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A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells.

Groysman M, Shoval I, Kalcheim C - Neural Dev (2008)

Bottom Line: Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above.In the latter condition, cells emigrated while arrested at G1.Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. mayagr@ekmd.huji.ac.il

ABSTRACT

Background: Neural crest progenitors arise as epithelial cells and then undergo a process of epithelial to mesenchymal transition that precedes the generation of cellular motility and subsequent migration. We aim at understanding the underlying molecular network. Along this line, possible roles of Rho GTPases that act as molecular switches to control a variety of signal transduction pathways remain virtually unexplored, as are putative interactions between Rho proteins and additional known components of this cascade.

Results: We investigated the role of Rho/Rock signaling in neural crest delamination. Active RhoA and RhoB are expressed in the membrane of epithelial progenitors and are downregulated upon delamination. In vivo loss-of-function of RhoA or RhoB or of overall Rho signaling by C3 transferase enhanced and/or triggered premature crest delamination yet had no effect on cell specification. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced crest emigration without affecting cell proliferation. These treatments altered neural crest morphology by reducing stress fibers, focal adhesions and downregulating membrane-bound N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above. Since delamination is triggered by BMP and requires G1/S transition, we examined their relationship with Rho. Blocking Rho/Rock function rescued crest emigration upon treatment with noggin or with the G1/S inhibitor mimosine. In the latter condition, cells emigrated while arrested at G1. Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

Conclusion: Rho-GTPases, through Rock, act downstream of BMP and of G1/S transition to negatively regulate crest delamination by modifying cytoskeleton assembly and intercellular adhesion.

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Rho/Rock signaling stabilize membrane-bound N-cadherin while inhibiting neural crest (NC) delamination. (A) In control explants, N-cadherin (red) is expressed in the membrane of epithelial and epithelioid cells flattening onto the substrate but is downregulated upon delamination in mesenchymal cells. (B) N-cadherin disappears from the membrane of Y27632-treated cells that exhibit a mesenchymal phenotype even close to the neural tube (NT). (C) Lysophosphatidic acid (LPA) strenghtens membranous N-cadherin immunoreactivity while inhibiting epithelial-to-mesenchymal transition (EMT). (D) These effects of LPA are reverted by co-treatment with Y27632. (E,F) Inhibition of N-cadherin cleavage by GI254023X keeps membrane-associated N-cadherin and prevents NC EMT; both effects are reverted by inhibiting Rock activity. Note in (F) that membrane N-cadherin staining is either fragmentary or absent. Insets show low magnification of phase contrast images. In all panels, the NT explant is to the top. (G,H) Treatment with LPA/pluronic gel in vivo prevents the normal downregulation of N-cadherin from adherens junctions (arrowhead in (H)) that is seen in control gel-treated embryos (arrows in (G)) at dissociated levels of the axis. (I and inset) Loss of RhoB function by transfection of N19-RhoB/green fluorescent protein (GFP) prematurely downregulates N-cadherin from adherens junctions in the apical NT when compared to the untreated side (arrowhead pointing at the apical side of the NT expressing N19/GFP but devoid of N-cadherin). Note that electroporation at more ventral domains of the NT was without effect on N-cadherin staining (ventral to the arrow), consistent with the dorsally restricted expression of RhoB. DM, dermomyotome; Scl, sclerotome. Bar: 15 μM (A-F); 500 μM (insets in E,F); 26 μM (G,H); 14 μM (I); 65 μM, inset in (I).
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Figure 8: Rho/Rock signaling stabilize membrane-bound N-cadherin while inhibiting neural crest (NC) delamination. (A) In control explants, N-cadherin (red) is expressed in the membrane of epithelial and epithelioid cells flattening onto the substrate but is downregulated upon delamination in mesenchymal cells. (B) N-cadherin disappears from the membrane of Y27632-treated cells that exhibit a mesenchymal phenotype even close to the neural tube (NT). (C) Lysophosphatidic acid (LPA) strenghtens membranous N-cadherin immunoreactivity while inhibiting epithelial-to-mesenchymal transition (EMT). (D) These effects of LPA are reverted by co-treatment with Y27632. (E,F) Inhibition of N-cadherin cleavage by GI254023X keeps membrane-associated N-cadherin and prevents NC EMT; both effects are reverted by inhibiting Rock activity. Note in (F) that membrane N-cadherin staining is either fragmentary or absent. Insets show low magnification of phase contrast images. In all panels, the NT explant is to the top. (G,H) Treatment with LPA/pluronic gel in vivo prevents the normal downregulation of N-cadherin from adherens junctions (arrowhead in (H)) that is seen in control gel-treated embryos (arrows in (G)) at dissociated levels of the axis. (I and inset) Loss of RhoB function by transfection of N19-RhoB/green fluorescent protein (GFP) prematurely downregulates N-cadherin from adherens junctions in the apical NT when compared to the untreated side (arrowhead pointing at the apical side of the NT expressing N19/GFP but devoid of N-cadherin). Note that electroporation at more ventral domains of the NT was without effect on N-cadherin staining (ventral to the arrow), consistent with the dorsally restricted expression of RhoB. DM, dermomyotome; Scl, sclerotome. Bar: 15 μM (A-F); 500 μM (insets in E,F); 26 μM (G,H); 14 μM (I); 65 μM, inset in (I).

Mentions: Previously, we reported that inhibition of ADAM 10-dependent cleavage of N-cadherin with GI254023X, which maintains the full-length protein in a membrane-bound conformation, prevented NC delamination [31]. Here we show that treatment with GI254023X also results in a stable cytoskeleton rich in F-actin stress fibers, similar to the phenotype of LPA-treated NTs. Importantly, application of Y27632 reversed both GI254023X-induced effects (Additional file 3E,F). These observations underscore an interaction between N-cadherin and Rho/Rock via regulation of F-actin dynamics. To directly explore this issue, we stained neural primordia for N-cadherin. In control explants, N-cadherin protein was strongly expressed in the membrane of epithelial progenitors as well as in the epithelioid cells flattening on the substrate. N-cadherin immunoreactivity was, however, lost from delaminating cells that adopted a mesenchymal phenotype (Figure 8A) [31]. In contrast, in the presence of Y27632, the cells adjacent to the NTs were already devoid of membrane-associated N-cadherin and appeared separated from each other, suggesting they lost intercellular adhesions prematurely (Figure 8B; see also Figure 3 and Additional file 1). Consistent with this observation, electroporation of N19-RhoB or C3 resulted in rapid downregulation of N-cadherin protein from adherens junctions in the transfected dorsal hemi-NT in ovo (Figure 8I, arrowhead), yet had no effect further ventrally where endogenous RhoB is absent (Figure 8I, arrow and ventralward, and data not shown). Reciprocally, LPA maintained and even upregulated N-cadherin membrane expression whereas co-treatment with Y27632 rescued NC delamination and also reduced N-cadherin immunoreactivity (Figures 8C,D and 9G). Consistent with the explant data, in ovo treatment with LPA, which inhibited NC emigration (Figure 6), maintained N-cadherin associated with adherens junctions in the dorsal NT at axial levels where N-cadherin has been normally downregulated (Figure 8G,H) [31]. Likewise, treatment with GI254023X, which inhibited NC emigration, maintained membrane-associated N-cadherin in explants; when co-treatment with Y27632 was performed, membranous N-cadherin was either completely lost or fragmentary and NC cells underwent EMT (Figure 8E,F).


A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells.

Groysman M, Shoval I, Kalcheim C - Neural Dev (2008)

Rho/Rock signaling stabilize membrane-bound N-cadherin while inhibiting neural crest (NC) delamination. (A) In control explants, N-cadherin (red) is expressed in the membrane of epithelial and epithelioid cells flattening onto the substrate but is downregulated upon delamination in mesenchymal cells. (B) N-cadherin disappears from the membrane of Y27632-treated cells that exhibit a mesenchymal phenotype even close to the neural tube (NT). (C) Lysophosphatidic acid (LPA) strenghtens membranous N-cadherin immunoreactivity while inhibiting epithelial-to-mesenchymal transition (EMT). (D) These effects of LPA are reverted by co-treatment with Y27632. (E,F) Inhibition of N-cadherin cleavage by GI254023X keeps membrane-associated N-cadherin and prevents NC EMT; both effects are reverted by inhibiting Rock activity. Note in (F) that membrane N-cadherin staining is either fragmentary or absent. Insets show low magnification of phase contrast images. In all panels, the NT explant is to the top. (G,H) Treatment with LPA/pluronic gel in vivo prevents the normal downregulation of N-cadherin from adherens junctions (arrowhead in (H)) that is seen in control gel-treated embryos (arrows in (G)) at dissociated levels of the axis. (I and inset) Loss of RhoB function by transfection of N19-RhoB/green fluorescent protein (GFP) prematurely downregulates N-cadherin from adherens junctions in the apical NT when compared to the untreated side (arrowhead pointing at the apical side of the NT expressing N19/GFP but devoid of N-cadherin). Note that electroporation at more ventral domains of the NT was without effect on N-cadherin staining (ventral to the arrow), consistent with the dorsally restricted expression of RhoB. DM, dermomyotome; Scl, sclerotome. Bar: 15 μM (A-F); 500 μM (insets in E,F); 26 μM (G,H); 14 μM (I); 65 μM, inset in (I).
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Figure 8: Rho/Rock signaling stabilize membrane-bound N-cadherin while inhibiting neural crest (NC) delamination. (A) In control explants, N-cadherin (red) is expressed in the membrane of epithelial and epithelioid cells flattening onto the substrate but is downregulated upon delamination in mesenchymal cells. (B) N-cadherin disappears from the membrane of Y27632-treated cells that exhibit a mesenchymal phenotype even close to the neural tube (NT). (C) Lysophosphatidic acid (LPA) strenghtens membranous N-cadherin immunoreactivity while inhibiting epithelial-to-mesenchymal transition (EMT). (D) These effects of LPA are reverted by co-treatment with Y27632. (E,F) Inhibition of N-cadherin cleavage by GI254023X keeps membrane-associated N-cadherin and prevents NC EMT; both effects are reverted by inhibiting Rock activity. Note in (F) that membrane N-cadherin staining is either fragmentary or absent. Insets show low magnification of phase contrast images. In all panels, the NT explant is to the top. (G,H) Treatment with LPA/pluronic gel in vivo prevents the normal downregulation of N-cadherin from adherens junctions (arrowhead in (H)) that is seen in control gel-treated embryos (arrows in (G)) at dissociated levels of the axis. (I and inset) Loss of RhoB function by transfection of N19-RhoB/green fluorescent protein (GFP) prematurely downregulates N-cadherin from adherens junctions in the apical NT when compared to the untreated side (arrowhead pointing at the apical side of the NT expressing N19/GFP but devoid of N-cadherin). Note that electroporation at more ventral domains of the NT was without effect on N-cadherin staining (ventral to the arrow), consistent with the dorsally restricted expression of RhoB. DM, dermomyotome; Scl, sclerotome. Bar: 15 μM (A-F); 500 μM (insets in E,F); 26 μM (G,H); 14 μM (I); 65 μM, inset in (I).
Mentions: Previously, we reported that inhibition of ADAM 10-dependent cleavage of N-cadherin with GI254023X, which maintains the full-length protein in a membrane-bound conformation, prevented NC delamination [31]. Here we show that treatment with GI254023X also results in a stable cytoskeleton rich in F-actin stress fibers, similar to the phenotype of LPA-treated NTs. Importantly, application of Y27632 reversed both GI254023X-induced effects (Additional file 3E,F). These observations underscore an interaction between N-cadherin and Rho/Rock via regulation of F-actin dynamics. To directly explore this issue, we stained neural primordia for N-cadherin. In control explants, N-cadherin protein was strongly expressed in the membrane of epithelial progenitors as well as in the epithelioid cells flattening on the substrate. N-cadherin immunoreactivity was, however, lost from delaminating cells that adopted a mesenchymal phenotype (Figure 8A) [31]. In contrast, in the presence of Y27632, the cells adjacent to the NTs were already devoid of membrane-associated N-cadherin and appeared separated from each other, suggesting they lost intercellular adhesions prematurely (Figure 8B; see also Figure 3 and Additional file 1). Consistent with this observation, electroporation of N19-RhoB or C3 resulted in rapid downregulation of N-cadherin protein from adherens junctions in the transfected dorsal hemi-NT in ovo (Figure 8I, arrowhead), yet had no effect further ventrally where endogenous RhoB is absent (Figure 8I, arrow and ventralward, and data not shown). Reciprocally, LPA maintained and even upregulated N-cadherin membrane expression whereas co-treatment with Y27632 rescued NC delamination and also reduced N-cadherin immunoreactivity (Figures 8C,D and 9G). Consistent with the explant data, in ovo treatment with LPA, which inhibited NC emigration (Figure 6), maintained N-cadherin associated with adherens junctions in the dorsal NT at axial levels where N-cadherin has been normally downregulated (Figure 8G,H) [31]. Likewise, treatment with GI254023X, which inhibited NC emigration, maintained membrane-associated N-cadherin in explants; when co-treatment with Y27632 was performed, membranous N-cadherin was either completely lost or fragmentary and NC cells underwent EMT (Figure 8E,F).

Bottom Line: Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above.In the latter condition, cells emigrated while arrested at G1.Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. mayagr@ekmd.huji.ac.il

ABSTRACT

Background: Neural crest progenitors arise as epithelial cells and then undergo a process of epithelial to mesenchymal transition that precedes the generation of cellular motility and subsequent migration. We aim at understanding the underlying molecular network. Along this line, possible roles of Rho GTPases that act as molecular switches to control a variety of signal transduction pathways remain virtually unexplored, as are putative interactions between Rho proteins and additional known components of this cascade.

Results: We investigated the role of Rho/Rock signaling in neural crest delamination. Active RhoA and RhoB are expressed in the membrane of epithelial progenitors and are downregulated upon delamination. In vivo loss-of-function of RhoA or RhoB or of overall Rho signaling by C3 transferase enhanced and/or triggered premature crest delamination yet had no effect on cell specification. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced crest emigration without affecting cell proliferation. These treatments altered neural crest morphology by reducing stress fibers, focal adhesions and downregulating membrane-bound N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above. Since delamination is triggered by BMP and requires G1/S transition, we examined their relationship with Rho. Blocking Rho/Rock function rescued crest emigration upon treatment with noggin or with the G1/S inhibitor mimosine. In the latter condition, cells emigrated while arrested at G1. Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

Conclusion: Rho-GTPases, through Rock, act downstream of BMP and of G1/S transition to negatively regulate crest delamination by modifying cytoskeleton assembly and intercellular adhesion.

Show MeSH
Related in: MedlinePlus