Functional conservation of Pax6 regulatory elements in humans and mice demonstrated with a novel transgenic reporter mouse
© Tyas et al; licensee BioMed Central Ltd. 2006
Received: 08 February 2006
Accepted: 04 May 2006
Published: 04 May 2006
The Pax6 transcription factor is expressed during development in the eyes and in specific CNS regions, where it is essential for normal cell proliferation and differentiation. Mice lacking one or both copies of the Pax6 gene model closely humans with loss-of-function mutations in the PAX6 locus. The sequence of the Pax6/PAX6 protein is identical in mice and humans and previous studies have shown structural conservation of the gene's regulatory regions.
We generated a transgenic mouse expressing green fluorescent protein (GFP) and neomycin resistance under the control of the entire complement of human PAX6 regulatory elements using a modified yeast artificial chromosome (YAC). Expression of GFP was studied in embryos from 9.5 days on and was confined to cells known to express Pax6. GFP expression was sufficiently strong that expressing cells could be distinguished from non-expressing cells using flow cytometry.
This work demonstrates the functional conservation of the regulatory elements controlling Pax6/PAX6 expression in mice and humans. The transgene provides an excellent tool for studying the functions of different Pax6/PAX6 regulatory elements in controlling Pax6 expression in animals that are otherwise normal. It will allow the analysis and isolation of cells in which Pax6 is activated, irrespective of the status of the endogenous locus.
Pax6 is a transcription factor containing an N-terminal DNA binding domain, a paired domain, separated by a glycine-rich linker sequence from a second DNA binding domain, a homeodomain, and a C-terminal proline-serine-threonine-rich transregulatory domain. It is highly conserved in very diverse species. In mammals, it is expressed during development in the eye, in specific regions of the CNS, in the nasal placodes and olfactory epithelium and in the pancreas [1–3]. Haploinsufficiency for Pax6 function (Pax6+/-) in the mouse results in the Small eye (Sey) phenotype . Homozygotes (Pax6-/-) die perinatally with no eyes and many brain abnormalities [3–14]. PAX6 haploinsufficiency also causes eye and brain defects in humans [15, 16].
Normal development requires not only that Pax6 be present in certain cells at certain times but also that it be present in the correct amounts. Schedl et al.  showed that severe eye abnormalities are caused not only by under-expression but also by over-expression of Pax6 in mice. Bishop et al. [18, 19] and Muzio et al.  provided evidence that graded expression of Pax6 across the developing neocortex of mice is essential for the correct specification of its major areas. Such findings imply that Pax6 expression is tightly regulated and that different levels are maintained in different regions as they grow.
Initial characterization of transgenic mice
Y593 was successfully modified as illustrated in Fig. 1 and as described in Methods to generate Y1123. This YAC was used to generate transgenic mice. Three fertile transgenic founders, named DTy22, DTy42 and DTy54, were identified. They all appeared phenotypically normal and successfully transmitted a tau-GFP-expressing Y1123 transgene to their offspring. PCR with primers marked in Fig. 1 (sequences in Methods) was used to examine the minimum extent of incorporation of Y1123: the results indicated that only DTy54 had incorporated at least the majority of Y1123, whereas DTy22 and DTy42 had incorporated truncated versions. Neither DTy22 nor DTy42 recapitulated the full expression pattern of Pax6. It was possible to identify DTy54 and DTy42 transgenic mice using a hand-held torch emitting blue light, of the wavelength required to excite GFP, and an appropriate filter, as described in . This revealed GFP expression in the eyes of living DTy54 and DTy42 mice; the eyes of DTy22 mice did not express GFP. DTy42 mice expressed GFP only in the eyes. Using DTy54 mice, Y1123 was crossed into embryos that were either Pax6+/- or Pax6-/-. Unlike the unmodified YAC Y593 , Y1123 produced no rescue of either the eye or brain defects in these mutants, confirming its predicted lack of function.
Numbers of copies of integrated transgenes This was estimated from the average of the ratios between fluorescence intensities using primers for human PAX6 and 0.5 × the fluorescence intensities using primers for mouse Pax3 or Pax6 in qPCR reactions on a constant amount of DNA from each of a series of embryos. Each qPCR reaction was repeated three times on each animal.
Average ratios (± SD)
Numbers of animals
PAX6/0.5 × Pax3
PAX6/0.5 × Pax6
0.8 ± 0.2
0.9 ± 0.4
1.3 ± 0.5
1.0 ± 0.5
1.4 ± 0.5
1.2 ± 0.5
Expression of tau-GFP in DTy54 mice
Analysis of DTy54 brains with flow cytometry
In DTy54, the modified YAC that had integrated into the mouse genome did not affect the endogenous Pax6 locus, unlike an alternative strategy involving the insertion of a reporter gene into the endogenous locus . The YAC1123 transgene can be crossed onto mice with any Pax6 status (e.g. Pax6+/+, Pax6+/-, Pax6-/-, Pax6loxP/loxP) to identify and isolate those cells in which Pax6 is being activated by upstream factors. In addition to generating a useful new tool for understanding the role of Pax6, our results demonstrate that the elements regulating the human PAX6 gene present in Y1123 and Y593  are necessary and sufficient to recapitulate accurately the expression of Pax6 in mice. This indicates that these elements are not only structurally [24, 25] but also functionally highly conserved. In their original study of mice containing human PAX6-expressing YACs, Schedl et al.  suggested functional conservation of the regulatory elements controlling the human and mouse genes on the basis that the human locus is able to complement the Sey mutation in mouse. The introduction of PAX6-producing transgenes corrected the eye defects in heterozygotes and rescued homozygotes from perinatal death. It remained unclear, however, how accurately the human regulatory elements reproduce the pattern of endogenous mouse Pax6 expression. Although Y593 must have caused re-expression of the missing factor in those cells that normally express it, thereby rescuing their abnormal phenotypes, additional ectopic expression from Y593 might have gone undetected. Our current work complements that of Schedl et al.  by demonstrating a remarkable conservation of function of the Pax6/PAX6 regulatory elements in the two species.
This work provides further evidence that the Pax6/PAX6 regulatory elements are highly conserved not only structurally but also functionally in mice and humans. Y1123 provides an excellent tool for studying the functions of different Pax6/PAX6 regulatory elements and will allow the analysis and isolation of cells in which Pax6 is activated, irrespective of the status of the endogenous locus.
Generation of the DTy54 transgenic mouse
All work on mice followed current Home Office (UK) regulations stipulated in the Animals (Scientific Procedures) Act 1986. An overview of the strategy is illustrated in Fig. 1. We inserted a tau-GFP reporter cassette and a neomycin resistance cassette, linked by an internal ribosomal entry site (IRES), in frame into the translation start site in exon 4 of the PAX6 gene in YAC Y593  by homologous recombination using a yeast URA3 selectable marker. The manipulated YAC (named Y1123) was then used to generate transgenic mice.
Integration of YAC Y593 into yeast window strain W3
Before modifying the parental YAC Y593 with the reporter construct, it was introduced into a yeast window strain. This was necessary because Y593 co-migrates with similar sized endogenous yeast chromosomes in pulse field gel electrophoresis, making it difficult to isolate from the endogenous chromosomes. Each window strain contains defined alterations in its karyotype, which provide a large size interval, or window, devoid of endogenous chromosomes [32, 33]. Window strain W3 was mated with Y593 using the kar-cross method [34, 35]. Y593, in addition to the PAX6 gene locus, contains the genes allowing yeast cells to produce adenine and tryptophan. By removing adenine hemisulfate salt and tryptophan from the growth medium, it was possible to select for yeast colonies expressing these genes and, therefore, containing Y593.
Integration of reporter cassette into Y593 by homologous recombination
Once Y593 had been moved into the window strain it was transformed with the bacterial construct pDT-1 using modified lithium acetate yeast transformation. W3 has a defective URA3 gene and is unable to survive pyrimidine starvation. Since pDT-1 contained the yeast gene URA3 (Fig. 7), any cell harbouring Y593 into which pDT-1 had recombined survived pyrimidine starvation and was selected by the omission of uracil from the growth medium.
Several colonies were picked and screened for the likely presence of a complete pDT-1 using PCR for parts of pDT-1 distant from the URA3 gene and with Southern blots. Of 18 clones screened, one showed correct first round recombination. This clone was grown in the presence of 5-fluoroorotic acid (5-FOA), which prevents yeast cells containing the URA3 gene from growing and provides selection for removal of the URA3 gene by internal homologous recombination [38, 39]. Nine clones were picked from the 5-FOA plate, designated 1121 to 1129, and Southern blots were done to identify correct clones. One clone (1123) was identified as correct, giving a success rate of about 11%. PCR combined with restriction digests on some of the PCR products was used to confirm that the individual parts of the reporter cassette were present in the clone. The junction between PAX6 and tauGFP was checked by sequencing in both directions, confirming that the PAX6 ATG in exon 4 was followed immediately by tauGFP.
Microinjection of Y1123 and initial assessment of transgenic mice
Y1123 DNA was isolated for microinjection using alternating contour-clamped homogeneous electric field pulse field gel electrophoresis . Injected one-cell embryos (from crosses of C57Bl/6 and CBA mice) were either replaced immediately into pseudopregnant female mice or first cultured overnight until two-cell. About 5% of injected one-cell embryos were born. Subsequent breeding was such that all mice carrying Y1123 studied here were hemizygous for the transgene. Southern blotting with a full-length cDNA probe was used to confirm that the modified PAX6 coding region had integrated. PCR with primers shown in Fig. 1 was used to confirm the extent of incorporation of Y1123. The primer sequences were:
FISH on blood smears, with cosmid FAT5 as a probe (marked as CFAT5 in Fig. 1) and methods described in Schedl et al. , was used to search for possible multiple integration sites. Quantitative PCR (qPCR; QuantiTect SYBR Green PCR Kit, Qiagen) was used to identify the number of copies of Y1123 present in the genome, using three sets of primers, one specific for human PAX6, one specific for mouse Pax6 and one specific for mouse Pax3. The latter two sets were standards (detecting genes with copy numbers of 2) against which to compare the intensity of the product from Y1123. The three sets of primers were as follows: (i) human PAX6 specific primers (PAX6HumF CCGTGTGCCTCAACCGTA, PAX6HumR CACGGTTTACTGGGTCTGG); (ii) mouse Pax6 specific primers (Pax6MouF CGCAAATACACCTTTGCTCA, Pax6MouR GAGGGTTTCCTGGATCTGG); (iii) mouse Pax3 specific primers (Pax3MouF AAGCAGCGCAGGAGCAGAACC, Pax3MouR CCTCGGTAAGCTTCGCCCTCT). These three sets of primers allowed the amplification of sequences of similar length with similar reaction kinetics. Conditions were such that the fluorescence intensity was related linearly to the amount of starting DNA; once this had been established, 300 ng of DNA was used in each qPCR reaction. By comparing the fluorescence generated with sets two and three against the fluorescence generated when the same amount of DNA was used with set one, the number of PAX6 gene copies was calculated. Each reaction was repeated three times on each of a series of embryos.
Assessing the transgene's expression
Pregnant females were killed at various ages by cervical dislocation and embryos were fixed overnight in ice cold 4% paraformaldehyde and embedded in 4% low melting point agarose (the day of the vaginal plug was designated E0.5). Vibratome sections were cut at 200 μm, counterstained with TOPRO3 (Molecular Probes, NL), mounted on glass slides and imaged using a Leica confocal microscope. For immunohistochemistry, tissue was fixed overnight in 4% paraformaldehyde, transferred to 15% sucrose, embedded in 7.5% gelatin/15% sucrose in phosphate buffered saline and placed in 30% sucrose overnight. Cryostat sections (15 μm) were cut and transferred to 20% goat serum in phosphate buffered saline containing 0.1%Triton-X for 30 min at room temperature. Sections were incubated with mouse anti-Pax6 ascites (1:5000; Developmental Studies Hybridoma Bank) and rabbit anti-GFP antibody (1:10000; Abcam) overnight at 4°C and for 1 hr at room temperature the following day. Secondary antibodies were goat anti-mouse and goat anti-rabbit Alexa fluor 568 and 488 respectively (1:150; Molecular Probes), applied for 1 hr at room temperature.
Telencephalic tissue from E14.5 wild-type and DTy54 embryos were dissociated with papain (Papain Dissociation System, Worthington Biochemical). Cells in suspension were analysed on a Beckman-Coulter XL flow cytometer (10,000–20,000 cells were analysed per sample).
bacterial artificial chromosome
downstream regulatory region
fluorescent in situ hybridization
green fluorescent protein
internal ribosomal entry site
yeast artificial chromosome.
We thank Linda Wilson for help with confocal microscopy, Clare Huxley for help with isolation of YAC DNA, Anne Seawright for help with FISH analysis, and Manchester University for injecting the oocytes with the YAC. Financial support was from an MRC Scholarship (DT), an Edinburgh College of Medicine Scholarship (CC), and from MRC, BBSRC and Wellcome Trust grants to JM and DP.
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