Altering HIF-1α through 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure affects coronary vessel development.

Differential tissue hypoxia drives normal cardiogenic events including coronary vessel development. This requirement renders cardiogenic processes potentially susceptible to teratogens that activate a transcriptional pathway that intersects with the hypoxia-inducible factor (HIF-1) pathway. The potent toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause cardiovascular defects by way of reduced myocardial hypoxia, inhibition of angiogenic stimuli, and alterations in responsiveness of endothelial cells to those stimuli. Our working hypothesis is that HIF-1 levels and thus HIF-1 signaling in the developing myocardium will be reduced by TCDD treatment in vivo during a critical stage and in particularly sensitive sites during heart morphogenesis. This inadequate HIF-1 signaling will subsequently result in outflow tract (OFT) and coronary vasculature defects. Our current data using the chicken embryo model showed a marked decrease in the intensity of immunostaining for HIF-1α nuclear expression in the OFT myocardium of TCDD-treated embryos. This area at the base of the OFT is particularly hypoxic during normal development; where endothelial cells initially form a concentrated anastomosing network known as the peritruncal ring; and where the left and right coronary arteries eventually connect to the aortic lumen. Consistent with this finding, anomalies of the proximal coronaries were detected after TCDD treatment and HIF-1α protein levels decreased in a TCDD dose-dependent manner

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

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

Kelly procedure for exstrophy or epispadias patients: Anatomical description of the pudendal neurovasculature

IntroductionAdequate penile length in males with bladder exstrophy or epispadias is a major challenge. Kelly previously described a surgical technique of a single stage reconstruction for patients with exstrophy or epispadias that potentially achieves significant penile lengthening by completely detaching the insertion of the corpora cavernosa from the ischiopubic rami. However, because of the possibility of damage to the pudendal neurovascular supply that may lead to partial or complete penile loss, this technique has not gained popularity. The aim of this study is to describe the surgical anatomic relationship of the pudendal neurovascular bundle (NVB) to the ischiopubic rami and to determine a safer approach to dissection during the Kelly procedure.MethodsWe performed meticulous dissection in three formalin-fixed and one fresh adult male cadavers to demonstrate the anatomical relationships between the pudendal neurovascular supply of the penis and the cavernosal insertion to the ischiopubic ramus.Results and discussionWe demonstrated the relationships and distance between the NVB and the area of separation between the crus and the ischiopubic ramus at the level of the periosteum. The insertion of the crus to the ischiopubic ramus is inferior lateral, whereas the NVB lies at a superior medial position. This anatomical relationship is best visualized when the dissection is carried out starting from the distal portion of the NVB and proceeding proximally. This area of the periosteum is avascular and the NVB can be preserved safely as long as the dissection is conducted at that subperiosteal level. Based on this cadaver dissection study, we suppose that detaching the corporal cavernosa from the pubic bones at the subperiosteal level allows for a safe distance to be maintained from the pudendal nerve at all times. We believe that if a surgeon performs the dissection inferiorly and laterally, the corpora cavernosa can be safely detached from the ischiopubic ramus and injury to the pudendal vessels and nerve can be avoided. However, it must be noted that there are limitations to applying the results from this study of normal, adult cadavers to the anatomy of children and adolescents with exstrophy or epispadias, who form the largest proportion of patients who are candidates for this procedure.ConclusionThis anatomical study demonstrates the relationship between the pudendal NVB, the crus, and the ischiopubic ramus. We demonstrated how the separation of the crus from the ischiopubic periosteum might be performed more safely

<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

<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