RNA in situ hybridization shows foxg1a expression in developing and regenerating posterior lateral line tissue. (A-D) Wholemount RNA in situ hybridization of foxg1a in wild-type zebrafish posterior lateral line primordium at 28 hpf (A), and in neuromasts at 2 dpf (B), 5 dpf (C), and 8 dpf (D). (F-G) Wholemount RNA in situ hybridization of foxg1a in wild-type zebrafish neuromasts during regeneration following neomycin (NEO) exposure at 5 dpf. (E) 3 h post-NEO, (F) 1-day post-NEO, and (G) 3 days post-NEO. (H-J′) Confocal projections of wild-type neuromasts showing hair cells labeled with Tg(myo6:GFP) (green), foxg1a expression with HCR fluorescent in situ hybridization (red) and nuclei labeled with DAPI (blue) at 5 dpf (H), 1 day-post NEO exposure (I), and 3 days-post NEO (J). Quantification of HCR foxg1a fluorescence intensity in arbitrary units (A.U.) in the whole neuromasts (K), hair cells (hc; K′), and support cells (sc; K″). n=15 NMs (nine larvae) 5 dpf, n=16 NMs (nine larvae) 1 day-post NEO, and n=18 NMs (nine larvae) 3 days-post NEO). All data presented at mean±s.d. Kruskal–Wallis test with Dunn's multiple comparisons. Scale bars: 20 µm.

Loss of Foxg1a results in slower posterior lateral line primordium migration and delayed neuromast formation. (A-B″) Confocal projections of time lapse video of posterior lateral line migration primordium at 0, 453, and 906 min in heterozygous (A-A″) and foxg1a^a266 mutant embryos (B-B″). Scale bar: 100 µm. (C,D) Kymograph of time lapse video of posterior lateral line migration in heterozygous (C) and foxg1a^a266 mutant embryos (D). (E) Quantification of average primordium velocity during migration, n=7 embryos per condition. (F,G) Live images of Tg(myo6:GFP)-labeled neuromasts in heterozygous (F) and foxg1a^a266 mutant embryos (G) at 2 dpf, white arrow indicates location of truncated primordium migration and terminal cluster neuromasts at the end of the tail are indicated by tc. (H) Quantification of NM number at 2 dpf, n=12 embryos per condition. (I,J) Live images of Tg(myo6:GFP)-labeled neuromasts at 5 dpf in heterozygous (I) and foxg1a^a266 mutant larvae (J). (K) Quantification of NM number at 5 dpf, n=12 larvae per condition. Data presented as mean±s.d., Mann–Whitney U-test. Scale bars: 20 µm.

Loss of Foxg1a function reduces hair cell development and regeneration. (A) Timeline of FM1-43FX exposure and live imaging at 5 dpf. (B-C″) Live confocal projections of Tg(myo6:GFP)-labeled (green) and FM1-43FX-labeled (magenta) neuromasts in heterozygous (B-B″) and foxg1a^a266 mutant larvae (C-C″) at 5 dpf, white arrows indicate co-labeling of myo6:GFP and FM1-43FX and green arrowheads indicate only myo6:GFP labeling. (D-D″) Quantification of myo6(GFP)+ hair cells (D), FM1-43FX+ hair cells (D′), and the percentage of FM1-43FX+ hair cells (D″) at 5 dpf. n=17 neuromasts (11 larvae) heterozygous sibling and n=19 neuromasts (ten larvae) foxg1a^a266 mutants. (E) Timeline of FM1-43FX exposure and live imaging at 8 dpf. (F-G″) Live confocal projections of Tg(myo6:GFP)-labeled (green) and FM1-43FX-labeled (magenta) neuromasts in heterozygous (F-F″) and foxg1a^a266 mutant larvae (F-G″) at 8 dpf. (H-H″) Quantification of myo6(GFP)+ hair cells (H), FM1-43FX+ hair cells (H′), and the percentage of FM1-43FX+ hair cells (H″) at 8 dpf. n=16 neuromasts (eight larvae) heterozygous sibling and n=17 neuromasts (nine larvae) foxg1a^a266 mutants. (I) Time line of NEO-exposure, regeneration, and FM1-43 labeling. (J-K″) Live confocal projections of Tg(myo6:GFP)-labeled (green) and FM1-43FX-labeled (magenta) neuromasts in heterozygous (J-J″) and foxg1a^a266 mutant larvae (K-K″) following regeneration at 8 dpf. (L-L″) Quantification of Tg(myo6:GFP)+ hair cells (L), FM1-43FX+ hair cells (L′), and the percentage of FM1-43FX+ hair cells (L″) following regeneration. n=14 neuromasts (seven larvae) heterozygous sibling and n=15 neuromasts (eight larvae) foxg1a^a266 mutants. (M-N′″) Confocal projections of 8 dpf larvae showing hair cells labeled with α-Oto antibody (green), support cells labeled with α-Sox-2 antibody (red), and nuclei labeled with DAPI in heterozygous sibling (M-M′″) and foxg1a^a266 (N-N′″) neuromasts. (O-O″) Quantification of α-Oto+, α-Sox-2 +, and DAPI+ cells. n=19 neuromasts (ten larvae) heterozygous sibling and n=20 neuromasts (ten larvae) foxg1a^a266 mutants. (P-Q′″) Confocal projects of 8dpf larvae following NEO-exposure and regeneration showing hair cells labeled with α-Oto antibody (green), support cells labeled with α-Sox-2 antibody (red), and nuclei labeled with DAPI in heterozygous sibling (P-P′″) and foxg1a^a266 (Q-Q′″) neuromasts. (R-R″) Quantification of regenerated α-Oto+, α-Sox-2 +, and DAPI+ cells. n=17 neuromasts (nine larvae) heterozygous sibling and n=18 neuromasts (nine larvae) foxg1a^a266 mutants. Data presented as mean ±s.d., Mann–Whitney U-test. Scale bars: 20 µm.

BrdU incorporation in foxg1a mutants is reduced during neuromast maturation. (A) Timeline of BrdU incorporation between 2 dpf and 5 dpf. (B-C′″-E,F′″,H-I′″,L-M′″) Confocal projections of heterozygous sibling and foxg1a^a266 embryos expressing Tg(myo6:GFP) (green) following 24 h windows of BrdU (red) exposure and nuclei labeled with DAPI (blue) between 2-5 dpf. White arrows indicate representative myo6:GPF+ hair cells co-labeled with BrdU. Yellow arrowheads indicate representative neuromast cells labeled with BrdU. (B-B′″) 3 dpf heterozygous sibling and (C-C′″) foxg1a^a266 mutant neuromast exposed to BrdU from 2-3 dpf. (D-D″) Quantification of heterozygous sibling and foxg1a^a266 hair cells (D), total BrdU incorporation (D′), and percent of BrdU to hair cells (D″) at 3 dpf. n=17 neuromasts (nine larvae) heterozygous sibling, n=16 neuromasts (eight larvae) foxg1a^a266. (E-F′″) 4 dpf heterozygous sibling (E-E′″) and foxg1a^a266 mutant (F-F′″) neuromasts exposed to BrdU from 3-4 dpf. (G-G″) Quantification of heterozygous siblings and foxg1a^a266 hair cells (G), total BrdU incorporation (G′), and index of BrdU to hair cells (G″) at 4 dpf. n=19 neuromasts (nine larvae) heterozygous sibling, n=15 neuromasts (eight larvae) foxg1a^a266. (H-I′″) 5 dpf heterozygous sibling (H-H′″) and foxg1a^a266 mutant (I-I″) neuromasts exposed to BrdU from 4-5 dpf. (J-J″) Quantification of heterozygous siblings and foxg1a^a266 hair cells (J), total BrdU incorporation (J′), and index of BrdU to hair cells (J″) at 5dpf. n=15 neuromasts (eight larvae) heterozygous sibling, n=17 neuromasts (eight larvae) foxg1a^a266. (K) Timeline of NEO expose at 5 dpf, followed by 24 h of BrdU incubation and then regeneration through to 8 dpf and fixation. (L-M′″) Confocal projections at 3 days-post NEO treatment at 8 dpf; hair cells and labeled with Tg(myo6:GFP) (green), proliferating cells are labeled by BrdU-incorporation (red), and nuclei labeled with DAPI (blue) in heterozygous sibling (L-L′″) and foxg1a^a266 mutant (M-M′″) neuromasts. (N-N″) Quantification of hair cells (N), BrdU-labeled cells (N′), and % of BrdU+ hair cells (N″). n=21 neuromasts (7 larvae) per condition. All data presented at mean±s.d. Mann–Whitney U-tests. Scale bars: 20 µm.

Support cell populations are unaffected by loss of Foxg1a during development. (A) Schematic of a neuromast showing peripheral mantle cells (blue), dorsoventral cells (purple), and anterior-posterior cells (yellow). (B) Timeline of UV photo-conversion of nlsEos and fixation of larvae at 5 dpf. (C-C′) Confocal projection of photo-converted Tg(sfrp1a:nlsEos)-expressing support cells in 5 dpf heterozygous sibling (C) and foxg1a^a266 larvae (C′). (D) Quantification of Tg(sfrp1a:nlsEos)-positive dorsoventral support cells. n=14 neuromasts (eight larvae) heterozygous sibling, n=13 neuromasts (six larvae) foxg1a^a266. (E-E′) Confocal projection of Tg(sost:nlsEos)-expressing dorsoventral support cells in 5 dpf heterozygous sibling (E) and foxg1a^a266 larvae (E′). (F) Quantification of Tg(sost:nlsEos)-positive dorsoventral support cells. n=23 neuromasts (eight larvae) per condition. (G-G′) Confocal projections of FISH HCR for tnfsf10l3 in 5 dpf heterozygous sibling (G) and foxg1a^a266 larvae (G′). (H) Quantification of tnfsf10l3+ cells. n=17 neuromasts (eight larvae) heterozygous, n=11 neuromasts in foxg1a^a266. (I) Timeline of photo-conversion and regeneration to 8 dpf after NEO-exposure at 5 dpf. (J-J′) Confocal projection of photo-converted Tg(sfrp1a:nlsEos)-expressing support cells in 8 dpf heterozygous sibling (J) and foxg1a^a266 larvae (J′). (K) Quantification of Tg(sfrp1a:nlsEos)-positive support cells. n=22 neuromasts (eight larvae) heterozygous sibling, and n=27 neuromasts (ten larvae) foxg1a^a266. (L-L′) Confocal projection of photo-converted Tg(sost:nlsEos)-expressing dorsoventral support cells in 8 dpf heterozygous sibling (L) and foxg1a^a266 larvae (L′). (M) Quantification of Tg(sost:nlsEos)-positive support cells. n=28 neuromasts (ten larvae) heterozygous sibling, n=27 neuromasts (nine larvae) foxg1a^a266. (N-N′) Confocal projections of HCR fluorescent in situ for tnfsf10l3 in 8 dpf heterozygous sibling (N) and foxg1a^a266 larvae (N′). (O) Quantification of tnfsf10l3+ cells. n=20 neuromasts (ten larvae) heterozygous sibling larvae, and n=11 neuromasts in seven foxg1a^a266 larvae. All quantification data presented as mean±s.d. Mann–Whitney U-test. Scale bars: 20 µm.

RNA in situ hybridization of NM cell markers. DIC images of wholemount RNA in situ hybridization showing mRNA expression in NMs at 5 dpf in heterozygous sibling (het sib) and foxg1a^a266 larvae, lower lefthand numbers indicate the number of larvae with expression in neuromasts over the total number analyzed. Central cell markers: isl1a expression in 11/12 het sib (A) and 1/14 foxg1a^a266 larvae (A′), six1a expression in 13/13 het sib (B) and 12/12 foxg1a^a266 larvae (B′), six1b expression in 11/11 het sib (C) and 11/11 foxg1a^a266 larvae (C′), and gata2a expression in 7/7 het sib (D) and 7/7 foxg1a^a266 larvae (D′). Dorsoventral and anterior-posterior (DV/AP) cell markers: sost expression in 11/11 het sib (E) and 9/10 foxg1a^a266 larvae (E′) and tnfs10l3 expression in 11/11 het sib (F) and 7/8 fox1ga^a266 larvae (F′). Expression of Notch/Delta pathways markers: notch3 expression in 10/11 het sib (G) and 10/10 foxg1a^a266 larvae (G′), her7 expression in 4/7 het sib (H) and 5/7 foxg1a^a266 larvae (H′), her4.1 expression in 7/7 het sib (I) and 7/7 foxg1a^a266 larvae (I′), lfng expression in 4/6 het sib (J) and 6/6 foxg1a^a266 larvae (J′), deltaD expression in 10/12 het sib (K) and 10/10 foxg1a^a266 larvae (K′), and atoh1a expression in 11/12 het sib (L) and 7/8 foxg1a^a266 larvae (L′). Canonical Wnt pathway: wnt10a expression in 8/10 het sib (M) and 9/13 foxg1a^a266 larvae (M′), wnt2 expression in 6/6 het sib (N) and 7/7 foxg1a^a266 larvae (N′), and ctnnb1 expression in 6/6 het sib (O) and 7/9 foxg1a^a266 larvae (O′). Scale bars: 20 µm.

α-Islet1 antibody-labeled cells are reduced in foxg1a^a266 mutants during development and regeneration. Confocal projections of neuromasts showing hair cells labeled with myo6:GFP (green), central cells and hair cells labeled with α-Isl1 antibody (red), and nuclei labeled with DAPI (blue). Examples of hair cells that are both myo6:GFP+ and α-Isl1+ are marked with yellow arrowheads, hair cells that are myo6:GFP+ and α-Isl1- are marked by green arrows, and cells that are α-Isl1+ and myo6:GFP- are marked with white asterisks. (A-B″) Heterozygous sibling and foxg1a^a266 mutants neuromasts at 2 dpf. (C-C′″) Quantification of myo6:GFP+ hair cells, α-Isl1+ cells, cells labeled with both myo6:GFP and α-Isl1, and cells labeled with only α-Isl1, n=17 NMs (nine embryos) heterozygous siblings and 15 NMs (eight embryos) foxg1a^a266 mutants. (D-E″) Heterozygous sibling and foxg1a^a266 mutant neuromasts at 5 dpf. (F-F′″) Quantification of myo6:GFP+ hair cells, α-Isl1+ cells, cells labeled with both myo6:GFP and α-Isl1, and cells labeled with only α-Isl1, n=16 neuromasts (eight larvae) heterozygous siblings and 18 neuromasts (nine larvae) foxg1a^a266 mutants. (G) Timeline of NEO exposure at 5 dpf and fixation at 3 h-post NEO (5 dpf), 1 day-post NEO (6 dpf), 2 days-post NEO (7 dpf), and complete regeneration (8 dpf). (H-I″) Heterozygous sibling and foxg1a^a266 mutants neuromasts at 3 h-post NEO (5 dpf). (J-J′″) Quantification of myo6:GFP+ hair cells, α-Isl1+ cells, cells labeled with both myo6:GFP and α-Isl1, and cells labeled with only α-Isl1, n=20 neuromasts (ten larvae) heterozygous siblings and 15 neuromasts (eight larvae) foxg1a^a266 mutants. (K-L″) Heterozygous sibling and foxg1a^a266 mutant neuromasts at 1 day-post NEO (6 dpf). (M-M′″) Quantification of myo6:GFP+ hair cells, α-Isl1+ cells, cells labeled with both myo6:GFP and α-Isl1, and cells labeled with only α-Isl1, n=14 neuromasts (seven larvae) heterozygous siblings and 15 neuromasts (eight larvae) foxg1a^a266 mutants. (N-O″) Heterozygous sibling and foxg1a^a266 mutants neuromasts at 2 days-post NEO (7 dpf). (P-P′″) Quantification of myo6:GFP+ hair cells, α-Isl1+ cells, cells labeled with both myo6:GFP and α-Isl1, and cells labeled with only α-Isl1, n=11 neuromasts (seven larvae) heterozygous siblings and 13 neuromasts (eight larvae) foxg1a^a266 mutants. (Q-R″) Heterozygous sibling and foxg1a^a266 mutants neuromasts at complete regeneration NEO (8 dpf). (S-S′″) Quantification of myo6:GFP+ hair cells, α-Isl1+ cells, cells labeled with both myo6:GFP and α-Isl1, and cells labeled with only α-Isl1, n=17 neuromasts (nine larvae) heterozygous siblings and 19 neuromasts (nine larvae) foxg1a^a266 mutants. All data presented at mean±s.d., Mann–Whitney U-test. Scale bars: 20 µm.

α-Islet1 antibody and sost:nlsEos label differentially label regenerating hair cells. Confocal projections of neuromasts showing hair cells labeled with myo6:GFP (green), central cells and hair cells labeled with α-Isl1 antibody (red), and sost:nlsEos cells (blue). Examples of hair cells that are both myo6:GFP+ and sost:nlsEos are marked with cyan arrows, hair cells that are both myo6:GFP+ and α-Isl1+ are marked with yellow arrowheads, hair cells that are myo6:GFP+, α-Isl1+, and sost:nlsEos+ are marked by white arrows, and cells that are α-Isl1+ and sost:nlsEos+ are marked with purple asterisks. (A) Timeline of UV photoconversion of sost:nlsEos cells at 5 dpf, homeostasis for 3 days, and fixation at 8 dpf. (B-C′″) Heterozygous sibling and foxg1^a266 mutants neuromasts at 8 dpf. (Dⁱ-D^vii) Quantification of myo6:GFP+ hair cells (Dⁱ), α-Isl1+ cells (Dⁱⁱ), sost:nlsEos+ cells (Dⁱⁱⁱ) cells labeled with both myo6:GFP and α-Isl1 (D^iv), hair cells labeled with myo6:GFP and sost:nlsEos (D^v), cells labeled with sost:nlsEos and α-Isl1 (D^vi), and cells labeled with myo6:GFP, α-Isl1, and sost:nlsEos (D^vii), n=15 neuromasts (eight larvae) heterozygous siblings and 14 neuromasts (eight larvae) foxg1^a266 mutants. (E) Timeline of UV photoconversion of sost:nlsEos cells and NEO exposure at 5 dpf, regeneration for 3 days, and fixation at 8 dpf. (F-G′″) Heterozygous sibling and foxg1^a266 mutants neuromasts at 8 dpf. (Hⁱ-H^vii) Quantification of myo6:GFP+ hair cells (Hⁱ), α-Isl1+ cells (Hⁱⁱ), sost:nlsEos+ cells (Hⁱⁱⁱ) cells labeled with both myo6:GFP and α-Isl1 (H^iv), hair cells labeled with myo6:GFP and sost:nlsEos (H^v), cells labeled with sost:nlsEos and α-Isl1 (H^vi), and cells labeled with myo6:GFP, α-Isl1, and sost:nlsEos (H^vii), n=15 neuromasts (eight larvae) heterozygous siblings and 14 neuromasts (nine larvae) foxg1^a266 mutants. All data presented at mean±s.d. Significance was determined with Mann–Whitney U-tests. Scale bars: 20 µm.