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The C. elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory

Behrooz Esmaeili1, Jennifer M. Ross2,*, Cara Neades1, David M. Miller, III2 and Julie Ahringer1,{dagger}

1 Wellcome CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
2 Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232-2175, USA
* Present address: University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA



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  		Fig. 1. VAB-7 is expressed in DB motoneurones, as well as in posterior mesodermal and epidermal precursors. Immunostaining of VAB-7 in (A) 100 cell stage embryo and (B) bean stage embryo is in mesodermal and epidermal prescursors. (C) Two-fold embryo. VAB-7 is found in five posterior epidermal cells (hyp8-hyp11; arrowhead), seven DB motoneurones in the ventral nerve cord (arrow) and two unidentified neurones in the head. One DB and the two neurones in the head are not visible in this image. (D) A threefold embryo showing VAB-7 (green) and unc-25::lacZ, a DD motoneurone marker (red). (E) An L1 larva showing expression of VAB-7 in all seven DB motoneurones. Anterior is towards the left. Scale bar: 10 µm.

 


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  		Fig. 2. vab-7 specifies DB posterior axonal polarity. (A) Morphologies of DA and DB motoneurones. DA (nine) and DB (seven) cell bodies are located in the ventral nerve cord (VNC). Representative DA (blue) and DB (red) neurones are composed of dendritic processes in the VNC, circumferential commissures, and axons extending along the dorsal nerve cord (DNC). Note dendritic and axonal projections from DA (anterior) and DB (posterior) adopt opposite trajectories. (B,C) The cell bodies of DA and DB motoneurones visualized with unc-129::GFP. (B) Wild type. (C) A vab-7(e1562) mutant; both DA and DB are present. (High background in A,B is due to unc-129::GFP expression in intestinal cells.) (D-G) Dorsal views of adult animals. Anterior is towards left. Arrows point to the DNC and arrowheads identify DB commissures. (D) DB motor axons project posteriorly in the DNC of a wild-type animal. (E) vab-7 is required for posterior DB axonal outgrowth. In vab-7(e1562), DB motor axons adopt a DA-like anterior trajectory. Mutations in unc-4 (F) and unc-37 (G) restore posterior DB axonal polarity. Scale bars: in B, 50 µm in B,C; in D, 10 µm in D-F; in G, 4 µm.

 


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  		Fig. 3. vab-7 represses unc-4 in DB motoneurones. (A) Wild-type expression of unc-4::GFP in DA motoneurones of an L1 larva. (B) unc-4::gfp is ectopically expressed in the DBs in a vab-7(e1562) background. (C) Wild-type expression of unc-4::lacZ in DAs of an L1 larva (D) unc-4::lacZ is repressed in DAs of an L1 larvae by ectopic VAB-7 expession from unc-3::vab-7 transgene (weIs10). ß-Galactosidase staining is retained in a cluster of neurones at the anterior end of the VNC in which unc-3::vab-7 is not expressed Anterior is towards the left in all panels. Scale bars: in A, 10 µm in A,B; in C, 2.5 µm in C,D.

 


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  		Fig. 4. acr-5 expression in DB neurones is induced by VAB-7 repression of unc-4. (A-D) acr-5::gfp in lateral views of L1 larvae. (A) acr-5::gfp is expressed in wild-type DB motoneurones but is absent (B) from DBs in a vab-7(e1562) mutant. (C) acr-5::gfp is ectopically expressed in DA neurones of an unc-4(e120) mutant. (D) acr-5::gfp expression is restored to the DBs by genetic removal of unc-4 in the unc-4(e120);vab-7(e1562) double mutant. (E) acr-5::gfplacZ detected by anti-ß-galactosidase antibody in an adult hermaphrodite expressing ectopic VAB-7 from weIs10. acr-5::gfplacZ, which is normally only in DBs and VBs, is induced in DAs, VAs and ASs. (F) unc-17::gfp expression of animal in E used to identify the neurones. Anterior is towards the left. Scale bar: 10 µm.

 


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  		Fig. 5. Ectopic VAB-7 is sufficient to promote posterior axonal outgrowth in cholinergic motoneurones. Dorsal views of unc-17/cha-1::gfp staining of DA, DB and AS axons entering the DNC of adult animals. (A) DA and AS motor axons project anteriorly in the wild type; DB axons are posteriorly directed. (B) An unc-3::vab-7 transgene (weIs10) drives ectopic VAB-7 expression in ventral cord motoneurones and redirects AS and DA axons to adopt a posterior trajectory. Yellow arrow points to DNC. Anterior is towards the left. Scale bar: 15 µm.

 


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  		Fig. 6. Fasiculation of both dorsal and ventral nerve cords depends on vab-7 activity. (A-E) unc-17/cha-1::gfp was used to visualize cholinergic motoneurone processes in adults. (A) Ventral view of wild type. Arrows point to normal right and left process bundles in the VNC. (B) Ventral view of vab-7(e1562) showing highly defasciculated VNC (arrows). (C) Dorsal view of wild type. Arrowhead points to the DNC; arrows point to dorsal sub-lateral nerve cords. (D) Dorsal view of defasciculated DNC in vab-7(e1562) mutant (arrowheads). (E) Ectopic expression of VAB-7 in weIs10 restores normal fasiculation to the DNC in a vab-7(e1562) mutant. (F) Expression of unc-129::gfp in vab-7(e1562), showing normal fasiculation of DA and DB neurones in the VNC. Anterior is towards the left. Scale bars: in A, 15 µm in A-C,E,F.

 


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  		Fig. 7. Specification of DA versus DB motoneurones fate. UNC-4 blocks expression of DB genes in DA motoneurones. It is not known if UNC-4 also acts to promote expression of DA-specific genes. VAB-7 specifies DB traits (posterior axonal polarity, acr-5 expression) by repressing UNC-4 and by acting in a parallel UNC-4-independent pathway. The posterior trajectory of DB motoneurones may also depend on another unknown pathway (?) that does not involve VAB-7. Nerve cord fasciculation is uniquely dependent on VAB-7 function.

 




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