreatic enhancer with a consensus Pbx binding sequence (mutant Rc); this led to the expected result of stronger Prep1–Pbx1 binding (Fig. 4C). The protein–DNA complex on RΔ and Rc probes was abolished in the presence of
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contrast, lens expression of the two mutant transgenic constructs was at least as strong as that of the wild-type transgene. Thus, the in vivo effects of Meis site mutations on Pax6 pancreatic enhancer activity directly correlate with the effects of these mutations on Prep1–Pbx1 DNA binding in vitro. We next tested the effect of a 6-base mutation in the Pbx site (mutant mR6). In contrast to the wild-type transgene, this mutation reduced but did not abolish pancreatic enhancer activity in all 9 independent lines assayed. This result is consistent with the biochemical experiment which produced a similar result (see Fig. 4B). Taken together, these results support the hypothesis that both Meis and Pbx sites are required for optimal transcriptional activity of the Pax6 pancreatic enhancer. Meis and Pbx bind the Pax6 pancreatic enhancer in a relatively weak manner compared to their binding to the consensus composite Meis–Pbx site described in the literature. This observation raises the question of whether the endogenous Pax6 enhancer sequence, which deviates from the consensus both in sequence and in the relative orientation of the Meis and Pbx sites, is functionally equivalent to the consensus sequence (Fig. 5A). To address this question, we replaced the 16 bp Fig. 3. Prep and Pbx proteins preferentially bind to the Pax6 pancreatic enhancer. Probes containing the wild-type Meis–Pbx site in the Pax6 pancreatic enhancer (Wild Type) form complexes with either Meis1 and Pbx1, or Meis2 and Pbx1, but not with Meis1, Meis2, Prep1 or Pbx1 individually. Much stronger binding is observed for the Prep1 and Pbx1 complex, whose formation is blocked by anti-Pbx1 or anti-Prep1 antibodies, but not by anti-TGIF antibody. For the consensus Meis–Pbx probe (Consensus), equally strong Meis1–Pbx1, Meis2–Pbx2 and Prep1–Pbx1 binding is observed, validating the protein activities in the experiments employing the wild-type probe.
anti-Prep1 and anti-Pbx1 antibodies, demonstrating that the protein complexes indeed consisted of Prep1 and Pbx1 (Fig. 4D). In summary, both the Meis and Pbx sites in the Pax6 pancreatic enhancer contribute to the stabilization of a Prep1– Pbx1 complex on DNA. Pax6 pancreatic enhancer activity in mice requires a functional Meis–Pbx site Although we identified several pancreatic endocrine cell lines that expressed Meis1, Meis2, Prep1 and Pax6, none supported the activity of the Pax6 pancreatic enhancer upon transfection. Not surprisingly, we found that neither Prep1– Pbx1 nor Meis1–Pbx1 transactivates the Pax6 pancreatic enhancer in any of these cell culture systems (data not shown). To test the functional requirement of the Meis–Pbx site for Pax6 pancreatic enhancer activity during development, we performed mutational analyses in transgenic mice. We employed constructs containing both the Pax6 pancreatic and lens enhancers, so that lens expression could serve as an internal standard for changes in pancreatic expression. When a single base mutation in the Meis site (mutant m5) was introduced into construct P0–3.9LacZ in transgenic mice, pancreatic expression was significantly reduced in all 11 independent lacZ reporter expressing lines (Figs. 5A, B). Additionally, mutation of 5 bases in the Meis site (mutant mC) completely abolished enhancer activity in the pancreas in all 5 expressing lines (Fig. 5). In
Fig. 4. Meis and Pbx sites are required for Prep1 and Pbx1 binding and transactivation. (A) Sequences of Meis and Pbx site mutations. (B) Mutations in Meis sites disrupt the binding activity of Prep1 and Pbx1 in EMSA. Note that mutant mC completely abolishes Prep1 and Pbx1 binding. (C) Mutations in Pbx sites weaken Prep1/Pbx1 binding. (D) Protein complexes that form on RΔ and Rc are sensitive to Prep1 and Pbx1 antibodies.
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Fig. 5. The Meis–Pbx site is required for Pax6 pancreatic enhancer activity in transgenic mice. (A) Schematic diagram of the transgenic constructs. The Meis and Pbx sites and the mutants tested are highlighted. (B) Representative E10.5 transgenic embryos carrying the designated transgenes. Note that, whereas lens expression is preserved, pancreatic expression is diminished in mutant embryos containing the m5, mC and mR6 constructs (arrows). Replacement of the wildtype Meis–Pbx sequence with the consensus sequence (Tg-con) results in retention of transgene expression in the pancreas (con). *Weak pancreatic expression was detectable in the m5 and mR6 embryos.
Meis–Pbx site in the Pax6 pancreatic enhancer LacZ reporter construct with a 10 bp Meis–Pbx consensus sequence (Tg-con) taken from the literature (Chang et al., 1997) and then assayed LacZ reporter activity of the Tg-con transgene in transgenic mice at E10.5. Pancreatic expression similar to that seen with the Pax6 pancreatic enhancer was observed in 4 out of 4 of these Tg-con transgenics. In addition, immunohistochemistry showed co-localization of the LacZ and Pax6 expressing cells in the E10.5 pancreas (Fig. 5B). Therefore, during early embryonic development, a Meis–Pbx consensus sequence can functionally substitute for an indispensable region of the Pax6 pancreatic enhancer. Pax6 is down-regulated in the Pbx1 and Pbx2 knockout pancreas To test whether Pbx1 is required for Pax6 pancreatic expression, we first re-examined pancreatic development in Pbx1 deficient mouse embryos (Kim et al., 2002). At E14.5, the number of Pax6 expressing cells and their qualitative intensity
were strongly reduced in E14.5 Pbx1 mutants. This was in contrast to the robust expression of Pdx1 found in Pbx1 mutant pancreatic epithelium, suggesting that Pax6 down-regulation is not due to general deficiency of pancreatic progenitors (Fig. 6A). Indeed, the ratio of Pax6/Pdx1 positive cells was significantly reduced in Pbx1 mutant embryos (Fig. 6D). Endocrine cell differentiation defects in Pbx1 mutants were also revealed by decreased insulin and glucagon expression, as well as by a reduction in the number of Islet-1 positive cells (Fig. 6A and data not shown). These results confirm a previous demonstration that Pbx1 is required for proper pancreatic development (Kim et al., 2002) but additionally demonstrate that Pbx1 is required at 14.5 for the maintenance of pancreatic Pax6 expression. We next analyzed Pax6 pancreatic expression in E10.5 Pbx1 mutants. Strong expression of Pax6, glucagon, islet-1 and Pdx1 was detected in both wild-type and Pbx1 mutant pancreatic epithelium, and the Pax6/Pdx1 cell number ratio was unchanged (Figs. 6B–D and data now shown). This demonstrates that islet cell differentiation is not significantly disrupted by Pbx1 mutation during early pancreatic development. In E10.5 Pbx1−/−Pbx2+/− compound embryos, however, we observed severe pancreatic hypoplasia and significant reduction in the number of Pax6 positive cells and Pax6/Pdx1 ratio (Figs. 6C, D). More importantly, the level of Pax6 expression in the remaining endocrine cells was also down-regulated. This was shown more clearly by RNA in situ hybridization, which revealed that Pax6 expression was lower in Pbx1−/−Pbx2+/− pancreatic buds than in wild-type controls (Fig. 6C). As control, Pax6 expression in the neural tube remained unchanged in the Pbx1−/−Pbx2+/− mutants. Therefore, Pbx1 and Pbx2 together regulate Pax6 expression during early pancreatic development. Discussion Role of Meis family members In this paper, we have defined a minimal 237 bp enhancer for embryonic Pax6 pancreatic expression. Transgenic experiments further demonstrate that the Meis–Pbx binding site in this enhancer is critical for its activity and that a Meis–Pbx consensus sequence can substitute for the endogenous binding site in this regard. We also show that the Meis and Pbx family members, Prep1 and Pbx1 respectively, can bind this element cooperatively. It is likely that other Meis and Pbx family members, such as Prep2 and Pbx2, can also bind this element cooperatively. We found that Prep2 acted interchangeably with Prep1 in EMSA (data not shown), and based on sequence similarity, Pbx2 should act similarly to Pbx1. Moreover, both Prep2 and Pbx2 share co-expression with Pax6 in the developing pancreatic epithelium at E10.5, when the enhancer is active. Many Meis and Pbx family members are expressed during pancreatic development, and members of each family recognize closely related DNA sequences. However, we observed that the Meis family members Prep1 and Prep2 form substantially stronger binding complexes with Pbx1 on the Pax6 pancreatic enhancer than do Meis1 and Meis2. These
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Fig. 6. Pax6 expression is reduced in Pbx1 and Pbx2 mutant pancreata. (A) In E14.5 Pbx1 mutant pancreata, marked reductions in Pax6, Islet-1, glucagon and insulin expression are observed. However, Pdx1 expression remains relatively unchanged. (B) Pax6 and glucagon are expressed in E10.5 Pbx1 mutant pancreata, but downregulated in Pbx1−/−Pbx2+/− mutants. The pancreatic epithelium is marked by strong Pdx1 protein expression and outlined with dotted lines. (C) Pax6 mRNA level in the pancreas is reduced in E10.5 Pbx1−/−/Pbx2+/− mutant as shown by RNA in situ hybridization. The same pancreas section was stained for glucagon to mark alpha cells. Pax6 expression in the neural tube on the same sections is not changed. (D) Quantification of E10.5 and E14.5 Pax6+/Pdx1+ cell ratios. Specific loss of Pax6 expressing cells is observed in Pbx1−/− mutants at E14.5 and Pbx1−/−Pbx2+/− mutants at E10.5.
results likely reflect different DNA binding sub-specificities among Meis family proteins and, taken with their colocalization with Pax6, suggest that Prep1 and Prep2 are the most critical Meis factors regulating Pax6 during pancreatic development. Further analysis of Prep1 and Prep2 mutant mice should clarify the role of these genes in Pax6 embryonic pancreatic expression. Role of Pbx family members Previous studies have identified an important role for Pbx1 in pancreas development. Genetic ablation of Pbx1 results in severe pancreatic hypoplasia and β cell dysfunction (Kim et al., 2002). Consistent with this phenotype, binding sites for Pbx1 and its co-factors, Pdx1 and Meis/Prep family proteins, have been found in both endocrine and exocrine genes, including somatostatin, glucagon and elastase (Herzig et al., 2000; Peers et al., 1995; Swift et al., 1998). In addition, Pbx1 and Pdx1 genetically interact in glucose homeostasis, and a Pdx1 mutant lacking Pbx1 binding ability fails to support the normal migration and proliferation of pancreatic islet cells (Dutta et al., 2001; Kim et al., 2002). Therefore, Pbx1, together with Meis and Pdx1, directly regulates β cell function in the pancreas. Nevertheless, study of Pbx1 knockout mice has also shown that
Pbx1 is required for both endocrine and exocrine cell differentiation, suggesting that Pbx1 also regulates pancreatic developmental genes. In this study, we present biochemical and transgenic data showing that the critical developmental regulatory gene Pax6 is a direct target of Pbx1 during islet genesis. Thus, Pbx1 acts at multiple levels during pancreas development. The analysis of Pbx1–Pbx2 compound knockout embryos also demonstrates that both Pbx1 and Pbx2 are required for Pax6 regulation. In E14.5 Pbx1 mutant pancreatic islets, we observed significant reductions in the number of Pax6 positive cells relative to the number of Pdx1 positive cells; this was also accompanied by a down-regulation in insulin and glucagon. Although we found little change in Pax6 expression in Pbx1 mutant pancreatic epithelium at E10.5, in Pbx1−/−Pbx2+/− mutants, we observed a significant loss of Pax6 positive cells in the pancreatic bud. Moreover, Pax6 transcript levels in islet progenitors were also much reduced. Since Pbx1/2 inactivation leads to profound pancreatic defects, we cannot rule out the possibility that the loss of Pax6 expression is due to pleiotropic effects of the Pbx1/2 mutation on pancreatic development. However, the reduction of Pax6 mRNA levels shows that Pbx1 and Pbx2 must regulate Pax6 at the transcriptional level. Thus, our results are consistent with a model whereby Pax6 is a direct
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downstream target of Pbx1, Pbx2 or both in pancreatic development. In addition, although we were unable to detect Pbx3 expression in the E10.5 pancreas by whole mount in situ hybridization, Pbx3 has been detected in Pdx1 positive cells in microarray studies (Table S4 in Gu et al., 2004) and also at later stages of pancreatic development (Di Giacomo et al., in press). Of note, while transgenic analysis demonstrates the critical importance of the Meis–Pbx binding site for endogenous Pax6 pancreatic enhancer activity, Pax6 expression is not completely eliminated in Pbx1−/−Pbx2+/− compound mutant embryos. This discrepancy could be accounted for by genetic redundancy from the remaining copy of Pbx2 or by a different Pbx gene family during pancreatic epithelial development. Thus, in principle, all three Pbx genes may contribute to the regulation of Pax6 pancreatic expression. Conserved role for Meis in Pax6 regulation We previously showed that Meis1 and Meis2 are direct upstream regulators of Pax6 in vertebrate lens development where they act upon a Meis site juxtaposed to a non-Pbx cofactor site in the Pax6 lens placode enhancer (Zhang et al., 2002). In this paper, we have extended the requirement for a Meis-site-dependent regulatory mechanism to embryonic Pax6 pancreatic expression. It is notable that, although the Pax6 lens and pancreatic enhancers share Meis binding sites, the Meis cofactors that bind these two elements are likely different. In the Pax6 pancreatic enhancer, Pbx protein is the requisite binding partner of Meis, but in the Pax6 lens enhancer, Pbx does not bind DNA even in the presence of Meis protein. Not surprisingly, therefore, inactivation of Pbx1 disrupts Pax6 expression in the pancreas but not in the lens (this paper and data not shown). In fact, the Pax6 lens and pancreatic enhancers do not share any other apparent homologous sequence except their respective Meis sites. This suggests that Meis may interact with different sets of transcription factors in lens and pancreas. Therefore, the conserved regulation of Pax6 by Meis proteins may be accomplished in different organs by changes in the identities of the Meis co-factors, which may then further define the different tissue-specific activities of these enhancers. Acknowledgments The authors gratefully thank Drs. M. Cleary, A. Buchberg, M. Featherstone, K. Gross, J. Habener and C. Wright for embryos, antibodies and cell lines and Drs. Francesco Blasi and Susan Bonner-Weir for helpful discussions. We are also indebted to Terence Capellini and Dr. Elisabetta Ferretti for donating Pbx1/2 compound mutant embryos. SR was supported by a fellowship from the Canadian Institutes of Health Research. The work was supported by grants from the American Diabetes Foundation (1-04-RA-117 to XZ) and the NIH (RO1 DK065791 to RLM). References Ahlgren, U., Jonsson, J., Edlund, H., 1996. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/ PDX1-deficient mice. Development 122, 1409–1416.
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Genomes & Developmental Control
Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development Xin Zhang a , Sheldon Rowan b , Yingzi Yue b , Shaun Heaney b , Yi Pan a , Andrea Brendolan c , Licia Selleri c , Richard L. Maas b,⁎ a
Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 W. Walnut St., IB244, Indianapolis, IN 46202, USA b Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA c Department of Cell and Developmental Biology, Cornell University, Weill Medical School, New York, NY 10021, USA Received for publication 6 February 2006; revised 13 June 2006; accepted 21 June 2006 Available online 27 June 2006
Abstract Pancreatic development depends on the transcription factor Pax6, which controls islet cell differentiation and hormone production. To understand the regulation of Pax6 pancreatic expression, we have identified a minimal Pax6 pancreatic enhancer and show that it contains a composite binding site for Meis and Pbx homeoproteins. We further show that Meis proteins are expressed during pancreatic development, and together with Pbx, are able to form a synergistic binding complex on the Pax6 pancreatic enhancer. When tested in transgenic mice, both the Meis and Pbx sites are essential for Pax6 pancreatic enhancer activity, and the composite site can be functionally replaced by a consensus Meis–Pbx sequence. In addition, analysis of Pbx1 and Pbx2 knockout mice demonstrates that, during pancreatic islet formation, Pax6 expression becomes dependent upon Pbx1 and Pbx2 function. As Meis homeoproteins have been previously demonstrated to regulate Pax6 expression during lens development, these results suggest a conserved mechanism of Pax6 regulation by Meis homeoproteins in two different organs. © 2006 Elsevier Inc. All rights reserved. Keywords: Pax6; Meis; Prep1; Pbx1; Pbx2; Transcription factors; Pancreas; Development enhancer; DNA binding; Mouse
Introduction Vertebrate pancreatic cells derive from a common pool of pluripotent progenitors. A complex network of interacting transcription factors, including the paired domain and homeodomain containing transcription factor Pax6, underlies endocrine cell differentiation during pancreatic development. Several lines of evidence support the functional role of Pax6 in pancreatic development. In the Pax6 mutant, Sey1Neu, all endocrine cell types are reduced in number (Sander et al., 1997). In the Pax6−/− knockout, glucagon-producing α cells are absent and islet structure is disrupted (St-Onge et al., 1997). Interestingly, this is compensated for by a significant increase in the number of ghrelin expressing ε cells (Heller et al., 2004; Prado et al., 2004). Glucose intolerance and diabetes have also been observed in patients with PAX6 mutations (Yasuda et al., ⁎ Corresponding author. E-mail address: [email protected] (R.L. Maas). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.06.030
2002). The sensitivity of pancreatic development to Pax6 gene dosage is further demonstrated by transgenic mice overexpressing Pax6, which develop diabetes and pancreatic adenomas (Yamaoka et al., 2000). Pax6 is a key regulator of islet hormone gene expression. The promoter regions of the genes encoding glucagon, insulin and somatostatin all contain Pax6 binding sites, and Pax6 can activate transcription of these genes in cell culture (Andersen et al., 1999; Ritz-Laser et al., 1999; Sander et al., 1997). As a consequence, hormone production is substantially reduced in the Pax6 mutant pancreas, and mice with a pancreas specific knockout of Pax6 die soon after birth from overt diabetes (Ashery-Padan et al., 2004; Sander et al., 1997). Pax6 can also interact with other transcription factors in the pancreas. For example, the transactivation ability of Pax6 on the glucagon promoter is significantly enhanced by its direct interaction with Cdx-2/3 and Maf (Hussain and Habener, 1999; Planque et al., 2001; Ritz-Laser et al., 1999). More recently, Pax6 binding to Pax4 and Pdx1 control sequences has been demonstrated, and
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reduction of Pdx1 expression was observed in Pax6 mutant mice (Ritz-Laser et al., 2002; Samaras et al., 2002; Wang et al., 2004). Thus, Pax6 also plays a direct role in the regulation of key pancreatic regulators. During pancreatic development, a number of transcription factors are known to act earlier than Pax6. These include Hb9 and Pdx1 (also known as Ipf1, Idx-1 and Stf-1), two homeodomain proteins expressed in the pre-pancreatic endoderm (Ahlgren et al., 1996; Guz et al., 1995; Harrison et al., 1999; Li et al., 1999; Miller et al., 1994; Offield et al., 1996). Loss of Hb9 expression blocks dorsal but not ventral pancreatic bud formation from the endoderm (Harrison et al., 1999; Li et al., 1999). Meanwhile, abrogation of Pdx1 results in an arrest of pancreatic development at the bud stage (Ahlgren et al., 1996; Jonsson et al., 1994; Offield et al., 1996). A conditional gene inactivation experiment reveals that Pdx1 is required for the differentiation of both islet and acinar cells in later development (Ahlgren et al., 1998). Another pancreatic bud specific transcription factor is P48 (also known as Ptf1a), which can bias gut epithelium toward pancreatic instead of duodenal fates (Kawaguchi et al., 2002; Krapp et al., 1998). The TALE (three amino acid loop extension) family homeoprotein Pbx1 is a known binding partner of Pdx1, and both exocrine and endocrine cell differentiation are severely disrupted in the Pbx1 mutant (Dutta et al., 2001; Kim et al., 2002; Peers et al., 1995; Swift et al., 1998). The first endocrine lineage specific gene is Neurogenin3 (Ngn3), which is only transiently expressed in the pancreatic epithelium (Apelqvist et al., 1999; Jensen et al., 2000; Schwitzgebel et al., 2000). Nevertheless, inactivation of Ngn3 results in a complete loss of endocrine cells (Gradwohl et al., 2000). Conversely, ubiquitous expression of Ngn3 converts the entire pancreas to an endocrine fate (Apelqvist et al., 1999; Schwitzgebel et al., 2000). Expressed slightly later than Ngn3, the LIM homeodomain protein Islet-1 is expressed in newly differentiated endocrine cells, and its function is clearly demonstrated by its mutant phenotype, which includes a failure of all endocrine cells to differentiate (Ahlgren et al., 1997). At the onset of Pax6 expression, other endocrine-cell-specific transcription factors also appear in the pancreas. These include NeuroD1, Nkx2.2, Pax4, Nkx6.1 and Brn4, all of which affect endocrine cell subtype specification (Hussain et al., 2002; Naya et al., 1997; Sander et al., 2000; Sosa-Pineda et al., 1997; Sussel et al., 1998). Many of these genes are candidates to encode direct regulators of Pax6 during pancreatic development. In a recent study of vertebrate eye development, we uncovered a role for the Meis family homeoproteins Meis1 and Meis2 in controlling Pax6 lens placode expression (Zhang et al., 2002). In this role, Meis recognizes a conserved sequence in the Pax6 lens placode enhancer and forms a multi-protein complex with currently unknown co-factor(s) to directly regulate Pax6 lens placodal expression. To search for direct upstream regulators of Pax6 in the pancreas, we previously identified a Pax6 pancreatic enhancer which resides on a 450 bp element 1.9 kb upstream of the Pax6 P0 promoter (Zhang et al., 2003). In transgenic mice, this enhancer is specifically active in the embryonic pancreas where its
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expression recapitulates endogenous Pax6 expression. In the current study, we have further refined the Pax6 pancreatic enhancer to a minimal 237 bp element. Biochemical studies show that this enhancer contains an evolutionarily conserved composite Meis–Pbx binding site, which exhibits distinct binding activities for different Meis family members. By transgenic analysis, we demonstrate that this Pax6 regulatory sequence that binds Meis and Pbx is required for Pax6 pancreatic enhancer activity in vivo and that it can be replaced by a consensus Meis–Pbx site. Finally, genetic deletion of Pbx1 and Pbx2 results in the down-regulation of Pax6 expression during pancreatic development. Therefore, Pbx and Meis homeoproteins collectively regulate Pax6 expression in the pancreas. Materials and methods Transgenic constructs All transgenic constructs were derived from the plasmid P0–3.9LacZ, which contains a 4.3 kb Pax6 genomic sequence that includes the Pax6 lens ectoderm enhancer, the Pax6 embryonic pancreatic enhancer, the Pax6 P0 promoter and a LacZ reporter gene (Zhang et al., 2002). For mapping the Pax6 pancreatic enhancer, a 1782 bp NsiI–XhoI fragment of the P0–3.9LacZ vector was shortened to 284 bp (PCR amplified using primers: 5′-TAAGATGCATGGCTACCGGGAGAACCTGACTCAGGC-3′ and 5′-AGCCCTCGAGCTTGTGCGGATCCCTGCAACCCAGC-3′); 237 bp (PCR primers: 5′-CTTAATGCATGGTGACCCCAACGCCCGCTCCCTCTCGCC3′ and 5′-AGCCCTCGAGCTTGTGCGGATCCCTGCAACCCAGC-3′); 167 bp (PCR primers: 5′-GAGCATGCATAAGCGTTGGCCTGGAGCTCGGGAGGGC-3′ and 5′-AGCCCTCGAGCTTGTGCGGATCCCTGCAACCCAGC-3′); or 246 bp (PCR primers: 5′-TAAGATGCATGGCTACCGGGAGAACCTGACTCAGGC-3′ and 5′-TGCTCTCGAGCGAAGCTGGAAACGCCACAGCC-3′). For transgenic constructs m5, mC, mR6 and Tg-con, the respective nucleotides were modified using a QuickChange site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing.
Mice Transgenic mice were generated as described previously (Zhang et al., 2002). Pbx1 and Pbx2 knockout mice are maintained in C57BL/6 and Black Swiss background respectively (Capellini et al., 2006; Kim et al., 2002). We also analyzed Pbx1 embryos from the original colony maintained by Dr. Michael Cleary (Stanford University) and consistently observed a similar pancreatic phenotype.
RT-PCR Pancreatic tissue was dissected in ice-cold PBS and immediately placed in liquid nitrogen. RNA was isolated from tissue extracts using a kit (RNA isolation kit, Qiagen), and reverse transcription was carried out according to the manufacturer's instructions (Invitrogen). The primers used for PCR are, Meis1 (forward: 5′AACCTCAACGCATCCGCAGGC-3′; reverse: 5′-GATTGACCAGTCAAATCGAGC-3′), Meis2 (forward: 5′-GGCATCCACTCGTTCAGGAGGA-3′, reverse: 5′-GATAGACCAGTCCAACCGAGC-3′), Meis3 (forward: 5′-CAGTGGTCTGTACATCTGGGG-3′, reverse: 5′-GATTGACCAGTCTAATCGCAC-3′), Prep1 (forward: 5′-CAGACTCAGCCCTACAACAGGG-3′, reverse: 5′-GCTTGATGCCAGCAACCCAGA-3′), Prep2 (forward: 5′-CAGCCTCAGCACTCCAGCAGGG-3′, reverse: 5′-GCTTGATGCCAGCAACCCAGA-3′).
EMSA EMSA was performed as described (Zhang et al., 2002). The probe for the region containing the Meis–Pbx site in the wild-type Pax6 pancreatic element is 5′-GCCCTTTATTGACAGACAGATAGATAAGCTGG-3′. The consensus
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Meis–Pbx binding site probe is 5′-CGAAGCCGGACCTGTCAATCATAGAA3′. The probes were made by annealing oligonucleotides of complementary sequence. Antibodies for DNA binding interference assays were anti-Pbx1, anti-Prep1 and anti-TGIF (all from Santa Cruz Biotechnology, Santa Cruz, CA). Meis1, Meis2, Pbx1, Prep1 and Prep2 proteins were made by in vitro transcription and translation using a kit (Promega). Expression vectors were kindly provided by Dr. Neal Copeland (Meis1, Meis2) (NCI, Frederick), Dr. Galvin Swift (Pbx1) (UT Southwestern, Dallas), Dr. Kenneth Gross (Prep1) (Roswell Park Cancer Institute, Buffalo) and Dr. Mark Featherstone (Prep2) (McGill Cancer Center, Montreal).
Histology, in situ hybridization and immunohistochemistry X-gal staining, in situ hybridization and immunohistochemistry were performed as previously described (Zhang et al., 2003). The following antibodies were used: mouse anti-Pax6 and mouse anti-Islet-1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-Pax6 (Covance, Berkeley, CA), mouse anti-insulin and rabbit antiglucagon (BioGenex, San Ramon, CA), guinea pig anti-Glucagon (Linco, St. Charles, MO), rabbit anti-Meis1 and anti-Meis2 (kind gift from Dr. Arthur Buchburg, Jefferson Medical College), rabbit anti-Pbx1/2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Pdx1 (kind gift from Dr. Chris Wright, Vanderbilt University), rabbit anti-IDX-1 (kind gift from Dr. Joel Habener, MGH, Harvard Medical School) and rabbit anti-Prep2 (kind gift from Dr. Mark Featherstone, McGill Cancer Center). Rabbit and guinea pig Prep1 antibodies were raised against the C-terminal 102 amino acids of mouse Prep1 fused to GST using the expression vector pGEX-6P-3 (Amersham Bioscience). Both rabbit and guinea pig polyclonal antibodies were raised by Covance and affinity-purified. The resulting anti-Prep1 antibodies were tested by Western blot and immunostaining and specifically recognized Prep1 in transfected but not control cells. To determine the Pax6/Pdx1 ratio, mouse embryos were sectioned serially at 10 μm increments and stained with Pax6 and Pdx1 antibodies. The total number of Pax6 and Pdx1 positive cells was counted on every 6th section for E14.5 embryos or on at least 5 sections for E10.5 embryos. At least 3 embryos of each genotype were analyzed. The statistical significance was calculated using Student's t test.
Results The minimal Pax6 embryonic pancreatic enhancer contains a composite Meis–Pbx binding site To study the transcriptional regulation of Pax6, we first determined the minimal sequence required for proper Pax6 pancreatic enhancer activity in transgenic mice. To do this, a LacZ reporter gene was fused downstream of 4.3 kb of murine Pax6 genomic sequence, containing the endogenous Pax6 promoter (P0), the embryonic pancreatic enhancer (Pan) and the lens ectoderm enhancer (Lens) (Zhang et al., 2002). After two consecutive deletions to the previously determined 450 bp Pax6 pancreatic enhancer element, the resulting 237 bp sequence fully retained pancreatic expression in all 5 E10.5 transgenic embryos analyzed (Figs. 1A, B). Further deletion of 70 bp from the 5′ end or of 38 bp from the 3′ end of the 237 bp sequence, however, abolished all pancreatic enhancer activity. This was not due to transgene integration effects since lens expression was not affected. Therefore, this 237 bp region is both necessary and sufficient for Pax6 pancreatic enhancer activity. Comparison with the genomes of mouse, human and Fugu fish revealed that, although the 96 bp at the 3′ end of the enhancer is conserved
Fig. 1. The minimal Pax6 pancreatic enhancer contains a conserved Meis–Pbx site. (A) The Pax6 lens ectoderm enhancer (Lens), Pax6 embryonic pancreatic enhancer (Pan) and the Pax6 P0 promoter are shown in the 3.9 kb Pax6 genomic sequence. The Meis–Pbx site lies at the beginning of the human/ mouse/fugu fish homologous region (the 5′ boundary denoted by a vertical line). For transgenic analysis, the number of β-galactosidase expressing lines is shown together with the total number of independent transgenic embryos. B: BamHI, E: EcoRI, H: HindIII. (B) Transient transgenic embryos were collected at E10.5, and the reporter gene activities in pancreas detected by X-gal staining (arrow). Note that lens expression is preserved in each transgenic line, while truncation of the minimal 237 bp element by 70 bp at the 5′-end (167 bp construct) or by 38 bp at the 3′-end (246 bp construct) results in loss of pancreatic expression.
between all three species, the 5′ portion of the 237 bp element is homologous only between human and mouse. This suggests the existence of mammalian specific regulatory elements that control embryonic Pax6 pancreatic expression.
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Analysis of the Pax6 pancreatic enhancer sequence revealed a perfect consensus Meis binding site (5′-TGACAG-3′) at the 5′ end of homologous region between human, mouse and Fugu fish (Chang et al., 1997; Shen et al., 1997) (Fig. 1A). Consistent with evolutionary significance, this Meis binding site is perfectly conserved between human, mouse and fugu fish (Miles et al., 1998). This observation is particularly intriguing in light of our previous finding that the Pax6 embryonic lens enhancer also contains a Meis binding site (5′-TGACAA-3′) and is regulated by Meis family members (Zhang et al., 2002). In the developing lens, the Meis protein forms a nuclear protein complex with additional unknown co-factors to regulate Pax6 expression. Further sequence analysis failed to identify any additional homology between the lens and pancreatic Pax6 enhancers. However, immediately downstream of the murine pancreatic Meis site is a region (5′-AGATAGAT-3′) which closely resembles the consensus Pbx binding site (5′-TGATTGAT-3′) (Van Dijk et al., 1993) (Fig. 1A). In addition, previous studies have established that Meis and Pbx can form functional heterodimers and bind DNA synergistically (Berthelsen et al., 1998; Chang et al., 1997), suggesting a model in which Meis and Pbx cooperatively bind the Pax6 pancreatic enhancer to regulate Pax6 expression. Meis and Pbx family members are expressed throughout pancreatic development The vertebrate Meis family has five members, Meis1, Meis2, Meis3, Prep1 and Prep2 (Berthelsen et al., 1998; Fognani et al., 2002; Haller et al., 2002; Moskow et al., 1995; Nakamura et al., 1996). These proteins all contain an N-terminal Meis domain, which mediates the interaction with Pbx, and a C-terminal homeodomain that can bind DNA. To determine whether Meis family members are expressed in the pancreas, we first performed RT-PCR to examine Meis transcripts in the whole pancreas. In both postnatal day P3 and adult pancreata, Meis1, Meis2 and Prep1 were clearly expressed (Fig. 2A). In embryonic E14.5 pancreata, transcripts of all five Meis family genes were detected (Fig. 2A). Consistent with this finding, we also observed Meis1, Meis2, Prep1 and Prep2 protein expression in the developing E10.5 pancreas by immunostaining (Figs. 2B–E). The antibodies used were specific to each of the four proteins, as demonstrated by their unique staining patterns in the spinal cord (data not shown). At E10.5, Meis1 and Meis2 were strongly expressed in pancreatic mesenchyme and only weakly co-localized with scattered Pax6 positive pancreatic epithelial cells (Figs. 2B, C). In contrast, Prep1 protein was ubiquitously expressed in pancreatic epithelial cells, thus overlapping with Pax6 positive cells (Fig. 2D). Prep2 showed strong pancreatic mesenchymal expression, but also colocalized with Pax6 positive epithelial cells (Fig. 2E). These results were further confirmed by RNA in situ hybridization (data not shown). We next examined Pbx gene expression in the E10.5 pancreas. Adjacent pancreatic sections from E10.5 embryos were analyzed for Pax6, Pbx1, Pbx2 and Pbx3 transcripts by
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RNA in situ hybridization and for total Pbx protein by immunofluorescence with a pan-Pbx reactive antibody (Figs. 2F–I). While Pax6 was detected only in clusters of islet cells at this stage (Fig. 2F), Pbx1 and Pbx2 were widely expressed in the pancreatic region (Figs. 2G, H). Pbx1 expression was detected at high levels in pancreatic mesenchyme and apparent only at lower levels in pancreatic epithelium (Fig. 2G). Pbx2 was expressed in both the epithelial and mesenchymal compartments (Fig. 2H), while Pbx3 pancreatic expression was not detected at this stage by this method (data not shown). Consistent with these results, a pan-Pbx antibody detected expression of Pbx proteins in both pancreatic epithelial and mesenchymal cells, and a subset of Pbx positive epithelial cells co-expressed Pax6 (Fig. 2I). Prior studies have also detected Pbx1 and Pbx3 expression in developing islet cells (Gu et al., 2004; Kim et al., 2002). Taken together, we conclude that Pax6, Meis and Pbx family members exhibit overlapping expression in the developing endocrine pancreas. Prep1 and Pbx1 bind the Pax6 pancreatic enhancer synergistically Our previous studies indicated that both Meis1 and Meis2 have the potential to regulate Pax6 expression in the lens (Zhang et al., 2002). We thus performed DNA electrophoretic mobility shift assays (EMSAs) to test whether these two proteins could also bind the Pax6 pancreatic enhancer element. Interestingly, neither Meis1, Meis2 nor Pbx1 (lane 4) alone bound a 26 bp oligonucleotide containing the putative Meis– Pbx site (Fig. 3 and data not shown). However, inclusion of both Meis1 and Pbx1 (lane 2) or Meis2 and Pbx1 (lane 3) proteins in the EMSA resulted in a moderately abundant shift in DNA mobility, indicating the formation of binary protein complexes on the DNA (Fig. 3). Moreover, the synergistic binding between Meis1 or 2 and Pbx1 was abolished by an anti-Pbx antibody but not by a control anti-Prep1 antibody (data not shown). Thus, Meis1 or 2 and Pbx1 cooperatively bind the Pax6 pancreatic enhancer in vitro. We next tested the DNA binding activity of Prep1. While both Meis1 and Meis2 bound the Pax6 enhancer element in conjunction with Pbx1, a markedly stronger binding was observed when Prep1 and Pbx1 were tested together (Fig. 3, lane 6 vs. lanes 4 and 5). The Prep1–Pbx1 complex was sensitive to antibodies against Prep1 or Pbx (lanes 7 and 8), but not to a control anti-TGIF antibody (lane 9; TGIF is another closely related TALE family homeoprotein). Similarly, we also observed strong cooperative binding of Prep2 and Pbx1 to the 26 bp probe (data not shown). The differential binding affinities exhibited by Meis1, Meis2, Prep1 and Prep2 are surprising because PCR-based binding site selection experiments suggest that Meis1 and Prep1 prefer identical consensus sequences (Berthelsen et al., 1998; Chang et al., 1997). Indeed, Meis1, Meis2 and Prep1 also bound equally strongly with Pbx1 protein on a probe containing a Meis–Pbx composite site (5′TGATTAGCAG-3′) taken from the literature (Fig. 3; lanes 11, 12 and 15) (Chang et al., 1997). Therefore, the preferential binding of Prep1 and Pbx1 or of Prep2 and Pbx1 to the Pax6
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Fig. 2. Expression analysis of Meis and Pbx family members during pancreatic development. (A) RT-PCR analysis was carried out for adult, day P3 and E14.5 mouse pancreas. In the adult and P3 pancreas, Meis1, Meis2 and Prep1 are expressed. In E14.5 pancreata, all 5 Meis family genes were detected. Pax6, β-actin and ribosomal L19 transcripts were present in all three samples. The nominally equivalent efficiencies of the PCR primers for the different Meis family genes were established by testing equal amounts of plasmid DNA for each gene as template (data not shown). (B–E) At E10.5, immunohistochemistry detected Meis1 and Meis2 proteins mainly in pancreatic mesenchyme, while Prep1 and Prep2 proteins are present throughout the pancreas and co-localize with Pax6 in islet cells (yellow nuclei). (F–G) Pax6, Pbx1 and Pbx2 transcripts and protein detected by RNA in situ hybridization and immunohistochemistry. Circled regions depict the boundary of the pancreatic epithelium containing prospective islet cells. Pbx1 expressed is detected at higher levels in pancreatic mesenchyme relative to epithelium, whereas Pbx2 is expressed in both compartments. The pan-Pbx antibody detects co-expression of Pax6 (red fluorophore) and Pbx (green fluorophore) in islet cells (yellow nuclei).
pancreatic enhancer is due to the unique sequence of this element, and not to a difference in protein concentrations or activities. Especially when taken with the results from preceding expression experiments, these results strongly suggest that Prep1 and Prep2 are the most likely Meis family candidates to be involved in regulation of the Pax6 embryonic pancreatic enhancer. Mutational analysis of the Meis–Pbx site We next undertook an in vitro mutational analysis of the Prep1 and Pbx1 composite binding site in the Pax6 pancreatic enhancer sequence (Fig. 4A). In EMSAs, whereas mutation of 2 bases 5′ to the Meis site did not affect Prep1–Pbx1 binding, replacement of 5 of the 6 bases of the Meis binding site (mutant C) completely abolished complex formation in vitro (Fig. 4B and data not shown). Moreover, as further evidence that the Meis site is required for Prep1–Pbx1 binding, point mutations
(mutant m5 and mutant m1) within the Meis site significantly inhibited protein–DNA complex formation (Fig. 4B). In the 8 bp Pbx site, mutation of 2 bases (mutant R2), 4 bases (mutant R4) or 6 bases (mutant mR6) progressively reduced the binding affinity of the Prep1–Pbx1 complex to DNA (Fig. 4C). However, deletion from the probe of all 8 bases comprising the Pbx1 site (mutant RΔ) did not abolish complex binding, which is thus relatively independent of the sequence of the Pbx site (Fig. 4C). This finding is surprising considering that Pbx1 protein is critically required for Prep1 to bind to the Pax6 pancreatic enhancer (see Fig. 3) and suggests that Pbx1 contributes to the stability of the binding complex by inducing a conformational change in Prep1. Lastly, we replaced the Pbx site in the Pax6 pancreatic enhancer with a consensus Pbx binding sequence (mutant Rc); this led to the expected result of stronger Prep1–Pbx1 binding (Fig. 4C). The protein–DNA complex on RΔ and Rc probes was abolished in the presence of
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contrast, lens expression of the two mutant transgenic constructs was at least as strong as that of the wild-type transgene. Thus, the in vivo effects of Meis site mutations on Pax6 pancreatic enhancer activity directly correlate with the effects of these mutations on Prep1–Pbx1 DNA binding in vitro. We next tested the effect of a 6-base mutation in the Pbx site (mutant mR6). In contrast to the wild-type transgene, this mutation reduced but did not abolish pancreatic enhancer activity in all 9 independent lines assayed. This result is consistent with the biochemical experiment which produced a similar result (see Fig. 4B). Taken together, these results support the hypothesis that both Meis and Pbx sites are required for optimal transcriptional activity of the Pax6 pancreatic enhancer. Meis and Pbx bind the Pax6 pancreatic enhancer in a relatively weak manner compared to their binding to the consensus composite Meis–Pbx site described in the literature. This observation raises the question of whether the endogenous Pax6 enhancer sequence, which deviates from the consensus both in sequence and in the relative orientation of the Meis and Pbx sites, is functionally equivalent to the consensus sequence (Fig. 5A). To address this question, we replaced the 16 bp Fig. 3. Prep and Pbx proteins preferentially bind to the Pax6 pancreatic enhancer. Probes containing the wild-type Meis–Pbx site in the Pax6 pancreatic enhancer (Wild Type) form complexes with either Meis1 and Pbx1, or Meis2 and Pbx1, but not with Meis1, Meis2, Prep1 or Pbx1 individually. Much stronger binding is observed for the Prep1 and Pbx1 complex, whose formation is blocked by anti-Pbx1 or anti-Prep1 antibodies, but not by anti-TGIF antibody. For the consensus Meis–Pbx probe (Consensus), equally strong Meis1–Pbx1, Meis2–Pbx2 and Prep1–Pbx1 binding is observed, validating the protein activities in the experiments employing the wild-type probe.
anti-Prep1 and anti-Pbx1 antibodies, demonstrating that the protein complexes indeed consisted of Prep1 and Pbx1 (Fig. 4D). In summary, both the Meis and Pbx sites in the Pax6 pancreatic enhancer contribute to the stabilization of a Prep1– Pbx1 complex on DNA. Pax6 pancreatic enhancer activity in mice requires a functional Meis–Pbx site Although we identified several pancreatic endocrine cell lines that expressed Meis1, Meis2, Prep1 and Pax6, none supported the activity of the Pax6 pancreatic enhancer upon transfection. Not surprisingly, we found that neither Prep1– Pbx1 nor Meis1–Pbx1 transactivates the Pax6 pancreatic enhancer in any of these cell culture systems (data not shown). To test the functional requirement of the Meis–Pbx site for Pax6 pancreatic enhancer activity during development, we performed mutational analyses in transgenic mice. We employed constructs containing both the Pax6 pancreatic and lens enhancers, so that lens expression could serve as an internal standard for changes in pancreatic expression. When a single base mutation in the Meis site (mutant m5) was introduced into construct P0–3.9LacZ in transgenic mice, pancreatic expression was significantly reduced in all 11 independent lacZ reporter expressing lines (Figs. 5A, B). Additionally, mutation of 5 bases in the Meis site (mutant mC) completely abolished enhancer activity in the pancreas in all 5 expressing lines (Fig. 5). In
Fig. 4. Meis and Pbx sites are required for Prep1 and Pbx1 binding and transactivation. (A) Sequences of Meis and Pbx site mutations. (B) Mutations in Meis sites disrupt the binding activity of Prep1 and Pbx1 in EMSA. Note that mutant mC completely abolishes Prep1 and Pbx1 binding. (C) Mutations in Pbx sites weaken Prep1/Pbx1 binding. (D) Protein complexes that form on RΔ and Rc are sensitive to Prep1 and Pbx1 antibodies.
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Fig. 5. The Meis–Pbx site is required for Pax6 pancreatic enhancer activity in transgenic mice. (A) Schematic diagram of the transgenic constructs. The Meis and Pbx sites and the mutants tested are highlighted. (B) Representative E10.5 transgenic embryos carrying the designated transgenes. Note that, whereas lens expression is preserved, pancreatic expression is diminished in mutant embryos containing the m5, mC and mR6 constructs (arrows). Replacement of the wildtype Meis–Pbx sequence with the consensus sequence (Tg-con) results in retention of transgene expression in the pancreas (con). *Weak pancreatic expression was detectable in the m5 and mR6 embryos.
Meis–Pbx site in the Pax6 pancreatic enhancer LacZ reporter construct with a 10 bp Meis–Pbx consensus sequence (Tg-con) taken from the literature (Chang et al., 1997) and then assayed LacZ reporter activity of the Tg-con transgene in transgenic mice at E10.5. Pancreatic expression similar to that seen with the Pax6 pancreatic enhancer was observed in 4 out of 4 of these Tg-con transgenics. In addition, immunohistochemistry showed co-localization of the LacZ and Pax6 expressing cells in the E10.5 pancreas (Fig. 5B). Therefore, during early embryonic development, a Meis–Pbx consensus sequence can functionally substitute for an indispensable region of the Pax6 pancreatic enhancer. Pax6 is down-regulated in the Pbx1 and Pbx2 knockout pancreas To test whether Pbx1 is required for Pax6 pancreatic expression, we first re-examined pancreatic development in Pbx1 deficient mouse embryos (Kim et al., 2002). At E14.5, the number of Pax6 expressing cells and their qualitative intensity
were strongly reduced in E14.5 Pbx1 mutants. This was in contrast to the robust expression of Pdx1 found in Pbx1 mutant pancreatic epithelium, suggesting that Pax6 down-regulation is not due to general deficiency of pancreatic progenitors (Fig. 6A). Indeed, the ratio of Pax6/Pdx1 positive cells was significantly reduced in Pbx1 mutant embryos (Fig. 6D). Endocrine cell differentiation defects in Pbx1 mutants were also revealed by decreased insulin and glucagon expression, as well as by a reduction in the number of Islet-1 positive cells (Fig. 6A and data not shown). These results confirm a previous demonstration that Pbx1 is required for proper pancreatic development (Kim et al., 2002) but additionally demonstrate that Pbx1 is required at 14.5 for the maintenance of pancreatic Pax6 expression. We next analyzed Pax6 pancreatic expression in E10.5 Pbx1 mutants. Strong expression of Pax6, glucagon, islet-1 and Pdx1 was detected in both wild-type and Pbx1 mutant pancreatic epithelium, and the Pax6/Pdx1 cell number ratio was unchanged (Figs. 6B–D and data now shown). This demonstrates that islet cell differentiation is not significantly disrupted by Pbx1 mutation during early pancreatic development. In E10.5 Pbx1−/−Pbx2+/− compound embryos, however, we observed severe pancreatic hypoplasia and significant reduction in the number of Pax6 positive cells and Pax6/Pdx1 ratio (Figs. 6C, D). More importantly, the level of Pax6 expression in the remaining endocrine cells was also down-regulated. This was shown more clearly by RNA in situ hybridization, which revealed that Pax6 expression was lower in Pbx1−/−Pbx2+/− pancreatic buds than in wild-type controls (Fig. 6C). As control, Pax6 expression in the neural tube remained unchanged in the Pbx1−/−Pbx2+/− mutants. Therefore, Pbx1 and Pbx2 together regulate Pax6 expression during early pancreatic development. Discussion Role of Meis family members In this paper, we have defined a minimal 237 bp enhancer for embryonic Pax6 pancreatic expression. Transgenic experiments further demonstrate that the Meis–Pbx binding site in this enhancer is critical for its activity and that a Meis–Pbx consensus sequence can substitute for the endogenous binding site in this regard. We also show that the Meis and Pbx family members, Prep1 and Pbx1 respectively, can bind this element cooperatively. It is likely that other Meis and Pbx family members, such as Prep2 and Pbx2, can also bind this element cooperatively. We found that Prep2 acted interchangeably with Prep1 in EMSA (data not shown), and based on sequence similarity, Pbx2 should act similarly to Pbx1. Moreover, both Prep2 and Pbx2 share co-expression with Pax6 in the developing pancreatic epithelium at E10.5, when the enhancer is active. Many Meis and Pbx family members are expressed during pancreatic development, and members of each family recognize closely related DNA sequences. However, we observed that the Meis family members Prep1 and Prep2 form substantially stronger binding complexes with Pbx1 on the Pax6 pancreatic enhancer than do Meis1 and Meis2. These
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Fig. 6. Pax6 expression is reduced in Pbx1 and Pbx2 mutant pancreata. (A) In E14.5 Pbx1 mutant pancreata, marked reductions in Pax6, Islet-1, glucagon and insulin expression are observed. However, Pdx1 expression remains relatively unchanged. (B) Pax6 and glucagon are expressed in E10.5 Pbx1 mutant pancreata, but downregulated in Pbx1−/−Pbx2+/− mutants. The pancreatic epithelium is marked by strong Pdx1 protein expression and outlined with dotted lines. (C) Pax6 mRNA level in the pancreas is reduced in E10.5 Pbx1−/−/Pbx2+/− mutant as shown by RNA in situ hybridization. The same pancreas section was stained for glucagon to mark alpha cells. Pax6 expression in the neural tube on the same sections is not changed. (D) Quantification of E10.5 and E14.5 Pax6+/Pdx1+ cell ratios. Specific loss of Pax6 expressing cells is observed in Pbx1−/− mutants at E14.5 and Pbx1−/−Pbx2+/− mutants at E10.5.
results likely reflect different DNA binding sub-specificities among Meis family proteins and, taken with their colocalization with Pax6, suggest that Prep1 and Prep2 are the most critical Meis factors regulating Pax6 during pancreatic development. Further analysis of Prep1 and Prep2 mutant mice should clarify the role of these genes in Pax6 embryonic pancreatic expression. Role of Pbx family members Previous studies have identified an important role for Pbx1 in pancreas development. Genetic ablation of Pbx1 results in severe pancreatic hypoplasia and β cell dysfunction (Kim et al., 2002). Consistent with this phenotype, binding sites for Pbx1 and its co-factors, Pdx1 and Meis/Prep family proteins, have been found in both endocrine and exocrine genes, including somatostatin, glucagon and elastase (Herzig et al., 2000; Peers et al., 1995; Swift et al., 1998). In addition, Pbx1 and Pdx1 genetically interact in glucose homeostasis, and a Pdx1 mutant lacking Pbx1 binding ability fails to support the normal migration and proliferation of pancreatic islet cells (Dutta et al., 2001; Kim et al., 2002). Therefore, Pbx1, together with Meis and Pdx1, directly regulates β cell function in the pancreas. Nevertheless, study of Pbx1 knockout mice has also shown that
Pbx1 is required for both endocrine and exocrine cell differentiation, suggesting that Pbx1 also regulates pancreatic developmental genes. In this study, we present biochemical and transgenic data showing that the critical developmental regulatory gene Pax6 is a direct target of Pbx1 during islet genesis. Thus, Pbx1 acts at multiple levels during pancreas development. The analysis of Pbx1–Pbx2 compound knockout embryos also demonstrates that both Pbx1 and Pbx2 are required for Pax6 regulation. In E14.5 Pbx1 mutant pancreatic islets, we observed significant reductions in the number of Pax6 positive cells relative to the number of Pdx1 positive cells; this was also accompanied by a down-regulation in insulin and glucagon. Although we found little change in Pax6 expression in Pbx1 mutant pancreatic epithelium at E10.5, in Pbx1−/−Pbx2+/− mutants, we observed a significant loss of Pax6 positive cells in the pancreatic bud. Moreover, Pax6 transcript levels in islet progenitors were also much reduced. Since Pbx1/2 inactivation leads to profound pancreatic defects, we cannot rule out the possibility that the loss of Pax6 expression is due to pleiotropic effects of the Pbx1/2 mutation on pancreatic development. However, the reduction of Pax6 mRNA levels shows that Pbx1 and Pbx2 must regulate Pax6 at the transcriptional level. Thus, our results are consistent with a model whereby Pax6 is a direct
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downstream target of Pbx1, Pbx2 or both in pancreatic development. In addition, although we were unable to detect Pbx3 expression in the E10.5 pancreas by whole mount in situ hybridization, Pbx3 has been detected in Pdx1 positive cells in microarray studies (Table S4 in Gu et al., 2004) and also at later stages of pancreatic development (Di Giacomo et al., in press). Of note, while transgenic analysis demonstrates the critical importance of the Meis–Pbx binding site for endogenous Pax6 pancreatic enhancer activity, Pax6 expression is not completely eliminated in Pbx1−/−Pbx2+/− compound mutant embryos. This discrepancy could be accounted for by genetic redundancy from the remaining copy of Pbx2 or by a different Pbx gene family during pancreatic epithelial development. Thus, in principle, all three Pbx genes may contribute to the regulation of Pax6 pancreatic expression. Conserved role for Meis in Pax6 regulation We previously showed that Meis1 and Meis2 are direct upstream regulators of Pax6 in vertebrate lens development where they act upon a Meis site juxtaposed to a non-Pbx cofactor site in the Pax6 lens placode enhancer (Zhang et al., 2002). In this paper, we have extended the requirement for a Meis-site-dependent regulatory mechanism to embryonic Pax6 pancreatic expression. It is notable that, although the Pax6 lens and pancreatic enhancers share Meis binding sites, the Meis cofactors that bind these two elements are likely different. In the Pax6 pancreatic enhancer, Pbx protein is the requisite binding partner of Meis, but in the Pax6 lens enhancer, Pbx does not bind DNA even in the presence of Meis protein. Not surprisingly, therefore, inactivation of Pbx1 disrupts Pax6 expression in the pancreas but not in the lens (this paper and data not shown). In fact, the Pax6 lens and pancreatic enhancers do not share any other apparent homologous sequence except their respective Meis sites. This suggests that Meis may interact with different sets of transcription factors in lens and pancreas. Therefore, the conserved regulation of Pax6 by Meis proteins may be accomplished in different organs by changes in the identities of the Meis co-factors, which may then further define the different tissue-specific activities of these enhancers. Acknowledgments The authors gratefully thank Drs. M. Cleary, A. Buchberg, M. Featherstone, K. Gross, J. Habener and C. Wright for embryos, antibodies and cell lines and Drs. Francesco Blasi and Susan Bonner-Weir for helpful discussions. We are also indebted to Terence Capellini and Dr. Elisabetta Ferretti for donating Pbx1/2 compound mutant embryos. SR was supported by a fellowship from the Canadian Institutes of Health Research. The work was supported by grants from the American Diabetes Foundation (1-04-RA-117 to XZ) and the NIH (RO1 DK065791 to RLM). References Ahlgren, U., Jonsson, J., Edlund, H., 1996. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/ PDX1-deficient mice. Development 122, 1409–1416.
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