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fraction of mouse tbx4 lung mesenchyme specific enhancer (lme)  (GenScript corporation)

 
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    GenScript corporation fraction of mouse tbx4 lung mesenchyme specific enhancer (lme)
    a Schematic model of tracheoesophageal segregation. b Transverse sections of Nkx2.1 null mouse embryos and littermate controls. Sections were stained for Sox2 ( green ), <t>Tbx4</t> ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal <t>mesoderm.</t> Asterisks indicate nonspecific background signal of blood cells in dorsal aorta. n = 3/3 embryos per genotype. c Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos and littermate controls. Sections were stained by Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. n = 3/3 embryos per genotype. d Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos-, and littermate controls. Sections were stained by Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. n neural tube, a aorta, Es Esophagus, Tr Trachea, Tr–E Trachea–Esophageal tube. Scale bar, 40 μm.
    Fraction Of Mouse Tbx4 Lung Mesenchyme Specific Enhancer (Lme), supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
    fraction of mouse tbx4 lung mesenchyme specific enhancer (lme) - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells"

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17969-w

    a Schematic model of tracheoesophageal segregation. b Transverse sections of Nkx2.1 null mouse embryos and littermate controls. Sections were stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. Asterisks indicate nonspecific background signal of blood cells in dorsal aorta. n = 3/3 embryos per genotype. c Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos and littermate controls. Sections were stained by Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. n = 3/3 embryos per genotype. d Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos-, and littermate controls. Sections were stained by Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. n neural tube, a aorta, Es Esophagus, Tr Trachea, Tr–E Trachea–Esophageal tube. Scale bar, 40 μm.
    Figure Legend Snippet: a Schematic model of tracheoesophageal segregation. b Transverse sections of Nkx2.1 null mouse embryos and littermate controls. Sections were stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. Asterisks indicate nonspecific background signal of blood cells in dorsal aorta. n = 3/3 embryos per genotype. c Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos and littermate controls. Sections were stained by Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. n = 3/3 embryos per genotype. d Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos-, and littermate controls. Sections were stained by Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. n neural tube, a aorta, Es Esophagus, Tr Trachea, Tr–E Trachea–Esophageal tube. Scale bar, 40 μm.

    Techniques Used: Staining

    a Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Nkx2.1 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos. b Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos per genotype. c Transversal section of mouse embryo at E10.5. Section were stained for Axin2 ( green ), Nkx2.1 ( magenta ) and DAPI ( blue ) by RNAscope experiment. Arrowheads indicate Axin2 + cells. n = 2/2 embryos. d Transverse sections of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and littermate controls at E10.5. Upper panels show sections stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Lower panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). Arrowhead indicates Tbx4 + cells. Arrows indicate Nkx2.1 + cells. n = 3/3 embryos per genotype. e Three-dimensional imaging of whole trachea and esophagus tissue at E16.5. Cartilage morphology and smooth muscle architecture in the tracheas of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates. Whole trachea and esophagus were stained for Sox9 ( green ) and SMA ( magenta ). n = 3/3 embryos per genotype. f Model of tracheal architecture in Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates based on e . g Integrative Genomics Viewer (IGV) snapshot of mm10 (chr11:85,884,521-85,904,766) showing mouse tbx4 lung mesenchyme specific element (LME) and compiled ENCODE data of ATAC-seq E14.5 lung (ENCSR335VJW), H3K27Ac E14.5 lung (ENCSR452WYC), H3K4me1 E15.5 lung (ENCFF283EBS), EP300 postnatal day (PND) 0 lung and vertebrate conservation (Phastcons). Numbers indicate fold enrichment over input (ChIP-seq). CisBP and Jaspar predicted Tcf/Lef-binding sites (highlighted in green, region: mm10, chr11:85,893,703-85,894,206) are localized at the ATAC-seq and p300 peaks that are conserved among most vertebrates. Sequence in red shows the Tcf/Lef-binding sites mutated. Eso Esophagus, Lu Lung, Tr Trachea, Tr–E Tracheoesophageal tube. Scale bar: 40 μm ( a , b ), 50 μm ( c , d ), 300 μm ( e ).
    Figure Legend Snippet: a Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Nkx2.1 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos. b Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos per genotype. c Transversal section of mouse embryo at E10.5. Section were stained for Axin2 ( green ), Nkx2.1 ( magenta ) and DAPI ( blue ) by RNAscope experiment. Arrowheads indicate Axin2 + cells. n = 2/2 embryos. d Transverse sections of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and littermate controls at E10.5. Upper panels show sections stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Lower panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). Arrowhead indicates Tbx4 + cells. Arrows indicate Nkx2.1 + cells. n = 3/3 embryos per genotype. e Three-dimensional imaging of whole trachea and esophagus tissue at E16.5. Cartilage morphology and smooth muscle architecture in the tracheas of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates. Whole trachea and esophagus were stained for Sox9 ( green ) and SMA ( magenta ). n = 3/3 embryos per genotype. f Model of tracheal architecture in Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates based on e . g Integrative Genomics Viewer (IGV) snapshot of mm10 (chr11:85,884,521-85,904,766) showing mouse tbx4 lung mesenchyme specific element (LME) and compiled ENCODE data of ATAC-seq E14.5 lung (ENCSR335VJW), H3K27Ac E14.5 lung (ENCSR452WYC), H3K4me1 E15.5 lung (ENCFF283EBS), EP300 postnatal day (PND) 0 lung and vertebrate conservation (Phastcons). Numbers indicate fold enrichment over input (ChIP-seq). CisBP and Jaspar predicted Tcf/Lef-binding sites (highlighted in green, region: mm10, chr11:85,893,703-85,894,206) are localized at the ATAC-seq and p300 peaks that are conserved among most vertebrates. Sequence in red shows the Tcf/Lef-binding sites mutated. Eso Esophagus, Lu Lung, Tr Trachea, Tr–E Tracheoesophageal tube. Scale bar: 40 μm ( a , b ), 50 μm ( c , d ), 300 μm ( e ).

    Techniques Used: Staining, RNAscope, Imaging, Control, ChIP-sequencing, Binding Assay, Sequencing

    a In situ hybridization for Wnt2 mRNA during tracheoesophageal segregation. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm at E9.5 and E10.5. n = 2/2 embryos. b Transverse sections of Shh Cre , Wls flox/flox mouse embryos and littermate controls at E10.5. Left panels show sections stained with Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Right panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. c In situ hybridization for Wnt2 mRNA in Shh Cre , Wls flox/flox mouse embryos and littermate controls at E9.5. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm. n = 2/2 embryos. d Refined model of tracheoesophageal segregation and tracheal mesodermal differentiation. e In situ hybridization for Wnt7b mRNA in mouse embryo at E10.5. Arrowhead indicates Wnt7b + cells. n = 2/2 embryos. Eso Esophagus, Lu Lung, Tr Trachea. Scale bar; 40 μm ( a – c ), 50 μm ( e ).
    Figure Legend Snippet: a In situ hybridization for Wnt2 mRNA during tracheoesophageal segregation. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm at E9.5 and E10.5. n = 2/2 embryos. b Transverse sections of Shh Cre , Wls flox/flox mouse embryos and littermate controls at E10.5. Left panels show sections stained with Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Right panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. c In situ hybridization for Wnt2 mRNA in Shh Cre , Wls flox/flox mouse embryos and littermate controls at E9.5. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm. n = 2/2 embryos. d Refined model of tracheoesophageal segregation and tracheal mesodermal differentiation. e In situ hybridization for Wnt7b mRNA in mouse embryo at E10.5. Arrowhead indicates Wnt7b + cells. n = 2/2 embryos. Eso Esophagus, Lu Lung, Tr Trachea. Scale bar; 40 μm ( a – c ), 50 μm ( e ).

    Techniques Used: In Situ Hybridization, Expressing, Staining

    a Experimental design to generate tracheal mesoderm from mESCs. b Differentiating cells from mESCs at day 5. Cells were stained for Foxf1 ( magenta ) and Gata4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two independent experiments. c qRT-PCR for LPM markers of mESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from mESCs at day 6. Cells were stained for Tbx4 ( green ) and Foxf1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of at least two independent experiments. e qRT-PCR for respiratory mesoderm marker expression of mESC-derived trachea mesodermal cells. ( n = 3 independent wells). Experiments were repeated at least twice. f Diagram showing the constructs utilized in luciferase experiments containing Tbx4- − − LME Tbx4-LME wild-type containing five Tcf/Lef-binding sites and Mutant. mESC-derived LPMs were transfected with wild-type or mutant Tbx4 -LME during 4hrs following by respiratory induction in presence or absence of CHIR99021. g Luciferase assay examining the activation of Tbx4 -LME wt and mutant in response to 3 μM CHIR99021. P -values were provided by two-sided Tukey’s multiple comparison. *** p < 0.0001 ( n = 3 from independent wells from a single experiment). h Differentiating cells from mESCs at day 12. Cells were stained for Acta2 ( magenta ) and Sox9 ( green ). The asterisk indicates Sox9 + /SMA - chondrocyte aggregates. Images are representative of two independent experiments. i qRT-PCR for Sox9 and Acta2 expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated twice. j Differentiating cells from mESCs at day 12. Chondrocytes were stained with Alcian blue. The asterisk indicates one of the chondrocyte aggregates. Images are representative of two independent experiments. k Differentiating cells from mESCs at day 12. Cells were stained for Col1a1 ( magenta ) and Aggrecan ( green ). Image is representative of two independent experiments. l Differentiating cells from mESCs at day 12. Cells were stained for Tagln ( magenta ) and Col2a1 ( green ). Image is representative of two independent experiments. Each column shows the mean with S.D. Scale bar; 50 μm. Source data for b, c, d, e, g, i are provided in Source data file.
    Figure Legend Snippet: a Experimental design to generate tracheal mesoderm from mESCs. b Differentiating cells from mESCs at day 5. Cells were stained for Foxf1 ( magenta ) and Gata4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two independent experiments. c qRT-PCR for LPM markers of mESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from mESCs at day 6. Cells were stained for Tbx4 ( green ) and Foxf1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of at least two independent experiments. e qRT-PCR for respiratory mesoderm marker expression of mESC-derived trachea mesodermal cells. ( n = 3 independent wells). Experiments were repeated at least twice. f Diagram showing the constructs utilized in luciferase experiments containing Tbx4- − − LME Tbx4-LME wild-type containing five Tcf/Lef-binding sites and Mutant. mESC-derived LPMs were transfected with wild-type or mutant Tbx4 -LME during 4hrs following by respiratory induction in presence or absence of CHIR99021. g Luciferase assay examining the activation of Tbx4 -LME wt and mutant in response to 3 μM CHIR99021. P -values were provided by two-sided Tukey’s multiple comparison. *** p < 0.0001 ( n = 3 from independent wells from a single experiment). h Differentiating cells from mESCs at day 12. Cells were stained for Acta2 ( magenta ) and Sox9 ( green ). The asterisk indicates Sox9 + /SMA - chondrocyte aggregates. Images are representative of two independent experiments. i qRT-PCR for Sox9 and Acta2 expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated twice. j Differentiating cells from mESCs at day 12. Chondrocytes were stained with Alcian blue. The asterisk indicates one of the chondrocyte aggregates. Images are representative of two independent experiments. k Differentiating cells from mESCs at day 12. Cells were stained for Col1a1 ( magenta ) and Aggrecan ( green ). Image is representative of two independent experiments. l Differentiating cells from mESCs at day 12. Cells were stained for Tagln ( magenta ) and Col2a1 ( green ). Image is representative of two independent experiments. Each column shows the mean with S.D. Scale bar; 50 μm. Source data for b, c, d, e, g, i are provided in Source data file.

    Techniques Used: Staining, Quantitative RT-PCR, Derivative Assay, Marker, Expressing, Construct, Luciferase, Binding Assay, Mutagenesis, Transfection, Activation Assay, Comparison

    a Experimental design to generate tracheal mesoderm from hESCs. b Differentiating cells from hESCs at day 2. Cells were stained for FOXF1 ( magenta ) and GATA4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two experiments. c qRT-PCR for LPM marker expression of hESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from hESCs at day 5. Cells were stained for TBX4 ( green ) and FOXF1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. e qRT-PCR for respiratory mesoderm marker expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated at least twice. f Differentiating cells from hESCs at day 10. Cells were stained for TBX4 ( green ) and NKX6.1 ( magenta ). Images are representative of three wells in a single experiment. White arrows; TBX4 + /NKX6.1 + mesodermal cells. White arrowheads; TBX4 + /NKX6.1 − mesodermal cells. Grey arrows; TBX4 − /NKX6.1 + mesodermal cells. g The rate of differentiated cells at day 10. % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. h Differentiating cells from hESCs with or without CHIR99021 at day 10. Cells were stained for ACTA2 ( magenta ) and SOX9 ( green ). Images are representative of at least two experiments. i qRT-PCR for SOX9 and ACTA2 expression of hESC-derived trachea mesodermal cells with different doses of CHIR99021 ( n = 3 independent well). Experiments were repeated twice. j Differentiating cells from hESCs at day 10. Chondrocytes were stained with Alcian blue. The asterisk indicates a chondrocyte aggregate. Images are representative of two experiments. k Differentiating cells from hESCs at day 10. Cells were stained for COL1A1 ( magenta ) and AGGRECAN ( green ). Images are representative of two experiments. l Differentiating cells from hESCs at day 10. Cells were stained for TAGLN ( magenta ) and COL2A1 ( green ). Images are representative of two experiments. Each column shows the mean with S.D. ( n = 3). Scale bar; 50 μm ( d, f, h, j, k, l ), 100 μm ( b ). Source data for b, c, d, e, g, i are provided in Source data file.
    Figure Legend Snippet: a Experimental design to generate tracheal mesoderm from hESCs. b Differentiating cells from hESCs at day 2. Cells were stained for FOXF1 ( magenta ) and GATA4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two experiments. c qRT-PCR for LPM marker expression of hESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from hESCs at day 5. Cells were stained for TBX4 ( green ) and FOXF1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. e qRT-PCR for respiratory mesoderm marker expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated at least twice. f Differentiating cells from hESCs at day 10. Cells were stained for TBX4 ( green ) and NKX6.1 ( magenta ). Images are representative of three wells in a single experiment. White arrows; TBX4 + /NKX6.1 + mesodermal cells. White arrowheads; TBX4 + /NKX6.1 − mesodermal cells. Grey arrows; TBX4 − /NKX6.1 + mesodermal cells. g The rate of differentiated cells at day 10. % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. h Differentiating cells from hESCs with or without CHIR99021 at day 10. Cells were stained for ACTA2 ( magenta ) and SOX9 ( green ). Images are representative of at least two experiments. i qRT-PCR for SOX9 and ACTA2 expression of hESC-derived trachea mesodermal cells with different doses of CHIR99021 ( n = 3 independent well). Experiments were repeated twice. j Differentiating cells from hESCs at day 10. Chondrocytes were stained with Alcian blue. The asterisk indicates a chondrocyte aggregate. Images are representative of two experiments. k Differentiating cells from hESCs at day 10. Cells were stained for COL1A1 ( magenta ) and AGGRECAN ( green ). Images are representative of two experiments. l Differentiating cells from hESCs at day 10. Cells were stained for TAGLN ( magenta ) and COL2A1 ( green ). Images are representative of two experiments. Each column shows the mean with S.D. ( n = 3). Scale bar; 50 μm ( d, f, h, j, k, l ), 100 μm ( b ). Source data for b, c, d, e, g, i are provided in Source data file.

    Techniques Used: Staining, Quantitative RT-PCR, Marker, Expressing, Derivative Assay

    Related Articles

    Synthesized:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Clone Assay:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Plasmid Preparation:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Transfection:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Staining:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    RNAscope:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Imaging:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Control:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    ChIP-sequencing:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Binding Assay:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Sequencing:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    In Situ Hybridization:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Expressing:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Quantitative RT-PCR:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Derivative Assay:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Marker:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Construct:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Luciferase:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Mutagenesis:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Activation Assay:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Comparison:

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells
    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.



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    GenScript corporation fraction of mouse tbx4 lung mesenchyme specific enhancer (lme)
    a Schematic model of tracheoesophageal segregation. b Transverse sections of Nkx2.1 null mouse embryos and littermate controls. Sections were stained for Sox2 ( green ), <t>Tbx4</t> ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal <t>mesoderm.</t> Asterisks indicate nonspecific background signal of blood cells in dorsal aorta. n = 3/3 embryos per genotype. c Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos and littermate controls. Sections were stained by Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. n = 3/3 embryos per genotype. d Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos-, and littermate controls. Sections were stained by Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. n neural tube, a aorta, Es Esophagus, Tr Trachea, Tr–E Trachea–Esophageal tube. Scale bar, 40 μm.
    Fraction Of Mouse Tbx4 Lung Mesenchyme Specific Enhancer (Lme), supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Schematic model of tracheoesophageal segregation. b Transverse sections of Nkx2.1 null mouse embryos and littermate controls. Sections were stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. Asterisks indicate nonspecific background signal of blood cells in dorsal aorta. n = 3/3 embryos per genotype. c Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos and littermate controls. Sections were stained by Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. n = 3/3 embryos per genotype. d Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos-, and littermate controls. Sections were stained by Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. n neural tube, a aorta, Es Esophagus, Tr Trachea, Tr–E Trachea–Esophageal tube. Scale bar, 40 μm.

    Journal: Nature Communications

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

    doi: 10.1038/s41467-020-17969-w

    Figure Lengend Snippet: a Schematic model of tracheoesophageal segregation. b Transverse sections of Nkx2.1 null mouse embryos and littermate controls. Sections were stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. Asterisks indicate nonspecific background signal of blood cells in dorsal aorta. n = 3/3 embryos per genotype. c Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos and littermate controls. Sections were stained by Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate Tbx4 + tracheal mesoderm. n = 3/3 embryos per genotype. d Transverse sections of Shh Cre , Ctnnb1 flox/flox mouse embryos-, and littermate controls. Sections were stained by Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. n neural tube, a aorta, Es Esophagus, Tr Trachea, Tr–E Trachea–Esophageal tube. Scale bar, 40 μm.

    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Techniques: Staining

    a Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Nkx2.1 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos. b Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos per genotype. c Transversal section of mouse embryo at E10.5. Section were stained for Axin2 ( green ), Nkx2.1 ( magenta ) and DAPI ( blue ) by RNAscope experiment. Arrowheads indicate Axin2 + cells. n = 2/2 embryos. d Transverse sections of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and littermate controls at E10.5. Upper panels show sections stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Lower panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). Arrowhead indicates Tbx4 + cells. Arrows indicate Nkx2.1 + cells. n = 3/3 embryos per genotype. e Three-dimensional imaging of whole trachea and esophagus tissue at E16.5. Cartilage morphology and smooth muscle architecture in the tracheas of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates. Whole trachea and esophagus were stained for Sox9 ( green ) and SMA ( magenta ). n = 3/3 embryos per genotype. f Model of tracheal architecture in Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates based on e . g Integrative Genomics Viewer (IGV) snapshot of mm10 (chr11:85,884,521-85,904,766) showing mouse tbx4 lung mesenchyme specific element (LME) and compiled ENCODE data of ATAC-seq E14.5 lung (ENCSR335VJW), H3K27Ac E14.5 lung (ENCSR452WYC), H3K4me1 E15.5 lung (ENCFF283EBS), EP300 postnatal day (PND) 0 lung and vertebrate conservation (Phastcons). Numbers indicate fold enrichment over input (ChIP-seq). CisBP and Jaspar predicted Tcf/Lef-binding sites (highlighted in green, region: mm10, chr11:85,893,703-85,894,206) are localized at the ATAC-seq and p300 peaks that are conserved among most vertebrates. Sequence in red shows the Tcf/Lef-binding sites mutated. Eso Esophagus, Lu Lung, Tr Trachea, Tr–E Tracheoesophageal tube. Scale bar: 40 μm ( a , b ), 50 μm ( c , d ), 300 μm ( e ).

    Journal: Nature Communications

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

    doi: 10.1038/s41467-020-17969-w

    Figure Lengend Snippet: a Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Nkx2.1 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos. b Transverse sections of LEF1 EGFP reporter mouse embryos at E9.5 to E11.5. Sections were stained for EGFP ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Arrowheads indicate GFP + cells. n = 3/3 embryos per genotype. c Transversal section of mouse embryo at E10.5. Section were stained for Axin2 ( green ), Nkx2.1 ( magenta ) and DAPI ( blue ) by RNAscope experiment. Arrowheads indicate Axin2 + cells. n = 2/2 embryos. d Transverse sections of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and littermate controls at E10.5. Upper panels show sections stained for Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Lower panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). Arrowhead indicates Tbx4 + cells. Arrows indicate Nkx2.1 + cells. n = 3/3 embryos per genotype. e Three-dimensional imaging of whole trachea and esophagus tissue at E16.5. Cartilage morphology and smooth muscle architecture in the tracheas of Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates. Whole trachea and esophagus were stained for Sox9 ( green ) and SMA ( magenta ). n = 3/3 embryos per genotype. f Model of tracheal architecture in Dermo1 Cre , Ctnnb1 flox/flox mouse embryos and control littermates based on e . g Integrative Genomics Viewer (IGV) snapshot of mm10 (chr11:85,884,521-85,904,766) showing mouse tbx4 lung mesenchyme specific element (LME) and compiled ENCODE data of ATAC-seq E14.5 lung (ENCSR335VJW), H3K27Ac E14.5 lung (ENCSR452WYC), H3K4me1 E15.5 lung (ENCFF283EBS), EP300 postnatal day (PND) 0 lung and vertebrate conservation (Phastcons). Numbers indicate fold enrichment over input (ChIP-seq). CisBP and Jaspar predicted Tcf/Lef-binding sites (highlighted in green, region: mm10, chr11:85,893,703-85,894,206) are localized at the ATAC-seq and p300 peaks that are conserved among most vertebrates. Sequence in red shows the Tcf/Lef-binding sites mutated. Eso Esophagus, Lu Lung, Tr Trachea, Tr–E Tracheoesophageal tube. Scale bar: 40 μm ( a , b ), 50 μm ( c , d ), 300 μm ( e ).

    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Techniques: Staining, RNAscope, Imaging, Control, ChIP-sequencing, Binding Assay, Sequencing

    a In situ hybridization for Wnt2 mRNA during tracheoesophageal segregation. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm at E9.5 and E10.5. n = 2/2 embryos. b Transverse sections of Shh Cre , Wls flox/flox mouse embryos and littermate controls at E10.5. Left panels show sections stained with Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Right panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. c In situ hybridization for Wnt2 mRNA in Shh Cre , Wls flox/flox mouse embryos and littermate controls at E9.5. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm. n = 2/2 embryos. d Refined model of tracheoesophageal segregation and tracheal mesodermal differentiation. e In situ hybridization for Wnt7b mRNA in mouse embryo at E10.5. Arrowhead indicates Wnt7b + cells. n = 2/2 embryos. Eso Esophagus, Lu Lung, Tr Trachea. Scale bar; 40 μm ( a – c ), 50 μm ( e ).

    Journal: Nature Communications

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

    doi: 10.1038/s41467-020-17969-w

    Figure Lengend Snippet: a In situ hybridization for Wnt2 mRNA during tracheoesophageal segregation. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm at E9.5 and E10.5. n = 2/2 embryos. b Transverse sections of Shh Cre , Wls flox/flox mouse embryos and littermate controls at E10.5. Left panels show sections stained with Sox2 ( green ), Tbx4 ( magenta ), and DAPI ( blue ). Right panels show sections stained for Nkx2.1 ( magenta ) and DAPI ( blue ). n = 3/3 embryos per genotype. c In situ hybridization for Wnt2 mRNA in Shh Cre , Wls flox/flox mouse embryos and littermate controls at E9.5. Arrowheads indicate Wnt2 expression in the ventrolateral mesoderm. n = 2/2 embryos. d Refined model of tracheoesophageal segregation and tracheal mesodermal differentiation. e In situ hybridization for Wnt7b mRNA in mouse embryo at E10.5. Arrowhead indicates Wnt7b + cells. n = 2/2 embryos. Eso Esophagus, Lu Lung, Tr Trachea. Scale bar; 40 μm ( a – c ), 50 μm ( e ).

    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Techniques: In Situ Hybridization, Expressing, Staining

    a Experimental design to generate tracheal mesoderm from mESCs. b Differentiating cells from mESCs at day 5. Cells were stained for Foxf1 ( magenta ) and Gata4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two independent experiments. c qRT-PCR for LPM markers of mESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from mESCs at day 6. Cells were stained for Tbx4 ( green ) and Foxf1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of at least two independent experiments. e qRT-PCR for respiratory mesoderm marker expression of mESC-derived trachea mesodermal cells. ( n = 3 independent wells). Experiments were repeated at least twice. f Diagram showing the constructs utilized in luciferase experiments containing Tbx4- − − LME Tbx4-LME wild-type containing five Tcf/Lef-binding sites and Mutant. mESC-derived LPMs were transfected with wild-type or mutant Tbx4 -LME during 4hrs following by respiratory induction in presence or absence of CHIR99021. g Luciferase assay examining the activation of Tbx4 -LME wt and mutant in response to 3 μM CHIR99021. P -values were provided by two-sided Tukey’s multiple comparison. *** p < 0.0001 ( n = 3 from independent wells from a single experiment). h Differentiating cells from mESCs at day 12. Cells were stained for Acta2 ( magenta ) and Sox9 ( green ). The asterisk indicates Sox9 + /SMA - chondrocyte aggregates. Images are representative of two independent experiments. i qRT-PCR for Sox9 and Acta2 expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated twice. j Differentiating cells from mESCs at day 12. Chondrocytes were stained with Alcian blue. The asterisk indicates one of the chondrocyte aggregates. Images are representative of two independent experiments. k Differentiating cells from mESCs at day 12. Cells were stained for Col1a1 ( magenta ) and Aggrecan ( green ). Image is representative of two independent experiments. l Differentiating cells from mESCs at day 12. Cells were stained for Tagln ( magenta ) and Col2a1 ( green ). Image is representative of two independent experiments. Each column shows the mean with S.D. Scale bar; 50 μm. Source data for b, c, d, e, g, i are provided in Source data file.

    Journal: Nature Communications

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

    doi: 10.1038/s41467-020-17969-w

    Figure Lengend Snippet: a Experimental design to generate tracheal mesoderm from mESCs. b Differentiating cells from mESCs at day 5. Cells were stained for Foxf1 ( magenta ) and Gata4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two independent experiments. c qRT-PCR for LPM markers of mESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from mESCs at day 6. Cells were stained for Tbx4 ( green ) and Foxf1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of at least two independent experiments. e qRT-PCR for respiratory mesoderm marker expression of mESC-derived trachea mesodermal cells. ( n = 3 independent wells). Experiments were repeated at least twice. f Diagram showing the constructs utilized in luciferase experiments containing Tbx4- − − LME Tbx4-LME wild-type containing five Tcf/Lef-binding sites and Mutant. mESC-derived LPMs were transfected with wild-type or mutant Tbx4 -LME during 4hrs following by respiratory induction in presence or absence of CHIR99021. g Luciferase assay examining the activation of Tbx4 -LME wt and mutant in response to 3 μM CHIR99021. P -values were provided by two-sided Tukey’s multiple comparison. *** p < 0.0001 ( n = 3 from independent wells from a single experiment). h Differentiating cells from mESCs at day 12. Cells were stained for Acta2 ( magenta ) and Sox9 ( green ). The asterisk indicates Sox9 + /SMA - chondrocyte aggregates. Images are representative of two independent experiments. i qRT-PCR for Sox9 and Acta2 expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated twice. j Differentiating cells from mESCs at day 12. Chondrocytes were stained with Alcian blue. The asterisk indicates one of the chondrocyte aggregates. Images are representative of two independent experiments. k Differentiating cells from mESCs at day 12. Cells were stained for Col1a1 ( magenta ) and Aggrecan ( green ). Image is representative of two independent experiments. l Differentiating cells from mESCs at day 12. Cells were stained for Tagln ( magenta ) and Col2a1 ( green ). Image is representative of two independent experiments. Each column shows the mean with S.D. Scale bar; 50 μm. Source data for b, c, d, e, g, i are provided in Source data file.

    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Techniques: Staining, Quantitative RT-PCR, Derivative Assay, Marker, Expressing, Construct, Luciferase, Binding Assay, Mutagenesis, Transfection, Activation Assay, Comparison

    a Experimental design to generate tracheal mesoderm from hESCs. b Differentiating cells from hESCs at day 2. Cells were stained for FOXF1 ( magenta ) and GATA4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two experiments. c qRT-PCR for LPM marker expression of hESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from hESCs at day 5. Cells were stained for TBX4 ( green ) and FOXF1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. e qRT-PCR for respiratory mesoderm marker expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated at least twice. f Differentiating cells from hESCs at day 10. Cells were stained for TBX4 ( green ) and NKX6.1 ( magenta ). Images are representative of three wells in a single experiment. White arrows; TBX4 + /NKX6.1 + mesodermal cells. White arrowheads; TBX4 + /NKX6.1 − mesodermal cells. Grey arrows; TBX4 − /NKX6.1 + mesodermal cells. g The rate of differentiated cells at day 10. % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. h Differentiating cells from hESCs with or without CHIR99021 at day 10. Cells were stained for ACTA2 ( magenta ) and SOX9 ( green ). Images are representative of at least two experiments. i qRT-PCR for SOX9 and ACTA2 expression of hESC-derived trachea mesodermal cells with different doses of CHIR99021 ( n = 3 independent well). Experiments were repeated twice. j Differentiating cells from hESCs at day 10. Chondrocytes were stained with Alcian blue. The asterisk indicates a chondrocyte aggregate. Images are representative of two experiments. k Differentiating cells from hESCs at day 10. Cells were stained for COL1A1 ( magenta ) and AGGRECAN ( green ). Images are representative of two experiments. l Differentiating cells from hESCs at day 10. Cells were stained for TAGLN ( magenta ) and COL2A1 ( green ). Images are representative of two experiments. Each column shows the mean with S.D. ( n = 3). Scale bar; 50 μm ( d, f, h, j, k, l ), 100 μm ( b ). Source data for b, c, d, e, g, i are provided in Source data file.

    Journal: Nature Communications

    Article Title: Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

    doi: 10.1038/s41467-020-17969-w

    Figure Lengend Snippet: a Experimental design to generate tracheal mesoderm from hESCs. b Differentiating cells from hESCs at day 2. Cells were stained for FOXF1 ( magenta ) and GATA4 ( green ), respectively. % was calculated from randomly chosen 3 fields. Images are representative of two experiments. c qRT-PCR for LPM marker expression of hESC-derived LPM ( n = 3 independent wells). Experiments were repeated at least twice. d Differentiating cells from hESCs at day 5. Cells were stained for TBX4 ( green ) and FOXF1 ( magenta ). % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. e qRT-PCR for respiratory mesoderm marker expression of hESC-derived trachea mesodermal cells ( n = 3 independent wells). Experiments were repeated at least twice. f Differentiating cells from hESCs at day 10. Cells were stained for TBX4 ( green ) and NKX6.1 ( magenta ). Images are representative of three wells in a single experiment. White arrows; TBX4 + /NKX6.1 + mesodermal cells. White arrowheads; TBX4 + /NKX6.1 − mesodermal cells. Grey arrows; TBX4 − /NKX6.1 + mesodermal cells. g The rate of differentiated cells at day 10. % was calculated from randomly chosen three fields. Images are representative of three wells in a single experiment. h Differentiating cells from hESCs with or without CHIR99021 at day 10. Cells were stained for ACTA2 ( magenta ) and SOX9 ( green ). Images are representative of at least two experiments. i qRT-PCR for SOX9 and ACTA2 expression of hESC-derived trachea mesodermal cells with different doses of CHIR99021 ( n = 3 independent well). Experiments were repeated twice. j Differentiating cells from hESCs at day 10. Chondrocytes were stained with Alcian blue. The asterisk indicates a chondrocyte aggregate. Images are representative of two experiments. k Differentiating cells from hESCs at day 10. Cells were stained for COL1A1 ( magenta ) and AGGRECAN ( green ). Images are representative of two experiments. l Differentiating cells from hESCs at day 10. Cells were stained for TAGLN ( magenta ) and COL2A1 ( green ). Images are representative of two experiments. Each column shows the mean with S.D. ( n = 3). Scale bar; 50 μm ( d, f, h, j, k, l ), 100 μm ( b ). Source data for b, c, d, e, g, i are provided in Source data file.

    Article Snippet: The fraction of mouse Tbx4 lung mesenchyme specific enhancer (LME) (mm10, chr11:85,893,703-85,894,206, GenScript, ID U3154EL200-3) 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 150 μl of Opti-MEM (Thermo Fisher Scientific, 31985088) with 2 μl of Lipofectamine Stem (Thermo Fisher Scientific, STEM00003) and 1 μg of pGL4.23 (luc2/minP) containing a fraction of mouse Tbx4 -LME or Tbx4 -LME containing mutated Tcf/Lef sites.

    Techniques: Staining, Quantitative RT-PCR, Marker, Expressing, Derivative Assay