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First published online 14 January 2009
doi: 10.1242/dev.027334

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A role for Syndecan-4 in neural induction involving ERK- and PKC-dependent pathways

Department of Cell and Developmental Biology, Faculty of Life Sciences, University College London, Gower Street, London WC1E 6BT, UK.

View larger version (58K): [in this window] [in a new window]   Fig. 1. Dynamic expression of Syn4 in the neural plate region. Whole-mount in situ hybridisation analysis of Syn4 and Sox expression. (A) Lateral view of a stage 10.5 Xenopus embryo showing Syn4 expression in the dorsal marginal zone. Dorsal (d) to the right; ventral (v), left; animal pole to the top. (B) Fate map of a stage 10.5 embryo, shown in the same orientation as in A. NP, prospective neural plate; m, prospective mesoderm. (C) At stage 12, Syn4 expression (arrowheads) is restricted to the dorsal region of the embryo (orientation as in A). (D) Fate map of a stage 12 embryo, shown in the same orientation as in C. NP, neural plate. (E) At stage 14, Syn4 expression is seen in the neural plate, but is absent from the dorsal midline (arrowhead). Dashed line indicates the plane of the section in G. (F) Stage 14 embryo showing Sox2 expression. The expression pattern is similar to that of Syn4 in E. Dashed line indicates the plane of the section in H. Arrowhead, dorsal midline. (G) Section of a stage 14 embryo, showing Syn4 expression. No expression is observed at the midline or in mesoderm. (H) Section of a stage 14 embryo, showing Sox2 expression (I) At stage 16, Syn4 expression is seen in the neural plate. (J) Sox2 expression at stage 16.

View larger version (119K): [in this window] [in a new window]   Fig. 2. Syn4 is required for neural induction. Sox2 and Nrp1 expression was analysed by whole-mount in situ hybridisation. (A-H) MO-injected samples analysed at stage 14. The lineage tracer in shown in the insets. (A) Syn4 MO injected in one animal blastomere of an 8-cell stage Xenopus embryo. Note the inhibition of Sox2 expression on the injected (right-hand) side (60%, n=45). (B) Similar to A, but injection with control MO. No effect on Sox2 expression is observed (0%, n=57). (C) Syn4 MO was injected into the dorsal animal blastomere (A1) at the 32-cell stage. Note the inhibition of Sox2 expression in the injected region (arrowhead) (55%, n=50). (D) Syn4 mRNA mutated in the MO sequence region was co-injected with Syn4 MO into the A1 blastomere of a 32-cell stage embryo. Note the rescue of the expression of Sox2 (8%, n=66). (E) Syn4 MO injected into one animal blastomere of an 8-cell stage embryo. Note the inhibition of Nrp1 expression on the injected side (95%, n=42). (F) No inhibition is observed with the control MO (0%, n=21). (G) Control-morpholino-injected ectoderm was grafted into an uninjected embryo. Sox2 expression is normal (100%, n=10). Black dashed line indicates the anterior border of Sox2 expression. White dashed line outlines the graft. Inset shows the position of the graft by its fluorescence. (H) Syn4 MO-injected ectoderm was grafted into an uninjected embryo. Sox2 expression is absent from the grafted area (asterisk; 70%, n=10). Inset shows the position of the graft by its fluorescence. (I-N) Analysis of Sox2 expression at stage 11. (I) Expression of Sox2 is initially observed in the dorsal region (arrowhead). (J) Expansion of Sox2 expression by BMP inhibition [BMP antagonists: truncated BMP receptor (tBR) and chordin (chd) mRNA]. (K) Early induction of Sox2 was eliminated by co-injection of Syn4 MO (69%, n=50). (L) Control animal caps, showing no expression of Sox2. (M) Animal caps taken from embryos injected as in J, showing weak upregulation of Sox2. (N) Animal caps taken from embryos as in K. Co-injection of Syn4 MO blocks Sox2 induction.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Syn4 overexpression neuralises ectoderm. (A-D) Anterior view, dorsal to the top (A,B); ventral view (C,D). (A,C) 500 pg of NLS-β-gal mRNA was injected into the A4 blastomere of a 32-cell stage Xenopus embryo, and the expression of Sox2 (A) and epidermal keratin (Epk) (C) was analysed by in situ hybridisation. (B,D) 1 ng of Syn4 mRNA was injected into the A4 blastomere of a 32-cell stage embryo, and the expression of Sox2 (C; 96% of induction, n=87) and Epk (D; 80% of inhibition, n=70) was analysed. (E,F) Higher magnification of embryos injected as in B,D. Arrows indicate nuclei stained with X-Gal (blue). (G-J) Dorsal (G,I) and ventral (H,J) views of embryos injected as in B with Syn4 mRNA. No ectopic expression of MyoD (0%) or Xbra (0%) was observed. Inset in H shows injected fluorescein. (K-N) Animal cap (AC) assay for Sox2 (K,L) or Epk (M,N) expression. (K,M) Control caps express only Epk. (L,N) Syn4 mRNA-injected animal caps express Sox2 and downregulate Epk.

View larger version (79K): [in this window] [in a new window]   Fig. 4. The FGF/MAPK pathway is required for neural induction by Syn4. (A-F) Sox2 expression analysed by whole-mount in situ hybridisation in Xenopus animal caps. (A) Control caps. No Sox2 expression. (B) Syn4-injected caps express Sox2. (C) Co-injection of Syn4 and a dominant-negative form of FGF receptor 1 (XFD-1) inhibits Sox2 expression. (D) Syn4-injected caps treated with 40 µM SU5402 (in DMSO) do not show Sox2 expression. (E) Syn4-injected caps treated with 80 µM U0126 do not express Sox2. (F) Syn4-injected caps treated with 80 µM U0124 show expression of Sox2.(G) Animal caps were injected as indicated (Cont, control) and samples taken for western blot analysis of MAPK phosphorylation at the equivalent of stage 12. Antibodies against MAPK or phosphorylated MAPK (p-MAPK) can recognise both p42 and p44 as a single band. Each experiment was repeated three times with at least 50 animal caps.

View larger version (123K): [in this window] [in a new window]   Fig. 5. Membrane translocation of PKC by FGF is inhibited by Syn4. Xenopus animal caps analysed by confocal microscopy after injection/treatment as indicated. (A-C) Control animal cap shows cytoplasmic localisation of PKC. (D-F) Animal caps injected with Syn4 mRNA. PKC shows cytoplasmic distribution. (G-L) Phorbol ester (PMA; G-I) or FGF2 (J-L) triggers the translocation of PKC into the membrane. (M-O) Syn4 inhibits the translocation of PKC activated by FGF2.

View larger version (100K): [in this window] [in a new window]   Fig. 6. Inhibition of PKC or activation of PKC is essential for neural induction by Syn4. (A-H) Sox2 expression in Xenopus embryos injected, as indicated, into A4 blastomeres at the 32-cell stage. (A-D) Ventral view, dorsal to the top. Insets on left show a dorsal view. (A) Dominant-negative Xenopus PKC (DN-PKC). Note the ectopic Sox2 induction (48%, n=71). (B) Co-injection of Syn4 and Xenopus full-length PKC mRNAs represses the ectopic expression of Sox2 (18%, n=91). Inset on the right shows the ventral ectopic Sox2 expression induced by Syn4 mRNA (95%, n=85). (C) Human (h) PKC mRNA can induce ectopic Sox2 expression (68%, n=92). (D) Co-injection of hPKC and PKC mRNAs shows inhibition of neural induction (20%, n=124). (E) Co-injection of hPKC with control MO (CMO). Arrowhead indicates the ectopic expression of Sox2 (70%, n=68). (F) Co-injection of hPKC mRNA with Syn4 MO. Sox2 induction is not observed in the injected cells (arrowhead) (23%, n=51). (G) Co-injection of Syn4 and a dominant-negative form of PKC (DN-PKC-EGFP) inhibits Sox2 expression (15%, n=51). (H) Injection of PKC-EGFP mRNA. Ectopic induction of Sox2 is similar to that upon PKC injection, showing that EGFP does not affect the activity of the fusion protein. (I-N) Confocal images of animal caps injected as indicated. PKC-EGFP mRNA was injected into both blastomeres at the 2-cell stage. At the 16-cell stage, Syn4 or control MO and membrane Cherry mRNA were injected into one blastomere. (I-K) PKC-EGFP spontaneously localises at the membrane, colocalising with membrane Cherry. (L-N) The distribution of Syn4 MO can be identified by the fluorescence of mCherry. Note that cells with a high level of Syn4 MO (asterisks) exhibit a low level of PKC-EGFP in the membrane.

View larger version (56K): [in this window] [in a new window]   Fig. 7. The PKC-dependent pathway of neural induction is mediated by the Rac/JNK/AP-1 pathway. (A) RT-PCR of the indicated genes using RNA from Xenopus whole embryos (WE, lane 1), animal caps (AC, lane 2), animal caps expressing PKC (PKC, lane 3) or animal caps expressing PKC and treated with the inhibitor U0126 (+U0126, lane 4). Note that Sox2 is induced by PKC even when MAPK is inhibited (lane 4). (B) Western blot of control- or PKC-injected animal caps, detecting c-Fos, phosphorylated MAPK (p-MAPK) and MAPK. Neuralisation of the animal caps correlates with an increase in c-Fos levels, whereas p-MAPK is unchanged. (C) Rac activation assay. (Left) Rac1-His recombinant protein (24 kDa; 20 ng) was loaded and detected by anti-Rac antibody as a positive control. (Right) The same volume of reaction mix was loaded for each condition. Lane 1 (AC+GDP), negative control; lane 2 (AC+GTP), positive control that shows the total amount of Rac protein in the animal cap samples; lane 3 (AC), endogenous active Rac present in animal caps at stage 11.5; lane 4 (Syn4), endogenous active Rac present in animal caps at stage 11.5 injected with 500 pg of Syn4 mRNA (note that Syn4 abolishes endogenous Rac activity); lane 5 (Syn4+GTP), total amount of Rac protein after Syn4 injection. The experiment was repeated three times. (D-I) Whole-mount in situ hybridisation analysis of embryos injected, as indicated, into the A4 blastomere of 32-cell stage embryos. (D) Human (h) PKC mRNA induces ectopic Sox2 expression (68%, n=92). (E) Co-injection of constitutively active Rac and hPKC inhibits ectopic Sox2 expression (21% of induction, n=34). (F) Injection of constitutively active Rac does not induce Sox2 expression (0%, n=32). (G) Dominant-negative Rac1 mRNA induces ectopic ventral Sox2 expression (90%, n=40). (H,I) Ventral view of embryos injected with c-Fos-GR mRNA and activated with dexamethasone (DEX) at stage 10.5. Ectopic Sox2 expression is observed only after DEX treatment (I), being absent when no DEX is added (H; inset shows dorsal side). (J-O) In situ hybridisation for Sox2 in animals caps. (J) Control animal caps show no Sox2 expression. (K) Animal caps from embryos injected with chordin (chd) mRNA show Sox2 expression. (L) Animal caps from embryos injected with chordin, constitutive Rac and c-Fos-GR mRNA but without adding DEX. Activation of Rac leads to inhibition of Sox2. (M) Similar to L, but c-Fos-GR is activated by DEX treatment. Rescue of Sox2 expression is observed. (N) Injection of c-Fos-GR mRNA, but without adding DEX. (O) c-Fos-GR-injected animal caps activated with DEX show an increase in Sox2 expression.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Model of neuralisation by Syn4. (A) The Syn4/ERK-dependent pathway. The glycosaminoglycans (GAGs) in the extracellular domain of Syn4 activate the FGF/MAPK pathway. The activation of this pathway can lead to mesoderm induction, but also contributes to neural induction, probably though the inhibition of Smad1. (B) The Syn4/PKC-dependent pathway. The intracellular domain of Syn4 inhibits PKC and activates PKC. The inhibition of PKC is required for the recruitment of PKC to the membrane and its binding to Syn4. Activated PKC inhibits Rac activity. Rac activates JNK, which phosphorylates c-Jun and inhibits the formation of the c-Jun/c-Fos dimers that form part of the AP-1 transcriptional regulator complex. Thus, the inhibition of Rac by Syn4/PKC leads to the activation of the AP-1 complex that controls the transcription of preneural genes

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