Mouse aphakia (ak) is a recessive phenotype that spontaneously occurs in the 129/Sv-SlJ strain and is characterized by small eyes that lack a lens. We have recently identified a homeobox-containing gene, Pitx3, and have shown that it is expressed in the developing lens and maps to chromosome 19 close to ak in mouse. Human PITX3 gene was found to underlie anterior segment dysgenesis and cataracts. We have now obtained the entire sequence of the mouse Pitx3 gene including 10 kb of the 5′ region and 5 kb of the 3′ region. Of several microsatellite repeat regions identified within the Pitx3 sequence, one was informative for linkage analysis. No recombination was observed between ak and the Pitx3 marker, indicating that these two loci are closely linked (0.2 ± 0.2 cM). Additionally, Pitx3 transcripts were not detected in the ak/ak mice either in the lens placode or at later developmental stages of the lens by in situ hybridization. Since no differences were previously found between ak/ak and wild-type sequences in the Pitx3 coding region, we hypothesized that an etiologic mutation is located in the promoter or other regulatory regions. To test this hypothesis we studied the 5′ flanking region of the Pitx3 gene. This analysis revealed a deletion of 652 bp located 2.5 kb upstream from the start point of the Pitx3 5′ UTR sequence in ak/ak mice. The deletion co-segregated with the ak mutation and was not detected in 16 samples from 10 different mouse strains including the founder strains. Analysis of the 652 bp region identified sequences similar to consensus binding sites for transcription factors AP-2 and Maf that were shown to play a critical role in lens determination. These lines of evidence suggest that the abnormal ocular development in the aphakia mouse is due to the deletion upstream of the Pitx3 gene.
Received 4 February 2000; Revised and Accepted 14 April 2000.
Human cataracts represent one of the major causes of treatable blindness, accounting for nearly half of all vision impairment worldwide (1). Congenital cataracts are less common (the estimated prevalence is 1 to 6 cases per 10 000 live births) but are more difficult to treat and are therefore responsible for a significant number of blind individuals in the pediatric age group (2). Moreover, healthy lens embryology is essential for the normal development of other ocular structures, particularly in the anterior chamber of the eye, that play an important role in normal vision. Analysis of factors underlying normal lens development is difficult in humans; therefore mouse models are often used in these studies.
In addition, the embryonic lens of vertebrates provides a useful model for studying gene regulation of tissue development from tissue induction to maturation. During development, the vertebrate lens is induced upon contact between presumptive retina (optic vesicle) and surface ectoderm. As a result, the ectoderm thickens into a placode that will first form a vesicle and later differentiate into a lens. To date, more than 46 mouse mutations have been described that result in lens abnormalities with some that affect early lens development (reviewed in ref. 3). Genetic factors responsible for the majority of these phenotypes are still to be determined.
Mouse aphakia (ak) is a recessive phenotype that spontaneously occurred in the 129/Sv-SlJ strain and was first described by Varnum and Stevens in 1968 (4). The phenotype of ak/ak newborn mice is characterized by small eyes that lack a lens as well as anterior segment structures. These anomalies are caused by an arrest of lens development at the stage of lens vesicle formation around embryonic days 10.5–11 (4,5). In the ak/ak mutants, there is a persistence of the lens stalk at embryonic day 12.5 interrupting the corneal mesenchyme and leading to a permanent close contact between the developing cornea and other ocular tissues without the formation of an anterior chamber (5,6). There are no other systemic abnormalities reported in these mice.
Transcription factors in general and homeodomain-containing proteins in particular are fundamental for the genetic control of different basic developmental processes. We have recently identified a homeobox-containing gene, Pitx3, and showed that it is expressed in the developing lens and maps to chromosome 19 close to ak (7). The human homolog of this gene, PITX3, which maps to the 10q24–25 region, was found, when mutated, to cause anterior segment dysgenesis with cataract and congenital total cataract in two unrelated families (8). All these facts—location, expression pattern and conserved function of the PITX3/Pitx3 gene during mammalian lens development—implicated it as being a strong candidate for the ak phenotype. Surprisingly, a search for mutations failed to identify any significant differences in the coding and exon–intron junction regions of Pitx3 between ak and wild-type genomic DNA (7) as well as mRNA (J. Graw, unpublished results) sequences.
In this paper we report close linkage between ak and Pitx3 gene, altered expression of Pitx3 in ak/ak mice and identification of a deletion in a 5′ flanking region of the Pitx3 gene, which is specific for ak mice and is not seen in two strains constituting the background of this mutation as well as eight other mouse strains. Therefore we suggest that altered expression of the Pitx3 gene in ak mice is most likely a result of the deletion and leads to an abnormal phenotype in these mice.
Genomic sequence of the mouse Pitx3 gene: analysis of the promoter region and identification of neighboring genes Gbf1 and Cig30
The genomic sequence of the Pitx3 gene was obtained by sequencing of BAC genomic clones: complete sequence of introns and ~10 kb of a 5′ flanking region and ~5 kb of a 3′ flanking region was identified. The gene was found to span 12.6 kb in genomic sequence and to consist of four exons of 125, 129, 210 and 927 bp for exons 1–4, respectively, and three introns of 10729, 196 and 379 bp for introns 1–3, respectively (Fig. 1A). The genomic structure of the Pitx3 gene resembles the other Pitx genes, Pitx1 and Pitx2, with the homeobox region being interrupted by an intron at position 46 of the homeodomain and the large first intron separating the first and second exons (9). Analysis of the Pitx3 genomic sequence was performed by comparison with other sequences in GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/blast.cgi ). Identification of potential transcription units was performed by GRAIL analysis and identification of consensus binding sites for different transcription factors was performed using MatInspector Version 2.2 software (http://transfac.gbf-braunschweig.de/cgi-bin/matSearch/ matsearch.pl ) as well as manually.
Analysis of the genomic sequence identified two murine genes in close proximity to the Pitx3 gene: the Gbf1 gene, a mouse homologue of the human GBF1, a ubiquitously expressed gene of the Sec7 domain family (10), and the Cig30 gene, encoding a brown adipose tissue membrane glycoprotein (11). The Gbf1 gene sequence was identified in the 5′ flanking region of the Pitx3 gene at a distance of 4.2 kb from the Pitx3 5′ UTR start point; the direction of the transcription of the Gbf1 gene is opposite to that of the Pitx3 gene, hence the 5′ regions of these two genes overlap (Fig. 1A). Four exons of the Cig30 gene were identified in the 4.5 kb region immediately downstream of the Pitx3 gene but oriented in the opposite direction. In fact, the 3′ UTR sequences of the Cig30 and Pitx3 genes are complementary over the last 10 nucleotides. This is in agreement with a previous report (12). No other existing or potential transcription units were identified within 22 kb of Pitx3 genomic sequence.
The DNA located upstream and surrounding the transcription initiation sites typically confers promoter activity for a gene. As a transcription start point (tsp), the exact 5′ end of Pitx3 mRNA from mouse embryonic carcinoma was used (7). Analysis of the 5′flanking region upstream of this tsp identified the closest potential CAAT and TATA box sequences at positions –205 to –200 and –86 to –80, and at positions –1590 to –1584 and –1560 to –1552 (Fig. 1B). Also, sequences similar to consensus binding sites for some tissue-specific transcription factors were identified: several motifs similar to the AP-2 consensus sequence 5′-CCCCAGG-3′ (13) or 5′-CCCAGCCC-3′ (14); several sequences similar to the alphaCE2 element, 5′-TGCTGACC-3′, that was originally identified in the alpha A-crystallin gene promoter and was shown to bind Maf transcription factors (13,15); and two motifs similar to binding sites for forkhead-7 (16) (Fig. 1B). The AP-2 and Maf proteins were found to be crucial for normal formation of the craniofacial region and play an important role in lens development (17–22).
Pitx3 gene and aphakia are closely linked
In previous reports, a distance of 1.8 cM between ak and Pitx3 was deduced from two independent crosses (5). In order to perform a detailed linkage analysis of Pitx3 and ak/ak, we identified seven microsatellite repeat regions within the Pitx3 genomic sequence (Table 1) and tested five of them for polymorphisms between ak/ak and the two mouse strains, AKR and JF1, which were previously used for fine mapping of the ak mutation (5). Three markers did not reveal any differences between ak/ak, AKR and JF1 mice; only the Pitx3-CT marker was informative with the JF1 strain. Therefore this marker was used to search for recombination events in the existing backcross panel of 416 F2 animals from matings (ak/ak × JF1)F1 × ak/ak (5). In particular, all animals were checked for recombinations within the interval between the markers D19Mit19 and D19Mit38 spanning the entire ak-critical region of ~8 cM. No recombination was observed between ak and the Pitx3-CT marker placing them at a distance of 0.2 ± 0.2 cM from each other. In addition, the markers D19Mit7, D19Mit8 and D19Mit102 were used in the same set of animals to refine the previous mapping data. The results led us to conclude that the marker D19Mit8 is also very close to ak; however, D19Mit102 (one recombination) and D19Mit7 (six recombinations including one double crossover) have to be placed more proximal to ak (Fig. 2A). The actual mapping positions of ak and polymorphic markers are summarized in Figure 2B.
Moreover, the markers Pitx3-CTT and CTAC did not give a signal in the ak/ak mutants, but yielded strong signals in all tested wild-type mice suggesting that an ak-specific deletion might be present in the corresponding region (see below). Therefore, another set of primers was designed spanning the entire region including both repetitive sequences. Using these novel primers, all the ak/ak mutants revealed a shorter fragment than the wild-types: heterozygotes showed the long wild-type band and the short ak band. This strain difference also segregated in the JF1 backcross together with the ak mutation; no recombination could be detected. In conclusion, all these mapping experiments demonstrated a close linkage of ak to Pitx3 and, moreover, suggested a deletion in the region of the CTAC and CTT repeats.
Expression of the Pitx3 gene is altered in aphakia mice
The aphakia mice are characterized by severe ocular abnormalities (Fig. 3). Varnum and Stevens originally reported in 1968 (3) that the first visible abnormalities in ak lens development are seen at the early lens vesicle stage in 10.5- to 11-day embryonic ak mice, with further development of the lens and the eye being grossly disturbed. Later, Zwaan and Kirkland (23) noted that distinct anomalies are already seen at the lens placode stage in 10- to 10.5-day embryonic ak mice. Therefore, we studied expression of the Pitx3 gene in the eye in detail to see whether its expression coincides with the onset of anomalies in ak mice and to better assess the role of Pitx3.
Sections at the level of the eye from embryonic day 10 to postnatal day 1 were studied by in situ hybridization in wild-type mice. Adult eye RNA was analyzed by RT–PCR. Expression was first strongly detected in the late lens placode stage shortly before the start of its invagination towards the optic cup (Fig. 4). Later in development expression was seen in the lens pit and in the primary fiber cells that obliterate the lumen of the lens vesicle (Fig. 4). In the maturing lens of day 13–15 embryos the most abundant transcript is present at the equator regions as described previously (7). In the postnatal and in the adult mouse eye, Pitx3 expression is still present at low levels (data not shown).
RT–PCR analysis of Pitx3 expression was performed using total RNA extracted from whole day 10 and day 15 embryos of wild-type, ak/+ and ak/ak mice. Pitx3-specific primers were used with forward and reverse primers located in exons 1 and 2, respectively, in order to eliminate false positives from genomic DNA (see Materials and Methods). Pitx3 expression was detected in day 15 wild-type and ak/+ embryos while it was altered in three ak/ak embryos studied: completely absent in one ak/ak embryo (data not shown) and largely diminished in two (Fig. 5A). Both Cig30 and Gbf1 RNAs were detected in the day 15 embryos at a level indistinguishable between ak/ak and wild-type mice. Northern blot analysis of the Pitx3 expression was performed on the day 15 total RNA by hybridization with a radioactive probe containing partial Pitx3 cDNA sequence. Distinct signal corresponding to the Pitx3 transcript was detected in wild-type RNA but not in the ak/ak RNA after a 5-day exposure (Fig. 5B). Longer exposure allowed weak detection of the Pitx3 transcript in ak/ak RNA but its level was largely diminished in comparison with the wild-type (Fig. 5B).
In order to assess specific sites of Pitx3 expression in ak/ak embryos, in situ hybridization on sections was performed on day 10–16 ak/ak and wild-type embryos. While expression was missing in ak/ak embryos (Fig. 4A–P: embryonic days 10 and 14 are shown), in the wild-type embryos Pitx3 expression was detected in the lens placode and, at later stages of lens development, in the head muscles, tongue, midbrain region, mesenchyme and around the vertebrae and sternum. These observations were all in good agreement with the pattern described previously (7,8).
In order to see whether expression of other Pitx genes is altered in ak/ak embryos, expression of Pitx1 and Pitx2 was studied by RT–PCR (Pitx1, Pitx2) and in situ hybridization (Pitx1). The Pitx1 and Pitx2 genes demonstrate overlapping patterns of expression with the Pitx3 gene as has been shown for many homeobox genes belonging to the same family (7,24). Transcripts for both genes were found to be present at indistinguishable levels in the total RNA extracted from embryonic day 15 ak/ak and wild-type mice (RT–PCR data not shown). There were also no significant differences in the Pitx1 gene expression studied by in situ hybridization in embryos ranging from embryonic days 10–16 between ak and wild-type mice (Figure 4Q–T: day 12.5 is shown). Pitx1 was present in all of the sites of its normal expression and without any visible difference in the amount of transcript between the two mice: tongue, midbrain, developing pituitary and gut region. The only expression that appeared to be altered was expression in the developing lens where the Pitx1 gene is normally co-expressed with Pitx3 gene starting from the late lens vesicle stage around embryonic days 11.5–12 (E.V. Semina and R. Reiter, unpublished results). The ak/ak embryos did not show any detectable Pitx1 expression in the lens, which is most likely attributable to the evidently disturbed lens development in ak/ak embryos by this stage and therefore the lack of cells that would normally express the Pitx1 gene. It is also possible that the Pitx3 and Pitx1 genes are involved in a cascade of mutual activation in the lens as has been shown for some other paralogous homeobox genes (25–27).
Identification of a deletion in the 5′ region of Pitx3 gene in aphakia mice
Detailed sequence analysis of the Pitx3 5′ sequence in the ak/ak mice identified a 652 bp deletion located ~2.5 kb from the start point of Pitx3 cDNA (Fig. 5C and D). This deletion was not detected in the DNA from mouse strains 129/Sv-SlJ (129S1/Sv-+p +Tyr-c MgfSl-J/+ according to the new nomenclature) and C57BLKSrepresenting the genetic background of this mutation (4; B.A. Richards-Smith and D. Schroeder, personal communication) as well as eight other strains tested to date (Fig. 5C). Analysis of the 652 bp region revealed several motifs similar to the AP-2, Maf and forkhead 7 [now referred to as FOXL1 (28)] binding site consensus sequences (13–16) (Fig. 1B) that play a crucial role during early ocular development. This suggests that the loss of binding capacity for at least some of these transcription factors might lead to a loss of Pitx3 transcripts, at least at the time during development when the eyes are being formed.
Other possible upstream regulators of Pitx3 ocular expression include Pax6, Six3, Eya1, Eya2 and Sox1–3 genes (29–34). No binding sites for Pax6 or Sox proteins were found in the 652 bp deletion fragment by either MatInspector program or visual examination.
Over the past few years, significant advances have been made in understanding the biology of eye development and in unraveling its molecular basis (34–36). Studies of mouse ocular mutants were particularly useful for the identification of genes that control these diverse morphogenetic processes (6,36).
Development of the vertebrate eye is dependent upon the complex interaction and coordinated development of tissues deriving from surface ectoderm, neural ectoderm, neural crest and mesodermal mesenchyme (35,37,38). The eye field is one of the first regions to be defined in the anterior neural plate. This region evaginates from the forebrain during the neural folding process to become the optic vesicle, which contacts with the overlying ectoderm and induces it to develop into the lens placode, which, in turn, invaginates and separates from the ectoderm to form the lens vesicle. Structures of the anterior segment of the eye such as the corneal stroma bounded by an endothelium, iris, ciliary body, trabecular meshwork and sclera are developed upon separation of the lens vesicle from the corneal ectoderm and include large contributions from neural crest mesenchyme cells (39).
In the aphakia mice, the first lens abnormalities are seen at the late lens placode–early lens vesicle stage at days 10.5–11.0 of embryonic life (4,23). The lumen of the mutant lens vesicle fills up with cells released from the lens epithelium (4). Abnormal lens morphogenesis in ak mice results in a club-shaped elongated lens structure that remains attached to the surface epithelium by a persistent connecting stalk. Failure of the lens to separate from the cornea arrests the development of the anterior chamber, which is never formed in ak mice. In ak mice the primary and secondary lens fiber cells are not produced and thus the abnormal lens structure dissolves with time so that the lumen of the eye globe becomes filled with the folding retinal tissue.
By analysis of the ak/or and ak/fi compound mutants, it was suggested that the ak gene exerts its function exclusively in the lens (40). Several candidate genes were studied for their potential role in the ak phenotype including Pax2, Fgf8 and Chuk1 and were excluded either by linkage or sequence analysis (5). Previously, we mapped the homeodomain-containing transcription factor gene Pitx3 to the vicinity of ak, but failed to identify any mutations in the Pitx3 coding region by sequencing analysis of the ak genomic DNA (7) or RT–PCR analysis of its mRNA (J. Graw, unpublished results). Subsequently, the human PITX3 gene was found to be involved in anterior segment dysgenesis with cataracts in one family and dominant congenital cataracts in another (8).
In this study, we report an analysis of the entire sequence of the Pitx3 gene, which comprises 12.6 kb in genomic sequence. The Pitx3-containing region on mouse chromosome 19 was found to be extremely gene-rich as two other genes were identified in a partially overlapping tail-to-tail (Cig30) and head-to-head (Gbf1) orientation to the Pitx3 gene at a distance of –10 bp and +4.2 kb, respectively, from the known Pitx3 cDNA sequences. The GBF1 gene encodes a putative guanine nucleotide exchange factor that is likely to play a housekeeping role and is ubiquitously expressed in humans (10), while the Cig30 gene encodes the membrane glycoprotein, which appears to have a role in the recruitment of brown adipose tissue and shows a restricted expression pattern (11,12). Both GBF1 and Cig30 genes are expressed during embryonic development, as is the Pitx3 gene, therefore their transcripts may form RNA duplexes, if present in the same cell.
We identified several repetitive elements in the Pitx3 genomic sequence that enabled us to map the Pitx3 gene directly in the ak backcross. No recombination events were found between ak and Pitx3 in 416 animals, placing them at distance of 0.2 ± 0.2 cM. Additionally, Pitx3 expression was found to be largely diminished in ak/ak mice affecting all of the sites in which it is normally present. No expression was detected in the lens placode and lens pit or later in the lens remnants by in situ hybridization in the ak/ak animals. This led us to suggest that the etiologic mutation might be located in the 5′ region of the gene and that it affects a promoter or other regulatory elements. Such mutations were described for a variety of human and mouse phenotypes (41–44). Therefore we studied the Pitx3 promoter region in the ak/ak genomic DNA and identified a deletion of 652 nucleotides (Δ652) residing at a distance of 2.5 kb from the start of the Pitx3 cDNA. Multiple mouse strains were tested for the presence of this deletion including 129/Sv-SlJ and C57B6KS, which represent the background of this mutation. The original ak mutation occurred spontaneously in the 129/Sv-SlJ strain, which was later crossed to C57B6KS several times to obtain more viable mice (4) and was maintained as such in the Jackson Laboratory. None of the 10 mouse strains was found to have the deletion.
The deleted 652 bp fragment contains several sequences that are similar to binding sites for ocular transcription factors. Combining the sequence analysis of the deleted region with the Pitx3 expression data in the ak/ak mice, there is striking evidence for enhancer/promoter activity in this particular region. Several motifs similar to binding consensus sequences for AP-2 and Maf transcription factors were identified in this region. The AP-2 transcription factors belong to a family of retinoic acid-responsive proteins and were shown to be required for early morphogenesis of the lens vesicle (21). A pan-specific antibody that recognizes all three AP-2 proteins detected a strong signal in the developing lens starting from the lens placode stage and in neural-crest-derived mesenchymal cells around the developing eye (21). This expression pattern resembles that of the Pitx family homeodomain transcription factor genes—developing lens for Pitx3 and Pitx1 genes, and neural-crest-derived mesenchyme for Pitx2 and Pitx1—indicating that AP-2 transcription factors might be involved in regulation of the Pitx genes during ocular development. Moreover, the AP-2α-deficient mice exhibit ocular phenotypes ranging from anophthalmia to defects in the developing lens involving persistent adhesion of the lens to the overlying surface ectoderm, which is similar to the aphakia phenotype.
The Maf family genes encode transcription factors containing a basic region/leucine zipper domain; these proteins play an important role in lens development as supported by expression and mutant studies in mice (45–48). The L-maf products are first seen in the lens placode and are restricted to lens cells, which is similar to Pitx3 expression (47). Ectopic expression of L-maf converts chick embryonic ectodermal cells and cultured cells into lens fibers suggesting that this factor regulates the expression of multiple genes expressed in the lens. It was also recently shown that in the C-maf-deficient mice, lens fiber cells fail to elongate resulting in a small and hollow lens cavity and a microphthalmia phenotype (45). Both AP-2 and Maf proteins represent good candidates for upstream regulators of Pitx3 expression in the developing lens. Hence, removal of their binding sites from the Pitx3 promoter region in ak/ak mice, as a result of the Δ652 deletion, may be responsible for altered expression of Pitx3 in these animals. It would be interesting to investigate whether Pitx3 expression is altered in the AP-2- and Maf-deficient mice.
Also, putative binding sites were identified for proteins of the forkhead/winged-helix family of transcription factors in the deleted region (16). The forkhead genes serve as regulatory keys in embryogenesis (49); their role in ocular development has just started to be elucidated (50,51).
There are several mouse mutations that result in the formation of an abnormal lens vesicle that often remains attached to the corneal ectoderm leading to an overall disorganized eye development (3,6). In addition to the Ap-2-deficient and ak/ak mice mentioned above, this phenotype is also present in at least five other independent mutations in the mouse: dominant mouse mutations Small eye = Pax6 (52), Coloboma (53), Cat4a (54), recessive mutations dysgenetic lens (55) and lens aplasia (56,57). In humans, anterior segment dysgenesis and particularly Peters’ anomaly disorders resemble these mouse phenotypes. These disorders are characterized by corneal defects sometimes associated with keratolenticular adhesion, cataract or even aphakia (58–60). Both PITX3/Pitx3 and PAX6/Pax6 genes in mouse, which are involved in aphakia and small eye, respectively (this paper and ref. 61), were found to be involved in related disorders in humans (8,62), suggesting conservation of the gene function in ocular development. Identification of cellular partners of the Pitx3 gene may reveal genes responsible for similar murine phenotypes and may also lead to the isolation of human genes responsible for defects in the anterior segment of the eye and lens development.
In this paper we present several lines of evidence suggesting that abnormal ocular development in mouse aphakia is caused by a mutation in the promoter region of the Pitx3 gene. First, we show that the ak and Pitx3 genes are closely linked; second, we demonstrate that the expression of Pitx3 gene is altered in ak/ak mice; third, analysis of the promoter region of the Pitx3 gene identified a deletion in ak/ak DNA, which is not present in the parental strains of ak/ak as well as eight other mouse strains tested; and fourth, several sequences similar to consensus binding sites for AP-2 and Maf transcription factors that were shown to regulate a cascade of genes involved in lens development, were identified in the deleted region. It further implicates the Pitx family of homeodomain-containing transcription factors as important determinants of lens and anterior segment development in mammals (8,9,63–66).
This investigation also demonstrates that extended mutational analysis of an appropriate candidate gene may disclose etiologic mutations in regulatory regions well outside traditional gene boundaries. The mutation now provides a substrate for the investigation of the role of other factors in lens and anterior segment development.
MATERIALS AND METHODS
The ak/ak mice used in this study were obtained from the Jackson Laboratory and then housed by the Transgenic Core Facility at the University of Iowa, which employs an animal management program that is recommended by the American Association for Accreditation of Laboratory Animal Care and meets NIH guidelines regarding the care and use of laboratory animals. The original breeding pair, consisting of a heterozygous ak/+ female and homozygous ak/ak male, were bred with each other several times to obtain homozygous animals. They were outcrossed to C57BL/6 mice several times in order to obtain more viable animals and then crossed with each other to obtain ak/ak mice. The ak/ak embryos were collected at embryonic days 9–16 from ak/ak females impregnated by ak/ak males.
For histological analysis, embryos from a homozygous ak line were fixed in Carnoy solution and embedded in JB4 (Polysciences Europe, Eppelheim, Germany). Sections (2 µm) were cut and stained with 0.013% methylene blue and 0.035% basic fuchsin in 20% ethanol. Sections were analyzed under a Zeiss Axioplan microscope and documented with a high-resolution CCD camera (ProGres 3008; Carl Zeiss Jena, Jena, Germany). Pictures were adjusted for brightness, contrast and color balance in Adobe Photoshop 5.0.
Identification and analysis of genomic sequence
The BAC genomic clone carrying an insert that contained the entire Pitx3 gene was identified by screening the Mouse BAC Library (Research Genetics) clones for the presence of the Pitx3-specific product. The product was obtained by PCR-amplification of the library’s DNA using standard conditions (7) and specific primers that corresponded to the 5′ and 3′ regions of the gene: Pitx3-5′F, ctgcctgcgctccagaa; Pitx3-5′R, agtgcgtcccgcagcag; Pitx3-3′F, gcaggtctgtggatccatc; Pitx3-3′R, tgcgagggaaaaggccctct. The BAC DNA was isolated using a Qiagen plasmid DNA isolation kit following the manufacturer’s protocols (Qiagen). Sequencing of the introns and the 5′ and 3′ regions was performed automatically using an ABM Sequencer and Pitx3-specific primers were designed from the cDNA sequence. Contigs were assembled using Sequencer 3.0 software.
Female homozygous ak mice were mated to male JF1 mice and the heterozygous outcross animals were backcrossed to homozygous ak mice (5). The resulting backcross mice were classified for the absence or presence of the ak phenotype and genotyped for the Pitx3 polymorphic markers (see Table 1). Genomic DNA was isolated from tail clips of the backcross offspring. For comparison, genomic DNA was also isolated from wild-type AKR and JF1 mice. Hot-start PCR was performed as described previously (5). Additionally, in all cases 2 µl DMSO were added to the reaction mixture; final MgCl2 concentration and annealing temperature were as indicated in Table 1. Pitx3-CT products were analyzed using 15% polyacrylamide gel. Primers for PCR amplification of the markers D19Mit7, D19Mit8 and D19Mit102 were obtained from the NCBI homepage (http://www.ncbi.nlm.nih.gov/dbSTS/index.html ) and applied in the existing (ak/ak × JF1)F1 × ak/ak backcross (5). Annealing temperature of the markers D19Mit7 and D19Mit102 was 50°C, and 55°C for D19Mit8.
Expression studies: RT–PCR, northern blot and in situ hybridization analyses
Total RNA was prepared from embryos using the RNA extraction kit from Qiagen in accordance with the protocols supplied by the manufacturer. Total RNA (5 µg) was transcribed to the first strand of cDNA using Advantage RT-for-PCR kit from Clontech. PCR cycling after the RT step was performed with primers specific for the gene of interest and designed to flank the intron region in order to assure specific amplification of the cDNA. Products were analyzed by electrophoresis on a 1.5% agarose gel and, if needed, extracted from the gel using the gel-extraction kit from Qiagen and sequenced. Total RNA (15 µg) extracted from the day 15 wild-type and ak/ak embryos was denatured, separated on 1.2% formaldehyde gel and blotted onto Hybond N membrane (Amersham). To prepare a probe for hybridization, DNA insert containing nucleotides 30–716 of Pitx3 cDNA (7) was released from the plasmid vector by digestion with NotI and EcoRI, isolated by electrophoresis in agarose gels and subsequently purified using a column gel purification kit from Qiagen. DNA fragments were radioactively labeled with [32P]dCTP using random-primed DNA labeling kit (Boehringer Mannheim) and hybridized with the blot using conditions recommended by the manufacturer (Amersham). The in situhybridization on embryos and sections was performed as described previously (7).
Screening of the genomic sequence for mutations
Approximately 4 kb of the 5′ region of the Pitx3 gene was PCR amplified from the genomic DNA of wild-type, ak/+ and ak/ak mice using extended PCR conditions as described previously (9) and the following specific primers: Pitx3-5′-set1 (PCR product = 1140 bp), forward, aattcggcccgtgcaatctt, and reverse, taatcccagcacttgagagg; Pitx3-5′-set2 (PCR product = 1183 bp), forward, ctcgaactcaggtctgtctg, and reverse, gtgccctgatttgtcttcat; Pitx3-set3 (PCR product = 971 bp), forward, attagaggtcgttcaggatg, and reverse, taattgaggccttgggctct; Pitx3-set4 (PCR product = 701 bp), forward, aagacagacagcgacaagtg, and reverse, aaactccatggagggaggtc. The DNA fragments were prepared for automated sequencing by separation of the PCR products on 1% agarose gel, followed by extraction using a Gel Extraction kit (Qiagen) as described previously (9).
The following mouse strains were tested: AKR, JF1 (GSF, National Research Center for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany), C57B6J, Mus spretus, CAST, B33alpha, 129/Sv-SlJ, C57B6KS, DBA, B1 (University of Iowa).
The 5′ region sequence of Pitx3 gene was submitted to GenBank under accession no. AF224268.
We would like to thank Bonnie Ludwig for help with sequencing, Karmen Munson and Dawn Cady for animal care and Erika Bürkle for expert technical assistance during PCR-based linkage analysis. We are also grateful to B.A. Richards-Smith and D. Schroeder from the Jackson Laboratory for providing us with DNA samples and information about mouse strains used in this study. This work was supported by NIH-NEI grant EY12384 to J.C.M.
To whom correspondence should be addressed. Tel: +1 319 335 6566; Fax: +1 319 335 6970; Email: email@example.com
|Name||Position||Repeat unit||Forward primer||Reverse primer||Product size||Tm (°C)||MgCl2|
|Pitx3_2a||5′ region||(CT) (AC)||ttgaagccagcctgtgcttc||tactgtaactccagctccag||177||55||1.5|
|Name||Position||Repeat unit||Forward primer||Reverse primer||Product size||Tm (°C)||MgCl2|
|Pitx3_2a||5′ region||(CT) (AC)||ttgaagccagcctgtgcttc||tactgtaactccagctccag||177||55||1.5|
aHighly polymorphic markers.
aTm, annealing temperature; MgCl2, final concentration; nt, not tested.