SerpinE2 and HtrA1 are expressed in the neural crest

Xenopus embryos were analyzed by whole-mount in situ hybridization.

(A-C) Anterior view of embryos at stage 17. The brackets point to pre-migratory NC cells on each side of the neural plate. The numbers label the Twist-expressing cranial NC segments: 1, mandibular; 2, hyoid; 3, anterior branchial; 4, posterior branchial. Arrowheads show SerpinE2 and HtrA1 transcripts in the trunk NC. The stippled lines indicate the level of sections in A’-C’.

(A’-C’) Transversally hemisectioned embryos. SerpinE2 and HtrA1 signals appear in the NC (strippled circle lines). Note that SerpinE2 is also expressed in the inner sensorial layer of the neural plate and underlying notochord, whereas HtrA1 expression is more abundant in the outer ependymal layer of the neural plate.

(D-F) Lateral view of embryos at stage 26. SerpinE2 and HtrA1 are expressed in Twist+ NC cell streams (1-4). Transcripts of both genes can also be seen in the brain, eye and otic placode.

(D’-F’) Magnification of embryos. Arrowheads demarcate SerpinE2 transcripts near the front (E’) and HtrA1 transcripts at the rear end (F’) of the migrating NC cell collectives in the anterior branchial arch (3) and posterior branchial arch (4).

(G, H) Summary of gene expression domains. At stage 17, SerpinE2 is transcribed in ventral and HtrA1 in dorsal cells of the pre-migratory NC (G). At stage 26, SerpinE2 is expressed in leader cells and HtrA1 in follower cells of migrating NC streams (H). ey, eye; hb, hindbrain; NC, neural crest; np, neural plate; nt, notochord; ot, otic placode.

Knockdown of SerpinE2 causes defects in neural crest-derived dorsal fin, melanocyte, and craniofacial skeleton structures and inhibits migration of neural crest cells

(A) Scheme of extirpation. Dorsal view of Xenopus embryo at stage 17, from which NC tissue was removed on both sides.

(B,C) Tadpole embryo at stage 40 following NC excision (B) and sibling control (C). Note the small head, absence of dorsal fin tissue (arrowheads) and the reduced number of melanocytes (arrow) resulting from NC extirpation in A.

(D) Unaffected tadpole after microinjection with control-MO into all animal blastomeres at the 8-cell stage.

(E) SerpinE2-MO causes a reduction of head tissue, dorsal fin structures (arrowheads) and melanocytes (arrow).

(F) Co-injection of SerpinE2-MO and 2 ng non-targeted Flag-SerpinE2 mRNA restores a normal phenotype.

(G-I) Ventral view of cartilaginous skeleton extracted from embryos at stage 46 after Alcian Blue staining. Note that SerpinE2 knockdown specifically reduces mandibular, hyoid and branchial structures.

(J) Scheme for microinjections in K-W. MOs and mRNAs were injected together with 100 pg nlacZ mRNA as lineage tracer (red nuclei) into one dorsal animal blastomere of embryos at the 8-cell stage. The injected side is marked with a star.

(K-T) Anterior view of neurula embryos. Neither control-MO nor SerpinE2-MO affect Twist and Sox9 expression in NC cells in the head and trunk (arrowhead) at stage 18 (K-N). SerpinE2-MO inhibits the EMT of Foxd3+ and Snail2+ NC cells (arrows) at stage 20, whereas the control-MO and SerpinE2-5MM-MO have no effect (O-T).

(U-W) Lateral view of stage 26 embryos. SerpinE2-MO, but not control-MO, leads to defective migration of Snail1+ NC cells (arrow) on the injected side. 333 pg Flag-SerpinE2 mRNA rescues NC migration in the SerpinE2-morphant embryo.

br, branchial crest segment; ey, eye primordium; hy, hyoid crest segment; ma, mandibular crest segment; MO, morpholino oligonucleotide; NC, neural crest; ot, otic vesicle. Doses of injected MOs per embryo were 40 ng (D-I) and 10 ng (K-W). Indicated phenotypes were shown in B, 10/11; D, 89/90; E, 64/83; F, 122/132; G, 83/88; H, 73/77; I, 77/87; K, 29/29; L, 24/26; M, 7/7; N, 10/11; O, 9/9; P, 7/7; Q, 7/9; R, 7/8; S, 8/9; T, 9/11; U, 10/10; V, 13/15; W, 9/9.

HtrA1 protease inhibits neural crest-derived structures and reduces neural crest migration

Embryos were injected into all animal blastomeres (A-H) or a single dorsal animal blastomere (I-P) at the 8-cell stage.

(A-D) Tadpoles at stage 40. HtrA1 mRNA causes reduction of head tissue, dorsal fin structures (arrowheads) and melanocytes (arrow), whereas HtrA1(S307A) and Flag-HtrA11′SP mRNA have no effect.

(E-H) Ventral view of isolated skulls at stage 46. Note that HtrA1 mRNA, but not HtrA1(S307A) and Flag-HtrA11′SP mRNAs, diminishes cranial cartilage structures.

(I-K) Anterior view of embryos at stage 18. Stars demarcate the injected sides. Neither 65 pg HtrA1 mRNA nor 10 ng HtrA1-MO do affect the specification of Twist+ cranial NC cells.

(L,M) Anterior view of embryos at stage 20. 65 pg HtrA1 mRNA reduces the EMT of Foxd3+ cranial NC cells (arrow) but does not affect the specification of trunk NC cells (arrowhead).

(N-P) Lateral view of embryos at stage 26. The migration of NC cells is reduced by 65 pg HtrA1 mRNA (arrow) but not affected by 10 ng HtrA1-MO.

br, branchial crest segment; hy, hyoid crest segment; ma, mandibular crest segment. Unless otherwise noted, the mRNA doses of HtrA1 and derived constructs per embryo were 100 pg. Indicated phenotypes were shown in B, 98/100; C, 74/83; D, 84/87; E, 57/58; F, 17/21; G, 72/77; H, 67/78; I, 31/31; J, 51/53; K, 59/60; L, 28/31; M, 23/26; N, 9/10; O, 21/21; P, 58/79 embryos.

Overexpression of HtrA1 and knockdown of SerpinE2 inhibit cranial neural crest cell migration and adhesion to fibronectin in vitro

(A) Scheme of migration experiment. The cranial neural crest was explanted from uninjected or injected embryos at stage 17 and cultured on a fibronectin-covered plastic plate.

(B-E’’’) Time lapse of cell migration in CNC explants after culturing for 0, 4 or 7 hours. Note collective cell migration (open arrowheads) in uninjected controls and explants injected with control-MO, whereas HtrA1 mRNA and SerpinE2-MO block migration (filled arrowheads). In B’’-E’’, the surface areas of explants at 0 hours (blue) and 4 hours (red) were determined by ImageJ and superimposed.

(F) Quantification of initial CNC migration. Indicated is the surface ratio of explants 4 hours versus 0 hours after plating. 12 explants were analyzed per sample.

(G) Scheme of adhesion experiment. Upon injection of eGFP mRNA, CNC explants were dissociated in Ca2+- and Mg2+-free medium, and single cells were cultured on a fibronectin plate.

(H-K) Single eGFP-labelled CNC cells after 1 hour culture. Note adhering cells with extended cytoplasmic processes (open arrowheads) in control sample and after co-injection with control-MO, whereas HtrA1 mRNA and SerpinE2-MO prevent adhesion causing injected cells to acquire a round phenotype (filled arrowheads).

(L) Quantification of CNC adhesion. Indicated is the ratio of adherent cells relative to the control. 6 explants were analyzed per sample.

CNC, cranial neural crest; GFP, green fluorescent protein. Embryos were injected with 100 pg mRNAs and 40 ng MOs. Two independent experiments were performed.

HtrA1 inhibits neural crest migration as an extracellular protease

Embryos were injected into a single dorsal animal blastomere at the 8-cell stage. A star labels the injected side. Twist expression demarcates the NC in embryos at stage 20 (B-G; anterior view) and stage 26 (B’-G’; lateral view).

(A) Overview of wild type (top) and mutant (bottom) HtrA1 protein constructs.

(B-E’) HtrA1 mRNA, but neither Flag-HtrA11′SP nor HtrA1(S307A) mRNAs, reduces EMT and migration of NC cells on the injected side (arrow). Note that the diffusible HtrA1 protein reduces NC cell migration to a lower extent also on the non-injected side.

(F-G’) Both HtrA1-myc and HtrA11′PDC-myc mRNAs reduce NC EMT and migration (arrows). br, branchial segment; hy, hyoid segment; ma, mandibular segment. If not otherwise indicated, injected mRNA doses per embryos are 65 pg. For quantification of NC migration defects, see Supplementary Figure S5A,B.

SerpinE2 functions in neural crest cell migration in an HtrA1- and Sdc4-dependent manner

mRNAs and morpholino oligonucleotides (MOs, 10 ng) and were injected into one dorsal animal blastomere at the 8-cell stage. Embryos are shown in anterior view (stage 20, injected side labelled with a star, B-M) and lateral view (stage 26, B’-M’).

(A) Overview of wild type (left) and mutant (right) SerpinE2 protein constructs.

(B,B’) 4 ng Flag-SerpinE2 mRNA has no effect on the migration of Twist+ NC cells.

(C,C) Flag-HtrA1 inhibits NC cell migration robustly. Arrows label defects on the injected sides.

(D-F’) SerpinE2 mRNA, but neither Flag-SerpinE21′SP nor SerpinE2pm mRNA, rescues normal EMT and migration of NC cells upon co-injection with Flag-HtrA1.

(G,G’) Sdc4 mRNA restores normal NC migration in Flag-HtrA1-injected embryos.

(H-J’) SerpinE2-MO, blocks EMT and migration of Twist+ NC cells (arrow) on the injected side, while control-MO and SerpinE2-5MM-MO have no effect.

(K-M’) Flag-SerpinE2 mRNA, HtrA1-MO, and Sdc4 mRNA restore normal NC migration in SerpinE2-morphant embryos.

(N) Proposed mechanism for the regulation of NC migration by SerpinE2, HtrA1 and Syndecan-4. Injected mRNA doses per embryos are 333 pg (Flag-SerpinE2) and 450 pg (Sdc4). For quantification of NC migration defects, see Supplementary Figures S3A,B and S7A,B.

Model for a proteolytic pathway of SerpinE2 and HtrA1 that regulates collective neural crest migration

(A) SerpinE2 stimulates collective NC migration by a double-inhibitory mechanism involving the secreted serine protease HtrA1 and its proteolytic substrates Syndecan-4 and Fibronectin.

(B) Opposing gradients of SerpinE2 and HtrA1 activities regulate the migration in a collective of NC cells. High SerpinE2 and low HtrA1 levels coincide with abundant focal adhesion sites and polymerized actin that drive mesenchymal migration at the leading edge.

(C) SerpinE2 anchored to the heparan sulfate chains of the transmembrane proteoglycan Syndecan-4 protects the integrity of focal adhesions at the leading front and allows collective cell migration to occur (left side). Unbound HtrA1 triggers the proteolytic cleavage of Syndecan-4 and degrades the matrix protein Fibronectin (middle), causing loss of cell-matrix adhesion at the rear end of the NC cell collective (right side).

HS, heparan sulfate; NC, neural crest; Sdc4, Syndecan-4.

Antisense morpholino oligonucleotides

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