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First published online 23 March 2005
doi: 10.1242/dev.01781

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Commissural neuron identity is specified by a homeodomain protein, Mbh1, that is directly downstream of Math1

Rie Saba¹, Jane E. Johnson² and Tetsuichiro Saito^1,*,

¹ Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
² Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9111, USA

View larger version (60K): [in a new window]   Fig. 1. Mbh1 expression in mouse embryos. Lateral (A,C,E) and dorsal (B,D,F) views of whole-mount in situ hybridization of E10.5 (A-D) and E11.5 (E,F) embryos with antisense cRNA probes for Math1 (A,B) and Mbh1 (C-F). Arrowheads indicate the ventral telencephalon (only in E), dorsal diencephalon and mesencephalon, which expressed Mbh1 but not Math1. Scale bars: in A, 1 mm for A-D; in E, 1 mm for E,F.

View larger version (45K): [in a new window]   Fig. 2. Identification of an enhancer to drive lacZ expression in the dorsal spinal cord. (A) Schematic representation of the genomic structure of Mbh1 and Mbh1/lacZ transgenes. The orange boxes indicate exons, and translation start site (ATG) is designated as nucleotide number 1. The blue and green boxes represent lacZ and the ß-globin promoter/lacZ construct (BGZA), respectively. The 3' fragment was reversed in Tg6. The number of transgenic (#TgM) and ß-gal+ (#expression) embryos are shown in the table; athe two embryos expressed lacZ at low levels in the midbrain. Lateral (B) and dorsal (C-E) views of representative ß-gal+ embryos harboring Tg2 (B,C), Tg4 (D) and Tg5 (E). Transverse sections of the embryos carrying Tg2 (F) and Tg4 (G). Arrowheads indicate ß-gal+ ventral funiculi. (H) Transverse section of the embryo carrying Tg4 was immunostained with antibodies against Math1 (red) and ß-gal (green). Scale bars: in B, 1 mm for B-E; in F, 100 µm for F,G; in H, 50 µm.

View larger version (38K): [in a new window]   Fig. 3. An E-box 3' of the Mbh1 gene was required for lacZ expression in the spinal cord. (A) Structure of the transgenes, and the number of transgenic and ß-gal+ embryos recorded. The 3' fragments were reversed in Tg7, Tg10 and Tg13. Restriction enzyme sites used for cloning are indicated: Ba, BamHI; Kp, KpnI; Na, NarI, Ps, PstI; Sm, SmaI; Xb, XbaI. aThe three embryos expressed lacZ in the midbrain, floor plate or whole body. blacZ was expressed in the branchial arch or midbrain. cThe three embryos expressed lacZ in the telencephalon, limb or skin. Expression levels of lacZ in these ectopic sites (a-c) were low. dSeven embryos also expressed lacZ in the midbrain and somite. (B-D) Dorsal views of representative ß-gal+ embryos harboring Tg8 (B), Tg14 (C) and Tg18 (D). An arrow and an arrowhead indicate the ß-gal+ midbrain and somite, respectively. Scale bars: in B, 1 mm for B,C; in D, 1 mm. (E) Nucleotide sequence comparison of the 123-base sequence among the three species. Residues identical in the three species are boxed.

View larger version (60K): [in a new window]   Fig. 4. Math1 was necessary for Mbh1 expression in the spinal cord. Transverse sections at brachial (A-C,E-G) and forebrain levels (D,H) of E11.5 Math1+/+ (A-D) and Math1–/– (E-H) mouse embryos were hybridized with antisense cRNA probes for Math1 (A,E), Mbh1 (B,D,F,H) and Lhx9 (C,G). Loss of Mbh1 and Lhx9 expression was observed through all axial levels of the spinal cord in all Math1–/–embryos (n=3). Scale bar: 100 µm.

View larger version (55K): [in a new window]   Fig. 5. Misexpression of Math1 induced ectopic expression of Mbh1 in the spinal cord. Two days after electroporation, mouse embryos were collected at E13.5. Transverse sections at brachial levels of the electroporated spinal cord were hybridized with antisense cRNA probes for Math1 (A), Mbh1 (B) and Lhx9 (C). Right sides of sections were transfected with Math1. Arrowheads indicate endogenous Mbh1+ and Lhx9+ domains. Ectopic expression of Mbh1 and Lhx9 was also detected at E12.5, one day after electroporation (data not shown), and through all axial levels of the spinal cord at both E12.5 and 13.5 in all electroporated embryos (n=4). Misexpression of EYFP did not induce expression of either Mbh1 or Lhx9 (data not shown). Scale bar: 100 µm.

View larger version (18K): [in a new window]   Fig. 6. Math1 bound the Mbh1 enhancer containing the E-box in the spinal cord. (A) ChIP demonstrating binding of endogenous Math1 to the enhancer. Formaline-cross-linked chromatin from the E10.5 spinal cord was incubated without (–) and with rabbit polyclonal antibodies against Math1 and neurofilament. (B) Misexpressed Math1 also bound the enhancer. One day after electroporation of Math1-HA, cross-linked chromatin from the E12.5 spinal cord was incubated without (–) and with a rat monoclonal anti-HA antibody and IgG. Immunoprecipitates were analyzed by PCR using primers specific to the Mbh1 enhancer (upper panels) and to the region 5.8 kb upstream of the E-box as a negative control (lower panels). Each input represents DNA purified from the chromatin before immunoprecipitation. These data are representative of two (A) and four (B) independent experiments. Arrows and arrowheads indicate the amplified 502 bp and 546 bp DNA fragments, respectively.

View larger version (31K): [in a new window]   Fig. 7. Transcriptional activation by Math1 in the spinal cord. The E11.5 spinal cord was electroporated with Tg12 and either EYFP (A-D) or EYFP/Math1 (E-H), and stained with X-gal, two days after electroporation. (A,B,E,F) Dorsal views of the spinal cord. (C,D,G,H) Transverse sections at brachial levels. Dark (A,C,E,G) and illuminated (B,D,F,H) views to show transfected (EYFP+) and ß-gal+ cells, respectively. Upper (in A,B,E,F) and right (in C,D,G,H) sides were transfected with the genes. Arrow, ß-gal+ cell; arrowheads, commissural axons. Scale bars: in A, 1 mm for A,B,E,F; in C, 100 µm for C,D; in G, 100 µm for G,H. (I) Quantitative analysis of transcriptional activation by Math1. Two days after electroporation, cell extracts were prepared from EYFP+ portions. After normalization to EYFP fluorescence, the data are expressed as ß-gal+ activity relative to the activity obtained by transfection of EYFP alone. Error bars indicate standard error of at least three independent experiments.

View larger version (74K): [in a new window]   Fig. 8. Mbh1 function as a transcriptional repressor. Transverse sections at brachial levels of the spinal cord, two days after electroporation at E11.5 of EYFP alone (A), EYFP/Mbh1 (B), EYFP/Math1 (C), EYFP/En-Mbh1 (D), EYFP/VP16-Mbh1 (E) and EYFP/Math1/VP16–Mbh1 (F). Similar patterns of EYFP+ axons were observed through all axial levels of the spinal cord in all electroporated EYFP+ embryos (n=20, 32, 20, 10, 10 and 10 for EYFP, EYFP/Mbh1, EYFP/Math1, EYFP/En-Mbh1, EYFP/VP16-Mbh1 and EYFP/Math1/VP16–Mbh1, respectively). Arrowheads indicate EYFP+ commissural axons. Scale bar: 100 µm.

View larger version (8K): [in a new window]   Fig. 9. Transcriptional cascade to generate commissural neurons downstream of Math1.