Subcellular mislocalization and defective ATR/Chk1 activation link to defects in HSPCs in the <i>topbp1</i>^{<i>cas003</i>} mutants.

<p>(A) Confocal imaging analysis of anti-FLAG immunostaining (green) and DAPI (blue) in HeLa cells transfected with FLAG tagged TopBP1^{<b><i>WT</i></b>} (WT), TopBP1^{<b><i>cas003</i></b>} (<i>cas003</i>) and TopBP1^{<b><i>cas003-NLS</i></b>} (<i>cas003</i>-NLS). TopBP1^{<b><i>WT</i></b>} is mainly localized in the nucleus (left column), cytosol mislocalization of TopBP1^{<b><i>cas003</i></b>} (middle column) can be corrected by the additional SV40 NLS on its C-terminus (TopBP1^{<b><i>cas003-NLS</i></b>}, right column). Scale bars represent 25 μm. (B-F) WISH analysis of hematopoiesis in siblings and <i>topbp1</i>^{<b><i>cas003</i></b>} mutant embryos with Tol2-transposase mediated <i>topbp1</i>^{<b><i>WT</i></b>} and <i>topbp1</i>^{<b><i>cas003-NLS</i></b>} transgenesis at 4dpf, which are quantified in F (the numbers of embryos are shown above). Defective <i>c-myb</i> expression in <i>topbp1</i>^{<b><i>cas003</i></b>} mutants can be rescued by <i>topbp1</i>^{<b><i>cas003–NLS</i></b>} as well as <i>topbp1</i>^{<b><i>WT</i></b>}. (B-C, B’-C’) 28/32 siblings and 12/15 <i>topbp1</i>^{<b><i>cas003</i></b>} mutants show indicated WISH results. (D-E, D’-E’) WISH results of well rescued mutants. (B’-E’) Enlarged views of CHT region in the left column. (G) Western blot with pChk1 antibody in control morphants and hydroxyurea (HU) treated control or <i>topbp1</i> morphants. The morphants were treated with 250mM HU or mock from 60hpf to 76hpf. <i>topbp1</i> knockdown could abrogate the Chk1 phosphorylation in the tail region upon HU treatment. (H) Immunoblotting analysis showing reduced phospho-Chk1 level in tail region of <i>topbp1</i>^{<b><i>cas003</i></b>} mutants upon HU treatment, comparing to that in wild-type siblings. The embryos were treated with 250mM HU or mock from 60hpf to 76hpf. (I) Schematic diagram of variant forms of TopBP1, including wild-type (WT), <i>cas003</i>, <i>cas003</i>-NLS, ΔAAD, W1156R, R122E and R669E mutation. The regions associated with ATR activation and Rad9 or MDC1 interactions are indicated. After Tol2-mediated transient transgenesis of variant forms of TopBP1 into <i>topbp1</i>^{<b><i>cas003</i></b>} mutant embryos, quantitative analysis of the <i>c-myb</i> expression in the CHT region at 4dpf was performed for the evaluation of rescue capability (n>20). “+” (rescue); “-” (not rescue effect). Except WT and <i>cas003</i>-NLS, all TopBP1 mutation forms are unable to rescue hematopoietic defects in <i>topbp1</i>^{<b><i>cas003</i></b>} mutants.