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a HCR-FISH of PTCH1 in an E3.5 chick foregut slice 24 h after implantation of an Affi-Gel bead loaded with <t>recombinant</t> mouse <t>SHH</t> protein. b Comparison of mesenchymal cell density near the implanted bead (with or without SHH) and the contralateral side. N = 7 biological replicates for control, N = 5 for SHH. P -values are calculated by a paired t -test. c PIV analysis of an E3.75 GFP chick slice with an implanted SHH-bead. The color wheel indicates the directions of PIV vectors. d Live imaging of an SHH-bead-implanted GFP chick slice overlaid with cellular trajectories colored by their average speeds. e Average velocity vectors of individual cell trajectories in the blue and red boxes of ( d ). Vectors are scaled with the speed and color-coded by their directions according to the color wheel. The dashed circle marks the bead. f Immunofluorescence of GM130 (GOLGA2) in a chick foregut section with a SHH bead. g , h Distributions of mesenchymal cell orientation in slices implanted with control beads ( g , N = 4 biological replicates) and SHH beads ( h , N = 6 biological replicates). Data are presented as mean ± SD. P -values are calculated by Chi-square test against uniform distribution (df = 3 for Control, df = 4 for SHH bead). i , j In ovo electroporation of plasmids encoding mCherry ( i ), SHH ( i ), or HHIP ( j ) into the right epithelium of the chick foregut. Frontal views of the foreguts by stereo microscopy show the effective electroporation ( i ). Immunofluorescence of SHH ( i ) and HCR-FISH of HHIP and PTCH1 ( j ) confirmed the efficient expression of the constructs. Arrows indicate the asymmetrical deformation of the epithelium induced by SHH overexpression. The dashed lines bisect the foregut lumen as a reference for measuring epithelial distances to the midline. k Comparisons of right (electroporated) and left (non-electroporated) epithelium-to-midline distances with control (mCherry, N = 5 biological replicates), SHH ( N = 7 biological replicates), and HHIP ( N = 6 biological replicates) electroporation. Data are presented as mean ± SD. P -values are calculated by One-way ANOVA with Dunnett’s test. l Working model of TES initiation. SHH secreted from the foregut epithelium induces the dorsal sub-epithelial mesenchyme’s migration towards the epithelium, generating bilateral compressive force essential to TES. Created in BioRender. Yan, R. (2025) https://BioRender.com/cdwttt9 . Scale bars: 50 µm, except those marked on the images.
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a HCR-FISH of PTCH1 in an E3.5 chick foregut slice 24 h after implantation of an Affi-Gel bead loaded with <t>recombinant</t> mouse <t>SHH</t> protein. b Comparison of mesenchymal cell density near the implanted bead (with or without SHH) and the contralateral side. N = 7 biological replicates for control, N = 5 for SHH. P -values are calculated by a paired t -test. c PIV analysis of an E3.75 GFP chick slice with an implanted SHH-bead. The color wheel indicates the directions of PIV vectors. d Live imaging of an SHH-bead-implanted GFP chick slice overlaid with cellular trajectories colored by their average speeds. e Average velocity vectors of individual cell trajectories in the blue and red boxes of ( d ). Vectors are scaled with the speed and color-coded by their directions according to the color wheel. The dashed circle marks the bead. f Immunofluorescence of GM130 (GOLGA2) in a chick foregut section with a SHH bead. g , h Distributions of mesenchymal cell orientation in slices implanted with control beads ( g , N = 4 biological replicates) and SHH beads ( h , N = 6 biological replicates). Data are presented as mean ± SD. P -values are calculated by Chi-square test against uniform distribution (df = 3 for Control, df = 4 for SHH bead). i , j In ovo electroporation of plasmids encoding mCherry ( i ), SHH ( i ), or HHIP ( j ) into the right epithelium of the chick foregut. Frontal views of the foreguts by stereo microscopy show the effective electroporation ( i ). Immunofluorescence of SHH ( i ) and HCR-FISH of HHIP and PTCH1 ( j ) confirmed the efficient expression of the constructs. Arrows indicate the asymmetrical deformation of the epithelium induced by SHH overexpression. The dashed lines bisect the foregut lumen as a reference for measuring epithelial distances to the midline. k Comparisons of right (electroporated) and left (non-electroporated) epithelium-to-midline distances with control (mCherry, N = 5 biological replicates), SHH ( N = 7 biological replicates), and HHIP ( N = 6 biological replicates) electroporation. Data are presented as mean ± SD. P -values are calculated by One-way ANOVA with Dunnett’s test. l Working model of TES initiation. SHH secreted from the foregut epithelium induces the dorsal sub-epithelial mesenchyme’s migration towards the epithelium, generating bilateral compressive force essential to TES. Created in BioRender. Yan, R. (2025) https://BioRender.com/cdwttt9 . Scale bars: 50 µm, except those marked on the images.
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a HCR-FISH of PTCH1 in an E3.5 chick foregut slice 24 h after implantation of an Affi-Gel bead loaded with <t>recombinant</t> mouse <t>SHH</t> protein. b Comparison of mesenchymal cell density near the implanted bead (with or without SHH) and the contralateral side. N = 7 biological replicates for control, N = 5 for SHH. P -values are calculated by a paired t -test. c PIV analysis of an E3.75 GFP chick slice with an implanted SHH-bead. The color wheel indicates the directions of PIV vectors. d Live imaging of an SHH-bead-implanted GFP chick slice overlaid with cellular trajectories colored by their average speeds. e Average velocity vectors of individual cell trajectories in the blue and red boxes of ( d ). Vectors are scaled with the speed and color-coded by their directions according to the color wheel. The dashed circle marks the bead. f Immunofluorescence of GM130 (GOLGA2) in a chick foregut section with a SHH bead. g , h Distributions of mesenchymal cell orientation in slices implanted with control beads ( g , N = 4 biological replicates) and SHH beads ( h , N = 6 biological replicates). Data are presented as mean ± SD. P -values are calculated by Chi-square test against uniform distribution (df = 3 for Control, df = 4 for SHH bead). i , j In ovo electroporation of plasmids encoding mCherry ( i ), SHH ( i ), or HHIP ( j ) into the right epithelium of the chick foregut. Frontal views of the foreguts by stereo microscopy show the effective electroporation ( i ). Immunofluorescence of SHH ( i ) and HCR-FISH of HHIP and PTCH1 ( j ) confirmed the efficient expression of the constructs. Arrows indicate the asymmetrical deformation of the epithelium induced by SHH overexpression. The dashed lines bisect the foregut lumen as a reference for measuring epithelial distances to the midline. k Comparisons of right (electroporated) and left (non-electroporated) epithelium-to-midline distances with control (mCherry, N = 5 biological replicates), SHH ( N = 7 biological replicates), and HHIP ( N = 6 biological replicates) electroporation. Data are presented as mean ± SD. P -values are calculated by One-way ANOVA with Dunnett’s test. l Working model of TES initiation. SHH secreted from the foregut epithelium induces the dorsal sub-epithelial mesenchyme’s migration towards the epithelium, generating bilateral compressive force essential to TES. Created in BioRender. Yan, R. (2025) https://BioRender.com/cdwttt9 . Scale bars: 50 µm, except those marked on the images.
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Image Search Results


a HCR-FISH of PTCH1 in an E3.5 chick foregut slice 24 h after implantation of an Affi-Gel bead loaded with recombinant mouse SHH protein. b Comparison of mesenchymal cell density near the implanted bead (with or without SHH) and the contralateral side. N = 7 biological replicates for control, N = 5 for SHH. P -values are calculated by a paired t -test. c PIV analysis of an E3.75 GFP chick slice with an implanted SHH-bead. The color wheel indicates the directions of PIV vectors. d Live imaging of an SHH-bead-implanted GFP chick slice overlaid with cellular trajectories colored by their average speeds. e Average velocity vectors of individual cell trajectories in the blue and red boxes of ( d ). Vectors are scaled with the speed and color-coded by their directions according to the color wheel. The dashed circle marks the bead. f Immunofluorescence of GM130 (GOLGA2) in a chick foregut section with a SHH bead. g , h Distributions of mesenchymal cell orientation in slices implanted with control beads ( g , N = 4 biological replicates) and SHH beads ( h , N = 6 biological replicates). Data are presented as mean ± SD. P -values are calculated by Chi-square test against uniform distribution (df = 3 for Control, df = 4 for SHH bead). i , j In ovo electroporation of plasmids encoding mCherry ( i ), SHH ( i ), or HHIP ( j ) into the right epithelium of the chick foregut. Frontal views of the foreguts by stereo microscopy show the effective electroporation ( i ). Immunofluorescence of SHH ( i ) and HCR-FISH of HHIP and PTCH1 ( j ) confirmed the efficient expression of the constructs. Arrows indicate the asymmetrical deformation of the epithelium induced by SHH overexpression. The dashed lines bisect the foregut lumen as a reference for measuring epithelial distances to the midline. k Comparisons of right (electroporated) and left (non-electroporated) epithelium-to-midline distances with control (mCherry, N = 5 biological replicates), SHH ( N = 7 biological replicates), and HHIP ( N = 6 biological replicates) electroporation. Data are presented as mean ± SD. P -values are calculated by One-way ANOVA with Dunnett’s test. l Working model of TES initiation. SHH secreted from the foregut epithelium induces the dorsal sub-epithelial mesenchyme’s migration towards the epithelium, generating bilateral compressive force essential to TES. Created in BioRender. Yan, R. (2025) https://BioRender.com/cdwttt9 . Scale bars: 50 µm, except those marked on the images.

Journal: Nature Communications

Article Title: Convergent flow-mediated mesenchymal force drives embryonic foregut constriction and splitting

doi: 10.1038/s41467-025-65644-9

Figure Lengend Snippet: a HCR-FISH of PTCH1 in an E3.5 chick foregut slice 24 h after implantation of an Affi-Gel bead loaded with recombinant mouse SHH protein. b Comparison of mesenchymal cell density near the implanted bead (with or without SHH) and the contralateral side. N = 7 biological replicates for control, N = 5 for SHH. P -values are calculated by a paired t -test. c PIV analysis of an E3.75 GFP chick slice with an implanted SHH-bead. The color wheel indicates the directions of PIV vectors. d Live imaging of an SHH-bead-implanted GFP chick slice overlaid with cellular trajectories colored by their average speeds. e Average velocity vectors of individual cell trajectories in the blue and red boxes of ( d ). Vectors are scaled with the speed and color-coded by their directions according to the color wheel. The dashed circle marks the bead. f Immunofluorescence of GM130 (GOLGA2) in a chick foregut section with a SHH bead. g , h Distributions of mesenchymal cell orientation in slices implanted with control beads ( g , N = 4 biological replicates) and SHH beads ( h , N = 6 biological replicates). Data are presented as mean ± SD. P -values are calculated by Chi-square test against uniform distribution (df = 3 for Control, df = 4 for SHH bead). i , j In ovo electroporation of plasmids encoding mCherry ( i ), SHH ( i ), or HHIP ( j ) into the right epithelium of the chick foregut. Frontal views of the foreguts by stereo microscopy show the effective electroporation ( i ). Immunofluorescence of SHH ( i ) and HCR-FISH of HHIP and PTCH1 ( j ) confirmed the efficient expression of the constructs. Arrows indicate the asymmetrical deformation of the epithelium induced by SHH overexpression. The dashed lines bisect the foregut lumen as a reference for measuring epithelial distances to the midline. k Comparisons of right (electroporated) and left (non-electroporated) epithelium-to-midline distances with control (mCherry, N = 5 biological replicates), SHH ( N = 7 biological replicates), and HHIP ( N = 6 biological replicates) electroporation. Data are presented as mean ± SD. P -values are calculated by One-way ANOVA with Dunnett’s test. l Working model of TES initiation. SHH secreted from the foregut epithelium induces the dorsal sub-epithelial mesenchyme’s migration towards the epithelium, generating bilateral compressive force essential to TES. Created in BioRender. Yan, R. (2025) https://BioRender.com/cdwttt9 . Scale bars: 50 µm, except those marked on the images.

Article Snippet: 10 μL of 2 mg/mL recombinant mouse SHH N-terminus (R&D Systems, 464-SH) was dropped on the lid of a plastic dish placed on ice.

Techniques: Recombinant, Comparison, Control, Imaging, Immunofluorescence, In Ovo, Electroporation, Microscopy, Expressing, Construct, Over Expression, Migration