RESEARCH ARTICLE 1433
Development 136, 1433-1442 (2009) doi:10.1242/dev.028472
C. elegans mig-6 encodes papilin isoforms that affect distinct
aspects of DTC migration, and interacts genetically with
mig-17 and collagen IV
Takehiro Kawano1, Hong Zheng1, David C. Merz1, Yuji Kohara2, Katsuyuki K. Tamai3, Kiyoji Nishiwaki3 and
Joseph G. Culotti1,4,*
The gonad arms of C. elegans hermaphrodites acquire invariant shapes by guided migrations of distal tip cells (DTCs), which occur in
three phases that differ in the direction and basement membrane substrata used for movement. We found that mig-6 encodes long
(MIG-6L) and short (MIG-6S) isoforms of the extracellular matrix protein papilin, each required for distinct aspects of DTC migration.
Both MIG-6 isoforms have a predicted N-terminal papilin cassette, lagrin repeats and C-terminal Kunitz-type serine proteinase
inhibitory domains. We show that mutations affecting MIG-6L specifically and cell-autonomously decrease the rate of postembryonic DTC migration, mimicking a post-embryonic collagen IV deficit. We also show that MIG-6S has two separable functions –
one in embryogenesis and one in the second phase of DTC migration. Genetic data suggest that MIG-6S functions in the same
pathway as the MIG-17/ADAMTS metalloproteinase for guiding phase 2 DTC migrations, and MIG-17 is abnormally localized in mig6 class-s mutants. Genetic data also suggest that MIG-6S and non-fibrillar network collagen IV play antagonistic roles to ensure
normal phase 2 DTC guidance.
INTRODUCTION
Little is known about the dynamic extracellular matrix (ECM)
processes that are evoked to ensure the orchestrated cell movements
and proliferation required to produce organs of the proper shape, size
and organization. However, during the past decade, several
proteinases, including matrix metalloproteinases (MMPs) and
ADAM family metalloproteinases, have been found to be necessary
for normal morphogenesis and for cancer metastasis by remodeling
the ECM (Chang and Werb, 2001; McFarlane, 2003).
We are using genetics to understand the molecular mechanisms
that regulate a readily monitored cell migration in vivo – the
migration of the two C. elegans hermaphrodite distal tip cells
(DTCs) (Nishiwaki, 1999; Su et al., 2000). The sequential threephase migration pattern of these two cells determines the final shape
of each of the two U-shaped hermaphrodite gonad arms (Fig. 1A).
The DTCs are born post-embryonically near one another in the
ventral mid-body during the first larval stage. Their phase 1
migration comprises a longitudinal migration in opposite directions
away from the mid-body using the ventral body muscle basement
membrane as a substratum for migration. During phase 2, the DTCs
turn and migrate across the lateral epidermal basement membrane
towards the dorsal body wall muscles. During phase 3, the DTCs
reorient again and migrate centripetally on the dorsal body
muscles – back towards the mid-body – where they normally stop
(Fig. 1A).
1
Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto, M5G 1X5,
Canada. 2Genome Biology, National Institute of Genetics, Mishima, Shizuoka 4118540, Japan. 3RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan.
4
Department of Molecular Genetics, University of Toronto, Toronto, M5S 1AB,
Canada.
*Author for correspondence (e-mail: [email protected])
Accepted 20 February 2009
Among the first genes known to affect DTC migration is unc-6,
which encodes a secreted repulsive cue (UNC-6/netrin) for axon and
DTC migrations that occur along the dorsoventral (DV) axis, as well
as unc-5 and unc-40/DCC, which encode receptors that mediate the
repulsive effects of UNC-6/netrin (Chan et al., 1996; Hedgecock et
al., 1987; Leung-Hagesteijn et al., 1992). In mutants of these genes,
the DTCs frequently fail to initiate the phase 2 migration, but if they
do initiate it, this migration appears normal. This raises the question
of what molecular mechanisms are required to guide the phase 2
DTC migration after it is initiated by UNC-6, UNC-5 and UNC-40
activities?
We have found two major phenotypic classes of mig-6 mutant
alleles. Class-l mutations [aka mig-6(l)] hinder the rate of DTC
movement during all phases of its migration, whereas class-s
mutations [aka mig-6(s)] alter DTC guidance during the second
(ventral to dorsal) phase of its migration. The nature of mutational
lesions, RNAi, and rescue by endogenous and cell specific
expression all show the mig-6 class-s and class-l mutations affect the
function of the two alternatively spliced mRNAs, mig-6S and mig6L, which encode MIG-6S and MIG-6L proteins, respectively.
We cloned mig-6 and found that it is the previously reported c-ppn
gene (registered as ppn-1 in Wormbase) of C. elegans (Kramerova
et al., 2000), which is highly related to genes encoding the secreted
multi-component ECM proteins Drosophila papilin and Manduca
sexta lacunin (Kramerova et al., 2000; Nardi et al., 1999). Previous
histological studies have shown that papilin and lacunin are
constituents of basement membranes and suggest that they have
roles in the morphogenesis of epithelial tissues. Single papilin
orthologues are found in C. elegans and Drosophila genomes. In this
report, we use the name mig-6, the first published name for this gene
(Hedgecock et al., 1987) and now the official gene name in
Wormbase.
DTC migration defects and consequent gonad phenotypes of gon1 and mig-17 mutants, which both encode secreted ADAMTS
metalloproteinases (Blelloch and Kimble, 1999; Nishiwaki et al.,
DEVELOPMENT
KEY WORDS: C. elegans, Cell migration, MIG-17/ADAMTS, MIG-6/papilin, Collagen IV
1434 RESEARCH ARTICLE
Development 136 (9)
MATERIALS AND METHODS
Mutant strains
mig-6 class-l alleles: oz alleles were provided by Dr T. Schedl, Washington
University; mig-6(e1931) was provided by the Caenorhabditis Genetics
Center.
mig-6 class-s alleles: mig-6(ev701) was obtained from an EMS-induced
(Brenner, 1974) screen for dominant DTC mutants. Other mig-6(s) alleles
were obtained in F2 screens. A heterozygote of mig-6(ev788) was isolated
by sib-selection from a formaldehyde-induced deletion library (Johnsen and
Baillie, 1988). ev788 deletes nucleotides 490 in exon 4 to 1543 in exon 6
(gaatctggaaacttctacta.......tgagcaagagaagttcgaca). Class-s and null alleles
were out-crossed four times and dpy-11(e224) mig-6 doubles were made and
balanced by translocation eT1 (III, V) or nT1[qIs51] (IV, V) (Edgley et al.,
2006). dpy-11 mig-6(ev788)/nT1[qIs51] segregates non-Dpy heterozygous
worms with DTC defects and arrested embryos. All other strains were
provided by the Caenorhabditis Genetics Center.
Genetic mapping and rescue of mutant phenotypes
mig-6 was mapped between two deficiencies, sDf35 and sDf20, and 30
cosmid clones (kind gifts from the Sanger Centre) in this region were tested
for their ability to rescue the mig-6(s) and mig-6(l) mutants by injecting 10
μg/ml of each DNA with 50 μg/ml of sur-5::gfp co-transformation marker.
In rescue experiments designed to identify the mig-6 gene, class-s mutant
DTC defects were scored as clear patches in the body – typically caused by
altered DTC migration patterns, which rarely occur in wild-type animals
(Hedgecock et al., 1987). In all other rescue experiments, three classes of
DTC defects (see Results) were scored using Nomarski optics to examine
the shape of gonad arms.
To score rescue of lethality and sterility, lines heterozygous for the
transgene and for dpy-11(e224) mig-6 were established [dpy-11(e224) was
used in initial rescue experiments and later found to further reduce viability].
Homozygous dpy-11 mig-6(s) segregants were scored as rescued for lethality
(designated ‘+’) if broods of transgenic animals were greater than 100
(broods of non-transgenic siblings were always less than 100). Homozygous
dpy-11 mig-6(l) segregants are like mig-6(l) homozygotes in that they
normally have no progeny (n=500 individuals); therefore, when a strain
segregated dpy-11 mig-6(l) homozygotes that in the presence of the
transgene produced any progeny at all (typically 20% of transgenic animals
did this), the transgene was scored as rescuing (designated ‘+’).
2000), are similar or identical to mig-6(l) and mig-6(s) alleles,
respectively. Intergenic non-complementation suggests that mig-6S
and mig-17 act in the same mechanistic pathway to ensure normal
phase 2 DTC guidance: consistent with immunostaining results
that suggest MIG-6S regulates MIG-17 basement membrane
localization. Additional genetic evidence indicates that MIG-6S is
antagonistic to non-fibrillar network collagen IV in guiding phase 2
DTC migrations. These data suggest that MIG-6 regulates distinct
aspects of DTC migration by dynamically regulating the abilities of
specific proteinases to remodel different basement membranes
encountered during sequential phases of DTC movements.
Genetics
Animals doubly heterozygous for emb-9 and mig-6(ev701) were made by
crossing dpy-11 mig-6(ev701)/nT1[qIs51](VI, V) to unc-36(e251)emb9(g23cg46)/qC1 dpy-19 (e1259) glp-1(q339) III. F1 non-GFP-expressing
male progeny [unc-36(e251) emb-9(g23cg46)/++; dpy-11 mig-6(ev701)/++]
were crossed into dpy-18(e364)/eT1 III; unc-46(e177) sDf20/eT1[let500(s2165)]V (Edgley et al., 2006). F1 Unc L4 larvae {unc-36(e251) emb9(g23cg46)/eT1 III; dpy-11 mig-6 (ev701)/eT1 [let-500(e2165)]V} were
cloned and the genotype was confirmed by outcrossing to N2 males and
finding that F1s segregate Dpy and non-Dpy non-Unc animals with phase 2
DTC migration defects. Control strain unc-36(e251)/eT1 III; dpy-11 mig6(ev701)/eT1[let-500] V was made by the same protocol. dpy-5 unc-13/++ I;
dpy-11 mig-6(e1931)/++ V males were crossed into smg-1(r861) I to produce
dpy-5 unc-13/smg-1 I; dpy-11 mig-6(e1931)/++ V. smg-1 self-progeny were
homozygosed and their Dpy progeny were checked for sterility and DTC
defects. Ten out of 200 Dpy progeny were non-sterile and confirmed dpy-11
mig-6(e1931)/dpy-11 + recombinants.
Microscopy
Worms were observed and photographed using a Leica DMRA2
microscope equipped with Hamamatsu ORCA-ER digital camera.
Lengths along the core of gonadal arms from the tip of the anterior gonad
to the tip of posterior gonad were measured using OpenLab software
(Improvision).
cDNA and minigene constructs
To make a full-length cDNA of mig-6S, the 5⬘ upstream region was
amplified by PCR from a wild-type cDNA pool using an SL1 primer and a
reverse primer in exon 5, then spliced to the partial cDNA yk5c8 (from the
DEVELOPMENT
Fig. 1. mig-6 class-l mutations reduce the rate of DTC migration.
(A) Schematic of an adult C. elegans hermaphrodite: DTCs (green),
distal gonad arms with no eggs (gray), proximal arms with eggs (gray
ovals), spermathecae (red) and fertilized eggs (white ovals) are shown.
(B) Images of gonad arms and DTC (arrows) relative to the vulva
(triangle) of N2 (left panels) (in two focal planes for the adult) and classl mig-6(oz113) (right panels) at mid-L3, mid-L4 and adult stages.
Asterisks indicate posterior. Scale bars: 50 μm. Black bar applies to top
four panels. (C) Lengths of anterior and posterior gonad arms of
individual animals (from a partially synchronized population) are plotted
against developmental stage, as determined by vulva shape. Rates of
movement for mutants (grouped) and controls were obtained by the
slope of the line, as determined by the least squares method.
MIG-6/papilin and cell migration in C. elegans
RESEARCH ARTICLE 1435
Table 1. Hermaphrodites DTC migration defects in mig-6 class-s and null alleles
% Anterior DTC migration defects
Strain
mig-6 (s) homozygotes
ev700
ev701
ev721
k177
mig-6 (s) heterozygotes
ev700/+
ev701/+
ev721/+
k177/+
mig-6(s)/mig-6(l)
ev701/e1931
ev788/eT1†
% Posterior DTC migration defects
Phase 2D
Phase 2V
Phase 2M
Normal
Phase 2D
Phase 2V
Phase 2M
Normal
n*
6
5
4
9
15
19
32
8
72
69
49
61
7
7
15
22
6
5
2
3
11
6
16
3
70
77
60
77
13
12
22
17
53
122
108
61
3
0
n.d.
0
32
0
n.d.
0
0
0
n.d.
0
65
100
96
100
7
18
17
8
24
32
32
3
36
26
34
2
33
24
17
87
45
148
65
108
1
7
3
1
1
1
95
91
19
10
32
3
23
9
26
78
108
87
*Number of animals scored.
†
Putative null, ev788, homozygotes of which are lethal. eT1 is a balancer.
n.d., not determined. The total of phase 2D, 2V, and 2M defects is 4%.
GFP reporters and in situ hybridizations
A 210 bp genomic PCR fragment 5⬘ to and including the start codon was
linked to the 5⬘ adjacent 5 kb KpnI-MunI fragment of mig-6(+).This was
sub-cloned into the KpnI site of gfp expression vector pPD95.79 (from Dr
A. Fire). Protocols for a large scale in situ hybridization of C. elegans larvae
can be found at http://nematode.lab.nig.ac.jp/method/insitu_larvae.html.
Molecular biology
Standard procedures were used. Mutational lesions were determined by
direct sequencing of PCR amplified cDNA fragments.
RNAi
cDNA fragments were PCR amplified using T7-tagged gene-specific
primers and the M13-20 universal primer. Primer sequences are available on
request. dsRNA preparations (0.2-0.5 mg/ml) were injected into intestine or
pseudocoelom of wild-type adults. The progeny of the injected animals were
examined for lethality and DTC defects.
For RNAi of (1) mig-6S, (2) mig-6L or (3) both, the following fragments
were subcloned into the pPD129.36 RNAi vector (kind gift of Dr A. Fire):
(1) an AvaI fragment of yk3e8 corresponding to the 3⬘ UTR of mig-6S
(within intron 11 of mig-6L) (Fire et al., 1998), (2) a HindIII-SpeI fragment
of yk257g8 (exons 14-15) and (3) a BsaBI-HindIII fragment of yk3e8 (exon
8). For RNAi by feeding, these were transfected into HT115 (DE3) bacteria
(Timmons et al., 2001). emb-9 RNAi utilized a clone from the RNAi
bacterial library (gift from Dr J. Ahringer).
Collagen IV temperature shift experiments
Temperature-sensitive emb-9(g34ts) collagen mutant animals were cultured
at 16°C until the late L2 stage, then cultured at 25°C for 8 hours. After an
additional 20 hours of culture at 16°C, L4 larvae were examined by
Nomarski optics for gonad defects.
Expression of MIG-17
A mig-17 promoter-driven translational fusion between mig-17 and gfp
(Nishiwaki et al., 2000) and an unc-119(+) co-transformation marker (from
Dr D. Pilgrim) were introduced into unc-119(e2498) by microparticle
bombardment (Praitis et al., 2001). By cloning non-Unc dauer worms,
several mig-17::gfp integrated transgenic lines were established and passed
into appropriate strains using standard genetic techniques.
Immunohistochemistry
For whole-mount immunostaining, worms were fixed in Bouin’s fixative
(Duerr, 2006). Frozen sections of synchronized early L4 worms were prepared
according to Kubota et al. (Kubota et al., 2006). Embryos were permeabilized
with alkaline hypochlorite and fixed with 3% paraformaldehyde (Miller and
Shakes, 1995). For blocking and antibody dilution, PBS containing 0.5%
Triton X-100 and 1% bovine serum albumin (Sigma) was used.
Rabbit polyclonal antibodies were raised against MIG-6 peptide
SGQKETGNWGPWVPE(C) (amino acids 70-85) and affinity purified.
Goat polyclonal anti-SAD-1 (Hung et al., 2007) and rabbit anti-EMB9/collagen IV antibodies were kind gifts from Dr M. Zhen (Mt. Sinai
Hospital, Toronto) and Dr J. M. Kramer (Northwestern U.), respectively.
Rabbit anti-GFP polyclonal antibody (A11122; used at 1:200 dilution) and
Alexa Fluor 568 phalloidin (used at 20 U/ml) were purchased from
Invitrogen. Secondary antibodies (Molecular Probes) were goat anti-rabbit
IgG-Alexa fluor 488 (used at 1:2000 dilution), goat anti-mouse IgG-Alexa
Fluor 546 (1:1000) or donkey anti-goat IgG-Alexa Fluor 594 (1:400).
RESULTS
Specific aspects of DTC migration are
differentially affected by two distinct classes of
mig-6 mutations
In class-l mig-6 mutants (e1931, oz113, oz159 and oz90) originally
described by Hedgecock et al. (Hedgecock et al., 1987), DTC
migration is extremely slow relative to the wild type, resulting in
foreshortened gonad arms (Fig. 1B,C). The rate of DTC migration
found in the mig-6(l) mutants is about one-third of the wild-type rate
(14 versus 38 micrometers per hour) (Fig. 1C). DTCs in mig-6(l)
mutants nevertheless initiate phase 2 migrations toward the dorsal
muscles. However, the ability of the DTCs to reach the dorsal
muscle with normal timing is impeded in these mutants. The distal
region of the gonad does not grow in diameter and looks like a small
appendage to the bulbous proximal arm (Fig. 1B). mig-6(l) mutant
animals are also completely sterile. These phenotypes are fully
penetrant and recessive for all four mig-6(l) alleles.
We also identified several semi-dominant, highly penetrant classs alleles of mig-6 in which the DTCs migrate at an approximately
normal rate but have specific defects in phase 2 migrations. In mig6(s) mutants, the DTCs appear to detach from ventral muscle bands
at the onset of their phase 2 migration, but their subsequent
migration usually follows one of three patterns in addition to the
DEVELOPMENT
National Institute of Genetics, Japan). For mig-6L, the above fragment was
then combined with partial cDNAs yk5c8 and yk257g8. A mig-6S
genomic/cDNA minigene construct (pZH125) was made by splicing yk5c8
(exons 5 to 11a) and a KpnI-BsrGI genomic fragment spanning a 5 kb region
5⬘ upstream to exon 5. The mig-6L genomic/cDNA minigene (pZH117) was
made by splicing yk3e8 (exons 8 to 11), yk257g8 (exon 11 to exon 18) and
a KpnI-BsaBI genomic fragment spanning a 5 kb region 5⬘ upstream to exon
8. To produce heterologous promoter driven minigenes, 3.0 kb of the lag-2
promoter (for pZH116 and pZH118) from pJK590 (gift from Dr J. Kimble)
and 2.2 kb of the myo-3 promoter (for pZH135 and pZH145) from pPD96.52
(gift from Dr A. Fire) were substituted for the endogenous 5⬘ regulatory
regions of mig-6S and mig-6L genomic/cDNA minigenes.
Fig. 2. mig-6 class-s mutations cause phase 2 DTC migration
defects. Broken and unbroken arrows trace phase 1 and subsequent
paths of DTC migration, respectively. DTCs in class-s mig-6(ev701)
heterozygous hermaphrodites show (A) phase 2D, (B) phase 2V and (C)
phase 2M (meandering – see Results) defects. The vulva (triangles) and
the posterior of the animal (asterisks) are also indicated. Scale bars:
20 μm.
wild-type pattern (Table 1). For the phase 2D pattern, DTCs migrate
diagonally until they reach the dorsal body muscles, which they
then follow back to the mid-body region (Fig. 2A). For the phase
2V pattern, DTCs start the phase 2 ventral to dorsal migration onto
the lateral epidermis normally, but quickly return to the ventral
body muscles and complete phase 3 by migrating centripetally
along these muscles back towards the mid-body (Fig. 2B). This
causes gonad arm defects roughly reminiscent of those observed in
mutants of unc-5, unc-6 and unc-40 (Hedgecock et al., 1990).
Finally, for the phase 2M category, the DTCs, after a diagonal phase
2 migration as in phase 2D defects, migrate towards mid-body but
meander along the DV axis of the lateral epidermis as they do (Fig.
2C). We suggest that this phenotype results from loss of precise
DTC guidance along the DV axis of the lateral epidermis during the
phase 2 migration.
mig-6(s) mutations are semi-dominant and, as heterozygotes,
show the same spectrum of DTC migration patterns as
homozygotes. In class-s heterozygotes, phase 2V and 2D patterns
are most prevalent, whereas meandering gonads (phase 2M) are less
frequent (especially anterior arms) than in homozygotes (Table 1).
Thus, we consider the meandering gonad phenotype to be caused by
a greater loss of mig-6 function. This is supported by mig-6S-specifc
RNAi experiments (see below).
mig-6 encodes two predicted ECM proteins with
putative matrix binding and proteinase inhibitory
domains
We mapped mig-6 to a short region of LGV and rescued the DTC
phenotypes with two overlapping cosmids (C32A1 and C37C3) then
with gene C37C3.6 (Fig. 3A). C37C3.6 encodes at least two
mRNAs, a short (C37C3.6a) and a long (C37C3.6b) form, herein
designated mig-6S and mig-6L, respectively (Fig. 3B). The
predicted 3⬘ ends of these transcripts are available as EST cDNA
clones (Y.K., National Institute of Genetics, Mishima, Japan).
Development 136 (9)
The two predicted transcripts share the same exon structure and
open reading frame from exon 1 into exon 11, but differ in their 3⬘
extensions. mig-6S includes the whole of exon 11, which encodes a
stop codon and about 320 base pairs of 3⬘ untranslated mRNA,
whereas mig-6L includes only part of exon 11 as this exon is spliced
to exon 12 using an alternative donor site.
Two transcripts of approximately the predicted sizes are detected
by Northern blot analysis (Fig. 3C). RNAase protection experiments
show that both transcripts are abundant in embryos and less
abundant during all larval stages (see Fig. S1 in the supplementary
material).
As shown in Fig. 3D, the predicted MIG-6 proteins bear a
common signal sequence for secretion at their N terminus, but no
predicted transmembrane domain, suggesting they are secreted. The
signal peptide is followed by thrombospondin type 1 (TSP1) repeats
(also known as TSRs), the first two of which are separated by a
cysteine rich spacer region (CR1), then a series of cysteine-rich
lagrin repeats (CR2) and six predicted Kunitz-type serine proteinase
inhibitor (KU) domains. The C terminus of MIG-6L contains five
additional Kunitz domains and a single predicted immunoglobulin
(Ig) C2-type domain (Fig. 3D). Secreted basement membrane
proteins with similar domain organization as MIG-6 are a lacunin
from the moth Manduca sexta (Nardi et al., 1999), and papilins from
Drosophila (Kramerova et al., 2000) and the nematode Haemonchus
contortus (Skuce et al., 2001). Genes encoding papilin related
proteins with similar overall domain organization are also found in
human and mouse genomes. These proteins differ in the number of
repeats of a given type of domain, having, for example, only a single
KU domain in the mammalian version.
In Drosophila, alternative papilin splice forms are known that
differ only in the number of KU and IgC2 domains they encode
(Kramerova et al., 2003). Whether mammalian papilin-like genes
encode multiple mRNAs and proteins is unknown. The N-terminal
TSP1 repeats and the intervening cysteine rich CR1 domain,
together termed the ‘papilin cassette’, have highest evolutionary
conservation among related proteins (38% identity and 54%
similarity between MIG-6 and Drosophila papilin, and 36%
identity and 50% similarity between MIG-6 and human or mouse
papilin) (Fig. 3D). Comparisons among these related molecules
have been described previously (Kramerova et al., 2000; Nardi et
al., 1999).
MIG-6L and MIG-6S have separate functions in DTC
migration as determined by isoform-specific RNAi
To explore stage-specific requirements for the two mig-6 transcripts,
we compared RNAi of specific transcripts by injection (affecting
embryonic and post-embryonic expression in the subsequent
generation), and by feeding starting as L1 larvae (affecting postembryonic expression in the same generation) (Table 2). RNAi of
mig-6L by either method phenocopies the mig-6(l) mutant DTC
defects (Fig. 1B). This suggests that mig-6L alone normally encodes
class-l functions that are required post-embryonically for a wild-type
rate of DTC migration.
Although ~60% of self-fertilization progeny of mig-6(s)
homozygotes survive to adulthood, RNAi of mig-6S by injection
causes severe embryonic and early larval lethality allowing only
about 1-2% of animals to survive to adulthood. Thus, RNAi is more
potent at reducing embryonic MIG-6S activity than are any of the
mig-6(s) mutations, probably because they all cause amino acid
substitutions that primarily affect postembryonic DTC migrations
(see below). This mig-6S RNAi effect is also indistinguishable from
the effect of injecting dsRNA that targets both transcripts (Table 2),
DEVELOPMENT
1436 RESEARCH ARTICLE
MIG-6/papilin and cell migration in C. elegans
RESEARCH ARTICLE 1437
Fig. 3. Cloning and characterization of the mig-6/papilin
gene. (A) mig-6 was mapped to the region between two
deficiencies, sDf35 and sDf20, on LG V. (B) Schematic
drawing of cosmid clones (white bars with single black
region denoting the mig-6 gene) and subclones (unbroken
lines representing the KpnI and FspI fragments), with rescue
results with these DNA fragments shown on the right and
exon/intron organization of mig-6 shown below (exons and
3⬘ UTRs are black and white boxes, respectively). Rescue of
mig-6 class-s (ev701) lethality and mig-6(e1931) sterility were
determined as described in the Materials and methods. These
experiments were internally controlled by scoring transgenic
Dpy animals and their non-transgenic Dpy siblings. The
number of rescuing transgenic lines over the number of lines
tested is indicated in parentheses. DTC defects, which in
mig-6(s) and mig-6(l) alleles manifest as clear patches in the
body, were also rescued (denoted ‘+’). (C) Northern blot
analysis of 3 μg of mRNA from a mixed-stage population of
wild-type C. elegans. A probe predicted to hybridize to both
mig-6S and mig-6L detected two bands (lane 1). Longer
exposure with a mig-6L-specific probe (exon 14-17) shows a
single band at 7.7 kb (lane 2). (D) Schematic diagrams of
predicted protein structures of MIG-6S and MIG-6L with sites
of molecular lesions found in class-l (blue) and class-s (red)
alleles. The extent of the ev788 deletion is indicated. The
region corresponding to the peptide used for raising an
antibody is indicated by an underlying red bar.
suggesting that the null phenotype is lethal. To further examine this
possibility, we isolated a predicted null allele (Fig. 3D) by
formaldehyde-induced direct deletion (Materials and methods). mig6(ev788) is deleted for a genomic segment ranging from nucleotide
490 in exon 4 to 1543 in exon 6. All possible splicing of the mutant
transcript is predicted to produce an early out-of-frame protein. As
predicted from the RNAi experiments, all ev788 homozygotes are
embryonic or early larval lethals.
RNAi of mig-6S by feeding, which should affect post-embryonic
functions of MIG-6S in the same generation, phenocopies the phase
2D and 2V DTC defects predominant in class-s heterozygotes but at
a lower penetrance and with rare meandering DTC defects (Table 2).
The reduced penetrance and severity (i.e. rarity of phase 2M defects)
may occur because of incomplete disruption of the mig-6S message
by this method. In fact, predicted enhancement of RNAi efficacy by
feeding in an rrf-3 mutant background (Simmer et al., 2002)
enhances the frequency of meandering DTCs (Table 2). These
results suggest that mig-6S alone encodes class-s functions required
post-embryonically for properly executing the second phase DTC
migration and verify that meandering DTC defects reflect a greater
loss of mig-6S protein.
Because ~40% of the fertilized eggs laid by homozygous class-s
hermaphrodites die in embryogenesis and as L1 larvae (see Table S1
in the supplementary material), it was surprising to find that 132 of
526 progeny (25%) from class-s mig-6(ev701) heterozygotes were
viable mutant homozygotes that became adults, the majority of
Table 2. Transcript-specific RNA interference
Injection
Feeding
Feeding
Target
Genotype
Development
Class-l
Class-s
n
mig-6S and L
mig-6S
mig-6L
mig-6S and L
mig-6S
mig-6L
mig-6S
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
rrf-3
Embryonic/L1 arrest
Embryonic/L1 arrest
Normal
Normal
Normal
Normal
Normal
–
–
100%
100%
0%
100%
0%
–
–
–
–
0%(A), 6%(P)*
6%(A), 37%(P) †
>100
>100
152
>100
63
*Phase 2V and D defects are predominant.
†
Phase 2M defect is predominant.
DEVELOPMENT
DTC migration defects
RNAi method
1438 RESEARCH ARTICLE
Development 136 (9)
which had meandering DTC defects. This suggests that development
of these mutants is largely normal but is defective in a way that
specifically affects post-embryonic DTC migrations.
mig-6 class-s alleles are anti-morphs
The DTC migration defects in most of the class-s mutant
heterozygotes are more penetrant than in heterozygotes of the
putative null allele ev788 but exhibit a similar spectrum of
phenotypes (Table 1). If ev788 is a true null (see below), the higher
penetrance of defects in class-s heterozygotes (e.g. ev701/+) must
result from a greater than 50% (half dose) loss of mig-6(+) activity
and indicates that these mutants are anti-morphs for DTC migration
(i.e. they interfere with wild-type activity).
mig-6S and mig-6L have different tissue specific
expression profiles
In principle, it is possible that the differential expression of mig-6S
and mig-6L, as opposed to different inherent abilities of the proteins
they encode, are responsible for the differences in their function. In
situ hybridization with a mig-6L-specific probe shows that mig-6L
is restricted to the DTCs (Fig. 4A) and what are likely to be
coelomocytes (not shown, but see Fig. 4E); however, a probe that
detects both transcripts revealed additional expression in body wall
muscles (Fig. 4B). Transcriptional reporters for mig-6 revealed
expression in DTCs, body wall muscles, CAN neurons, head
mesodermal cells, GLR cells and coelomocytes (Fig. 4C-E). This
expression pattern is consistent with the in situ hybridization data;
however, it does not distinguish whether mig-6S is normally
expressed in the DTCs, as we know to be the case for mig-6L.
Developmental change in MIG-6 localization
MIG-6S localizes to embryonic muscles (Fig. 4F). In double
immunostaining experiments with anti-myosin and anti-MIG-6
antisera, MIG-6 appears to be localized near the muscle surface (not
Fig. 4. Expression of mig-6 encoded mRNAs and proteins. (A,B) In
situ hybridization using a mig-6L-specific probe showing DTC (d)
specific expression (A), and using a probe common for mig-6S and mig6L showing expression in the body wall muscles and other unidentified
cells (B). (C-E) From L2 to adult stages, a mig-6 promoter::gfp
transcriptional reporter construct is expressed in DTCs (d) (C), in body
wall muscles (m), in the CAN neuron (can) (D), in the head mesodermal
cell (hmc), in GLR cells (glr) and in coelomocytes (cc) (E). (F,G) AntiMIG-6 antibody staining of late embryos (released from their shells)
shows MIG-6 protein is expressed by body wall muscle primordium
(green) in the wild type (F) but not in mig-6(ev788) (G), whereas control
antibody, anti-SAD-1 (orange) stains nerve ring and ventral nerve cord
in both strains. (H) In early larval stages, anti-MIG-6 antibody stains
basement membranes of the gonad primordium (g), pharynx (ph) and
intestine (i). This staining persists throughout larval and adult stages.
(I,J) Anti-MIG-6 antibody stains basement membrane of the gonad and
migrating DTCs (d) of L3 (I) and L4 (J) stages. Enlargement in I shows
staining on the DTC. (K,L) The same basement membranes and DTCs
are stained by anti-MIG-6 antibody in class-s mig-6(ev700) (K) and in
mig-6(k177), which displays a phase 2M DTC defect (L) (see also Fig. S2
in the supplementary material). Vulvae are indicated by white triangles.
Asterisks indicate posterior. Dorsal is upwards in all panels. Scale bars:
20 μm.
shown). In the mig-6(ev788) putative null allele there is no
expression by muscle (Fig. 4G) even though the epitope is near the
N terminus and therefore little else is predicted to be encoded by this
allele (Fig. 3D) (and Materials and methods).
DEVELOPMENT
mig-6(l) mutations affect only MIG-6L KU and IgC2
function, whereas mig-6(s) mutations affect
ancillary domains
We determined molecular lesions for eight mig-6 alleles. Three class-l
mutations introduce a premature stop codon predicted to delete the
MIG-6L-specific KU repeats and the C-terminal IgC2 motif (Fig. 3D).
Combined with the finding that RNAi silencing of mig-6L produces
a class-l mutant phenotype and that a clone including only mig-6S
(pZH96) is not sufficient to rescue mig-6(e1931) class-l mutants (Fig.
3B), we conclude that MIG-6L is solely responsible for the DTC
migration functions that are defective in mig-6(l) mutants.
In C. elegans early stop codons, like those observed for the classl alleles, may trigger nonsense-mediated decay of mRNAs.
However, the smg-1(r861) mutation, which is known to suppress
this decay (Blelloch and Kimble, 1999), did not suppress the mig6(e1931) class-l DTC migration phenotype, as smg-1(r861); dpy-11
mig-6(e1931) was 100% sterile (n=190) and had class-l mutant DTC
defects. The tested allele (e1931) is a nonsense mutation that
eliminates the last three Kunitz domains and the IgC2 domain (Fig.
3D), suggesting that at least one of these Kunitz domains and/or
IgC2 is essential for MIG-6L function.
In contrast to class-l alleles, all class-s alleles are missense
mutations that affect a TSP-1 repeat or a CR1 domain in the papilin
cassette (Kramerova et al., 2000), or a cysteine-rich lagrin repeat
(Fig. 3). Previous studies found that the ‘papilin cassette’ and the
CR1 region within the cassette are required for tight binding to ECM
(Kuno and Matsushima, 1998).
MIG-6/papilin and cell migration in C. elegans
RESEARCH ARTICLE 1439
Table 3. Rescue of mig-6 mutant classes by heterologous promoter driven mig-6 minigenes
% Rescue of DTC migration defects*
Transgene
mig-6p::mig-6S (pZH125)
mig-6p::mig-6L (pZH117)
lag-2p::mig-6S (pZH116)
lag-2p::mig-6L (pZH118)
myo-3p::mig-6S (pZH135)
myo-3p::mig-6L (pZH145)
Rescue of lethality
class-l (e1931)
class-s/+ (ev701/+)
class-s (ev701)
class-s (ev701)
0
67
0
45
0
0
60
80
59
51
58
42
19
0
2
0
0
0
+
–
+/–
–
–
–
*The percent rescue was calculated by (Pc–Pt)/Pc ⫻ 100, where Pc and Pt means the percentages of worms that show the defects in the mutants without the transgene and
with the transgene, respectively. For each experiment, at least 100 animals were scored. DTC defects (as in Figs 1 and 2) and lethality were scored as described in the
Materials and methods.
MIG-6S functions cell non-autonomously and MIG6L functions cell autonomously in DTCs to
regulate their migration
A genomic DNA construct (pZH95), including regulatory
sequence and sequence encoding both mig-6 transcripts rescued
the lethality and DTC migration defects of the null allele to near
wild-type levels (see Table S2 in the supplementary material). A
genomic/cDNA minigene construct (pZH125) encoding MIG-6S
could not rescue class-l mutant sterility (not shown) or DTC
defects (Table 3), but could partially rescue viability and DTC
defects of mig-6(s) homozygotes (slightly) and mig-6(s)
heterozygotes (substantially) (Table 3). A genomic/cDNA
minigene encoding MIG-6L (pZH117) partially rescued the DTC
defects of the class-l homozygotes, but not homozygous class-s
DTCs or lethality (Table 3); however, substantial rescue of classs heterozygote DTC defects by mig-6L did occur (Table 3). These
data indicate that when expressed under the control of nearly
identical endogenous regulatory sequences, MIG-6S cannot
substitute for MIG-6L but MIG-6L can partially substitute for
MIG-6S in guiding phase 2 DTC migrations.
To explore the significance of mig-6 expression in DTCs and in
muscles, we engineered mig-6S or mig-6L minigenes under the
control of either of two heterologous 5⬘ regulatory regions. The
lag-2 promoter (lag-2p) drives expression primarily in the
hermaphrodite DTCs (Blelloch and Kimble, 1999; Nishiwaki et al.,
2000), whereas the myo-3 promoter (myo-3p) drives expression in
all of the body-wall muscles (Okkema et al., 1993). For each
transgene, rescuing activity was examined in class-s and class-l
mutants. We found that only pZH118 [lag-2p::mig-6L] rescues the
mig-6(l) mutant DTC migration defects to roughly the same extent
as the mig-6L minigene driven by endogenous promoter (pZH117 in
Table 3). These results indicate that MIG-6L functions cell
autonomously in the DTCs to regulate the rate of their migration and
are consistent with the in situ hybridizations, which show that mig6L expression in larvae is limited largely to DTCs (Fig. 4A). Also
as predicted, MIG-6S cannot substitute for MIG-6L when expressed
in the DTCs (pZH116 in Table 3).
Although mig-6S is normally expressed in body wall muscles,
neither the myo-3p::mig-6S nor the myo-3p::mig-6L hybrid minigenes
rescued the homozygous class-s or class-l DTC phenotypes.
Transgenic animals that carry both myo-3p::mig-6S and lag-2p::mig6S also failed to rescue the homozygotes (data not shown).
Intergenic non-complementation between mig-6
class-s and mig-17 mutations suggests they act in
the same pathway to promote normal phase 2
DTC migrations
Mutants of mig-17 display DTC defects that mimic DTC defects of
mig-6(s) homozygotes, but at lower penetrance. Although the mig17 mutant defects are recessive, animals that are doubly
heterozygous for mig-6 class-s alleles ev701 or k177, and the mig17 putative null allele, k174, show higher penetrance (Fig. 5A) and
expressivity (higher frequency of type 2M DTC migrations) of
defects than respective mig-6(s) heterozygotes. This intergenic noncomplementation suggests that MIG-6S functions in the same
pathway (but see Yook et al., 2001) as MIG-17, a known ADAMTS
metalloproteinase that is required (like MIG-6S) to guide phase 2
DTC migrations.
mig-6(s) mutations affect distribution of the MIG17 metalloproteinase
As shown previously using anti-GFP antibodies (Nishiwaki et al.,
2000), MIG-17::GFP is distributed evenly with moderate intensity
along the gonad basement membrane in wild-type animals (Fig.
5B,C). We found that in many mig-6(ev700) class-s mutant animals,
MIG-17::GFP is distributed unevenly along the surface of the gonad,
with high expression of the protein concentrated at variable places
along the gonad arms (Fig. 5D). In some but not all cross-sections
of mig-6(s) mutant animals, regions of abnormally high interspersed
with regions of abnormally low expression are observed in spaces
between gonad and body muscle bands or hypodermis (Fig. 5E). In
other sections, MIG-17::GFP appears within the gonad arm (not
shown). Western blots (see Fig. S3 in the supplementary material)
show that the amount of MIG-17 protein is not dramatically changed
in mig-6(ev700), suggesting that MIG-6S does not significantly
affect the overall level of MIG-17, but does prevent overaccumulation in variable regions of the gonad arms. Whether this is
a direct or an indirect effect of mutant MIG-6S proteins on MIG-17
is unknown.
The mig-6 class-l gonad phenotype is mimicked by
mutations in the emb-9 collagen IV gene
Previous anecdotal reports of the effects of collagen mutations on
DTC migration (Cassada et al., 1981) prompted us to examine
collagens as possible targets of MIG-6 activity. Collagen type IV is
one of the major components of specific basement membranes in C.
elegans and other animals. Two EMB-9/collagen IV alpha 1 chains
and one LET-2/collagen IV alpha 2 chain normally form a single
heterotrimeric collagen. Glycine substitution mutants in Gly-X-Y
repeats of the EMB-9 or LET-2 exhibit temperature-sensitive
embryonic lethality, caused by reduced secretion and assembly of
collagen type IV into basement membranes (Gupta et al., 1997). We
DEVELOPMENT
MIG-6 in larvae also localizes to intestine, pharynx and gonad
basement membranes (Fig. 4H-J). There is no obvious abnormal
localization (either intracellular or extracellular) of MIG-6 proteins
in mig-6(s) mutants (Fig. 4K,L); however, functionally relevant
concentration differences could have been missed by this
technique.
1440 RESEARCH ARTICLE
Development 136 (9)
Fig. 5. Effect of mig-6s mutations on MIG-17::GFP distribution. (A) Double heterozygotes of mig-6 and mig-17 display enhancement of class-s
DTC defects. Black and gray bars represent percentages of anterior and posterior DTCs, respectively, with defective migrations (total of phase 2D,
2V and 2M defects – see Fig. 2). Lines on the end of the bars indicate standard errors of the expected mean. Numbers of animals scored are
indicated towards the right. Asterisks indicate comparisons in which P<0.001 by the chi-square test. (B-E) Localization of MIG-17::GFP is visualized
by anti-GFP antibody staining of whole-mount animals (B,D) and frozen cross-sections (green in C,E). Lower panels are Nomarski images of the
same animal in the upper panel. MIG-17::GFP in a control genetic background displays uniform distribution on the gonad surface in lateral view (B)
and on the gonad, intestine and body wall muscle periphery in cross-section (arrowheads in C). MIG-17::GFP in class-s mig-6(ev700) displays patchy
staining along the phase 2 DTC path of a gonad arm (D) and outside of gonad membranes (arrows) in cross-section (E). Staining on the remaining
gonad and intestine surfaces is weaker in the mutant genetic background (arrowhead in E) than in the wild type (C) (equal exposure times were
used). Eight out of 15 animals in the mutant background showed accumulation of MIG-17::GFP away from the gonad and gut surfaces (arrow),
whereas only one of 28 animals in the wild-type background showed this pattern. Phalloidin staining of body wall muscle actin filaments is orange
in cross-sections. Positions of intestine (i) and gonad arms (g) are indicated. Scale bars: 20 μm.
mig-6 class-s mutations are partially rescued by a
reduction in functional collagen IV
Given the above data, we attempted to examine the relationship of
MIG-6S with collagen IV function. Suppression of the phase 2 DTC
defect was partial when RNAi was used to reduce EMB-9/collagen
IV in mig-6(ev701) heterozygotes, but effectively complete when
the wild-type dose of emb-9 was halved in heterozygotes for the
putative null (Gupta et al., 1997) allele emb-9(g23cg46) (Fig. 6C).
A lesser but substantial suppression was also caused by let-2
temperature-sensitive mutations (Fig. 6C). Thus, a reduction in
collagen IV protein (by RNAi or a heterozygous null) or a reduction
in collagen IV folding and secretion (caused by point mutation) can
suppress mig-6 class-s DTC defects. These results indicate that
collagen IV acts antagonistically to MIG-6S in an extracellular
mechanism that affects phase 2 DTC migration. We did not observe
any difference in EMB-9 distribution between wild type and
mig-6 or mig-17 mutants using anti-EMB-9 antibodies for
immunostaining; however, functionally relevant concentration
differences could have been missed by this technique.
DISCUSSION
MIG-6/papilin is essential for embryogenesis, DTC
migration and gonadogenesis
Papilin is a basement membrane protein that is essential for
embryonic development, as shown by RNAi knockdown
experiments in Drosophila and C. elegans (Kramerova et al., 2000).
In the present study, we identified two different classes of MIG6/papilin proteins and analyzed their functions by class-specific
mutations and RNAi. Our results indicate that both of the MIG6/papilin isoforms, MIG-6S and MIG-6L, function specifically to
regulate DTC migration and MIG-6S also affects embryogenesis.
There are several reasons to believe that the DTC and embryonic
functions of the MIG-6 proteins are separate. First, the class-specific
RNAi by feeding, which affects MIG-6 post-embryonic function in
the same generation, can produce either class-s or class-l mutant
DTC phenotypes. Second, all DTC phenotypes can be rescued to
some degree by post-embryonic expression of one or the other of the
two isoforms. Finally, the DTC function of MIG-6S proteins is
genetically separable from the observed function of MIG-6S in
embryogenesis as class-s mutant heterozygotes have normal sized
broods with one-quarter mutant homozygous progeny that manifest
DTC defects. Taken together, these results indicate that mutations
that affect either MIG-6 isoform can cause specific defects in postembryonically derived basement membrane structures required for
DTC migration without affecting basement membrane functions
required for embryogenesis.
MIG-6L/papilin may act with collagen IV to
promote a normal rate of DTC migration
We have found that the two isoforms of MIG-6/papilin are made
by different cell types in C. elegans and normally have distinctly
different functions in DTC migration. Class-l mutations, which
eliminate the three C-terminal KU and the single IgC2 C-terminal
domains (predicted to only affect MIG-6L), appear to specifically
reduce the rate of DTC migration to about one-third that of the
wild type. Other more subtle gonad defects are likely in class-l
mutants as they are sterile and their distal gonad arms have a
greatly reduced cross-sectional diameter compared with the
proximal arm.
There are several reasons to believe that mig-6(l) mutations
primarily affect the gonad basement membrane. Most importantly,
MIG-6L is expressed by DTCs as they migrate and can act cell
autonomously in larvae to promote DTC migration even though it is
predicted to be a secreted protein. It is possible that the MIG-6L
DEVELOPMENT
exposed the emb-9(g34ts) mutants to the non-permissive
temperature for 8 hours of L2 to L3 larval development at the time
when the DTCs initiate their phase 1 migration. emb-9(g34ts)
hermaphrodite animals so treated have a normal morphology, but
their gonads phenocopy the mig-6(l) mutant gonad morphology,
suggesting a possible functional/regulatory connection between
MIG-6L and EMB-9 (Fig. 6A,B).
Fig. 6. Effect of collagen IV mutations. (A,B) Temperature-sensitive
collagen IV mutant emb-9(g23) cultured at non-permissive temperature
from L2 to L4 larval stages showed slow gonad elongation (B), which is
similar to class-l defect of mig-6(e1931) (A). Scale bars: 20 μm.
(C) Reduction of collagen IV alpha1 by emb-9-specific RNAi (by feeding)
or by combining with a heterozygote for the emb-9 null allele
suppressed the class-s DTC phenotype of ev701 heterozygotes, as did a
mutation of let-2/collagen IV alpha2. Balancer eT1 (aka unc-36) and
eT1* (equals eT1 with a let-500 mutation in the balancing
translocation) significantly enhances the penetrance of DTC migration
defect in class-s heterozygote for unknown reasons. Black and gray
bars represent percentages of anterior and posterior DTCs, respectively,
that show defective migrations (total of phase 2D, 2V and 2M defects).
Numbers of animals scored are indicated towards the right. Asterisks
indicate comparisons in which P<0.001 by the Chi-square test.
specific C-terminal region may include a retention signal or a motif
(e.g. one or more KU domains and/or the IgC2 domain) that help
localize it to the gonad basement membrane.
Because emb-9/collagen IV alpha 1 have been suggested to affect
DTC migration (Cassada et al., 1981), we further characterized the
DTC migration defects caused by inactivating emb-9 during DTC
migration. The phenotypic similarity of post-embryonically induced
DTC defects in emb-9 and in mig-6(l) mutants is striking. As both
MIG-6L and EMB-9 are produced by the DTC, it is tempting to
speculate that MIG-6L promotes post-embryonic collagen IV
function, and that it could do so by downregulating the activity of
proteinases that degrade collagen IV as is known for ADAM
proteinases in other animals (Roy et al., 2004).
Because the phenotypes of gon-1 mutants, mig-6(l) mutants and
post-embryonically induced DTC defects in emb-9 mutants are all
highly penetrant, we could not test genetic interactions between
them. Furthermore, gon-1/+; mig-6(e1931)/+ double heterozygotes
are normal; therefore, additional studies will be required to elucidate
the molecular mechanisms of cell surface remodelling required for
a normal rate of DTC migration.
RESEARCH ARTICLE 1441
MIG-6S positively regulates the distribution of
MIG-17 in gonad basement membranes and
possibly affects collagen IV function
The class-s point mutations affect both long and short isoform amino
acid sequences. However, RNAi experiments indicate that it is
alteration of the short isoform that causes a defect in phase 2 DTC
migration. Unlike MIG-6L, expression of MIG-6S in the DTCs did
not significantly rescue class-s homozygote DTC defects [possibly
owing to adverse effects of anti-morphic mig-6(s) alleles], but did
significantly rescue heterozygotes. This suggests that MIG-6S, like
MIG-17 (Nishiwaki et al., 2000), may affect DTC migration cell
non-autonomously; however, the tissue of origin for the most
prominent effect of MIG-6S on DTC migration is still unknown.
Genetic interactions between mig-6(s) and mig-17/
metalloproteinase mutants suggest that they act in the same
mechanistic pathway used to guide phase 2 DTC migrations. MIG17 is an ADAMTS metalloproteinase secreted as a pro-form from
body wall muscle. As reported previously (Ihara and Nishiwaki,
2007), MIG-17::GFP localizes to the entire gonad and intestinal
surface after the initiation of the phase 2 DTC migration. We found
that in mig-6(s) mutant animals MIG-17::GFP did not consistently
show uniform distribution along gonad and intestine surfaces but
instead it often showed discontinuous overaccumulations along
these organs (see Fig. 5 legend). These results suggest that mutant
MIG-6S does not efficiently retain MIG-17 in the gonad basement
membrane. The lack of MIG-17 in this basement membrane could
then cause phase 2 DTC migration defects similar to mig-17 mutants
(Kubota et al., 2006; Nishiwaki et al., 2004). The abnormal
distribution of MIG-17 in many mig-6(s) mutant animals suggests
that MIG-6S plays a role in regulating MIG-17 basement membrane
localization; however, whether this involves a physical interaction
(direct or indirect) between these proteins or results from an
unrelated abnormality in DTC function caused by mig-6(s)
mutations is unknown.
MIG-6S and MIG-17 could modify interactions between the DTC
and the lateral epidermal basement membrane, which is normally
used as a substratum to support and guide phase 2 DTC migrations.
Consistent with a functional or regulatory relationship between
MIG-6 proteins and basement membrane proteins is the finding that
emb-9 RNAi, as well as emb-9/collagen IV alpha 1 and let2/collagen IV alpha 2 mutations, clearly suppresses mig-6(s) mutant
DTC defects. Thus, genetic reduction of mutant or wild-type forms
of collagen IV are both able to suppress mig-6(s) defects, suggesting
that losing mutant forms of collagen IV [which are abnormally
retained in the endoplasmic reticulum (Gupta et al., 1997) and might
have caused co-retention of MIG-6 mutant protein] does not
contribute to the suppression. The suppression observed is therefore
reminiscent of the ability of fbl-1 (fibulin) mutations to suppress
gon-1/adamts proteinase and mig-17 mutant DTC defects
(Hesselson et al., 2004; Kubota et al., 2004) and suggests that
secreted forms of collagen IV and MIG-6S act antagonistically,
either in parallel mechanistic pathways (like fibulin and GON-1) or
in the same pathway.
The genetic and phenotypic characterization of mig-6, mig-17
and collagen IV mutants reported here shows that MIG-6S and
MIG-17 act antagonistically with secreted collagen IV to guide
phase 2 DTC migrations and suggests that MIG-6L and collagen
IV act together to promote a normal rate of DTC migration. We
speculate that the abnormal distribution of MIG-17 in mig-6 classs mutants causes an overabundance of normally or abnormally
localized collagen IV, which hinders phase 2 DTC guidance. This
model predicts that reducing the collagen IV level or altering its
DEVELOPMENT
MIG-6/papilin and cell migration in C. elegans
supramolecular structure could also rescue the DTC migration
defects of mig-17 mutants. In fact, one of the suppressors of mig17 is a let-2 allele that has a mutation in the non-collagenous (NC)
domain of the collagen IV alpha 2 chain (Ohkura and Nishiwaki,
personal communication). NC domains are involved in forming a
supramolecular network among triple helical protomers and this
allele may have lost the ability to form this network. According
to this model, EMB-9/collagen IV could be a target for
inactivation by MIG-17/ADAMTS acting with MIG-6S. The
possible pairing of the MIG-6S isoform with the MIG-17
metalloproteinase to regulate collagen IV extracellular activity
could be a theme shared with MIG-6L and the GON-1 ADAMTS,
except the latter pair would promote collagen IV function and the
former pair would negatively regulate it or antagonize it in some
other way. We therefore speculate further that collagen IV levels
in gonad basement membrane may need to be regulated
oppositely in phase 1 and phase 2 DTC migration, and that this is
accomplished by the two isoforms of MIG-6.
We thank Drs T. Schedl, A. Coulson, J. Kimble, A. Spence, A. Fire, J. Ahringer,
D. Pilgrim, W. Hung and R. Steven for materials; the Caenorhabditis Genetics
Center for strains; Drs H. Qadota, R. Ikegami, G. Dalpe, B. Nash and N. LevyStrumpf for their help and suggestions; and Dr S. Cordes, J. Yu, L. Zhang and
L. Brown for invaluable technical support. We also thank Drs M. Zhen and J.
M. Kramer for anti-SAD-1 and anti-EMB-9 antibodies, respectively. This work
was supported by grants to J.G.C. from the Canadian Institutes of Health
Research (MOP-77722 and MT-13207) and the NIH (NS41397). J.G.C. holds a
CRC Chair in Molecular Neurogenetics (#950-202224). T.K. was partially
supported by a fellowship from the Uehara Memorial Foundation, Japan.
Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/9/1433/DC1
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