Six more strains strengthen and narrow the EMMA interval.

<p>The EMMA algorithm was applied to collateral number for the 21 strain data set, ie, 6 new strains (red strains in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone-0031910-g002" target="_blank">Figure 2</a>) plus 15 from Zhang et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910-Zhang1" target="_blank">[9]</a>. ∼49,000 high quality imputed SNPs under the <i>Canq1</i> peak were tested. The previous highly significant region was strengthened and narrowed (p<9×10⁻⁶, 4 SNPs). Black bars indicate the most (Group A) and second most (Group B) significant regions in the previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910-Wang1" target="_blank">[15]</a>. Mapping allowed fewer than three SNPs with missing genotypes.</p

Collateral number for 21 inbred strains and heatmap of their SNPs within the EMMA region.

<p><b>Left</b>, Heatmap of known and imputed SNPs for 21 strains in EMMA region (blue, SNP same as B6; green, different from B6; blank, genotype unknown). <b>Right</b>, Collateral number per hemisphere for 21 strains. Names in red denote newly phenotyped strains, black names are strains from Zhang et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910-Zhang1" target="_blank">[9]</a>. N = 8–10/strain. Dashed line, reference to B6.</p

Genome-wide mapping of collateral number in 123 SWRxSJL-F2 mice.

<p><b>Top panel</b>, Collateral number per hemisphere among 15 strains of mice (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone-0031910-g002" target="_blank">Figure 2</a>). <b>Middle panel</b>, Green = known and high-quality imputed (p>0.90) SNPs common to both strains and BALB/c. White, known and low-quality SNPs different between the two strains, including two common regions with “no useful data”, per JAX website (red). <b>Lower panel</b>, LOD profiling using single QTL model. Locations of genotyping SNPs are shown as ticks on the abscissa. 95% confidence level (dashed line) was estimated using 1000 permutations. Insert shows the range of collateral number between the MCA and ACA trees per hemisphere on the abscissa (bin range = 3–14), and number of mice having a given collateral number bin on the ordinate (range 2–23).</p

Collateral extent is not altered in <i>Itgal, IL4 or IL4ra</i> deficient mice relative to host strains.

<p><b>A, </b><i>Itgal^−/−</i> and <i>Il4^−/−</i> mice are B6 while <i>Il4ra^−/</i>⁻ (receptor alpha) are Bc background. Number and diameter are not significantly different among <i>Itgal^−/−</i>, <i>Il4 ^−/−</i> and B6, nor between <i>Il4ra^−/−</i> and Bc (t-tests). <b>B,</b> Territory (size) of ACA, MCA, and PCA trees are same in B6 and Bc but differ in <i>Itgal^−/−</i> and <i>Il4ra^−/</i>⁻, confirming dissociation of collateral and tree territory phenotypes among inbred mouse strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910-Zhang1" target="_blank">[9]</a>. <b>C,D,</b> Bleeding and infarct volume of knockouts are not different from B6; re-bleeding in <i>Il4^−/− is</i> not different from B6 but shorter in <i>Itgal^−/−</i> (<i>t</i>-tests).</p

Genes having differential expression either between strains or among different embryonic days.

<p>Expression assay by NanoString nCounter. 3 RNA samples of pia at each time point for each strain, with each sample composing of ≥8 embryos from ≥2 litters (18 samples, ∼200 embryos). Transcript number for each gene normalized to mean transcript number for 6 housekeeping genes: <i>βactin</i>, Gapdh, <i>Tubb5, Hprt1, Ppia, Tbp</i>.</p>*<p>Gene number same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone-0031910-g006" target="_blank">Figure 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910.s010" target="_blank">Table S2</a>.</p>†<p><b>I</b>, genes in EMMA region; <b>II</b>, genes in 95% CI of Chr 7 QTL; <b>III</b>, angiogenic-related genes located elsewhere in genome; <b>IV</b>, proliferation-related genes located elsewhere in genome.</p>‡<p>Fold change for Bc vs B6 if positive and B6 vs Bc if negative.</p>§<p>Bonferroni-adjusted p values (p-value÷150, for 150 genes/splice forms assayed) from 2-way ANOVA for 2 strains (20 genes p<0.05) and 3 embryonic days (6 genes p<0.05).</p

Collateral extent in CXB11.HiAJ (CxB11) confirms importance of <i>Canq1</i>.

<p><b>A</b>, Inheritance patterns of <i>Cang1</i> on chromosome 7 in CxB11 recombinant inbred line derived from BALB/c (Bc)×C57BL/6 (B6). Y axis, SNP positions (Build 37). CxB11 inherits from B6 the segment (B6, grey; Bc, black) that includes the EMMA region and extends through <i>Itgal</i>. <b>B</b>, Collateral number is intermediate between B6 and Bc in CxB11 and significantly different from B6xBc-F1. <b>C</b>, Territory of cerebral cortex supplied by the ACA, MCA, and PCA trees. B6 and Bc are identical (confirms Zhang et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910-Zhang1" target="_blank">[9]</a>), while CxB11 has smaller MCA and PCA and larger ACA territory than B6. These differences do not correlate with variation in collateral number or diameter in these or 15 other strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910-Zhang1" target="_blank">[9]</a>. Numbers of mice given in parentheses in this and other figures.</p

Fold changes between BALB/c and C57BL/6 in mRNA levels of genes within <i>Canq1</i> and selected angiogenic- and proliferation/aging-related genes at embryonic day E14.5, E16.5 and E18.5.

<p>The numbers 1–150 at top denote gene numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone-0031910-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031910#pone.0031910.s010" target="_blank">Table S2</a>. Y-axis, fold changes; Bc vs B6 if positive, B6 vs Bc if negative. Genes 1–127 located in 95% CI of <i>Canq1</i> are arranged in order of physical location. <b>I</b>, genes within the EMMA region (132.356–132.528 Mb); <b>II</b>, genes in 95% CI of <i>Canq1</i>; <b>III</b>, angiogenic-related genes; <b>IV</b>, proliferation-related genes. Black curve, negative logarithm Bonferroni-adjusted p-values (p-value÷150) for strain effect in 2-way ANOVA; *p<0.05, **p<0.01, ***p<0.001 (÷150). Letters above bars denote significance derived from p-values for 2-way ANOVA for strain after Bonferroni inequality corrected <i>post-hoc</i> tests; A, p.strain<0.001, B, p.strain<0.01.</p

Phenotype_data_FINAL

see readm

Mst1 Directs Myosin IIa Partitioning of Low and Higher Affinity Integrins during T Cell Migration

<div><p>Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of <i>Mst1</i>-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. <i>Mst1</i> deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the anterior and posterior membrane during T cell migration.</p></div

Mst1 is dispensable for integrin-dependent T cell polarization by required for CCL19-induced migration.

<p><b>A</b>) Wt and <i>Mst1^h/h</i> CD4 T cells were seeded into slide chambers pre-coated with 100 ng/mL ICAM-1-Fc prior to stimulation with CCL19. Polarization of CD44 to the uropod in comparison to LFA-1 expression was visualized by confocal microscopy. <b>B</b>) Computational scoring of CD44 and LFA-1 clustering during live imaging of wt and <i>Mst1^h/h</i> CD4 T cells on ICAM-1 coated chamberslides stimulated for 30 minutes with 100 ng/mL CCL19 in presence of 0.08 ng/mL Alexa647-anti-CD11a/LFA-1 (M17/4) and Alexa488-anti-CD44. For each time point, 99–166 individual cells were analyzed for receptor clustering. Student's t-test was performed to compare clustering efficiency for Mst1^wt and Mst1^h/h T cells. <b>C</b>) Transmigration of purified wt and <i>Mst1^h/h</i> CD4 T cells in response to 100 ng/mL CCL19 through 3 μm or 5 μm pores pre-coated with BSA or ICAM-1 Fc. Data is displayed as mean ± SEM of triplicate samples in a single experiment representative of 3–5 independent experiments. Student's t-test was performed to compare migration efficiency for Mst1^wt and Mst1^h/h T cells, * p<0.002, ** p<0.0001. <b>D</b>) CCR7 expression was determined by flow cytometry.</p