|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 18 March 2009
doi: 10.1242/dev.028472
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto, M5G 1X5,
Canada.
2 Genome Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540,
Japan.
3 RIKEN 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: culotti{at}mshri.on.ca)
Accepted 20 February 2009
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: C. elegans, Cell migration, MIG-17/ADAMTS, MIG-6/papilin, Collagen IV
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; 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 three-phase 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).
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 mig-6L, 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 gon-1 and
mig-17 mutants, which both encode secreted ADAMTS metalloproteinases
(Blelloch and Kimble, 1999;
Nishiwaki et al., 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.
|
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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 `+').
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)emb-9(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[let-500(s2165)]V (Edgley et
al., 2006). F1 Unc L4 larvae {unc-36(e251) emb-9(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 mig-6(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
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.
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-EMB-9/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 |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), 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 class-s
alleles of mig-6 in which the DTCs migrate at an approximately normal
rate but have specific defects in phase 2 migrations. In mig-6(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 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 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).
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 post-embryonic 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.
|
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),
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).
mig-6(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.
|
) 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 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.
|
In C. elegans early stop codons, like those observed for the
class-l 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 mig-6(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 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 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).
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.
MIG-6S functions cell non-autonomously and MIG-6L 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 class-s 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.
|
; 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 mig-6L 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::mig-6S 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
mig-17 mutant defects are recessive, animals that are doubly
heterozygous for mig-6 class-s alleles ev701 or
k177, and the mig-17 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
non-complementation 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 MIG-17 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
over-accumulation 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 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).
|
) 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 |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). In the present study, we identified two different classes
of MIG-6/papilin proteins and analyzed their functions by class-specific
mutations and RNAi. Our results indicate that both of the MIG-6/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 post-embryonically 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 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.
|
), 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.
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. MIG-17 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 let-2/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 class-s 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 supramolecular structure could also rescue the DTC migration defects of mig-17 mutants. In fact, one of the suppressors of mig-17 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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/dev.028472/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Blelloch, R. and Kimble, J. (1999). Control of
organ shape by a secreted metalloprotease in the nematode Caenorhabditis
elegans. Nature
399,586
-590.[CrossRef][Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Cassada, R., Isnenghi, E., Culotti, M. and von Ehrenstein,
G. (1981). Genetic analysis of temperature-sensitive
embryogenesis mutants in Caenorhabditis elegans. Dev.
Biol. 84,193
-205.[CrossRef][Medline]
Chan, S. S., Zheng, H., Su, M. W., Wilk, R., Killeen, M. T.,
Hedgecock, E. M. and Culotti, J. G. (1996). UNC-40, a C.
elegans homolog of DCC (Deleted in Colorectal Cancer), is required in
motile cells responding to UNC-6 netrin cues. Cell
87,187
-195.[CrossRef][Medline]
Chang, C. and Werb, Z. (2001). The many faces
of metalloproteases: cell growth, invasion, angiogenesis and metastasis.
Trends Cell Biol. 11,S37
-S43.[Medline]
Duerr, J. S. (2006). Immunohistochemistry. In
WormBook (ed. The C. elegans Research
Community). WormBook 1-61.
Edgley, M. L., Baillie, D. L., Riddle, D. L. and Rose, A. M.
(2006). Genetic balancers. In WormBook
(ed. The C. elegans Research Community). WormBook1
-32.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S.
E. and Mello, C. C. (1998). Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans.
Nature 391,806
-811.[CrossRef][Medline]
Gupta, M. C., Graham, P. L. and Kramer, J. M.
(1997). Characterization of alpha1(IV) collagen mutations in
Caenorhabditis elegans and the effects of alpha1 and alpha2(IV)
mutations on type IV collagen distribution. J. Cell
Biol. 137,1185
-1196.
Hedgecock, E. M., Culotti, J. G., Hall, D. H. and Stern, B.
D. (1987). Genetics of cell and axon migrations in
Caenorhabditis elegans. Development
100,365
-382.[Abstract]
Hedgecock, E. M., Culotti, J. G. and Hall, D. H.
(1990). The unc-5, unc-6, and unc-40 genes
guide circumferential migrations of pioneer axons and mesodermal cells on the
epidermis in C. elegans. Neuron
4, 61-85.[CrossRef][Medline]
Hesselson, D., Newman, C., Kim, K. W. and Kimble, J.
(2004). GON-1 and fibulin have antagonistic roles in control of
organ shape. Curr. Biol.
14,2005
-2010.[CrossRef][Medline]
Hung, W., Hwang, C., Po, M. D. and Zhen, M.
(2007). Neuronal polarity is regulated by a direct interaction
between a scaffolding protein, Neurabin, and a presynaptic SAD-1 kinase in
Caenorhabditis elegans. Development
134,237
-249.
Ihara, S. and Nishiwaki, K. (2007).
Prodomain-dependent tissue targeting of an ADAMTS protease controls cell
migration in Caenorhabditis elegans. EMBO J.
26,2607
-2620.[CrossRef][Medline]
Johnsen, R. C. and Baillie, D. L. (1988).
Formaldehyde mutagenesis of the eT1 balanced region in Caenorhabditis
elegans: dose-response curve and the analysis of mutational events.
Mutat. Res. 201,137
-147.[Medline]
Kramerova, I. A., Kawaguchi, N., Fessler, L. I., Nelson, R. E.,
Chen, Y., Kramerov, A. A., Kusche-Gullberg, M., Kramer, J. M., Ackley, B. D.,
Sieron, A. L. et al. (2000). Papilin in development: a
pericellular protein with a homology to the ADAMTS metalloproteinases.
Development 127,5475
-5485.[Abstract]
Kramerova, I. A., Kramerov, A. A. and Fessler, J. H.
(2003). Alternative splicing of papilin and the diversity of
Drosophila extracellular matrix during embryonic morphogenesis.
Dev. Dyn. 226,634
-642.[CrossRef][Medline]
Kubota, Y., Kuroki, R. and Nishiwaki, K.
(2004). A fibulin-1 homolog interacts with an ADAM protease that
controls cell migration in C. elegans. Curr.
Biol. 14,2011
-2018.[CrossRef][Medline]
Kubota, Y., Sano, M., Goda, S., Suzuki, N. and Nishiwaki, K.
(2006). The conserved oligomeric Golgi complex acts in organ
morphogenesis via glycosylation of an ADAM protease in C. elegans.
Development 133,263
-273.
Kuno, K. and Matsushima, K. (1998). ADAMTS-1
protein anchors at the extracellular matrix through the thrombospondin type I
motifs and its spacing region. J. Biol. Chem.
273,13912
-13917.
Leung-Hagesteijn, C., Spence, A. M., Stern, B. D., Zhou, Y., Su,
M. W., Hedgecock, E. M. and Culotti, J. G. (1992). UNC-5, a
transmembrane protein with immunoglobulin and thrombospondin type 1 domains,
guides cell and pioneer axon migrations in C. elegans.
Cell 71,289
-299.[CrossRef][Medline]
McFarlane, S. (2003). Metalloproteases: carving
out a role in axon guidance. Neuron
37,559
-562.[CrossRef][Medline]
Miller, D. and Shakes, D. (1995).
Immunofluorescence microscopy. In Caenorhabditis elegans: Modern
Biological Analysis of an Organism, vol.48
(ed. H. Epstein and D. Shakes), p.365
. New York: Academic Press.[CrossRef]
Nardi, J. B., Martos, R., Walden, K. K., Lampe, D. J. and
Robertson, H. M. (1999). Expression of lacunin, a large
multidomain extracellular matrix protein, accompanies morphogenesis of
epithelial monolayers in Manduca sexta. Insect Biochem.
Mol. Biol. 29,883
-897.[CrossRef][Medline]
Nishiwaki, K. (1999). Mutations affecting
symmetrical migration of distal tip cells in Caenorhabditis elegans.
Genetics 152,985
-997.
Nishiwaki, K., Hisamoto, N. and Matsumoto, K.
(2000). A metalloprotease disintegrin that controls cell
migration in Caenorhabditis elegans. Science
288,2205
-2208.
Nishiwaki, K., Kubota, Y., Chigira, Y., Roy, S. K., Suzuki, M.,
Schvarzstein, M., Jigami, Y., Hisamoto, N. and Matsumoto, K.
(2004). An NDPase links ADAM protease glycosylation with organ
morphogenesis in C. elegans. Nat. Cell Biol.
6, 31-37.[CrossRef][Medline]
Okkema, P. G., Harrison, S. W., Plunger, V., Aryana, A. and
Fire, A. (1993). Sequence requirements for myosin gene
expression and regulation in Caenorhabditis elegans.
Genetics 135,385
-404.[Abstract]
Praitis, V., Casey, E., Collar, D. and Austin, J.
(2001). Creation of low-copy integrated transgenic lines in
Caenorhabditis elegans. Genetics
157,1217
-1226.
Roy, R., Wewer, U. M., Zurakowski, D., Pories, S. E. and Moses,
M. A. (2004). ADAM 12 cleaves extracellular matrix proteins
and correlates with cancer status and stage. J. Biol.
Chem. 279,51323
-51330.
Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet,
M. L., Fire, A., Ahringer, J. and Plasterk, R. H. (2002).
Loss of the putative RNA-directed RNA polymerase RRF-3 makes C.
elegans hypersensitive to RNAi. Curr. Biol.
12,1317
-1319.[CrossRef][Medline]
Skuce, P. J., Newlands, G. F., Stewart, E. M., Pettit, D.,
Smith, S. K., Smith, W. D. and Knox, D. P. (2001). Cloning
and characterisation of thrombospondin, a novel multidomain glycoprotein found
in association with a host protective gut extract from Haemonchus
contortus. Mol. Biochem. Parasitol.
117,241
-244.[CrossRef][Medline]
Su, M., Merz, D. C., Killeen, M. T., Zhou, Y., Zheng, H.,
Kramer, J. M., Hedgecock, E. M. and Culotti, J. G. (2000).
Regulation of the UNC-5 netrin receptor initiates the first reorientation of
migrating distal tip cells in Caenorhabditis elegans.
Development 127,585
-594.[Abstract]
Timmons, L., Court, D. L. and Fire, A. (2001).
Ingestion of bacterially expressed dsRNAs can produce specific and potent
genetic interference in Caenorhabditis elegans.
Gene 263,103
-112.[CrossRef][Medline]
Yook, K. J., Proulx, S. R. and Jorgensen, E. M.
(2001). Rules of nonallelic noncomplementation at the synapse in
Caenorhabditis elegans. Genetics
158,209
-220.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  S. S. Apte A Disintegrin-like and Metalloprotease (Reprolysin-type) with Thrombospondin Type 1 Motif (ADAMTS) Superfamily: Functions and Mechanisms J. Biol. Chem., November 13, 2009; 284(46): 31493 - 31497. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
