Fig. 6

Subcellular mislocalization and defective ATR/Chk1 activation link to defects in HSPCs in the topbp1^cas003 mutants.

(A) Confocal imaging analysis of anti-FLAG immunostaining (green) and DAPI (blue) in HeLa cells transfected with FLAG tagged TopBP1^WT (WT), TopBP1^cas003 (cas003) and TopBP1^cas003-NLS (cas003-NLS). TopBP1^WT is mainly localized in the nucleus (left column), cytosol mislocalization of TopBP1^cas003 (middle column) can be corrected by the additional SV40 NLS on its C-terminus (TopBP1^cas003-NLS, right column). Scale bars represent 25 µm. (B-F) WISH analysis of hematopoiesis in siblings and topbp1^cas003 mutant embryos with Tol2-transposase mediated topbp1^WT and topbp1^cas003-NLS transgenesis at 4dpf, which are quantified in F (the numbers of embryos are shown above). Defective c-myb expression in topbp1^cas003 mutants can be rescued by topbp1^cas003–NLS as well as topbp1^WT. (B-C, B′-C′) 28/32 siblings and 12/15 topbp1^cas003 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 topbp1 morphants. The morphants were treated with 250mM HU or mock from 60hpf to 76hpf. topbp1 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 topbp1^cas003 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), cas003, cas003-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 topbp1^cas003 mutant embryos, quantitative analysis of the c-myb expression in the CHT region at 4dpf was performed for the evaluation of rescue capability (n>20). "+" (rescue); "-" (not rescue effect). Except WT and cas003-NLS, all TopBP1 mutation forms are unable to rescue hematopoietic defects in topbp1^cas003 mutants.