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First published online May 8, 2009
doi: 10.1242/10.1242/dev.032276

Development 136, 1919-1928 (2009)
Published by The Company of Biologists 2009
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SDF1/CXCR4 signalling regulates two distinct processes of precerebellar neuronal migration and its depletion leads to abnormal pontine nuclei formation

Yan Zhu1,*, Tomoko Matsumoto1, Sakae Mikami2,{dagger}, Takashi Nagasawa2 and Fujio Murakami1,*

1 Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan.
2 Department of Medical Systems Control, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.


Figure 1
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  		Fig. 1. The meninges are required for the marginal migration of PCN. (A) Schematics of the developing mouse precerebellar system. (B) Schematics of in vitro electroporation into the lower rhombic lip (LRL) and an open-book organotypic culture. Electroporation was performed at E12.5, a stage when inferior olivary (IO) neurons have already left the LRL. (C) After 2 days, many GFP-positive cells emigrated from the LRL towards the floor plate (white line) on an organotypically cultured hindbrain. The image corresponds to the box in B. (D,E) The migratory stream was confined to the pial surface when the hindbrain was cultured with the meninges as shown on a transverse section (arrowheads in D), whereas removal of the meninges caused many cells to migrate submarginally (arrow in E). IV, fourth ventricle; CB, cerebellum; l, lateral; m, medial; p, pial; v, ventricular. Scale bars: 400 µm in C; 200 µm in E.

 

Figure 2
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  		Fig. 2. CXCR4 expression in migrating PCN and SDF1 expression in the meninges. Adjacent sections at various axial levels of mouse hindbrains were subjected to in situ hybridisation for Mbh2 (A,C,E,G,I,K) and Cxcr4 (B,D,F,H,J,L). (A-D) At E13.5, Cxcr4 was expressed in a stream of cells (B, arrowheads in D) resembling the posterior extramural stream (PES) labelled by Mbh2 (A, arrowheads in C). (C,D) Higher-magnification of the boxed areas in A and B, respectively. (E-L) Cxcr4 was also expressed in the anterior extramural stream (AES) at E13.5 (E,F, arrows). Note that Cxcr4 was expressed in the ventricular zone (B,F). At E15.5, Cxcr4 expression diminished in the PES, which started to aggregate into LRN (compare H with G), and continued in the migrating AES cells (arrows in I,J). At E17.5, Cxcr4 was not expressed in pontine nuclei (PN) (compare L with K). (M-P) PAX6 and CXCR4 double immunohistochemistry showing co-localization of CXC4 protein and PAX6, a PCN marker, in the PES (M,N) and AES (O,P) at E14.5. The insets are merged higher-magnification images of the boxed areas in N and P. (Q,R) Double immunohistochemistry for SDF1 and PAX6 on sections neighbouring M and O, respectively, showing the spatial relation between SDF1 protein and migrating PCN. Scale bars: 400 µm in F,J,L; 200 µm in D; 300 µm in P; 75 µm in the insets and Q.

 

Figure 3
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  		Fig. 3. PCN from LRL migrate preferentially towards co-cultured meninges or recombinant SDF1 protein. (A) Schematic of the explant co-culture in matrigel. (B) Schematic illustrating the quantification method. (C-H) DAPI staining of explant co-cultures to reveal the nuclei of cells. Dotted red lines delineate the borders of the co-cultured meninges (Mng) or collagen blocks to the right. LRL-derived cells migrated preferentially towards the co-cultured meninges (C,G), or towards an SDF1-embedded collagen block (E), but showed no preference towards a control collagen block (F). The attraction elicited by the meninges was abrogated by 20 µM AMD3100 (D), a specific inhibitor of CXCR4, or in co-cultures with Sdf1-/- meninges (H). (I) Quantification of each condition. *P<0.0001, **P<0.005, ***P<0.0005; Mann-Whitney U-test. Scale bar: 200 µm.

 

Figure 4
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  		Fig. 4. PCN migration is markedly disrupted in Cxcr4-/- mice. The migration of PCN and subsequent nuclei formation were analysed by Mbh2 in situ hybridisation on transverse sections of E14.5 (A) and E16.5 (B) hindbrains. (A) The schematic shows a wild-type E14.5 hindbrain with the AES and PES depicted in green and the approximate axial levels of the illustrated sections indicated. (a,b) The frontier of the AES turning ventrally in the E14.5 wild type (a, arrow) was not observed in a corresponding section from Cxcr4-/- (b, asterisk). (c,d) At posterior AES, PCN migrated marginally from the LRL in the wild type (c, arrow), whereas in Cxcr4-/- most Mbh2-positive cells left the marginal stream heading straight towards the midline (d, arrow). (e,f) Within PES, many PCN migrated at a distance from the pial surface in Cxcr4-/- (f, arrow), as opposed to marginally in the wild type (e, arrow). These results suggest that PCN derailed from marginal migration in Cxcr4-/-. (B) The schematic shows a wild-type E16.5 hindbrain with precerebellar nuclei depicted in green and the approximate axial levels of the illustrated sections indicated. (a,b) Prominent and symmetrical PN at the level of pontine flexure seen in the wild type (a, arrow) appeared much smaller and asymmetrical in Cxcr4-/- (b, arrow). (c,d) Posterior to PN, where no Mbh2-positive cells were found medially in the wild type (c), an ectopic pontine-like cluster emerged in Cxcr4-/- (d, arrow). Loose trails of Mbh2-positive cells appeared to link between the LRL and the cluster (d, arrowheads). (e-h) By contrast, the ECN (f, arrow) and LRN (h, arrow) in Cxcr4-/- were comparable in size and position to those of the wild type (e and g, arrows). Scale bar: 400 µm.

 

Figure 5
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  		Fig. 5. Multiple posterior pontine clusters with bilaterally asymmetrical distribution are present in Cxcr4-/- mice. Mbh2 in situ hybridisation was performed on whole-mount E16.5 wild-type, Cxcr4+/- and Cxcr4-/- hindbrains. (A,B) A wild-type (A) and a heterozygotic (B) hindbrain showed bilaterally symmetrical PN anteriorly and LRN posteriorly (asterisks). (C,D) Two Cxcr4-/- hindbrains presented here had reduced PN at their presumptive positions (notched arrowheads), accompanied by an emergence of multiple ectopic clusters. One large ectopic cluster across the midline was located deeply (arrowheads). The other ectopic clusters adjacent to the midline, as well as their migratory streams, were located superficially (arrows) and displayed left-right asymmetry. (E) The nature of these ectopic clusters was confirmed by Mbh2 signals on sections. Seven sections approximately 200 µm apart spanning posteriorly from the deep ectopic cluster (arrowheads) to the more anterior superficial clusters (arrows) are shown. (F) Schematic summarizing the results from sections and whole mounts to show the two abnormal behaviours in pontine neuron migration in Cxcr4-/-: (a) pontine neurons departed from the marginal stream and headed straight to the ventral midline to form the deep ectopic cluster; (b) pontine neurons migrated marginally but departed from the anterior path prematurely to form the superficial clusters. Scale bars: 800 µm in D; 400 µm in E.

 

Figure 6
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  		Fig. 6. CXCR4 functions cell-autonomously in the migrating PCN. Egfp or Cxcr4 (with co-expressed Egfp) was introduced into the LRL of wild-type (A,B,E,F) or Cxcr4-/- (C,D,G,H) mouse embryos by in utero electroporation at E12.5. (A-D) Transverse sections of E14.5 samples after EGFP and laminin (labels the meninges) double immunohistochemistry. Insets show views of whole sections. Egfp electroporation labelled a marginal PES directly abutting the laminin-positive meninges in the wild type (A). Expression of Cxcr4 in the wild type did not affect the appearance of the PES (B). In Cxcr4-/-, PES appeared broadened, with many cells migrating at a distance from the pial surface (C). This defect was rescued by expressing Cxcr4 in Cxcr4-/- LRL (D). (E-H) Whole-mount E16.5 hindbrains after electroporation at E12.5. Asterisks indicate the gV rootlets. In the wild type, expressing either Egfp (E) or Cxcr4 (F) labelled similar profiles: a largely ipsilateral PN and the stereotypic anterior path of the AES (arrows in E and F). Egfp electroporation in Cxcr4-/- labelled the posterior type I and type II clusters (G, arrowhead and open arrowhead, respectively). Note that the characteristic anterior migratory path was missing. Cxcr4 expression in Cxcr4-/- LRL restored PN in their normal position as well as the anterior migratory path (H, arrow). (I-J''') Transverse sections at indicated axial levels in G and H, respectively. Higher-magnification of the boxed areas in I and J are shown in I'-I''' and J'-J'''. (I) In Cxcr4-/- expressing only EGFP, many GFP-labelled cells were located in the type I ectopic cluster, many of which were PAX6-positive (inset). But in Cxcr4-/- expressing CXCR4, few labelled cells were found in the type I ectopic cluster. Scale bars: 400 µm in A-D,I-I''',J-J'''; 800 µm in E-H.

 

Figure 7
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  		Fig. 7. Corticospinal tract axons extend ectopic collateral branches towards the ectopic pontine clusters. (A) Schematic depicting motor corticospinal tract (CST) in a parasagittal plane of an early postnatal brain (see O'Leary and Terashima, 1988Go). The box outlines the approximate area shown in B,C,E,F. (B,C) The DiI-labelled CST in a P5 Cxcr4fl/fl hindbrain shows stereotypic collateral branching initiated from the CST segment overlying PN. PN were labelled by PAX6 immunoreactivity (C). No notable site-specific collateral branching occurred from the trunk of CST posterior to PN. (D) Higher-magnification of the PN region. (E-H) In a Wnt1-Cre;Cxcr4fl/fl hindbrain, ectopic collateral branching occurred at multiple locations along the trunk of CST in the hindbrain, each one of which was correlated with a PAX6-positive ectopic pontine cluster. Two such sites (arrows in F) are shown at higher magnification in G and H. pd, pyramidal decussation. Scale bars: 200 µm.

 

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© The Company of Biologists Ltd 2009