- Title
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Notch signaling restricts FGF pathway activation in parapineal cells to promote their collective migration
- Authors
- Wei, L., Al Oustah, A., Blader, P., Roussigné, M.
- Source
- Full text @ Elife
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rbpja/b morphants phenocopy mindbomb mutants. (A–B’) Confocal sections showing the expression of gfi1ab (red) at 48 hpf in control embryos (A, n = 33) and in two representative embryos injected with 4 ng of rbpja/b morpholinos (MO) (B, B’); sections are merged with nuclear staining (gray) to highlight the epiphysis (white circle) and parapineal (yellow circle). Embryo view is dorsal, anterior is up; scale bar = 10 µm. (C–D) Dot plots showing the number of Tg(dusp6:d2EGFP) positive parapineal cells (C) and the mean position of parapineal cells highlighted by nuclei staining (Topro-3) (D) at 32 hpf in embryos injected with 4 ng rbpja/b MO (orange dots; n = 36) or in controls (blue dots; n = 28). (E–F) Dot plots showing the mean position (E) or the number (F) of gfi1ab expressing parapineal cells in non-injected controls (blue dots, n = 33) or in rbpja/b MO injected embryos (orange dots, n = 46) at 48 hpf. Both the migration (distance from the midline; p-value=0.0001 in Welch t-test on absolute value) and laterality (left orientation of migration; p-value=0.0002 on Wilcoxon test) are affected in rbpja/b morphants (E). The number of gfi1ab+ cells is also increased in rbpja/b morphants (F, *** p-value<0.0001; Welch t-test). Data are representative of three (A–B’, E–F) or two experiments (C–D). |
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(A–B) Confocal sections showing the expression of gfi1ab (red) in embryos treated with DMSO (A; n = 24) or 100 µM LY411575 (B; n = 32) from 22 to 32 hpf and fixed at 48 hpf, merged with nuclear staining (gray). Embryo view is dorsal, anterior is up; epiphysis (white circle) and parapineal (yellow circle); scale bar = 10 µm. (C–D) Dot plots showing the number (C) and the mean position (D) of gfi1ab expressing parapineal (PP) cells at 48 hpf in embryos treated with DMSO (controls, blue dots, n = 47), with 30 µM LY411575 (light red dots, n = 31) or 100 µM LY411575 (red dots, n = 32) from 22 hpf to 32 hpf, with mean ± SEM; *** p-value=0.0003. (E–H) Confocal sections showing the expression of gfi1ab (red) merged with nuclear staining (gray) at 48 hpf, in wild-type (+/+) (E, F) or mib+/- embryos (G, H) treated with DMSO (E, n = 23 or G, n = 26) or with LY411575 (F, n = 25 and H, n = 34) from 22 to 32 hpf. (I–N) Upper panels show a schematic of the LY411575 treatment timeline (yellow box, 22 to 32 hpf for I-K or 8 to 22 hfp for dot plots L-N), and the time window corresponding to when the parapineal initiates its migration (blue box, 28 to 30 hpf). Dot plots showing the number (I, L) and the mean position (J, M) of gfi1ab expressing cells at 48 hpf, or the number of Tg(dusp6:d2EGFP) expressing cells at 32 hpf (K, N), in the parapineal of DMSO-treated wild-type (+/+, light blue dots; I-J, n = 23; L-M, n = 12; K, n = 28; N, n = 18), DMSO-treated mib+/- heterozygote (dark blue dots; I-J, ± = 26; L-M, n = 17; K, n = 21; N, n = 21), LY411575-treated wild-type ( light red dots; I-J, n = 25; L-M, n = 11; K, n = 23, N, n = 19) and LY411575-treated mib+/- (dark red dots, I-J, n = 34 or L-M, n = 16; K, n = 29, N, n = 26); each dot represents a single embryo. Mean ± SEM is shown in I, K, L, N; *** p-value<0.0001, in Wilcoxon test and Welch t-test. In J and M, there is no defect in migration per se (ns p-value in Welch t-test on absolute value) but LY411575 treatment from 8 to 22 hpf (M) triggers a laterality defect (increased number of embryos with a parapineal on the right). Data are representative of three (E–H, I–K) or two experiments (A–D and L–N). |
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(A–F) Confocal sections showing the expression of Tg(dusp6 :d2EGFP) (A-B, green), sox1a (C-D, red) or tbx2b (E-F, red) at 36 hpf, in control embryos (A, n = 16; C, n = 12; E, n = 5) or in Tg(hsp70l:Gal4), Tg(UAS:myc-notch1a-intra) embryos (B, n = 16; D, n = 17; F, n = 6) following a heat-shock (HS) at 26 hpf; sections are merged with nuclei staining (gray). (G–L) Dot plots showing the number (G) and the mean intensity fluorescence (H) of Tg(dusp6 :d2EGFP) expressing parapineal cells, the number of sox1a (I) and tbx2b (J) expressing parapineal cells, or the mean position of parapineal cells highlighted by Topro-3 nuclei staining (K) and tbx2b + parapineal cells (L) in controls (blue dots) or in NICD expressing embryos (orange dots) at 36 hpf following heat shock at 26 hpf. (M–R) Confocal sections showing the expression of sox1a (M–N), gfi1ab (O–P) or tbx2b (Q–R) (red) merged with nuclei staining (gray), at 48 hpf, in the epithalamia of control (M, n = 17; O, n = 27; Q, n = 17) or Tg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra) double transgenic embryos (N, n = 17; P, n = 25; R, n = 16), following heat-shock (HS) at 26 hpf. The expression of sox1a and gfi1ab is lost or decreased while tbx2b expression is unchanged in the parapineal of NICD expressing embryos. (S–X) Dot plots showing the number of sox1a (S), gfi1ab (T) and tbx2b (U) expressing parapineal cells at 48 hpf in controls (blue dots) or in embryos expressing NICD after heat shock at 26 hpf (orange dots) and the corresponding mean position of the cells (V–X) when expression was detected (number of sox1a + or gfi1ab+ cells > 0). In confocal sections, embryo view is dorsal, anterior is up; epiphysis (white circle) and parapineal gland (yellow circle); scale bar = 10 µm. Mean ± SEM is indicated on dot plots G-J and S-U; *** p-value<0.0001, * p-value<0.05 in Welch t-test and Wilcoxon test. For migration dot plots, p-value<0.01 (L) or p-value<0.001 (K and V–X) in pairwise Wilcoxon test and Welch t-test on absolute values. Data are representative of three (O, P, T, W) or two experiments (A–D, G–I, M–N, Q–R, S, U, V, X); data based on tbx2b expression at 36 hpf (E–F, J, L) represents one experiment. |
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