Fig. 7.

SSCs secrete mucin-like molecule(s). (A) Lectin blot for PNA conjugated to horseradish peroxidase (PNA-HRP) of an agarose gel loaded with samples (reduced and alkylated). Secretions from wild-type embryos with cement gland (WT + CG), put through a filter to remove species with molecular weight lower than 100 kDa, shows two bands positive for PNA (blue and red arrows). For secretions from wild-type embryos with cement gland excised (WT - CG), there is only a single band evident (red arrow). Cement gland lysate alone gave predominantly the upper band, whereas aspirating directly from the skin gave predominantly the lower band. (B) Lectin blot for PNA-HRP of unreduced (-DTT) and reduced (+DTT) samples taken from secretions of wild-type embryos with cement gland removed. Reduced samples migrate much further in the gel. (C) Lectin blot for PNA-HRP comparing levels of PNA positive material secreted into the media in control and foxa1 morpholino-injected embryos. There is a clear reduction in the lower band (red arrow), whereas the upper band (blue arrow) is of a similar level. (D) Chart showing results of CsCl density gradient centrifugation on samples obtained from the media of embryos with cement gland excised. Fractions of decreasing density were harvested and tested for PNA-reactivity in slot blots. Intensity measurements for PNA reactivity were recorded for each fraction and plotted as shown. (E) Timecourse of otogelin-like mRNA in situ hybridisation shows epidermal staining at four stages. There is strong staining in the SSCs at stage 32 (zoom, inset). (F) Otogelin-like (green) in situ hybridisation combined with PNA-Alexa-568 (red) staining, at stage 32, shows colocalisation in SSCs and goblet cells (at a lower level). Lower image is a magnification of the area in the box of the upper image. Scale bars: in E, 100 μm and 10 μm (stage 32 image, inset); in F, 100 μm (upper) and 20 μm (lower).