(A) Schematic showing markers of acinar and duct cells at E12 and E15. (B) E14 SLG stained for AQP5, SOX2 and E-cadherin (ECAD). Scale bar is 20 µm. (C) Immunostaining of E14 SLG for SOX2, nerves …
(A–D) SMG+SLG from Krt14CreERT2; Sox2fl/fl and wild-type (WT) embryos in which recombination was induced before gland ontogenesis at E10.5 and 11.5. (A) Representative brightfield images of Krt14CreE…
Source data relating to Figure 2C.
qPCR analysis of E16.5 Krt14CreERT2; Sox2fl/fl and wild-type (WT) SMG+SLG for genes involved in acinar differentiation, ductal differentiation and innervation, with expression normalised to Rsp29 and the WT. n = 3 embryos per genotype. s.d. = standard deviation.
Source data relating to Figure 2D.
Quantification of cells expressing acinar or ductal markers, cleaved caspase-3 or Ki67 in acini of E16.5 in Krt14CreERT2; Sox2fl/fl and wild-type (WT). n = 2–4 glands/genotype and cells were counted in 3–4 acini/gland. s.d. = standard deviation.
Source data relating to Figure 2F.
SMG+SLG from Krt14CreERT2; Rosa26mTmG and Krt14CreERT2; Rosa26mTmG; Sox2fl/fl were immunostained for SOX10 and AQP5 and GFP+ cells expressing SOX10 and AQP5 were quantified and expressed as a percentage of total positive cells. n = 3 glands/genotype and cells were counted in 3–4 acini/gland. s.d. = standard deviation.
Source data relating to Figure 2G.
qPCR for enrichment of Sox10 in SOX2 ChIP. n = 20 pooled SLG, average three experiments. s.d. = standard deviation.
Representative images of Krt14CreERT2; Sox2fl/fl and wild-type (WT) SMG+SLG at E13 (A; scale bar is 100 μm), E13.5 (B; top and bottom panels; scale bars are 100 and 50 μm, respectively) and E16.5 (C;…
Source data relating to Figure 2—figure supplement 1D.
Quantification of acini in Krt14CreERT2; Sox2fl/fl and wild-type (WT) glands at E13.5, with WT set to 100%. n = 3–7. s.d. = standard deviation.
Source data relating to Figure 2—figure supplement 1E.
Quantification of acini in Krt14CreERT2; Sox2fl/fl and wild-type (WT) glands at E16.5, with WT set to 100%. n = 3–7. s.d. = standard deviation.
Source data relating to Figure 2—figure supplement 1F.
qPCR analysis of gene expression in Krt14CreERT2; Sox2fl/fl and wild-type (WT) glands at E13.5. Data were normalized to Rsp29 and WT. n = 3–4 SMG+SLG per genotype. s.d. = standard deviation.
Source data relating to Figure 2—figure supplement 1G.
qPCR analysis of gene expression in Krt14CreERT2; Sox2fl/fl and wild-type (WT) glands at E16.5. Data were normalized to Rsp29 and WT. n = 3–4 SMG+SLG per genotype. s.d. = standard deviation.
(A–C) Representative images of SMG+SLG from WT, Krt14CreERT2; Sox2fl/fl; Rosa26mTmG or Krt14CreERT2; Sox2fl/fl immunostained for the ductal marker KRT19, apoptotic marker CASP3, proliferation marker …
Source data relating to Figure 3E.
Quantification of the number of CASP3+ cells in acini of E11.5 Krt14CreERT2; Sox2fl/fl and wild-type (WT) glands cultured for 60 hr ± Z-VAD-FMK. n = 3 glands per treatment and cells were counted in 3–4 acini per gland. Data are the mean of three biological replicates and two experiments. s.d. = standard deviation.
Source data relating to Figure 3F.
Quantification of the number of acini of E11.5 Krt14CreERT2; Sox2fl/fl and wild-type (WT) glands cultured for 60 hr ± Z-VAD-FMK. n = 3 glands per treatment. Data are means of three biological replicates and two experiments. s.d. = standard deviation.
(A–C) E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion (nerves) and subjected to immunofluorescent analysis (A). Glands were immunostained for markers of duct cells (KRT19, KRT7), …
Source data relating to Figure 4B.
E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion (nerves). The number of acini were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.
Source data relating to Figure 4C.
E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion (nerves) and subjected to immunofluorescent analysis. The number of AQP5+ and SOX10+ cells were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.
Source data relating to Figure 4E.
E11.5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr. The number of acini were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.
Source data relating to Figure 4F.
E11.5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr and qPCR performed. Data were normalized to Rsp29 and the WT. Data are means of three biological replicates and three experiments. s.d. = standard deviation.
(A–B) E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. White dashed line outlines acini. Arrows indicate double positive SOX2+EdU+ cells. Scale bar is 50 µm. The number of SOX2+, EdU+ and …
Source data relating to Figure 5B.
E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. The number of SOX2+, EdU+ and SOX2+EdU+ cells were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.
Source data relating to Figure 5C.
E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP (10 µM). The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS, normalized to control and expressed as percentage of total ECAD+ cells. s.d. = standard deviation.
Source data relating to Figure 5G.
E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and the number of AQP5+ and KRT19+ cells counted. Counts were normalized to the control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.
Source data relating to Figure 5H.
E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and subjected to qPCR analysis. Data were normalized to Rsp29 and control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.
(A) Flow cytometry plots show gating strategy to analyze SOX2+ cells versus SOX2- cells (left) and gating strategy to analyze percentage of SOX2+/SOX2- cells that express the muscarinic receptor …
Source data relating to Figure 5—figure supplement 1C.
E14 mouse SLGs cultured for 4 hr with DMSO or the muscarinic inhibitor 4-DAMP (10 µM) and subjected to gene profiling by qPCR. Data were normalized to Rsp29 and control values (DMSO). s.d. = standard deviation.
Source data relating to Figure 5—figure supplement 1E.
E14 mouse SLGs cultured for 24 hr with DMSO or the muscarinic inhibitor 4-DAMP (10 µM). The number of AQP5+, KRT19+, SOX10+, Ki67+ remaining in SLG cultured with or without 4-DAMP for 24 hr were quantified. n = 2 SLG per treatment and cells were counted in 3–4 end acini per gland. s.d. = standard deviation.
Source data relating to Figure 5—figure supplement 1F.
E14 mouse SLGs cultured for 4 hr with PD 168393 (20 μM), KN-93 (15 μM) or vehicle (water; Control) were subjected to gene profiling by qPCR. Data were normalized to Rsp29 and control values. Data are means of three to four SGs per treatment/genotype. s.d. = standard deviation.
(A) Immunofluorescent analysis of TUBB3+ nerves as well as SOX2-, SOX10-, CHRM1- and EGFR-expressing cells in fetal human submandibular (SMG) or sublingual (SLG) at 16–24 w. E-cadherin (ECAD) marks …
Source data relating to Figure 6E.
Human fetal SLG explants cultured with murine E13 PSG or mesenchyme for 7 days were subjected to gene profiling by qPCR. Data were normalized to GAPDH and control (+ mesenchyme). Data are means of six biological replicates, two individual experiments. s.d. = standard deviation.
Source data relating to Figure 6F.
qPCR analysis of fetal human SLG (22–23 w) dissociated cells cultured ± CCh for 48 hr. Data were normalized to GAPDH and control (-CCh). Data are means of six biological replicates, two individual experiments. s.d. = standard deviation.
Source data relating to Figure 6G.
qPCR analysis of fetal human SLG (22–23 w) explants cultured ± CCh for 72 hr. Data were normalized to GAPDH and control (-CCh). Data are means of six biological replicates, two individual experiments. s.d. = standard deviation.
Source data relating to Figure 6H.
Analysis of fetal human SLG (22–23 w) dissociated cells cultured ± CCh for 48 hr. The number of ECAD+SOX2+ and ECAD+SOX2+Ki67+ cells were measured by FACS as a percentage of total ECAD+ cells. Each # represents an independent experiment. s.d. = standard deviation.
(A) Immunofluorescent analysis of nerves (TUBB3), acinar (SOX2, SOX10, CD44, and AQP3) and ductal (KRT7, KRT5, KIT, KRT14) cells in fetal human submandibular (SMG), sublingual (SLG) and parotid (PG) …
Sequences for mouse primers used for qPCR.
Gene targ | Forward primer sequence | Reverse primer sequence |
---|---|---|
Aqp3 | CTGCCCGTGACTTTGGACCTC | CGAAGACACCAGCGATGGAACC |
Aqp5 | TCTACTTCTACTTGCTTTTCCCCTCCTC | CGATGGTCTTCTTCCGCTCCTCTC |
Ascl3 | GACAGGCTCTCGGTCTTCG | CATCTGTGTAAGAGGCCGGTA |
Atoh1 | GAGTGGGCTGAGGTAAAAGAGT | GGTCGGTGCTATCCAGGAG |
Bax | TGAAGACAGGGGCCTTTTTG | AATTCGCCGGAGACACTCG |
Bbc3 | AGCAGCACTTAGAGTCGCC | CCTGGGTAAGGGGAGGAGT |
Calm1 | TGGGAATGGTTACATCAGTGC | CGCCATCAATATCTGCTTCTCT |
Calr | GAAGCTGTTTCCGAGTGGTTT | GCACAATCAGTGTGTATAGGTGT |
Cnn1 | AAACAAGAGCGGAGATTTGAGC | TGTCGCAGTGTTCCATGCC |
Ccnd1 | CATCCATGCGGAAAATCGTGG | AAGACCTCCTCTTCGCACTTC |
Cdh1 | GACTGGAGTGCCACCACCAAAGAC | CGCCTGTGTACCCTCACCATCGG |
Cdkn1a | CCCCCAATCGCAAGGATTCTT | CTTGGTTCGGTGGGTCTGTC |
Chrm1 | TCCCAAGGCTCACCCAGATGTC | GCTCTGTGTGCTTTATTCTGTTGTTTCC |
Chrm3 | CATAGCACCATCCTCAACTCTACCAAG | GGGCATTTCTCTCTACATCCATAGTCC |
Dcpp1 | TGGTGGGGTATTATGTGGGCA | GGGATCGTTAGGGAAGCTAGA |
Dcpp2 | ATGGGCCAATGTAGATGCTC | CCCAAGAGGCAACAGTAGGA |
dNp63 | TTGTACCTGGAAAACAATG | GCATCGTTTCACAACCTCG |
Etv4 | GGTCCTGTGTTCTTGGTGCTGTG | GGTCCTGTGTTCTTGGTGCTGTG |
Etv5 | AAGCCCTTCAAAGTGATAGCGGAGAC | GTGTCCACAAACTTTCCTCTTTCTGTCAACT |
Egfr | ACACTACGCCGCCTGCTTCAAGAG | ACTGTGCCAAATGCTCCCGAACCC |
Fgf1 | GCACCGTGGATGGGACAAGGGACAGGAG | CACTTCGCCCGCACTTTCCGCACTGAG |
Fgf7 | CTCTACAGGTCATGCTTCCACC | ACAGAACAGTCTTCTCACCCT |
Fgf10 | TCTTCCTCCTCCTCGTCCTTCTCCTCTCCTTCC | CCGCTGACCTTGCCGTTCTTCTCAATCG |
Fgfr2b | TGGCTCTGTTCAATGTGACGGAGATGGATG | AGGCGCTTGCTGTTTGGGCAGGAC |
Kit | TGGTTGTGGTTGTTGTTGTTGTTG | GAAGGCTTGTTCCGAAGTGTAGAC |
Krt5 | TCCTGTTGAACGCCGCTGAC | CGGAAGGACACACTGGACTGG |
Krt7 | CGCCGCTGAGTGTGGACATCG | CTGGCTGCTCTTGGCTGACTTCTG |
Krt8 | GGAGGAGAGCAGGCTGGAGTC | TGGTGCGGCTGAAAGTGTTGG |
Krt14 | CCTCATCCTCTCAATTCTCCTCTGGCTCTC | CTTGGTGCGGATCTGGCGGTTGG |
Krt15 | GCTGCTACATGCTGCTCAGGCTTAGG | CCAGGAAGGACAAGGGTCAAGTAAAGAGAGTG |
Krt19 | GCCACCTACCTTGCTCGGATTG | GTCTCTGCCAGCGTGCCTTC |
Mist1 | GCTGACCGCCACCATACTTAC | TGTGTAGAGTAGCGTTGCAGG |
Muc19 | CTGGGTCTGGAAGTAGAAGTA | TCTAAGCCACAGAAGGAGAT |
Nrtn | CGCTACCACACGCTGCAAGAG | TCCCACACTTATGTGAAGTCAGTTCTC |
Pip | GGGTCTCTCATTCACATTCAGTG | TGATCTCCTGATTTTCCTGTGCT |
Pmaip1 | GCAGAGCTACCACCTGAGTTC | CTTTTGCGACTTCCCAGGCA |
Ptch1 | CACCCAGAAAGCAGACTACCCGAATATC | TCTCCTCCAGCATGACATACTTCACATTG |
Prol1 | CACCTAAGCCTAGCACCTCTA | ACTTCCAAAACACTTCCGCAAAT |
Rab3d | TACTATCGCGGAGCTATGGGT | TTTGATCTGCGTAGCCCAGTC |
Rps29 | GGAGTCACCCACGGAAGTTCGG | GGAAGCACTGGCGGCACATG |
Smgc | TGGCTCTGCAACACAACAGT | GGCGAAAAGCTCCCAGGTAA |
Sox2 | CAGCATGTCCTACTCGCAGCAG | TGGAGTGGGAGGAAGAGGTAACC |
Sox10 | ATCAGCCACGAGGTAATGTCCAAC | ACTGCCCAGCCCGTAGCC |
Spdef | AAGGCAGCATCAGGAGCAATG | CTGTCAATGACGGGACACTG |
Syn2 | TAGACTGCTGTGGAGGTGAA | GCTCTGAAAGGTAAAGGTAACTG |
Trp53 | CTCTCCCCCGCAAAAGAAAAA | CGGAACATCTCGAAGCGTTTA |
Tubb3 | CCAGAGCCATCTAGCTACTGACACTG | AGAGCCAAGTGGACTCACATGGAG |
Vacht | GAGTGGGAGATGGGCATGGTTTGG | GCAGGCAGGTACGACGCAAGAG |
Vip | TCCAGTGATAGGTACTCCATCTC | CATCCATAGCACACGCAGAA |
Zeb1 | GCTGGCAAGACAACGTGAAAG | GCCTCAGGATAAATGACGGC |
Zeb2 | ATTGCACATCAGACTTTGAGGAA | ATAATGGCCGTGTCGCTTCG |
Sequences for human primers used for qPCR.
Gene | Forward primer sequence | Reverse primer sequence |
---|---|---|
AQP3 | GTTTCTGTGTATGTGTATGTCTGCCTTT | CGTCCCACTGCTCCTACTTATGT |
CDH1 | AGGTGACAGAGCCTCTGGATAGA | TGGATGACACAGCGTGAG AGA |
CD44 | CTGCCGCTTTGCAGGTGTA | CATTGTGGGCAAGGTGCTATT |
CHRM1 | ACCTCTATACCACGTACCTG | TGAGCAGCAGATTCATGACG |
CHRM3 | ATCGGTCTGGCTTGGGTC | CCCGGAGGCACAGTTCTC |
EGFR | TGGCAGGTACAGTAGGATAA | CAAGTCAGTCTAACGCTCAT |
GAPDH | CAGCCTCAAGATCATCAGCA | TGTGGTCATGAGTCCTTCCA |
KIT | GCAGAGGAAGTGGAAGGCATCAG | TCAGTGAGACAGTAGCATTATGGAAGGT |
KRT5 | CGTGCCGCAGTTCTATATTCT | ACTTTGGGTTCTCGTGTCAG |
KRT7 | TCCGCGAGGTCACCATTAAC | GCTCTGTCAACTCCGTCTCAT |
KRT8 | AAGGATGCCAACGCCAAGTT | CCGCTGGTGGTCTTCGTATG |
KRT14 | ATCCAGAGATGTGACCTCCTC | CTCAGTTCTTGGTGCGAAGG |
KRT19 | GTCTGCCTCCAAGGTCCTCTGA | TCTACCCAGAAGACACCCTCCAAA |
MIST1 | CGGATGCACAAGCTAAATAACG | GCCGTCAGCGATTTGATGTAG |
SOX2 | TGGCGAACCATCTCTGTGGT | GGAAAGTTGGGATCGAACAAAAGC |
SOX10 | TCATCCCTTCAATGCCCCCT | TGCGTCTCAAGGTCATGGAGG |
Sequences for primers used for SOX10 ChIP-qPCR.
Primer | Forward primer sequence | Reverse primer sequence |
---|---|---|
A | GTGGAGGTTTGTTGATGGA | TTTGCGATGGGAGAGTCTGA |
B | ACAGTCAGAACCTGTTGCCT | TGATACCTACTGCAGGCTGC |
C | GCAGCCTGCAGTAGGTATCA | CTTCTTGAAGAGTAGGGC |
D | AAAAGACAGGAACTGCCCTG | AAGGGTGCCTTCACTGAGAA |
E | GATAGTGGGGACACAAAGAG | TCCTAATTCACTGGGCTCTG |
F | TCTTGTTCGGGGCCTTGAAA | ATGCTTGCTGCTCCGTCCCT |
G | AGACATCAATGAGCAGCAGG | CGCACACACACACTTTCCTA |