bsx expression depends on Nkx2.1, Nkx2.4, and Otp transcription factors. (a–l) Lateral views of wildtype and mutant (genotypes as indicated above panels) 4 dpf (a–d), 3 dpf (e–h,k,l) or 2 dpf (i,j) embryos, stained by in situ hybridization for bsx expression. All images are minimum intensity projections of 50 brightfield focal planes (distance 1 μm). Anterior is to the left. Scale bar (a–l): 100 μm. The ratio of observed phenotype and total analyzed embryos per genotype [n (phenotype)/n (total)] is indicated in right bottom corner of each panel. Arrowheads indicates bsx expression in the telencephalic septal region; small arrows indicate bsx expression which might correspond to “PTv” region; asterisk in (d) indicates tissue which might correspond to residual basal hypothalamus (bHyp) but which was not confirmed through marker gene expression in nkx2.1, nkx2.4a, nkx2.4b triple mutants; asterisks in (j,l) indicate ectopic bsx expressing cells. (m) Schematic drawing of a lateral view of bsx expressing forebrain regions of a zebrafish embryo 3–4 dpf illustrating which of the bsx expression domains depend on Otp or Nkx2.1 and Nkx2.4 transcription factors as indicated by color code. Anterior at left. For abbreviations see list.

bsx expression within the bHyp in relation to expression of neural progenitor markers and neuropeptidergic genes. Dorsal view of confocal sections of zebrafish embryos at 2 dpf (a–b′′), 3 dpf (c–e′′) or 4 dpf (f–g′′) after double-fluorescent whole-mount in situ hybridization using probes as indicated. Maximum intensity Z-projections of 30 (a–b′′,d–d′′), 40 (f–g′′), 50 (c–c′′), or 70 (e–e′′) single confocal planes (1 μm steps) are shown. Schematics on the right show lateral view of the bHyp for 2–4 dpf embryos indicating which planes were selected for Z-projections in the panels indicated. Expression of bsx is detected further away from the ventricle [white dashed line in (a–b′′)] than expression of neuronal progenitor markers (a–b′′). bsx expression colocalizes in neurons expressing Th [anti-Th immunostain; (c–c′′)] or neuropeptidergic transcripts (d–g′′). Scale bar in (a) for all images: 50 μm. Schematics in the top right represents a model of the zebrafish forebrain highlighting the ventricular recesses. Anterior at left. For abbreviations see list.

Forebrain patterning is normal in bsx mutants. (a–p) Lateral views of 2 dpf zebrafish embryo heads stained by in situ hybridization using probes as indicated. No differences in expression of any marker was observed between wildtype and bsx mutant embryos. All images are minimum intensity projections of 40 brightfield focal planes (distance 1 μm). Anterior is to the left. Scale bar in (a) for all images: 50 μm. Anterior at left. Number n of embryos analyzed (wildtype) = 3 (a,e,h,i); 5 (b,m); 6 (c,p); 10 (d); 9 (f,n); 14 (g); 8 (j,k,o); 12 (l). n (bsx mutants) = 3 (a); 11 (b,g); 4 (c,j); 12 (d); 5 (e,l,o); 9 (f,n,p); 8 (h,i); 10 (k); 6 (m).

agrp and penka expression domains in the terminal tuberal hypothalamus are lost in bsx mutant embryos. Lateral (a,b,e,f) and dorsal views (c,d,g,h) of the ventral forebrain in 3 dpf embryos stained by in situ hybridization using probes as indicated. All images are minimum intensity projections of 40 brightfield focal planes (distance 1 μm). Scale bar in (a) for all images: 50 μm. Anterior at left. Schematics to the right show location of agrp (red) or penka (green) expressing cells as observed by stainings and indicate the focal planes which are shown in dorsal view pictures and schematics. Cells with Bsx-dependent expression are circled. Numbers n (phenotype shown)/n (total analyzed) as indicated.

vip, npb, and trh expression domains in mamillary hypothalamic regions are lost in bsx mutant embryos. Lateral (a,b,e,f,i,j) and dorsal views (c,d,g,h,k,l) of the ventral forebrain in 3 dpf embryos stained by in situ hybridization using probes as indicated. All images are minimum intensity projections of 40 brightfield focal planes (distance 1 μm). Scale bar in (a) for all images: 50 μm. Anterior at left. Schematics to the right show location of vip (blue), npb (magenta), or trh (red) expressing cells as observed by stainings and indicate the focal planes which are shown in dorsal view pictures and schematics. Cells with Bsx-dependent expression are circled. Numbers n (phenotype shown)/n (total analyzed) as indicated.

crhb, crhbp and uts1 expression domains in the secondary prosencephalon are lost in bsx mutant embryos. Lateral (a,b,e,f,k,l) and dorsal views (c,d,g–j,m,n) of the ventral forebrain in 3 dpf embryos stained by in situ hybridization using probes as indicated. All images are minimum intensity projections of 40 brightfield focal planes (distance 1 μm). Scale bar in (a) for all images: 50 μm. Anterior at left. Schematics to the right show location of crhb (blue), crhbp (magenta), or uts1 (green) expressing cells as observed by stainings and indicate the focal planes which are shown in dorsal view pictures and schematics. Cells with Bsx-dependent expression are circled. Numbers n (phenotype shown)/n (total analyzed) as indicated.

vmat2 expression, hdc expression and 5-HT immunoreactivity in the posterior recess region are reduced or absent in bsx mutant embryos. Lateral (a,b,e,f) and dorsal views (c,d,g,h) of the ventral forebrain in 3 dpf embryos stained by in situ hybridization using probes as indicated. (i,j) Dorsal view of the ventral forebrain in 3 dpf embryos immunostained using 5-HT (green) and HuC antibody (magenta). Images show minimum intensity projections of 40 brightfield focal planes (a–h; 1 μm steps) or maximum intensity projections of 40 confocal planes (i,j; 1 μm steps). Scale bars in (a) for (a–h) and in (i) for (i) and (j): 50 μm. Anterior at left. Schematics to the right show location of vmat2 (green) and hdc (red) expressing cells as observed in stainings and indicate the focal planes which are shown in dorsal view pictures and schematics. Cells with Bsx-dependent expression are circled. Numbers n (phenotype shown)/n (total analyzed) as indicated.

nts and nos1 expression in the posterior recess region is lost in bsx mutant embryos. Lateral (a,b,e,f) and dorsal views (c,d,g,h) of the ventral forebrain in 3 dpf embryos stained by in situ hybridization using probes as indicated. All images are minimum intensity projections of 40 brightfield focal planes (distance 1 μm). Scale bar in (a) for all images: 50 μm. Anterior at left. Schematics to the right show location of nts (red) or nos1 (green) expressing cells as observed in stainings and indicate the focal planes which are shown in dorsal view pictures and schematics. Cells with Bsx-dependent expression are circled. Numbers n (phenotype shown)/n (total analyzed) as indicated.

Distribution of Bsx-dependent neuronal cell types. Schematic representation of a sagittal view of the ventral forebrain in 3–4 dpf embryos showing bsx expression domains in gray. Only Bsx-dependent neuronal cells are shown and color coded as indicated. The number of symbols reflects the spatial extend of the affected domain but not cell numbers For abbreviations see list. Anterior at left.