doi: 10.1242/10.1242/dev.00518
UNC-71, a disintegrin and metalloprotease (ADAM) protein, regulates motor axon guidance and sex myoblast migration in C. elegans
Xun Huang1,
Peng Huang2,
Matthew K. Robinson 2,*,
Michael J. Stern2 and
Yishi Jin 1,3,
1 Department of Molecular, Cellular and Developmental Biology, Sinsheimer
Laboratories, University of California, Santa Cruz, CA 95064, USA
2 Department of Genetics, Yale University School of Medicine, New Haven, CT
06520-8005, USA
3 Howard Hughes Medical Institute, University of California, Santa Cruz, CA
95064, USA
* Present address: Fox Chase Cancer Center, Medical Oncology, 7701 Burholme
Avenue, Philadelphia, PA 19111, USA
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Fig. 1. Cloning of unc-71. (A) unc-71 corresponds to
Y37D8A.13/adm-1. Rescue of unc-71 pleiotropies: movement
(Unc); egg-laying (Egl); and axon morphology of the D neurons (Max). +,
rescue;–no rescue; *, the unc-71 minigene NH#1092
was used for these experiments. (B) UNC-71 domain structures. UNC-71 contains
an inactive metalloprotease domain. A consensus active zinc-binding catalytic
site sequence is shown for comparison. The underlined residues are required
for zinc binding. The conserved residues in the disintegrin loop are
highlighted in red, and lesions of two unc-71 mutations are shown
above. The positions of other unc-71 mutations are indicated as
color-coded bars.
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Fig. 2. unc-71 expression pattern. (A-C,E) Expression patterns of
Punc-71GFP. GFP is seen in the excretory cell (arrow in A), the
excretory gland (arrow in B), in some neurons in the head (arrowhead in B),
one of which is AVG interneuron (arrow in C), and in hypodermis (arrow in E)
flanking the vulva in adult animals. (D) Punc-71GFP is not expressed
in the ventral cord motoneurons; the GFP present is from the processes of the
interneurons in the head (arrow points to the ventral cord). (F,G) UNC-71::GFP
may be localized to the cell membrane. Punc-115UNC-71::GFP expression
is seen in the nerve ring (arrow in F) and along the nerve cords(arrow in G).
(H,I)
Pe15*2UNC-71::GFP
expression in hypodermis is seen near the nerve cords (arrow in H,I).
Arrowhead in H indicates the vulva hypodermis. *Likely GFP
aggregates. Scale bars: 20 µm in A,F-I; 50 µm in B-E.
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Fig. 3. The type D motoneuron axon guidance defects in unc-71 mutants.
(A-J) Axonal morphology of the type D neurons visualized by
juIs76[Punc-25GFP] marker. (A,B) Low magnification of wild-type (A)
and unc-71 (B) animals. unc-71 animals have axonal gaps in
the dorsal cord (arrows). (C,D) L1 larvae of wild type (C) and unc-71
(D) animals. Arrows show DD2 and DD5 commissures, which
did not grow out in unc-71 animals. (E,F) Ventral views of wild-type
(E) and unc-71 (F) animals. In wild-type animals, DD and VD ventral
processes form a tight bundle (arrow in E); in unc-71 animals,
defasciculation is seen as two separated bundles (arrow in F). (G,H) Dorsal
views of wild-type (G) and unc-71 (H) animals. (G) All commissures
(arrowhead) reach the dorsal cord (arrow) from the same side in wild-type
animals. (H) In unc-71 animals, commissures (arrowheads) reach the
dorsal cord (arrow) from both sides (left side, narrow arrowhead; right side,
arrowhead) and some do not reach the dorsal cord (asterisk). (I,J) Ventral
view of wild-type and unc-71 animals expressing
nuIs25[Pglr-1GFP] in the interneurons. In wild-type animals, the
ventral cord processes of these interneurons form a tight bundle (arrow in I);
in unc-71 they are defasciculated (arrow in J). (K,L) Schematics of
VD13 neuron morphology viewed using juIs76 marker (K) and
AWC neuron morphology viewed by kyIs136[Pstr-2GFP] marker (L) in
wild-type and unc-71 animals. Numbers represent the percentage of
particular phenotypes. Scale bars: 20 µm in C,D; 50 µm in A,B,E-J.
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Fig. 5. Axon guidance phenotypes in the double mutants. (A-F) Lateral view of
juIs76[Punc-25GFP] in different genetic backgrounds. In wild type (A)
and ina-1(gm144) (C) animals commissures (arrow) all reach the dorsal
cord. In unc-71, fewer commissures (arrow) grow out and some stop
short (arrowhead in B). (D) In unc-71(ju156); ina-1(gm144) animals,
even fewer commissures (arrow) grow out. (E) In unc-6(ev400) animals,
few commissures (arrow) grow out but all are defective. (F) In
unc-71(ju156); unc-6(ev400) animals, no commissures grow out of the
ventral cord. Arrow indicates the lateral positions where commissures would be
found in unc-6 mutants. (G-J) Ventral view of juIs76 in
different genetic backgrounds. (G) DD and VD ventral processes are
fasciculated forming a tight bundle (arrow) in wild-type animals. (H,I) DD and
VD ventral processes are defasciculated in unc-71(ju156) and
ina-1(gm144) animals as two separated processes (arrow). (J) The
ventral cord (arrows) is largely disorganized in unc-71(ju156);
ina-1(gm144) animals. (K-N) DA and DB motoneurons in different genetic
backgrounds viewed by juIs14[Pacr-2GFP]. In wild type, all
commissures reach the dorsal cord (arrowhead in K); in unc-71(ju156)
occasionally one commissure does not reach the dorsal cord (arrowhead) but
instead runs laterally (arrow in L); in unc-6(ev400) none of the
commissures reaches the dorsal cord (arrowhead in M) and all run laterally
(arrow in M); in unc-71(ju156); unc-6(ev400) double mutants none of
the commissures reaches the dorsal cord (arrowhead) and all run laterally
(arrow in N), which is similar to unc-6 alone (M). Scale bars: in A,
50 µm for A-J; in K, 50 µm for K-N.
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Fig. 4. Genetic interactions of unc-71 with ADAMs, integrins,
unc-6 and other signaling molecules. (A) unc-71 acts
independently of the other three C. elegans ADAMs. Percentages of
dorsal guidance defects were quantified as
Table 4. (B) Axonal
fasciculation defects in unc-71 were strongly enhanced by mutations
in ina-1 and pat-3 but not inb-1. The percentages
of fasciculation defects were quantified by counting the number of ventral
cord processes from VD3-12 that were separated from the DD ventral
cord processes, divided by 10. (C) Axonal defects in double mutants of
unc-71 with mutants in the unc-6/netrin signaling pathway.
Percentages of dorsal guidance defects were quantified as
Table 4. Bars represent
standard deviation. *P<0.001, Student's
t-test, compared with unc-71 single mutant control.
**P>0.05, Student's t-test, compared with
unc-71 single mutant control.
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© The Company of Biologists Ltd 2003
