Figure 3

Figure 3

CRISPR/Cas9 targeting of the pax5 gene and features of leukemic E::R;pax5mut fish. (a) Structure of the zebrafish Pax5 protein has five conserved functional domains: paired (PD), octapeptide (OP), homeo (HD), transactivation (TAD), and inhibitory (ID) domains. Brackets indicate the boundaries of the eleven encoding exons. Red arrowheads mark the Cas9 cut positions in exon 3 and exon 5, and the respective sequences of the sgRNA target sites are shown. (b) Phenotypic and histological analysis of E::R;pax5mut zebrafish. Images depict lateral views of representative control UAS:GFP zebrafish (b(I)) and leukemic zebrafish (b(II)). The E::R;pax5mut leukemic zebrafish developed subcutaneous bleedings in the ventral body region (highlighted by a red frame in b(II)). An inset provides a close-up view of the bleeding (marked with red asterisks) (b(II)). Top views of the entire kidneys of UAS:GFP and E::R;pax5mut leukemic zebrafish are shown (b(III–IV)). The kidney of the leukemic zebrafish exhibited enlargement along its entire length (b(IV)). Giemsa staining of a peripheral blood smear from the UAS:GFP fish (b(V)) revealed normal nucleated erythrocytes, while Giemsa staining of the leukemic blood smear (b(VI)) showed the presence of clusters of lymphoblasts. Hematoxylin and Eosin staining of the sagittal paraffin sections of tissues from UAS:GFP fish (b(VII,IX,XI,XIII)) and leukemic fish (b(VIII,X,XII,XIV)) highlighted the presence of lymphoblasts in the kidney marrow (b(VIII)), muscle tissue (b(X), arrows), epidermis (b(XII), arrows), and liver (b(XIV), arrows). Scale bars: (b(V,VI)): 10 µm; (b(VII–XIV)): 100 µm. (c) Flow cytometry plot (right) showing cell populations separated by their light scatter characteristics in the whole kidney marrow of leukemic E::R;pax5mut fish, along with the UAS:GFP control plot (left). The predominant cell population in the leukemic fish exhibited light scatter characteristics similar to the precursor cell fraction in the control group. (d) Western blot analysis was performed using protein extracted from kidney marrow cells of UAS:GFP fish, non-leukemic E::R knock-in fish, and 6 out of 10 leukemic E::R;pax5mut fish (#4–#9). Primary antibodies targeting human RUNX1 and β-Actin were used. E::R protein was detected in all tumor samples but not in the samples from the UAS:GFP control or non-leukemic E::R knock-in fish. The uncropped blots are shown in Supplementary Materials.