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Gene/Clone	Species	Stage	Anatomy Item	Experimenter
foxa1	xenopus

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Experiment details for foxa1

Cibois M et al. (2015) Assay

BMP signalling controls the construction of vertebrate mucociliary epithelia.

Gene	Clone	Species	Stages	Anatomy
foxa1.L		laevis	NF stage 14	epidermis , secretory epithelial cell

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Fig. 3. BMPpathway overactivation affects cell specification in the developing Xenopus epidermis. Stage 9 Xenopus embryos were injected in the blastocoel with either BSA (A-M) or 2-7 ng recombinant BMP4 (A′-M′). (A,A′) SEM of the epidermis at stage 37 revealed the severe decrease in the numbers of MCCs (red arrowhead), ionocytes (yellow arrowhead) and SSCs (green arrowhead). (B,B′) At tailbud (stage 30), BMP4-injected embryos showed normal morphology but substantially fewer α-tubulin-positive MCCs, as revealed by whole-mount in situ hybridisation (WISH). (C,C′) Both the differentiated MCC marker acetylated tubulin (white) and the ionocytemarker foxi1e (green)were lost in sectioned tailbud (stage 25)BMP4-injected embryos. (D,D′)WISHrevealed that the differentiated ionocyte marker v1awas strongly decreased in stage 25BMP4-injectedembryos. (E-F′) The differentiatedSSCsmarkers serotonin (red in E,E′) andtryptophan hydroxylase-1 (tph1, red in F,F′)were lost or strongly decreased in stage 25BMP4-injected embryos. (G,G′) In sections of stage 25BMP4-injected embryos, P63 (white), amarker of the non-intercalatinginner layercells,was lost,whereasthe goblet cellmarker intelectin-1 (green)wasupregulated. otogelin (redin H,H′,J,J′)andtrim29(red in I,I′), two other markers of goblet cells, were unaffected. (K-M′) Stage 14 embryos were analysed by WISH in order to stain MCCs for foxj1 (K,K′), ionocytes for foxi1e (L,L′) and SSCs for foxa1 (M,M′). (B,B′,D-F′,J-M′) Whole-mount embryos; (C,C′,G-I′) cryosectioned embryos. (B-M′) All markers were revealed by chromogenic or fluorescent in situ hybridisation, except for acetylated tubulin, serotonin and P63, which were revealed by immunofluorescence. (C,C′,E-I′) Nuclei were stained with DAPI. (B,B′,D-F′,K-M′) The number of embryos showing the phenotype among the total number of embryos examined is indicated.

Gene	Clone	Species	Stages	Anatomy
foxa1.L		laevis	NF stage 14	epidermis , epithelial cell , secretory epithelial cell

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Fig. 5. BMP inhibition promotes MCC, ionocyte and SSC fates in the developing Xenopus epidermis. (A-O′) Eight-cell stage Xenopus embryos were injected in one animal ventral blastomere (fated to become only epidermis) with either 500 pg GFP mRNA alone (Cntl) or with 500 pg GFP mRNA and BMP2, BMP4 and BMP7 morpholinos (BMP MOs, 10 ng each) and were analysed at stage 14 (A-F′) or 25 (G-O′). GFP immunostaining was used to identify the injected cells. Injection of BMP MOs resulted in an increase in the numbers of stage 14 inner layer cells expressing markers for committed MCCs ( foxj1 and MCI, red in A,A′ and B,B′, respectively), for committed ionocytes ( foxi1e, red in C,C′) and for committed SSCs ( foxa1, red in D,D′). Conversely, injection of BMP MOs led to a severe decrease in the expression levels of the goblet cell markers otogelin and trim29 (red in E,E′ and F,F′, respectively). When analysed at stage 25, embryos injected with BMP MOs showed an increase in the numbers of both α-tubulin-positive MCCs (white in G,G′,I,I′) and foxi1e-positive ionocytes (red in H,H′,I,I′), together with a decrease in the expression levels of the outer layer goblet cell markers intelectin-1 (red in J,J′.L,L′) and 5G7 (white in K-L′) and of the inner layer non-intercalating cell markers P63 (white in M,M′,O,O′) and α-dystroglycan (red in N,N′,O,O′). (A-F′,M-O′) Cryosectioned embryos; (G-L′) Whole-mount embryos. (P-S) Quantification of the different inner layer cellular populations in injected epidermal clones at stage 25. Shown are the percentages of MCCs (P), ionocytes (Q), SSCs (R) and P63-positive inner non-intercalating cells (S) among injected, GFP-positive cells. The increase in the number of MCCs, ionocytes and SSCs in BMP morphants was significant (Student’s t-test). No significant variation was observed for P63-positive cells. Error bars indicate s.d.

Gene	Clone	Species	Stages	Anatomy
foxa1.L		laevis	NF stage 14	epidermis , epithelium , secretory epithelial cell

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Fig. 8. TheBMPpathway controls dll1 expression in the developing Xenopus epidermis. (A-D′) Embryos injected in the blastocoel at stage 9 with either BSA or BMP4 were probed for dll1 expression at stage 12 or 25. BMP4-injected embryos showed a strong and persistent upregulation of dll1 within the epidermal inner layer. (E-F′) Eight-cell stage embryos were injected in a ventral ectoderm precursor blastomere with GFP mRNA alone (control) or with GFP mRNA and an mRNA encoding dominant-negative Smad5 (dnSmad5). Stage 10 embryos were sectioned and hybridised with a probe against dll1 and an antibody against GFP. The amount of dll1 signal (red) that colocalised with GFP fluorescence (green) was lower when GFP was co-injected with dnSmad5, indicating that dnSmad5 cell-autonomously decreases dll1 expression. (G,G′) The dll1 signal (red channel fluorescence) was measured in areas of equal size within (inj) or outside (non inj) of the injected clones in control (G) and dnSmad5-injected (G′) embryos. Signals were compared pairwise within each section, confirming the significant decrease in dll1 signal in dnSmad5-injected cells (Wilcoxon test). The middle bar indicates the median, and the outlier bars delimit the lower and upper quartiles. (H-J′) Embryos were injected at stage 9 with BSA (H,I,J), or with BMP4 at stage 9 (H′,I′,J′) or 11 (H′,I′,J′), then probed for dll1 2 h after injection (H-H′) or at stage 12 (I-I′) and for α-tubulin at stage 25 (J-J′). BMP4 caused ectopic dll1 activation and the loss of MCCs when injected at stage 9 but not at stage 11. (K-V) Cryosectioned embryos were hybridised with probes against dll1 and the MCC early marker foxj1 (K-N), the ionocyte early marker foxi1e (O-R) or the SSC early marker foxa1 (S-V) at stages 11, 12 and 14, respectively. dll1 colocalised with foxj1 at stage 11, with foxi1e at stage 12 and with foxa1 at stage 14.