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First published online 23 March 2005
doi: 10.1242/dev.01798

Development 132, 2047-2056 (2005)
Published by The Company of Biologists 2005
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Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord

Yu-Qiang Ding1,*, Ji-Young Kim1, Yong-Sheng Xu1, Yi Rao2,{dagger} and Zhou-Feng Chen1,{ddagger}

1 Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology, School of Medicine, Washington University Pain Center, St Louis, MO 63110, USA
2 Anatomy and Neurobiology, School of Medicine, Washington University Pain Center, St Louis, MO 63110, USA



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  		Fig. 1. Organization of the dorsal horn circuitry and identification of two waves of neuronal migration in the dorsal spinal cord by BrdU labeling. (A) Schematic diagram of the spinal cord circuitry. The dorsal horn can be divided into several laminae. Laminae I-II (green) consist of small neurons in the superficial dorsal horn, whereas laminae III-V (red) contain large neurons in the deep dorsal horn. Proprioceptive afferents (light pink) innervate the motoneurons in the ventral horn via a medial entry route. In the dorsal horn, while mechanoreceptive afferents (red) project to laminae III-IV, nociceptive afferents (green) project mainly to laminae I-II. (B,C) Distribution of E10.5 BrdU-labeled cells in E12.5 (B) and E13.5 (C) dorsal horns. There are no BrdU-labeled cells in the superficial layer of E13.5 dorsal horns (arrow in C). (D) Quantitative comparisons of E10.5 BrdU-labeled cells between the dorsal and ventral horn at E11.5, E12.5 and E13.5. The percentages of BrdU-labeled cells in the dorsal half of the spinal cord (black bar) decreases (79.5%, 62.7% and 9.4%) progressively, while those of the labeled cells in the ventral half (white bar) increases accordingly. (E) E11.5 BrdU-labeled cells in E13.5 spinal cord. (F) E12.5 BrdU-labeled cells in E14.5 spinal cord. Scale bars: 100 µm in B-D,F.

 


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  		Fig. 2. Expression of Dcc in early-born dorsal interneurons. (A) Immunocytochemical staining of Dcc of E11.5 neural tube shows Dcc expression in the region lining the VZ of the neural tube. (B) Double immunocytochemical staining of Dcc (green) and Isl1 (red) in E11.5 dorsal spinal cord shows Isl1 in the nuclei and Dcc on membrane and in axons. (C) Higher magnification of B. Some Isl1+ cells were not stained for Dcc (arrow in C). (D) Double staining of Dcc (green) and Lmx1b (red). (E) Higher magnification of (D). Some Dcc+ cells do not express Lmx1b (arrowhead in E). (F) Double staining of Dcc (green) and Pax2 (red; dI4 and dI6 marker). (G) Higher magnification of dI4 region. (H) Higher magnification of dI6 region. Double arrowheads indicate double labeled-neurons. Scale bars: 100 µm in A; 50 µm in B,D,F; 25 µm C,E,G,H.

 


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  		Fig. 3. Impaired ventral migration of early-born neurons in Dcc–/– mutants. (A,B) Nissl staining of the dorsal horn of wild-type embryo (A) and DCC–/– mutant (B) embryos. Arrow in (B) indicates an area that is lightly stained with Nissl in the medial part of laminae I-II. (C,D) Immunocytochemical staining of Lmx1b in the dorsal horn of wild-type (C) and Dcc–/– mutant (D) embryos shows Lmx1b+ neurons in laminae I-II (arrow and arrowhead in C) and the region lacking Lmx1b staining (arrow in D). (E,F) X-gal staining of E14.5 Math1+/– (E) and Math1+/–/Dcc–/– embryos (F) shows an absence of Math1+ cells in the dorsal horn (arrow) of Math1+/– embryos in contrast to the ectopic Math1+ cells in the medial superficial dorsal horn in Dcc–/– mutants (arrow in F). Arrowheads indicate Math1+ cells in the intermediate region of the spinal cord. (G,H) Isl1 staining of wild-type (G) and Dcc–/– mutant (H) embryos shows the ectopic Isl1+ cells in the dorsal horn of Dcc–/– mutants (arrow in H) when compared with wild-type control. Arrowheads indicate Isl1+ cells in the intermediate region of the spinal cord. (I,J) E10.5 BrdU-labeled neurons are detected in the dorsal half of the spinal cord of wild-type (I) and Dcc–/– mutant (J) embryos at E14.5. Arrows indicate a cluster of BrdU-labeled neurons in the medial superficial dorsal horn of the mutant (J), but not in wild-type (I). Arrowheads indicate BrdU-labeled neurons at the intermediate region of wild-type and Dcc–/– mutant embryos. (K,L). E11.5 BrdU-labeled neurons are seen in the dorsal horn of wild-type (K) and Dcc–/– mutant (L) embryos. Arrows indicate an absence of BrdU-labeled neurons in the medial superficial dorsal horn of the mutants. Scale bars: 100 µm.

 


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  		Fig. 4. Expression of early-born neuron-specific markers in wild-type and Dcc–/– mutants. (A,B) Math1 immunocytochemical staining of E10.5 wild-type (A) and Dcc–/– mutant (B) dorsal neural tubes shows an absence of Math1+ cells in more ventral aspect of the neural tube of Dcc–/– mutant (arrowhead in B) when compared with wild-type control (arrowhead in A). (C,D) Foxd3 (dI2 marker) expression in E10.5 wild-type (C) and Dcc–/– mutant (D) neural tubes detected by in situ hybridization. Foxd3+ cells are absent in more ventral aspect of the neural tube, as indicated by arrowhead in D. (E,F) Isl1 staining of E11.5 wild-type (E) and Dcc–/– mutant (F) neural tubes. More Isl1+ cells are present in the dorsal part of the neural tube (arrow in F). (G,H) Double staining of Lmx1b (green; dI5 marker) and Pax2 (red; dI4, dI6 marker) in E10.5 wild-type (G) and Dcc–/– mutant (H) neural tubes. Arrowhead in H indicates dorsally migrated Lmx1b+ cells. (I,J) Phox2a staining of E10.5 wild-type (I) and Dcc–/– mutant (J) neural tubes. Arrows and arrowheads in I,J indicate normal and abnormal positions of Phox2a+ neurons, respectively. Scale bars: 100 µm.

 


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  		Fig. 5. Forced expression of Dcc in the dorsal spinal cord. (A,B) Detection of EGFP in the dorsal spinal cord electroporated with EGFP only (A) and Egfp/Dcc (B) plasmids. There are a few neurons located in the ventral horn of the spinal cord after the electroporation of Egfp/Dcc (arrowheads in B). (C,D) Higher magnification of A,B indicates the superficial dorsal horn. Brackets outline laminae I-II region. In the spinal cord electroporated with EGFP, EGFP+ cells are found in laminae I-II, but few Egfp/Dcc+ cells are present in the corresponding region (D). (E) Dcc immunostaining of Egfp/Dcc electroporated side of the dorsal horn. Arrows indicate Dcc+ neurons in the same region. (F) Arrows indicate EGFP+ neurons. (G) Dcc and Egfp are colocalized in the same cells (arrows). (H-J) Counting of EGFP+ and Egfp/Dcc+ cells in the spinal cord (H), ventral horn (I) and laminae I-II (J). For cell counting in the ventral horn, Egfp/Dcc+ or EGFP+ cells located ventral to the center of the central canal were included, as shown in A,B. For cell counting in laminae I-II, the morphology of the spinal cord is revealed by Hoechst counterstaining (red color in A-D), and labeled cells in laminae I-II, as shown in C,D were counted. Student's t-test was used when making comparisons, *P<0.0001. cc, central canal; VH, ventral horn. Scale bars: 200 µm in A,B; 100 µm in C,D; 25 µm in E-G.

 


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  		Fig. 6. Abnormal projection of proprioceptive afferents in Dcc–/– mutants. (A,B) 2H3 immunocytochemical staining indicates an absence of 2H3+ afferent projections to the ventral horn in E14.5 Dcc–/– mutant spinal cord (B) when compared with wild-type control (A) (double arrowheads in A,B). 2H3+ afferents are aberrantly located in the medial superficial dorsal horn of Dcc–/– mutant (arrows in B). (C,D) Double immunocytochemical staining of 2H3 and Isl1 in E13.5 wild-type (C) and Dcc–/– mutant (D) dorsal horns shows that 2H3+ afferents (arrows in C,D) appear to follow the pattern of Isl1+ staining (arrowheads in C,D). (E,F) DiI labeling of E15.5 wild-type (E) and Dcc–/– mutant (F) spinal cords shows no proprioceptive afferents growing towards the ventral horn in Dcc–/– mutant (arrowheads in E,F). Asterisks indicate the heavily labeled superficial dorsal horn by DiI. Scale bars: 100 µm.

 


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  		Fig. 7. Abnormal projection of proprioceptive afferents in Dcc–/– mutants at E18.5. (A,B) Immunocytochemical staining of Lmx1b shows the distribution of Lmx1b+ neurons in the superficial dorsal horn of the wild-type mice (A), and the region lacking Lmx1b+ neurons (arrow in B) in the dorsal horn of Dcc–/– mutants at E18.5. (C,D) 2H3 staining shows the absence of 2H3+ primary afferents to the ventral horn (double arrowhead in D) and its abnormal location in the superficial dorsal horn (arrow in D) in Dcc–/– mutants (D), compared with wild-type mice (C). (E,F) TrkA immunostaining shows the absence of TrkA+ primary afferent in the medial superficial dorsal horn in Dcc–/– mutants (arrow in F). In wild-type mice, TrkA+ fibers are evenly distributed in the superficial dorsal horn (E). (G,H) DiI labeling shows a dramatic reduction of proprioceptive projection to the ventral horn (large arrow in H) and abnormal projection to the midline region (small arrow in H) of Dcc–/– mutant, compared with wild-type mice (G). Broken white lines outline the ventral horn of the spinal cord. Scale bars: 100 µm.

 


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  		Fig. 8. Early-born neurons and Sema3a repel TrkA+ afferents in the dorsal spinal cord. (A,B) TrkA immunocytochemical staining of E14.5 wild-type (A) and Dcc–/– mutant (B) spinal cords shows an absence of TrkA + afferents in the medial superficial dorsal horn of Dcc–/– mutant (arrow in B) when compared with wild-type control (arrow in A). Brackets in A-D indicate superficial dorsal horn. (C,D) In situ hybridization in wild-type (C) and Dcc–/– mutant (D) dorsal horn shows the ectopic Sema3a in the medial superficial dorsal horn of the mutant (arrow in D) when compared with wild-type control (arrow in C). (E) Immunocytochemical staining of TrkA+ afferents (red) and EGFP (green; arrow in E) in Sema3a+EGFP co-electroporated spinal cord. (F) Strong Sema3a expression in electroporated spinal dorsal horn. Arrow indicates introduced Sema3a expression, whereas arrowhead indicates endogenous Sema3a expression in the ventral horn. (G,H) Higher magnifications of E, showing TrkA staining of the contralateral (G) and electroporated sides (H) of the spinal cord. On the contralateral side of the dorsal horn, strong TrkA staining is detected in laminae I-II (arrowheads in G), but on the electroporated side much less TrkA staining is seen in laminae I-II (arrowheads in H). (I) Quantitative analysis of TrkA-positive areas. Asterisk indicates a significant difference in the area of TrkA-positive staining. *P<0.001. Areas of TrkA+ fibers are expressed by k µm2; k=1000. Co, contralateral side; Ep, electroporated side. Scale bars: 200 µm in E,F; 100 µm in A-D,G,H.

 

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© The Company of Biologists Ltd 2005