Fig. 1

Mucus is transported from the surface into club cells. a Oblique section through the skin of an adult zebrafish, after 2 h of incubation in Alexa594 WGA (magenta). Cells are visualized by SYTO 9 (green), which binds nucleic acids. WGA binding is visible on the surface (arrow), and in goblet cells (red arrowhead). b Reconstructed transverse section through a z-stack of the fish in a. WGA (magenta) is restricted to the surface. c Oblique section through the skin of a fish, after 2 h label in WGA followed by 2 days in clean water. Punctate label is visible in superficial cells (white arrow), goblet cells (red arrowheads) and in club cells (yellow arrowheads; see inset). d Oblique section through another fish after transient label with WGA and a 2-day chase, showing label in elongated cells (white and cyan arrowheads). e, f Reconstructed transverse sections through the z-stack of the fish in d, showing the location of the labeled cell (white arrowhead) below a club cell. A superficial goblet cell is indicated (red arrowhead). g An Alexa594 WGA labeled cell (magenta; white arrowhead) inside a club cell. The club cell ER is indicated by the yellow arrowhead. h, i Reconstructed transverse sections through the club cell with the second cell. j A SYTO 9 labeled fish. The white arrowhead indicates a second cell inside the club cell. The club cell nucleus (yellow arrowhead) is morphologically distinct. k, l Transverse sections through the club cell. m–o Alexa-568 Phalloidin labeling to visualize f-actin. The club cell contains a second cell (white arrowhead) with cortical labeling. n, o Transverse sections through the club cell. p–x Two different focal planes of the skin of a zebrafish, following transient label of mucus by Alexa594-WGA and 2 days incubation in clean water. p–r show a deeper plane of focus, with a WGA-containing cell that may be blebbing (yellow arrowheads); s–u show a shallower plane with the club cell nucleus visible (cyan arrowhead). t The cytoplasm of the club cell is filled with Alexa594 WGA (red arrowhead). v–x Reconstructed transverse view through the z-stack. The club cell nucleus is indicated by the cyan arrowhead, while the WGA labeled cell is indicated by the yellow arrowhead. Club cells without internalized cells are indicated by the green arrowhead. Scale bar = 10 µm