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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Genesis. 2012 May 14;50(10):766–774. doi: 10.1002/dvg.22036

Generation of conditional alleles for Foxc1 and Foxc2 in mice

Amy Sasman 1, Carey Nassano-Miller 1, Kyoo Seok Shim 1, Hyun Young Koo 1, Ting Liu 1, Kathryn M Schultz 1, Meredith Millay 1, Atsushi Nanano 2, Myengmo Kang 2, Takashi Suzuki 2, Tsutomu Kume 1,*
PMCID: PMC3435482  NIHMSID: NIHMS375794  PMID: 22522965

Abstract

The Forkhead box transcription factors Foxc1 and Foxc2 are crucial for development of the eye, cardiovascular network, and other physiological systems, but their cell type–specific and post-developmental functions are unknown, in part because conventional (i.e., whole-organism) homozygous-null mutations of either factor result in perinatal death. Here, we describe the generation of mice with conditional-null Foxc1flox and Foxc2flox mutations that are induced via Cre-mediated recombination. Mice homozygous for the unrecombined alleles are viable and fertile, indicating that the conditional alleles retain their wild-type function. The embryos of Foxc1flox or Foxc2flox mice crossed with Cre-deleter mice that are homozygous for the recombined allele (i.e., Foxc1Δ/Δ or Foxc2Δ/Δ embryos) lack expression of the corresponding gene and show the same developmental defects observed in conventional homozygous mutant embryos. We expect these conditional mutations to enable characterization of the cell-type specific functions of Foxc1 and Foxc2 in development, disease, and adult animals.

Keywords: conditional knockout, gene targeting, Cre recombination, Foxc1, Foxc2

Introduction

FoxC1 and FoxC2 are closely related members of the Fox (Forkhead box) family of transcription factors (Benayoun et al., 2011; Hannenhalli and Kaestner, 2009). Mutations of human FOXC1 are associated with Axenfeld-Rieger anomaly (ARA), a dominantly inherited syndrome characterized by dysgenesis of the anterior chamber in the eye and congenital glaucoma [Online Mendelian Inheritance in Man (OMIM) no. 601090] (Mears et al., 1998; Nishimura et al., 1998), and human FOXC2 mutations lead to the autosomal dominant syndrome Lymphedema-distichiasis (LD), which is characterized by the obstruction of lymphatic drainage in the limbs and the growth of an extra set of eyelashes (OMIM no. 602402) (Fang et al., 2000; Petrova et al., 2004; Tammela et al., 2005). There is also evidence that some humans heterozygous for mutations in FOXC1 or FOXC2 have congenital heart defects (Fang et al., 2000; Maclean et al., 2005; Winnier et al., 1999), whereas FoxC2 is implicated in tumor angiogenesis and metastasis (Kume, 2012; Mani et al., 2007; Sano et al., 2010).

In mice, Foxc1 and Foxc2 expression largely overlap in many embryonic tissues derived from the non-axial (i.e., paraxial, intermediate, and lateral plate) mesoderm (Hiemisch et al., 1998; Iida et al., 1997; Kume et al., 2000b; Kume et al., 1998; Kume et al., 2001; Sasaki and Hogan, 1993; Seo et al., 2006; Seo and Kume, 2006; Winnier et al., 1997; Winnier et al., 1999). Foxc1 and Foxc2 are also expressed in neural-crest derivatives, including cells in the pharyngeal arches, the endocardial cushions of the cardiac outflow tract, the meninges and the periocular mesenchyme (Gage et al., 2005; Gitler et al., 2004; Kume et al., 1998; Seo et al., 2012; Siegenthaler et al., 2009; Winnier et al., 1999) Mice that are homozygous for either a spontaneous mutation in Foxc1 (congenital hydrocephalus, Foxc1ch) or an engineered null mutation (Foxc1lacZ) die prenatally or perinatally with identical phenotypic abnormalities (Gruneberg, 1943; Hong et al., 1999; Kume et al., 1998), including hemorrhagic hydrocephalus and multiple skeletal, ocular, genitourinary and cardiovascular defects (Kume, 2009). Similar skeletal, genitourinary, and cardiovascular defects, as well as pre- and perinatal death, occur in Foxc2-null mutant mice (Iida et al., 1997; Kume et al., 2000b; Kume et al., 2001; Takemoto et al., 2006; Winnier et al., 1997; Winnier et al., 1999).

Heterozygous Foxc1- (Foxc1+/−) and Foxc2- (Foxc2+/−) mice survive to adulthood, but most of the compound heterozygous offspring from crosses of Foxc1+/− and Foxc2+/− mice (i.e., Foxc1; Foxc2 heterozygotes) die pre- or perinatally with cardiovascular and genitourinary abnormalities similar to those associated with each individual homozygous-null mutation (Kume et al., 2000b; Kume et al., 2001; Seo et al., 2006; Seo and Kume, 2006; Smith et al., 2000; Wilm et al., 2004; Winnier et al., 1999). Furthermore, our group has shown that the severity of the mutant phenotype, including the abnormalities in blood- and lymphatic-vessel formation, is dependent on the cumulative “dose” of functional Foxc1 and Foxc2 genes; abnormalities are more extensive in Foxc1−/−; Foxc2−/− mice than in Foxc1+/−; Foxc2−/− mice or Foxc1−/−; Foxc2+/− mice (Seo et al., 2006; Seo and Kume, 2006). Thus, Foxc1 and Foxc2 functionally overlap and cooperatively regulate many aspects of embryonic development in a gene-dose dependent manner (Kume, 2009, 2010). However, these experiments were performed with conventional (i.e., global) knockout mice, so the cell type–specific functions of Foxc1 and Foxc2 expression remain to be elucidated. Mice carrying a conditional Foxc knockout mutation, crossed with (inducible) Cre mice, may also live longer than mice with conventional Foxc mutations and, consequently, could be analyzed in postnatal development and disease. For these reasons, we generated lines of mice with conditionally mutant alleles of these genes. These mouse lines will be useful to define the particular roles of Foxc1 and Foxc2 in specific cell lineages during embryonic development as well as in various pathological conditions in adults.

Results and Discussion

The entire coding region of Foxc1 is located within a single exon and contains no introns. To generate the conditional Foxc1-null (Foxc1flx) allele in mice, a genomic DNA fragment containing the coding exon of Foxc1 was isolated from the RPCI-22 129S6/SvEvTac mouse BAC library; loxP sequences were inserted into the 5’ upstream region and the 3’ untranslated region (UTR) to flank the coding region, and an frt-flanked neor expression cassette was inserted immediately upstream of the 5’ loxP sequence to enable positive selection (Fig. 1a). The resulting target vector was transfected into TL1 mouse embryonic stem (ES) cells by electroporation; then, the ES cell colonies were selected with G418 and gancyclovior, screened via southern blot hybridization with the 5’ probe (Fig. 1b), and verified via rehybridization with the 3’ probe (data not shown). Chimeric mice were generated by microinjecting Foxc1flx-neo/+ ES cells (Fig. 1b) into C57BL/6 blastocysts, and germline transmission was verified via obtaining F1 Foxc1flx-neo/+ offspring from crossing chimeric and Black Swiss mice. The F1 Foxc1flx-neo/+ mice were crossed with FLPe mice (Rodriguez et al., 2000) to remove the neor cassette in the germ line; then, the Foxc1flx/+ offspring were interbred, and F2 mice were genotyped via PCR after weaning (Fig. 1c). The homozygous Foxc1flx/flx mice appeared normal, were fertile, and were born in the expected Mendelian ratio.

Fig. 1.

Fig. 1

Fig. 1

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Generation of the conditional Foxc1flx allele in mice. (a) The Foxc1 gene (top) contains a single protein-coding exon (black box) flanked by 5’ and 3’ UTRs (open boxes). Arrowheads identify the locations of the primers used for PCR genotyping. The 5’ and 3’ probes are indicated by bars. B, BamHI; Bg, BglII; K, KpnI; Xb, XbaI. (b) Southern blot analysis of ES cell clones with the 5’ probe after BglI digestion. (c) PCR genotyping of a representative offspring from interbreeding between two Foxc1flx/+ mice; the wild-type and Foxc1flx alleles produce 230-bp and 330-bp PCR products, respectively. (d) PCR genotyping of three offspring from breeding between Foxc1flx/+ mice and EIIa-Cre mice. The Foxc1Δ allele produces a 500-bp PCR product, and the asterisk indicates partial deletion of the Foxc1flx allele.

To verify that expression of the Foxc1-null allele could be induced via Cre-mediated recombination, Foxc1flx/+ mice were crossed with EIIa-Cre mice (Lakso et al., 1996), which carry a Cre transgene that induces the expression of Cre recombinase in the germ cells. The Foxc1+/Δ offspring of this cross (Fig. 1d) were interbred, and Foxc1Δ/Δ embryos were examined to confirm that Foxc1 expression was completely abolished (Fig. 2a). This observation is consistent with previously published results from our group, in which tissue-specific deletion of Foxc1 in vascular endothelial cells was achieved by crossing Foxc1flx mice with endothelial Tie2-Cre mice (Hayashi and Kume, 2008; Seo et al., 2012). The Foxc1Δ/Δ mutant embryos also displayed the hydrocephalic and abnormal ocular phenotypes (Fig. 2b-iii, iv) observed in conventional Foxc1-null mutant embryos (Kidson et al., 1999; Kume et al., 1998).

Fig. 2.

Fig. 2

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Analysis of conditional Foxc1Δ/Δ embryos at E15.5. (a) Quantitative RT-PCR analysis of Foxc1 expression in wild-type (WT) and conditional Foxc1Δ/Δ embryos. (b) Images of (i, ii) WT and (iii, iv) conditional Foxc1Δ/Δ embryos. Arrows identify (iii) the enlarged cerebral hemisphere (hydrocephalus) and (iv) abnormally thickened cornea observed in conditional Foxc1Δ/Δ embryos. The conditional Foxc1Δ/Δ embryo also (iv) lacked an anterior chamber in the eye. ac, anterior chamber. Scale bar = 100 µm.

Conditional Foxc2-null mice were generated in a similar manner. Like Foxc1, Foxc2 is coded within a single exon and contains no introns. A targeting vector containing the conditional Foxc2flx allele and a neor expression cassette (Fig. 3a) was generated and transfected into mouse ES cells; then, ES cell colonies were selected and screened via Southern blot hybridization (Fig. 3b), and Foxc2flx-neo/+ ES cells were injected into blastocysts to generate chimeric mice with germ-line Foxc2flx-neo expression. The chimeric mice were crossed with Black Swiss mice to obtain Foxc2flx-neo/+ F1 mice, which were subsequently bred with FLPe mice to generate Foxc2flx/+ mice, and then the Foxc2flx/+ mice were interbred (Fig. 3c). Homozygous Foxc2flx/flx mice were born at the expected Mendelian ratio and appeared normal. Foxc2flx/+ mice were bred with EIIa-Cre mice to produce Foxc2+/Δ mice (Fig. 3d); then, the Foxc2+/Δ mice were interbred, and the lack of Foxc2 expression in Foxc2Δ/Δ embryos was confirmed (Fig. 4a). The Foxc2Δ/Δ embryos also had cardiac defects, such as ventricular septal defects and thinner myocardium (Fig. 4b-iii, iv), similar to those reported in conventional Foxc2−/− mutant embryos (Iida et al., 1997; Winnier et al., 1999).

Fig. 3.

Fig. 3

Fig. 3

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Generation of the conditional Foxc2flx allele in mice. (a) The Foxc2 gene (top) contains a single protein-coding exon (black box) flanked by 5’ and 3’ UTRs (open boxes). Arrowheads identify the locations of primers used for PCR genotyping. The probe is indicated by a bar. H, HindIII, M, MfeI, Nd, NdeI, Nh, NheI; Xb, XbaI. (b) Southern blot analysis of ES cell clones with the probe after XbaI digestion. (c) PCR genotyping of a representative offspring from interbreeding between two Foxc2flx/+ mice. The wild-type and Foxc2flx alleles give 300 bp and 400 bp PCR products, respectively. (d) PCR genotyping of three offspring from breeding between Foxc2flx/+ mice and EIIa-Cre mice. The Foxc2Δ allele gives a 505-bp PCR product, and the asterisk indicates partial deletion of the Foxc2flx allele.

Fig. 4.

Fig. 4

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Analysis of conditional Foxc2Δ/Δ embryos at E13.5. (a) Quantitative RT-PCR analysis of Foxc2 expression in wild-type (WT) and conditional Foxc2Δ/Δ embryos. (b) Images of (i, ii) WT and (iii, iv) conditional Foxc2Δ/Δ embryos. (ii, iv) show the high power image of the regions indicated in i and iii, respectively. A ventricular septal defect (arrow in iii) and abnormally thin myocardium (double arrows in iv) were observed in conditional Foxc2Δ/Δ mutant embryos. Scale bars = 500 µm in B and 100 µm in C.

To determine where Foxc1 and Foxc2 are expressed in the adult, we isolated CD31+ endothelial cells from the kidney, heart, and lung of adult wild-type mice and performed real-time RT-PCR analysis (Fig. 5). Both Foxc1 and Foxc2 were highly expressed in CD31+ vascular endothelial cells in all three organs, particularly the heart. To a lesser extent, they were also expressed in CD31- cell populations, which could include fibroblasts and other types of nonvascular cells. Based on these results, the newly generated conditional Foxcflx mice can be used to analyze the postnatal role of Foxc1 and Foxc2 in vascular endothelial cells by crossing these mice with tamoxifen-inducible Cre mouse lines (Gothert et al., 2004; Wang et al., 2010).

Fig. 5.

Fig. 5

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Foxc1 and Foxc2 gene expression in endothelial cells. Real-time RT-PCR was performed to determine the levels of Foxc1 and Foxc2 in CD31+ endothelial cells isolated from mouse kidney, heart, and lung. Gene expression is relative to 18S rRNA. Values are mean ± SD of three independent experiments.

Collectively, the results reported here demonstrate that we have successfully generated conditional Foxc1-null and Foxc2-null mutant mouse lines, that the Foxc1flx and Foxc2flx alleles retain the function of their corresponding wild-type genes, and that the inactive Foxc1-null and Foxc2-null alleles can be induced via Cre-mediated recombination. When used with currently available techniques for restricting Cre expression to individual cell lineages or various stages of development, we expect these conditional Foxc mutant lines to enable characterization of the tissue-type and cell-type-specific functions of Foxc1 and Foxc2 in many developmental processes and disease, as well as the function of Foxc1 and Foxc2 in adult animals. Therefore, our conditional mutant mouse lines will be a powerful tool in elucidating the mechanisms by which Foxc1 and Foxc2 function in various embryonic and pathological processes.

Materials and Methods

Mice

EIIa-Cre (Stock 003724) (Lakso et al., 1996) and Flpe deleter (Stock 005703) (Rodriguez et al., 2000) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained under pathogen-free conditions as approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All animal procedures were reviewed and approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC) and comply with all applicable laws and guidelines.

Foxc1 and Foxc2 targeting vectors

Clones containing genomic DNA of the Foxc1 and Foxc2 locus were obtained from a 129S6/SvEvTac BAC library (RPCI-22) (BACPAC Resources, Children’s Hospital Oakland, CA), and loxP sites were inserted into the 5’ upstream and 3’ downstream regions. For the Foxc1 vector, a neomycin resistance cassette (neor) was placed 5′ to the first loxP site, and a Herpes Simplex Virus-thymidine kinase cassette (tk) was placed at the end of the 5’ homology region; for the Foxc2 vector, the selection cassettes were placed in the 3’ downstream region (neor) and at the end of the 3’ homology region (tk). The neor cassettes were flanked by two FRT sites to facilitate deletion by Flp recombinase. The resulting vectors were verified by sequencing.

Targeted ES cells and generation of chimeric mice

Linearized targeting vectors (100 µg) for Foxc1flx-neo or Foxc2flx-neo were electroporated into TL1 ES cells (129S6), and then successfully transfected cells were selected via resistance to G418 and gancyclovir. Double-resistant ES cell colonies were screened by Southern blot analysis as described previously (Kume et al., 2000a; Kume et al., 1998); one clone containing the Foxc1flx-neo-targeted allele was identified out of 350 screened clones, and two clones containing the Foxc2flx-neo-targeted allele were identified out of 251 screened clones. To confirm the correct targeting of the homologous regions, the Foxc1 and Foxc1flx-neo alleles were detected by using a 5′ probe and a 3’ probe after BglII digestion; the Foxc1 allele was detected as 8.5-kb (5’) and 12.5-kb (3’) bands, and the Foxc1flx-neo allele was detected as 5.2-kb (5’) and 16-kb (3’) bands. The Foxc2 and Foxc2flx-neo alleles were detected by using a probe after XbaI digestion and after MfeI/NdeI digestion; the Foxc2 allele was detected as 12.6-kb (5’) and 7.6-kb (3’) bands, and the Foxc2flx-neo allele was detected as 7.3-kb (5’) and 14.8-kb (3’) bands.

Targeted ES cells for Foxc1flx-neo or Foxc2flx-neo were injected into host (C57BL/6) blastocysts and produced germline chimeras. Chimeric male mice were mated with Black Swiss (Taconic) females and the offspring were genotyped by PCR with the primers described below; then, the Foxc1flx-neo/+ or Foxc2flx-neo/+ F1 mice (on a mixed, 129 × Black Swiss, genetic background) were bred with Flpe deleter mice to generate conditional Foxc1flx/+ or Foxc2flx/+ mice.

Genotyping

Embryonic tissues or tail tips were lysed by proteinase K (Roche), diluted in TE (10:1) buffer for 2 hours at 55 °C; then, the proteinase was inactivated at 94 °C for 15 min. The conditional-mutant mice were genotyped by performing PCR with primers for Foxc1flx (c1-F1, 5’-ATTTTTTTTCCCCCTACAGCG-3’; c1-R1, 5’-ATCTGTTAGTATCTCCGGGTA-3’) or Foxc2flx (c2-F1, 5’-CTCCTTTGCGTTTCCAGTGA-3’; c2-R1, 5’-ATTGGTCCTTCGTCTTCGCT-3’), and the Cre-mediated deletion of Foxc1 (Foxc1Δ) or Foxc2 (Foxc2Δ) was confirmed by performing PCR with primers for Foxc1Δ (c1-F2, 5’-ACTGCTGACTCTGGTACTTC-3’; c1-R1, 5’-ATCTGTTAGTATCTCCGGGTA-3’) or Foxc2Δ (c2-F1, 5’-CTCCTTTGCGTTTCCAGTGA-3’; c2-R2, 5’-CAACAGGGGCAACAAGTCC-3’).

Embryo analysis

Embryos were obtained at several different stages of development, with embryonic day 0.5 (E0.5) defined as the time when the vaginal plug was detected. Gross evaluations of embryos and histological analyses were performed as described previously (Kume et al., 2000a, 2000b; Kume et al., 1998; Kume et al., 2001; Seo et al., 2006); images of whole embryos were obtained with a DFC320 camera (Leica).

Endothelial cell isolation

WT male mice were anesthetized and perfused with PBS. The heart, lungs, and kidneys were dissected in cold DMEM/20% FBS/1% penicillin-streptomycin, minced well with a razor blade, and then enzymatically digested in 1 mg/mL type I collagenase at 37° for 40 min. The tissues were then mechanically dissociated by trituration and filtered (70 um). The cell suspension was incubated with CD31-Dynabeads, prepared with rat anti-mouse CD31 (BD Pharmingen, clone MEC 13.3) and sheep anti-rat IgG Dynabeads (Invitrogen), for 1 hr at 4°. After extensive washing with 0.1% FBS/2 mM EDTA/PBS, the bead-bound endothelial cells and the negative fraction were resuspended in 1 mL of Trizol (Invitrogen).

RNA isolation and real-time RT-PCR

For analysis of embryonic gene expression, total RNA was isolated from the tail, which includes the presomitic mesoderm and somites where Foxc1 and Foxc2 are expressed, with the Absolutely RNA Nanoprep Kit (Stratagene), and cDNA was synthesized with iScript reverse transcriptase (Bio-Rad) as directed by the manufacturer’s instructions. Real-time RT-PCR was performed with a Bio-Rad iCycler, Absolute SYBR Fluoroscein Mix, and primer sets for the respective genes. Peptidylprolyl isomerase A (Ppia) was used as an internal standard for mRNA expression. The primers used in this study were as follows: Ppia-sense, 5’-CAAATGCTGGACCAAACACA-3’; Ppia-anti, 5’-TGCCATCCAGCCATTCAGTC-3’; Foxc1-sense: 5’-TTCTTGCGTTCAGAGACTCG-3’; Foxc1-anti: 5’-TCTTACAGGTGAGAGGCAAGG-3’; Foxc2-sense, 5’-GCAACCCAACAGCAAACTTTC-3’; Foxc2-anti, 5’-GACGGCGTAGCTCGATAGG-3’.

For analysis of gene expression in endothelial cells isolated from adult mice, total RNA was isolated using TRIzol Reagent (Invitrogen). cDNA was synthesized with Taqman Reverse Transcription kit (Applied Biosystems) and amplification was performed on a Taqman 7500 (Applied Biosystems). The relative expression of each gene was calculated by the ddCt comparative threshold method and normalized to endogenous 18S rRNA expression. The 18S primers and probe used in this study were as follows: 18S sense, 5’-ACGAGACTCTGGCATGCTAACTAGT-3’, 18S anti, 5’-CGCCACTTGTCCCTCTAAGAA-3’, 18S probe, 5’-ACGCGACCCCCGAGCGGT-3’.

Statistical analyses

Data are shown as mean and SD. Statistical significance was determined by Student’s t test (two-tailed). The analyses were conducted with GraphPad Prism.

Acknowledgements

We thank Drs. Takeshi Kurita, Anees Fatima, and Seungwoon Seo for their help with the analysis of Foxc mutant embryos and their technical assistance. We thank W. Kevin Meisner, PhD, ELS, for editorial support. This work was supported by US National Institutes of Health grants HL074121 and EY019484 to T.K and was made possible through the use of the Vanderbilt University Transgenic Mouse and ES Cell Core.

Footnotes

Gene symbols for human Foxc are presented with all uppercase letters (e.g., FOXC1), the first letter is capitalized for mouse genes (e.g., Foxc1), and the first and subclass letters are capitalized for chordates (e.g., FoxC1) (Kaestner et al., 2000).

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