Normal contribution of <i>Hnrpll</i>^thu/thu NKT cells, but a cell autonomous decrease in NK1.1 in the mixed bone marrow chimeras.

<p>Irradiated C57BL/6 mice were reconstituted with an equal mixture of CD45.1-marked wild-type bone marrow and CD45.2-marked <i>Hnrpll</i>^thu/thu bone marrow cells. After hemopoietic reconstitution, recipient mice were analysed for B, NKT and conventional T cell populations. (A) Graph shows the ratio of CD45.2⁺ wild type and <i>Hnrpll</i>^thu/thu derived T cells to CD45.2⁺ wild type and <i>Hnrpll</i>^thu/thu derived B cells in the spleen of chimeric recipient mice. (B) Graph shows the ratio of CD45.2⁺ wild type and <i>Hnrpll</i>^thu/thu derived NKT cells to CD45.2⁺ wild type and <i>Hnrpll</i>^thu/thu derived B cells in the spleen of chimeric recipient mice. (C, D) Representative overlay histograms comparing the expression of NK1.1 on wild type (shaded grey) and <i>Hnrpll</i>^thu/thu (black line) derived NKT cells in the (b) thymus and (c) spleen of individual recipient mice from mixed bone marrow chimeras. Graphs show the geometric mean florescence intensity (MFI) of NK1.1 in the CD45.2⁺ wild type or <i>Hnrpll</i>^thu/thu derived NKT cells in the (b) thymus and (c) spleen of individual recipient mice from mixed bone marrow chimeras. (n = 6 for wild type and n = 5 for <i>Hnrpll</i>^thu/thu). Bars represent the mean ± s.d. values.</p

hnRNPLL is required for the splicing of CD45 isoforms in NKT cells.

<p>Representative overlay histograms of (A) CD45 and (B) CD45RA, CD45RB and CD45RC expressions on wild type (+/+) (shaded grey) and <i>Hnrpll</i>^thu/thu (thu/thu) (black line) CD4 T cells and NKT cells in the thymus (Data are representative of two independent experiments with n = 2–3 mice per group in each).</p

The <i>Hnrpll</i> mutation does not affect the up-regulation of CD122 in the developmental stages of NKT cell.

<p>(A) Representative overlay histograms showing the expression of CD122 on the surface of NKT cells from the thymus of a wild type and <i>Hnrpll</i>^thu/thu mouse. (B) Representative overlay histograms comparing CD122 expression on stage 1 (CD44^lo NK1.1^lo), stage 2 (CD44^hi NK1.1^lo) and stage 3 (CD44^hi NK1.1^hi) NKT cells in the thymus in wild type (left) and <i>Hnrpll</i>^thu/thu mice (right). (C) Graph shows the geometric mean fluorescence intensity (MFI) of CD122 expression on stage 2 and 3 NKT cells and DP thymocytes in the thymus and NKT cells and TCRβ⁺ cells in the spleen. Bars represent the mean value of each group ± s.d. from one of two independent experiments with a group of n = 3–5 mice per group in each. (D) <i>In vitro</i> survival of NKT cells culture in the presence of varying concentrations of IL-15. Data shows the percentage of viable cells by 7-AAD exclusion after 3 days of culture for wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) thymic NKT cells. Graph from one of three independent experiments represents the average of viable cells recovered from duplicate cultures with 2 or 3 mice per group.</p

The <i>Hnrpll</i> mutation does not disrupt cytokine production by thymic, splenic and hepatic NKT cells.

<p>(A) Representative histograms showing intracellular cytokine production by α-GalCer-CD1d tetramer⁺ TCRβ⁺ NKT cells from wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) mice that were either resting (shaded grey) or following activation with PMA and ionomycin (black line). (B) Frequency of cytokine producing cells of total NKT cells in the thymus of wild type and <i>Hnrpll</i>^thu/thu mice. (C) Frequency of cytokine producing cells of total NKT cells in the spleen of wild type and <i>Hnrpll</i>^thu/thu mice. (D) Frequency of cytokine producing cells of total NKT cells in the liver of wild type and <i>Hnrpll</i>^thu/thu mice. Bars represent the mean value of each group ± s.d. from one of two independent experiments with a group of n = 3–5 mice per group in each.</p

Normal numbers of NKT cell in the thymus and periphery of <i>Hnrpll</i>^thu/thu mice despite of reduced number of conventional T cells in the periphery.

<p>(A) Graphs show absolute number of CD4 and CD8 conventional T cells in wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) mice (n = 9 for wild type and n = 8 for <i>Hnrpll</i>^thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values. (B) Representative flow cytometric dot plots of NKT cells (α-GalCer-loaded CD1d tetramer⁺ TCRβ⁺) in the thymus (Thy) and spleen (Spl) in wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) mice. (C) Graph shows absolute number of NKT cells in the different tissues of wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) mice (n = 12 for wild type and n = 11 for <i>Hnrpll</i>^thu/thu analysed in three independent experiments). Bars represent the mean ± s.d. values. (D) Graph shows the ratio of absolute number of NKT cells to absolute number of TCRβ⁺ conventional T cells (n = 12 for wild type (+/+) and n = 11 for <i>Hnrpll</i>^thu/thu (thu/thu) analysed in three independent experiments). Bars represent the mean ± s.d. values. (E) Representative flow cytometric dot plots comparing the expression of CD4 versus TCRβ on CD1d-tetramer⁺ TCRβ⁺ NKT cells in the thymus, and spleen of wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) mice. (F) Graph shows absolute number of CD4⁺ and CD4⁻ NKT cell subsets in the thymus, spleen and liver of individual wild type (+/+) and <i>Hnrpll</i>^thu/thu (thu/thu) mice (n = 9 for wild type and n = 8 for <i>Hnrpll</i>^thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values.</p

Phenotypic defects observed in homozygous mice from the <i>Etv5</i> allelic series.

<p>n/a: not analysed due to embryonic lethality.</p

Levels of <i>Etv5</i> mRNA in postnatal day 3 testes (A), kidney (B) and spleen (C) in <i>Etv5^sco/sco</i> and wild-type (WT) mice.

<p>(D) ETV5 immunoblotting showed an absence of full length and truncated proteins in the mutant spleen. HPRT was used as a loading control. Levels of <i>Cxcr4</i> (E) and <i>Ccl9</i> (F) mRNAs in <i>Etv5^sco/sco</i> and WT postnatal day 3 testes. A–C, E–F, WT values were set as 100%. <i>n</i> = 3 mice per group, **p&lt;0.01, ***p&lt;0.001 (unpaired <i>t</i>-test, two-tailed).</p

Incidence of developmental abnormalities in <i>Etv5^sco/sco</i> adult mice.

<p>Incidence of developmental abnormalities in <i>Etv5^sco/sco</i> adult mice.</p

The SCO mouse line carries a missense mutation within the <i>Etv5</i> gene.

<p>(A) Schematic of the mouse <i>Etv5</i> gene and the location of the identified mutation. Exon positions are based on the ENSMUST00000079601 transcript. (B) ETV5 protein and the location of the missense amino acid.</p

<i>Etv5</i> mutant (<i>Etv5^sco/sco</i>) males are sterile due to the progressive loss of germ cells.

<p>(A) Testis of 8 weeks-old <i>Etv5^sco/sco</i> and a wild-type (WT) littermate. (B) Testis weight to body weight ratio of <i>Etv5^sco/sco</i> mice compared to WT littermates. <i>n</i> = 12 per group. *p&lt;0.05 (unpaired <i>t</i>-test, two-tailed). (C–F) PAS staining of 4–8 weeks-old testes of <i>Etv5^sco/sco</i> and WT mice.</p