Dubaissi E, Rousseau K, Lea R, Soto X, Nardeosingh S, Schweickert A, Amaya E, Thornton DJ, Papalopulu N.

097820/Z/11/Z Wellcome Trust , G1000450 Medical Research Council , WT 088785/Z/09/Z Wellcome Trust , WT 090868Z/09/Z Wellcome Trust , Wellcome Trust

	Fig. 1. foxa1 marks a new epidermal cell type. (A) Double fluorescence in situ hybridisation and antibody staining for a ciliated cell marker (α-1-tubulin, red), an ionocyte marker (atp6v1a, green) and a goblet cell marker (anti-Xeel, grey) on stage 32 embryos. DAPI (blue) is used to mark each cell. At least one cell type (yellow circles) is not stained by these markers. (B) Chromogenic in situ hybridisation for foxa1 on stage 25 embryos shows scattered, spotted epidermal distribution. (C) foxa1 is epidermally expressed in a subset of DAPI-positive cells. (D) foxa1 expression (grey) completes the epidermal staining when added to markers of the other cell types at stage 32. Scale bars: 20 μm in A,C,D; 500 μm in B.
	Fig. 2. foxa1 cells intercalate from inner to outer layer at mid-tailbud stages. (A) Sections of stage 22 and stage 32 embryos stained for DAPI (blue) and foxa1 (red) by fluorescent in situ hybridisation shows that expression changes from inner to outer layer as the embryo develops. (B) Transplant of MR-labelled (red) outer layer epidermal tissue on to FLDX-labelled (green) host embryo and stained using antibodies for ciliated cell marker, acetylated α-tubulin (AcTub, blue) and ionocyte marker, V1a (grey) at stage 32. A cell type (yellow arrows) intercalates from inner to outer layer in addition to ciliated cells and ionocytes. (C) Transplant of FLDX-labelled (green) outer layer epidermal tissue on to CB-labelled (blue) host embryo and stained by fluorescent in situ hybridisation for foxa1 (red) at stage 32. (D) Transplant of FLDX-labelled (green) outer layer epidermal tissue on to MR-labelled (red) host embryo and stained with anti-Xeel antibody (blue). Scale bars: 50 μm in A,B,D; 15 μm in C.
	Fig. 3. Small cells with large vesicles containing secretory material. (A) Small cells (yellow circles) are not labelled by markers for ciliated cells (anti-AcTub), ionocytes (anti-V1a) or goblet cells (anti-Xeel). Membranes are marked with membrane GFP (mGFP). (B) SEM shows small cells with large apical openings and secretory material highlighted with an arrow. Sections imaged by TEM show vesicles at the apical membrane containing a dark core surrounded by lighter material. Highlighted with a yellow arrow is a vesicle deeper within the cytoplasm that may represent an immature vesicle. Scale bars: in A, 10 μm; in B, 2 μm (SEM), 1 μm (TEM, low magnification) and 0.5 μm (TEM, high magnification).
	Fig. 4. Molecular profile of the new cell type. (A) In situ hybridisation using an antisense probe to the itpkb transcript reveals a scattered, punctate epidermal pattern. (B) Double fluorescence in situ hybridisation for foxa1 and itpkb shows colocalisation of the two transcripts. (C) Antibody staining for Itpkb with mGFP shows localisation in the small cells with large apical vesicles. (D) PNA conjugated to Alexa Fluor-568 colocalises with foxa1. (E) PNA together with mGFP staining shows strong labelling of material in the large vesicles. Staining is also evident at a lower level in vesicles of goblet cells (yellow arrow). (F) Antibody staining to the carbohydrate epitope HNK-1 together with mGFP shows specific expression in the vesicles of the small cells. (G) Co-staining for HNK-1 and PNA shows that they are present in the same cell. Scale bars: 500 μm in A; 50 μm in B; 5 μm in C,E-G; 15 μm in D.
	Fig. 5. FoxA1 is a regulator of SSC development. (A) The foxa1 gene has two exons and one intron. An antisense morpholino oligo (red) was designed against the splice junction of exon 1 and intron 1. Loss of foxa1 transcript is evident with foxa1 MO compared with controls (MOC) by RT-PCR (using primers either side of the splice site), indicating knockdown of expression. A higher band was evident for the foxa1 morphants, which is indicative of an unspliced transcript. There is no difference in the intensity of bands for the ubiquitously expressed gene ornithine decarboxylase (ODC). (B) Representative images of stage 32 embryos showing the frequency of each cell type after applying MOC or foxa1 morpholino. Ciliated cells were marked by α-1-tubulin, SSCs by itpkb, ionocytes by atp6v1a and goblet cells by antibody to Xeel. The total number of cells in a defined area was determined by DAPI staining of nuclei and a ratio for each marker relative to the total number of cells was determined as shown in the chart. The mean ratio for ten embryos is shown for each sample. Error bars represent s.e.m. Student�s t-test, P<0.01 (**). Scale bars: 50 μm.
	Fig. 6. Depletion of SSCs leaves embryos susceptible to infection. (A) Representative images showing embryo death following foxa1 knockdown. Supplementing the media with the broad-spectrum antibiotic, gentamicin (20 μg/ml) leads to a greater level of survival. n=7 experiments. (B) Quantitation of the survival rate. Error bars represent s.e.m. Student�s t-test, **P<0.01. Scale bars: 2 mm.
	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).
	Fig. 8. Impact of otogelin-like knockdown on the epidermis. (A) The otogelin-like gene is predicted to have up to 79 exons and 78 introns. An antisense morpholino oligo (red) was designed against the splice junction of exon 1 and intron 1. Loss of otogelin-like transcript is evident with otogelin-like MO compared with controls (MOC) by RT-PCR, indicating knockdown of expression. There is little difference in the intensity of bands for the ubiquitously expressed gene, ornithine decarboxylase (ODC). (B) Comparison of PNA staining in otogelin-like morphants compared with controls (MOC). Right panels are higher magnification images of the flank epidermis. (C) TEM images of goblet cells and SSCs in controls (MOC) and otog-like MO treated embryos. Note absence of dark core in the vesicles of the SSCs. Scale bars: in B, 250 μm (left panels) and 50 μm (right panels); in C, 1 μm.
	Fig. 9. Overview of Xenopus epidermal cell types and secretions. The epidermis of late tailbud/tadpole embryos has four cell types: ciliated cells, ionocytes, goblet cells and small secretory cells. Otogelin-like is a major secretory glycoprotein secreted from goblet cells and SSCs. Goblet cells also secrete the lectin, Xeel. SSCs also secrete another granular material in their vesicles that has yet to be identified. Other innate defence molecules found in secretions include vitellogenin, apolipoprotein b, complement factors (C3 and C9) and FCGBP/FCGBP-like proteins.
	Fig. S1. Time course comparison and expression pattern of new cell type. Time course of expression for early markers of ciliated cells (foxj1), ionocytes (foxi1) and the new cell type (foxa1). Ciliated cells arise first in the epidermis appearing at stage 11, ionocytes next appear at stage 12 and finally the new cell type is expressed in the epidermis in a scattered distribution between stages 13-14. Scale bars: 300 μm.
	Fig. S2. Itpkb is not found in ciliated cells, ionocytes or goblet cells. Itpkb shows staining in a cell type independent of ciliated cells (A), ionocytes (B) and goblet cells (C) by fluorescent in situ hybridization combined with antibody staining. Ciliated cells are marked with the anti-acetylated α-tubulin (AcTub) antibody, ionocytes with an atp6v1a probe, and goblet cells with an anti-Xeel antibody. Note that (A) and (B) represent the same embryo and staining for itpkb but with anti-AcTub and atp6v1a, respectively. Scale bars: 50 μm.
	Fig. S3. Muc5e, a cement gland specific Mucin. (A) Addition of PNA-Alexa Fluor-568 to live embryos at stage 32 shows strong staining of the mucus-like material that emanates from the cement gland. (B) Fluorescent in situ hybridization for muc5e (red) and DAPI (blue) staining of nuclei shows exclusive expression of muc5e in the cement gland. Scale bars: 200 μm (A) and 100 μm (B).
	Fig. S4. Analysis of cell death in the epidermis of foxa1morphants. (A) TUNEL stains of epidermis in controls and foxa1 morphants at stage 21 and stage 33. The frequency of cells in a defined area on the flank epidermis was counted and the means determined as shown in the chart. n=11 embryos (MOC st. 21), n=9 embryos (foxa1 MO st. 21 and MOC st. 33), n=8 embryos (foxa1 MO st. 33). (B) In situ hybridization for itpkb in controls and Foxa1 morphants at stage 21 and stage 33. The frequency of cells in a defined area on the flank epidermis was counted and the means determined as shown in the chart. n=9 embryos (MOC st. 21 and foxa1 MO st. 33), n=10 embryos (foxa1 MO st. 21 and MOC st. 33). Error bars represent s.e.m. Student�s t-test, P<0.01 (**) and P<0.001 (***). Scale bars: 250 μm.
	Fig. S5. Foxa1-HA misexpression. (A) Representative images showing misexpression of 10 pg HA-tagged foxa1 mRNA (and lineage tracer β-gal mRNA) in one side of the embryo and probed by in situ hybridization for SSC marker, otog-like, at stage 33. Chart shows comparison of mean number of SSCs in a defined area of the flank epidermis (otog-like positive) on injected and uninjected sides (n=9 embryos). Error bars represent s.e.m. Student�s t-test, P<0.05 (*). Scale bars: 500 μm. (B) Representative image showing embryo at stage 33 injected with higher dose (100 pg) of foxa1-HA mRNA and lineage tracer, membrane GFP (mGFP, green). PNA (red) is largely absent from areas where mGFP and foxa1-HA are misexpressed. The mGFP cells are numerous and form �masses�. Right panel is a higher magnification image of area enclosed in white box in the middle panel. Scale bars: 200 μm (left and middle panel) and 30 μm (right panel).
	Fig. S6. Foxi1-HA misexpression increases ionocytes but does not affect survival. (A) Representative images of an uninjected control and an embryo injected at the one cell stage with foxi1-HA mRNA (100 pg) and β-gal mRNA. Embryos were fixed at stage 30 and probed for the ionocyte marker gene, atp6v1a. Chart shows comparison of mean number of atp6v1a positive cells in a defined area on the flank epidermis of controls and foxi1-HA overexpressed embryos (n=5 embryos each). (B) Chart showing survival rates of controls and foxi1-HA misexpressed embryos both with and without supplementing with the antibiotic, Gentamicin (10 μg/ml). Results of three independent experiments. Error bars represent s.e.m. Student�s t-test, P<0.001 (***). Scale bars: 500 μm
	Fig. S7. Impact of foxa1 knockdown on epidermal integrity. Representative images of MOC-treated and foxa1 MO-treated embryos expressing membrane GFP at stage 22 and stage 32. Scale bars: 30 μm

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