Fig. 8

Fig. 8 A. Overlay of DIC and fluorescent image of live 24 hpf wild-type host, in which endogenous mesendoderm was chemically suppressed, and which was transplanted with polster cells (precursors of hatching gland cells) of a wild-type donor labeled with membrane-bound GFP (via transgene Tg(Ola.Actb:Hsa.hras-egfp)). B. Magnified view of fluorescently labeled hatching gland cells in A. C. Magnified view of fluorescently labeled wild-type hatching gland cells whose precursors had been transplanted into a spint2 morphant host, as in A. D. Magnified view of fluorescently labeled spint2 morphant hatching gland cells whose precursors had been transplanted into a wild-type host, as in A. E. Stills of a time-lapse recording of GFP-labeled spint2 morphant donor cells in a wild-type host at 30 hpf in 5 ​min intervals. Red arrowhead points to a cell undergoing cell death. F–I. Immunofluorescence of Cdh1 (E-cadherin) in hatching gland cells at 18 hpf. In whole mounts, cell contacts between wild-type hatching gland cells show continuous Cdh1 (red) localization (F), whereas in spint2^fr49/fr49 mutants, Cdh1 localization is disrupted (G). Immunofluorescence on sections showing comparable Cdh1 levels (green) at apical and basal membranes of wild-type (H) and spint2 morphant (I) embryos but reduced Cdh1 levels at lateral membranes (indicated by arrowheads) between hatching gland cells of spint2 morphant. J. Tukey box and whiskers plot showing a decreased ratio of the mean intensity of CDH1 on lateral versus apical cell junctions of HGCs in spint2 morphant embryos compared to wild-type controls; N ​= ​3 embryos, n ​= ​36–38; ∗∗∗ indicates p value of 0.0002 determined via Student’s t-test. K–N. Transmission electron microscopy (TEM) sections of wild-type (K) and spint2 morphant (L) embryos at 16 hpf, and of wild-type (M) and spint2fr49/fr49 mutant (N) embryos at 19 hpf, with intercellular spaces false-colored in red. Interspaces between hatching gland cells (HGC) and peridermal cells (PC) of spint2 morphant and wild-type control are unaltered (K,L), whereas interspaces between adjacent hatching gland cells (HGC) are increased in spint2^fr49/fr49 embryo compared to wild-type sibling (M,N).