Requirement of neuropilin 1-mediated Sema3A signals in patterning of the sympathetic nervous system
Takahiko Kawasaki1,2,*,
Yoko Bekku1,*,
Fumikazu Suto1,2,
Takashi Kitsukawa3,
Masahiko Taniguchi5,
Ikuko Nagatsu6,
Toshiharu Nagatsu7,
Kazuo Itoh8,
Takeshi Yagi2,4 and
Hajime Fujisawa1,2,
1 Group of Developmental Neurobiology, Division of Biological Science, Nagoya University Graduate School of Science, Chikusa-ku, Nagoya 464-8602, and
2 CREST, JST (Japan Science and Technology Corporation), 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
3 Laboratory of Specification Mechanisms 1, National Institute for Basic Biology, and
4 Laboratory of Neurobiology and Behavioral Genetics, National Institute for Physiological Science, Myodaiji, Okazaki 444, Japan
5 Department of Biochemical and Molecular Biology, Graduate School of Medicine, Tokyo University, Hongo, Tokyo 113-0033, Japan
6 Department of Anatomy and
7 Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan
8 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Machikaneyama 1-16, Osaka 560-0043, Japan
* These authors equally contributed to the paper
View larger version (118K):
[in a new window]
|
Fig. 1. Dislocation of TH-positive sympathetic neurons in the neuropilin 1 and Sema3A mutant embryos. (A-F) Frontal sections of the wild-type (A), neuropilin 1 homozygous mutant (B) and Sema3A homozygous mutant (C) embryos at E12.5, immunostained with anti-TH antibody. (D,F) High-magnification views of the boxed areas in A and B, respectively. (E) A high-magnification view of the medial part of an embryo in a section adjacent to B. Arrows in C indicate TH-positive neurons in the upper arm and axilla. (G) A double immunostaining of a section from the Nrp-1–/– embryo at E12.5, with anti-TH antibody (red) and anti-neurofilament antibody 2H3 (green). (H,I) The wild-type (H) and homozygous neuropilin 1 mutant (I) embryos at E12.5, immunostained in whole-mount with anti-TH antibody. Arrows indicate TH-positive neurons at ectopic positions. nt, the neural tube; da, the dorsal aorta; SCG, the superior cervical ganglion; fl, the forelimb. Scale bar: 200 µm (A-C); 200 µm (D,E); 100 µm (F,G); 500 µm (H,I).
|
|
View larger version (14K):
[in a new window]
|
Fig. 2. Quantitative analysis on the distribution of TH-positive sympathetic neurons. The average number of TH-positive neurons per section was calculated from around 20 serial frontal sections that were made at the cervical level of embryos at E12.5. Open and filled columns indicate the number of TH-positive neurons located at the original sites of the sympathetic ganglia and ectopic positions, respectively. (A) neuropilin 1 mutant embryos. 100%, 99.4% and 51.2% of TH-positive neurons are located at the original sites of the sympathetic ganglia in the wild-type embryo (1 embryo), heterozygous mutant embryos (2 embryos) and homozygous mutant embryos (3 embryos), respectively. (B) The Sema3A mutant embryos. 97.6% and 35.9% of TH-positive neurons are located at the original sites of the sympathetic ganglia in the wild-type embryos (3 embryos) and homozygous mutant embryos (2 embryos), respectively.
|
|
View larger version (38K):
[in a new window]
|
Fig. 3. Migratory pathways of neural crest cells within the sclerotome. (A) Neural crest cells from the wild-type embryo at E8.5 in vitro, immunostained with a neural crest cell-specific monoclonal antibody 4E9R (red) and anti-neuropilin 1 antibody (green). (B-E) Distribution of neural crest cells within the sclerotomes at the cervical level of E9.0 embryos. (B) A diagram indicating 9 regions of the sclerotome defined by different colors. (C-E) Histograms showing relative number (percentages) of neural crest cells contained within each region of the sclerotome shown in B, in the wild-type (C), neuropilin 1 homozygous mutant (D) and Sema3A homozygous mutant (E) embryos. d, dorsal; v, ventral; r, rostral; c, caudal; nt, neural tube; n, notochord; s, sclerotome. Scale bar: 50 µm in A.
|
|
View larger version (99K):
[in a new window]
|
Fig. 4. Distribution of sympathetic neuron precursors, spinal nerve fibers, and Sema3A transcripts in E9.25 embryos. (A,B) In situ hybridization analysis on frontal sections of the wild-type (A) and neuropilin 1 homozygous mutant (B) embryos, using an antisense cRNA probe for Mash1. Arrows indicate cells with the Mash1 transcripts. (C,D) Detection of spinal nerve fibers by immunostaining with 2H3 in sections adjacent to A and B. 2H3-positive fibers (arrows) are limited around the neural tube (nt). The staining in blood cells and the internal surface of the digestive tracts is non-specific. (E) In situ hybridization analysis on a frontal section of a wild-type embryo, using an anti-sense cRNA probe for Sema3A. An arrow indicates the notochord. da, dorsal aorta; fl, the forelimb; dm, dermamyotome; nt, neural tube. Scale bar: 100 µm (A-E).
|
|
View larger version (99K):
[in a new window]
|
Fig. 5. Effect of Sema3A on migration of dorsal root ganglia neurons and sympathetic neurons in vitro. (A-D) Co-cultures of the dorsal root ganglia explants from the wild-type (A,B) and homozygous neuropilin 1 mutant (C,D) embryos at E12.5 with the control HEK293T cells (A,C) and the Sema3A-secreting HEK293T cells (B,D) (2,000 cells/cm2 each). (E) A cultured sympathetic ganglion neuron, immunostained with anti-neuropilin 1 antibody (green) and anti-TH antibody (originally red but yellow after merging). (F-I) Co-culture of the sympathetic ganglion explants from the wild-type (F,G) and homozygous neuropilin 1 mutant (H,I) embryos at E12.5 with the control HEK293T cells (F,H) and the Sema3A-secreting HEK293T cells (G,I) (2,000 cells/cm2 each). Cultures were immunostained with anti-TH antibody. (J-M) Co-culture of the sympathetic ganglion explants from the wild-type embryos at E12.5 with the control HEK293T cells (J,L) or the Sema3A-secreting HEK293T cells (K,M) (800 cell/cm2 each). The cultures were immunostained with anti-TH antibody (red) and 2H3 (green). J and K are phase-contrast micrographs. L and M correspond to the boxed regions in J and K, respectively. Arrows in A-D, J and K indicate the HEK293T cells. Scale bar: 100 µm (A-D); 25 µm (E); 200 µm (F-I); 200 µm (J,K); 200 µm (L,M).
|
|
View larger version (99K):
[in a new window]
|
Fig. 6. Effects of Sema3A on morphology of sympathetic ganglion explants and neurites. The sympathetic ganglion explants from the wild-type embryos at E12.5 were co-cultured with the control HEK293T cells (A-C) or the Sema3A-secreting HEK293T cells (D-F) (800 cells/cm2 each). Phase-contrast micrographs of the explants at 12 hour (A,D), 24 hour (B,E) and 48 hour (C,F) in culture. Arrows indicate the HEK293T cells. Scale bar: 100 µm (A-F).
|
|
View larger version (21K):
[in a new window]
|
Fig. 7. Possible roles of Sema3A in migration and arrest of sympathetic neuron precursors. (A) In the wild-type embryo, Sema3A (blue) production in the dermamyotome (dm), forelimb (fl) and paraxial mesenchymal tissue forms a concentration gradient. Sympathetic neuron precursors (red spots), which have migrated through the ventral migratory pathway (a green arrow), are arrested at the bottom of the Sema3A gradient, the lateral part of the dorsal aorta (da). (B,C) Sympathetic neuron precursors in the neuropilin 1-deficient embryo (B) and the Sema3A-deficient embryo (C) do not stop at the lateral part of the dorsal aorta but migrate further along the permissive route. nt, neural tube; n, notochord.
|
|
© The Company of Biologists Ltd 2002
