Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of either RhoA or RhoB activities enhances neural crest (NC) delamination in ovo. (A-C) Transverse sections at a dissociated somite level showing the emigration of control green fluorescent protein (GFP)-expressing (A,A'), N19-RhoB-expressing (B,B') and GAP-RhoB-expressing (C,C') NC cells (green). Note in (A',B',C') that most delaminating progenitors are also bromo-deoxyuridine (BrdU)+ (red; arrows pointing at double-labeled cells). (D,D') Transverse section at a similar level as in (A-C) of a hemi-NT that received N19-RhoB (green) and was further stained for HNK-1 (red in (D)) and FoXD3 mRNA (D' and inset). Transfected emigrating cells are positive both for HNK-1 and FoXD3 (arrows). (E, F) Two transverse sections of embryos that received N19-RhoA (green) and were further processed for HNK-1 immunostaining (E, red) or FoXD3 in situ hybridization (F and inset). Note that delaminating cells that received N19-RhoA also express HNK-1 or FoxD3 (arrows). Note as well that the transfected hemi-NT cells lost their normal pseudostratified appearance and are rounded. (G-I) Transverse sections through the caudal segmental plate (cSP) level (G), rostral segmental plate (rSP) level (H), and recently formed epithelial somite (ES) (I) of embryos that received N19-RhoA. Note premature delamination of NC progenitors expressing N19-rhoA/GFP (arrows) on a background of Hoechst nuclear stain (blue) or co-expressing Snail2 mRNA (inset in (I)). No delamination from these axial levels is observed upon transfection of control GFP (not shown). Note that at these very caudal levels of the axis, electroporation was predominantly dorsal due to positioning of the electrodes at a slightly more rostral level in order not to damage the gastrulating area of the axis. (J) Quantification of NC delamination (*p < 0.05, **p < 0.01, ***p = 0.0001). DS, dissociating somite; ES, epithelial somite; s.d., standard deviation; SP, segmental plate. Bar: 45 μM (A-F); 53 μM (G-I); 83 μM, insets in (D,E,I).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2577655&req=5

Figure 5: Inhibition of either RhoA or RhoB activities enhances neural crest (NC) delamination in ovo. (A-C) Transverse sections at a dissociated somite level showing the emigration of control green fluorescent protein (GFP)-expressing (A,A'), N19-RhoB-expressing (B,B') and GAP-RhoB-expressing (C,C') NC cells (green). Note in (A',B',C') that most delaminating progenitors are also bromo-deoxyuridine (BrdU)+ (red; arrows pointing at double-labeled cells). (D,D') Transverse section at a similar level as in (A-C) of a hemi-NT that received N19-RhoB (green) and was further stained for HNK-1 (red in (D)) and FoXD3 mRNA (D' and inset). Transfected emigrating cells are positive both for HNK-1 and FoXD3 (arrows). (E, F) Two transverse sections of embryos that received N19-RhoA (green) and were further processed for HNK-1 immunostaining (E, red) or FoXD3 in situ hybridization (F and inset). Note that delaminating cells that received N19-RhoA also express HNK-1 or FoxD3 (arrows). Note as well that the transfected hemi-NT cells lost their normal pseudostratified appearance and are rounded. (G-I) Transverse sections through the caudal segmental plate (cSP) level (G), rostral segmental plate (rSP) level (H), and recently formed epithelial somite (ES) (I) of embryos that received N19-RhoA. Note premature delamination of NC progenitors expressing N19-rhoA/GFP (arrows) on a background of Hoechst nuclear stain (blue) or co-expressing Snail2 mRNA (inset in (I)). No delamination from these axial levels is observed upon transfection of control GFP (not shown). Note that at these very caudal levels of the axis, electroporation was predominantly dorsal due to positioning of the electrodes at a slightly more rostral level in order not to damage the gastrulating area of the axis. (J) Quantification of NC delamination (*p < 0.05, **p < 0.01, ***p = 0.0001). DS, dissociating somite; ES, epithelial somite; s.d., standard deviation; SP, segmental plate. Bar: 45 μM (A-F); 53 μM (G-I); 83 μM, insets in (D,E,I).

Mentions: Since C3 transferase blocks activity of all Rho proteins, we next monitored the in vivo effects of inhibiting either RhoA or RhoB separately. Inhibition was achieved by overexpression of N19-RhoA or N19-RhoB, which lack GTPase activity [58], or by the chimeric construct GAP-rhoB. The latter was prepared by fusing the RhoGAP domain of p190, a GTPase activating protein that accelerates intrinsic GTPase activity, with the carboxy-terminal hypervariable sequence of RhoB, which confers specificity to individual Rho proteins [59]. First, we monitored their effects on the F-actin cytoskeleton. To this end, neural primordia were electroporated and then explanted. Whereas control-GFP-treated cells contained stress fibers as well as cortical actin in their periphery, NC cells that received mutant Rho constructs were devoid of stress fibers when compared to their untransfected neighbors and to control-GFP (Additional file 2, and data not shown). Next, we examined their effects on NC delamination in ovo. Both N19-RhoB and GAP-RhoB enhanced NC delamination opposite both epithelial (3.4- and 3.6-fold over control, respectively) and dissociated somites (1.5- and 1.6-fold over control, respectively) (Figure 5A–C,J, and data not shown). As with C3 transferase (Figure 4), the effects were more pronounced at earlier stages when fewer control cells emigrated. Since the same embryos were analyzed for both axial levels, values monitored adjacent to epithelial somites stemmed from electroporations that attained the caudal segmental plate level. We assume, therefore, that the delay between transfection and onset of emigration, which is longest at this level, enabled a more efficient expression of the plasmids prior to the beginning of cell emigration and, hence, led to a greater effect. In addition, the delaminating GFP+ NC progenitors were also BrdU+ (Figure 5A,B,C, arrows), further extending the results obtained with C3 and altogether demonstrating that loss of Rho function has no adverse effect on G1-S transition. The identity of delaminating N19-RhoB/GFP+ progenitors was additionally assessed by co-staining with HNK-1 and in situ hybridization with FoxD3. The N19-RhoB/GFP+ emigrating cells co-expressed both HNK-1 and FoxD3 markers, substantiating their NC identity (Figure 5D,D' and inset).


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 either RhoA or RhoB activities enhances neural crest (NC) delamination in ovo. (A-C) Transverse sections at a dissociated somite level showing the emigration of control green fluorescent protein (GFP)-expressing (A,A'), N19-RhoB-expressing (B,B') and GAP-RhoB-expressing (C,C') NC cells (green). Note in (A',B',C') that most delaminating progenitors are also bromo-deoxyuridine (BrdU)+ (red; arrows pointing at double-labeled cells). (D,D') Transverse section at a similar level as in (A-C) of a hemi-NT that received N19-RhoB (green) and was further stained for HNK-1 (red in (D)) and FoXD3 mRNA (D' and inset). Transfected emigrating cells are positive both for HNK-1 and FoXD3 (arrows). (E, F) Two transverse sections of embryos that received N19-RhoA (green) and were further processed for HNK-1 immunostaining (E, red) or FoXD3 in situ hybridization (F and inset). Note that delaminating cells that received N19-RhoA also express HNK-1 or FoxD3 (arrows). Note as well that the transfected hemi-NT cells lost their normal pseudostratified appearance and are rounded. (G-I) Transverse sections through the caudal segmental plate (cSP) level (G), rostral segmental plate (rSP) level (H), and recently formed epithelial somite (ES) (I) of embryos that received N19-RhoA. Note premature delamination of NC progenitors expressing N19-rhoA/GFP (arrows) on a background of Hoechst nuclear stain (blue) or co-expressing Snail2 mRNA (inset in (I)). No delamination from these axial levels is observed upon transfection of control GFP (not shown). Note that at these very caudal levels of the axis, electroporation was predominantly dorsal due to positioning of the electrodes at a slightly more rostral level in order not to damage the gastrulating area of the axis. (J) Quantification of NC delamination (*p < 0.05, **p < 0.01, ***p = 0.0001). DS, dissociating somite; ES, epithelial somite; s.d., standard deviation; SP, segmental plate. Bar: 45 μM (A-F); 53 μM (G-I); 83 μM, insets in (D,E,I).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Inhibition of either RhoA or RhoB activities enhances neural crest (NC) delamination in ovo. (A-C) Transverse sections at a dissociated somite level showing the emigration of control green fluorescent protein (GFP)-expressing (A,A'), N19-RhoB-expressing (B,B') and GAP-RhoB-expressing (C,C') NC cells (green). Note in (A',B',C') that most delaminating progenitors are also bromo-deoxyuridine (BrdU)+ (red; arrows pointing at double-labeled cells). (D,D') Transverse section at a similar level as in (A-C) of a hemi-NT that received N19-RhoB (green) and was further stained for HNK-1 (red in (D)) and FoXD3 mRNA (D' and inset). Transfected emigrating cells are positive both for HNK-1 and FoXD3 (arrows). (E, F) Two transverse sections of embryos that received N19-RhoA (green) and were further processed for HNK-1 immunostaining (E, red) or FoXD3 in situ hybridization (F and inset). Note that delaminating cells that received N19-RhoA also express HNK-1 or FoxD3 (arrows). Note as well that the transfected hemi-NT cells lost their normal pseudostratified appearance and are rounded. (G-I) Transverse sections through the caudal segmental plate (cSP) level (G), rostral segmental plate (rSP) level (H), and recently formed epithelial somite (ES) (I) of embryos that received N19-RhoA. Note premature delamination of NC progenitors expressing N19-rhoA/GFP (arrows) on a background of Hoechst nuclear stain (blue) or co-expressing Snail2 mRNA (inset in (I)). No delamination from these axial levels is observed upon transfection of control GFP (not shown). Note that at these very caudal levels of the axis, electroporation was predominantly dorsal due to positioning of the electrodes at a slightly more rostral level in order not to damage the gastrulating area of the axis. (J) Quantification of NC delamination (*p < 0.05, **p < 0.01, ***p = 0.0001). DS, dissociating somite; ES, epithelial somite; s.d., standard deviation; SP, segmental plate. Bar: 45 μM (A-F); 53 μM (G-I); 83 μM, insets in (D,E,I).
Mentions: Since C3 transferase blocks activity of all Rho proteins, we next monitored the in vivo effects of inhibiting either RhoA or RhoB separately. Inhibition was achieved by overexpression of N19-RhoA or N19-RhoB, which lack GTPase activity [58], or by the chimeric construct GAP-rhoB. The latter was prepared by fusing the RhoGAP domain of p190, a GTPase activating protein that accelerates intrinsic GTPase activity, with the carboxy-terminal hypervariable sequence of RhoB, which confers specificity to individual Rho proteins [59]. First, we monitored their effects on the F-actin cytoskeleton. To this end, neural primordia were electroporated and then explanted. Whereas control-GFP-treated cells contained stress fibers as well as cortical actin in their periphery, NC cells that received mutant Rho constructs were devoid of stress fibers when compared to their untransfected neighbors and to control-GFP (Additional file 2, and data not shown). Next, we examined their effects on NC delamination in ovo. Both N19-RhoB and GAP-RhoB enhanced NC delamination opposite both epithelial (3.4- and 3.6-fold over control, respectively) and dissociated somites (1.5- and 1.6-fold over control, respectively) (Figure 5A–C,J, and data not shown). As with C3 transferase (Figure 4), the effects were more pronounced at earlier stages when fewer control cells emigrated. Since the same embryos were analyzed for both axial levels, values monitored adjacent to epithelial somites stemmed from electroporations that attained the caudal segmental plate level. We assume, therefore, that the delay between transfection and onset of emigration, which is longest at this level, enabled a more efficient expression of the plasmids prior to the beginning of cell emigration and, hence, led to a greater effect. In addition, the delaminating GFP+ NC progenitors were also BrdU+ (Figure 5A,B,C, arrows), further extending the results obtained with C3 and altogether demonstrating that loss of Rho function has no adverse effect on G1-S transition. The identity of delaminating N19-RhoB/GFP+ progenitors was additionally assessed by co-staining with HNK-1 and in situ hybridization with FoxD3. The N19-RhoB/GFP+ emigrating cells co-expressed both HNK-1 and FoxD3 markers, substantiating their NC identity (Figure 5D,D' and inset).

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