|
Fig. 3 Notch signaling pathway plays a role in FEVPs proliferation at the LPM.
Loss of grl function by morpholino injection is shown in panels (A) to (D). Panels (A) and (D) are grl MO injected, panels (B) and (C) are uninjected fish, and panel C2 is control MO injected fish. In panels (A) and (B), still images of Tg(fli1a:EGFP) grl-MO injected (A) or (B) uninjected Tg(fli1a:EGFP) 16 som embryos are shown. These images were part of a series of images that were taken under a fluorescence microscope at every 10 min from 15 to 21 hpf, which was reconstituted to movie clips (Movie S1 & S2). The grl-MO injected (A) clearly shows fewer fli+ cells at 16 som. The same embryo group was also subjected to phospho-histone H3 antibody staining at 10 som (C & D). E shows quantification of number of phospho-histone H3+ cell at midline (C: center = inside 50 µm bar) in uninjected (UI, dark blue bar), control MO (cont-MO, green bar), gridlock MO (grl MO, light blue bar), and in the same samples at LPM (P: periphery = outside 50 µm bar). Error bars represent SEM (n = 8). NS - not significant and ** p<0.001. (F-H) are control MO-injected, Su(H)DBM (a gridlock blocker) mRNA-injected and notchICD mRNA injected embryos stained for etsrp at 10 som. The Su(H)DBM mRNA-injected embryo shows dramatic reduction of etsrp+ cells at 10 som (F) while notchICD mRNA injected embryo (H) shows induction of etsrp+ cells at 10 som compared to uninjected embryo (G). (I) qPCR analysis showed that KD of grl by MO or dominant negative (Su(H)DBM) approach resulted in reduction of transcript level of etsrp but not vegf and actin (Error bar is SEM, n = 3, p<0.001). (J-N) Embryos treated with indicated chemicals from 10-14 hpf, subjected to whole mount ISH for scl and etsrp markers, and flat mounted. (J-L) ISH for scl in posterior LPM of embryos treated with vehicle control DMSO (J) or the γ -secretase inhibitors DAPT (K) or WPE-III-31C [17] (L). Bracket indicates the midline convergence segment of the gamma loop of Gering et al. [54] The posterior scl expression domain is thickened, and expanded anteriorly in embryos treated with γ-secretase inhibitors. (M-N) ISH for etsrp in posterior LPM of embryos treated with vehicle control DMSO (M) or DAPT (N). Etsrp expression domain is expanded anteriorly. (O-P) Dorsal view of Su(H)DBM mRNA- injected embryo at 16 som (P) showed dramatic mispatterning of flk+ cells in trunk compared to that of uninjected embryo (O). (See also Figure S1 and Movie S1 and Movie S2). The numbers on the top right panel of all ISH embryos in this figure indicate number of embryos out of total number displaying that particular phenotype.