Isolation of a new homeobox gene belonging to the Pitx/Rieg family: expression during lens development and mapping to the aphakia region on mouse chromosome 19
Isolation of a new homeobox gene belonging to the Pitx / Rieg family: expression during lens development and mapping to the aphakia region on mouse chromosome 19Elena V. Semina1, Rebecca S. Reiter2 and Jeffrey C. Murray1,2,*
Departments of 1Pediatrics and 2Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA
Received July 2, 1997;Revised and Accepted August 25, 1997
We recently reported the positional cloning of a homeobox gene involved in the pathogenesis of Rieger syndrome, RIEG1, and its mouse homolog, Rieg1. Rieg1 (also independently described as Pitx2) is highly homologous to the Ptx1/Potx gene product, suggesting that there may be additional members of this novel Pitx family. The Pitx genes play an important role in eye, tooth, pituitary and umbilical region development as evidenced by Rieger syndrome and iris hypoplasia phenotypes, resulting from mutations in the RIEG1 gene and by expression studies. In order to characterize further the Pitx gene family we searched mouse cDNA libraries to identify additional members. A new gene was isolated which encodes a homeoprotein with strong homology to the otherPitx proteins and 97-100% identity in the homeodomain itself, suggesting that this is a third member of the family, Pitx3. In whole mount in situ hybridization on mouse embryos ranging from 8.5 to 11.5 days post-coitum (d.p.c.), Pitx3 mRNA was seen only in the developing lens starting at day 11. Hybridization on cross-sections revealed strong signals in the lens vesicle in 11 d.p.c. embryos and throughout the lens, particularly in the anterior epithelium and equator region in 15 d.p.c. embryos. Pitx3 was mapped close to aphakia on mouse chromosome 19. The aphakia homozygous mouse is characterized by small eyes lacking a lens, which fail to develop beyond 11 d.p.c. These data make Pitx3 a strong candidate gene for the aphakia phenotype in the mouse and suggest a role for the human homolog in congenital lens malformations.
The homeobox-containing transcription factor genes encode homeodomain (HD)-containing proteins, which play a key role in coordination of gene activity and determination of cell fate in the development of organisms as diverse as yeast, plants, insects and mammals. The HD proteins share remarkable evolutionary conservation of both protein structure and function. Some homeobox genes are organized in clusters in the genome, e.g. HOM-C in insects and nematodes and HOX in mammals. The mammalian cluster is duplicated two or three times to form four copies with up to 14 identifiable types of genes in each Hox cluster (1 ). Based on their HD sequence similarities and positions within the cluster, the Hox genes are grouped into paralogs. Corresponding paralogs in different Hox clusters are often expressed in similar patterns and can be functionally redundant.
Other homeobox genes are dispersed throughout the genome. According to their HD similarities, they are grouped into families consisting of several members with overlapping expression patterns and possible redundancy in function (2 ,3 ). Similarity in the biochemical activity of different members of the same family was elegantly demonstrated by Hanks et al. in rescuing the En-1 mutant phenotype in mouse by replacing the En-1 coding sequences with En-2 sequences through gene targeting (4 ).
Previously we reported the positional cloning of the homeobox gene RIEG1, responsible for Rieger syndrome (RIEG; 5 ) and iris hypoplasia (6 ) in humans. RIEG is characterized by abnormalities of the anterior chamber of the eye, dental hypoplasia, craniofacial dysmorphism and umbilical abnormalities (7 -9 ). Ocular anomalies are the most characteristic and debilitating features of RIEG, leading to glaucoma in >50% of affected individuals. Murine Rieg1 [independently isolated and published under the name Pitx2 (10 ) and Otlx2 (11 )] mRNA was detected in the periocular mesenchyme, maxillary and mandibular epithelium, umbilical vessels, Rathke's pouch and limb mesenchyme (5).
The human RIEG1 and murine Rieg1/Pitx2 genes have strongest homology to the recently discovered Ptx1/POTX gene (12 ,13 ). Rieg1/Pitx2 and Ptx1/POTX encode proteins with almost identical HDs (with only two out of 60 amino acids differing) and other regions of similarity, suggesting that they are members of the same family (5 ,10 ). In addition, Rieg1/Pitx2 and Ptx1/POTX have significantly overlapping expression patterns. To characterize further this new homeobox-containing gene family we screened mouse cDNA libraries with a probe comprising the homeobox region of Rieg1/Pitx2 and identified a third member of the family, Pitx3. Expression studies on 8.5-11.5 days post-coitum (d.p.c.) mouse embryos revealed that the new gene is exclusively expressed in the eye of an embryo and prominently so in the developing lens.
Pitx3 was mapped to mouse chromosome 19, close to aphakia. The aphakia homozygous mouse is characterized by small eyes lacking a lens and closed eyelids. These data make Pitx3 a strong candidate gene for causing the aphakia phenotype in the mouse and suggest a role for the human homolog in congenital lens malformations.
Mouse day 15 embryo (Novagene) and embryonic carcinoma (Stratagene) cDNA libraries were screened with a probe containing the homeobox region of Rieg1/Pitx2. Three distinct clones were identified from the 3 * 106 clones examined. These clones were sequenced and analyzed to construct a cDNA contig, designated r13-2, which was then sequenced in both directions.
The cDNA was 1392 bp in length, with a putative polyadenylation signal at positions 1352-1358 and a poly(A) tail (Fig. 1 a). The first methionine associated with the longest open reading frame was found at position 134 of the cDNA. The region 5' of the first methionine comprises 80% G/C content, which is characteristic of the 5'-untranslated regions (UTR) of vertebrate genes. The protein ascribed to the open reading frame consists of 302 amino acids, which is comparable to proteins encoded by other genes from this family, i.e. the 271 and 317 amino acid isoforms generated by alternative splicing from Rieg1/Pitx2 (10) and the 315 amino acid Ptx1/POTX (12).
The Pitx3 gene was found to consist of four exons of 121, 130, 203 and 918 bp each (Fig. 1 b). Sequencing of the exon-intron junctions revealed the standard donor and acceptor site sequences (Fig. 1 b). The homeobox region of the gene is interrupted by an intron located in the same position as in other genes from this family (Rieg1/Pitx2 and Ptx1/Potx), between codons for amino acids 46 and 47 of the HD.
We used a 1 kb fragment spanning the 5'-UTR and most of the coding sequence of Pitx3 cDNA as a template for preparation of riboprobes for in situ hybridization. Whole mount in situ hybridization on 8.5, 9, 10, 11 and 11.5 d.p.c. mouse embryos revealed a strong signal in the developing lens beginning on day 11 (Fig. 3 ). Hybridization on sections at the eye level of day 11 and 15 embryos further evidenced Pitx3 expression in the developing lens. At 11 d.p.c. the strong signal is present in the lens vesicle (Fig. 4 ), with expression throughout the developing lens at 15 d.p.c., including the fiber cells, but most notably in the anterior epithelium and in the equator (bow) regions of the lens. Pitx3 expression was also detected in the eye muscles and the eyelid in day 15 embryos (Fig. 4 ).
A single-strand conformational variant between C57BL/6J and Mus spretus was identified for the 212 bp PCR product spanning nt 455-666 of the cDNA. Interspecific backcross panels from The Jackson Laboratory, (C57BL/6J M.spretus)F1 * C57BL/6J (BSB) and (C57BL/6J M.spretus)F1 * M.spretus (BSS), were then used to map Pitx3 within the mouse genome (Fig. 5 ). Pitx3 maps to the distal half of mouse chromosome 19 between markers D19Mit27 and D19Mit4, positioned 32.8 and 35 cM respectively from the proximal end of the chromosome according to the MIT map and 43 and 48 cM according to the Mouse Genome Database (MGD) map. The combined data from two crosses show proximal-D19Mit27-4/188 or 2.66 ± 1.17 cM-Pitx3-2/188 or 1.06 ± 0.75 cM-D19Mit4-distal.
The Pitx3 gene fragments were amplified from genomic DNA of mouse strain B6 * C57BL/ks-ak, homozygous for the aphakia mutant allele. Oligonucleotides for PCR were designed to amplify the entire coding region with exon-intron junction sequences. The amplified fragments of the aphakiaPitx3 gene were then analyzed by single-strand conformation variant analysis and sequencing. No variant band or sequence difference in comparison to normal have been found.
We report the isolation and characterization of a new member of the Pitx/Rieg homeobox-containing transcription factor gene family, Pitx3. We suggest that Pitx3 is the third member of this family, along with Ptx1/Potx and Rieg1/Pitx2, because of its strong homology to the proteins encoded by these genes. At the amino acid level, 97% identity with Ptx1/Potx and 100% identity with Rieg1/Pitx2 was found in the HD regions, with significant homology extending into the N-and C-terminal regions from the HD.
The HD is a DNA binding motif which is characteristic and strongly conserved among the HD proteins. The specificity of HD binding to its target is based on distinct DNA binding properties of the HD sequence and assembly with other proteins. Protein-protein interactions can involve both specific residues within the HD itself and other regions of the protein, as has been suggested for the regions immediately C- and N-terminal to the HD (18 ).
The Pitx proteins are found to be most closely related to the Caenorhabditis elegans Unc-30 protein, Drosophila orthodenticle (Otd) and its murine homologs Otx1 and Otx2 and the Paired-like proteins Drg11, al, Cart-1, Prx-1, Prx-2 and Chx-10 (for comparisons see 5 ,12 ). The Paired-like proteins were found to share a 14 amino acid motif, located 3' of the HD, with the Pitx proteins (5; recently designated OAR, 19 ), which may represent a DNA binding element or a site of protein-protein interaction playing a role in the specificity of the HD protein function.
The expression of Pitx3 is markedly distinct from that of the two other Pitx genes. Rieg1/Pitx2 and Ptx1/Potx have very similar temporal and spatial expression, overlapping in the maxilla, mandible, Rathke's pouch, eye, umbilicus, midgut region and the limbs (5 ,13 ) and beginning early in development; 8 d.p.c. embryos exhibit strong signal in the head and trunk (13 ; Semina and Reiter, unpublished data). Pitx3 mRNA, on the other hand, was first detected in 11 d.p.c. embryos in a survey of embryos from 8.5-11.5 d.p.c. and the signal was restricted to the developing lens (Fig. 3 ). Future studies should explore Pitx3 expression sites in older embryos and in adult mice.
The development of the lens is a well-studied process and includes several stages. The formation of the lens placode (by 10 d.p.c. in mouse embryo) is induced by the neuroepithelium of the optic vesicle after establishment of close contact between the optic vesicle and overlying surface ectoderm at 9.5-9.75 d.p.c. (16 ,17 ). The lens placode starts to invaginate in the 10.5 d.p.c. embryo and the lens cup rapidly deepens within the next few hours. The closure of the lens cup and detachment of the lens vesicle from the surface occur by 11-11.25 d.p.c. At 11.5 d.p.c. formation of lens fibers begins, with continued elongation of the fibers leading to occlusion of the lens cavity before the end of 13 d.p.c. By day 13 the lens has a similar configuration to that of the adult organ (16 ,17 ).
During lens development the peripheral epithelial cells proliferate anterior to the lens equator (or bow region), where they subsequently differentiate into the lens fiber cells. Differentiation into lens fiber cells includes cell elongation and loss of subcellular organelles, cessation of DNA replication and of cell division and synthesis of fiber cell-specific proteins, such as various crystallins (20 ).
Many genes are involved in lens formation. Pax-6, a master gene in eye development, has been implicated in the various mouse and rat Smalleye (Sey) mutant phenotypes (21 ) as well as in human aniridia (22 ). Pax-6 is expressed in all stages of lens development. A histological analysis of murine homozygous Sey mutants revealed that the optic vesicles grow out but there is no lens induction (23 ). Tissue transplantation experiments in a rat Sey mutant demonstrated that homozygous rSey ectoderm loses its lens-forming competence early in development (24 ). Several papers have demonstrated that Pax-6 is involved in the regulation of lens-specific expression of the crystallin genes (25 ). These results suggests that Pax-6 is involved in lens induction and subsequent development and differentiation.
Other homeobox genes involved in early lens development include Prox1 (related to Drosophilaprospero), which is expressed in the early lens placode and later throughout the lens, especially in the bow region, in chicken (26 ) and in the developing lens in mouse (27 ) and human (26 ). Murine Six3 is expressed in the optic vesicle and lens (28 ). In Xenopus, the Six3 transcript was detected in the anterior neural plate, a region involved in lens induction. The ectopic expression of murine Six3 in fish embryos resulted in ectopic lens formation in the area of the otic vesicle (29 ). Chicken GH6 is expressed in the lens epithelium at stage 23, which is roughly equivalent to mouse 11 d.p.c. (30 ). Mouse Msx2 and Emx1 are expressed in the lens, as reviewed by Beebe (31 ). In addition, some Sox homeodomain proteins have been shown to be involved in lens-specific activation of crystallin genes in mouse (32 ).
The Pitx3 gene was mapped to mouse chromosome 19 in the aphakia (ak) region. ak is a recessive mutation, described originally by Varnum and Stevens in 1968 (33 ). Although affected mice demonstrated reduced eye size and overall ocular disorganization, it appears that the primary cause of the phenotype is abnormal lens development. Unlike the Sey mutation effect on lens induction at the lens placode stage (23 ), the ak lens develops normally until the lens cup stage. The first abnormalities are noted slightly before closure of the lens cup at 10.5-11 d.p.c. (16 ,17 ): there is a progressive accumulation of cells, released by the lens epithelium, in the lumen of the lens cup, so that by the time of lens vesicle formation the lumen is entirely obliterated. Frequently the lens vesicle does not detach from the ectoderm. After 11 d.p.c. no further organization of the lens takes place; the cells at the posterior wall never elongate to become lens fibers. The lens abnormality is accompanied by a number of defects in other parts of the eye. The eye remains smaller than normal and the neural retina undergoes multiple foldings, filling the vitreous space, so that the lens vesicle becomes more bent and elongated than normal. The cornea, iris, pupillary opening and vitreous never differentiate properly (15 -17 ). These abnormalities are thought to be secondary to and dependent on abnormal lens development because the same features are seen when the lens has been ablated by targeted expression of a cytotoxic gene (34 ) or surgical removal (35 ). In addition to noted morphological abnormalities in ak mice, delayed appearance of some specific lens proteins has been described. A small amount of [alpha]-crystallin was found in the ak lens vesicle at 14 d.p.c. rather than at 11 d.p.c., while the [gamma]- and [beta]-crystallins were absent from abnormal lenses of 10-18 d.p.c. embryos (36 ).
Pitx3 represents a strong candidate gene for the ak phenotype in mouse. First, Pitx3 mRNA is present at high levels in the lens vesicle at 11 d.p.c., where the morphological abnormalities in ak lenses have been reported, and in day 15 mouse embryos Pitx3 is expressed throughout the lens, particularly in the anterior lens epithelium and equator region, where cytodifferentiation of lens fibers takes place. Second, Pitx3 was localized to the same region on mouse chromosome 19, where ak has been mapped. Although we have not identified any mutations in the ak mouse Pitx3 gene sequence by sequencing and SSCP analysis, there is still a chance that a mutation alters a regulatory region or alternative exon sequence not yet identified. The possibility of another gene being responsible for ak also cannot be discounted. Detailed expression studies of Pitx3 in the developing eye in normal and ak mice may further adjudicate the candidacy of this gene.
Human congenital aphakia is a rare anomaly, classified as primary, in which no lens `anlage' has developed, or secondary, in which a lens has developed to some degree but then is expelled in utero (37 ). Genetic loci responsible for this condition have not yet been identified. Several cases where aphakia was associated with Peter's anomaly (3 -40 ) and one case with aniridia (41 ) have been reported, thus admitting the possibility that some mutations in PAX6 could be responsible for this more severe phenotype, as PAX6 mutations were found in patients with Peter's anomaly (42 ) and aniridia (22 ). Moreover, environmental factors, such as exposure to rubella virus during pregnancy, may play a role in some aphakia cases (43 ,44 ). Conversely, human congenital cataracts are fairly common and variable conditions that have been only partially assigned to specific chromosomes and could be candidate phenotypes for mutations in the human homolog of the mouse Pitx3 gene.
No human phenotypes containing eye, and specifically lens, defects have been localized to the long arm of chromosome 10, which is syntenic to the Pitx3 mapping region on mouse chromosome 19. Should future mapping studies localize ocular phenotypes to this region, then the human PITX3 gene would be a logical candidate. Further, its interactions with genes such as the crystallins and the keratins that might predispose to cataracts or other eye abnormalities would also be important to elucidate. Descriptions of the gene sequence, expression pattern and genetic mapping of the Pitx3 gene provide an opportunity for better understanding the role that transcription factors play in both mouse and human ocular development.
Mouse cDNA libraries [embryonic carcinoma (Stratagene) and day 15 embryo (Novagene)] were screened with a random prime radiolabeled (Boehringer) probe comprising the homeobox region of Rieg1 (5). Hybridization was carried out in standard hybridization buffer at 50oC for 18 h, followed by several washes at room temperature and a 1 h wash at 55oC in 0.1% SSC, 0.5% SDS. Plasmids from positive plaques were isolated from their Uni-Zap XR or [lambda]EXloxR hosts by in vivo excision with R408 helper phage as described by the manufacturer. Bluescript plasmids containing cDNA inserts were sequenced and analyzed using the BLASTN and GRAIL search engines.
A 1 kb fragment spanning the 5'-region of Pitx3 cDNA and most of the coding sequence (nt 25-1026) was used to create probes for in situ hybridization. From linearized cDNA, 35S- or digoxigenin-labeled RNA probes were synthesized using either T3 or T7 RNA polymerase. NIH Swiss mice from Harlan (Indianapolis, Indiana), staged according to Theiler (45 ), were used.
Embryonic mice for radioactive in situ hybridization on sections were fixed overnight at 4oC in 4% paraformaldehyde in PBS, dehydrated, cleared in Histosol (National Diagnostics) and embedded in Paraplast Plus (Oxford). Seven micron sections were cut and mounted on Superfrost-plus slides (Fisher) with DEPC-treated water, dried overnight at 40oC and then stored at room temperature. Before use, the slides were baked at 60oC overnight and then processed for in situ hybridization as described by Sassoon and Rosentha (46 ). Slides were hybridized overnight at 50oC, washed in 5* SSC at 50oC to remove coverslips and then washed in 2* SSC, 50% formamide at 60oC. Autoradiography was with Kodak NTB-2 emulsions. Embryonic mice for whole mount in situ hybridization were fixed and processed according to a modification of the method of Harland (47 ).
The BSS and BSB interspecific backcross panels were obtained from The Jackson Laboratory (Bar Harbor, ME). Each panel contains 94 backcross animals plus parental controls (48 ). Complete haplotype data for these crosses are available electronically at http://www.jax.org/resources/documents/cmdata.
A 212 bp fragment of Pitx3 sequence was amplified with the following primers: 5'-tgtggttcaagaaccggc-3', forward; 5'-ttgaccgagttgaaggcgaa-3', reverse. Amplification was carried out in a PCR thermocycler (Perkin-Elmer Cetus), with a single 4 min, 94oC stage followed by 30 cycles comprising three 30 s steps at 94, 55 and 72oC each, in 10 [mu]l total volume of 1 [mu]l Boehringer PCR buffer, 2.5 pmol each primer, 2 mM each dNTP and 0.25 U Taq polymerase (Boehringer). PCR products were heated for 4 min at 95oC and electrophoresed for 4 h at 20 W through a fan-cooled gel composed of 3.3 ml 10* TBE, 1.37 ml glycerol, 13.75 ml MDE mix (from FMC), 36.6 ml water, 220 [mu]l 10% APS and 22 [mu]l TEMED. After silver staining, the gels were visually inspected and genotyped. Genotypes were submitted to The Jackson Laboratory for interpretation.
BAC genomic clones for Pitx3 were isolated by screening of a mouse BAC genomic library obtained from Research Genetics with a PCR product containing a specific sequence from the 3'-UTR of the gene amplified from 5'-gcaggtctgtggatccat-3' and 5'-aaaggccctcttcgaagc-3' forward and reverse primers respectively. Genomic structure of Pitx3 was identified by primer walking between cDNA and genomic clones.
DNA samples of the mouse strain B6 * C57BL/ks-ak with genotype ak/ak were obtained from Mouse DNA Resources of The Jackson Laboratory. Oligonucleotides used for PCR amplification of the Pitx3 coding regions and adjacent introns were: 5'-cgcactagacctccctcc-3' and 5'-ggttatcatcactctcgctc-3', forward and reverse respectively, for exon 1; 5'-aggaattccttgaggcccct-3' and 5'-ctcatgtcagggtagcgatt-3' for intron 1-exon 2; 5'-aggacggctctctgaagaa-3' and 5'-ccgcagagtcaccagcta-3' for exon 2-intron 2; 5'-tgacagcctttctcggatac-3' and 5'-ttgaccgagttgaaggcgaa-3' for intron 2-exon 3; 5'-tactcgtacggcaactgg-3' and 5'-acgagggcaagccagtcta-3' for exon 3. Genomic DNA from the aphakia mouse was amplified in a PCR thermocycler (Perkin-Elmer Cetus), with a single 4 min, 94oC stage followed by 30 cycles comprising three 45 s steps at 94, 55 and 72oC respectively, in 10 [mu]l total volume of 1 [mu]l Boehringer 10* PCR buffer, 2.5 pmol each primer, 2 mM each dNTP and 0.25 U Taq polymerase (Boehringer). Each PCR product was sequenced with its generating primers using an ABI PRISM 373 DNA Sequencer. The sequences were compared with the normal sequence.
The GenBank accession number for the Pitx3 cDNA sequence is AF005772.
We thank Sally Camper and Phillip Gage for helpful discussions of the material. Jim Lin provided assistance with in situ hybridization and Bonnie Ludwig with the sequencing. We also would like to thank Lucy B.Rowe (The Jackson Laboratory) for excellent assistance with mapping data analysis. This work was funded by CARC grant DE-09170 and DERC grant DK-25295 from the NIH.
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*To whom correspondence should be addressed at: Department of Pediatrics, The University of Iowa, 200 Hawkins Drive, W229-1 GH, Iowa City, IA 52242, USA. Tel: +1 319 335 6897; Fax: +1 319 335 6970; Email: jeff-murray@uiowa.edu
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