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First published online 12 April 2006
doi: 10.1242/dev.02354

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Classic cadherins regulate tangential migration of precerebellar neurons in the caudal hindbrain

Hiroki Taniguchi^1,2,*, Daisuke Kawauchi^3,, Kazuhiko Nishida^2,3 and Fujio Murakami^1,2,3,

¹ Division of Behavior and Neurobiology, National Institute for Basic Biology, Myodaijicho, Okazaki 444-8585, Japan.
² CREST, Japan Science and Technology, Japan.
³ Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

View larger version (119K): [in a new window]   Fig. 1. mRNA expressions of classic cadherins in the marginal and submarginal streams of the migrating precerebellar neurons in the myelencephalon. Transverse sections of the E15 rat caudal hindbrain hybridized with antisense digoxigenin-labeled riboprobes for Cad6 (A), Ncad (B), Cad8 (C) and Cad11 (D), and sense digoxigenin-labeled probes for Cad11 (E). Signals of cadherin mRNAs are seen in both marginal (arrow) and submarginal (arrowhead) streams with antisense probes (A-D), whereas sense probes produce no significant signals (E). Scale bar: 400 µm.

View larger version (95K): [in a new window]   Fig. 2. Electroporation-based gene transfer in the flat whole-mount culture of the embryonic rat medulla. (A) Schematic diagrams showing the procedure to make the flat whole-mount preparation of the hindbrain into which expression vectors are transferred. The DNA solution containing expression vectors and a dye is injected into the fourth ventricle. The head is cut, immersed in PBS and electroporated using forceps-shaped electrodes. The hindbrain is dissected out, cut at the dorsal midline and opened in the medium. The medulla is excised from it by cutting at the red lines and placed on the membrane in an open book configuration. Fixed preparations are sectioned along the blue line to make cryostat sections for immunostaining. (B-D) Transverse sections of 2 div explants immunostained with an anti-pan-cadherin Ab. An egfp cDNA is introduced into the explants. Lower (B,C) and higher (D) magnification. EGFP-positive migrating neurons express cadherin proteins. VM, ventral midline. (E-G) Transverse sections of 2 div explants expressing EGFP+CAD11-FLAG (E), EGFP+cN390-FLAG (F) and EGFP-NCAD(t)+DsRed (G). FLAG signal is detected with an anti-FLAG antibody. Most of the cells labeled with fluorescent reporter proteins express exogenous cadherin constructs. Scale bars: 200 µm in B and C; 50 µm in D-G.

View larger version (118K): [in a new window]   Fig. 3. Overexpression of DN cadherin constructs but not full-length cadherin constructs slows down the migration of LRN/ECN neurons. Flat whole-mount preparations of rat hindbrains electroporated with expression vectors encoding EGFP (A), EGFP+CAD11 (B), EGFP+NCAD (C), EGFP+cN390 (D) and EGFP+EGFP-NCAD(t) (E) at 3 div. (Left column) Lower magnification views of flat whole-mount preparations in which migrating neurons are labeled with EGFP. (Middle and right columns) Higher magnification views of contralateral and ipsilateral sides, respectively, in the flat whole-mount preparations in the left column. Many migrating neurons with EGFP, EGFP+CAD11 and EGFP+NCAD that have crossed the ventral midline are seen on the contralateral side (middle column in A-C). By contrast, fewer cells with EGFP+cN390 and EGFP+EGFP-NCAD(t) are observed in the contralateral part, apart from the ventral midline (middle column in D and E). Denser cell clumps are seen on the ipsilateral side of the preparations with DN cadherins (right column in D and E) compared with the control preparations with only EGFP or the preparations with exogenous full-length cadherins (right column in A-C). Broken lines labeled VM indicate the position of the ventral midline. Scale bars: 400 µm in left column; 50 µm in middle and right columns.

View larger version (37K): [in a new window]   Fig. 4. Quantification of the effect of cadherin constructs on the migration of LRN/ECN neurons in the flat whole-mount preparations. (A) Schematic diagrams showing the method for quantification (see detail in text). (B) Bar graph indicating the mean distance between the VM and the forefront neuron in the rectangle with more than 10 neurons, which is the farthest from the ventral midline. n=10, one-way ANOVA, P<0.0001; Dunnett's Multiple Comparison Test, *P<0.01.

View larger version (163K): [in a new window]   Fig. 5. The delay in the migration of LRN/ECN neurons expressing DN cadherin constructs is observed before crossing of the ventral midline by control neurons expressing only EGFP. Flat whole-mount preparations electroporated with expression vectors encoding EGFP (A,B), EGFP+EGFP-NCAD(t) (C,D) at 1.5 div. (B,D) Enlarged views of migrating neurons on the ipsilateral side near the FP in A,C, respectively. Whereas the control neurons with only EGFP reach the FP at this time (A,B), the neurons expressing EGFP+EGFP-NCAD(t) do not (C,D). Broken lines labeled VM indicate the position of the ventral midline. Scale bars: 400 µm in A,C; 50 µm in B,D.

View larger version (73K): [in a new window]   Fig. 6. Overexpression of a DN cadherin construct disrupts the migration of LRN/ECN neurons in vivo. E15.5 mouse hindbrains electroporated with Venus YFP (A,C,E,G) and EGFP-NCAD(t) (B,D,F,H) at E12.5. Lower magnification views from dorsal (A,B) and ventral (C,D) side. The right lower rhombic lip is exclusively labeled with fluorescent markers. PEMS and AEMS corresponding to the migratory streams of LRN/ECN neurons and of pontine gray and reticulotegmental nuclei neurons, respectively, are seen in the ventral views. Rostral is towards the left. Asterisks represent the labeled side. Cb, cerebellum; AEMS, anterior extramural migratory stream; PEMS, posterior extramural migratory stream. Higher magnification views of ipsilateral (E,F) and contralateral (G,H) sides. More control neurons are seen on the contralateral side in controls, whereas more neurons expressing EGFP-NCAD(t) are found on the ipsilateral side. Broken lines labeled VM indicate the position of the ventral midline. Scale bars: 1 mm in A-D; 200 µm in E-H.

View larger version (82K): [in a new window]   Fig. 7. Migration of LRN/ECN neurons slowed by cadherin function blocking causes ectopic ipsilateral formation of nuclei. Transverse sections of E18.5 mouse hindbrains electroporated with Venus YFP (A,C,E) and EGFP-NCAD(t) (B,D,F) at E12.5. Lower magnification views (A,B). LRN but not ECN is included in these sections owing to the angle of sectioning. Squares correspond to the higher magnification views in C-F. Asterisks represent the labeled side. Higher magnification views of ipsilateral (C,D) and contralateral (E,F) sides. EGFP-NCAD(t) neurons form a nucleus ipsilaterally unlike control YFP neurons. Scale bars: 600 µm in A,B; 150 µm in C-F.

View larger version (71K): [in a new window]   Fig. 8. Blocking of the cadherin function by DN cadherin has no effect on TAG1 expression in LRN/ECN neurons. Transverse sections of 2 div explants expressing EGFP (A-C) and EGFP+EGFP-NCAD(t) (D-F) immunostained with an anti-TAG1 antibody. Confocal images of EGFP signals (A,D), TAG1 signals (B,E) and merged signals (C,F). EGFP-positive cells express TAG1 in both conditions. Scale bar: 50 µm.

View larger version (77K): [in a new window]   Fig. 9. The attraction of LRN/ECN neurons by the FP is not disturbed by overexpression of DN cadherin. The dorsal medulla electroporated with EGFP (A) or EGFP+EGFP-NCAD(t) (B) is co-cultured with an FP explant in collagen gels. In both conditions, many neurons migrate towards the FP explant. Scale bar: 400 µm.