Program of the 2001 International Worm Meeting - Sternberg Lab ...

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142. A Constitutively Active
UNC-40/DCC Reveals Downstream
Signaling Components
Zemer Gitai 1 , Erik Lundquist 2 , Marc
Tessier-Lavigne 1 , Cori Bargmann 1
1 HHMI, UCSF, 513 Parnassus Ave, San
Francisco, CA 94143-0452
2 Univeristy of Kansas, Lawrence, KS 66045
UNC-6/Netrin attracts and repels
circumferentially migrating axons in C. elegans.
UNC-6/Netrin is expressed ventrally and
mediates attraction via the receptor
UNC-40/DCC. Ventral migration of numerous
axons including the AVM touch cell axon is
disrupted in unc-6 and unc-40 mutants. In order
to identify signaling events subsequent to
UNC-40 activation, we have attempted to
generate a constitutively active UNC-40
transgene by replacing the extracellular and
transmembrane domains with a myristoylation
signal. When this transgene, designated
MYR::UNC-40, is expressed by the mec-7
promoter, a variety of phenotypes were induced
in the touch cells. These include exuberant axon
outgrowth and branching, defective axon
guidance, and lamellipodial extensions from cell
bodies. These effects are independent of
endogenous UNC-6 and UNC-40. Similar
phenotypes were also observed when
MYR::UNC-40 was expressed in other neurons
that utilize UNC-40 in axon guidance. Thus we
believe MYR::UNC-40 represents a
ligand-independent, gain-of-function receptor.
We have identified several suppressors of
MYR::UNC-40 that may represent components
of the UNC-40 signaling pathway. The
suppressors include loss-of-function alleles of
unc-115 (an actin-binding protein with LIM
domains), unc-34/Enabled, and ced-10/Rac.
These three genes have all been implicated in
signaling to the actin cytoskeleton.
Loss-of-function mutations in all three genes
can affect axon guidance, including ventral
migration of the AVM touch cell axon. In
unc-40; unc-115 double mutants, the AVM
axon guidance defect is not enhanced compared
to single mutants, further suggesting that
UNC-115 functions in the endogenous UNC-40
pathway. The fact that UNC-34/Enabled may be
downstream of UNC-40/DCC is particularly
interesting given that Enabled may also be
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downstream of the SAX-3/Robo receptor, which
mediates axon repulsion. This result suggests a
possible bifunctional role for UNC-34/Enabled
in signaling cascades that both attract and repel
axons.
Biochemical and cell biological experiments are
currently underway to determine whether
UNC-40 physically associates with UNC-115,
UNC-34, or CED-10. Structure/function
experiments are also being conducted to
determine which residues of the UNC-40
cytoplasmic domain are responsible for
signaling to its effectors. We are also searching
for new components of the UNC-40 signaling
pathway by screening for additional suppressors
of MYR::UNC-40. One additional mutant of
interest is ky612, which was identified in a
screen for mutants with HSN axon guidance
defects. ky612 is a recessive mutant that
phenocopies UNC-40 gain-of-function in HSN.
Molecular identification of ky612 may provide
insights into inhibitory components of the
UNC-40 signaling pathway.