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. Scale bar, 0.5 mm.

Knockdown of SerpinE2 mimics the phenotype of neural excision 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 (open 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. Highlighted are the pigmented melanocytes (arrow) and the intact dorsal fin (filled arrowheads).

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

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

(G) 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.

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

(R-T) Lateral view of stage 26 embryos. A single injection of control-MO does not affect the migration of Snail1+ NC cells (filled arrowhead). SerpinE2-MO leads to a migration defect on the injected side (open arrowhead). 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 (H-T). Indicated phenotypes were shown in B, 10/11; D, 89/90; E, 64/83; F, 122/132; H, 29/29; I, 24/26; J, 7/7; K, 10/11; L, 9/9; M, 7/7; N, 7/9; O, 7/8; P, 8/9; Q, 9/11; R, 10/10; S, 13/15; T, 9/9. Scale bar, 0.5 mm.

HtrA1 protease inhibits the formation of 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-HtrA1ΔSP mRNA have no effect.

(E-G) 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. (H,I) Anterior view of embryos at stage 20. 65 pg HtrA1 mRNA reduces the EMT of Foxd3+ cranial NC cells (arrowheads) but does not affect the specification of trunk NC cells (arrows).

(J-L) Lateral view of embryos at stage 26. The migration of NC cells (arrowheads) is reduced by 65 pg HtrA1 mRNA 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, 31/31; F, 51/53; G, 59/60; H, 28/31; I, 23/26; J, 9/10; K, 21/21; L, 58/79 embryos; at least two independent experiments.

Scale bar, 0.5 mm.

HtrA1 overexpression and SerpinE2 knockdown decrease cartilaginous elements and craniofacial structures

Xenopus embryos were injected into four animal blastomeres at the 8-cell stage with a total of 100 pg mRNA and 40 ng morpholino oligonucleotides (MOs).

(A-D’) Tadpoles at stages 40/41 after whole-mount in situ hybridization in lateral (A-D) and ventral view (A’-D’). Note that HtrA1 mRNA and SerpinE2-MO reduce Sox9 expression, whereas control-MO has no effect on the labelled cartilaginous elements.

(E-K) Ventral view of cartilaginous skeleton extracted from embryos at stage 46 after Alcian Blue staining. The dorsal ethmoid-trabecular cartilage was removed for better visibility. Note that HtrA1 mRNA, but not HtrA1(S307A) and Flag-HtrA1ΔSP mRNAs, diminishes craniofacial structures (E-H). SerpinE2-MO, but not control-MO nor a combination of SerpinE2-MO and Flag-SerpinE2 mRNA, reduce head skeleton structures (I-K).

(L) Scheme of the cartilaginous skeleton at stage 46 in ventral view. Indicated is the contribution of neural crest streams to the craniofacial skeleton elements. cb, ceratobranchial; ch, ceratohyal; et, ethmoid-trabecular; mc, Meckel’s cartilage; pq, palatoquadrate.

Indicated phenotypes were shown in A, 11/11; B, 17/19; C, 12/13; D, 15/17; E, 57/58; F, 17/21; G, 72/77; H, 67/78; I, 83/88; J, 73/77; K, 77/87 embryos; at least two independent experiments. Scale bar, 0.5 mm.

HtrA1 overexpression of and SerpinE2 knockdown 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 (filled arrowheads) in uninjected controls and explants injected with control-MO, whereas HtrA1 mRNA and SerpinE2-MO block migration (open arrowheads). In B’’-E’’, the surface areas of explants at 0 hours (blue) and 4 hours (red) were determined by ImageJ and superimposed. Scale bar, 0.2 mm

(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; two independent experiments.

(G) Scheme of adhesion experiment. Upon injection of eGFP mRNA, CNC eplants 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 (filled 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 (open arrowheads). Scale bar, 0.02 mm.

(L) Quantification of CNC adhesion. Indicated is the ratio of adherent cells relative to the control. Analysis of n>1600 cells from at least 6 explants per sample; two independent experiments. CNC, cranial neural crest; GFP, green fluorescent protein. Embryos were injected with 100 pg mRNAs and 40 ng MOs.

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-HtrA1ΔSP nor HtrA1(S307A) mRNAs, reduces EMT and migration of NC cells on the injected side (arrowheads). 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 HtrA111PDC-myc mRNAs reduce NC EMT and migration.

br, branchial segment; hy, hyoid segment; ma, mandibular segment. Injected mRNA doses per embryos are 65 pg. For quantification of NC migration defects, see Figure 6-figure supplement 1A,B.

SerpinE2 and HtrA1 interact with Sdc4 in neural crest cell migration

mRNAs 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-G) and lateral view (stage 26, B’-G’).

(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 (filled arrowheads).

(C,C) Flag-HtrA1 inhibits NC cell migration robustly on the injected sides (open arrowheads).

(D-F’) SerpinE2 mRNA, but neither Flag-SerpinE2ΔSP 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.

If not otherwise indicated, injected mRNA doses per embryos are 65 pg (Flag-HtrA1), 333 pg (Flag-SerpinE2 derived constructs) and 450 pg (Sdc4). For quantification of NC migration defects, see Figure 7-figure supplement 1A,B.

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

mRNAs and morpholino oligonucleotides (MOs, 10 ng) 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-G) and lateral view (stage 26, B’-G’).

(A) Proposed mechanism for the regulation of NC migration by SerpinE2, HtrA1 and Syndecan-4. (B-D’) 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.

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

Injected mRNA doses per embryos are 333 pg (Flag-SerpinE2) and 450 pg (Sdc4). For quantification of NC migration defects, see Figure 8-figure supplement 1A,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 directed migration in a collective of NC cells. The SerpinE2/HtrA1 pair contributes to the formation of a chemoattractant gradient that guides the NC stream towards a source of FGF signals in the target tissue. 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).

FGF, fibroblast growth factor; HS, heparan sulfate; NC, neural crest; Sdc4, Syndecan-4.