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First published online 24 January 2007
doi: 10.1242/dev.02791

Development 134, 857-865 (2007)
Published by The Company of Biologists 2007
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Neuropilin asymmetry mediates a left-right difference in habenular connectivity

Yung-Shu Kuan1, Hung-Hsiang Yu2, Cecilia B. Moens2 and Marnie E. Halpern1,*

1 Carnegie Institution of Washington, Department of Embryology, 3520 San Martin Drive, Baltimore, MD 21218, USA.
2 Howard Hughes Medical Institute and Division of Basic Science, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Box 19024 Seattle, WA 98109, USA.


Figure 1
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  		Fig. 1. Differential dorsoventral innervation of the IPN by the left and right habenulae. (A) Dorsolaterial view of the habenulo-IPN projections in a 4-day-old zebrafish larva. DiI (red) and DiO (green) were injected into the left and right habenula, respectively. The yellow signal is a visual artifact owing to the superimposition of the differentially-labeled L-R habenulae in this orientation. (B-D) Higher magnification of A confirms that the left habenula innervates dorsal (d) and ventral (v) IPN, whereas the right habenula only projects ventrally. (E-P) Lateral views of (E-H) 2-, (I-L) 3- and (M-P) 4-day-old larvae labeled with anti-Lov (red) and anti-Ron (green) antibodies. F-H, J-L and N-P are higher magnifications of E, I and M, respectively. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Asymmetric expression of nrp1a in the dorsal diencephalon. (A,B) Left-sided expression of nrp1a (black arrow) is detected by 2 days (A) and increases by 4 days (B). (C) nrp1a expression (red) overlaps with the dense neuropil of the left habenula revealed by anti-acetylated tubulin immunolabeling (green) at 4 days. (D,E) The nrp1a-expressing neurons (red) are a subset of the Lov-immunoreactive population (D, green) and are also distinct from Ron-immunoreactive cells (E, green) of the left habenula (arrowheads). (F-H) Expression of (F) nrp1b, (G) nrp2a and (H) nrp2b was not detected in the habenular nuclei. (I,J) Laterality of nrp1a expression (orange) correlates with parapineal position (otx5 expression, black arrowheads) in (I) mock-injected (94±2% left bias, n=89) and (J) spaw MO-injected 4-day-old larvae (56±2% sinistral and 44±2% dextral, n=96). A-C and F-J are dorsal views of the brain; C and D are lateral views of composite Z-stack confocal images through the left habenula. Left (L) and right (R) sides of the brain are indicated. Scale bars: 50 µm.

 

Figure 3
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  		Fig. 3. Innervation of the dorsal IPN requires Nrp1a. Dorsal views of habenular nuclei (A,B,G,H) and lateral views of the IPN (C,D,E,F,I) of 4-day-old larvae. (A-D) Mock-injected (A,C) or Nrp1a MO-injected (B,D) larvae were labeled with anti-Lov (red) and anti-Ron antibody (green). (E,F,I) Mock-injected (E), Nrp1a MO-injected (F) or parapineal-ablated (I) larvae were labeled at 4 days with DiI (red) in the left habenula. (G) All control-ablated larvae (n=27) had an intact parapineal (black arrowhead) as confirmed by otx5 expression (blue), and strong nrp1a expression (orange) in the left habenula. (H) In the majority of parapineal-ablated larvae, nrp1a expression (orange) was greatly reduced in the left habenula (~71%, n=39). Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Sema3D depletion disrupts innervation of the dorsal IPN. Larvae were stained with anti-Lov (red) and anti-acetylated tubulin (green) at 4 days. (A-H) Dorsal views of the brain in control (A), Nrp1a (B), Sema3Aa (C), Sema3Ab (D), Sema3D (E), Sema3Fa (F), Sema3Ga (G), or Sema3Gb (H) MO-injected embryos. Optimal concentrations of MOs (see Table 1) showed no effect on general brain morphology. (I-V) Dorsal views of the habenular nuclei (I-K) and lateral views of the IPN (L-V) of (I,L,O) mock-injected larvae or larvae injected with antisense MOs for (J,M,P) Sema3D, (K,N,Q) Sema3D and Nrp1a, (R) Sema3Aa, (S) Sema3Ab, (T) Sema3Fa, (U) Sema3Ga or (V) Sema3Gb. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. Dose-dependence and synergism of Nrp1a and Sema3D MOs. Innervation of the IPN was assayed by anti-Lov immunolabeling and confocal microscopy and the number of larvae lacking dorsal projections (% abnormal dorsal IPN innervation) was determined for each experimental condition. Error bars represent the s.e.m. from at least three independent injection experiments. For a given MO, high MO refers to the concentration that caused morphological defects and lethality in a large proportion of larvae (above 50%); optimal MO concentrations did not produce morphological defects or lethality above mock-injected larvae; low MO concentrations were at least 25% less effective than the optimal MO concentration at causing defective IPN innervation. Co-injection of low MO amounts for Nrp1a and Sema3D had a significantly greater effect than injection of either MO alone. For the total number of larvae assayed and other experimental details see Table 1.

 

Figure 6
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  		Fig. 6. Overexpression of sema3D causes ectopic left habenular projections. (A-F) Lateral views of the 4-day-old larval IPN from (A,D) mock-injected embryos (n=91) or embryos injected with (B,E) sema3D mRNA (n=64), or (C,F) sema3Gb mRNA (n=69). Larvae were stained with anti-Lov antibody (red) and anti-Ron antibody (green) in A-C, or labeled with DiI (red) in the left habenula in D-F. (G,H) Lateral views of (G) 2- and (H) 4-day-old larvae double-labeled with sema3D antisense RNA probe and anti-Lov antibody. sema3d transcripts (red) are abundant in the brain region dorsal to the IPN, as visualized by Lov+ habenular projections (green). Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. Model of Nrp1a-Sema3D-mediated guidance of left habenular axons. (A) Sema3D attracts Nrp1a-expressing growth cones of left habenular axons toward the dorsal IPN. Following Nrp1a (B) or Sema3D (C) depletion, left habenular efferents innervate only the ventral IPN, similar to right habenular neurons that do not express nrp1a. (D) Sema3D overexpression can result in ectopic habenular projections that extend beyond the dorsal IPN.

 

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© The Company of Biologists Ltd 2007