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Conditional Creation and Rescue of Nipbl -Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects

View Article: PubMed Central - PubMed

ABSTRACT

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

No MeSH data available.


Related in: MedlinePlus

FLEX alleles allow successive toggling between mutant and wildtype genotypes and phenotypes.A. Schematic of EUCE313f02 (NipblFLEX) allele from which the NipblFLEX/+ mouse line and allelic series are derived. The rsFlp-Rosa-βgeo cassette is inserted 14.5 kbp downstream of Nipbl Exon 1 on Chromosome 15. B. In the NipblFLEX allele, the splice acceptor (SA) in the cassette traps Nipbl expression, resulting in termination of Nipbl expression after exon 1 and expression of the β-geo reporter for the trapped  allele. Adult NipblFLEX/+ mice are smaller than wildtype littermates: Image is of 4-wk-old male littermates. Scatter plot shows weights of 12-wk-old NipblFLEX/+ mice (red, n = 3: 1 female, 2 males) and wildtype littermates (black, n = 8: 4 females, 4 males) from 3 litters. Ubiquitous expression of β-geo was detected by X-gal staining in E10.5 NipblFLEX/+ embryos. Histogram shows mean ± SEM of relative Nipbl expression, assessed by qRT-PCR, in kidneys of E17.5 NipblFLEX/+ (n = 8) and wildtype littermates (n = 6); asterisk: p < 0.05 by Student’s t test. C. Mating NipblFLEX/+ mice with mice carrying universal Flp recombinase inverts the SA-βgeo-pA at heterotypic recognition targets (frt and F3 sites) and simultaneously excises cognate recognition sites, resulting in progeny carrying the NipblFlox/+ allele. Inversion allows normal splicing between the endogenous Nipbl splice sites (Exon 1 to Exon 2), thereby yielding a phenotypically wildtype allele. NipblFlox/+ mice are similar to wildtype littermates in size: Image is of 3-wk-old male littermates; scatter plot shows weights of 11-wk-old NipblFlox/+ mice (red, n = 18: 4 female; 14 male) compared to wildtype littermates (black, n = 19: 4 female; 15 male) from 5 litters. Expression of β-geo is not detected by X-gal staining in E10.5 NipblFlox/+ embryos. Histogram shows qRT-PCR analysis of relative Nipbl expression in brain tissue of E17.5 in NipblFlox/+ (n = 8) versus wildtype littermates (n = 7), plotted as in B; p > 0.05, Student’s t test. D. Mating NipblFLEX/+ mice with mice carrying a universal Cre recombinase causes recombination of the NipblFLEX allele (at LoxP and lox5171 recognition sites), resulting in progeny carrying the NipblFlrt allele. NipblFlrt/+mice are phenotypically wildtype: Image is of male NipblFlrt/+ and wildtype littermates at 3 wk of age showing no apparent difference in body size. Scatter plot shows weights of 12-wk-old NipblFlrt/+ mice (red, n = 19: 6 female; 13 male) and wildtype littermates (black, n = 10: 3 female; 7 male) from 3 litters. Expression of β-geo was not detected by X-gal staining in E10.5 NipblFlrt/+ embryos. qRT-PCR results show relative Nipbl expression in kidneys of E17.5 NipblFlrt/+ (n = 6) compared to wildtype littermates (n = 6), plotted as in B; p > 0.05 by Student’s t test. E. Cre-mediated recombination of mice carrying the NipblFlox allele, obtained by crossing NipblFlox/+ mice with Nanog-Cre hemizygous mice, results in re-inversion of the SA-βgeo-pA cassette and re-trapping of Nipbl expression. Resulting progeny (NipblFIN/+ mice) are phenotypically mutant, and survive poorly, with only 13 NipblFIN/+ mice (4%) surviving to weaning age out of 315 total pups born (significantly less than the expected 25% survival, p < 0.001 by Chi-square analysis). Adult NipblFIN/+ mice are smaller than wildtype littermates: Image is of 6-wk old males; scatter plot shows weights of 8-wk-old NipblFIN/+ mice (red, n = 11: 4 females; 7 males) compared to wildtype littermates (black, n = 7: 3 females; 4 males) from 16 litters. Ubiquitous expression of β-geo is detected by X-gal staining. qRT-PCR results show reduced Nipbl expression in brains of E17.5 NipblFIN/+ (n = 7) compared to wildtype littermates (n = 6), plotted as in B; asterisk: p < 0.05, Student’s t test. Scale bars = 1 mm for all panels. Frt (purple triangles), F3 (green triangles), loxP (orange triangles) and lox5171 (yellow triangles); SA, splice acceptor; β-geo, β-galactosidase/neomycin phosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence.
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pbio.2000197.g002: FLEX alleles allow successive toggling between mutant and wildtype genotypes and phenotypes.A. Schematic of EUCE313f02 (NipblFLEX) allele from which the NipblFLEX/+ mouse line and allelic series are derived. The rsFlp-Rosa-βgeo cassette is inserted 14.5 kbp downstream of Nipbl Exon 1 on Chromosome 15. B. In the NipblFLEX allele, the splice acceptor (SA) in the cassette traps Nipbl expression, resulting in termination of Nipbl expression after exon 1 and expression of the β-geo reporter for the trapped allele. Adult NipblFLEX/+ mice are smaller than wildtype littermates: Image is of 4-wk-old male littermates. Scatter plot shows weights of 12-wk-old NipblFLEX/+ mice (red, n = 3: 1 female, 2 males) and wildtype littermates (black, n = 8: 4 females, 4 males) from 3 litters. Ubiquitous expression of β-geo was detected by X-gal staining in E10.5 NipblFLEX/+ embryos. Histogram shows mean ± SEM of relative Nipbl expression, assessed by qRT-PCR, in kidneys of E17.5 NipblFLEX/+ (n = 8) and wildtype littermates (n = 6); asterisk: p < 0.05 by Student’s t test. C. Mating NipblFLEX/+ mice with mice carrying universal Flp recombinase inverts the SA-βgeo-pA at heterotypic recognition targets (frt and F3 sites) and simultaneously excises cognate recognition sites, resulting in progeny carrying the NipblFlox/+ allele. Inversion allows normal splicing between the endogenous Nipbl splice sites (Exon 1 to Exon 2), thereby yielding a phenotypically wildtype allele. NipblFlox/+ mice are similar to wildtype littermates in size: Image is of 3-wk-old male littermates; scatter plot shows weights of 11-wk-old NipblFlox/+ mice (red, n = 18: 4 female; 14 male) compared to wildtype littermates (black, n = 19: 4 female; 15 male) from 5 litters. Expression of β-geo is not detected by X-gal staining in E10.5 NipblFlox/+ embryos. Histogram shows qRT-PCR analysis of relative Nipbl expression in brain tissue of E17.5 in NipblFlox/+ (n = 8) versus wildtype littermates (n = 7), plotted as in B; p > 0.05, Student’s t test. D. Mating NipblFLEX/+ mice with mice carrying a universal Cre recombinase causes recombination of the NipblFLEX allele (at LoxP and lox5171 recognition sites), resulting in progeny carrying the NipblFlrt allele. NipblFlrt/+mice are phenotypically wildtype: Image is of male NipblFlrt/+ and wildtype littermates at 3 wk of age showing no apparent difference in body size. Scatter plot shows weights of 12-wk-old NipblFlrt/+ mice (red, n = 19: 6 female; 13 male) and wildtype littermates (black, n = 10: 3 female; 7 male) from 3 litters. Expression of β-geo was not detected by X-gal staining in E10.5 NipblFlrt/+ embryos. qRT-PCR results show relative Nipbl expression in kidneys of E17.5 NipblFlrt/+ (n = 6) compared to wildtype littermates (n = 6), plotted as in B; p > 0.05 by Student’s t test. E. Cre-mediated recombination of mice carrying the NipblFlox allele, obtained by crossing NipblFlox/+ mice with Nanog-Cre hemizygous mice, results in re-inversion of the SA-βgeo-pA cassette and re-trapping of Nipbl expression. Resulting progeny (NipblFIN/+ mice) are phenotypically mutant, and survive poorly, with only 13 NipblFIN/+ mice (4%) surviving to weaning age out of 315 total pups born (significantly less than the expected 25% survival, p < 0.001 by Chi-square analysis). Adult NipblFIN/+ mice are smaller than wildtype littermates: Image is of 6-wk old males; scatter plot shows weights of 8-wk-old NipblFIN/+ mice (red, n = 11: 4 females; 7 males) compared to wildtype littermates (black, n = 7: 3 females; 4 males) from 16 litters. Ubiquitous expression of β-geo is detected by X-gal staining. qRT-PCR results show reduced Nipbl expression in brains of E17.5 NipblFIN/+ (n = 7) compared to wildtype littermates (n = 6), plotted as in B; asterisk: p < 0.05, Student’s t test. Scale bars = 1 mm for all panels. Frt (purple triangles), F3 (green triangles), loxP (orange triangles) and lox5171 (yellow triangles); SA, splice acceptor; β-geo, β-galactosidase/neomycin phosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence.

Mentions: To make it possible to investigate the roles of different cell lineages in the development of heart defects in Nipbl-deficient mice, we developed a Nipbl allelic series based on embryonic stem (ES) cells bearing a “conditional-invertible” (FLEX, or Flip-Excision [33,53,54]) gene trap in the Nipbl locus. We tested several Nipbl-gene-trapped ES cells that are available through public repositories, and ultimately selected, verified, and generated mice using ES cells bearing the NipblGt(EUCE313f02)Hmgu allele (MGI: 4374347, hereafter known as NipblFLEX), which is depicted in Fig 2A (see also S2 Fig). The gene-trap vector in these cells is inserted into intron 1 (14.5 kb downstream of exon 1) of the Nipbl gene, the same intron in which the gene-trap vector was inserted in the ES cells we used previously to generate Nipbl+/- mice [22].


Conditional Creation and Rescue of Nipbl -Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects
FLEX alleles allow successive toggling between mutant and wildtype genotypes and phenotypes.A. Schematic of EUCE313f02 (NipblFLEX) allele from which the NipblFLEX/+ mouse line and allelic series are derived. The rsFlp-Rosa-βgeo cassette is inserted 14.5 kbp downstream of Nipbl Exon 1 on Chromosome 15. B. In the NipblFLEX allele, the splice acceptor (SA) in the cassette traps Nipbl expression, resulting in termination of Nipbl expression after exon 1 and expression of the β-geo reporter for the trapped  allele. Adult NipblFLEX/+ mice are smaller than wildtype littermates: Image is of 4-wk-old male littermates. Scatter plot shows weights of 12-wk-old NipblFLEX/+ mice (red, n = 3: 1 female, 2 males) and wildtype littermates (black, n = 8: 4 females, 4 males) from 3 litters. Ubiquitous expression of β-geo was detected by X-gal staining in E10.5 NipblFLEX/+ embryos. Histogram shows mean ± SEM of relative Nipbl expression, assessed by qRT-PCR, in kidneys of E17.5 NipblFLEX/+ (n = 8) and wildtype littermates (n = 6); asterisk: p < 0.05 by Student’s t test. C. Mating NipblFLEX/+ mice with mice carrying universal Flp recombinase inverts the SA-βgeo-pA at heterotypic recognition targets (frt and F3 sites) and simultaneously excises cognate recognition sites, resulting in progeny carrying the NipblFlox/+ allele. Inversion allows normal splicing between the endogenous Nipbl splice sites (Exon 1 to Exon 2), thereby yielding a phenotypically wildtype allele. NipblFlox/+ mice are similar to wildtype littermates in size: Image is of 3-wk-old male littermates; scatter plot shows weights of 11-wk-old NipblFlox/+ mice (red, n = 18: 4 female; 14 male) compared to wildtype littermates (black, n = 19: 4 female; 15 male) from 5 litters. Expression of β-geo is not detected by X-gal staining in E10.5 NipblFlox/+ embryos. Histogram shows qRT-PCR analysis of relative Nipbl expression in brain tissue of E17.5 in NipblFlox/+ (n = 8) versus wildtype littermates (n = 7), plotted as in B; p > 0.05, Student’s t test. D. Mating NipblFLEX/+ mice with mice carrying a universal Cre recombinase causes recombination of the NipblFLEX allele (at LoxP and lox5171 recognition sites), resulting in progeny carrying the NipblFlrt allele. NipblFlrt/+mice are phenotypically wildtype: Image is of male NipblFlrt/+ and wildtype littermates at 3 wk of age showing no apparent difference in body size. Scatter plot shows weights of 12-wk-old NipblFlrt/+ mice (red, n = 19: 6 female; 13 male) and wildtype littermates (black, n = 10: 3 female; 7 male) from 3 litters. Expression of β-geo was not detected by X-gal staining in E10.5 NipblFlrt/+ embryos. qRT-PCR results show relative Nipbl expression in kidneys of E17.5 NipblFlrt/+ (n = 6) compared to wildtype littermates (n = 6), plotted as in B; p > 0.05 by Student’s t test. E. Cre-mediated recombination of mice carrying the NipblFlox allele, obtained by crossing NipblFlox/+ mice with Nanog-Cre hemizygous mice, results in re-inversion of the SA-βgeo-pA cassette and re-trapping of Nipbl expression. Resulting progeny (NipblFIN/+ mice) are phenotypically mutant, and survive poorly, with only 13 NipblFIN/+ mice (4%) surviving to weaning age out of 315 total pups born (significantly less than the expected 25% survival, p < 0.001 by Chi-square analysis). Adult NipblFIN/+ mice are smaller than wildtype littermates: Image is of 6-wk old males; scatter plot shows weights of 8-wk-old NipblFIN/+ mice (red, n = 11: 4 females; 7 males) compared to wildtype littermates (black, n = 7: 3 females; 4 males) from 16 litters. Ubiquitous expression of β-geo is detected by X-gal staining. qRT-PCR results show reduced Nipbl expression in brains of E17.5 NipblFIN/+ (n = 7) compared to wildtype littermates (n = 6), plotted as in B; asterisk: p < 0.05, Student’s t test. Scale bars = 1 mm for all panels. Frt (purple triangles), F3 (green triangles), loxP (orange triangles) and lox5171 (yellow triangles); SA, splice acceptor; β-geo, β-galactosidase/neomycin phosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence.
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pbio.2000197.g002: FLEX alleles allow successive toggling between mutant and wildtype genotypes and phenotypes.A. Schematic of EUCE313f02 (NipblFLEX) allele from which the NipblFLEX/+ mouse line and allelic series are derived. The rsFlp-Rosa-βgeo cassette is inserted 14.5 kbp downstream of Nipbl Exon 1 on Chromosome 15. B. In the NipblFLEX allele, the splice acceptor (SA) in the cassette traps Nipbl expression, resulting in termination of Nipbl expression after exon 1 and expression of the β-geo reporter for the trapped allele. Adult NipblFLEX/+ mice are smaller than wildtype littermates: Image is of 4-wk-old male littermates. Scatter plot shows weights of 12-wk-old NipblFLEX/+ mice (red, n = 3: 1 female, 2 males) and wildtype littermates (black, n = 8: 4 females, 4 males) from 3 litters. Ubiquitous expression of β-geo was detected by X-gal staining in E10.5 NipblFLEX/+ embryos. Histogram shows mean ± SEM of relative Nipbl expression, assessed by qRT-PCR, in kidneys of E17.5 NipblFLEX/+ (n = 8) and wildtype littermates (n = 6); asterisk: p < 0.05 by Student’s t test. C. Mating NipblFLEX/+ mice with mice carrying universal Flp recombinase inverts the SA-βgeo-pA at heterotypic recognition targets (frt and F3 sites) and simultaneously excises cognate recognition sites, resulting in progeny carrying the NipblFlox/+ allele. Inversion allows normal splicing between the endogenous Nipbl splice sites (Exon 1 to Exon 2), thereby yielding a phenotypically wildtype allele. NipblFlox/+ mice are similar to wildtype littermates in size: Image is of 3-wk-old male littermates; scatter plot shows weights of 11-wk-old NipblFlox/+ mice (red, n = 18: 4 female; 14 male) compared to wildtype littermates (black, n = 19: 4 female; 15 male) from 5 litters. Expression of β-geo is not detected by X-gal staining in E10.5 NipblFlox/+ embryos. Histogram shows qRT-PCR analysis of relative Nipbl expression in brain tissue of E17.5 in NipblFlox/+ (n = 8) versus wildtype littermates (n = 7), plotted as in B; p > 0.05, Student’s t test. D. Mating NipblFLEX/+ mice with mice carrying a universal Cre recombinase causes recombination of the NipblFLEX allele (at LoxP and lox5171 recognition sites), resulting in progeny carrying the NipblFlrt allele. NipblFlrt/+mice are phenotypically wildtype: Image is of male NipblFlrt/+ and wildtype littermates at 3 wk of age showing no apparent difference in body size. Scatter plot shows weights of 12-wk-old NipblFlrt/+ mice (red, n = 19: 6 female; 13 male) and wildtype littermates (black, n = 10: 3 female; 7 male) from 3 litters. Expression of β-geo was not detected by X-gal staining in E10.5 NipblFlrt/+ embryos. qRT-PCR results show relative Nipbl expression in kidneys of E17.5 NipblFlrt/+ (n = 6) compared to wildtype littermates (n = 6), plotted as in B; p > 0.05 by Student’s t test. E. Cre-mediated recombination of mice carrying the NipblFlox allele, obtained by crossing NipblFlox/+ mice with Nanog-Cre hemizygous mice, results in re-inversion of the SA-βgeo-pA cassette and re-trapping of Nipbl expression. Resulting progeny (NipblFIN/+ mice) are phenotypically mutant, and survive poorly, with only 13 NipblFIN/+ mice (4%) surviving to weaning age out of 315 total pups born (significantly less than the expected 25% survival, p < 0.001 by Chi-square analysis). Adult NipblFIN/+ mice are smaller than wildtype littermates: Image is of 6-wk old males; scatter plot shows weights of 8-wk-old NipblFIN/+ mice (red, n = 11: 4 females; 7 males) compared to wildtype littermates (black, n = 7: 3 females; 4 males) from 16 litters. Ubiquitous expression of β-geo is detected by X-gal staining. qRT-PCR results show reduced Nipbl expression in brains of E17.5 NipblFIN/+ (n = 7) compared to wildtype littermates (n = 6), plotted as in B; asterisk: p < 0.05, Student’s t test. Scale bars = 1 mm for all panels. Frt (purple triangles), F3 (green triangles), loxP (orange triangles) and lox5171 (yellow triangles); SA, splice acceptor; β-geo, β-galactosidase/neomycin phosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence.
Mentions: To make it possible to investigate the roles of different cell lineages in the development of heart defects in Nipbl-deficient mice, we developed a Nipbl allelic series based on embryonic stem (ES) cells bearing a “conditional-invertible” (FLEX, or Flip-Excision [33,53,54]) gene trap in the Nipbl locus. We tested several Nipbl-gene-trapped ES cells that are available through public repositories, and ultimately selected, verified, and generated mice using ES cells bearing the NipblGt(EUCE313f02)Hmgu allele (MGI: 4374347, hereafter known as NipblFLEX), which is depicted in Fig 2A (see also S2 Fig). The gene-trap vector in these cells is inserted into intron 1 (14.5 kb downstream of exon 1) of the Nipbl gene, the same intron in which the gene-trap vector was inserted in the ES cells we used previously to generate Nipbl+/- mice [22].

View Article: PubMed Central - PubMed

ABSTRACT

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

No MeSH data available.


Related in: MedlinePlus