MLL is expressed on the yolk of lyz:MLL-ENL and lyz:MLL-AF9 injected embryos.

Diagram of the lyz:MLL-ENL/AF9 construct used in the study (A). The α-crystallin promoter drives expression of EGFP in the lens of lyz:MLL-ENL/AF9 injected embryos (B,C). MLL expression (all panels) was identified on the yolk of lyz:MLL-ENL and lyz:MLL-AF9 embryos (arrows) but was absent from controls at 30 hpf (D-F), 48 hpf (G-I), 72 hpf (J-L) and 7 dpf (M-O).

Accumulation of MLL positive cells on the yolk results from migration defects and not cardiovascular or hematopoietic defects.

Average heart rates of control and lyz:MLL-ENL injected embryos at 48 hpf (A, N = 10). MLL (yolk) and lyz expression in control (B,C) and MLL-ENL expressing embryos (D,E). The number of lyz positive cells in the CHT in control and lyz:MLL-ENL injected embryos at 72 hpf (F, N = 12–18). MLL/Tal1 (G-I, arrows–tal1, red arrowheads-MLL, 24 hpf) and MLL/runx1/cmyb (J-L, black arrowheads-runx1/cmyb, red arrowheads-MLL, 36 hpf) expression were analyzed in control (G,J, N = 15–30), lyz:MLL-ENL (H,K, N = 13–30), and lyz:MLL-AF9 (I,L, N = 17–25) embryos. Still images at 0, 5 and 10 minutes from spi1b:GFP timelapse videos for control (M) and lyz:MLL-AF9 (N) embryos. Statistical analysis was conducted using student’s t-test. * p<0.05. Anterior to the left.

MLL expression colocalized with mpx, spi1b, and mpeg expression on the yolk of lyz:MLL-ENL injected embryos at 48 and 72 hpf.

Expression of lyz, mpx, spi1b, and mpeg in control (A,C,E,G) and lyz:MLL-ENL injected (B,D,F,H) embryos at 48 hpf. Black arrows indicate expression in the CHT, arrowheads indicate expression on the yolk (A-H). The number of cells on the yolk expressing lyz, mpx, spi1b, and mpeg was assessed at 48 hpf (I, N = 10–46). MLL colocalization with lyz (0%), mpx (56.5% +/- 18.9%), spi1b (63.9% +/- 20.0%), and mpeg (71.4% +/- 16.1%) was determined at 48 hpf (J-M, N = 17–42) and with lyz (0%), mpx (61.8% +/- 19.7%), spi1b (80.7% +/- 14.7%), and mpeg (76.4% +/- 9.6%) at 72hpf (N-Q, N = 15–63). Blue, red, and purple bars indicate the percentage of cells stained on the yolk that were myeloid single positive (blue arrows), MLL single positive (red arrows), or myeloid marker/MLL double positive (purple arrows), respectively (J-Q). Statistical analysis was conducted using student’s t-test. * p<0.05, ** p<0.01, NS = not significant. Anterior to the left.

MLL expression colocalized with mpx, spi1b, and mpeg expression on the yolk of lyz:MLL-AF9 injected embryos at 48 and 72 hpf.

Expression of lyz, mpx, spi1b, and mpeg in control (A,C,E,G) and lyz:MLL-AF9 injected (B,D,F,H) embryos at 48 hpf. Black arrows indicate expression in the CHT, arrowheads indicate expression on the yolk (A-H). The number of cells on the yolk expressing lyz, mpx, spi1b, and mpeg was assessed at 48 hpf (I, N = 31–62). MLL colocalization with lyz (0%), mpx (41.9% +/- 14.0%), spi1b (47.1% +/-14.1%), and mpeg (53.4% +/- 13.6%) was determined at 48 hpf (J-M, N = 37–52) and with lyz (0%), mpx (36.6% +/- 15.4%), spi1b (49.8% +/- 12.0%), and mpeg (58.8% +/- 16.0%) at 72hpf (N-Q, N = 26–34). Blue, red, and purple bars indicate the percentage of cells stained on the yolk that were myeloid single positive (blue arrow), MLL single positive (red arrow), or myeloid marker/MLL double positive (purple arrow), respectively (J-Q). Statistical analysis was conducted using student’s t-test. * p<0.05, ** p<0.01, NS = not significant. Anterior to the left.

Myeloid targeted MLL-ENL induced expression of cdk9 and bcl2 on the yolk.

Cdk9 was expressed on the yolk of lyz:MLL-ENL injected embryos (B lateral; B’, ventral) compared to control embryos (A,A’). Quantitative analysis of cdk9 positive cells on the yolk (C, N = 28). Bcl2 expression was observed on the yolk of lyz:MLL-ENL injected embryos (E lateral; E’, ventral) compared to control embryos (D,D’). Quantitative analysis of bcl2 positive cells on the yolk (F, N = 35). Statistical analysis was conducted using student’s t-test. ** p<0.01. Anterior to the left.

Myeloid targeted MLL-AF9 induced expression of cdk9 and bcl2 on the yolk.

Cdk9 was expressed on the yolk of lyz:MLL-AF9 injected embryos (B lateral; B’, ventral) compared to no expression on the yolk in control embryos (A,A’). Quantitative analysis of cdk9 positive cells on the yolk (C, N = 31). Bcl2 expression was observed on the yolk of lyz:MLL-AF9 injected embryos (E lateral; E’, ventral) compared to control embryos (D,D’). Quantitative analysis of bcl2 positive cells on the yolk (F, N = 30). Statistical analysis was conducted using students t-test. ** p<0.01. Anterior to the left.

Dose dependent effect of Venetoclax and Flavopiridol on myelopoiesis.

Embryos were treated with 100nM or 500nM of Venetoclax (A-M) or 100nM and 500nM Flavopiridol (N-Z) and assessed for expression of lyz (A-C, N-P, N = 10), mpx (D-F, Q-S, N = 16–19), mpeg (G-I, T-V, N = 20), and cmyb (J-L, W-Y, N = 16–20) by counting the number of positive cells expressing each marker in the CHT (M,Z). Arrows indicate marker gene expression in the CHT. Statistical analysis was conducted using one-way ANOVA followed by Tukey HSD. * p<0.05, ** p<0.01, NS = not significant. Anterior to the left.

Venetoclax and Flavopiridol co-treatment significantly reduced MLL expression on the yolk of lyz:MLL-ENL and lyz:MLL-AF9 injected embryos.

Lyz:MLL-ENL injected embryos were incubated with 200nM DMSO (A, N = 82), 200nM Venetoclax (B, N = 76), 200nM Flavopiridol (C, N = 84) or 200nM Venetoclax and Flavopiridol (D, N = 98) at 24 hpf and the number of MLL positive cells was assessed at 72 hpf (E). Lyz:MLL-AF9 injected embryos were incubated with 200nM DMSO (F, N = 96), 200nM Venetoclax (G, N = 105), 200nM Flavopiridol (H, N = 102) or 200nM Venetoclax and Flavopiridol (I, N = 95) at 24 hpf and the number of MLL positive cells was assessed at 72 hpf (E,J). Lyz:MLL-ENL (K,L) and lyz:MLL-AF9 (M,N) injected embryos were assessed for expression of tp53 and mcl1a by qPCR (N = 2). Statistical analysis was conducted using one-way ANOVA followed by Tukey HSD. * p<0.05. Anterior to the left.

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