First published online 18 October 2006
doi: 10.1242/dev.02648
Development 133, 4549-4559 (2006)
Published by The Company of Biologists 2006
The Caenorhabditis elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated commissural motor axon guidance
Mark Lucanic1,
Maureen Kiley2,
Neville Ashcroft3,
Noelle L'Etoile1,4 and
Hwai-Jong Cheng1,2,*
1 Center for Neuroscience, and Cell and Developmental Biology Graduate Group,
University of California, Davis, CA 95616, USA.
2 Section of Neurobiology, Physiology and Behavior, College of Biological
Sciences, and Department of Pathology and Laboratory Medicine, School of
Medicine, University of California, Davis, CA 95616, USA.
3 Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1
9RR, UK.
4 Department of Psychiatry and Behavioral Sciences, University of California,
Sacramento, CA 95817, USA.
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Fig. 1. max-2 is required for ventral cord commissural motoneuron
guidance. (A,B) A schematic of the DD and VD cell bodies
(solid dots) and commissural axons in wild-type (A) and
max-2(cy2) (B) animals. In each panel, a cross-section is on
the left and a lateral view of the entire animal is on the right. (C-H)
Confocal images of representative animals: anterior is to the left and dorsal
is up. (C,D) First larval stage (L1) animals before the formation of the VD
neurons. In wild-type animals (C), the dorsal commissures of the DD neuron all
reach and enter the dorsal cord, but the DD commissures in
max-2(cy2) animals (D) often fail to reach the dorsal cord.
(E,F) L4 animals, after the migration of the VD commissures. In wild-type
animals, all commissures reach the dorsal cord (E). In
max-2(cy2) animals, many commissures fail to reach the
dorsal cord (F). All animals in C-F are in the
oxIs12[Punc-47::GFP]X background to visualize the
DD and VD motoneurons. (G,H) max-2 animals have defects in the DA and
DB motoneurons. Shown here are examples of commissural axons in wild-type (G)
and max-2(cy2) animals (H) at early L4 stage. All animals in
G,H are in the evIs82[Punc-129::GFP] background to visualize
the DA and DB neurons. Scale bar: 10 µm.
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Fig. 2. max-2 is a single gene that includes the two predicted ORFs
F18A11.4 and Y38F1A.10. (A) A diagram of a genomic region on the
distal right arm of chromosome II (LGII) that contains the max-2
gene. Cosmid and YAC coverage in this region are diagrammed with black bars.
The boxed region of the schematic is enlarged and shows the region that
contains the two predicted ORFs F18A11.4 and Y38F1A.10 (boxed region is not to
scale). (B) A diagram of the max-2 genomic organization
(upper) and the minigene construct (lower) used for the rescue experiments in
(C). The boxed area indicates the region deleted in the
max-2(nv162) allele, and the asterisk indicates the site of
the point mutation identified in the max-2(cy2) allele. (C)
max-2 mutants are rescued by injecting the rescue construct depicted
in B. The graph shows the percentage of the DD and VD motor commissures that
fail to reach the dorsal cord (DC). Two independent transgenic lines
expressing the minigene construct
znex130-131[Pmax-2::max-2(cDNA)] are shown here;
non-transgenic siblings that lack the transgene are also shown for
znex131. (D) max-2(cy2) and
max-2(nv162) show similar severity in the defects of dorsal
guidance of commissural axons. Injecting double-stranded RNA (dsRNA) of the
max-2 gene causes less severe axon guidance defects that are
otherwise similar to the max-2 mutants. The numbers (n) of
animals used for each experiment are shown; bars represent the standard error
(C,D).
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Fig. 3. max-2 expression is dynamic and is required cell autonomously
for dorsal commissure guidance. (A-E) The expression pattern of
max-2 from znex135[Pmax-2::GFP] transgenic animals.
Promoter element (5'-3') contains the 5 kb region from 5557 to 262
on cosmid F18A11. (A-C) At the comma and later embryonic stages, GFP
expression is mainly in the anterior region of the embryo and along the
ventral cord. (D) After hatching, GFP is expressed in the pharynx and several
head and tail neurons. Expression of GFP in the ALM neurons (arrow) and the
postembryonic PVD neuron can also be seen. (E) A young adult animal, showing
GFP expression in the postembryonic AVM neuron (arrow and left inset) and the
elaborate dendritic connections of a mature PVD neuron (right inset).
(F) A max-2 cDNA expressed in the DD and VD motoneurons
cell-autonomously rescues the max-2(cy2) defects in these
neurons. For the Punc-25::max-2 rescue, the data presented are the
combined data from four independently generated transgenic lines. A
Punc-47::max-2 rescue line (znex133) and its non transgenic
siblings (znex133 sibs) are also shown here. The unc-25
promoter used for this experiment contained 2 kb of the 5' region
through the start codon. The unc-47 promoter used for this experiment
contained 1.7 kb of the region 5' to the start codon through the 50th
codon. The numbers (n) of animals used for each experiment are shown;
bars represent the standard error in F. Asterisks indicate a P-value
of less than 0.001 (Student's t-test). In all pictures, anterior is
to the left and dorsal is up. Scale bar: 10 µm.
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Fig. 4. Sequence analysis of the three C. elegans p21-activated
kinases. (A) An amino acid sequence comparison of the kinase
domains from the three C. elegans PAKs and representative PAKs from
other species. The asterisk indicates the site of the
max-2(cy2) mutation. (B) A diagram of the structural
organization of the three C. elegans PAKs. The red boxes indicate the
P21-binding domain, the blue box indicates the site of a clearly conserved
autoinhibitory domain, the green ovals indicate the kinase domain and the
vertical lines indicate the sites of putative SH3-interacting (PXXP) domains.
(C) An unrooted phylogenetic tree of the entire amino acid sequence of
all the known PAKs from C. elegans (Ce), D. melanogaster
(Dm) and H. sapiens (Hs). The group I/A and group II/B PAKs are boxed
with blue and green, respectively. This tree was generated with the ClustalW
alignment tool from the European Bioinformatics Institute
(www.ebi.ac.uk/clustalw/).
Sequences for max-2 and pak-2 were deposited in GenBank as
DQ523832 and DQ523831, respectively.
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Fig. 5. max-2;pak-1 double mutants are phenotypically similar to
unc-73 and double rac mutants. (A-D) Representative
morphology of the DD and VD neurons in unc-73, rac and PAK mutants.
Arrows point to displaced VD neurons and open arrowheads point to abnormal
commissures. (A) A wild-type animal. (B) An
unc-73(e936)I animal. (C) A
ced-10(n1993)IV;mig-2(mu28)X
animal. (D) A
max-2(cy2)II;pak-1(ok448)X
animal. (E) DD and VD commissural guidance defects in PAK mutants.
(F) P cell migration defects in PAK, rac and unc-73 mutants.
The numbers (n) of animals used for each experiment are shown; bars
represent the standard error in E,F. All animals are in the
oxIs12[Punc-47::GFP]X. Scale bar: 10 µm.
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Fig. 6. pak-1 and max-2 are differentially required for
commissural axon guidance. Genetic interaction studies on the DD and VD
commissural guidance defects in PAK mutants and other mutants in genes that
are known to play roles in the unc-6/netrin commissural guidance
pathway. (A) Axon guidance defects of rac mutants are greatly enhanced
by a loss of max-2 but not by a loss of pak-1. (B)
The defects caused by constitutively active
rac(GF)s can be suppressed by the loss of
pak-1. The asterisk denotes a P-value less than 0.001
(Student's t test). (C) The defects caused by constitutively
active rac(GF)s are enhanced by a loss of function
in the max-2 gene. The max-2 data was also independently
confirmed with dsRNA as in the ced-10(GF) result. (D)
max-2(cy2) enhances the defects of mutants from genes that
are known to play roles in the unc-6/netrin commissural guidance
pathway. The numbers (n) of animals used for each experiment are
shown; bars represent the standard error.
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Fig. 7. MAX-2 is required for the axon guidance effects induced by ectopic
expression of UNC-5 in the anterior touch cell receptor neurons.
(A-C) DIC images of adult animals after fixation and staining for the
presence of lacZ. In all images, dorsal is up and anterior is to the
left. Black arrowheads mark the dorsal cord, white arrowheads mark touch
receptor cell bodies and black arrows mark touch receptor cell axons. For
clarity not all cell bodies and axons present are marked in each image.
Animals
[evIs41[Pmec7::lacZ,Pmec7::unc-5];pag-1(ls2)]
that ectopically express UNC-5 and lacZ under the control of the
mec-7 promoter have the anterior touch receptor cell axons (ALMs and AVM)
guided to the dorsal cord (A). (B,C) In a max-2 background, the
guidance effects from ectopic expression of UNC-5 are partially suppressed.
The image in B is two superimposed images from different focal planes of the
same animal. Occasionally we observed ventral migration of the AVM axon in a
max-2 background, as shown in C. We never observed this in the
ectopic UNC-5 strain alone. (D) A graphical representation of the
quantification of ALM and AVM guidance in the two populations. The numbers
(n) of animals used for each experiment are shown; bars represent the
standard error. The asterisk denotes a P-value less than 0.001
(Student's t-test). Scale bar: 10 µm.
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