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First published online 30 May 2006
doi: 10.1242/dev.02427
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1 Howard Hughes Medical Institute, Waksman Institute and Department of Molecular
Biology and Biochemistry, Rutgers The State University of New Jersey,
Piscataway, NJ 08854, USA.
2 Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ
08854, USA.
3 Department of Biology, Texas A&M University, College Station, TX
77843-3258, USA.
¶ Author for correspondence (e-mail: irvine{at}waksman.rutgers.edu)
Accepted 4 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dachs, Myosin, Leg, Wing, fat, Growth, Protocadherin, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Klein,
2001; Lawrence and Struhl,
1996), including Notch, Wnt, Egf, Hedgehog and Bmp pathways.
Imaginal discs have proven to be an outstanding model for identifying and
characterizing components of these pathways. Recent work has led to the
suggestion that disc growth and patterning are also dependent upon a new
signaling pathway, the Fat pathway.
fat encodes a large protocadherin
(Mahoney et al., 1991).
Genetic studies in Drosophila have identified three crucial
requirements for fat during imaginal disc development. First,
fat is required to limit wing growth, as mutation of fat
causes disc overgrowth (Bryant et al.,
1988). Second, fat acts cell-autonomously to influence an
intercellular signaling process between distal and proximal wing cells that
establishes a ring of Wingless (Wg) expression in the proximal wing
(Cho and Irvine, 2004). Third,
fat plays a crucial role in the establishment of tissue polarity
(Casal et al., 2002;
Rawls et al., 2002;
Strutt and Strutt, 2002;
Yang et al., 2002).
The molecular basis for the influence of fat on these processes
has not been determined, but genetic studies have identified genes that
function together with fat (Adler
et al., 1998; Casal et al.,
2002; Cho and Irvine,
2004; Rawls et al.,
2002; Strutt and Strutt,
2002; Yang et al.,
2002). Two, four-jointed (fj) and
dachsous (ds), act genetically upstream of fat in
the regulation of tissue polarity (Yang et
al., 2002), and act non-autonomously to influence the expression
of Wg in the proximal wing (Cho and
Irvine, 2004). ds encodes a protocadherin
(Clark et al., 1995) that
appears to participate in heterophilic interactions with Fat
(Ma et al., 2003;
Matakatsu and Blair, 2004;
Strutt and Strutt, 2002).
These observations suggest that Ds and Fat might bind each other to mediate
intercellular signaling, with Ds acting as a ligand and Fat as a receptor.
fj encodes a protein found in both secreted
(Villano and Katz, 1995) and
Golgi-resident (Strutt et al.,
2004) forms, and might influence Fat-Dachsous interactions.
A third gene that has been genetically linked to fat is
dachs, which was first described by Bridges and Morgan
(Bridges and Morgan, 1919). The
original dachs mutant allele, d1, results in
reduction of wing and leg growth similar to that observed in alleles of
fj and ds (Waddington,
1940; Waddington,
1943). dachs also interacts genetically with fj
to influence leg segmentation and growth
(Buckles et al., 2001),
suggesting that its function is related to these Fat pathway components. More
recently, dachs was shown to be epistatic to fat both for
the regulation of wg expression in the proximal wing, and for
imaginal disc growth (Cho and Irvine,
2004). These observations suggest that dachs might act as
a downstream component of Fat signaling. However, the nature and extent of the
requirement for dachs has not been well understood because only one
allele was characterized, and the molecular nature of Dachs had not been
described.
Here, we present the first phenotypic characterization of strong mutations in dachs. These mutations define requirements for dachs in different fat-dependent processes. We also show that dachs encodes an unconventional myosin, and characterize its subcellular localization. Finally, we show that the localization or stability of Dachs at the membrane is influenced by Fat signaling, thus providing molecular evidence that Dachs is a downstream component of a Fat signaling pathway.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a stock
was constructed to contain two flanking markers for the area of interest,
black (b) and clot (cl), as well as a
source of P transposase (Robertson et al.,
1988). w; cl1 d1 b1/Cyo;
Sb
2-3/TM6B flies were crossed to a set of P element
insertion stocks (Fig. 5A).
Recombination in males is confined to these P insertion sites. For each
P-element, 22-28 crosses were set up and 1500-3000 progeny were screened.
dachs phenotypes were analyzed using the d1,
d210, dGC2 and dGC13
alleles, in homozygous, transheterozygous, and hemizygous combinations, using
Df(2L)N22-5 and Df(2L)ED623. We also examined
dGC13 ck13, fat8, fatG-rv,
dGC13 fat8, d1 fat8,
dGC13 fatG-rv, fjd1, dsUA071,
and ds36D mutant animals. UAS-d insertions were isolated on the second (UAS-d[2D], UAS-d[2A]) and third (UAS-d[2B]) chromosomes. UAS-d:V5 insertions were isolated on the second (UAS-d:V5[50-5], UAS-d:V5[18-4]) and third (UAS-d:V5[9-F], UAS-d:V5[18-2], UAS-d:V5[8-3]) chromosomes. Rescue and overexpression experiments were conducted by crossing to tub-Gal4, arm-Gal4, C765Gal4, ptc-Gal4 or da-Gal4. Ectopic expression clones were created by Flp-out using the UAS-d and UAS-d:V5 lines, as well as the following stocks: y w hs-Flp[122]; act>y+>Gal-4 UAS-GFP (AyGal4); UAS-fj; GS-ds; UAS-fat; UAS-fat; UAS-d:V5[9F]; UAS-fj; UAS-d:V5[9F]; and GS-ds; UAS-d:V5[9F].
Simple mutant clones were created using FLP-FRT mediated recombination, with the following stocks: y w; dGC13 FRT40A/CyO-GFP; y w; d1 FRT40A/CyO-GFP; y w; fat8 FRT40A/CyO-GFP; y w; fatG-rv FRT40A/L14; y w; dGC13 fat8 FRT40A/CyO-GFP; dGC13 ck13 FRT40A/CyO-GFP; y w; d1 fat8 FRT40A/CyO-GFP; y w hs-flp[122]; Ubi-GFP FRT40A; w hs-flp[122] f; M(2)25A P[f+30B] FRT40A; y w hs-flp[122]; y+ FRT40A/CyO; y w hs-flp[122]; M(2)25A Ubi-GFP FRT40A/CyO.
To examine Dachs:V5 expression in fat mutant clones we used y w; ftG-rv FRT40A; UAS-d:V5[9F]/L14 x y w hs-FLP; Ubi-GFP FRT40A; C765Gal4 and related stocks, substituting UAS-d:V5[18-2], tub-Gal4 or arm-Gal4.
Molecular biology
For RFLP analysis of dachs mutant DNA, probes were prepared by XL
PCR (Perkin-Elmer) based on sequence in GadFly across the 70 kb interval
indicated in Fig. 5. Candidate
genes were then amplified by PCR from wild-type and mutant DNA.
The identification of dachs was communicated to GenBank by F.K.
prior to publication (Accession Number AF405293). FlyBase
(Drysdale and Crosby, 2005)
currently lists three transcripts for dachs, d-RA, d-RB and d-RC.
d-RA (Accession Number NM_175991) differs slightly from the original
submission of AF405293, but is consistent with BDGP EST sequences and our own
more recent sequence analysis of cDNAs and RT-PCR products, and encodes the
product depicted in Fig. 5; the
entry for AF405293 has thus been corrected to match d-RA. d-RB and d-RC
correspond to Myo29D transcripts reported by Tzolovsky et al.
(Tzolovsky et al., 2002), but
we were unable to detect them by RT-PCR from larval total RNA. dachs
cDNAs were recovered from a third instar cDNA library enriched for imaginal
discs (Brown and Kafatos,
1988). We also prepared cDNA by RT-PCR from total RNA using either
Superscript II reverse transcription (GibcoBRL) or OneStep RT-PCR kit
(Qiagen). For construction of UAS-dachs, cDNA fragments were prepared
by RT-PCR and then cloned into pGEM-T (Promega) to make a cDNA corresponding
to nucleotides 52-3915 of d-RA, which was then verified by DNA sequencing.
Plasmid pUAST-d was constructed by cloning nucleotides 71-3915 of
dachs into pUAST using EcoRI and NotI; the start
codon begins at nucleotide 71. A CCACC Kozak sequence was introduced in front
of the ATG. V5-tagged Dachs was created by replacing the stop codon and
3' UTR of dachs with V5 and His6 sequences from
pMTBip/V5-His (Invitrogen).
Histology, Immunohistochemistry and western blotting
Imaginal discs were fixed and stained as described previously
(Cho and Irvine, 2004), using
as primary antibodies rabbit anti-V5 (1:1000, Novus), mouse anti-V5 (1:200,
Invitrogen), rat anti-Fat intracellular domain (1:100. H. McNeill), mouse
anti-Wg (1:800, 4D4, DSHB), rat anti-Ser (1:1000)
(Papayannopoulos et al.,
1998), goat anti-ß-gal (1:1000, Biogenesis), rat anti-Elav
(1:40, 7E8A10, DSHB), mouse anti-Dac (1:40, DSHB), rat anti-DE-Cad (1:40,
DSHB), mouse anti-Dll (1:400) (Duncan et
al., 1998) and mouse anti-Prospero (1:50, DSHB).
In situ hybridization was carried out as described previously
(Rauskolb and Irvine, 1999),
using a labeled antisense RNA probe corresponding to nucleotides 1710-3915 of
the predicted dachs transcript.
Western blotting was performed on larval tissue (wing, leg and haltere discs, attached to fragments of cuticle) boiled in SDS-PAGE loading buffer. The Dachs:V5 was detected with mouse anti-V5 (Invitrogen).
Examination of adult tissues
Mutant clones in adult flies were generated and analyzed as described by
Hao et al. (Hao et al., 2003),
except that flies were heat-shocked 0-72 hours AEL. Pupal legs were dissected
in PBS, fixed for 45 minutes in PLP (McClean and Nakane, 1974), and mounted in
fluorescent mounting medium. Polarity was scored in male abdominal segments
2-4. Mutant clones were marked with yellow, which is only scorable in
bristles; thus, we did not distinguish between autonomous and non-autonomous
effects in these experiments. We scored only clones that included more than
one bristle; the severity of the polarity phenotype did not correlate with
clone size among clones analyzed (2-11 bristles).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Waddington,
1940; Waddington,
1943). The reduction in wing length is most obvious in the middle
of the wing, as evidenced by the reduced distance between the cross veins
(Fig. 1B, compare with
1A). As each cell in the wing
normally secretes a single hair, the number and spacing between hairs serves
as a measure of cell number and size. Counting hairs between the crossveins in
d1 mutants revealed that the reduction in inter-crossvein
distance is associated with a corresponding decrease in cell number (not
shown). To better define requirements for dachs, additional alleles were characterized. The strength of these alleles was then evaluated by examining the phenotypes of homozygous, hemizygous and transheterozygous animals. This analysis identified two alleles, d210 and dGC13, that are stronger than d1, and another allele, dGC2, that is weak like d1 (Figs 1, 2; data not shown). Animals homozygous for d210 die by early larval stages, but this likely results from other mutations on the d210 chromosome, as dGC13 homozygotes survive until the end of pupal development, yet d210 behaves similarly to dGC13 in combinations with dGC13 and d1 (Figs 1, 2; data not shown), and molecular analysis (see below) predicts that dGC13 encodes a more severely truncated protein than d210. dGC13 animals make adult tissue, but usually fail to eclose from the pupal case (less than 1% of animals eclose), forming pharate adults that can be dissected out and examined. The dGC13 phenotype is similar in homozygotes and in transheterozygous combinations with dachs deficiencies (Figs 1, 2; data not shown), suggesting that it could be a null mutation.
dachs adult phenotypes
The legs of dGC13 mutants are more severely affected
than d1 mutants (Fig.
2). All of the intermediate and distal segments of the leg (femur
through tarsus) are noticeably shortened, and some tarsal segments are fused,
typically resulting in the formation of only three tarsal segments instead of
the normal five. Although each of the genes in the Fat pathway influence leg
development, their phenotypes are different, presumably reflecting their
distinct roles within the pathway (see Fig. S1 in the supplementary material).
In addition to a general reduction in length, in some cases no external leg
tissue is evident in dGC13 mutants, or the leg appears to
form a single mass of tissue. Pupal legs, however, are consistently shortened
but never absent or severely deformed. Examination of pupae suggested that the
legs absent phenotype actually derives from defects in the morphological
changes that occur during leg disc eversion
(Fig. 2L; data not shown).
Indeed, direct examination of dGC13 pharate adults
revealed that when external legs are absent, masses of leg tissue could be
found differentiating within the body cavity
(Fig. 2M). The disc eversion
phenotype was also observed in dGC2, but not in
d1. To further define the dachs leg phenotype, we
also characterized dGC13 mutants clones. These caused
reduced growth and fusions of tarsal segments similar to those observed in
mutant animals, but even large mutant clones were not associated with a
failure of disc eversion (not shown).
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Transcriptional targets of dachs and fat
In wing imaginal discs, clones of cells mutant for fat are
associated with upregulation of Wg expression in the proximal wing
(Cho and Irvine, 2004).
Conversely, clones of cells mutant for d1 exhibit a severe
loss of Wg expression in the proximal wing, but this loss of Wg is transient
(Cho and Irvine, 2004). To
investigate whether the transience of Wg loss in d1
results from remaining dachs activity, we examined
dGC13 mutant clones, but their affect on Wg expression was
similar - Wg is severely reduced at early third instar, but essentially normal
by late third instar (not shown).
Clones of cells mutant for fat have been reported to be associated
with upregulation of fj expression in eye imaginal discs
(Yang et al., 2002), and we
have observed that fat mutant clones are also associated with
induction of fj expression in leg (not shown) and wing
(Fig. 3D) imaginal discs, as
assayed by a fj-lacZ reporter. Importantly then, clones of cells
mutant for dGC13 are associated with decreased fj
expression in eyes and wings (Fig.
3A,C). However, the influence of dachs on fj
expression was detected during early and mid-third instar, but not late third
instar.
Loss of leg joints and reduced leg growth is characteristic of mutations in
genes in the Notch signaling pathway. To evaluate the potential relationship
between dachs phenotypes and Notch signaling, we investigated the
expression of the Notch ligand Serrate (Ser) in discs containing
dGC13 mutant clones. During third instar, loss of Ser
could not be detected (Fig.
3F). Some loss of Ser expression could be detected in pupal legs
in regions where dachs mutation is associated with leg segment
fusions (Fig. 3I), but it is
not clear in this context whether loss of Ser is a cause or a consequence of
the loss of leg tissue. Nonetheless, Fj
(Buckles et al., 2001) and Ds
(data not shown) can induce the expression of Ser and Delta in neighboring
cells when ectopically expressed in the leg. These observations suggest that
the Fat pathway does have a role in regulating Notch ligand expression during
leg development. To further evaluate this, we examined Ser expression in
clones of cells mutant for fat. fat mutant clones could be associated
with ectopic Ser expression, although this was preferentially observed in the
proximal leg (Fig. 3G). Within
the proximal leg (defined for these experiments as proximal to the
dachshund expression domain), 36/47 fat clones exhibited an
obvious upregulation of Ser expression. Thus, fat has a normal role
in repressing Ser expression during leg development. An influence of
fat on Notch ligand expression probably accounts for the occasional
outgrowths of leg tissue observed in fat mutants and in association
with fat mutant clones (Bryant et
al., 1988; Mahoney et al.,
1991) (Fig. 2I), as
these outgrowths appear similar to those observed upon ectopic Notch
activation (Rauskolb and Irvine,
1999).
Prior work has identified a set of broadly expressed genes, the leg gap
genes, that are responsible for the initiation of Notch ligand expression in
the leg (Rauskolb, 2001). To
position the action of Fat within the leg segmentation hierarchy, we examined
the expression of two key leg gap genes, dachshund and
Distal-less. Both of these genes were expressed normally within
fat mutant clones (not shown), suggesting that the action of Fat in
leg segmentation is downstream of these broadly expressed genes.
Epistasis of dachs to fat
dachs suppresses the consequences of fat mutation on wing
disc growth (Cho and Irvine,
2004). Moreover, even though dachs mutation does not lead
to permanent loss of Wg expression in the proximal wing, it nonetheless
completely suppresses the ability of fat mutation to induce ectopic
Wg expression (Cho and Irvine,
2004) (Fig. 4). To
further evaluate the hypothesis that dachs is a general downstream
component of a Fat pathway, we evaluated the ability of dachs to
suppress additional fat mutant phenotypes, and to suppress
fat phenotypes in other tissues.
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Another striking feature of fat mutant clones is their roundness
(Fig. 4A). Normally, clones of
cells adopt irregular, elongated shapes. However, genetic manipulations that
influence cell affinity cause clones to be rounder and smoother. Strikingly,
mutation of dachs also suppresses the roundness of fat
clones (Fig. 4C, see Table S1 in the supplementary material), suggesting that
dachs is required for an altered affinity of fat mutant
clones. A difference in cell affinity probably also accounts for the
appearance of internal vesicles of cuticular tissue within the legs of animals
containing fat mutant clones (Fig.
2I: 40/50 legs with fat clones had internal vesicles).
The appearance of these vesicles is suppressed by mutation of dachs
(Fig. 2J: 0/62 legs with
fat dachs clones had internal vesicles). Examination of clone size
also confirmed that the suppression of fat mutant overgrowth by
dachs is observed not only at the level of the whole disc
(Cho and Irvine, 2004), but
also within individual clones (Fig. 4C, see Table S1 in the supplementary
material). Altogether, these results indicate that dachs is epistatic
to fat for multiple phenotypes, including gene expression, growth and
cell affinity, and in multiple tissues.
Effects of dachs on tissue polarity
Another crucial function of fat is to regulate tissue polarity
(Casal et al., 2002;
Rawls et al., 2002;
Strutt and Strutt, 2002;
Yang et al., 2002). To
investigate the possibility that dachs influences polarity, we
examined dGC13 mutant animals and
dGC13 mutant clones. Planar polarity is evident throughout
most of the adult cuticle in the polarized orientation of hairs and bristles.
In fat mutants, the normal orientation is disturbed, and swirling
patterns of hairs and bristles occur in many tissues (e.g.
Fig. 5B). It has been reported
that d1 can have mild polarity phenotypes in the leg
(Held et al., 1986). We
examined dGC13 for polarity phenotypes in wings, legs,
abdomens and eyes. Only mild polarity phenotypes were observed. As in
wild-type animals, hairs in the abdomen point posteriorly
(Fig. 5D), most leg bristles
and wing hairs point distally (Fig.
2, Fig. 5I), and
most ommatidia are correctly oriented (Fig.
5K).
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),
dachs mutants have no obvious polarity phenotype
(Fig. 5D) and fat
dachs double mutants exhibit an intermediate phenotype
(Fig. 5E), in which there is
some irregularity in the orientation of abdominal hairs, but they are less
disturbed than in fat mutants. To provide a quantitative comparison,
we scored disturbances in polarity associated with clones of mutant cells in
abdominal segments. All (39/39) fat clones had polarity phenotypes
(Fig. 5C), whereas only 3%
(2/60) of dachs clones (Fig.
5G) and 54% (37/68) of fat dachs clones
(Fig. 5F) exhibited polarity
phenotypes. There was also a difference in the severity of the polarity
phenotypes, as all of the fat clones were associated with some hairs
at an angle greater than 90° relative to the normal orientation, whereas
26% (18/68) of fat dachs clones and 0% of dachs clones were
associated with hairs at an angle of 90° or greater. In the remaining
fat dachs clones (19/68), the degree of change in polarity was less
severe (Fig. 5F). Thus,
mutation of dachs partially suppresses polarity phenotypes associated
with mutation of fat in the abdomen.
Polarity in the eye is manifest in the specification of chiral forms of
ommatidia, which differ in their orientation and placement of photoreceptor
cells. Staining with a neural marker (Elav) and an R7 marker (Prospero)
illustrates the regular polarized orientation of ommatidia in the eye
(Fig. 5J). In
dGC13, some disorganization in ommatidial orientation is
observed, indicative of a mild polarity phenotype, but overall polarity is
again largely normal (Fig. 5K).
To examine the relationship of dachs to fat, we again
focused on animals containing clones of mutant cells. fat mutant
clones are consistently associated with strong polarity phenotypes
(Rawls et al., 2002;
Strutt and Strutt, 2002;
Yang et al., 2002), which
include completely reversed ommatidia (Fig.
5M, 32/33 fat clones included ommatidia rotated more than
90° away from normal), whereas dachs mutant clones exhibited mild
polarity phenotypes, with only slightly mis-rotated ommatidia
(Fig. 5L, 1/32 dachs
clones included ommatidia rotated more than 90° away from normal). fat
dachs double mutant clones appear similar to fat
(Fig. 5N, 28/33 fat
dachs clones included ommatidia rotated more than 90° away from
normal).
dachs encodes an unconventional myosin
Deficiency mapping and male recombination localized dachs to
29D1-2, between the most distal P element used for male recombination mapping
(7704) and the distal end of a small non-complementing deficiency
[Df(2L)N22-5; Fig.
6A]. Correlation with the genomic map
(FlyBase, 1999) gave an
interval of 
70 kb, containing 14 potential transcripts. We used RFLP
analysis throughout this interval to identify a single predicted gene,
CG10595, which contained an internal deletion in
dGC2, and a large insertion in d1 (not
shown). Subsequent analysis showed that this same candidate gene contained
mutations in two other d alleles (see below), providing strong
evidence that this locus encodes dachs.
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). The
central region of Dachs includes all of the defining features of a myosin head
domain, including a well-conserved ATP binding domain, actin-binding domain
and active thiol region (Fig.
6). However, in the middle of the head domain, just before the
actin-binding domain, is an unusual insertion of 44 amino acids that is not
found in any other Myosin. By blast analysis, the closest mammalian myosins
are members of the myosin V, X, and VII families. However, Dachs is not
clearly orthologous to any of these mammalian myosins, as the
Drosophila genome encodes other genes that appear to be more closely
related to mammalian myosins V, X, and VII
(Tzolovsky et al., 2002).
In addition to the head domain, unconventional myosins sometimes have an
N-terminal extension preceding the head domain, which is characteristic for
each class (Korn, 2000). Dachs
has an N-terminal extension of 235 amino acids that does not have significant
similarity to other proteins. As in other myosins, the head domain is followed
by neck and tail domains. In Dachs, the neck domain contains a single
calmodulin-type IQ-like motif (Fig.
6), the binding site for regulatory light chains. Dachs also
encodes a tail domain of 187 amino acids, which does not show extensive
similarity to any other proteins. Surprisingly, sequence analysis identifies a
potential transmembrane domain C-terminal to the IQ motif, with a predicted
type II orientation. This computer prediction has to be treated cautiously, as
there is no precedent for transmembrane myosins.
We sequenced the four dachs mutant alleles
(Fig. 6).
dGC2 contains a deletion that removes part of the
N-terminal extension while retaining the reading frame. d1
contains an insertion of the blood retrotransposon
(Bingham and Chapman, 1986) in
the tail domain. blood insertions have been reported to affect
transcript stability in other genes
(Bingham and Chapman, 1986).
dGC13 and d210 are both predicted to
encode proteins truncated within the head domain. dGC13
contains an 11 bp deletion near the N terminus of the head domain, causing a
frame shift followed closely by a stop codon, while d210
contains a point mutation that results in a stop codon in the active thiol
region (Fig. 6). If the myosin
head domain is required for dachs function, then both of these
mutations would be expected to encode non-functional alleles.
To gain further insight into potential functions of dachs, dachs expression was examined by in situ hybridization to embryos and imaginal discs. dachs mRNA is expressed broadly throughout embryonic and imaginal development, although at certain stages some local upregulation of dachs expression was observed (Fig. 7C). Early embryonic expression of dachs was near background until stage 9 (Fig. 7), which suggests that there is no significant maternal contribution of dachs mRNA.
Based on the identification of Dachs as a myosin, we considered the
possibility that dachs might be partially redundant with other
Drosophila myosins. In particular, crinkled encodes a
Drosophila Myosin VII family member
(Kiehart et al., 2004), and
exhibits a multiple wing hair phenotype, which is also sometimes observed in
tissue polarity mutants. To evaluate the possibility of redundancy between
dachs and crinkled, we examined double mutant clones. These
displayed both dachs and crinkled phenotypes, but the
phenotypes were not obviously more severe than in the respective single
mutants. Thus, loss of Wg expression in the proximal wing was still transient
(not shown). Moreover, clones of cells mutant for strong tissue polarity
mutants, like fat, can influence the polarity of surrounding
wild-type cells (Casal et al.,
2002), but the polarity of hairs surrounding dachs
crinkled mutant clones in the abdomen appeared normal
(Fig. 5H).
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High level expression of dachs, achieved using different
UAS-dachs insertions, or by raising animals at higher temperature,
can result in disruptions of normal wing and leg development. In the wing,
Dachs overexpression increased wing size and resulted in vein abnormalities
(Fig. 1J), while in the leg
occasional outgrowths of leg tissue and formation of internal vesicles were
observed (Fig. 2H). These
phenotypes resemble those observed in weak, viable alleles of fat
(Bryant et al., 1988),
consistent with the possibility that downregulation of dachs is a
crucial fat function. Examination of Dachs-expressing clones in adult
abdomens did not reveal any effects on polarity (not shown).
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Influence of Ds, Fj and Fat on Dachs localization
Genetic studies suggest that Dachs acts as a downstream component of a Fat
signaling pathway. However, it was conceivable that Dachs could instead act in
parallel to Fat. To further explore the functional connection between Dachs
and Fat signaling, we examined Dachs and Fat protein localization under
conditions where Fat pathway activity was altered, either by mutation of
fat, or by overexpression of Fj, Ds or Fat.
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;
Ma et al., 2003;
Matakatsu and Blair, 2004)
(Fig. 8D,E). However, when Ds
is overexpressed, Fat appears depleted from other membranes of neighboring
cells (Fig. 8E,F), suggesting
that the elevated Fat staining at the edge of Ds-expressing clones represents
Fat protein in neighboring cells recruited to cellular interfaces where it can
bind higher levels of Ds (Fig.
9D) (Cho and Irvine,
2004; Matakatsu and Blair,
2004). Conversely, when Four-jointed is overexpressed, Fat
staining can appear partially depleted from other membranes of cells just
inside the clone border (Fig.
8C,D). This suggests that the elevated Fat staining at the edge of
Fj-expressing clones represents Fat protein recruited from cells inside the
clone (Fig. 9C).
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Fj is expressed most strongly by distal wing cells, while Ds is expressed
most strongly by proximal wing cells
(Brodsky and Steller, 1996;
Clark et al., 1995;
Villano and Katz, 1995). Thus,
if Fj and Ds normally influence Dachs protein localization, there might be
some asymmetry in Dachs localization even in wild-type animals. Within the
middle of clones expressing Dachs:V5, polarization of staining can not be
assessed because the resolution of confocal microscopy can not distinguish
which of two neighboring cells is contributing membrane staining. However, at
the edges of Dachs:V5-expressing clones, all staining observed comes from a
single cell. Importantly then, examination of clones of Dachs:V5-expressing
cells in the wing pouch revealed that Dachs:V5 staining is generally stronger
along the distal sides of the clones, and weaker along the proximal sides
(Fig. 8A; 64/79 clones were
scored as having elevated Dachs on the distal side, 15/79 were scored as
having no significant difference, and 0/79 were scored as having stronger
Dachs:V5 on the proximal side). This observation implies that asymmetric
localization of Dachs in response to regulation of Fat occurs during normal
development, and at endogenous expression levels of Fj and Ds.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Here,
we have provided two types of evidence that confirm this suggestion. First,
dachs is epistatic to fat for multiple phenotypes in
multiple tissues, including gene expression, growth and cell affinity. Indeed,
with the notable exception of the influence of fat on tissue
polarity, all known fat mutant phenotypes are completely suppressed
by mutation of dachs. Second, we found that expression of regulators
of Fat, Fj and Ds, or of Fat itself, influence the localization or stability
of Dachs protein at the membrane, thus providing a molecular link from Fat to
Dachs.
A myosin in the Fat pathway
The predicted structure of Dachs is unique within the myosin superfamily,
and places Dachs in a new class of unconventional myosins. It has most
similarity to myosins V, VII, and X. This is intriguing, as a mammalian
protocadherin, Cdh23, has been functionally linked to myosin VIIa during the
development of sensory hair cells in the inner ear
(Boeda et al., 2002).
Within the myosin head region, the major conserved domains are all present,
suggesting that Dachs functions as a motor protein. However, it is also
possible that Dachs serves a structural or scaffolding role. For example, in
the Hedgehog pathway, a kinesin-related protein, Costal2, is thought to
function largely as a scaffold that brings together crucial kinases with their
substrates (reviewed by Ogden et al.,
2004).
The dGC2 mutation deletes part of the N terminal extension. As dGC2 mutants have relatively weak phenotypes, the N terminal extension might not be not essential for Dachs activity. Conversely, the severe phenotypes of alleles that truncate Dachs in the myosin head region imply that the myosin domain is essential. dGC13 in particular is predicted to eliminate almost all of the myosin head domain, and genetically it appears to act as a null allele.
Normal requirements for dachs in wing and leg growth
Characterization of new dachs alleles has provided an opportunity
to define more clearly the requirements for dachs. dachs is required
for normal wing and leg growth, although some appendage growth is
dachs independent. Importantly, the identification of dachs
as a downstream component of a Fat signaling pathway that influences growth
implies that the reduced growth in dachs mutants is reflective of a
normal role for a Fat pathway in growth promotion. That is, while fat
is a gene whose normal role can be thought of as to restrain growth, as mutant
tissue overgrows, we suggest that inhibition of Fat occurs during normal
development, and that this inhibition contributes to normal appendage growth,
as defined by the reduced growth of dachs mutants. Normal inhibition
of Fat activity would presumably be effected by the two known regulators of
Fat, Fj and Ds.
Whether available dachs mutations fully define the normal involvement of the Fat pathway in growth promotion is not yet clear. We cannot exclude the possibility that dachs is partially redundant with other proteins (e.g. other myosins), although this seems unlikely given the complete suppression of all non-polarity phenotypes of fat by dachs. It is also possible that dachs is required only for peak Fat signaling. This explanation is suggested by the observation that expression of the Fat target genes wg, Ser and fj is only partially or transiently lost in dachs mutants, yet the elevated or ectopic expression of these genes in fat mutants is completely eliminated by mutation of dachs.
Dachs and tissue polarity
The relatively mild tissue polarity phenotypes of dachs mutants,
and the inability of dachs mutation to completely suppress the
influence of fat on tissue polarity, contrast with the absolute
dependence of fat gene expression, growth and affinity phenotypes on
dachs. These observations suggest that there are two distinct Fat
pathways. One, crucially dependent on Dachs, influences gene expression,
growth and cell affinity, and another, partially independent of Dachs,
influences tissue polarity. Studies of the atrophin protein Grunge also
support the suggestion that there is a distinct Fat polarity pathway, as
Grunge interacts with Fat and influences tissue polarity
(Fanto et al., 2003), but does
not exhibit other phenotypes observed in fat mutants
(Cho and Irvine, 2004;
Fanto et al., 2003). Thus,
Dachs might act redundantly with another protein in a polarity pathway, but
non-redundantly in a pathway that influences gene expression. It should also
be noted that effects of dachs on gene expression might contribute to
the polarity phenotypes of dachs mutants. For example, fj is
regulated by dachs (Fig.
4), and fj has polarity phenotypes
(Casal et al., 2002;
Zeidler et al., 1999;
Zeidler et al., 2000).
The asymmetric localization of Dachs observed in wild-type wings, and the
influence of Fj and Ds on Dachs localization, have important implications for
tissue polarity. First, the asymmetric localization of Dachs is itself a form
of polarity, and its detection in third instar imaginal discs emphasizes that
these cells are polarized well before core polarity proteins such as Frizzled
and Dishevelled become asymmetrically localization in pupal wings (reviewed by
Eaton, 2003). A similar
conclusion can be drawn from the recent observation that fat and
ds influence the orientation of cell divisions in third instar discs
(Baena-Lopez et al., 2005).
Second, our observations identify an ability to induce asymmetric protein
localization as a mechanism through which the Fat pathway might influence
tissue polarity. Dachs is one target, but the Fat polarity pathway might
similarly involve asymmetric localization of other myosins, or of other types
of proteins, to affect tissue polarity.
How does Fat signaling affect Dachs?
Mutation of fat is associated with elevated Dachs staining at the
membrane, and overexpression of Fat decreases Dachs staining at the membrane.
Although this negative effect of Fat on Dachs is subject to the caveat that we
can only detect tagged overexpressed Dachs:V5, this tagged protein rescues
dachs mutants, and the effects of Fat on Dachs staining are
consistent with their opposite phenotypes and the epistasis of dachs
to fat. Manipulations of the expression of Fat regulators provide
further evidence that Fat regulates Dachs levels at the membrane, and
altogether our observations implicate Dachs as a crucial intracellular
component of a Fat signaling pathway (Fig.
9A,B).
The concomitant elevation of Fat staining and loss of Dachs staining observed at the perimeter of Fj-expressing clones is consistent with the conclusion that Fat can antagonize the localization or stability of Dachs at the membrane. Because the elevation of Fat is limited to the periphery of Fj-expressing clones, we hypothesize that it results from an influence of Fj on Fat-Ds interactions, rather than the expression of Fj per se. Tissue polarity studies have implied that Fj and Ds have opposite affects on Fat. Although it has not yet been determined whether Fj can directly modify Fat or Ds, the simplest explanation for the elevated Fat staining at the edge of Fj-expressing cells would be to propose that Fj modifies Ds to inhibit its interactions with Fat (Fig. 9B). In this case, Fat protein within Fj-expressing clones would be predicted to prefer to bind to Ds outside of the clone, and hence to accumulate at the clone perimeter, where it would then downregulate Dachs (Fig. 9C).
The interpretation of the elevated Dachs staining at the perimeter of Ds-expressing clones is more complex. Although Fat is elevated at the clone perimeter, the depletion of Fat from neighboring cells suggests that the elevated Fat staining largely reflects Fat outside of the clone, rather than in Ds-expressing cells (Fig. 8E,F; Fig. 9D). Given that dachs and fat influence transcriptional targets cell autonomously, and dachs acts genetically downstream of fat, the link between elevated Fat in one cell and elevated Dachs in a neighboring cells must be indirect. It might be that Ds can also influence Dachs localization, and does so in opposite fashion to Fat. According to this scenario, the elevated Fat staining in cells neighboring the clone would be reflective of high levels of Ds engaged by Fat at the clone perimeter, which would then recruit or stabilize Dachs at the membrane. However, mutation of ds did not result in any noticeable decrease of Dachs:V5 staining (data not shown). Alternatively, it might be that Fat antagonizes the accumulation of Ds within the same cell. High Fat accumulation at the edge of one cell could then result in low Fat accumulation at the edge of its neighbor through this hypothesized downregulation of Ds. In this case, the elevated Dachs accumulation at the edge of Ds-expressing clones would be a consequence of low levels of Fat. This model would also imply that asymmetric localization of Fat could be propagated from cell to cell, which could have important consequences for Fat pathway regulation. However, there is as yet no evidence that Fat is asymmetrically localized at wild-type levels of Fj and Ds expression.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/13/2539/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: RIKEN Center for Developmental Biology, 2-2-3
Minatojimaminamimachi, Chuo-ku, Kobe, 650-0047, Japan
Present address: PO Box 371, 29 Palms, CA 92277, USA
Present address: Fogarty International Center, N.I.H., 31 Center Drive MSC
2220, Bethesda, MD 20892-2220, USA
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