Differential expression and functional analysis of Pitx2 isoforms in regulation of heart looping in the chick

In vertebrates, the overall bilateral symmetry of the adult body is broken by the consistently asymmetric placement of various internal organs such as the heart, liver, spleen and gut, or asymmetric development of paired organs such as brain and lungs. The first morphological indication of the left-right asymmetry is manifested by the rightward looping of the developing heart. A great deal of research has been undertaken into the study of factors involved in left-right axial determination and dextral looping of the heart. Recent advances in this area have led to sophisticated and complex pathways of induction and regulation between numerous signaling molecules and transcription factors (Harvey, 1998; Ramsdell and Yost, 1998). The first demonstration of left-right asymmetric gene expression, preceding organogenesis, was seen in the chick embryo and includes a cascade of signals involving activin receptor IIa (cAct-IIa), sonic hedgehog (Shh) and Nodal (Levin et al., 1995). During early gastrulation, the asymmetric expression of cAct-IIa on the right side of the node restricts Shh expression to the left side of the node of a chick embryo. The asymmetric Shh expression is then responsible for the asymmetric expression of Nodal to the left lateral plate mesoderm (LPM). It was recently found that caronte, a member of the Cerberus/Dan gene family, mediates Shh induction of Nodal by antagonizing a repressive activity of BMPs (Rodriguez-Esteban et al., 1999; Yokouchi et al., 1999; Zhu et al., 1999). Studies in other vertebrate species. including mouse and Xenopus, indicate that the asymmetrical expression of Nodal in the left LPM is conserved across species and is crucial for establishing initial left-right asymmetry (Collignon et al., 1996; Hyatt et al., 1996; Lowe et al., 1996; Sampath et al., 1997). Downstream from this left side signaling cascade reside lefty1 and lefty2 (Meno et al., 1996; Meno et al., 1997;

Meno et al., 1998; Rodriguez-Esteban et al., 1999; Yokouchi et al., 1999), and Pitx2 (Campione et al., 1999; Logan et al., 1998; Piedra et al., 1998; Ryan et al., 1998; St Amand et al., 1998; Yoshioka et al., 1998). Retinoic acid, which is involved in the left-right patterning (Chen and Solursh, 1992; Smith et al., 1997), has also been shown to regulate the expression of left side-specific genes in a pathway downstream or parallel to Shh across different species (Chazaud et al., 1999; Tsukui et al., 1999; Wasiak and Lohnes, 1999; Zile et al., 2000). Several other genes also exhibit asymmetrical expression and participate in the regulation of left-right axis formation, as summarized by King and Brown (King and Brown, 1999), including Fgf8, Snail-related gene and Nkx3.2 (Issac et al., 1997; Sefton et al., 1998; Boettger et al., 1999; Meyers and Martin, 1999; Schneider et al., 1999; Rodriguez-Esteban et al., 1999; Capdevila et al., 2000).

Pitx2 is a member of the Pitx/RIEG family of homeobox containing genes which are closely related to the Drosophila gene bicoid (Semina et al., 1996, Mucchielli et al., 1996). Two additional family members Pitx1 and Pitx3 have also been identified (Lamonerie et al., 1996; Szeto et al., 1996; Lanctôt et al., 1997; Semina et al., 1997). PITX2 (also known as RIEG) was first identified in humans, and mutations in this gene have been shown to be responsible for the autosomal-dominant disorder, Rieger syndrome, which exhibits defects in the tooth and eye (Semina et al., 1996). PITX2 has also been identified as a downstream target of the human acute leukemia ALL1 gene (MLL1 – Human Gene Nomenclature Database), suggesting a role in tumorigenesis (Arakawa et al., 1998). A role for Pitx2 in vertebrate organogenesis has been suggested by its expression in developing Rathke’s pouch, eye, limb bud, heart, brain and tooth germ during embryogenesis (Semina et al., 1996; Mucchielli et al., 1996; Mucchielli et al., 1997; T. R. St Amand, PhD Thesis, Tulane University, 1999; St Amand et al., 2000), and has been further demonstrated by gene knockout experiments (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999).

A prominent function of Pitx2 in vertebrate organogenesis is its involvement in the regulation of left-right development. Pitx2 is expressed in the left side LPM and the left side of the cardiac tube and gut, and functions downstream of Shh/Nodal pathway in all vertebrate species examined (Meno et al., 1998; Logan et al., 1998; Piedra et al., 1998; Ryan et al., 1998; St Amand et al., 1998; Yoshioka et al., 1998; Bisgrove et al., 1999; Campione et al., 1999). Functional analysis indicated that Pitx2 may interpret and subsequently execute the left-right developmental program (reviewed in Yost, 1999). Ectopic expression of Pitx2 on the right LPM alters left-right developing of the heart and other internal organs (Logan et al., 1998; Ryan et al., 1998; Campione et al., 1999). On the other hand, loss of Pitx2 function in mouse by gene targeting also results in defects in the left-right asymmetry of internal organs, including heart (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999). Several Pitx2 isoforms have been isolated from human, mouse, chick, Xenopus and zebrafish (Gage and Camper, 1997; Arakawa et al., 1998; Logan et al., 1998; St Amand et al., 1998; Essner et al., 2000; Schweickert et al., 2000). Thus, the potential role of these Pitx2 isoforms in the regulation of left-right asymmetry has just begun to be elucidated (Essner et al., 2000; Schweickert et al., 2000).

We have cloned two chick Pitx2 isoforms (Pitx2a and Pitx2c), which correspond to the mouse and human isoforms of Pitx2 and differ only in their N-terminal region. In this study, we have analyzed their differential expression patterns during early chick embryonic development and their involvement in the regulation of heart looping. We revealed the presence of Pitx2a and Pitx2c but not Pitx2b in the developing chick embryos. Whole mount in situ hybridization using Pitx2c specific probe, in combination with RT-PCR, indicates that Pitx2c, but not Pitx2a, is restrictedly expressed in the left LPM. Although ectopic expression of either Pitx2a or Pitx2c to the right LPM via RCAS retroviral infection randomized heart looping direction, specific repression of Pitx2 isoforms by antisense oligonucleotide approach, as well as ectopic expression of dominant negative forms of RCAS-Pitx2 demonstrate that left-right asymmetry in developing chick embryo is regulated by Pitx2c.

Chick Pitx2a and Pitx2c were isolated simultaneously from a day 10 chick embryonic brain cDNA library using a mouse Pitx2 cDNA probe under low stringency condition, as described previously (St Amand et al., 1998). Briefly, about 1×10⁶ phage plaques were plated and hybridized with a mouse Pitx2 probe in 6× SSPE, 5× Denhardts, 50 mM phosphate, 0.1% SDS, 10 mM Na₄P₂O₇, 200 μg/ml salmon sperm DNA at 60°C. Membranes were then washed three times for 30 minutes each with 4× SSPE, 0.2% SDS before exposing to X-ray film.

Chick Pitx2c-specific probes were generated by PCR amplification of the first 306-bp of the 5′ region, including 76-bp 5′ UTR, from Pitx2c cDNA (St Amand et al., 1998). This region exhibits sequence diversity from Pitx2a. The PCR amplified fragment was subcloned into pBluescript KS. Pitx2 full-length probes were generated as described previously (St Amand et al., 1998). The Drosophila engrailed repressor sequence was released from the Slax 13 adaptor plasmid and subcloned into pBluescript KS. All probes were labeled with digoxigenin, as directed by the manufacturer (Boehringer Mannheim). Probe size and yield were determined by comparing with an RNA standard after agarose gel electrophoresis.

Whole-mount in situ hybridization was performed as described previously (St Amand et al., 1998) and signals were visualized using NBT/BCIP substrate (Boehringer Mannheim). For visualization, analysis and photographs, samples were dehydrated with glycerol and mounted onto slides. In the case of samples to be sectioned, the samples were gradually re-hydrated in PBS and dehydrated again in ethanol. Samples were then cleared with xylene followed by embedding in paraffin wax. Embryos were sectioned at 15 μm and mounted with Permount.

Northern blot analysis was performed using NorthernMax kit (Ambion, Austin, TX) according to manufacture’s instructions. Poly(A) RNA samples were extracted from whole chick embryos at stages 4, 8, 11, 21, 26 and 31 using the PolyATtract System 1000 (Promega Corp.; Madison, WI). About 2 μg of Poly(A) RNA was denatured in formamide based denaturing buffer and subject to electrophoresis through a 1% agarose-formaldehyde gel. RNA was then transferred to positively charged nylon membrane and immobilized by UV crosslink. A 1-Kb ³²P-labeled probe was used that covers both exon 5 and exon 6 which are present in all three Pitx2 isoforms. Hybridization and washes were carried out at appropriate temperatures and the blots were exposed to X-ray film.

To make RCAS-Pitx2 constructs, full-length Pitx2a and Pitx2c sequences were released from the pBluescript vectors by double digestion with BamHI and EcoRI, and were cloned into the Cla 12 adapter plasmid. The ClaI fragments containing Pitx2a and Pitx2c sequences were cloned into the RCAS retroviral vectors. The orientation was determined by PCR using an upper primer targeting on retroviral sequence and a lower one targeting on Pitx2 sequence and further by sequencing. To make RCAS-Pitx2-En^r constructs, DNA fragments with the open reading frames containing the entire N-terminal region and the homeodomain of Pitx2a and Pitx2c were generated by high fidelity PCR and cloned into the pBluescript vector. After sequencing, the fragments were released and inserted into a version of Slax 13 adaptor plasmid containing the Drosophila engrailed repressor domain sequence (En^r; provided by Dr C. Cepko of Harvard Medical School). The ClaI fragments harboring Pitx2 sequence with the C terminus replaced with the En^r domain were further cloned into the ClaI site of RCAS retroviral vectors. Generation and concentration of viral supernatant were carried out according to a protocol described previously (Logan and Francis-West, 1999). Retrovirus infection was performed on stage 3+ or stage 4 embryos (Hamburger and Hamilton, 1951) explanted in New culture (New, 1955). RCAS-Pitx2a and RCAS-Pitx2c viruses were targeted to the right side of the blastoderm by multiple points of injection, while RCAS-Pitx2a-En^r and RCAS-Pitx2c-En^r were targeted to the left side of the blastoderm. Injected embryos were returned to a 37°C incubator in a humid chamber, and were allowed to develop to stage 11 when heart looping has finished.

Antisense oligonucleotide treatment was carried out on stage 6-7 embryos explanted in New culture as described previously (Srivastava et al., 1995; Issac et al., 1997). The antisense oligonucleotides have sequences (Pitx2a, 5′-ACGCAGGCTGAGACAAGT-3′; Pitx2c, 5′- GGTGTCAGAGATAGTGTG-3′) that target to the specific N-terminal regions of Pitx2a and Pitx2c. A random oligonucleotide (5′-AGGCTCGAACTCAGACTT-3′) was used as a control. All the oligonucleotides used were synthesized as phosphorothioate derivatives and HPLC purified (IDT, Corralville, IA). Oligonucleotides were diluted to a concentration of 40 μM in lipofectAMINE (GibcoBRL). Approximate 10 μl of mixed lipofectAMINE was applied directly onto the cultured embryo within the ring. Embryos were allowed to develop to stage 11, and then scored for the heart looping direction.

The DNA fragments encoding the N-terminal regions (amino acids 2-100 for Pitx2c; amino acids 2-38 for Pitx2a) and the C terminus (identical in both isoforms; amino acids 161-333 in Pitx2c and amino acids 100-271 in Pitx2a) of Pitx2 were amplified from chick Pitx2a and Pitx2c cDNA plasmids and cloned into pBXG1 which contains the DNA-binding domain of GAL4 (amino acids 1-147) under the control of SV40 enhancer/promoter (Lillie and Green, 1989). The DNA fragments were inserted downstream of and in frame with the GAL4 DNA-binding domain. All constructs, named GAL4/Pitx2cN, GAL4/Pitx2aN and GAL4/Pitx2C, were confirmed by DNA sequencing. Transfection and chloramphenicol acetyltransferase (CAT) assay were carried out using P19 cells. Briefly, The GAL4/Pitx2 constructs were co-transfected into P19 cells respectively, with the pG ₅ ECAT reporter plasmid containing five GAL4-binding sites upstream of the adenovirus E1b minimal promoter and the CAT gene (Ma and Ptashne, 1987). As an internal control for transfection efficiency, a CMV-β-gal plasmid was included. Transfection was achieved by addition onto cells plated in a 60 mm culture dishes of a mixture of 1 μg GAL4/Pitx2, 1 μg pG₅ECAT, 1 μg CMV-β-gal and 12 μl of lipofectAMINE (GibcoBRL), according to the manufacturer’s instruction. Co-transfection of a plasmid (pBXGAL-II) expressing the GAL4 DNA-binding domain and activation region II (amino acid 768-881) with pG₅ECAT served as a positive control for the activation of the CAT reporter gene, while transfection of a SV40-CAT plasmid served as a positive control for the CAT assay. Transfection of pG₅ECAT with pBXG1 was included as a negative control. Transfected cells were cultured for 36 hours and cell extracts were prepared for CAT assay according to the standard protocol (‘Protocols and Applications Guide’, Promega). The CAT activities were determined by thin-layer chromatography (TLC) and scintillation counting. Each experiment was repeated at least three times.

To examine the expression of Pitx2 in the early developing embryo, chick embryos from stages 4 to 31 were collected, and total RNA extracted. To examine the differential expression of Pitx2 isoforms during the stages when heart looping direction is determined, head tissues, tissues containing only left LPM, and tissues containing right LPM and its adjacent extra-embryonic region from stage 8-10 embryos were isolated. Samples were treated with 2.25% trypsin/0.75% pancreatin on ice for 5 minutes prior to being transferred to a 1:1 solution of PBS and horse serum. Endoderm and ectoderm were then removed. Mesodermal tissues were collected and pooled together and total RNA extracted. Total RNA from same stage whole embryos was also extracted and used as positive control. Primers used for PCR detection of Pitx2 isoforms were 5′-CGATAACGGGTAATGGAG-3′ (upper primer targeting to exon 2, for both Pitx2a and Pitx2b), 5′- CTCTCTCTTCCTCCATTT-3′ (lower primer targeting to exon 6, for all isoforms), and 5′-GTCCTCTCGCCGATGAGT-3′ (upper primer targeting to exon 4, for Pitx2c). PCR was carried out under the following conditions: 94°C, 1 minute; 62°C, 1 minute; 72°C, 1 minute (30 cycles). As a control, Gapdh, a constitutively expressed mRNA, was amplified from each sample using the following primers: 5′- AGAGGTGCTGCCCAGAACATCATC-3′ (upper primer) and 5′- GTGGGGAGACAGAAGGGAACAGA-3′ (lower primer). The PCR profile used was as follows: 94°C, 1 minute; 60°C, 30 seconds; 72°C, 1 minute (30 cycles).

In our original screen of a chick embryonic brain cDNA library with a mouse Pitx2 probe, two Pitx2 cDNA isoforms containing a open reading frame were identified and named Pitx2a and Pitx2c, respectively. At the amino acid level, both Pitx2a and Pitx2c encode for a homeodomain-containing protein of 271 amino acids and 333 amino acids, respectively. The cDNA sequence of Pitx2c and its corresponding amino acid sequence have been reported in our previous studies (St Amand et al., 1998), while the amino acid sequence of Pitx2a is identical to that reported by Logan et al. (Logan et al., 1998). These two proteins differ by 77 amino acids at the 5′ region but are 100% identical from that point on, including 23 amino acids 5′ to the homeodomain, the homeodomain and the entire C terminus (Fig. 1). This region of identity also includes the fourteen amino acid motif in the C terminus, which is conserved among all members of the Pitx/Rieg family and some other selected homeodomain-containing transcription factors (Semina et al., 1998).

To confirm the presence of Pitx2 isoforms in developing chick embryo, Northern blot analysis was performed on RNA samples isolated from various developmental stages (Fig. 2). Poly(A) RNA was isolated from stage 4, 8, 11, 21, 26 and 31 embryos and analyzed by Northern blot using ³²P-labeled probe generated from chick Pitx2 exons 5 and 6, which are present in all isoforms. Two transcripts, approx. 1.6 and 2.3 kb, were detected in the samples from all the stages examined (Fig. 2A). To identify what specific isoforms these two transcripts are, RT-PCR was carried out on RNAs from the same stage embryos. It has been shown that the only difference between Pitx2b and Pitx2a is that Pitx2b has an additional 138 base exon 3 present between exons 2 and 5 (Schweickert et al., 2000). Therefore, an upper primer targeting to exon 2 and a lower primer targeting to exon 6 would cover both Pitx2a and Pitx2b. For Pitx2c, one pair of primers corresponding to exon 4, which is unique to Pitx2c, and exon 6 was used. As shown in Fig. 2B, only two PCR products, corresponding to the sizes expected for Pitx2a (303 bp) and Pitx2c (491 bp), respectively, were obtained. Sequencing analysis of these PCR products further confirmed the existence of Pitx2a and Pitx2c. Pitx2b is thus not present in the early chick embryos, at least at the stages examined. Similarly, it was noticed that that Pitx2b and Pitx2a were undetectable in zebrafish and Xenopus, respectively (Essner et al., 2000; Schweickert et al., 2000).

To determine the differential expression patterns of Pitx2a and Pitx2c during early chick embryogenesis, with particular interest in the establishment of the left-right axis, a 306 bp probe corresponding to the 5′ UTR and N-terminal coding region that are specific to Pitx2c was used. Since the sequence of 5′ UTR and the N-terminal coding region that are specific to the Pitx2a was too short (75 bp) to make an in situ hybridization probe, the expression pattern of Pitx2c could be only compared with that using full-length Pitx2 probe, as described previously (St Amand et al., 1998). By using whole- mount in situ hybridization, expression of Pitx2c was first detected at stage 5 (Fig. 3) with symmetrical expression in the hypoblast (data not shown). However, no expression was detected in the area opaca, as previously reported using a full- length Pitx2 at this stage (St Amand et al., 1998). By stage 8, asymmetric expression, characteristic of Pitx2, became evident and restricted to the left LPM, as confirmed by sections through these embryos (Fig. 3). Expression was also detected symmetrically in the head mesenchyme at this stage, but again no expression was detected in the area opaca. At stage 10, asymmetric expression of Pitx2c remained in the left one half of the embryo, including left side heart tube as the two primitive heart tubes fuse (data not shown). At the stage of heart looping (stage 11), Pitx2c expression was still restricted to the left LPM and the left half of the fused heart tube (Fig. 3). Sections through these samples reveal restricted expression in the left half epimyocardium of the fused heart tube (Fig. 3) as previously seen using a full-length Pitx2 probe (St Amand et al., 1998). We have previously reported the expression of Pitx2 in the angioblasts of blood islands and the endothelia of the blood vessels in the extra-embryonic regions (St Amand et al., 1998). However, through all stages analyzed, expression of Pitx2c was not detected in the extra-embryonic tissue, or in the endothelia and blood islands (Fig. 3). Therefore, these results suggest that Pitx2c be involved in the left-right axis determination and dextral looping of the heart, but not in the vasculogenesis during chick embryonic development. However, these observations do not rule out the possibility that Pitx2a is also expressed in the left LPM and may contribute to left-right development. To examine whether Pitx2a is also expressed in the left LPM, RT-PCR for Pitx2a was performed using RNA extracted from the left LPM collected from stage 8-10 embryos. In addition, Pitx2a and Pitx2c expression was also examined by RT-PCR in the head and extra-embryonic mesoderm containing the right LPM. The results demonstrated that both isoforms are expressed in the head region, but that only Pitx2c transcripts were detected in the left LPM, while Pitx2a transcripts were found exclusively in the extra- embryonic mesoderm (Fig. 4). Thus, Pitx2c contributes to Pitx2 expression in the left LPM.

Prior to functional analysis of Pitx2 isoforms in regulating left- right development, we asked whether both isoforms equally encode a transcriptional factor. To map the transactivation domain of Pitx2 products, we generated a series constructs in which the C-terminal region (from the amino acid immediately after the homeodomain to the last amino acid) of the Pitx2 and the N-terminal regions (from the second amino acid to the amino acid prior to the homeodomain) of Pitx2a and Pitx2c were fused in frame to the 147 amino acid DNA-binding domain of yeast GAL4. Transactivational activity of these domain was assayed by co-transfection of these constructs into cultured P19 cells with a CAT reporter plasmid containing five GAL4-binding sites and a basal promoter. As shown by TLC in Fig. 5, GAL4/Pitx2aN and GAL4/Pitx2cN fusion proteins did not activate CAT reporter gene expression, while GAL4/Pitx2C fusion protein activated CAT reporter gene expression. Similar results were obtained by scintillation counting (data not shown). Since the C-terminal region of Pitx2 is identical in both Pitx2a and Pitx2c, we thus conclude that both Pitx2a and Pitx2c encode transcriptional activator and their transcriptional activation function resides within the C-terminal region.

To analyze the function of Pitx2 isoforms in the left-right specification processes in which Pitx2 appears to be involved, RCAS-Pitx2a and RCAS-Pitx2c retroviruses were generated and used to direct ectopic expression of these isoforms to the right LPM of developing chick embryos, as described previously (Logan et al., 1998; Ryan et al., 1998). Stage 3+ or stage 4 chick embryos explanted in New culture were injected with either RCAS-Pitx2a or RCAS-Pitx2c retroviruses to the right LPM and cultured to stage 11 when heart looping completed. RCAS-alkaline phosphatase (RCAS-AP) retrovirus was used as a control. Only injected embryos that survived were harvested and scored for heart looping directions. Samples were randomly analyzed by whole-mount in situ hybridization to confirm viral misexpression. As shown in Fig. 6, ectopic expression of Pitx2 in the right LPM was observed, indicating the efficiency of RCAS infection. The results demonstrated that the injection of control viruses had no effect on heart looping (0/10). However, ectopic expression of Pitx2c to the right LPM resulted in 56% (19/34) of embryos with leftward heart looping (Fig. 6; Table 1). This result was expectable, since Pitx2c is expressed in the left LPM. Interestingly, ectopic expression of Pitx2a to the right LPM also resulted in reversal of heart looping but at a slightly less efficient level (41%; n=17) (Fig. 6; Table 1). Thus when ectopically expressed, both isoforms are able to randomize the direction of embryonic heart looping. This similar effect of ectopic expression of the two Pitx2 isoforms on heart looping could be explained by the fact that both isoforms encode transcriptional factors with similar transactivation activities, as demonstrated in Fig. 5.

As an alternative approach to analyze functional differences between Pitx2a and Pitx2c, we performed a loss-of-function experiment using antisense oligonucleotide approach. Stage 6-7 chick embryos explanted in New culture were treated with antisense oligonucleotides and allowed to develop to stage 11 at which time they were collected and scored morphologically for alterations in heart looping direction. A random oligonucleotide was used as a control. Embryos treated with control oligonucleotide exhibited a 13% (3/23) effect on heart looping similar to previous report (Isaac et al., 1997).

Treatment of embryos with antisense oligonucleotide specific to Pitx2c would be expect to eliminate Pitx2c expression in the left LPM. Random selection of these embryos for whole-mount in situ hybridization using a full-length Pitx2 probe confirmed the absence of Pitx2 expression in the left LPM (Fig. 7). However, expression of Pitx2 in the head mesoderm was not significantly affected (Fig. 7). This observation also supports the idea that Pitx2c contributes to the expression of Pitx2 in the left LPM. As expected, 43% (15/35) embryos treated with antisense oligonucleotide to Pitx2c exhibited a leftward heart looping (Fig. 7; Table 1). In contrast, embryos treated with antisense oligonucleotide specific to Pitx2a generated 19% (6/32) embryos with reversed heart looping (Table 1), a rate similar to that obtained after treatment with the control oligonucleotide. These results thus indicate that Pitx2c plays a crucial role in the regulation of left-right determination.

In order to confirm the results, we used another approach to perturb the function of Pitx2 isoforms in the left LPM. We generated putative dominant negative RCAS-Pitx2 constructs in which the transactivation domain (C-terminal region) of Pitx2a and Pitx2c was replaced with the repressor domain of the Drosophila Engrailed protein (En^r). It has been demonstrated before that a fusion protein containing a DNA-binding domain, such as a homeodomain, and with repressor domain of Engrailed can interfere with transcriptional activation by the wild-type protein (Furukawa et al., 1997; Bao et al., 1999). Thus, both Pitx2a-En^r and Pitx2c-En^r should exert a dominant negative effect to block transcriptional activation by Pitx2a or Pitx2c proteins, respectively. RCAS-Pitx2a-En^r and RCAS-Pitx2c-En^r retroviruses were targeted to the left LPM of stage 3+ or stage

4 embryos placed in New culture. Injected embryos were allowed to develop to stage 11 and scored for heart looping direction. The expression of Pitx2c-En^r in retrovirus infected embryos was confirmed by whole-mount in situ hybridization using a probe specific to the engrailed repressor domain (Fig. 8A). As we expected, ectopic expression of RCAS-Pitx2c-En^r randomized heart looping. In 19 embryos infected with RCAS- Pitx2c-En^r retrovirus, nine of them (47%) exhibited a leftward heart looping (Fig. 8B; Table 1). In contrast, ectopic expression of RCAS-Pitx2a-En^r to the left LPM did not affect rightward heart looping (Fig. 8C), with only two out of total 25 (8%) infected embryos exhibiting a leftward heart looping (Table 1). These dominant negative studies provide additional evidence for the involvement of Pitx2c but not Pitx2a in the regulation of heart looping in developing chick embryo.

Pitx2 has been demonstrated to play a crucial role in the development of left-right axis and the dextral looping of the heart. Its left-right asymmetric expressions and regulation by Nodal are highly conserved in vertebrates, including zebrafish, frog, chick and mouse (Capdevila et al., 2000). As a transcriptional factor downstream from the Shh-Nodal signaling pathway, Pitx2 products may execute the left-right developmental program. Several isoforms of Pitx2 have been isolated from human, mouse, chick, frog and zebrafish, but the distinct roles among these isoforms during embryogenesis are just begun to be elucidated (Essner et al., 2000; Schweickert et al., 2000). In this study, we examined the differential expression patterns of two Pitx2 isoforms, Pitx2a and Pitx2c, during early chick embryogenesis, and analyzed their function in the regulation of left-right development and heart looping.

By whole-mount in situ hybridization using a Pitx2c-specific probe, we showed that Pitx2c transcripts were detected symmetrically in the head mesoderm and asymmetrically in the LPM with restriction to the left LPM and the left side heart tube in the early chick embryo. However, Pitx2c expression was not found in the area opaca and extra-embryonic tissues where Pitx2 transcripts were detected in the prospective angioblasts of blood islands and the endothelia of the blood vessels using a full-length Pitx2 probe (St Amand et al., 1998). Using RT-PCR, we were able to confirm the in situ results and showed that Pitx2a is not expressed in the left LPM but is expressed in the head and extra-embryonic mesoderm. Thus, Pitx2c transcripts may primarily contribute to Pitx2 expression in the left LPM and left side heart tube. This conclusion is also supported by the observations that no Pitx2 transcripts could be detected in the left LPM after treatment with antisense oligonucleotides specific to Pitx2c. Consistent with our results, it was recently shown that Pitx2c, but not Pitx2a and Pitx2b, was asymmetrically expressed in the left LPM in both mouse and frog (Schweickert et al., 2000). However, it was reported that in the zebrafish, Pitx2c is expressed in the left dorsal diencephalon and developing gut while Pitx2a is restricted to the left heart primordium (Essner et al., 2000). Thus although the function of Pitx2 is conserved through vertebrates, different species may use different Pitx2 isoforms in the regulation of left-right development.

Being a member of the bicoid-like homeobox gene family, Pitx2 encodes a transcriptional factor that preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site (Amendt et al., 1998). In an effort to map the transactivation domains of Pitx2, it was shown that deletion of either N terminus or C terminus of human Pitx2a resulted in a decreased transactivation activity, but none of the deletions abolished transactivation activity completely (Amendt et al., 1999). It has been suggested that the both N-terminal and the C-terminal regions comprise transactivation domains (Amendt et al., 1999). However, by using a GAL4 DNA-binding/activation assay system, we have mapped the transactivation domain of Pitx2 exclusively to the C terminus. N termini from either Pitx2a or Pitx2c did not exhibit any transactivation activity in our assay system. We therefore suggest that the transactivation activity of Pitx2 resides in the C-terminal region that is identical in both Pitx2a and Pitx2c. The different conclusions drawn from previous results (Amendt et al., 1999) and our results are probably due to different assay systems used. Nevertheless, our results indicate that both Pitx2a and Pitx2c encode transcriptional activators.

It has been demonstrated previously that ectopic expression of Pitx2 induces reversal of heart looping in the chick and Xenopus embryos (Logan et al., 1998; Ryan et al., 1998; Campione et al., 1999). However, in these studies, different Pitx2 isoforms were not distinguished. In this study we have used both gain-of-function and loss-of-function approaches to examine the potentially distinct roles of these two Pitx2 isoforms in the regulation of left-right development in term of heart looping direction. Interestingly, when ectopically expressed in the right LPM, both Pitx2a and Pitx2c equally randomized heart looping directions, despite the fact that Pitx2c is predominantly expressed in the left LPM. Similar results were observed in Xenopus in which ectopic expression of either Pitx2a or Pitx2c was able to reverse heart laterality (Essner et al., 2000). This could be explained by the fact that both Pitx2a and Pitx2c contain an identical C terminus where the transactivation activity resides and may activate same set of downstream genes or execute similar downstream functions, although these downstream genes or functions remain to be identified. In contrast, results obtained from loss-of-function analysis indicate a distinct role for these two isoforms in the regulation of heart looping. Elimination of Pitx2c transcripts in the left LPM by specific antisense oligonucleotide randomized heart looping. However, embryos treated with antisense oligonucleotide specific to Pitx2a did not exhibit randomization of heart looping. These loss-of-function results were further confirmed by an alternative approach. When misexpressed, Pitx2a-En^r or Pitx2c-En^r, both of which can function as dominant negative forms, would perturb endogenous Pitx2a or Pitx2c function, respectively, by interfering their transcriptional activity. Indeed, Pitx2c-En^r, but not Pitx2a-En^r, was able to randomize heart looping when ectopically expressed in the early embryo. The dominant negative form of Pitx2a seems not to interfere with endogenous Pitx2c function. This is probably due to the significant difference in the N terminus of the two isoforms, which may interact with distinct transcriptional factors for specific DNA binding and transactivation activity. Pitx2c-En^r could compete with endogenous Pitx2c for these co-factors more efficiently than Pitx2a-En^r. It was reported that Pitx2 protein can form homodimers (Amendt et al., 1999). Therefore, an alternative explanation for the different effects of the dominant negative forms of Pitx2a and Pitx2c is that Pitx2c-En^r forms dimers with endogenous Pitx2c, and perturbs its function. Together with the differential expression patterns of Pitx2 isoforms, we conclude that Pitx2c plays a predominant role in the regulation of rightward heart looping in developing chick embryo.

We thank Drs Constance Cepko (Harvard Medical School) and John Epstein (University of Pennsylvania) for providing Slax 13 adaptor plasmid containing En^r sequence and the plasmids for GAL4 DNA- binding/activation assay. This work was supported by a Grand-in-Aid (9750104) from American Heart Association (Y. P. C.), a grant from Tulane Cancer Center (T. R. S.), and grants from NSF (IBN-9808126) and Kentucky Spinal Cord and Head Injury Trust to M. S. Q.