| Method Summary |
"Methods
Mice
All mouse experiments were approved by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch. Mice were handled in accordance with the ethics guidelines of the institute. Mice were housed in 18-23 °C with 40-60% humidity. A 12-hour light/12-hour dark cycle was used. Nkx2.1null, ShhCre, Dermo1Cre, Ctnnb1flox/flox, Wlsflox/flox mice were previously generated.
In all experiments, at least 3 embryos from more than 2 littermates were analyzed. All attempts for replicate were successful. Sample size was not estimated by statistical methods. No data was excluded in this study. All control and mutant embryos were analyzed. We did not distinguish the sex of the embryos. No blinding was done in this study.
Immunostaining
Mouse embryos were fixed by 4% Paraformaldehyde/PBS (PFA) at 4°C overnight. Specimens were dehydrated by ethanol gradient and embedded in paraffin. Paraffin sections (6-um) were de-paraffinized and rehydrated for staining. Detailed procedure and antibodies of each staining were listed in Supplementary Table 1.
In situ hybridization
Mouse embryos were fixed with 4%PFA/PBS at 4°C overnight, and then tracheas were dissected. Specimens were incubated in sucrose gradient (10, 20, 30%) and embedded in OCT compound. Frozen sections (12-um) were subjected to in situ hybridization. For Wnt2, 4, 5a, 7b probe construction, cDNA fragments were amplified by primers listed in Supplementary Table 2. These cDNA fragments were subcloned into pBluscript SK+ at EcoRI and SalI sites. For Wnt5b and 6 probes, pSPROT1-Wnt5b (MCH085322) and pSPROT1-Wnt6 (MCH000524) were linearized at SalI sites, The NIA/NIH Mouse 15K and 7.4K cDNA Clones were provided by the RIKEN BRC55-57. Antisense cRNA transcripts were synthesized with DIG labeling mix (Roche Life Science) and T3 or SP6 RNA polymerase (New England Biolabs Inc.). Slides were permeabilized in 0.1% Triton-X100/PBS for 30min and blocked in acetylation buffer. After pre-hybridization, slides were hybridized with 500ng/ml of DIG-labeled cRNA probes overnight at 65C. After washing with SSC, slides were incubated with anti-DIG-AP antibodies (1:1000, Roche Life Science, 11093274910). Sections were colored with BM-purple (Roche Life Science, 11442074001).
For RNAscope experiments, the RNAscope Multiplex Fluorescent v2 assays (Advanced Cell Diagnostics, 323110) were used. The detailed procedure and probes were listed on Supplementary Table 3
Cell culture
For mesodermal differentiation from mES cells, C57BL/6J-Chr 12A/J/NaJ AC464/GrsJ mES cells (The Jackson Laboratory) and EB3 cells (AES0139, RIKEN BioResorce Center) were used. C57BL/6J-Chr 12A/J/NaJ AC464/GrsJ mES cells were kindly provided by Kentaro Iwasawa and Takanori Takebe (Center for Stem Cell & Organoid Medicine (CuSTOM), Perinatal Institute, Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital, Cincinnati). EB3 was kindly provided by Dr. Hitoshi Niwa (Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics in Kumamoto University). Cells were maintained in 2i + leukemia inhibitory factor (LIF) media (1,000 units ml-1 LIF, 0.4uM PD0325901, 3uM CHIR99021 in N2B27 medium) on ornithine-laminin coated-dishes37. For mesodermal differentiation of mouse ES cells, cells were digested by TrypLE express (Thermo Fisher Scientific, 12604013) and seeded onto Matrigel-coated 12 well plate. EpiLC were induced by EpiLC differentiation medium (1% knockout serum, 20ng ml-1 Activin A, 12ng ml-1 FGF2, and 10uM Y27632 in N2B27 Medium) for 2 days. Lateral plate mesoderm was established by Loh’s protocol with some modification. EpiLC cells were digested by TrypLE express to single cells and seeded onto Matrigel-coated 12 well plate at the density of 6x105 cells per well. The cells around middle primitive streak was induced by LPM D2 medium composed of 2% B27 Supplement Serum free (Thermo Fisher Scientific, 17504044), 1 x GlutaMax (Thermo Fisher Scientific, 35050061), 20ng ml-1 basic FGF (Peprotech, AF-100-18B), 6μM CHIR99021 (Sigma Aldrich, SML1046), 40ng ml-1 BMP4 (R&D Systems, 5020-BP-010), 10ng ml-1 Activin A (Peprotech, PEP-120-14-10), 10μM Y27632 (Sigma Aldrich, Y0503) in Advanced DMEM (Thermo Fisher Scientific, 12491015) for 48 hours. After that, LPM was induced by LPM D4 medium composed of 2% B27 Supplement Serum free, 1 x GlutaMax, 2μM XAV939 (Sigma Aldrich, X3004), 2μM SB431542 (Merck, 616461), 30ng ml-1 human recombinant BMP4 in Advanced DMEM for 24 hours. At Day 5, respiratory mesenchyme was induced by Day5 medium composed of 2% B27 Supplement Serum free, 1 x GlutaMax, 1μM CHIR99021, 10ng ml-1 BMP4. Medium were freshly renewed every day.
H1 (NIHhESC-10-0043 and NIHhESC-10-0062), human embryonic stem cell, was provided by Cincinnati children’s hospital medical center Pluripotent Stem Cell Facility. Cells were maintained in mTeSR1 medium (Stem Cell Technologies) on Matrigel-coated plate. For differentiation of H1 cells to mesodermal cells, confluent cells were digested by Accutase to single cells and seeded onto Geltrex-coated 12well plate at the dilution of 1:20 – 1:18 in mTeSR1 with 1uM Thiazovivin (Tocris). Next day, the cells around middle primitive streak were induced by cocktails of 6μM CHIR99021 (Sigma Aldrich, SML1046), 40ng ml-1 BMP4 (R&D Systems, 5020-BP-010), 30ng ml-1 Activin A (Cell Guidance Systems), 20ng ml-1 basic FGF (Thermo Fisher Scientific) and 100nM PIK90 (EMD Millipore) in Advanced DMEM/F12 including 2% B27 Supplement minus vitamin A, 1% N2 Supplement, 10uM Hepes, 100UI mL-1 Penicillin/Streptomycin, 2mM L-glutamine for 24 hours. After that, LPM was induced by LPM D2 medium composed of 1μM Wnt C59 (Cellagen Technologies), 1μM A83-01 (Tocris), 30ng ml-1 human recombinant BMP4 in Advanced DMEM/F12 including 2% B27 Supplement minus V. A., 1 x N2 Supplement, 10uM Hepes, 100UI mL-1 Penicillin/Streptomycin, 2mM L-glutamine for 24 hours. To generate respiratory mesenchyme, we combined 3uM CHIR99021, 2uM Purmorphamine (Tocris), and 10ng ml-1 Bmp4 in Advanced DMEM/F12 medium including 2% B27 Supplement Serum free, 1 x N2 Supplement, 10uM Hepes, 100UI mL-1 Penicillin/Streptomycin, 2mM L-glutamine from Day 2 to Day10. Medium was freshly renewed everyday
Immunocytochemistry
At differentiating process, cells were fixed by 4% PFA for 10 minutes at room temperature. For intracellular staining, cells were permeabilized by 0.2% TritonX-100/PBS for 10 minutes at room temperature. After blocking the cells with 5% normal donkey serum or 0.3% Triron-X100/1% bovine serum albumin, cells were incubated with primary antibodies overnight at 4°C. Then, cells were incubated with secondary antibodies for 1hr at room temperature. Detailed procedure and antibodies of each staining were listed in Supplementary Table 4.
Luciferase reporter assay
The fraction of mouse Tbx4-lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3)22 or Tbx4-LME containing putative Tcf/Lef sites mutated (GenScript, ID U3154EL200-6) were synthesized and cloned into pGL4.23 (luc2/minP) vector (promega).
mESC-derived LPM cells were transfected at day 5 in 150µl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2µl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1µg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4-LME or Tbx4-LME containing mutated Tcf/Lef sites. Four hours after transfection the tracheal mesenchyme was induced using day5 medium in presence or absence of Wnt activator (3μM CHIR99021) and cells were cultured for 24 hours and then lysed and assayed using Dual-Luciferase Reporter Assay System (Promega, E1980).
Alcian blue staining
Cells were fixed in 4% PFA/PBS for 10 minute at room temperature. After washing with PBS, cells were incubated with 3% acetic acid for 3 minutes and then stained with 1% alcian blue/3% acetic acid for 20 minutes.
Quantitative RT-PCR
Total mRNA was isolated by using the Nucleospin kit (TaKaRa, 740955) according to manufacturer’s instruction. cDNA was synthesized by SuperTMScriptTM VILO cDNA synthesis kit (Thermo Fisher Scientific, 11754050). qPCR was performed by PowerUpTM SYBRTM Green Master Mix on QuantStudio 3 or 6. Primer sequences were listed on Supplementary Table 5 and 6. Data are expressed as a Fold Change and were normalized with undifferentiated cells expression.
Statistical analyses
Statistical analyses were performed with Excel2013 (Microsoft) or PRISM8 (GraphPad software). For multiple comparison, one-way ANOVA and two-tailed Tukey’s methods were applied. For paired comparison, statistical significance was determined by F-test and Student’s or Welch’s two-tailed t test.
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