Crucial roles of Robo proteins in midline crossing of cerebellofugal axons and lack of their up-regulation after midline crossing.
BACKGROUND
Robo1, Robo2 and Rig-1 (Robo3), members of the Robo protein family, are candidate receptors for the chemorepellents Slit and are known to play a crucial role in commissural axon guidance in the spinal cord. However, their roles at other axial levels remain unknown. Here we examine expression of Robo proteins by cerebellofugal (CF) commissural axons in the rostral hindbrain and investigate their roles in CF axon pathfinding by analysing Robo knockout mice.


RESULTS
We analysed the expression of Robo proteins by CF axons originating from deep cerebellar neurons in rodent embryos, focusing on developmental stages of their midline crossing and post-crossing navigation. At the stage of CF axon midline crossing, mRNAs of Robo1 and Robo2 are expressed in the nuclear transitory zone of the cerebellum, where the primordium of the deep cerebellar nuclei are located, supporting the notion that CF axons express Robo1 and Robo2. Indeed, immunohistochemical analysis of CF axons labelled by electroporation to deep cerebellar nuclei neurons indicates that Robo1 protein, and possibly also Robo2 protein, is expressed by CF axons crossing the midline. However, weak or no expression of these proteins is found on the longitudinal portion of CF axons. In Robo1/2 double knockout mice, many CF axons reach the midline but fail to exit it. We find that CF axons express Rig-1 (Robo3) before they reach the midline but not after the longitudinal turn. Consistent with this in vivo observation, axons elicited from a cerebellar explant in co-culture with a floor plate explant express Rig-1. In Rig-1 deficient mouse embryos, CF axons appear to project ipsilaterally without reaching the midline.


CONCLUSIONS
These results indicate that Robo1, Robo2 or both are required for midline exit of CF axons. In contrast, Rig-1 is required for their approach to the midline. However, post-crossing up-regulation of these proteins, which plays an important role in spinal commissural axon guidance, does not appear to be required for the longitudinal navigation of CF axons after midline crossing. Our results illustrate that although common mechanisms operate for midline crossing at different axial levels, significant variation exists in post-crossing navigation.
Tamada
Atsushi
A
National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan. tamada@brain.riken.jp
Kumada
Tatsuro
T
Zhu
Yan
Y
Matsumoto
Tomoko
T
Hatanaka
Yumiko
Y
Muguruma
Keiko
K
Chen
Zhe
Z
Tanabe
Yasuto
Y
Torigoe
Makio
M
Yamauchi
Kenta
K
Oyama
Hiroshi
H
Nishida
Kazuhiko
K
Murakami
Fujio
F
D000818:Animals; D001369:Axons / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D002531:Cerebellum / Q000196:embryology / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012249:Rhombencephalon / Q000196:embryology / Q000378:metabolism; D046509:Tissue Culture Techniques; D014162:Transfection; D000097905:Roundabout Proteins
D049452:Green Fluorescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; C516057:Robo2 protein, rat; C118348:Robo3 protein, mouse