Expression of Zbtb16 protein and zbtb16a/b mRNA in the zebrafish hindbrain. (A,B) Wholemount fluorescence micrographs showing staining with anti-Zbtb16 antibody (see Materials and Methods) at 14 hpf and 24 hpf. (C,F,I) Immunofluorescent staining with anti-Zbtb16 antibody at 14, 16 and 24 hpf. C and F show slices from confocal z-stack, I shows sum projection through z-stack. n≥5 embryos per stage. (D,E,G,H,J-O) HCR for zbtb16a and zbtb16b at 14, 16, 24, 30 and 44 hpf. Sum projections through the z-stack. n≥5 embryos per stage. The Tg[egr2b:H2B-Citrine] line expresses Citrine in rhombomeres (r) 3 and 5. Arrows indicate boundaries between high and low levels of Zbtb16 protein or zbtb16a/b mRNA expression. MB, midbrain; MHB, midbrain-hindbrain boundary; op, otic placode; ov, otic vesicle. Scale bars: 50 µm.

Expression of Zbtb16 during neurogenesis. (A-L) Immunofluorescence for Zbtb16 and HuC/D at 24 and 44 hpf. Dorsal views (A-C,G-I) and transverse sections (D-F,J-L) of the hindbrain. G-I are coronal slices at the level of the mantle zone. Arrows indicate postmitotic neurons (A,B), neurogenic zones (G,H) or Zbtb16+HuC/D double-positive cells (J,L). (M-P) Immunofluorescence for Zbtb16, HuC/D and GFAP at 44 hpf. Side views of the hindbrain, anterior to the left. Arrows indicate Zbtb16-positive migrating progenitors (M) or GFAP-positive glial fibres (O). (Q-V) Two-colour fluorescent ISH for neurog1 and neurod4 (Q-S) or zbtb16a and neurod4 (T-V) at 44 hpf. Side views of the hindbrain, anterior to the left. Arrows indicate overlap of zbtb16a and neurod4 expression. All images are slices from confocal z-stacks. mz, mantle zone; ov, otic vesicle; vz, ventricular zone. Scale bars: 50 µm.

Spatial pattern of neurogenesis in Zbtb16 mutants. (A-F) Colorimetric ISH for neurod4 in 30 hpf sibling control (n=121) and zbtb16a^−/− (n=7) embryos (A,B); 30 hpf sibling control (n=18) and zbtb16a^−/−;zbtb16b^−/− (n=8) embryos (C,D); 44 hpf sibling control (n=8) and zbtb16a^−/−;zbtb16b^−/− (n=8) embryos (E,F). (G-L,O-T) HCR for etv5b, neurod4 and neurog1 in 30 hpf sibling control (n=16) and zbtb16a^−/−;zbtb16b^−/− (n=11) embryos (G-L); 44 hpf sibling control (n=9) and zbtb16a^−/−;zbtb16b^−/− (n=12) embryos (O-T). Sum projections through confocal z-stack. (M,N,U,V) Profile plots of etv5b, neurod4 and neurog1 HCR signal across the hindbrain of representative sibling control (zbtb16a^+/+;zbtb16b^−/−) and zbtb16a^−/−;zbtb16b^−/− embryos; anterior to the left. Scale bars: 50 µm.

Spatial analysis of Fgf signalling in Zbtb16 mutants. (A,B) Colorimetric ISH for etv5b in sibling control (A; n=59) and zbtb16a^−/−;zbtb16b^−/− (B; n=25) embryos. Dorsal views of the hindbrain, wholemount. Arrows indicate rhombomere centres. (C,D) ISH for etv5b in sibling control (C; n=18) and zbtb16a^−/− (D; n=6) embryos. (E,F) Sibling control (E; n=88) and zbtb16a^−/−;zbtb16b^−/− (F; n=28) embryos treated with DMSO from 22 to 24 hpf and stained for etv5b. (G,H) Sibling control (G; n=73) and zbtb16a^−/−;zbtb16b^−/− (H; n=38) embryos treated with 2.5 µM SU5402 from 22 to 24 hpf and stained for etv5b. Three independent experiments. (I-L′) HCR for etv5b in 24 hpf; sibling control (n=14) and zbtb16a^−/−;zbtb16b^−/− (n=9) mutant embryos. 3D reconstructions of the z-stack, dorsal view (I,J); confocal z-stack slices (K-L). Arrows in I-L′ indicate the medial wall of the otic vesicle. (M,N) Colorimetric ISH for pax5 in 24 hpf sibling control (n=10) and zbtb16a^−/−;zbtb16b^−/− (n=11) embryos. (O,P) ISH for pax5 in 36 hpf sibling control (n=29) and zbtb16a^−/−;zbtb16b^−/− (n=11) embryos. Arrows in M-P show the antero-posterior extent of marker gene expression in the otic vesicle. MHB, midbrain-hindbrain boundary; ov, otic vesicle; r, rhombomere. Scale bars: 50 µm.

Expression of Fgf ligands in Zbtb16 mutants. (A,B) HCR for fgf20a in 24 hpf sibling control (n=26) and zbtb16a^−/−;zbtb16b^−/− (n=12) embryos. 3D reconstructions of the z-stack, dorsal view. (C,D) Colorimetric ISH for fgf20b in 24 hpf sibling control (n=15) and zbtb16a^−/−;zbtb16b^−/− (n=4) embryos. Arrows in A-D indicate fgf20-expressing neuronal clusters in rhombomere centres. (E-T) ISH time-course of fgf3 expression in the sibling control (14 hpf: n=19; 16 hpf: n=26; 18 hpf: n=53; 24 hpf: n=47; 30 hpf: n=10; 36 hpf: n=35; 44hpf: n=20; 48 hpf: n=43) and zbtb16a^−/−;zbtb16b^−/− (14 hpf: n=6; 16 hpf: n=10; 18 hpf: n=20; 24 hpf: n=12; 30 hpf: n=5; 36 hpf: n=13; 44 hpf: n=10; 48 hpf: n=12) hindbrain. Arrows in H indicate ectopic expression in r5/r6, arrow in L indicates ectopic expression in the posterior otic vesicle. MHB, midbrain-hindbrain boundary; r, rhombomere. Scale bars: 50 µm.

Partial rescue of the Zbtb16 mutant phenotype by Fgf3 knockdown. (A-B′) Colorimetric ISH for etv5b in sibling control (n=88) and zbtb16a^−/−;zbtb16b^−/− (n=40) embryos injected with standard control+p53 MO. (C-D′) ISH for etv5b in sibling control (n=83) and zbtb16a^−/−;zbtb16b^−/− (n=35) embryos injected with Fgf3 translation blocking MO+p53 MO. Three independent experiments. Dorsal views of the hindbrain, wholemount. r, rhombomere. Scale bars: 50 µm.

Early misexpression of Zbtb16 downregulates fgf3 in rhombomere 4. (A-C) Wholemount fluorescence micrographs of the tailbud of a wild-type embryo injected in one half with mRNA encoding myc-Zbtb16a and stained with anti-Zbtb16 and anti-Myc antibodies. No endogenous Zbtb16 staining is detected at this axial level at 13-14 hpf. (D-F) ISH for fgf3 followed by anti-Myc immunofluorescent staining in wild-type embryos (n=198) injected in one half with mRNA encoding myc-Zbtb16a. (G-I) ISH for fgf3 in wild-type embryos (n=126) injected in one half with mRNA encoding Citrine, showing Citrine fluorescence. Three independent experiments. MHB, midbrain-hindbrain boundary; r, rhombomere. Numbers (D,G; bottom left) indicate numbers of embryos that show dissimilar (D) or similar (G) levels of fgf3 expression in the left and right halves of r4. Scale bars: 50 µm.