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First published online 2 April 2008
doi: 10.1242/dev.015248

Development 135, 1703-1711 (2008)
Published by The Company of Biologists 2008
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Dynamic regulation of the expression of neurotrophin receptors by Runx3

Souichiro Nakamura1,*, Kouji Senzaki1,*, Masaaki Yoshikawa1, Mika Nishimura1, Ken-ichi Inoue2, Yoshiaki Ito2, Shigeru Ozaki1 and Takashi Shiga1,{dagger}

1 Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan.
2 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore.


Figure 1
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  		Fig. 1. Number of DRG neurons and DRG volume are decreased in P0 Runx3-/- mice with no differences in the number of caspase 3+ DRG cells between wild-type and Runx3-/- mice. (A,B) Islet1+ neurons in DRGs of wild-type (A) and Runx3-/- (B) mice at P0. (C,D) Quantitative analysis of the total number of Islet1+ DRG neurons (C) and the DRG volume (D) at the level of Th10 in wild-type (white bars) and Runx3-/- (black bars) mice at E13.5, E16.5 and P0. (E,F) Immunoreactivity (green) of caspase 3 in DRGs of wild-type (E) and Runx3-/- (F) mice at E17.5. The dotted line outlines the DRG. (G) Quantitative analysis of the number of caspase 3+ DRG cells in wild-type (white bars) and Runx3-/- (black bars) mice at E17.5 and P0. Data are shown as mean ±s.e.m.; *P<0.01. Scale bars: 100 µm.

 

Figure 2
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  		Fig. 2. Loss of expression of parvalbumin in Runx3-/- DRGs during development. (A-F) Photomicrographs indicate parvalbumin (PV) immunoreactivity (brown) at E14.5 (A,D), E18.5 (B,E) and P0 (C,F) in wild-type (A-C) and Runx3-/- (D-F) DRGs at the level of Th12. (G) Quantification of PV+ DRG neurons from E14.5 to P0. The number of PV+ neurons in wild-type (white squares) and Runx3-/- (black squares) mice are shown, and virtually no neurons express PV in Runx3-/- DRGs through these developmental stages. SC, spinal cord. Data are shown as mean±s.e.m.; *P<0.001. Scale bar: 100 µm.

 

Figure 3
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  		Fig. 3. Transient loss of TrkC expression in Runx3-/- DRGs during development. (A-J) Photomicrographs showing TrkC immunoreactivity (brown) at E11.5 (A,F), E13.5 (B,G), E14.5 (C,H), E18.5 (D,I) and P0 (E,J) in wild-type (A-E) and Runx3-/- (F-J) DRGs at the level of Th10. (K) Quantification of TrkC+ DRG neurons from E13.5 to P0. The number of TrkC+ DRG neurons was consistently smaller in Runx3-/- (black circles) than in wild type (white circles) mice between E13.5 and P0. (L-O) Immunoreactivity of TrkC in Runx3+/+; Bax-/- (L,M) and in Runx3-/-; Bax-/- (N,O) DRGs at the level of Th10. The DRGs of L and N are shown at higher magnification in M and O, respectively. TrkC was expressed in Runx3+/+; Bax-/- DRG neurons but not in Runx3-/-; Bax-/- DRG neurons. Data are shown as mean ±s.e.m.; *P<0.05, **P<0.01, ***P<0.001. Scale bars: 100 µm in J and in N for L,N; 50 µm in O for M,O.

 

Figure 4
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  		Fig. 4. DRG axon projections to the spinal cord, skin and muscle in wild-type and Runx3-/- mice at P0. (A-C) Representative photomicrographs of the immunoreactivity of TrkC (A',A''; green), PV (B',B''; brown) and Vglut1 (C',C''; brown) in the spinal cord of wild-type (A'-C') and Runx3-/- (A''-C'') mice. The spinal cords shown in A and B were from the same littermates, those in C from different littermates. The relative signal intensity of the immunoreactivity of TrkC (A'''), PV (B''') and Vglut1 (C''') in the spinal cords along the dorsoventral axis in wild-type (white squares) and Runx3-/- (black squares) mice. In total, six spinal cord sections from two to three animals of each genotype were analyzed. TrkC+ afferents project to the dorsal horn (DH), the intermediate zone (IZ) and the ventral horn (VH) in the wild-type mice (A',A'''), but not to the intermediate zone and the ventral horn in Runx3-/- mice (A'',A'''). PV+ afferents project to the ventral horn in the wild-type (B',B'''), but not in Runx3-/- mice (B'',B'''). Vglut1+ afferents project to the dorsal horn, the intermediate zone and the ventral horn in the wild type (C',C'''), but not to the ventral horn in Runx3-/- mice (C'',C'''). (D-G) Double staining of TrkC+ (D',E'; green) and TrkA+ (D'',E''; red), and of TrkC+ (F',G'; green) and CGRP+ (F'',G''; red) axons in the skin of wild-type (D,F) and Runx3-/- (E,G) mice from the same littermates. Arrowheads indicate TrkC+/TrkA+ or TrkC+/CGRP+ axons (yellow in D'''-G'''). (H-J) Immunoreactivity of TrkC (H,I''; green) and PV (I''; red) in the muscle of wild-type mice (merge in I'''), and of TrkC (J; green) in Runx3-/- mice. TrkC+ axons project to the muscle in wild-type but not in Runx3-/- mice. PV immunoreactivity was not observed in the muscle of Runx3-/- mice (data not shown). Data are shown as mean ±s.e.m.; *P<0.05, **P<0.01, ***P<0.001. Scale bars: 100 µm in A'',H,J; 50 µm in E''',G'''; 20 µm in I'''.

 

Figure 5
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  		Fig. 5. Changes in the number of DRG neuron subtypes in Runx3-/- mice. (A-G) Immunoreactivity (brown) of Vglut1 (A',A''), TrkA (B',B''), TrkB (C',C''), c-Ret (D',D''), calbindin-28K (CB; E',E''), somatostatin (SOM; F',F'') and CGRP (G',G'') in wild-type (A'-G') and Runx3-/- (A''-G'') mice. Boxed areas of immunoreactive neurons are shown at higher magnification in the insets at the left bottom of each panel. (A'''-G''') Quantification of the number of DRG neuron subtypes in wild-type (white bars) and Runx3-/- (black bars) mice. Data are shown as mean ±s.e.m.; *P<0.05, **P<0.001. Scale bar: 100 µm.

 

Figure 6
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  		Fig. 6. Coexpression of Runx3 and neuron subtype markers in the wild-type DRG. (A,B) Double staining of Runx3 (A',B'; green) and TrkC (A'',B''; red) at E13.5 (A) and P0 (B) in Th9 DRGs; (A''',B''') merge. (C) The ratio of Runx3+/TrkC+ DRG neurons at E13.5 and P0. (D) Triple staining of PV (a), Runx3 (b) and TrkC (c) in Th9 DRGs at P0; (d-f) merges. Some images are shown with pseudo coloring (A,B,D). (E,F) Double staining of Runx3 (red) in combination with TrkA (green) (E) and TrkB (green) (F) in Th10 DRGs at P0. (G,H) Double staining of Runx3 (green) and Runx1 (red) at E16.5 (G) and E18.5 (H) in Th10 DRGs. Data are shown as mean ± s.e.m.; *P<0.001. Scale bars: 100 µm.

 

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© The Company of Biologists Ltd 2008