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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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Inhibition of Rock activity rescues delamination of G1-arrested neural crest (NC) cells. (A,D,G,J) Phase contrast. (B,C,E,F,H,I,K,L) Bromo-deoxyuridine (BrdU) immunostaining. (C,F,I,L) Higher magnifications to appreciate the front of the delaminated NC cells. Inhibition of Rock with Y27632 enhanced delamination of BrdU+ NC cells ((D-F) compared to (A-C)). Mimosine blocked both BrdU incorporation and NC delamination following 9 h (G-I), whereas cotreatment with mimosine (Mim) and Y-27632 rescued emigration of G1-arrested, BrdU-negative NC cells (J-L). Bar: 45 μM (A,B,D,E,G,H,J,K); 24 μM (C,F,I,L).
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Figure 10: Inhibition of Rock activity rescues delamination of G1-arrested neural crest (NC) cells. (A,D,G,J) Phase contrast. (B,C,E,F,H,I,K,L) Bromo-deoxyuridine (BrdU) immunostaining. (C,F,I,L) Higher magnifications to appreciate the front of the delaminated NC cells. Inhibition of Rock with Y27632 enhanced delamination of BrdU+ NC cells ((D-F) compared to (A-C)). Mimosine blocked both BrdU incorporation and NC delamination following 9 h (G-I), whereas cotreatment with mimosine (Mim) and Y-27632 rescued emigration of G1-arrested, BrdU-negative NC cells (J-L). Bar: 45 μM (A,B,D,E,G,H,J,K); 24 μM (C,F,I,L).

Mentions: We previously showed that trunk NC cells delaminate in the S-phase of the cell cycle and that G1-S transition is a prerequisite for cell emigration [21]. Furthermore, we demonstrated that BMP acts through canonical Wnt signaling that stimulates G1/S transition and NC delamination [14]. To further investigate the relationship between Rho/Rock activity and the BMP-dependent cascade, we prevented G1/S transition with the plant amino-acid mimosine, which induces expression of p27, and asked whether inhibition of Rock activity by Y27632 would rescue G1/S transition and NC emigration. As previously shown [21], mimosine (600 μM) completely blocked both BrdU incorporation and NC delamination from neural primordia explanted for 9 h, the approximate length of one cell cycle (Figure 10A–C,G–I; N = 16). Treatment with Y27632 facilitated delamination of NC cells and had no adverse effect on BrdU incorporation, with a proportion of BrdU+ cells similar to that in controls (Figure 10A–F, and data not shown; N = 16). Notably, co-treatment with mimosine and Y27632 rescued delamination of NC cells, which subsequently dispersed from the explanted epithelium, but the emigrating cells were BrdU-negative, indicating they were still arrested at G1 (Figure 10J–L; N = 16). A similar picture was obtained when explants were treated with membrane-soluble C3 and mimosine (data not shown), further suggesting that Rho proteins via Rock act downstream of G1/S transition to modulate NC emigration.


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)

Inhibition of Rock activity rescues delamination of G1-arrested neural crest (NC) cells. (A,D,G,J) Phase contrast. (B,C,E,F,H,I,K,L) Bromo-deoxyuridine (BrdU) immunostaining. (C,F,I,L) Higher magnifications to appreciate the front of the delaminated NC cells. Inhibition of Rock with Y27632 enhanced delamination of BrdU+ NC cells ((D-F) compared to (A-C)). Mimosine blocked both BrdU incorporation and NC delamination following 9 h (G-I), whereas cotreatment with mimosine (Mim) and Y-27632 rescued emigration of G1-arrested, BrdU-negative NC cells (J-L). Bar: 45 μM (A,B,D,E,G,H,J,K); 24 μM (C,F,I,L).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577655&req=5

Figure 10: Inhibition of Rock activity rescues delamination of G1-arrested neural crest (NC) cells. (A,D,G,J) Phase contrast. (B,C,E,F,H,I,K,L) Bromo-deoxyuridine (BrdU) immunostaining. (C,F,I,L) Higher magnifications to appreciate the front of the delaminated NC cells. Inhibition of Rock with Y27632 enhanced delamination of BrdU+ NC cells ((D-F) compared to (A-C)). Mimosine blocked both BrdU incorporation and NC delamination following 9 h (G-I), whereas cotreatment with mimosine (Mim) and Y-27632 rescued emigration of G1-arrested, BrdU-negative NC cells (J-L). Bar: 45 μM (A,B,D,E,G,H,J,K); 24 μM (C,F,I,L).
Mentions: We previously showed that trunk NC cells delaminate in the S-phase of the cell cycle and that G1-S transition is a prerequisite for cell emigration [21]. Furthermore, we demonstrated that BMP acts through canonical Wnt signaling that stimulates G1/S transition and NC delamination [14]. To further investigate the relationship between Rho/Rock activity and the BMP-dependent cascade, we prevented G1/S transition with the plant amino-acid mimosine, which induces expression of p27, and asked whether inhibition of Rock activity by Y27632 would rescue G1/S transition and NC emigration. As previously shown [21], mimosine (600 μM) completely blocked both BrdU incorporation and NC delamination from neural primordia explanted for 9 h, the approximate length of one cell cycle (Figure 10A–C,G–I; N = 16). Treatment with Y27632 facilitated delamination of NC cells and had no adverse effect on BrdU incorporation, with a proportion of BrdU+ cells similar to that in controls (Figure 10A–F, and data not shown; N = 16). Notably, co-treatment with mimosine and Y27632 rescued delamination of NC cells, which subsequently dispersed from the explanted epithelium, but the emigrating cells were BrdU-negative, indicating they were still arrested at G1 (Figure 10J–L; N = 16). A similar picture was obtained when explants were treated with membrane-soluble C3 and mimosine (data not shown), further suggesting that Rho proteins via Rock act downstream of G1/S transition to modulate NC emigration.

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