<i>FLEX</i> alleles allow successive toggling between mutant and wildtype genotypes and phenotypes.

<p>A. Schematic of EUCE313f02 (<i>Nipbl</i>^<i>FLEX</i>) allele from which the <i>Nipbl</i>^{<i>FLEX/+</i>} mouse line and allelic series are derived. The rsFlp-Rosa-βgeo cassette is inserted 14.5 kbp downstream of <i>Nipbl</i> Exon 1 on Chromosome 15. B. In the <i>Nipbl</i>^<i>FLEX</i> allele, the splice acceptor (SA) in the cassette traps <i>Nipbl</i> expression, resulting in termination of <i>Nipbl</i> expression after exon 1 and expression of the <i>β-geo</i> reporter for the trapped null allele. Adult <i>Nipbl</i>^{<i>FLEX/+</i>} mice are smaller than wildtype littermates: Image is of 4-wk-old male littermates. Scatter plot shows weights of 12-wk-old <i>Nipbl</i>^{<i>FLEX/+</i>} mice (red, <i>n</i> = 3: 1 female, 2 males) and wildtype littermates (black, <i>n</i> = 8: 4 females, 4 males) from 3 litters. Ubiquitous expression of <i>β-geo</i> was detected by X-gal staining in E10.5 <i>Nipbl</i>^{<i>FLEX/+</i>} embryos. Histogram shows mean ± SEM of relative <i>Nipbl</i> expression, assessed by qRT-PCR, in kidneys of E17.5 <i>Nipbl</i>^{<i>FLEX/+</i>} (<i>n</i> = 8) and wildtype littermates (<i>n</i> = 6); asterisk: <i>p</i> < 0.05 by Student’s <i>t</i> test. C. Mating <i>Nipbl</i>^{<i>FLEX/+</i>} mice with mice carrying universal Flp recombinase inverts the SA-<i>βgeo</i>-pA at heterotypic recognition targets (frt and F3 sites) and simultaneously excises cognate recognition sites, resulting in progeny carrying the <i>Nipbl</i>^{<i>Flox/+</i>} allele. Inversion allows normal splicing between the endogenous <i>Nipbl</i> splice sites (Exon 1 to Exon 2), thereby yielding a phenotypically wildtype allele. <i>Nipbl</i>^{<i>Flox/+</i>} mice are similar to wildtype littermates in size: Image is of 3-wk-old male littermates; scatter plot shows weights of 11-wk-old <i>Nipbl</i>^{<i>Flox/+</i>} mice (red, <i>n</i> = 18: 4 female; 14 male) compared to wildtype littermates (black, <i>n</i> = 19: 4 female; 15 male) from 5 litters. Expression of <i>β-geo</i> is not detected by X-gal staining in E10.5 <i>Nipbl</i>^{<i>Flox/+</i>} embryos. Histogram shows qRT-PCR analysis of relative <i>Nipbl</i> expression in brain tissue of E17.5 in <i>Nipbl</i>^{<i>Flox/+</i>} (<i>n</i> = 8) versus wildtype littermates (<i>n</i> = 7), plotted as in B; <i>p</i> > 0.05, Student’s <i>t</i> test. D. Mating <i>Nipbl</i>^{<i>FLEX/+</i>} mice with mice carrying a universal Cre recombinase causes recombination of the <i>Nipbl</i>^<i>FLEX</i> allele (at LoxP and lox5171 recognition sites), resulting in progeny carrying the <i>Nipbl</i>^<i>Flrt</i> allele. <i>Nipbl</i>^{<i>Flrt</i>/+}mice are phenotypically wildtype: Image is of male <i>Nipbl</i>^{<i>Flrt/+</i>} and wildtype littermates at 3 wk of age showing no apparent difference in body size. Scatter plot shows weights of 12-wk-old <i>Nipbl</i>^{<i>Flrt/+</i>} mice (red, <i>n</i> = 19: 6 female; 13 male) and wildtype littermates (black, <i>n</i> = 10: 3 female; 7 male) from 3 litters. Expression of <i>β-geo</i> was not detected by X-gal staining in E10.5 <i>Nipbl</i>^{<i>Flrt/+</i>} embryos. qRT-PCR results show relative <i>Nipbl</i> expression in kidneys of E17.5 <i>Nipbl</i>^{<i>Flrt/+</i>} (<i>n</i> = 6) compared to wildtype littermates (<i>n</i> = 6), plotted as in B; <i>p</i> > 0.05 by Student’s <i>t</i> test. E. Cre-mediated recombination of mice carrying the <i>Nipbl</i>^<i>Flox</i> allele, obtained by crossing <i>Nipbl</i>^{<i>Flox/+</i>} mice with <i>Nanog-Cre</i> hemizygous mice, results in re-inversion of the SA-<i>βgeo</i>-pA cassette and re-trapping of <i>Nipbl</i> expression. Resulting progeny (<i>Nipbl</i>^FIN/+ mice) are phenotypically mutant, and survive poorly, with only 13 <i>Nipbl</i>^FIN/+ mice (4%) surviving to weaning age out of 315 total pups born (significantly less than the expected 25% survival, <i>p</i> < 0.001 by Chi-square analysis). Adult <i>Nipbl</i>^<i>FIN/+</i> mice are smaller than wildtype littermates: Image is of 6-wk old males; scatter plot shows weights of 8-wk-old <i>Nipbl</i>^<i>FIN/+</i> mice (red, <i>n</i> = 11: 4 females; 7 males) compared to wildtype littermates (black, <i>n</i> = 7: 3 females; 4 males) from 16 litters. Ubiquitous expression of <i>β-geo</i> is detected by X-gal staining. qRT-PCR results show reduced <i>Nipbl</i> expression in brains of E17.5 <i>Nipbl</i>^<i>FIN/+</i> (<i>n</i> = 7) compared to wildtype littermates (<i>n</i> = 6), plotted as in B; asterisk: <i>p</i> < 0.05, Student’s <i>t</i> test. Scale bars = 1 mm for all panels. Frt (purple triangles), F3 (green triangles), loxP (orange triangles) and lox5171 (yellow triangles); SA, splice acceptor; <i>β-geo</i>, <i>β</i>-galactosidase/neomycin phosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence.</p

Relationships between <i>Nipbl</i> genotype, embryo size, heart size, and ASDs.

<p>A. Table summarizing genotypes, heart size, body size and incidence of ASDs in different crosses. B. Rescued <i>Nipbl</i>^{<i>FLEX/+</i>}<i>;cTnt-Cre</i> embryos (<i>n</i> = 14) resembled their <i>Nipbl</i>^{<i>FLEX/+</i>} littermates (<i>n</i> = 22) in body size and were smaller than control littermates (<i>cTnt-Cre n</i> = 18, wildtype <i>n</i> = 31). C. Similar results were observed in rescued <i>Nipbl</i>^{<i>FLEX/+</i>}<i>;Sox17-2A-iCre</i> embryos (<i>n</i> = 22; <i>Sox17-2A-iCre n</i> = 16; <i>Nipbl</i>^{<i>FLEX/+</i>} <i>n</i> = 18; wildtype <i>n</i> = 25). D. <i>Nipbl</i>^{<i>Flox/+</i>}<i>;cTnt-Cre</i> (<i>n</i> = 15) were similar in overall body size to littermate controls (<i>Nipbl</i>^{<i>Flox/+</i>} <i>n</i> = 14, <i>cTnt-Cre n</i> = 30, wildtype <i>n</i> = 21). E. Similar results were observed in <i>Nipbl</i>^{<i>Flox/+</i>}<i>;Sox17-2A-iCre</i> (<i>n</i> = 13) when compared to littermate controls (<i>Sox17-2A-iCre</i>, <i>n</i> = 20; <i>Nipbl</i>^{<i>Flox/+</i>} <i>n</i> = 10; wildtype <i>n</i> = 19). Note that individual weights for each cross in B–E were normalized to the mean weight of <i>cTnt-Cre</i> controls (B, D), or <i>Sox17-2A-iCre</i> controls (C, E); black bars indicate normalized mean weight for each genotype. F. Ventricular volume analyses (graphed as box plots) show that the overall heart size of rescued <i>Nipbl</i>^{<i>FLEX/+</i>}<i>;cTnt-Cre</i> embryos (<i>n</i> = 7) were similar in size to <i>Nipbl</i>^{<i>FLEX/+</i>} heart size (<i>n</i> = 9) (Mann-Whitney U, <i>p</i> > 0.05). Control hearts (<i>cTnt-Cre</i>, <i>n</i> = 6) were significantly larger than the hearts of their <i>Nipbl</i>^{<i>FLEX/+</i>} and <i>Nipbl</i>^{<i>FLEX/+</i>}<i>;cTnt-Cre</i> littermates (asterisks: Mann-Whitney U, <i>p</i> < 0.05). G. Rescued <i>Nipbl</i>^{<i>FLEX/+</i>}<i>;Sox17-2A-iCre</i> embryo hearts (<i>n</i> = 5) were also similar in size to <i>Nipbl</i>^{<i>FLEX/+</i>} littermate heart size (<i>n</i> = 6) (Mann-Whitney U, <i>p</i> > 0.05) and significantly smaller than control hearts (<i>Sox17-2A-iCre</i>, <i>n</i> = 5) (asterisk: Mann-Whitney U, <i>p</i> < 0.05). H. Ventricular volume analysis show that the ventricle size of <i>Nipbl</i>^{<i>Flox/+</i>}<i>;cTnt-Cre</i> embryos (<i>n</i> = 9), which display a high frequency of heart defects, were similar in size to control hearts (<i>cTnt-Cre</i>, <i>n</i> = 9) (Mann-Whitney U, <i>p</i> > 0.05). I. <i>Nipbl</i>^{<i>Flox/+</i>}<i>;Sox17-2A-iCre</i> mutant hearts (<i>n</i> = 5), which also display a high frequency of heart defects, were also similar in size to control hearts (<i>Sox17-2A-iCre</i>, <i>n</i> = 5) (Mann-Whitney U, <i>p</i> > 0.05).</p

Restoration of <i>Nipbl</i> expression in different heart lineages rescues heart defects.

<p>MRI analysis of E17.5 hearts show that <i>Nipbl</i>^{<i>FLEX/+</i>} mice exhibit ASDs at a frequency of ~30% (yellow arrowheads in A–C, and D). The incidence of heart defects was significantly reduced in mice in which <i>Nipbl</i> was restored in all tissues (<i>Nipbl</i>^{<i>FLEX/+</i>}<i>;Nanog-Cre</i>), specifically in the <i>cTnt</i> domain (<i>Nipbl</i>^{<i>FLEX/+</i>}<i>;cTnt-Cre</i>), or specifically in the <i>Sox17</i> domain (<i>Nipbl</i>^{<i>FLEX/+</i>}<i>;Sox17-2A-iCre</i>) (D, asterisks: Chi-square, <i>p</i> < 0.05). Chi-square analysis on the incidence of heart defects between the three rescued lines indicated no significant difference from one another (Chi-square, <i>p</i> > 0.71). Progeny are on various backgrounds depending on parental backgrounds: <i>Nipbl</i>^{<i>FLEX/+</i>} (mixed), wildtype (CD-1), <i>cTnt-Cre</i> (CD-1), <i>Nanog-Cre</i> (C57Bl6/J), and <i>Sox17-2A-iCre</i> (C57Bl6/J); the incidence of heart defects in <i>Nipbl</i>^{<i>FLEX/+</i>} embryos from each of these crosses did not differ significantly (<i>p</i> > 0.89 by Chi-square analysis; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000197#pbio.2000197.s001" target="_blank">S1 Data</a>). Size bar = 500 μm. lv, left ventricle; rv, right ventricle</p

Creation of <i>Nipbl</i> deficiency in cardiac developmental lineages.

<p><i>Nipbl</i>^{<i>Flox/+</i>} mice were crossed with mice hemizygous for each of five indicated Cre-expressing transgenes. MRI analysis of hearts was performed at E17.5. A–D show that whether <i>Nipbl</i> was made deficient in all tissues (<i>Nanog-Cre</i>, A), specifically in cardiomyocytes (<i>cTnt-Cre</i>, B), or primarily in endoderm-derived tissues (<i>Sox17-2A-iCre</i>, C) or mixed cardiac lineages (<i>FoxA2-2A-iCre</i>, D), the incidence of CHDs (primarily ASDs) was approximately 30%. (Chi-square analyses indicate that frequencies of heart defects observed in embryos with <i>Nipbl</i> deficiency in experiments A–D do not differ significantly from each other [<i>p</i> > 0.4 for each pairwise comparison].) In contrast, control hearts (a mix of wildtype, <i>Nipbl</i>^{<i>Flox/+</i>}, and <i>Cre</i>+ littermates for each specific cross; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000197#pbio.2000197.s001" target="_blank">S1 Data</a>: Sample Numbers) had an incidence of heart defects ranging from 0%–5% (A–D). <i>Nipbl</i> deficiency in the <i>Wnt1</i> domain (neural crest) did not give rise to heart defects (E). CHDs occurred primarily in the form of ASDs of the ostium secundum type (yellow arrowheads); VSDs observed in A and D are of the perimembranous type. <i>p</i>-Values are from Chi-square analyses and are indicated for each corresponding cross. Scale bar = 500 μm. lv, left ventricle; rv, right ventricle.</p

<i>Nipbl</i>^{<i>FLEX/+</i>} and <i>Nipbl</i>^<i>+/-</i> mice develop heart defects at the same high frequency.

<p>A, B. Paraffin-sectioned hearts stained with H&E (A) and MRI-scanned hearts (B) show large atrial septal defects (yellow arrowheads) in <i>Nipbl</i>^<i>+/-</i> and <i>Nipbl</i>^{<i>FLEX/+</i>} mice, but not in wildtype or <i>Nipbl</i>^{<i>Flox/+</i>} mice. Scans and histology were performed on fixed tissue from E17.5 embryos. Scale bar = 500 μm. la, left atrium; lv left ventricle; ra, right atrium; rv right ventricle; S, septum. C. Summary table showing incidence of atrial septal defects (ASDs) and ventricular septal defects (VSDs) in hearts of <i>Nipbl</i>^<i>+/-</i>, <i>Nipbl</i>^{<i>FLEX/+</i>}, <i>Nipbl</i>^{<i>Flox/+</i>} mice and wildtype littermate embryos at E17.5. Asterisks: <i>p</i> < 0.01 by Chi-square analysis. Data were pooled from analyses of multiple crosses (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000197#pbio.2000197.s001" target="_blank">S1 Data</a>: Sample Numbers) and progeny are on various backgrounds depending on parental backgrounds: <i>Nipbl</i>^<i>+/-</i>, CD-1; <i>Nipbl</i>^{<i>FLEX/+</i>}, mixed; <i>Nipbl</i>^{<i>Flox/+</i>}, C57Bl6/J or mixed.</p

Haploinsufficiency for <i>Nkx2-5</i> increases the incidence and severity of heart defects in <i>Nipbl</i>-deficient embryos.

<p>A. <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> mice displayed ASDs (yellow arrowhead), VSDs (green arrowhead), and/or PTAs (black asterisk). B. Histogram showing increases in frequency and types of heart defects in <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> hearts compared to littermates of various genotypes; number of hearts observed with each type of defect is indicated. Data show that the overall incidence of defects in <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> hearts (83%) is significantly greater than in either <i>Nipbl</i>^<i>+/-</i> (30%, <i>p</i> = 0.011 by Chi square) or <i>Nkx2</i>.<i>5</i>^<i>+/-</i> (13%, <i>p</i> = 0.001 by Chi-square) hearts. C. Gross overview of the great vessels highlighting PTA in <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> mice. D. Heart position and morphology in a subset of <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> mice was drastically different than in littermates. Arrows indicate the position of the trachea (Tr), dotted lines originate at the pulmonary artery and ends at the apex of the heart; note drastic change in the angle of dotted line in <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> heart. E. Ventricular volumes for hearts of each genotype; horizontal bars indicate means. Ventricular volumes of <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> hearts (<i>n</i> = 6) were similar in size to <i>Nipbl</i>^<i>+/-</i> hearts (<i>n</i> = 5) (Mann-Whitney U, <i>p</i> > 0.05), whereas control hearts (wildtype <i>n</i> = 5 and <i>Nkx2-5</i>^<i>+/-</i>, <i>n</i> = 5) were significantly larger than the hearts of their <i>Nipbl</i>^<i>+/-</i> and <i>Nipbl</i>^<i>+/-</i><i>;Nkx2-5</i>^<i>+/-</i> littermates (red asterisks: Mann-Whitney U, <i>p</i> < 0.05). ao, aorta; ASD, atrial septal defect; la, left atrium; lv, left ventricle; pa, pulmonary artery; PTA, persistent truncus arteriosus; ra, right atrium; rv, right ventricle; Tr, trachea; VSD, ventricular septal defect; Size bar = 500 μm.</p

Domains of Cre expression in mice made <i>Nipbl</i>-deficient in different heart developmental lineages.

<p>A. Relevant patterns of Cre expression from the six Cre lines used in this study (<i>Nanog-Cre</i>, <i>Nkx2-5-Cre</i>, <i>cTnt-Cre</i>, <i>Sox17-2A-iCre</i>, <i>FoxA2-2A-iCre</i>, <i>Wnt1-Cre</i>), assessed on the <i>Td-Tomato-EGFP</i> reporter during early embryogenesis. Cre-mediated recombination is indicated by expression of EGFP. Note two embryos are shown in the <i>Nanog-Cre</i> panel; only the embryo on the right carries the <i>Nanog-Cre</i> allele and exhibits EGFP expression in the inner cell mass (icm). B–F. X-gal staining was performed to detect expression of <i>β-geo</i> in <i>Nipbl</i>^{<i>Flox/+</i>} embryos carrying one of the Cre transgenes. X-gal staining was assessed in whole embryos (at E8–10.5, left panels), whole hearts (at E10.5, middle panels in B-E and at E13.5 in <i>Nipbl</i>^{<i>Flox/+</i>}<i>;Wnt1-cre</i> hearts, right panel in F), and sectioned hearts (at E10.5, right panels B-E). C’–E’. Confocal images of E15.5 hearts generated from crosses of <i>Nipbl</i>^{<i>Flox/+</i>};<i>Td-Tomato-EGFP</i> mice with different Cre-expressing lines. (C’) <i>cTnt-Cre</i>: EGFP in myocardium including atrial septum. (D’) <i>Sox17-2A-iCre</i>: EGFP in endocardium, and small blood vessels. (E’) <i>FoxA2-2A-iCre</i>: EGFP in both endoderm (bronchi linings) and some mesoderm (muscle in ventricle); minimal EGFP expression in atrial septum (note that pattern is different from <i>Sox17-2A-iCre</i>). ao, aorta; av, atrioventricular valve; ba1, branchial arch; br, bronchi; cc, cardiac cushion; drg, dorsal root ganglion; en, endocardium; ep, epicardium; fp, floor plate; fg, foregut; h, heart; hg, hindgut; icm, inner cell mass; la, left atria; lv, left ventricle; mb, midbrain; mc, myocardium; oft, outflow tract; pa, pulmonary artery; ph, pharyngeal arches; ra, right atria; rv, right ventricle; som, somites; th, thymus; tr, trachea</p