Midline crossing is not required for subsequent pathfinding decisions in commissural neurons.
BACKGROUND
Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. While midline crossing is achieved through coordinated signaling of attractive and repulsive cues, this has never been demonstrated at the single cell level. Further, though growth cone responsiveness to guidance cues changes after crossing the midline, it is unclear whether midline crossing itself is required for subsequent guidance decisions in vivo. In the zebrafish, spinal commissures are initially formed by a pioneer neuron called CoPA (Commissural Primary Ascending). Unlike in other vertebrate models, CoPA navigates the midline alone, allowing for single-cell analysis of axon guidance mechanisms.


RESULTS
We provide evidence that CoPA expresses the known axon guidance receptors dcc, robo3 and robo2. Using loss of function mutants and gene knockdown, we show that the functions of these genes are evolutionarily conserved in teleosts and that they are used consecutively by CoPA neurons. We also reveal novel roles for robo2 and robo3 in maintaining commissure structure. When midline crossing is prevented in robo3 mutants and dcc gene knockdown, ipsilaterally projecting neurons respond to postcrossing guidance cues. Furthermore, DCC inhibits Robo2 function before midline crossing to allow a midline approach and crossing.


CONCLUSIONS
Our results demonstrate that midline crossing is not required for subsequent guidance decisions by pioneer axons and that this is due, in part, to DCC inhibition of Robo2 function prior to midline crossing.
Biology Department, Skidmore College, Saratoga Springs, NY 12866, USA. jbonner@skidmore.edu
Jennifer
J
Bonner
Michael
M
Letko
Oliver Brant
OB
Nikolaus
Lisa
L
Krug
Alexandria
A
Cooper
Benjamin
B
Chadwick
Phoebe
P
Conklin
Amy
A
Lim
Chi-Bin
CB
Chien
Richard I
RI
Dorsky
D000818:Animals; D030801:Animals, Genetically Modified; D001369:Axons / Q000187:drug effects / Q000502:physiology*; D000075389:DCC Receptor; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D055495:Neurogenesis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis; D013116:Spinal Cord / Q000166:cytology* / Q000196:embryology; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism; D000097905:Roundabout Proteins
D000075389:DCC Receptor; D049452:Green Fluorescent Proteins; D009025:Morpholines; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D029961:Zebrafish Proteins; C506552:dcc protein, zebrafish; C498889:robo2 protein, zebrafish; D000097905:Roundabout Proteins; C499626:robo3 protein, zebrafish; C037574:morpholine