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First published online 30 June 2004
doi: 10.1242/dev.01250

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Lmx1b controls the differentiation and migration of the superficial dorsal horn neurons of the spinal cord

Yu-Qiang Ding^1,*, Jun Yin¹, Artur Kania², Zhong-Qiu Zhao¹, Randy L. Johnson³ and Zhou-Feng Chen^1,

¹ Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology, Washington University School of Medicine Pain Center, St. Louis, MO 63110, USA
² Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10027, USA
³ Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 70030, USA

View larger version (119K): [in a new window]   Fig. 2. Morphology, distribution and migration of wild-type and Lmx1b mutant dorsal horn neurons. (A,C,E) Nissl staining of wild-type embryos. (B,D,F) Nissl staining of Lmx1b-/- embryos. (A,B) At E12.5, no major difference was found between wild-type (A) and mutant embryos (B). (C,D) At E15.5, in wild-type, laminae I-II and laminae III-V are distinguishable, brackets outline the domain of laminae I-II and III-V, respectively. In Lmx1b-/- embryo, no clear boundary between laminae I-II and III-V exists (bracket outlines the whole domain). (E,F) Lower magnification of C,D. In wild-type embryo, the dorsal funiculus is enlarged (E, arrow), whereas in Lmx1b mutant it is smaller (F, arrow). (G) Wild-type embryo at E14.5: neurons labeled with BrdU at E11.5 are not detected in the outer aspect of laminae I-II (inset indicating a high magnification). Bracket outlines the most superficial layer. (H) Lmx1b mutant embryo at E14.5: neurons labeled with BrdU at E11.5 are detected in the most superficial layer (bracket in the insert). (I,J) Higher magnification view of G,H showing the dorsal horn that is divided into the medial one-third (Med 1/3) and lateral two-thirds (Lat 2/3) region by lines for quantitative analysis. (K) Quantitative comparison of BrdU-labeled neurons in the medial one-third region and lateral two-thirds region between wild-type (white bar) and Lmx1b-/- embryos (black bar). Asterisks indicate significant difference using Student's t-test P<0.0001. Scale bars: 100 µm in A-D,H; 200 µm in E,F; 60 µm in inset in H.

View larger version (86K): [in a new window]   Fig. 1. Expression of Lmx1b and specification of early-born neurons in the spinal cord of Lmx1b-/- mutants. (A-C) Expression of Lmx1b in the dorsal horn detected by in situ hybridization. (A) Lmx1b is expressed in dI5 interneurons at E10.5 (arrow). (B) At E11.5, in addition to dI5 neurons (small arrow), Lmx1b is expressed in neurons emerging from the dorsal VZ (large arrow). (C) Lmx1b expression is concentrated in laminae I-II neurons at E17.5. (D) Detection of BrdU+ (blue)/LMX1B+ (brown) in the spinal cord of wild-type E12.5 embryos. Arrows in D show nascent neurons co-labeled with anti-LMX1B and anti-BrdU antibodies. (E,F) Detection of LBX1 (dI4-6 marker, red) and BRN3A (dI1-3 and dI5 marker, green) in wild-type (E) and Lmx1b mutant (F) embryos at E11. (G,H) Detection of LBX1 (red) and PAX2 (dI4 and dI6 marker, green) in wild-type (G) and Lmx1b mutant (H) spinal cord at E11. (I,J) Detection of LMX1B (red) and PHOX2A (green) in dI5 neurons at E11.5; arrow in I and arrowheads in J (higher magnification of I) indicate double-stained cells. (K,L) Detection of LBX1 (red) and PHOX2A (green) in wild-type (K) and Lmx1b mutant embryos (L) at E11.5. Arrows indicate PHOX2A+ cells. (M) Statistical comparison of the number of PHOX2A+ cells in wild-type (white bar) and Lmx1b mutant (black bar) embryos. Asterisks indicate significant difference using Student's t-test P<0.001. Scale bars: 100 µm in A-C,E-I,K,L; 50 µm in D; 25 µm in J. MN, motoneurons.

View larger version (98K): [in a new window]   Fig. 3. Expression of laminae I-II markers and rescue of Drg11 and Ebf3 expression by forced expression of Lmx1b in the dorsal horn of Lmx1b-/- embryos. (A,B) Drg11 expression in the dorsal horn of wild-type (A, arrow) and Lmx1b-/- (B, arrow) embryos. (C,D) Ebf3 expression is restricted to laminae I-II neurons in wild-type dorsal horn (arrow in C), but is absent in Lmx1b mutant embryos (arrow in D). (E,F) In wild-type embryos (E) Ebf1 is concentrated in laminae I-II (arrow in E,F), but its expression is markedly reduced in Lmx1b mutant embryos (F, arrow). (G,H) Rnx expression in laminae I-II neurons (arrows) of wild-type (G) and Lmx1b mutant embryos (H). (I,L-Q) Reactivation of Drg11 and Ebf3 expression in Lmx1b-/- spinal cord by exogenously introduced Lmx1b. (I) Lmx1b expression in Lmx1b-/- spinal cord after electroporation of Lmx1b-expression vectors. No Lmx1b was found in the contralateral side of the spinal cord. (J,K) Expression of EGFP control plasmids in Lmx1b-/- spinal cord. (K) No Lmx1b was detected in the electroporated side of Lmx1b-/- spinal cord after electroporation of EGFP. (L) Induction of Drg11 (arrow) in Lmx1b-/- spinal cord electroporated with Lmx1b expression vectors. No Drg11 staining was found in the contralateral side of the spinal cord (arrow). (M,N) Higher magnification of the regions indicated by arrows in L. Arrowheads indicate Drg11+ cells. (O) Induction of Ebf3 expression (arrow) in the dorsal horn of Lmx1b-/- embryos after electroporation. (P,Q) Higher magnification of the regions indicated by arrows in O. Arrowheads in P indicate Ebf3+ cells. Scale bars: 100 µm in A-H,I-K,L,O; 30 µm in M,N,P,Q.

View larger version (79K): [in a new window]   Fig. 4. Expression of dorsal horn-specific molecular markers in wild-type and Lmx1b-/- embryos at E14.5 (G-N) and E15.5 (A-E). (A,B) Hoxb6 expression in wild-type (A) and Lmx1b mutant embryos (B). (D,E) Spinal cord Hoxb8 expression is mainly evident in laminae I-II of wild type (D); however, in Lmx1b mutant embryo (E) its expression appears expanded ventrally. (C,F) Quantitative analysis of Hoxb6+ (C) and Hoxb8+ (F) neurons in wild-type (white bar) and Lmx1b-/- embryos (black bar). (G,H) Expression of Lbx1 in wild-type (G) and Lmx1b mutant embryos (H). Broken white lines outline the dorsal horn and arrowheads indicate mispositioned LBX1+ neurons in Lmx1b mutant embryos. (I) Quantitative analysis of the number of LBX1+ neurons between wild-type and Lmx1b mutant embryos. (J,K) BRN3A+ expression in wild-type (J) and Lmx1b-/- embryos (K) at E14.5. Arrowheads indicate the accumulation of BRN3A+ neurons around the midline (asterisk). (L) Quantitative comparison of the total number of BRN3A+ neurons between wild-type and Lmx1b-/- embryos. (M,N) PAX2 expression in wild-type (M) and Lmx1b mutant embryos (N). (O) Numbers of PAX2+ neurons in wild-type and Lmx1b mutant embryos. Asterisks indicate the midline region. Scale bars: 200 µm in A,B,D,E; 100 µm in G,H,J,K,M,N.

View larger version (45K): [in a new window]   Fig. 5. Comparison of Zic1, Zic2 and Zic4 expression in the dorsal horns of wild-type, Lmx1b-/- and Rnx-/- embryos at E14.5. (A,B,G,H) Zic1 expression in wild-type (A,G), Lmx1b mutant (B) and Rnx mutant embryos (H). (C,D,I,J) Zic4 expression in wild-type (C,I), Lmx1b mutant (D) and Rnx mutant embryos (J). (E,F,K,L) Zic2 expression in wild-type (E,K), Lmx1b mutant (F) and Rnx mutant (L) embryos. Arrows indicate the dorsal horn regions. Asterisks indicate the midline region. Scale bars: 100 µm.

View larger version (126K): [in a new window]   Fig. 6. MAP2 expression in laminae I-II neurons in the dorsal horn of wild-type and Lmx1b-/- embryos at E15.5. (A,B) MAP2 staining in laminae I-II of wild-type (bracket in A) and Lmx1b mutant embryos (bracket in B). (C,D) Higher magnification of A,B, respectively. Scale bars: 100 µm in A,B; 20 µm in C,D.

View larger version (81K): [in a new window]   Fig. 7. Aberrant migration and defective differentiation of the dorsal horn neurons in wild-type and Rnx-/- and Drg11-/- mutant embryos. (A) Neurons labeled with BrdU at E11.5 are not detected in the outer layer of laminae I-II (bracket) in wild-type embryo at E14.5 (inset). (B) Neurons labeled with BrdU at E11.5 are detected in the outer layer of laminae I-II in Rnx-/- embryo (inset, bracket). (C) Quantitative comparison of the number of BrdU+ neurons in the medial one-third (Med 1/3) and lateral two-thirds (Lat 2/3) regions between wild-type and Rnx-/- embryos (*P<0.001). (D,E) Comparison of the distribution pattern of neurons labeled with BrdU at E11.5 in the dorsal horn of E14.5 wild-type (D) and Drg11 mutants (E). (F) Quantitative analysis of the numbers of BrdU-labeled neurons in the medial and lateral regions of wild-type and Drg11 mutant embryos (*P<0.001). (G,H) MAP2 expression in the dorsal horn of wild-type (G, bracket) and Rnx mutant embryos (H, bracket). MAP2 expression of the dorsal horn in wild-type (I, bracket) and Drg11-/- mutant embryos (J, bracket). Scale bars: in B and C, 100 µm for A and D, respectively; 50 µm in insets in B and E; 100 µm in G-J.

View larger version (69K): [in a new window]   Fig. 8. Selective block of the ingrowth of cutaneous afferents in the dorsal spinal cord of Lmx1b-/- mutant embryos at E14.5. (A,B) Immunocytochemical detection of TrkA in the dorsal horn of wild-type (A) and Lmx1b-/- mutant (B) embryos. Arrows indicate the superficial laminae region. Brackets outline laminae I-II region. (C,D) Peripherin detection in the dorsal horn of wild-type (C) and Lmx1b mutant embryos (D) appears unchanged. Arrows indicate peripherin-labeled presumptive proprioceptive afferents. (E,F) DiI labeling of primary afferents in the dorsal horn of wild-type (E) and Lmx1b mutant embryos (F). Arrowheads indicate presumptive mechanoreceptor afferents and arrows indicate presumptive muscle proprioceptive afferents. Asterisks and brackets outline laminae I-II region. (G,H) Sema3c expression in wild-type (G) and Lmx1b mutant embryos (H). (I,J) Nrp1 expression in wild-type (I) and Lmx1b mutant embryos (J). (K,L) Plexin A2 expression in wild-type (K) and Lmx1b mutant embryos (L). (M,N) Slit1 expression in wild-type (M) and Lmx1b mutant embryos (N). (O,P) Robo2 expression in wild-type (O) and Lmx1b mutant embryos (P). (Q,R) Netrin 1 expression in wild-type (Q) and Lmx1b mutant embryos (R). Arrows in G-S indicate laminae I-II. Arrowheads in Q and R indicate the lateral region of laminae I-II. Scale bars: 200 µm in A, B; 100 µm in C-R.

View larger version (15K): [in a new window]   Fig. 9. Schematic diagram summarizing the transcriptional cascade that controls the development of laminae I-II circuitry. In this cascade, we hypothesize that an unidentified transcription factor X, cooperating with Lbx1, activates and maintains Lmx1b expression, which in turn maintains Rnx expression. Lmx1b most probably activates Drg11 expression directly, whereas Rnx helps to maintain Drg11 expression. Rnx is also required for maintaining Lmx1b expression. Lmx1b acts in part through Rnx and Drg11, and in part through other unknown genes to repress Zic1 and Zic4 expression (not shown). Lmx1b and Rnx maintain Ebf1 and Ebf3 expression in laminae I-II neurons (not shown). Together these transcription factors coordinate the migration and differentiation of the superficial dorsal horn neurons, and subsequent innervation of TrkA+ afferents.