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The winged-helix transcription factor Foxd3 suppresses interneuron differentiation and promotes neural crest cell fate

¹ Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA
² Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19014-6058, USA

View larger version (76K): [in a new window]   Fig. 1. Foxd3 expression in neural crest cells. (A-C) Immunohistochemical analysis of Foxd3 (green) and Pax3 (red) expression in E9.5 mouse embryos at thoracic (A) and cervical (B,C) levels. Foxd3 is expressed in presumptive premigratory and migrating neural crest cells and in dorsal midline cells. All the Foxd3 cells are also Pax3 positive (yellow). (D-G) Foxd3 expression in E10.5 mouse embryos. (D,E) Differentiating neurons in sensory (drg) and sympathetic ganglia (sg) were detected using an antibody to NeuN (red). (G) Peripheral axons were stained with an antibody to neuron specific-tubulin (TuJ1) (red). Foxd3 (green) is rapidly downregulated in differentiating sensory (D) and autonomic neurons (E) but is maintained in Schwann cell precursors that envelope the ganglia and axons. (F) Cross-sections of embryos pulsed with BrdU for 90 minutes, and triple stained with BrdU (red), Foxd3 (blue) and Islet1 (green). BrdU-positive/Foxd3-positive cells (pink) are found encompassing and within the DRG (arrows); however, none of these cells co-expressed Islet1 (green) a marker of postmitotic sensory neurons. (H,I) Foxd3 (green) is expressed in melanocyte precursors migrating beneath the ectoderm at E12.5 (arrows). (H) The ectodermal layer is stained with ß-catenin (red). (I) Foxd3-positive/Pax3-positive cells (yellow) envelope the dorsal root ganglia (arrowhead). Foxd3-positive crest cells are also observed migrating along a dorsolateral pathway beneath the skin (arrow). dm, dermomyotome; drg, dorsal root ganglion; mn, motor neurons; nt, neural tube; sc, spinal cord; sg, sympathetic ganglion; sk, skin; vr, ventral root.

View larger version (52K): [in a new window]   Fig. 2. Foxd3 expression in wild-type and Splotch mouse embryos. (A,B,E,F) Whole-mount in situ of Foxd3 in E10 wild-type (A,B) and Splotch (E,F) mouse embryos. (A) Foxd3 is expressed in migrating cranial neural crest cells (arrows) and in premigratory crest (arrowhead). (E) Foxd3 is expressed in cranial neural crest (arrow) but is absent in more caudal regions of the embryo (arrowhead). (B) Cross sections through caudal neural tube at the level of the arrowheads shows Foxd3 expression in the dorsal neural tube of wild-type embryos. (E) No Foxd3 expression is observed in the open neural tube of Splotch embryos. (C,D,G,H) Foxd3 protein expression (green) in the hindbrain (C,G) and caudal spinal cords (D,H) of wild-type (C,D) and Splotch (G,H) embryos. Foxd3-positive crest cells are observed in the head regions of Splotch (G) embryos at E9.5. In Splotch embryos, Foxd3 is expressed in hindbrain-derived crest (G, arrow) but not in trunk-derived crest (H, arrow). (I) Cross section through the anterior cervical neural tube of an E10 Splotch embryo showing Pax7 expression (red) in the dorsal midline (arrowhead). Migrating Foxd3-positive neural crest cells are present at this level (green, arrow). (J) Cross-section through the caudal neural tube of an E10 Splotch embryo. Pax7 (red) is not expressed in the dorsal midline (arrowhead) at spinal cord levels where neural crest cells are lacking (arrow). Foxd3 (green) is however expressed in a population of postmitotic ventral interneurons (I,J, asterisk). hb, hindbrain; sc, spinal cord.

View larger version (72K): [in a new window]   Fig. 3. Neural crest induction by Foxd3. (A-I) Electroporation of Foxd3Myc (A-F) and Foxd3IRES-EGFP (G-I) in neural tubes of stage 10 chick embryos. (A-C) Forty-eight hours after Foxd3Myc electroporation (stage 22), widespread expression of Foxd3Myc leads to ectopic expression of HNK-1 in one half of the trunk neural tube (A,B). Normally, HNK-1 is expressed only on migrating neural crest cells (arrow). At this stage, cells ectopically expressing Foxd3, HNK-1 (B) and Cad-7 (C) are seen migrating away from the neural tube (arrow). (D) Ectopic expression of HNK-1 is observed in the brachial neural tube of stage 18 embryos as early 24 hours after electroporation of Foxd3Myc. (E,F) Delamination and migration of ectopic Foxd3Myc+ cells is also observed at hindbrain levels of the neural tube (arrows). (G-I) Bright field images (G,H) of neural tubes electroporated with Foxd3IRES-EGFP (I). (H,I) High magnification showing the delamination and emigration of Foxd3Myc+ cells from the intermediate neural tube (arrows). fp, floor plate.

View larger version (93K): [in a new window]   Fig. 4. Effects of Foxd3Myc electroporation at stage 28. (A,B,D,E) Foxd3Myc-expressing cells have migrated extensively and can be seen encircling neurons in the dorsal root ganglia (A,D,E, arrows). Foxd3Myc+ cells do not co-express Brn3.0 (A) or Phox2a (B), which mark sensory and sympathetic ganglia, respectively. Ectopic Foxd3Myc+ cells do not express markers of postmitotic neurons, TuJ1 (D) and NeuN (E). (C) Ectopic expression of the Schwann cell marker, P0, in the neural tube induced by Foxd3 misexpression. Foxd3Myc+/P0+ cells are marked by arrows. (F) Electroporation with a control CS2-Myc expression vector. Note that Myc-tag expressing cells enter the dorsal root ganglia and differentiate as sensory neurons (arrow). drg, dorsal root ganglion; fp, floor plate; sc, spinal cord; sym, sympathetic ganglia.

View larger version (68K): [in a new window]   Fig. 5. Ectopic Foxd3 suppresses interneuron differentiation in the spinal cord. (A-L) Cross-sections of neural tubes 48 hours after electroporation of Foxd3Myc or Myc alone (I,L). Ectopic expression of Foxd3 (green) leads to the loss of Isl1/2-positive D2 interneurons (A,B), Lhx2/9-positive D1 interneurons (C) and Lhx1/5-positive D3 interneurons (D,E) in the neural tube. The arrowhead marks the Isl1/2-positive cells (B) and the Lhx2/9-positive cells (C) on the unelectroporated side of the neural tube and the arrow shows the loss of dorsal interneurons on the electroporated side. (F) Foxd3 expression also suppresses expression of Brn3.0 (red) in dorsal interneurons. A domain of Brn3.0-expressing cells is present in the dorsal spinal cord (arrow), but these cells do not co-express Foxd3. Foxd3-positive cells migrating from the dorsal spinal cord (arrowhead). (G,H) Ectopic Foxd3 suppresses Pax2-positive interneuron differentiation in the dorsal and intermediate spinal cord. Fewer Pax2-positive (H) cells are present on the electroporated side of the neural tube (arrow) and these cells are Foxd3-negative (G). (I) Control electroporation with a Myc expression vector, showing no reduction in Pax2-positive interneurons (red), many of which co-express Myc (yellow, arrow). (J,K) Ectopic Foxd3 suppresses Pax6 expression in interneuron precursors. (K) Fewer Pax6-positive precursors are present on the electroporated side of neural tube (arrow). (J) Foxd3-positive cells (green) do not express Pax6. (L) Electroporation of Myc does not reduce the Pax6 expression domain (arrow). fp; floor plate.

View larger version (43K): [in a new window]   Fig. 6. Comparison of Foxd3 and Slug misexpression in electroporated embryos. (A-C) Ectopic Foxd3 did not upregulate the premigratory crest markers, Slug (A), Cad-6B (B), and RhoB (C) at 24 hours (A,B) or 48 hours (C) after electroporation. (D-H) Ectopic Slug did not induce ectopic expression of RhoB (D) or HNK-1 (E), 48 hours after electroporation. Ectopic Slug-positive cells remained within the neural tube and the interneuron patterning remained normal as shown by Pax2 expression (F). (G) Alternate sections of immunohistochemical (G) and in situ (H) analyses showing ectopic Slug-positive cells (green) and Foxd3 mRNA, respectively. Ectopic expression of Slug did not upregulate Foxd3 expression (arrows), nor did it suppress the expression of endogenous Foxd3 within the ventral regions of the neural tube (arrowheads). (I) Co-electroporation of Foxd3 and Slug expression vectors showed no enhancement of cell migration in Foxd3-positive/Slug-positive cells (yellow), 24 hours after electroporation compared with cells expressing ectopic Foxd3 only (see Fig. 3D). fp, floor plate.

View larger version (73K): [in a new window]   Fig. 7. Comparison of Foxd3 expression with Pax3, Slug, Cad-6B and RhoB. Cross sections of stage 10 chick embryos showing gene expression at three caudal levels. Sections were taken at the level of the most caudal somite, (A,D,G,J,M) the level of the segmental plate, (B,E,H,K,N) and at the mid point of the neural folds (C,F,I,L,O). (A-F) Foxd3 (A-C) and Pax3 (D-F) expression was visualized by whole-mount in situ hybridization. (G-O) Sections showing Slug (G-I), Cad-6B (J-L) and RhoB (M-O) expression at equivalent levels. The panels that are shown for Slug, Cad-6B and RhoB at each AP level represent directly adjacent sections from the same embryo. These sections are carefully matched to those shown for Pax3 and Foxd3.

View larger version (17K): [in a new window]   Fig. 8. Schematic outlining the genetic regulation of neural crest cell differentiation. The genes that are not induced by Foxd3 in committed neural crest progenitor cells are italicized.