A-F. Sections of embryos stained for ctslb mRNA and tp63 protein. Hatching gland precursor cells are organized in a multi-layered cluster that is located below basal keratinocytes at 10 hpf (A) but above basal keratinocytes at 14 hpf (B) and 16 hpf (C). At 18 hpf, the multi-layered cluster has started to rearrange to a mono-layered organization, which is most advanced at the anterior side of the anlage, where hatching gland cells have started to spread onto the yolk sac (D). At 24 hpf, hatching gland cells are organized in single layer that is a continuous sheet in wild type (E) but has gaps in spint2 morphant (F). G-H. Sections of embryos transgenic for the peridermal marker Tg(krt4:egfp) stained for ctslb mRNA and eGFP protein at 24 hpf. Hatching gland cells are located below the peridermal cells in wild-type (G) and morphant (H) embryos. I–K. TEM sections of wild-type embryos at 16 hpf showing hatching gland cells (HGC, false-colored in blue) in between peridermal cells (PC, false-colored in green) and basal keratinocytes (BC, false-colored in red) at the leading edge of the sheet (I) and at the transition from a bi-to the mono-layered state (J,K). L. Stills from a 33 min DIC time-lapse recording (Supplementary movie 1) of the top layer of hatching gland cells next to the head region, starting at 16 hpf. False-coloring of a subset of hatching gland cells reveals cells intercalating from below. M. Schematic illustrating intercalation and migration events of hatching gland cells around 16 hpf. Hatching gland cells (HGCs) are marked in blue, peridermal cells (PCs) in green, and basal cells (BCs) in red.
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