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First published online 16 April 2008
doi: 10.1242/dev.017954

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Essential role for PDGF signaling in ophthalmic trigeminal placode induction

Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125, USA.

View larger version (70K): [in this window] [in a new window]   Fig. 1. PDGFR, PDGFRβ and Pax3 mRNA expression at st. 8 and 10. (A) PDGFR is expressed in the head and somites at st. 8 in whole mount. (B) In section, PDGFR is detected in the midbrain-level mesenchyme. (C) At st. 10, PDGFR is in neural crest and somites. (D) Migrating neural crest cells express PDGFR at the midbrain-level at st. 10. (E,F) PDGFRβ is strongly expressed in ectoderm and neural folds at st. 8. (G,H) PDGFRβ is maintained in ectoderm at st.10. (I,J) Pax3 is absent from presumptive opV trigeminal placode at st. 8. (K,L) Pax3 marks opV trigeminal placode ectoderm and early migrating neural crest at midbrain level. Broken white lines in whole mounts (A,C,E,G,I,K) indicate level of section (B,D,F,H,J,L). ECTO, ectoderm; MES, mesenchyme; NF, neural folds; NC, neural crest.

View larger version (64K): [in this window] [in a new window]   Fig. 2. PDGFA,C,D mRNA expression in st. 8 and 10 embryos. (A,B) PDGFA is strongly expressed in caudal neural folds at st. 8. (C-E) By st. 10, PDGFA is expressed in midbrain- and trunk-level ectoderm. (F-I) PDGFC is in presumptive midbrain-level ectoderm at st. 8-10. (J) In the trunk at st. 10, PDGFC is in somites, intermediate and lateral mesoderm. (K,L) PDGFD is strong in neural folds at st. 8, and adjacent ectoderm to lesser extent. (M-O) At st.10, PDGFD is in midbrain and trunk dorsal neural tube. (N') Magnification of ectoderm and neural crest. (N'') Magnification of dorsal neural tube. PDGFD is in migrating neural crest but absent from midbrain-level ectoderm. Broken white lines in whole mounts (A,C,F,H,K,M) indicate level of section (B,D,E,G,I,J,L,N,O). ECTO, ectoderm; MES, mesenchyme; NC, neural crest; NF, neural folds; NT, neural tube; S, somite.

View larger version (52K): [in this window] [in a new window]   Fig. 3. PDGFR signaling is necessary for opV trigeminal placode induction in vitro. (A) Diagram of explanted conjugates. Quail ectoderm, cultured in collagen on top of chick dorsal neural tube for 18 hours, expresses opV trigeminal placode marker Pax3. PDGFR inhibitor reduces Pax3 induction. Ectoderm-derived Pax3+ cells are distinguished from neural tubes cells using quail specific marker QCPN. (B) Quantification of reduction of Pax3+ induction in explants. Pax3+ cells are reduced by 58% and 84% by 10 nM and 100 nM PIII, respectively (control, n=15 explants; 10 nM, n=15; 100 nM, n=11). (C-N) Sections of control, 10 nM, and 100 nM PDGFR inhibitor (PIII) treated explants stained with Pax3 (green), QCPN (red) and DAPI (blue). Pax3/QCPN+ cells are placode derived (arrow). DAPI shows overall health of explants. (C) Many Pax3+ cells are induced in control explants. (G) 10 nM PIII dramatically reduces number of Pax3+ cells in ectoderm. (K) At 100 nM PIII, very few Pax3+ cells are detected. Error bar=s.e.m. *P<0.001. ecto, ectoderm; nt, neural tube.

View larger version (80K): [in this window] [in a new window]   Fig. 4. PDGFR signaling is necessary for opV trigeminal placode induction in vivo. Vehicle control (A,C,E) or 2.5 µm PIII (B,D,F) was focally injected into the right midbrain-level mesenchyme at st. 8. At st. 10, Pax3 (green) in ectoderm marks (arrows) induced opV trigeminal placode cells. DAPI (black or blue) indicates total cell number. (A) In control embryos, Pax3 is expressed in many cells in the ectoderm. (B) 2.5 µm PIII significantly reduces Pax3+ cells in the ectoderm. (A'-F') Higher magnification of white box in matched panels A-F.

View larger version (22K): [in this window] [in a new window]   Fig. 5. PIII injection reduces opV trigeminal placode induction in ovo, but does not alter proliferation, cell death or total cell number. (A) Vehicle or PIII solution was focally injected at st. 8 and placode induction assessed at st. 10. PIII (1 µm) (n=6) resulted in 71% reduction of Pax3 relative to controls (n=7). Concentrations of 2.5 and 5 µm PIII caused 67% and 84% reduction (n=8, n=6), respectively. Error bars are s.e.m. *P<0.001. (B) No differences in phospho-histone H3+ were noted between control and 2.5 µm PIII embryos (P>0.05; n=5 each). (C) TUNEL staining revealed no difference in cell death between control and 2.5 µm PIII-injected embryos (P>0.05; n=5 each). (D) Total cell number as assayed by DAPI was not different between control and 2.5 µm PIII-injected embryos (P>0.05; n=5 each).

View larger version (73K): [in this window] [in a new window]   Fig. 6. PIII injection reduces CD151, a marker for opV trigeminal placode cells. (A,B) Control embryo shows no difference between injected and uninjected sides (n=5). (C) Magnification of vehicle injected control. (D) A focal injection of 2.5 µm PIII solution causes large reduction in CD151 expression on the right side (n=9). (E) Section of 2.5 µm PIII embryo shows decrease in CD151 staining on injected side. (F) Magnification of 2.5 µm PIII embryo. Arrowhead indicates side of embryo injected; Inj, injected; ecto, ectoderm.

View larger version (62K): [in this window] [in a new window]   Fig. 7. dnPDGFRβ blocks Pax3 induction. (A-C) Control empty pCIG vector co-expresses Pax3 and GFP. (D-F) dnPDGFRβ-pCIG vector dramatically reduces the expression of Pax3. (G) Quantification of Pax3/GFP+ shows a greater than 50% reduction with expression of dnPDGFRβ (*P=0.036). (H) Quantification of transfection (GFP/DAPI+ cells) efficiency shows no significant difference between control and dnPDGFRβ (P>0.35). Arrows indicate double-positive Pax3/GFP+ cells.

View larger version (100K): [in this window] [in a new window]   Fig. 8. PDGFR signaling is necessary during induction for opV trigeminal ganglion neurogenesis. (A) A st. 14 control embryo shows Pax3 (green) cells condensing into ophthalmic lobe of the trigeminal ganglion. (B) Hu/NF (red) staining of same section as A shows many of the Pax3+ cells are neurons. (C) Merge of A,B plus DAPI (blue). (D) At st.14, many fewer Pax3+ have condensed in 2.5 µm PIII embryos (injected at st. 8). (E) Similarly, fewer Hu/NF+ neurons were generated. (F) Merge of D,E plus DAPI. Arrows indicate double-labeled Pax3/Hu/NF cells.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Inhibiting PDGFR at st. 8 but not st. 10 blocks neurogenesis. (A) At st. 8, 1 µm (n=3) and 2.5 µm PIII (n=5 embryos) reduced neurogenesis by 70% compared with controls (n=6). Average number of triple positive Pax3/NF/Hu+ cells (at least 10 sections) was counted and cells/section normalized to controls. Error bars are s.e.m.; *P<0.05; **P<0.01. (B) For injections at st. 10, control or 2.5 µm PIII showed no significant differences in Pax3+ (white bars), Hu/NF+ (gray bars) or Pax3/Hu/NF+ (black bars) (P>0.05; n=6 each). Thus, once opV trigeminal placode cells are specified, neurogenesis is not blocked by inhibiting PDGFR.

View larger version (58K): [in this window] [in a new window]   Fig. 10. PDGFD injection increases opV trigeminal placode size and neurogenesis. (A,C) Control injected section of midbrain at st. 11 shows Pax3 (green) in opV trigeminal placode. White line indicates ventral extent of opV trigeminal placode. (C) Nuclear DAPI (blue) staining of A. (B,D) PDGFD injection (250 ng/µl solution) increases the number of Pax3+ cells and ventral spread of placode (white line). (D) Nuclear DAPI (blue) staining of B. (E) Control injected section of midbrain at st. 15 shows Pax3 (green) in condensing ganglion. (F) Hu/NF (red) expression in same control section. (G) Merge plus DAPI (blue) in same control section. (H,I) PDGFD injection results in increased Pax3+ (green) cells (H) and neurogenesis as detected with Hu/NF (red) (I) in condensing opV trigeminal ganglion. (J) Merge plus DAPI (blue) in same PDGFD injected section. (K) PDGFD injection increases Pax3+ cells by 32% at st. 11 and Pax3/Hu/NF+ cells by 54% compared with controls (st. 11: control n=8, PDGFD n=10; st. 15: control n=6, PDGFD n=6). Error bars are s.e.m. *P<0.004. Arrows indicate Pax3+ cells (A-D), or Pax3/Hu/NF+ opV trigeminal neuron (E-J).

View larger version (13K): [in this window] [in a new window]   Fig. 11. Model of role of PDGF signaling in opV trigeminal placode induction. Secreted PDGF ligand from the neural folds is necessary for opV trigeminal placode induction at st. 8. By st. 10, many opV trigeminal placode cells are specified (Pax3+ in green). By st. 13-14, opV trigeminal placode cells begin to delaminate and condense to form regions of the opV trigeminal ganglion, expressing Pax3 (green), Hu and NF (red). TGP, opV trigeminal placode; TG, opV trigeminal ganglion.

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