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First published online 21 June 2006
doi: 10.1242/dev.02478

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The function of growth/differentiation factor 11 (Gdf11) in rostrocaudal patterning of the developing spinal cord

Department of Neuroscience, University of Virginia, 409 Lane Road, MR4, Room 5032, Charlottesville, VA 22908, USA.

View larger version (98K): [in a new window]   Fig. 1. Expression of Gdf11 in the neural tube causes rostral displacement of Hox expression domains. (A-H) Hoxc6-Hoxc10 expression in longitudinally sectioned (A-D), and in cross-sectioned (E-H) spinal cords taken from HH stage 25 embryos 3 days after Gdf11 electroporation. The electroporated side (on the right in all panels) is marked by GFP expression. White arrows indicate the caudal expression limit of Hoxc6 (A) and the rostral expression limits of Hoxc9 (C) and Hoxc10 (D) in the control side. Yellow arrows indicate the caudal expression limits of Hoxc6 (A) and Hoxc8 (B) in the electroporated side. (A,C) The same section double labeled with Hoxc6 and Hoxc9. Broken lines in A-D indicate the RC levels of sections (E-H). (E,G) The same section double labeled with Hoxc6 and Hoxc9; (F,H) the same section double labeled with Hoxc8 and Hoxc10. (I) A diagram depicts the normal Hox expression domains in the control (CTL) side and the rostral displacement of Hox expression domains in the Gdf11 electroporated side (EXP) of the spinal cord. Brackets indicate the RC levels of A-D. Position of the limbs is represented by gray-colored areas. Scale bar: 100 µm. (J-M) Hoxc6-Hoxc10 expression and (J'-M') GFP expression in HH stage 15-17 embryos 20-24 hours after Gdf11 electroporation. The electroporated side is towards the bottom of the panels. Red and green arrows indicate the rostral limit of Hoxc6-Hoxc10 expression in the electroporated and the control side, respectively. Scale bar: 0.5 mm.

View larger version (85K): [in a new window]   Fig. 2. Expression of Gdf11 in the neural tube results in rostral displacement of motor neuron column and pool positions. (A) Raldh2 and (B) Pea3 expression in the spinal cord and (C,D) neurofilament (3A10) staining in HH stage 29 embryos 5 days after Gdf11electroporation. The electroporated side (EXP) is on the right of all panels, while the control side (CTL) is on the left. Rostral is towards the top and caudal is towards the bottom. White brackets indicate the origins of the spinal nerves that innervate the forelimbs in C and the hindlimbs in D. Green asterisks mark the first (C) and the last (D) spinal nerves that innervate the thoracic levels. Broken black lines mark the rostral boundaries of the hindlimbs in D. Scale bars: 0.5 mm.

View larger version (84K): [in a new window]   Fig. 3. Expression of follistatin (Fst) in the neural tube causes caudal displacement of Hox protein expression domains. (A-H) Hoxc6-Hoxc10 expression in longitudinally sectioned (A-D), and in cross-sectioned (E-H) spinal cords taken from HH stage 25 embryos 3 days after Fst electroporation. The electroporated side (on the right in all panels) is marked by GFP expression. White arrows indicate the caudal expression limits of Hoxc6 (A), Hoxc8 (B) and Hoxc9 (C) in the control sides. Yellow arrow indicates the rostral expression limit of Hoxc10 (D) in the electroporated side. Broken lines in A-D indicate the RC levels of sections (E-H). (I) A diagram depicts the normal Hox expression domains in the control (CTL) side and the caudal displacement of Hox expression domains in the Fst electroporated side (EXP) of the spinal cord. Brackets indicate the RC levels of A-D. Position of the limbs is represented by gray-colored areas. Scale bar: 100 µm.

View larger version (89K): [in a new window]   Fig. 4. Expression of Fst in the neural tube results in caudal displacement of motor neuron column and pool positions. (A) Raldh2 and (B) Pea3 expression in HH stage 29 spinal cords 5 days after Fst electroporation. (C,D) Neurofilament (3A10) staining of HH stage 27 embryos 4 days after Fst electroporation. The electroporated side (EXP) is on the right of all panels while the control side (CTL) is on the left. Rostral is towards the top and caudal is towards the bottom. White brackets mark the origins of the spinal nerves that innervate the forelimbs in C and the hindlimbs in D. Green asterisks mark the first (C) and the last (D) spinal nerves that innervate the thoracic levels. Broken black lines mark the rostral boundaries of the hindlimbs in D. Scale bars: 0.5 mm.

View larger version (78K): [in a new window]   Fig. 5. Caudal displacement of Hox expression domains, motoneuron column and pool positions in Gdf11-/- spinal cord. (A-D) Hoxc8 and Hoxc10 expression in flat-mounted open-book preparations of spinal cords isolated from E12.5 control (A,C) and Gdf11-/- (B,D) mouse embryos. Rostral is towards the top and caudal is towards the bottom. The caudal boundary of Hoxc8 expression is indicated by arrows in A,B, while the Hoxc10 expression domain is located between arrows in C,D. (E-H) Raldh2 and Pea3 expression in flat-mounted open-book preparations of spinal cords isolated from E13.5 control (E,G) and Gdf11-/- (F,H) mouse embryos. Rostral is towards the top and caudal is towards the bottom. Arrows indicate the expression boundaries of Raldh2 and Pea3. Scale bars: 1 mm.

View larger version (152K): [in a new window]   Fig. 6. Caudal displacement and expansion of Hox protein expression domains in Gdf11-/- mouse embryos. (A-J) Hoxc6-Hoxc10 expression in cross-sectioned E12.5 ventral spinal cords isolated from Gdf11+/+ (A,C,E,G,I) and Gdf11-/- (B,D,F,H,J) mouse embryos. Motoneurons and dorsal root ganglia are marked by Isl1 or Isl1/2 expression. (K) A diagram depicts the Hox expression domains in Gdf+/+and Gdf-/- spinal cords. Broken lines indicate the RC levels in A-J. Position of the limbs is represented in gray. Scale bar: 100 µm.

View larger version (121K): [in a new window]   Fig. 7. Expression of a constitutively activated Smad2 (Smad2-2E) induces caudal Hox protein expression at the expense of rostral Hox protein expression. (A-C) Western analyses of pSmad1/5/8 and pSmad2/3 levels in neural plate explants treated with Gdf11 (A), in Gdf11 electroporated chick embryos (B) and in E9.5 Gdf11-/- mouse embryos (C). (D-M) Hoxc6-Hoxc10 expression in longitudinally sectioned (D-H) and in cross-sectioned (I-M) HH stage 25 spinal cords 3 days after Smad2-2E electroporation. The electroporated side (on the right in all panels) is marked by GFP expression. Enlarged image of boxed areas are shown below each panel. White arrows indicate the inhibition of Hoxc6 (D,I) and Hoxc8 (E,J) expression, and the induction of Hoxc9 (F,K) and Hoxc10 (G,L) expression by Smad2-2E expression. Double labeling of Hoxc6 and Hoxc9 (H,M) demonstrate the Hoxc9+ cells (white arrows) induced by Smad2-2E in the Hoxc6 domain do not express Hoxc6. (D,F,H) The same section; (I,K,M) the same section. Scale bars: 50 µm.

View larger version (24K): [in a new window]   Fig. 8. A model for Gdf11 function in controlling Hox gene expression and rostrocaudal identity in the spinal cord. (A) Normally, the onset of Gdf11 expression begins after most of the cervical progenitor cells leave the stem zone, and therefore, only progenitor cells designated for caudal levels receive the Gdf11 signal. Fgf and Gdf11 work together to define Hox gene expression domains in the caudal spinal cord. (B) Gdf11 electroporation increases the level of Gdf11 and, thus, results in a rostral displacement of Hoxc6-Hoxc10 domains. (C) In Gdf11-/- embryos, only the Fgf signal remains. This causes severe caudal displacement and expansion in Hoxc10 and Hoxc9 domains, respectively, and a lesser effect in Hoxc6 and Hoxc8 domains. (D) A model for Gdf11 function in the control of Hox gene expression and RC identity of the spinal cord. Smad2 mediates the function of Gdf11 in promoting caudal Hox gene expression. Although high levels of Gdf11 can activate the Smad1/5/8 pathway in vitro, the significance of this pathway in vivo requires further examination.