Boletín de Segovia: Número 15 - 1918 febrero 4

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Additional file 9: Data 1. of Analysis of in vivo single cell behavior by high throughput, human-in-the-loop segmentation of three-dimensional images

DNA morphologies of cells classified as G1-, S-, G2-, and M-phase based on DNA and EdU content. (PDF 3296 kb

Additional file 7: Figure S6. of Analysis of in vivo single cell behavior by high throughput, human-in-the-loop segmentation of three-dimensional images

Benchmarking of cell row counter accuracy for C. elegans gonadal arms. (A) Overlay of microscope image (white signal, derived from the DNA stain DAPI) with segmentations color-coded by cell row position from the distal end (left), as computed automatically using our counter. (B) Size of the MZ scored manually vs size computed through the automatic counter. A small amount of noise (0.5 cell rows) was added in order to aid visualization of overlapping data. Positions scored manually and automatically are in close agreement; average percent deviation is 9.4 %. Diagonal shown for reference in red. (PDF 152 kb

Boletín de Segovia: Número 38 - 1884 marzo 26

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Additional file 10: Figure S8. of Analysis of in vivo single cell behavior by high throughput, human-in-the-loop segmentation of three-dimensional images

Comparison of frozen or vibratome section segmentations. (A) Active contour segmentation of frozen sections is often inaccurate (red arrows) due to poor cell separation in the original image. (B) Vibratome sections enable clearer staining and thus more accurate segmentation (yellow arrows). (PDF 749 kb

<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

<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