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hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature.

Acevedo LM, Lindquist JN, Walsh BM, Sia P, Cimadamore F, Chen C, Denzel M, Pernia CD, Ranscht B, Terskikh A, Snyder EY, Cheresh DA - Stem Cell Reports (2015)

Bottom Line: Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions.Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature.Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

View Article: PubMed Central - PubMed

Affiliation: Moores Cancer Center, University of California, San Diego, La Jolla, CA, 92093, USA; Sanford Burnham Medical Research Institute, La Jolla, CA 92037, USA; Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA.

No MeSH data available.


Related in: MedlinePlus

T-cadherin, Expressed by NCs and VSMCs, Mediates NC Co-alignment and Direct Contact with VSMCs, a Requirement for AN Differentiation(A and B) T-CAD knockdown in NCs and VSMCs (with siRNA against T-cad [siTcad]) decreased NC (DiI, green) differentiation toward a (A) neuronal fate (CTB, blue) and (B) an AN fate (DDC, blue), as measured by co-localization (overlap of pixels) of NC and marker immunostaining. Knockdown in ECs had no effect. Data are presented as mean ± SEM (A, top). For CTB, a one-way ANOVA with Dunnett’s post-test was used to determine statistical significance from the control “NC only” group (n = 8 images) (n = 11 images for each siTcad-treated group, ∗p = 0.0164, ∗∗p = 0.0006). (B, top) For DDC, a one-way ANOVA with Tukey’s post-test was used to determine statistical significance from the “NC only” group (n = 8 wells) (n = 11 with three replicates for each siTcad treated group, ∗p = 0.0337, #p = 0.0262). Data are pooled from three independent experiments done in triplicate. (A and B, bottom) Representative images of staining for CTB or DDC. Scale bar represents 100 μm. Note that, with the loss of T-cad in NCs and VSMCs, though not in ECs, as a result of siTcad, expression of CTB (blue in A) and DDC (blue in B) is significantly reduced. (The green-encircled black regions represent the location of the Cytodex beads upon which ECs were coated in the 3D fibrin gel system; see Methods). For all experiments, siTcad efficiency was established through real-time PCR by isolating RNA from non-embedded excess cells 48-hr post transfection (Figure S6A).(C) In the transwell differentiation culture system schematized in Figure 4, addition of recombinant T-cad (rT-cad, 1 μg/ml), which binds and inhibits T-cad homotypic interaction, to NCs and VSMCs (top chamber), with ECs in the bottom chamber, inhibited NC differentiation toward an AN fate. (Top) Quantitation of DDC (red) and peripherin (blue) staining in NCs (DiI, green) (judged by degree of pixel overlap), treated with control (n = 6 transwells) or rT-cad (n = 5 transwells). Data are presented as mean ± SEM and are pooled from three separate experiments. A two-tailed Student’s t test was used to determine statistical significance. ∗p < 0.0001, # p < 0.0001. (Bottom) Representative images of transwells. Scale bar represents 100 μm.(D, top) Percentage of DDC (green) and U.E. lectin (red) overlap (right histograms) and length of contact/overlap between ECs and ANs (left histograms) in each image from our hESC differentiation model. A two-tailed Student’s t test was used to determine statistical significance. ∗p = 0.038, ∗∗p = 7.5e-6, error bars are ± SEM; n = 9 images per group from three independent experiments. (Bottom) Representative high magnification images of degree of DDC (green) alignment with ECs (U.E. lectin, red) in hESC cultures treated with control versus rT-cad. Scale bar represents 100 μm. Note that, in contrast to control conditions (white arrow), with the loss of T-cad action following the addition of rT-cad, NC-derivatives and vasculature no longer co-align (white arrowhead).
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fig6: T-cadherin, Expressed by NCs and VSMCs, Mediates NC Co-alignment and Direct Contact with VSMCs, a Requirement for AN Differentiation(A and B) T-CAD knockdown in NCs and VSMCs (with siRNA against T-cad [siTcad]) decreased NC (DiI, green) differentiation toward a (A) neuronal fate (CTB, blue) and (B) an AN fate (DDC, blue), as measured by co-localization (overlap of pixels) of NC and marker immunostaining. Knockdown in ECs had no effect. Data are presented as mean ± SEM (A, top). For CTB, a one-way ANOVA with Dunnett’s post-test was used to determine statistical significance from the control “NC only” group (n = 8 images) (n = 11 images for each siTcad-treated group, ∗p = 0.0164, ∗∗p = 0.0006). (B, top) For DDC, a one-way ANOVA with Tukey’s post-test was used to determine statistical significance from the “NC only” group (n = 8 wells) (n = 11 with three replicates for each siTcad treated group, ∗p = 0.0337, #p = 0.0262). Data are pooled from three independent experiments done in triplicate. (A and B, bottom) Representative images of staining for CTB or DDC. Scale bar represents 100 μm. Note that, with the loss of T-cad in NCs and VSMCs, though not in ECs, as a result of siTcad, expression of CTB (blue in A) and DDC (blue in B) is significantly reduced. (The green-encircled black regions represent the location of the Cytodex beads upon which ECs were coated in the 3D fibrin gel system; see Methods). For all experiments, siTcad efficiency was established through real-time PCR by isolating RNA from non-embedded excess cells 48-hr post transfection (Figure S6A).(C) In the transwell differentiation culture system schematized in Figure 4, addition of recombinant T-cad (rT-cad, 1 μg/ml), which binds and inhibits T-cad homotypic interaction, to NCs and VSMCs (top chamber), with ECs in the bottom chamber, inhibited NC differentiation toward an AN fate. (Top) Quantitation of DDC (red) and peripherin (blue) staining in NCs (DiI, green) (judged by degree of pixel overlap), treated with control (n = 6 transwells) or rT-cad (n = 5 transwells). Data are presented as mean ± SEM and are pooled from three separate experiments. A two-tailed Student’s t test was used to determine statistical significance. ∗p < 0.0001, # p < 0.0001. (Bottom) Representative images of transwells. Scale bar represents 100 μm.(D, top) Percentage of DDC (green) and U.E. lectin (red) overlap (right histograms) and length of contact/overlap between ECs and ANs (left histograms) in each image from our hESC differentiation model. A two-tailed Student’s t test was used to determine statistical significance. ∗p = 0.038, ∗∗p = 7.5e-6, error bars are ± SEM; n = 9 images per group from three independent experiments. (Bottom) Representative high magnification images of degree of DDC (green) alignment with ECs (U.E. lectin, red) in hESC cultures treated with control versus rT-cad. Scale bar represents 100 μm. Note that, in contrast to control conditions (white arrow), with the loss of T-cad action following the addition of rT-cad, NC-derivatives and vasculature no longer co-align (white arrowhead).

Mentions: To refine our investigation of how T-cad regulates vascular-mediated AN differentiation, we transiently knocked down T-cad in ECs, VSMCs, or NCs prior to embedding them in our 3D vascular tube model. Only loss of T-cad in NC or VSMCs led to a significant decrease in AN differentiation as measured by staining first with the pan-neuronal marker CTB (Figure 6A) and the AN marker DDC (Figure 6B). T-cad knockdown in ECs did not inhibit AN differentiation (Figures 6A and 6B). These findings are consistent with a role for T-cad in direct cell-cell interactions between the VSMC and NC. Similarly, T-cad knockdown in VSMCs or NCs inhibited differentiation of NCs in the transwell model, whereas knockdown in ECs had no such effect (Figure S6B). To further investigate the role of T-cad in AN determination, we utilized a soluble form of recombinant T-cad (rT-cad) known to selectively disrupt T-cad-mediated cell-cell adhesion (Fredette et al., 1996). Because direct cell-cell contact between NCs and VSMCs is critical for AN differentiation (Figure 4A), we examined the interaction between these cells in the transwell model. Soluble rT-cad as a receptor antagonist was added to the top chamber at the time of NC and VSMC cell addition, and ECs were plated in the bottom chamber. After 4 days in co-culture, soluble rT-cad treatment significantly decreased the number of DDC+ and peripherin+ neurons, suggesting that NC differentiation toward an AN fate had been restricted (Figure 6C).


hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature.

Acevedo LM, Lindquist JN, Walsh BM, Sia P, Cimadamore F, Chen C, Denzel M, Pernia CD, Ranscht B, Terskikh A, Snyder EY, Cheresh DA - Stem Cell Reports (2015)

T-cadherin, Expressed by NCs and VSMCs, Mediates NC Co-alignment and Direct Contact with VSMCs, a Requirement for AN Differentiation(A and B) T-CAD knockdown in NCs and VSMCs (with siRNA against T-cad [siTcad]) decreased NC (DiI, green) differentiation toward a (A) neuronal fate (CTB, blue) and (B) an AN fate (DDC, blue), as measured by co-localization (overlap of pixels) of NC and marker immunostaining. Knockdown in ECs had no effect. Data are presented as mean ± SEM (A, top). For CTB, a one-way ANOVA with Dunnett’s post-test was used to determine statistical significance from the control “NC only” group (n = 8 images) (n = 11 images for each siTcad-treated group, ∗p = 0.0164, ∗∗p = 0.0006). (B, top) For DDC, a one-way ANOVA with Tukey’s post-test was used to determine statistical significance from the “NC only” group (n = 8 wells) (n = 11 with three replicates for each siTcad treated group, ∗p = 0.0337, #p = 0.0262). Data are pooled from three independent experiments done in triplicate. (A and B, bottom) Representative images of staining for CTB or DDC. Scale bar represents 100 μm. Note that, with the loss of T-cad in NCs and VSMCs, though not in ECs, as a result of siTcad, expression of CTB (blue in A) and DDC (blue in B) is significantly reduced. (The green-encircled black regions represent the location of the Cytodex beads upon which ECs were coated in the 3D fibrin gel system; see Methods). For all experiments, siTcad efficiency was established through real-time PCR by isolating RNA from non-embedded excess cells 48-hr post transfection (Figure S6A).(C) In the transwell differentiation culture system schematized in Figure 4, addition of recombinant T-cad (rT-cad, 1 μg/ml), which binds and inhibits T-cad homotypic interaction, to NCs and VSMCs (top chamber), with ECs in the bottom chamber, inhibited NC differentiation toward an AN fate. (Top) Quantitation of DDC (red) and peripherin (blue) staining in NCs (DiI, green) (judged by degree of pixel overlap), treated with control (n = 6 transwells) or rT-cad (n = 5 transwells). Data are presented as mean ± SEM and are pooled from three separate experiments. A two-tailed Student’s t test was used to determine statistical significance. ∗p < 0.0001, # p < 0.0001. (Bottom) Representative images of transwells. Scale bar represents 100 μm.(D, top) Percentage of DDC (green) and U.E. lectin (red) overlap (right histograms) and length of contact/overlap between ECs and ANs (left histograms) in each image from our hESC differentiation model. A two-tailed Student’s t test was used to determine statistical significance. ∗p = 0.038, ∗∗p = 7.5e-6, error bars are ± SEM; n = 9 images per group from three independent experiments. (Bottom) Representative high magnification images of degree of DDC (green) alignment with ECs (U.E. lectin, red) in hESC cultures treated with control versus rT-cad. Scale bar represents 100 μm. Note that, in contrast to control conditions (white arrow), with the loss of T-cad action following the addition of rT-cad, NC-derivatives and vasculature no longer co-align (white arrowhead).
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fig6: T-cadherin, Expressed by NCs and VSMCs, Mediates NC Co-alignment and Direct Contact with VSMCs, a Requirement for AN Differentiation(A and B) T-CAD knockdown in NCs and VSMCs (with siRNA against T-cad [siTcad]) decreased NC (DiI, green) differentiation toward a (A) neuronal fate (CTB, blue) and (B) an AN fate (DDC, blue), as measured by co-localization (overlap of pixels) of NC and marker immunostaining. Knockdown in ECs had no effect. Data are presented as mean ± SEM (A, top). For CTB, a one-way ANOVA with Dunnett’s post-test was used to determine statistical significance from the control “NC only” group (n = 8 images) (n = 11 images for each siTcad-treated group, ∗p = 0.0164, ∗∗p = 0.0006). (B, top) For DDC, a one-way ANOVA with Tukey’s post-test was used to determine statistical significance from the “NC only” group (n = 8 wells) (n = 11 with three replicates for each siTcad treated group, ∗p = 0.0337, #p = 0.0262). Data are pooled from three independent experiments done in triplicate. (A and B, bottom) Representative images of staining for CTB or DDC. Scale bar represents 100 μm. Note that, with the loss of T-cad in NCs and VSMCs, though not in ECs, as a result of siTcad, expression of CTB (blue in A) and DDC (blue in B) is significantly reduced. (The green-encircled black regions represent the location of the Cytodex beads upon which ECs were coated in the 3D fibrin gel system; see Methods). For all experiments, siTcad efficiency was established through real-time PCR by isolating RNA from non-embedded excess cells 48-hr post transfection (Figure S6A).(C) In the transwell differentiation culture system schematized in Figure 4, addition of recombinant T-cad (rT-cad, 1 μg/ml), which binds and inhibits T-cad homotypic interaction, to NCs and VSMCs (top chamber), with ECs in the bottom chamber, inhibited NC differentiation toward an AN fate. (Top) Quantitation of DDC (red) and peripherin (blue) staining in NCs (DiI, green) (judged by degree of pixel overlap), treated with control (n = 6 transwells) or rT-cad (n = 5 transwells). Data are presented as mean ± SEM and are pooled from three separate experiments. A two-tailed Student’s t test was used to determine statistical significance. ∗p < 0.0001, # p < 0.0001. (Bottom) Representative images of transwells. Scale bar represents 100 μm.(D, top) Percentage of DDC (green) and U.E. lectin (red) overlap (right histograms) and length of contact/overlap between ECs and ANs (left histograms) in each image from our hESC differentiation model. A two-tailed Student’s t test was used to determine statistical significance. ∗p = 0.038, ∗∗p = 7.5e-6, error bars are ± SEM; n = 9 images per group from three independent experiments. (Bottom) Representative high magnification images of degree of DDC (green) alignment with ECs (U.E. lectin, red) in hESC cultures treated with control versus rT-cad. Scale bar represents 100 μm. Note that, in contrast to control conditions (white arrow), with the loss of T-cad action following the addition of rT-cad, NC-derivatives and vasculature no longer co-align (white arrowhead).
Mentions: To refine our investigation of how T-cad regulates vascular-mediated AN differentiation, we transiently knocked down T-cad in ECs, VSMCs, or NCs prior to embedding them in our 3D vascular tube model. Only loss of T-cad in NC or VSMCs led to a significant decrease in AN differentiation as measured by staining first with the pan-neuronal marker CTB (Figure 6A) and the AN marker DDC (Figure 6B). T-cad knockdown in ECs did not inhibit AN differentiation (Figures 6A and 6B). These findings are consistent with a role for T-cad in direct cell-cell interactions between the VSMC and NC. Similarly, T-cad knockdown in VSMCs or NCs inhibited differentiation of NCs in the transwell model, whereas knockdown in ECs had no such effect (Figure S6B). To further investigate the role of T-cad in AN determination, we utilized a soluble form of recombinant T-cad (rT-cad) known to selectively disrupt T-cad-mediated cell-cell adhesion (Fredette et al., 1996). Because direct cell-cell contact between NCs and VSMCs is critical for AN differentiation (Figure 4A), we examined the interaction between these cells in the transwell model. Soluble rT-cad as a receptor antagonist was added to the top chamber at the time of NC and VSMC cell addition, and ECs were plated in the bottom chamber. After 4 days in co-culture, soluble rT-cad treatment significantly decreased the number of DDC+ and peripherin+ neurons, suggesting that NC differentiation toward an AN fate had been restricted (Figure 6C).

Bottom Line: Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions.Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature.Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

View Article: PubMed Central - PubMed

Affiliation: Moores Cancer Center, University of California, San Diego, La Jolla, CA, 92093, USA; Sanford Burnham Medical Research Institute, La Jolla, CA 92037, USA; Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA.

No MeSH data available.


Related in: MedlinePlus