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First published online February 24, 2006
doi: 10.1242/10.1242/dev.02283

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Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration

Daisuke Kawauchi^1,2,*, Hiroki Taniguchi^3,, Haruyasu Watanabe¹, Tetsuichiro Saito^4,* and Fujio Murakami^1,

¹ Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
² SORST, Japan Science and Technology, Kawaguchi, Saitama 332-0012, Japan.
³ Division of Behavior and Neurobiology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan.
⁴ Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.

View larger version (87K): [in a new window]   Fig. 1. In vivo labelling of lRL-derived PC neurons by exo utero electroporation of EYFP. (A) Diagram outlining the method. Following injection of a plasmid carrying EYFP or Venus (left panel), electric pulses were applied. Red arrow indicates an EYFP-transfected region. (B,C) Dorsal (B) and ventral (C) views of the hindbrain (outlined) that was electroporated with EYFP at E12.5 and fixed at E14.5. There are two streams of labelled cells, the AEMS and PEMS (arrowheads in C). The cells in the AEMS take two separate routes (arrows in the inset of B). Because neurons in the PEMS begin their migration earlier, most labelled cells therein were located ventrally, leaving few cells behind (arrowhead in the inset of B). Rostral is towards the left. Asterisks show the EYFP-transfected side, the broken horizontal lines show the midline. (D,E) Transverse sections of EYFP-introduced embryos immunostained for EYFP (green, left panel), after Mbh2 in situ hybridization (purple, middle panel). The left, middle and right panels in D and E show the same field of view of metencephalon (D) and myelencephalon (E). In both, EYFP-labelled migrating cells expressed Mbh2 (arrowheads in insets of D and E). Broken vertical lines show the midline. Dorsal is upwards and EYFP-transfected side is towards the left. Cb, cerebellum. Scale bars: 500 µm in B and C; 250 µm in the inset of B; 100 µm in D; 200 µm in E; 55 µm for right panel insets in D,E.

View larger version (62K): [in a new window]   Fig. 2. Midline crossing and onset of nucleogenesis in the myelencephalon. (A) EYFP-labelled cells migrated tangentially towards the ventral midline (arrow) from the lRL (asterisk). (B) EYFP-labelled cells in the dorsal myelencephalon on the side ipsilateral to the injection. Labelled neurons that had left the lRL migrated along the dorsal margin (arrowheads). (C) EYFP-labelled neurons found near the ventral midline. The inset shows a higher-power view of the area outlined by the rectangle in C. Many labelled cells cross the ventral midline (arrowheads in the inset). Broken line indicates the midline. (D) A higher-power view of the area outlined by the rectangle in A. EYFP-labelled neurons do not form an aggregate ipsilaterally, although cells that appear to originate from the contralateral side as judged by Mbh2 expression do so. A-D are from E14.5 animals. (E-H) The direction of the EYFP-labelled cell migration in the regions of ECN (E) and LRN (G) changed from tangential to radial, with F and H showing higher-magnification views of the areas outlined in E and G, respectively. At E15.5, some of the labelled cells had begun to migrate radially (F,H), extending leading processes (arrowheads in F and H), and had aggregated within the ECN (arrow in E) and LRN (arrow in G). In each panel, EYFP was introduced into the left side. Dorsal is upwards. Scale bar: 200 µm for A,E,G; 50 µm for C,D; 25 µm for the inset in C; 20 µm for B,F,H.

View larger version (127K): [in a new window]   Fig. 3. Maturation of ECN and LRN. (A-C) The distribution of EYFP-labelled neurons at the level of ECN (A), rostral LRN (B) and caudal LRN (C). Many labelled cells are located in deeper regions of the myelencephalon. Arrowheads in A indicate axons of presumptive LRN neurons. (D-G) Transverse sections of E18.5 mice myelencephalon after immunostaining for EYFP (green) and Mbh2 in situ hybridization (purple). Medial is towards the left and dorsal is upwards. Higher-magnification views of the regions marked d-g. EYFP-labelled cells extended several dendrite-like processes that indicated maturation and expressed Mbh2 mRNA (the right panels). Arrows in each of the two panels in E and F indicate the same cells. EYFP was electroporated into the left lRL. Cb, cerebellum; mLRN, magnocellular part of LRN; pLRN, parvocellular part of LRN. Scale bars: in C, 200 µm for A-C; in right panel of G, 50 µm for D,G and 20 µm for E,F.

View larger version (88K): [in a new window]   Fig. 4. Ectopic formation of ECN and LRN caused by dominant-negative form of cadherins. (A-D) Dorsal (A,C) and ventral (B,D) views of E18.5 hindbrains (outlined) labelled by electroporation of pCAG-Venus (A,B) and a mixture of pCAG-Venus and pCAG-EGFP-Ncad(t) (C,D). Although control plasmid (Venus) did not affect nucleogenesis of ECN (A) and LRN (B), EGFP-Ncad(t) caused an increase in the size of ipsilateral aggregates (arrowheads in D). Asterisks indicate the labelled side. Rostral is towards the left. Areas outlined in lower panels of A-D correspond to the region shown in the upper panels. (E,F) Localization of EGFP-Ncad(t)-transfected cells (left panel) and Mbh2 signal (middle panel) in ipsilateral (E) and contralateral (F) LRN on transverse section of an E18.5 mouse. Many labelled cells were found in the ipsilateral but not in the contralateral LRN. Neurons in ectopic (ipsilateral) LRN (eLRN) expressed Mbh2 and were intermingled with untransfected LRN neurons (Venus-/Mbh2+) (right panel of E). Each of the three panels in E and F shows the same field. Insets in E are higher-magnification views of the area outlined in the left panel. eECN, ectopic ECN; eLRN, ectopic LRN. Scale bar: in D, 1mm for A-D; in F, 200 µm for E,F, and 50 µm for insets of E.

View larger version (61K): [in a new window]   Fig. 5. Midline crossing and ventricle-directed migration of PGN and RTN neurons. (A) EYFP-labelled cells tangentially migrating across the midline of the pons in an E14.5 mouse. Arrowheads indicate radially deflected cells. (B) At E15.5, some EYFP-labelled cells accumulated on the ipsilateral side, while some others were still crossing the midline. (C) EYFP-labelled cells that had turned to become ventricle orientated before (arrowheads on the left) and after (arrowhead on the right) midline crossing. E14.5. (D) Labelled cells within the pons. E15.5. (E-G) High-power views of the rectangular areas marked e-g in D. Ventricle-directed neurons extended a trailing and a leading process (arrowheads in E and F). (F) A cell that had just changed its migratory direction from tangential to ventricle (arrowheads). (G) An RTN neuron that extended several dendrite-like processes. EYFP was electroporated into the left lRL. Transverse sections. Dorsal is upwards. Broken lines in A-E represent the positions of the midline. Scale bar: 50 µm for A-C; 100 µm for D; 20 µm for E-G.

View larger version (114K): [in a new window]   Fig. 6. Expression of Mbh2 by EYFP-labelled cells that form clusters in the pons at E18.5. (A-D) EYFP-labelled cells at the level of the rostral (A,B) and caudal (C,D) pons. (B,D) Mbh2 expression in locations corresponding to A and C. PGN neurons located on the contralateral side were distributed only in the dorsal PGN (A), although RTN neurons were symmetrically distributed on both sides (C). (E-J) Higher-power views of the area outlined by rectangles e-j. Although the ventral region of the ipsilateral PGN was densely occupied by labelled cells (E), the corresponding region in the contralateral PGN contained axons (F, arrowheads). Most labelled neurons on both ipsilateral (G,I) and contralateral (H,J) sides expressed Mbh2 (G-J, right). Arrows in each panel of I and J indicate identical areas. (A,C,E,F) Immunostaining for EYFP; (B,D) Mbh2 in situ hybridization signals. (G-J) Immunostaining for EYFP (green) and Mbh2 in situ hybridization pattern (purple) in the same section. EYFP was electroporated into the left lRL. E18.5 mouse transverse sections. Dorsal is upwards. Scale bars: in F, 200 µm for A-D and 50 µm for E,F; in J, 50 µm for G,H and 20 µm for I,J.

View larger version (100K): [in a new window]   Fig. 7. Midline crossing of early-born PGN/RTN neurons. (A,B) EYFP-expressing PGN/RTN neurons (left panels) labelled with BrdU (middle panels). Right panels show merged views. BrdU was injected at E12.5 (A) or 14.5 (B). Transverse sections of the metencephalon from E18.5 mice. (C,D) High-power views of the area outlined by rectangles in A,B. Neurons labelled by EYFP-electroporation at E12.5 were also labelled by BrdU (white, arrowheads in the right panel of C) when BrdU had been injected at E12.5 (A,C, purple), but not at E14.5 (B,D, purple). E14.5 BrdU injection labelled EYFP-expressing cells occupying the ventral region of the ipsilateral PGN (right panel of B). (E) Quantification of BrdU labelling. Ordinate represents the number of BrdU-positive and EYFP-expressing cells (NBrdU) divided by the number of EYFP-expressing cells (Ntotal) in the contralateral pontine region. The day of BrdU injection is shown along the y-axis. Vertical bar represents s.e.m. Each column represents pooled data from five different embryos. (F) Diagram showing the quantification method. The mediolateral extent of the PGN (l) was defined as the distance from the midline to the lateral end of the PGN, as visualized by EYFP labelling on the ipsilateral side. All EYFP-labelled cells located within this distance from the midline on the contralateral side (i.e. the region shown by the red contour) were counted. The labelled side is towards the left. Both heavily and lightly labelled cells, in the region described in F, were counted for the analysis. Broken vertical lines in A and B show the midline. Scale bar: 200 µm for A,B; 50 µm for C,D.

View larger version (164K): [in a new window]   Fig. 8. EYFP-labelled neurons show ventricle-directed migration in close apposition to nestin-positive radial fibres. (A,B) EYFP-labelled cells (left panels) in the myelencephalon (A) and the metencephalon (B). Subsets of migrating EYFP-labelled PC neurons associated with nestin-positive fibres (purple). Some nestin-positive fibres also extended horizontally (arrowheads in B). (C,D) Left panels in C and D show higher-magnification views of the rectangular areas in A and B, respectively. Middle panels show nestin immunostaining. Arrowheads indicate radially extending leading processes of EYFP-labelled neurons apposed to nestin-positive fibres (middle panels) in the myelencephalon (C) and metencephalon (D). An arrow in the middle panel of D shows a tangentially extending fibre. An EYFP-labelled neuron in the pontine region (green) extended branched leading processes that were oriented tangentially and radially, with both branches closely apposed to nestin-positive fibres (arrowheads in right panel of D). EYFP was electroporated into the left lRL. (E,F) Electron micrographs, taken in the pontine region, of a presumptive PC neuron making contact with a radial fibre (arrowheads in E). F represents higher-magnification view of the rectangle in E. Asterisks in E and F indicate the nucleus of the neuron. The neuron contacts the radial fibre along a length of its plasma membrane. Transverse sections of E15.5 embryos. Right panels in A-D show a merged view with nestin immunostaining of the same section. The ventricle is upwards; the pia is downwards. LRN, lateral reticular nucleus; RF, radial fibre. Scale bars: in B, 50 µm for A,B; in D, 35.2 µm for C and 20 µm for D; in E, 2 µm; in F, 250 nm.

View larger version (40K): [in a new window]   Fig. 9. Summary diagram showing the process of nucleogenesis by PC neurons. All subsets of PC neurons show two distinct phases of migration, tangential and radial. (A) PGN/RTN neurons first migrate tangentially along the ventral margin of the metencephalon. They then migrate towards the ventricle, either before or after crossing the midline. In this phase, they migrate along nestin-positive radial fibres (red and grey). On arrival at their final destination, they stop migration and extend several dendrite-like processes (violet). In the myelencephalon (B), ECN and LRN neurons also initially execute tangential migration and cross the midline. They then migrate along radial fibres towards their final positions, as do PGN/RTN neurons. We hypothesize that PC neurons acquire responsiveness to transition-inducing cues expressed by radial fibres in specific locations (shown in red) and this acquisition takes place in a time-dependent manner (see changes in colour of migrating cells from green to dark blue).

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