Ets-1 Confers Cranial Features on Neural Crest Delamination

Neural crest cells (NCC) have the particularity to invade the environment where they differentiate after separation from the neuroepithelium. This process, called delamination, is strikingly different between cranial and trunk NCCs. If signalings controlling slow trunk delamination start being deciphered, mechanisms leading to massive and rapid cranial outflow are poorly documented. Here, we show that the chick cranial NCCs delamination is the result of two events: a substantial cell mobilization and an epithelium to mesenchyme transition (EMT). We demonstrate that ets-1, a transcription factor specifically expressed in cranial NCCs, is responsible for the former event by recruiting massively cranial premigratory NCCs independently of the S-phase of the cell cycle and by leading the gathered cells to straddle the basal lamina. However, it does not promote the EMT process alone but can cooperate with snail-2 (previously called slug) to this event. Altogether, these data lead us to propose that ets-1 plays a pivotal role in conferring specific cephalic characteristics on NCC delamination

<i>Ets-1</i> and <i>Snail-2</i> Cooperate to Achieve Delamination.

<p>(A–N) Analysis of the effects of <i>h-ets-1</i> and <i>snail-2</i> coelectroporation (A–H) and <i>snail-2</i> 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. <i>H-ets-1</i> and <i>snail-2</i>, electroporated together, are able to promote EMT and migratory NCCs identity. Conversely, <i>snail-2</i> 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, <i>snail-2</i> electroporation leads to massive ectopic activation of HNK-1 (M–N) all along the dorso-ventral axis of the neural tube.</p

<i>Ets-1</i> Misexpression Triggers Ectopic Delamination without Inducing Neural Crest Fate.

<p>(A–J) Analysis of the effects of <i>h-ets-1</i> 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 <i>h-ets-1</i> probe. At 24hpe, misexpression of <i>h-ets-1</i> 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 <i>snail-2</i> (K–O, dark blue), <i>foxd-3</i> (L–P, dark blue), <i>ap-2</i> (M–Q, dark blue), <i>sox-10</i> (N–R, dark blue) and <i>h-ets-1</i> (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 <i>h-ets-1</i> in head or trunk does not ectopically activate <i>snail-2</i>, <i>foxd-3</i>, <i>ap-2</i> or <i>sox-10</i> (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 <i>w375r</i> has no effect (V–X). drg, dorsal root ganglia; lum, lumen; ot, otic vesicle.</p

Trunk NCC Delamination Occurs Prematurely is Amplified and Prolonged by <i>h-ets-1</i> Misexpression.

<p>(A–a) Analysis of the effects of <i>h-ets-1</i> 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 <i>sox-10</i> (A–J, dark blue), <i>cadherin-6B</i> (O–R, dark blue) and <i>h-ets-1</i> (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 <i>sox-9</i> (V–W) and <i>sox-10</i> (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 <i>h-ets-1</i> caudally transfected embryos, <i>sox-10</i> 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 <i>cadherin-6B</i> expression (O–P). At 24hpe, at level where delamination is already completed on the control side (H–J, asterisks), <i>h-ets-1</i> expression prolongs delamination of a massive amount of <i>sox-10</i> (H–J, arrow heads) and HNK-1 (M–N) positive NCCs. At 48hpe, <i>h-ets-1</i> 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 <i>sox-9</i> (V–W), <i>sox-10</i> (X–Y) and keep a strong expression of N-Cadherin (Z–a). ot, otic vesicle.</p

Description of the Cranial Neural Crest Cells Delamination.

<p>(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.</p

<i>Ets-1</i> Misexpression Leads to Massive Cell Movements within the Neuroepithelium.

<p>(A–V) Analysis of the effects of <i>h-ets-1</i> 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 <i>cyclin-d1</i> 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. <i>H-ets-1</i> misexpression leads to ectopic activation of <i>cyclin-d1</i> expression without affecting equilibrium between cell proliferation (B–F) and cell differentiation (G–L). Ectopic <i>h-ets-1</i> 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.</p

Ets-1 confers cranial features on neural crest delamination.

<p>(A) Normal delamination of cranial NCCs. Premigratory and migratory NCCs expressing <i>ets-1</i> are in purple. (B) Normal delamination of trunk NCCs. Premigratory and migratory NCCs are in yellow. (C) Consequences of <i>ets-1</i> electroporation in trunk neural tube at dorsal and at intermediate to ventral levels. <i>Ets-1</i> electroporated cells are coloured in green. (D) Cell movements induced by <i>ets-1</i> expression. Proliferating cells are in grey, non-proliferating cells are in blue. <i>Ets-1</i> 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 <i>ets-1</i> and massively delaminate independently of G1/S transition (A) whereas trunk NCCs do not express <i>ets-1</i> and delaminate progressively as a cell population subjected to successful G1/S transition (B). When <i>ets-1</i> 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 <i>ets-1</i> converts trunk delamination into cranial-like emigration (C). Ectopic <i>ets-1</i> 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 <i>snail-2</i> are required to perform full delamination and promote EMT and cell migration. M, cell in mitosis.</p

<i>Ets-1</i> Misexpression Emancipates Trunk NCC Delamination from Subordination to Successful G1/S Transition.

<p>(A–N) Analysis of the effects of <i>h-ets-1</i> 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), <i>h-ets-1</i> 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 <i>h-ets-1</i> misexpression does not target the most dorsal territory (J–K, arrow heads) or when NCCs are transfected by <i>w375r</i> (L–N, arrow heads).</p

Ets-1 and Snail-2 cooperate to achieve the cranial NCC delamination.

<p>Ets-1 and Snail-2 cooperate to achieve the cranial NCC delamination.</p