Figure 1.
Description of the Cranial Neural Crest Cells Delamination.
(A–P) Transversal cryosections (10 µm) of normal chick embryos at stages 6s (A–D), 8s (E–H), 10s (I–L) and HH14 (M–P) at cranial (A–L) and trunk (M–P) levels. Sections were assayed for N-Cadherin expression by immunofluorescence (A, C, E, G, I, K, M, O). The actin microfilaments and the nuclei were stained by Phalloidin (B, C, F, G, J, K, N, O) and DAPI incorporation (D, H, L, P) respectively. During delamination of cranial NCCs there is a massive accumulation of cells in the dorsal part of the neural tube (A–D). In this cell population, colocalisation of N-Cadherin and Phalloidin is lost indicating that they undergo an EMT (C, G, K). By contrast, during trunk delamination, NCCs emigrate one by one. No particular distortion of the dorsal neural tube is detectable (M-P, arrow heads). (Q–V) Analysis of BrdU incorporation in cranial neural tube during and after NCC delamination. Transversal cryosections (5 µm) of stages HH8–9 embryos, during and after delamination, labeled by immunofluorescence using anti-BrdU antibody (Q, R, T, U). Nuclei are stained by DAPI. Percentages of BrdU positive cells in the different zones of the neural tube are represented in diagrams (S,V). Cranial NCCs are not synchronized in S-phase during delamination (Q–R) or migration (T–U). del, delaminating cells; sur, surrounding region; mid, midline region; mig; migrating cells.
Figure 2.
Ets-1 Expression Occurs Later and in a More Restricted Area than Those of Foxd3 and AP2.
(A–O') Whole in situ hybridization of normal chick embryos at stages 4s–13s using c-ets-1 (A–H), foxd-3 (F–J') and ap-2 (K–O') probes. (F–H) Embryos were cut along the rostro-caudal axis. The left and right sides of the neural tube were treated independently with c-ets-1 and foxd-3 probes. C-ets-1 expression (A–B, F–G) begins after foxd-3 (F–G) and ap-2 (K–L) expressions. It is restricted to cells leaving the neural tube (C–D, insets) and cranial region (compare E–E' to J–J' and O–O'). * indicates ets-1 expression in the sclerotome. r, rhombomere.
Figure 3.
Ets-1 Activity Is Required for Cranial but not Trunk Delamination.
(A–L) Analysis of cranial NCCs delamination in HH7-9 chick embryos electroporated by c-ets-1 DBD and harvested at 15 hours post electroporation (hpe). (A–D; G–J) Whole mount in situ hybridization using ap-2 probe. GFP expression in (A) and (G) indicates the electroporation zone. (C–D) and (I–J) are vibratome sections (30 µm) of embryos presented in (B) and (H) respectively. (E–F; K–L) Transversal cryosections (14 µm) labeled by immunofluorescence using anti-HNK-1 antibody and nuclear-stained by DAPI incorporation. Electroporated cells are detected by GFP expression. Pink broken lines in (L) outline the NCCs. (M–N) Analysis of NCC migration in embryos electroporated in the rhombencephalon at stage HH10+ when the delamination is in progress and harvested at 15hpe (stage HH14). (O–T) Analysis of NCC specification in chick embryos electroporated by c-ets-1 DBD at stage HH7-9 and harvested at 6hpe. GFP expression in (O), (Q) and (S) indicates the electroporation zone of the embryos presented in whole mount in situ hybridization using snail-2, foxd-3 and sox-9 probes in (P), (R) and (T) respectively. (U–W) Analysis of trunk NCCs delamination in chick embryos electroporated by c-ets-1 DBD harvested at 24hpe. Transversal cryosections (10 µm) were labeled by immunofluorescence using anti-HNK-1 (U), anti-Pax-7 (V) and anti-Pax-6 (W) antibodies. Expression of c-ets-1 DBD in the head leads to a decrease or a lack of ap-2 (B–D, H–J) and HNK-1 expression (E, K). At cellular level, there is either a reduction of the size of the NCC stream on the electroporated side (L) or even a lack of NCCs between the ectoderm and the neural tube (E; F, green staple) while on control side NCCs are normally localized (B–D, ap-2 staining; F, pink arrow heads; L, left pink lasso). Data shown in A–D, E–F, G–J, K–L come from four distinct embryos respectively. This effect is restricted to the delamination step since inhibition of ETS-1 activity after the delamination does not affect the migration (M–N). Finally, c-ets-1 DBD does not prevent NCC specification as snail-2 (O–P), foxd-3 (Q–R) and sox-9 (S–T) remain expressed in the neural folds on both electroporated and control sides. At trunk level, misexpression of c-ets-1 DBD has no effect on dorso-ventral patterning, or on NCCs delamination and migration (U–W). r, rhombomere.
Figure 4.
Trunk NCC Delamination Occurs Prematurely is Amplified and Prolonged by h-ets-1 Misexpression.
(A–a) Analysis of the effects of h-ets-1 misexpression in trunk dorsal neural tube assayed at 15hpe (A–E, O–R), 24hpe (F–N) and 48hpe (S–a). (A–J, O–R) Whole mount in situ hybridization using sox-10 (A–J, dark blue), cadherin-6B (O–R, dark blue) and h-ets-1 (A–E, O–R, light blue) probes. (K–N) Wholemount immunostaining using anti HNK1 antibody. (C–E, H–J, M–N, P, R) Vibratome sections (30 µm) of embryos presented in (B), (G), (L), (O') and (Q') respectively. (V–Y) In situ hybridization on transversal cryosections (20 µm) using sox-9 (V–W) and sox-10 (X–Y) probes. (Z–a) Transversal cryosections (10 µm) immunolabeled using anti-N-Cadherin antibody. Electroporated cells are detected by GFP expression (S–T, W, Y, a) or DAPI staining (U). At 15hpe in h-ets-1 caudally transfected embryos, sox-10 trunk NCCs delaminate precociously (A–B, E, arrow heads). Besides, more rostrally, the outflow is increased (C–D) compared to contralateral side and is associated with a loss of cadherin-6B expression (O–P). At 24hpe, at level where delamination is already completed on the control side (H–J, asterisks), h-ets-1 expression prolongs delamination of a massive amount of sox-10 (H–J, arrow heads) and HNK-1 (M–N) positive NCCs. At 48hpe, h-ets-1 transfected cells are still able to leave the dorsal neural tube as a multilayered wave (S–U) but they fail to express NCCs markers such as sox-9 (V–W), sox-10 (X–Y) and keep a strong expression of N-Cadherin (Z–a). ot, otic vesicle.
Figure 5.
Ets-1 Misexpression Emancipates Trunk NCC Delamination from Subordination to Successful G1/S Transition.
(A–N) Analysis of the effects of h-ets-1 misexpression in trunk dorsal neural tube on cell cycle assayed at 15hpe. Immunofluorescence labeling using anti-BrdU antibody of transversal cryosections (5 µm). Nuclei are stained by DAPI incorporation. Dotted lines in (C), (F), (I) indicate delaminating transfected area as defined by GFP expression (B, E, H). Trunk NCCs emigrating precociously from dorsal electroporated neural tube opposite segmental plate (A–C) or the first epithelial somite (D–F) are not synchronized in S-phase. Similarly, opposite dissociating somites (G–I), h-ets-1 misexpression in the dorsal neural tube leads to increased NCC delamination of a mix of BrdU positive and negative cells. In contrast, trunk NCCs are predominantly in S-phase when h-ets-1 misexpression does not target the most dorsal territory (J–K, arrow heads) or when NCCs are transfected by w375r (L–N, arrow heads).
Figure 6.
Ets-1 Misexpression Triggers Ectopic Delamination without Inducing Neural Crest Fate.
(A–J) Analysis of the effects of h-ets-1 misexpression in intermediate to ventral neural tube at 24hpe (A, C, E–H) and 48hpe (B, D, I–J); at head (A–B, E–F) and trunk (C–D, G–J) levels. (A–D) Vibratome sections (30 µm) of whole mount in situ hybridization using h-ets-1 probe. At 24hpe, misexpression of h-ets-1 leads to ectopic delaminations towards basal or luminal sides (A, C, arrow heads; dotted lines indicate the neural tube limit). At 48hpe, the phenomena is stronger, involving more cells leaving the neural tube in both head (B) and trunk (D) as compact bulges of cells. (E–J) Transversal cryosections (10 µm) labeled with anti-Laminin antibody. Electroporated cells degrade the basal lamina (arrow heads) before invading the ECM. (K–X) Analysis of NCC fate in ectopic delaminating cells. (K–R) Whole mount in situ hybridization with snail-2 (K–O, dark blue), foxd-3 (L–P, dark blue), ap-2 (M–Q, dark blue), sox-10 (N–R, dark blue) and h-ets-1 (O, P, Q, R, light blue) probes. Dotted lines in (K), (L), (M), (N) indicate the transfected area as defined by GFP expression (insets in K, L, M, N). (S–X) Immunofluorescence labeling with anti-HNK-1 antibody on transversal (S–T, V–W) and longitudinal (U, X) cryosections (10 µm). At 24hpe, misexpression of h-ets-1 in head or trunk does not ectopically activate snail-2, foxd-3, ap-2 or sox-10 (K–R). Furthermore, at 48hpe ectopic cells (including cells emerging from the dorsal part of the neural tube) never express HNK-1 (S–U, arrow heads). Misexpression of w375r has no effect (V–X). drg, dorsal root ganglia; lum, lumen; ot, otic vesicle.
Figure 7.
Ets-1 Misexpression Leads to Massive Cell Movements within the Neuroepithelium.
(A–V) Analysis of the effects of h-ets-1 misexpression in intermediate to ventral neural tube at 24hpe (A–D) and 48hpe (E–V). (A) Vibratome section (30 µm) of whole mount in situ hybridization using cyclin-d1 probe. (B–L, Q–V) Immunofluorescence on cryosections (10 µm) with anti-BrdU (B), anti-phosphohistoneH3 (C–F), anti-β3-Tubulin (G-I), anti Lim-1/2 (J–L), anti-Pax-6 (Q–S), anti-Pax-7 (T–V) antibodies. (M–P) Nuclei are stained with DAPI. H-ets-1 misexpression leads to ectopic activation of cyclin-d1 expression without affecting equilibrium between cell proliferation (B–F) and cell differentiation (G–L). Ectopic h-ets-1 expression provokes cell accumulation close to the basal side of the neural tube (M–P). Interestingly, cell recruitment is detectable even when the phenotype is not strong enough to lead to ectopic delamination (M–N). These cell movements of neuroepithelial cells occur along the apico-basal axis of the neural tube and do not disturb dorso-ventral patterning (Q–V). fp, floor plate; lum, lumen.
Figure 8.
Ets-1 Misexpression Promotes Delamination without Inducing Epithelium to Mesenchyme Transition.
(A–P) Analysis of the effects of h-ets-1 misexpression in intermediate to ventral neural tube at trunk levels at 24hpe (A–B, I–J) and 48hpe (C–H, K–P). Transversal cryosections (10 µm) labeled by immunofluorescence with anti-Fibronectin (A–D, G–H), anti-N-cadherin (I–L, O–P) antibodies, by DAPI incorporation (E, M) and by histological staining with May-Grünwald Giemsa (MGG) (F, N). H-ets-1 electroporated cells invade the extracellular matrix without producing Fibronectin (A–D, arrow heads) and remain strongly attached to each others by N-cadherin (I–L, arrow heads) at 24hpe (A–B, I–J) and 48hpe (C–D, K–L). Accumulations of N-Cadherin are observed within the core of the ectopic clusters at 48hpe (K–L, white arrows). Nuclear detection (E, M) and histological staining (F, N) of sections presented in (C) and (K) confirm the high cellular density in the ectopic clusters (E–F, M–N, white and black lines). Cells transfected by w375r exhibit normal behavior (G–H, O–P). Tr, trunk.
Figure 9.
Ectopic Electroporated Cells Are Still Attached by Functional Cell-Cell Junctions.
(A–J) Analysis of the effects of h-ets-1 misexpression in intermediate to ventral neural tube at 48hpe. Transversal cryosections (10 µm) labeled by immunofluorescence with anti-N-Cadherin antibody (A–C, red; G–J, blue). Actin microfilaments and nuclei are stained with Phalloidin (D–I) and DAPI incorporation (A–F) respectively. Electroporated cells detected by GFP expression (A, D, G) are organized around dots of high N-Cadherin expression (A–C, red dots, white arrows) or high Phalloidin staining (D–F, red dots, white arrows). N-Cadherin and Phalloidin perfectly match to each other (G–J, arrow heads) indicating that N-Cadherin expressed by the electroporated cells is involved in functional cell-cell junctions.
Figure 10.
Ets-1 and Snail-2 Cooperate to Achieve Delamination.
(A–N) Analysis of the effects of h-ets-1 and snail-2 coelectroporation (A–H) and snail-2 alone (I–N) in intermediate to ventral neural tube at 48hpe. Transversal cryosections (10 µm) labeled with anti-Laminin (A–B), anti-N-Cadherin (C–F, I–L) and HNK-1 (G–H, M–N) antibodies. Co-electroporated cells degrade the basal lamina (A–B), lose N-Cadherin expression (C–F, white arrow heads and dotted line), cell-cell junctions at the apical side (white bracket) and strongly express HNK1 (G–H). These cells emigrate from the tube as a population of dissociated cells. H-ets-1 and snail-2, electroporated together, are able to promote EMT and migratory NCCs identity. Conversely, snail-2 electroporation does not affect either N-Cadherin expression or distribution (I–L, white arrow heads). Electroporated cells are unable to undergo EMT and then remain in the neural tube. However, snail-2 electroporation leads to massive ectopic activation of HNK-1 (M–N) all along the dorso-ventral axis of the neural tube.
Figure 11.
Ets-1 confers cranial features on neural crest delamination.
(A) Normal delamination of cranial NCCs. Premigratory and migratory NCCs expressing ets-1 are in purple. (B) Normal delamination of trunk NCCs. Premigratory and migratory NCCs are in yellow. (C) Consequences of ets-1 electroporation in trunk neural tube at dorsal and at intermediate to ventral levels. Ets-1 electroporated cells are coloured in green. (D) Cell movements induced by ets-1 expression. Proliferating cells are in grey, non-proliferating cells are in blue. Ets-1 electroporated cells are dotted in green. Cell-cell junctions involving N-cadherin are represented by black centers. Nuclei in S-phase are colored in black. Basal lamina is represented by twisted red line. Cranial NCCs express ets-1 and massively delaminate independently of G1/S transition (A) whereas trunk NCCs do not express ets-1 and delaminate progressively as a cell population subjected to successful G1/S transition (B). When ets-1 expression is forced in the dorsal part of trunk neural tube, trunk NCCs delamination is greatly enhanced and cells emigrate as multilayered streams (C, green cells). Moreover, they lose their subjection to cell cycle progression indicating that ets-1 converts trunk delamination into cranial-like emigration (C). Ectopic ets-1 expression in ventral part of the neuroepithelium leads to massive cell movements without affecting cell proliferation or differentiation. Electroporated cells are accumulated close to the basal side of the neural tube and the basal lamina is degraded (C, D). These events are sufficient to initiate delamination. However, other factors such as snail-2 are required to perform full delamination and promote EMT and cell migration. M, cell in mitosis.
Figure 12.
Ets-1 and Snail-2 cooperate to achieve the cranial NCC delamination.