|Molecular Vision 2005;
Received 15 July 2005 | Accepted 15 October 2005 | Published 26 October 2005
Intracorneal positioning of the lens in Pax6-GAL4/VP16 transgenic mice
Wilbur R. Harrison,2 Ningna Xiao,2 Dongcai Liang,2
Paul A. Overbeek2
1Cancer Center, Creighton University, Omaha, NE; 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX
Correspondence to: Venkatesh Govindarajan, 256 Criss III, Cancer Center, 2500 California Plaza, Creighton University, Omaha, NE, 68178; Phone: (402) 280-1819; FAX: (402) 280-3817; email: firstname.lastname@example.org
Purpose: The purpose of this study was to establish a GAL4/VP16-based binary transactivation system that was active in the lens and corneal epithelium of transgenic mice.
Methods: We generated transgenic mice with the transcriptional transactivator GAL4/VP16 driven by a modified Pax6 promoter that is active in lens and corneal epithelial cells. We also generated and tested UAS-lacZ reporter mice. Wild type and transgenic mice were analyzed by histological, in situ, and Southern hybridization techniques.
Results: Five families (OVE1931, OVE1934, OVE1935, OVE1936, and OVE1937) that carry the Pax6-GAL4/VP16 transgene were generated. Unexpectedly, mice from three of the transgenic lines showed ocular abnormalities. In the family OVE1936, cataracts were seen in the heterozygous mice at the time of eyelid opening and homozygotes showed microphthalmia. Transgenic mice in families OVE1931 and OVE1937 appeared normal. Histological analysis of ocular sections of OVE1934, OVE1935, and OVE1936 homozygous transgenic mice showed intracorneal positioning of the lens. The corneal stromal cells were disorganized and there was no distinctive corneal endothelial layer. In situ hybridizations showed robust expression of the GALVP16 transgene in the lens and corneal epithelial cells of the OVE1934, OVE1935, and OVE1936, but not in OVE1931 or OVE1937 families. Bigenic embryos generated by mating the Pax6-GAL4/VP16 mice to the UAS-lacZ mice showed that the GAL4/VP16 transgenic protein is functional and can induce eye-specific expression of a UAS-lacZ reporter gene.
Conclusions: Our data suggest that (1) expression the GAL4/VP16 transgene induces changes in gene expression in lens cells, (2) that developmentally important genes are affected, and (3) that bigenic phenotypes will need to be interpreted with caution.
Binary strategies using a transcriptional transactivator to regulate target gene expression are widely used in Drosophila  and have previously been shown to function in transgenic mice . The purpose of this study was to establish a binary transactivation system to activate and regulate expression of transgenes in the lens and cornea. As part of our research on the role of fibroblast growth factor (FGF) signaling in regulation of lens and corneal development, we had recently generated transgenic embryos that express FGF-4, a member of the FGF family, in these tissues using a modified version of the Pax6 promoter (described in detail below). These mice died at birth due to severe craniofacial defects (data not shown). To establish stable families to perform more detailed studies, we decided to establish a GAL4/VP16 bigenic system (more details below) to regulate transgene expression in ocular tissues. In addition, some of the promoters that were used to target transgene expression to the embryonic lens and corneal epithelial cells showed either mosaic or low levels of transgene expression. It was expected that a bigenic system would allow us to circumvent this problem. The rationale was that once we establish a stable transgenic family with desired expression of the transactivator, we should be able to use the transactivator to induce expression of many different transgenes with a reproducible timing and pattern of expression. An additional advantage of using a binary system is that there are versions of the GAL4 transactivator available that can be regulated by steroid hormone treatment allowing reversible regulation of target gene expression .
Binary systems for inducible gene expression usually include two components: (1) a transactivator component that consists of a transcription factor under the control of a tissue-specific promoter and (2) a responder component where the transgene of interest is linked to a minimal promoter containing cis-elements that are targeted by the transactivator. When bigenic mice are generated by breeding, expression of the transactivator activates tissue-specific expression of the responder. GAL4/VP16 and tetracycline-regulated systems are well-known examples of bigenic systems [4,5]. The transactivator component in the GAL4/VP16 system is a fusion protein containing the DNA binding domain of the yeast transcription factor GAL4 and the transactivation domain of herpes simplex virus (HSV) VP16 protein under the control of a tissue-specific promoter. The responder component contains the upstream activating sequence (UAS) that is recognized by the GAL4 transcription factor and the transgene of interest. The GAL4/VP16 system has been previously used successfully in mice , Drosophila , plants , medaka , and zebrafish . To target expression of GAL4/VP16 expression to ocular tissues, we used a version of the Pax6 promoter that was designed to be active in lens epithelial cells, corneal epithelial cells, and lacrimal glands [9,10]. Five transgenic lines that carried the Pax6-GAL4/VP16 transgene (Figure 1) were established. These mice were not expected to have a phenotype. Unexpectedly, some of the transgenic lines showed abnormal development of the lens and the cornea. By histological and molecular analyses we provide evidence that GAL4/VP16 expression is sufficient to alter the normal pathway of ocular development. We also established the responder component of the binary transactivation system by generating six transgenic lines that carried the UAS-lacZ transgene (Figure 1). Bigenic embryos generated by mating the Pax6-GAL4/VP16 mice to the UAS-lacZ mice showed induction of lacZ expression in the lens, cornea, and conjunctiva suggesting that the GAL4/VP16 transgenic protein is functional and can induce eye-specific expression of a UAS-lacZ reporter gene.
Construction of minigenes and generation of transgenic mice
The Pax6 promoter/enhancer was made by linking a 1.3 kb HincII-NsiI (blunt) enhancer (-4000 to -2700 bp relative to the P0 transcription initiation site) to a 1.0 kb XhoI (blunt)-BglII fragment which contains the Pax6 P0 promoter (-930 to +70). The HincII-NsiI fragment contains lens/corneal epithelial enhancer sequences [9,10]. This modified Pax6 promoter/enhancer does not contain the pancreatic enhancer sequences present in the promoters described previously [9,10]. The GAL4/VP16 cDNA (provided by Dr. Sophia Tsai, Baylor College of Medicine, Houston, TX) was digested with KpnI (blunt-ended) and HindIII, and then inserted into EcoRV and HindIII restriction sites between the Pax6 promoter and the small t intron/polyadenylation sequences of SV40 (Figure 1). The GAL4/VP16 fusion protein encodes 147 amino acids (including the ATG) from the amino terminus of GAL4 from Saccharomyces cerevisiae, followed by a 7 amino acid linker, then by 78 amino acids from the carboxy terminus of HSV-1 VP16 (including the stop codon) and 150 bp of 3' UTR. The Pax6-GAL4/VP16 transgene was then released from the vector backbone by SpeI/NotI digestion, gel purified using the QiaexII gel extraction kit (Qiagen, Hilden, Germany) and used for microinjections.
The UAS-lacZ construct was made as follows; a UAS-thymidine kinase (TK) element (265 bp) was isolated from a UAS-TK-CAT construct (provided by Dr. Sophia Tsai, Baylor College of Medicine, Houston, TX) by digestion with HindIII and XhoI (blunt-ended). This fragment contains 6 tandem copies of the 17 bp UAS linked to a minimal thymidine kinase promoter. The vector pKS hsp lacZ pA (from Dr. Janet Rossant, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada) was digested with HindIII and BstEII (blunt-ended) to remove the hsp sequences, then ligated to the UAS-TK fragment to generate UAS-lacZ. This vector contains a Kozak consensus sequence and NcoI site at the translation start for lacZ. The injection fragment (3.8 kb) was isolated by digestion with KpnI and NotI.
Transgenic mice were generated by microinjection into one-cell stage FVB/N embryos. Potential transgenic mice were screened by PCR for SV40 or lacZ sequences. Animals were handled following the guidelines provided in US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Homozygosity of transgenic mice was confirmed by breeding studies and Southern blots. Breeding studies were performed by mating potential homozygotes (identified by microphthalmia) to nontransgenic mice. If 100% of the offspring were transgenic then the transgenic parent was considered a homozygote. Transgene copy number was determined by Southern blots (more details below) and mice with twice the number of transgene copies relative to their littermates were deemed to be homozygotes.
Histological analyses were performed using standard histological techniques as described previously . Histological assays were performed on multiple sections of two different individuals from 2 different litters of each of the different transgenic lines. The figures show representative samples from each line.
In situ hybridization
To analyze transgene expression, 35S-UTP-labeled riboprobes specific to the SV40 or GAL4/VP16 sequences of the transgene were generated (Figure 1). Expression of endogenous p57Kip2 and Pax6 was analyzed using 35S-UTP-labeled riboprobes made from the corresponding mouse cDNAs (provided by Dr. Steve Elledge, Baylor College of Medicine, Houston and Dr. Kathleen Mahon, Baylor College of Medicine, Houston, respectively). In situ hybridizations on tissue sections were done as described previously . After hybridization and washing, slides were dipped in Kodak NTB-2 emulsion, dried and exposed for 3-7 days at 4 °C. Following development and fixation, the slides were counterstained with hematoxylin. In situ hybridization assays were performed on multiple sections of two different individuals from 2 different litters of the different transgenic lines. The figures show representative samples from each line.
Genomic DNAs (10 μg) were digested with BamHI and resolved on a 0.8% agarose gel. The gels were blotted and probed with an 800 bp BglII fragment from the Pax6 promoter. Southern hybridizations were performed following standard procedures . Membranes were washed, set up in a Phosphorimager cassette (Storm860, Amersham, Piscataway, NJ) and exposed overnight. Phosphorimager scans were quantified using the ImageQuaNT software (Amersham, Piscataway, NJ). Transgene copy number was determined by comparing the intensity of the transgene concatamer band (4 kb) to the endogenous Pax6 gene (2 kb) within the same lane and mice with twice the number of transgene copies relative to their littermates were considered homozygotes. Southern blots were performed twice with reproducible results.
Detection of β-galactosidase activity
Embryos or heads of embryos of GAL4/VP16 and UAS-lacZ monogenic or bigenic mice were collected at appropriate time points during development and fixed for 2 h at 4 °C in 0.1 M phosphate buffer containing 2% paraformaldehyde and 0.2% glutaraldehyde. Following fixation, the tissue samples were rinsed thrice at room temperature in 0.1 M phosphate buffer containing 0.01% sodium deoxycholate, 0.02% NP-40, 2 mM MgCl2, and stained overnight at 4 °C in an X-Gal substrate solution (0.01% sodium deoxycholate, 0.02% NP-40, 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml X-gal in 0.1 M phosphate buffer).
In order to establish a bigenic system that can be used to give reproducible transgene expression in epithelial cells of the embryonic lens and the cornea we started by linking the coding sequences of a GAL4/VP16 chimeric transactivation protein  to a modified version of the Pax6 promoter that contained the lens/cornea/lacrimal gland enhancer linked to the proximal P0 promoter (Figure 1). This modified Pax6 promoter/enhancer does not contain the pancreatic enhancer sequences present in the promoters described previously [9,10]. The Pax6-GAL4/VP16 transgene was microinjected and six transgenic founders were generated (Table 1). These founders were used to establish 5 transgenic lines (OVE1931, OVE1934-OVE1937).
Transgenic mice in families OVE1931 and OVE1937 appeared normal. However, mice from other transgenic lines (OVE1934, OVE1935, and OVE1936) showed ocular abnormalities. Heterozygous mice in families OVE1934 and OVE1935 had normal looking eyes but homozygous mice exhibited cataracts and severe microphthalmia (described in more detail below). Homozygosity was confirmed by breeding studies and/or Southern blots. In family OVE1936, cataracts and microphthalmia were visible in heterozygous mice at the time of eyelid opening and homozygous mice exhibited a facial dysmorphology (data not shown) in addition to severe microphthalmia.
In order to assay for changes in ocular development and morphology, sections of heads of OVE1934, OVE1935 and OVE1936 embryos were sectioned and stained with hematoxylin and eosin (Figure 2). Nontransgenic embryos were used as controls (Figure 2A,E,I). Ocular abnormalities were not detected in heterozygous OVE1934 and OVE1935 embryos (Figure 2B,F). By E15, OVE1936 heterozygotes showed changes in the lens epithelium (Figure 2J, arrows). Postnatally, the lenses of heterozygous OVE1936 mice showed ruptured lens capsules (Figure 2N, green arrows). Homozygous OVE1934, OVE1935, and OVE1936 embryos all showed a similar pattern of significant changes in the development of the anterior segment (Figure 2C,D,G,H,K,L). The embryonic lenses were smaller and found embedded within the corneal stroma (Figure 2C,G,K, green arrow). Lens epithelial cells were disorganized (Figure 2D,H,L,P, red arrow). Corneal stromal cells were disorganized and there was no evidence of a corneal endothelial layer in the prenatal (compare Figure 2D to Figure 2A, Figure 2H to Figure 2E, Figure 2L to Figure 2I) or postnatal (compare Figure 2P to Figure 2M) eye. The hyaloid vasculature (hv) was unorganized in the homozygote embryos (Figure 2C,G,K). In the OVE1936 homozygote adults, retinal architecture was significantly altered (compare Figure 2P to Figure 2M). A developmental analysis in family OVE1934 showed that lens pit formation was delayed at E10.5 in homozygous embryos (Figure 3) and lens development was significantly altered by E12.5 (data not shown).
Because the ocular phenotypes in OVE1934 and OVE1935 were recessive, we considered the possibility that they were caused by random insertional mutations in endogenous genes that were needed for normal eye development. However, since three of the transgenic families (OVE1934-OVE1936) showed similar ocular changes with no evidence for pathological or developmental changes in any other tissues (data not shown), we felt that the defects were caused by the presence of the transgene rather than by random mutations. One hypothesis was that the ocular defects were caused by expression of the GAL4/VP16 transgene. However, another possible explanation was that the transgenic Pax6 regulatory sequences were present at such a high copy number that they were binding to and sequestering the transcription factors that normally regulate endogenous Pax6 expression. In order to assess this possibility, we first determined the transgene copy number by performing Southern blots (Figure 4). Genomic DNAs were digested with BamHI and hybridized to a probe (Figure 1) generated from the Pax6 promoter of the transgene. Transgene copy number was estimated by comparison of the intensity of the transgenic concatamer band (Figure 4, arrow) to the intensity of the endogenous Pax6 signal (Figure 4, asterisk) within the same lane. The highest copy number (29 copies) was found in family OVE1931. The 3 families with phenotypes (OVE1934, OVE1935, OVE1936) showed a relatively low copy number (3-6 copies). Family OVE1937 had a single copy. The Southern hybridization results also demonstrate that each family has a different transgene integration pattern.
In order to assay for changes in expression of the endogenous Pax6 gene, in situ hybridizations were performed using a 35S-labeled Pax6 specific riboprobe (Figure 5). The pattern and level of Pax6 expression was unaltered in the embryos of the OVE1931, OVE1934, OVE1935, and OVE1936 families compared to the nontransgenic embryos (Figure 5). Therefore endogenous Pax6 expression was not squelched by the Pax6-GAL4/VP16 transgenes. For comparison, we have targeted expression of 13 different transgenes (including transcription factors, secreted growth factors, and growth factor receptors) to the lens and cornea using the same Pax6 promoter and have generated more than 50 different transgenic lines (data not shown). These transgenic mice neither show a phenotype comparable to the Pax6-GAL4/VP16 transgenic mice nor do they show ocular abnormalities that reflect reduced levels of endogenous Pax6 expression (data not shown). All these results considered together suggest that the Pax6 promoter that we used for our studies does not squelch endogenous Pax6 gene expression. Therefore, we predicted that the ocular phenotype was caused by expression of the GAL4/VP16 transgene and consequently, families OVE1934, OVE1935, and OVE1936 would exhibit higher levels of transgene expression.
In order to compare the levels of expression, in situ hybridizations were performed using 35S-labeled riboprobes that hybridize to the SV40 polyA sequences (Figure 3A-D,F-J) or the GAL4/VP16 sequences of the transgene (Figure 3E). No transgene expression was detected in the lens or corneal epithelial cells of the OVE1931 and OVE1937 embryos (Figure 3B,H). Expression of the transgene was seen in retinal neuroblasts of all families except OVE1937, with the family OVE1935 having the highest level of expression. In family OVE1936, ectopic expression of the transgene was also detected in the periocular mesenchymal cells (Figure 3G) and in the neural tube (data not shown). Probes that hybridize to the SV40 polyA sequences or GAL4 sequences showed the same hybridization pattern (Figure 3D,E). In order to assess transgene expression at the onset of the appearance of the ocular defects, in situ hybridizations were performed at E10.5 for the family OVE1934. The homozygous embryo showed a higher level of transgene expression in addition to the delayed morphogenesis of the lens pit (Figure 3I,J).
The lenses of homozygous mice in the OVE1934 and OVE1935 families were smaller and contained fewer fiber cells. To examine if there was a delay in the initiation of lens fiber differentiation, expression of p57Kip2 was examined by in situ hybridization. Expression of p57Kip2, a cyclin dependent kinase inhibitor, is normally upregulated at the transition zone (Figure 6A,B, red arrowheads) and has been shown to be critical for cell cycle exit during the initiation of fiber differentiation . In homozygous OVE1934 and OVE1935 embryos, p57 expression was seen more posteriorly in the lens (Figure 6C,F,I,J) probably due to the anterior displacement of the lens relative to the differentiation signal from the retina. In Figure 6E, it can be seen that the lens stalk is still intact and the lens epithelial cells are contiguous with corneal epithelial cells.
In order to assess whether the GAL4/VP16 protein was functional and to establish the responder component of the binary transactivation system, we linked 6 tandem copies of the UAS, along with a minimal TK promoter to lacZ (Figure 1). This UAS-lacZ transgene was microinjected and 6 transgenic founders were generated (OVE1972, 1973, 1974, 1975, 1976, and 1977). In the families OVE1973, 1974, 1975, and 1976, evidence for lacZ expression was seen even in the absence of the GAL4-VP16 transactivator (data not shown). Bigenic embryos generated by mating these families to OVE1934 or OVE1936 mice showed induction of lacZ expression in the lens, cornea, and conjunctiva as detected by staining with X-gal (data not shown). Embryos in family 1977 showed no detectable staining with X-gal when they were monogenic, but bigenic embryos had blue eyes (Figure 7A-D). These bigenic embryos showed induction of lacZ expression in the lens (OVE1934 and OVE1936) and retina (OVE1931 and OVE1936) as detected by staining with X-Gal (Figure 7B-G). Family 1972 showed no evidence for lacZ expression, even in bigenic mice (data not shown). These results suggest that the GAL4-VP16 transgenic protein is functional and can induce eye-specific expression of a UAS-lacZ reporter gene.
Bigenic systems for regulating transgene expression in murine tissues present several advantages. However, for proper function the following conditions should be fulfilled : (1) the transactivator must regulate only the responder/target transgene and should not, by itself, alter expression of endogenous genes, (2) target transgene(s) must remain silent in the absence of the transactivator (i.e., cis-elements in the responder must not be recognized by mammalian transcription factors), and (3) target transgenes must be adequately induced by expression of the transactivator. The results of our study suggest that the GAL4/VP16 transactivator by itself is sufficient to alter ocular development and therefore, phenotypes of bigenic offspring generated using this transactivator need to be interpreted with caution.
Five transgenic families were generated with a Pax6-GAL4/VP16 transgene. Changes in ocular development were seen in three of the families. We considered three possible explanations for the alterations in lens and corneal development: (1) random insertional inactivation of genes essential for normal development of the lens and the cornea, (2) reduced endogenous Pax6 expression caused by squelching of the endogenous Pax6 promoter, or (3) direct effects of transgene expression. The first possibility is unlikely since similar changes were seen in transgenic families with different transgene integration sites (as confirmed by Southern hybridization results, Figure 4). The second model is also not supported by the data. The transgenic mice with the highest copy number (OVE1931) do not show altered ocular development. In addition, in situ hybridization studies show that Pax6 expression is unaffected in the three lines (OVE1934, OVE1935, and OVE1936) that show altered differentiation of ocular tissues. Furthermore, other transgenic families that were generated using the same Pax6 promoter do not show a comparable phenotype. The results of our study support, instead, the viewpoint that the interesting changes in ocular development are a result of GAL4/VP16 expression. We show that there is a correlation between transgene expression and ocular abnormalities in the different transgenic lines. The lines with transgene expression in the lens and cornea (OVE1934, OVE1935, and OVE1936) show an ocular phenotype whereas the lines with no expression in the lens (OVE1931 and OVE1937) do not show altered ocular development. In addition, the phenotype of the GAL4/VP16 transgenic mice does not match that of any known (or published accounts of) Pax6 mutants [15-21].
In four of the five transgenic lines, transgene expression was seen in a subset of neuroblasts in the retina. However, embryonic retinal development did not appear to be morphologically altered as a result of GAL4/VP16 expression. We have not assayed adult retinas for changes in terminal differentiation. Since expression of the transgene was detected in lens, cornea, and retinal cells in each of the affected families, it is not clear whether the phenotype reflects cell-autonomous consequences of GAL4-VP16 expression.
Though the identities of GAL4/VP16 targets in the lens and cornea are not yet clear, it appears that the phenotype requires a threshold level of GAL4/VP16 expression. In transgenic lines OVE1934 and OVE1935, development of the lens and the cornea was affected only in homozygous mice. In the transgenic line OVE1936, the lens was affected in both hetero- and homozygotes. The homozygotes showed more profound alterations in ocular development. Although the levels of GAL4/VP16 transgenic protein expression have not been examined in these lines, it is possible that there is a threshold for target gene activation by GAL4/VP16 and that this threshold is exceeded only in the homozygotes. These results are consistent with other studies that provide evidence for dose dependent intolerance to VP16 expression in pre-implantation mouse embryos  and dose-dependent toxicity of GAL4/VP16 in yeast and medaka fish [7,23].
The mechanism by which GAL4/VP16 expression interferes with lens morphogenesis is unclear, but is likely to be novel and therefore, informative. One possibility is that GAL4 binds to sequences in the mouse genome that lie in or near genes that can affect lens development. This serendipitous binding may lead to ectopic expression of genes that are not normally expressed in the lens or in the cornea. Such ectopic expression may then cause the ocular phenotype. Alternatively, GAL4/VP16 may bind to and alter the activity of endogenous transcription factors that are needed for normal eye development. These factors might control chromatin configuration or polymerase activity. Alternatively, VP16 may bind to lens-specific regulatory proteins such as FoxE3 or Pitx3. Although not within the realm of the experiments described in this manuscript, the transgenic mice could be used for co-immunoprecipitation experiments to determine which proteins of the lens are binding to VP16. In the future, both Gal4 and VP16 should be expressed individually in ocular tissues to determine whether one or the other, or the combination of the two, is responsible for the unique and interesting alteration in lens morphogenesis. Previous experiments have shown that the VP16 activation domain can interact with proteins that are critical for basal transcription such as TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex . VP16 has also been shown to interact with host cell factor (HCF), a chromatin-associated protein that has been implicated in regulation of multiple aspects of cell proliferation . Altered function of any or all of these factors could explain, at least in part, altered differentiation of the lens and corneal epithelial cells.
In conclusion, our studies provide evidence that lens detachment from the surface ectoderm and ocular development are significantly altered by expression of the GAL4/VP16 transactivator. We predict that the VP16 transactivation domain in some fashion alters expression and/or function of genes that are critical for lens and corneal development. Though some of the GAL4/VP16 mice show altered ocular development, the heterozygotes in lines OVE1934 and OVE1935 are likely to be useful for bigenic regulation of target transgene expression. The homozygotes in lines OVE1934, OVE1935, and OVE1936 provide new models for studies of ocular development and morphogenesis.
The authors thank Dr. Sophia Tsai (Baylor College of Medicine, Houston) for GAL4/VP16 and UAS-Tk cDNAs, Dr. Janet Rossant (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada) for the pKS hsp lacZ pA plasmid, Drs. Fred Pereira and Steve Chua for insightful comments and discussion and Dr. Patrick Swanson for help in using the phosphorimager. Supported by NEI grants EY10803 and EY10448 (PAO) and revenue from Nebraska cigarette taxes and Nebraska Tobacco Settlement Biomedical Research Development Fund awarded to Creighton University by the Nebraska Department of Health and Human Services (VG).
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