|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 1, 2006
doi: 10.1242/10.1242/dev.02370
Department of Cell and Developmental Biology, University of Colorado School of Medicine, PO Box 6511, Aurora, CO 80045, USA.
* Author for correspondence (e-mail: joan.hooper{at}uchsc.edu)
Accepted 17 March 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Key words: Hedgehog signaling, Gli, E3 ubiquitin ligase, Cubitus interruptus, Roadkill, Drosophila
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
;
Lum and Beachy, 2004).
Misregulation in the Hh pathway results in numerous pathologies ranging from
birth defects to cancers (Beachy et al.,
2004; McMahon et al.,
2003; Ruiz i Altaba et al.,
2002). Hh itself is a secreted protein that acts over a short
range, up to about 20 cell diameters. It often acts as a morphogen
(Heemskerk and DiNardo, 1994;
Jessell, 2000), where
differences in Hh levels across a field of cells generate distinct
developmental outputs.
Hh signaling is a tightly regulated cascade that acts through Gli-family
transcription factors to alter gene expression and reprogram cell fate
(Hooper and Scott, 2005;
Lum and Beachy, 2004). In
Drosophila, Cubitus interruptus (Ci) is the only Gli-family member,
and its regulation by Hh is post-transcriptional. Ci can assume three forms:
the latent form Ci155, the transcriptional repressor CiR and the
transcriptional activator CiA. Different levels of Hh generate different
ratios and/or levels of CiR and CiA. Because they vary in their responses to
CiR and/or CiA (Muller and Basler,
2000), various target genes exhibit different thresholds for
activation by Hh. Thus, orchestration of CiR and CiA is crucial to the action
of Hh as a morphogen.
Distinct pathways mediate the transformation of Ci155 into CiR and CiA, and
these pathways respond to different levels of Hh. The latent form Ci155 is
anchored in the cytoplasm by regulatory complexes that include the divergent
kinesin Costal2, the protein kinase Fused and the novel protein Suppressor of
fused [Su(fu)] (Sisson et al.,
1997; Stegman et al.,
2000). In the absence of Hh, the complex facilitates processing of
Ci155 to CiR. Ci155 is phosphorylated by cAMP-dependent Protein Kinase A
(PKA), Glycogen Synthase Kinase 3 (GSK3) and Casein Kinase 1 (CK1), then
ubiquitylated by a Cullin1-based E3 ubiquitin ligase, and cleaved to remove
regulatory and transcriptional activation domains
(Dai et al., 2003;
Jia et al., 2002;
Jiang and Struhl, 1998;
Price and Kalderon, 2002). The
resulting CiR, freed from the regulatory complex, translocates to the nucleus
where it represses transcription of Hh target genes
(Aza-Blanc et al., 1997;
Dominguez et al., 1996).
Hh acts through its receptor Patched (Ptc) and the transmembrane signal
transducer Smoothened (Smo) to strip PKA, CKI and GSK3 from the Ci regulatory
complex (Alcedo et al., 1996;
van den Heuvel and Ingham,
1996). Ci155 is no longer processed to CiR, and, instead,
accumulates in the cytoplasm. Prolonged and/or elevated stimulation by Hh
releases a second activity of Smo (Hooper,
2003), which prompts Fused kinase to inhibit Su(fu). A
biochemically uncharacterized CiA is released from the regulatory complex and
begins to activate transcription, while levels of Ci155 drop
(Ohlmeyer and Kalderon,
1998).
The level of Ci155 is important for correct responses to Hh; overexpression
of Ci155 can de-repress Hh target genes like decapentaplegic
(dpp) in the absence of Hh
(Dominguez et al., 1996;
Hepker et al., 1997). At least
three pathways, in addition to processing to Ci75, affect Ci155 turnover. In
very low Hh, the novel protein Debra shunts phospho-Ci155 to the lysosome for
degradation (Dai et al.,
2003). Without Debra, Ci155 accumulates and there is increased
expression of its targets dpp and ptc. When Hh is high,
Ci155 is no longer phosphorylated and other pathways come into play. In the
eye, Cullin3 (Cul3) mediates depletion of unphosphorylated Ci155 in the
presence of Hh (Ou et al.,
2002; Mistry et al.,
2004). Removing cul3 kills cells, so it is unknown
whether excess Ci155 has consequences when Hh is high and target genes are
already de-repressed. The Hect-domain protein Hyperplastic discs contributes
to Ci155 turnover, but whether this is regulated by Hh and which (if any) of
its pleiotropic effects are via Ci155 remains unclear
(Lee et al., 2002). Thus, Hh
controls levels of Ci155 via multiple mechanisms. What remains unclear is how
degradation of Ci155 is triggered by high Hh and whether this downregulation
is necessary for appropriate responses.
Here, we describe identification and characterization of the roadkill (rdx) gene. It is expressed in response to high levels of Hh and then downregulates Hh responses by lowering levels of Ci155. rdx-mediated attenuation of Hh signaling is essential for eye morphogenesis. There, rdx highlights a novel role for Hh in packing ommatidia into the hexagonal array. The Rdx protein belongs to a family of substrate-specific adaptors for Cul3-based E3 ubiquitin ligase, and associates with Ci155 in vivo. Thus, Rdx identifies a negative-feedback loop through which Hh limits its own responses by targeting Ci155 for degradation. These observations raise the possibility that vertebrate orthologs of Rdx may modulate activity of the Hh pathway through regulated degradation of Gli-family transcription factors.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
); UAS:Hh from P. Ingham
(Ingham and Fietz, 1995);
UAS:Ci5M from D. Kalderon (Price
and Kalderon, 1999); MS1096 from K. Basler
(Dominguez et al., 1996);
Lz:Gal4 from U. Banerjee (Flores
et al., 1998); and act5c:<y+<Gal4, UasGFP from C.
Doe (Ito et al., 1997).
UAS:Smo is described elsewhere
(Hooper, 2003). All other
stocks were obtained from the Bloomington Stock Center.
Clones in imaginal discs were induced using the FLP:FRT system
(Xu and Rubin, 1993). Germline
clones were generated using the FLP:FRT ovoD system
(Chou and Perrimon, 1996).
Transgene expression used the Gal4:Uas system
(Brand and Perrimon, 1993).
Genotypes
Tissue preparation and analysis
Cuticles were prepared and mounted as described previously
(Struhl, 1989). Hatch rates
were determined 48 hours after 4-hour egg collections by counting `empty'
vitelline membranes (hatched) and intact eggs (unhatched). Wings were mounted
in 50% Canada balsam and 50% methyl salicylate. Standard histological methods
were used for thin sections of adult eyes
(Bentrop et al., 1997) and for
SEM (Moses et al., 1989),
which was performed at UTSA or the University of Wyoming.
Immunofluorescence on imaginal discs followed standard procedures
(Hooper, 2003). Pupal retinas
were dissected on ice in PBS, fixed in 9.25% formaldehyde on ice for 1-2
hours, and processed for immunofluorescence as above. Antibodies were obtained
from the Developmental Hybridoma Studies Bank at the University of Iowa
(unless otherwise indicated) and used at the following dilutions: Arm, N27A1
at 1:10; Ci, 2A1 at 1:3 (R. Holmgren, Northwestern University); Elav, 7E8A10
at 1:3; En, 4D9 at 1:10; ß-Gal, rabbit at 1:1000 (ICN); Myc, 9E10
ascites 1:5000 (J. Heffler, UCHSC); Ptc, ApaI at 1:50 (I. Guerrero,
Universidad Autonoma, Madrid); and Senseless, guinea pig serum at 1:1000 (H.
Bellen, Baylor College). Species-specific secondary antibodies conjugated to
Alexa488, Alexa546 (Molecular Probes) and Cy5 (Jackson ImmunoResearch) were
diluted 1:400. Tissues were mounted in Permafluor (Immunon). To visualize
mitotic figures, embryos were fixed and rehydrated as for immunofluorescence,
incubated in Hoechst 33258 at 1 µg/ml in PBS in for 4 minutes, rinsed and
mounted in 1:1 glycerol:PBS. Confocal images were captured with Zeiss Pascal
LSM and manipulated with Adobe Photoshop.
In situ hybridization was carried out using digoxigenin-labeled, antisense
riboprobes, according to standard procedures
(Jiang et al., 1991;
Tautz and Pfeifle, 1989).
rdxA-specific probe used a 180 bp EcoRI fragment from exon
6. rdxE probe used an EcoR1/EcoRV fragment derived
from exon 5. Exon 11-13 probe used a PCR product from cDNA #5-2. Fragments
subcloned into Bluescript (Stratagene) were used for in vitro
transcription.
Wing imaginal discs were prepared for western blotting as described by
Ohlmeyer (Ohlmeyer and Kalderon,
1998). For immunoprecipitation, embryos were collected overnight,
heat shocked at 38°C for 1 hour, recovered at 25°C for 4 hours, and
then lysed and analyzed as described previously
(Ogden et al., 2003). Myc
(9E10) and control immunoprecipitates (FlagM2, Sigma) each used 1 µg
purified IgG. Ci was detected with affinity purified rabbit 3.3 antibody at
1:5000 (P. Ingham). 
-tubulin antibody (Sigma) was used at 1:20,000
dilution.
Mapping and sequencing
Recovered genomic sequence adjacent to the rdx1
P-element insertion (Bier et al.,
1993) and used to screen a Drosophila 0-24 hour embryonic
cDNA library (Clontech). A 0.8 kb EcoRV/EcoRI fragment from
the 3' end of the longest cDNA was used to rescreen the library.
Restriction mapping of the five recovered cDNAs revealed two splice variants
corresponding to RdxE and RdxA. cDNA sequence (CU Cancer Center DNA Sequencing
& Analysis Core) was compiled with Sequencher software (Gene Codes). EMS
mutations were identified by PCR amplifying genomic DNA from homozygous
mutants (Laird et al., 1991),
followed by cloning and sequencing
(Maniatis and Sambrook, 1989).
Putative mutations were confirmed with independent PCR products. Splicing of
rdx6 was analyzed by sequencing RT-PCR products of mRNAs
derived from homozygous mutants. Chromatograms were evaluated manually;
sequence downstream of splice variations was read by scoring primary and
secondary peaks. Northern and Southern blotting followed standard procedures
(Maniatis and Sambrook,
1989).
Rdx-expressing transgenes were generated by inserting a
BamHI-XhoI fragment containing a RdxA cDNA (EST LD08515)
into the BglII-XhoI sites of pUAST
(Brand and Perrimon, 1993). For
MycRdxA, PCR was used to replace the 5' UTR and start codon with a
Kozack sequence, a start codon and a Myc epitope. The correct sequence was
confirmed and the modified RdxA cDNA was cloned into the
KpnI-XbaI sites of pUAST. Transgenic flies were obtained by
standard procedures (Spradling and Rubin,
1982). Transgene activity was confirmed by rescue of rdx
mutants; expression either ubiquitously or in the pattern of ptc
rescued viability of rdx1, rdx1/rdx6
and rdx4 /rdx6, but not
rdx5/rdx6.
|
RESULTS AND DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
We used northern blots (not shown) to confirm the presence of transcripts
corresponding to rdxA and rdxE in embryos. A probe unique to
rdxE (exon 5) detected transcripts of 
4 kb, while probe specific
to rdxA (exon 6) detected the cluster of transcripts 2.1-2.5 kb.
A probe derived from the cluster of downstream exons detected both the cluster
of transcripts 2.1-2.5 kb and the larger transcript 4 kb. Levels of
all transcripts were highest in 0- to 2-hour embryos. Most importantly, the 4
kb transcript corresponding to rdxE comprised a significant
proportion of all rdx forms in 4- to 8-hour embryos. Thus,
rdxE is confirmed as a fifth transcript of CG9924/rdx.
|
; van den Heuvel,
2004; Xu et al.,
2003). rdx is the only gene in the Drosophila
genome that encodes a protein containing both of these domains. There are two
orthologs in the human genome: Speckle-type POZ protein
(Nagai et al., 1997) at
17q21.33 and LOC339745 (GenBank, Accession number AK021919) at 2q22.1.
|
The double stripes of rdx expression during germ band extension
are reminiscent of those of patched (ptc), the canonical Hh
responsive gene. Using Engrailed (En) to mark the Hh-expressing cells, we
found that the segmentally repeated stripes of rdx were centered
around Hh during stage 8/9 (not shown), while the double stripes of
rdx during stage 10/11 flanked cells expressing Hh
(Fig. 2J,K). Thus, rdx
is expressed in cells known to be responding to Hh. To test whether expression
of rdx requires Hh signaling, we examined rdx expression in
ciCe mutant embryos. This allele produces a truncated Ci
protein that mimics CiR and constitutively represses Hh target genes
(Methot and Basler, 1999).
rdx expression in ciCe embryos was lost in the
segmented ectoderm by stage 11, though Hh-independent expression remained in
neuroblasts and in the ectoderm of A9 (Fig.
2H). To test whether Hh signaling is sufficient to activate
rdx expression, we used ptc mutant embryos to activate Hh
signaling throughout the anterior compartment
(Ingham et al., 1991). In
ptc mutants, rdx expression filled the anterior compartment
in the ectoderm, and was also extensively activated in the mesoderm
(Fig. 2I). Thus, rdx
is a Hh target gene; it is normally expressed in cells adjacent to those
expressing Hh, and Hh signaling is both necessary and sufficient to activate
rdx expression.
rdx alleles
Five additional alleles of rdx were identified by failure to
complement the enhancer trap allele rdx1. The molecular
lesions are mapped onto rdxE in
Fig. 1B. A LacW
insertion near the start of exon 5 is responsible for
rdx1, as precise excision of the P-element restored
viability (not shown). rdx2 and rdx3
(gift of T. Kornberg) are also LacW insertions with lethality and
ß-galactosidase (ß-gal) expression patterns similar to
rdx1. By Southern blotting, their insertions are within
100 nucleotides of the start of exon 5 (not shown). rdx4
is a complex rearrangement (not shown) that was generated by imprecise
excision of rdx1. rdx5 and
rdx6 derived from an EMS mutagenesis
(Mortin et al., 1992).
rdx6 contains a point mutation in the splice donor between
exon 12 and exon 13. Using RT-PCR, this allele generated mRNAs that retain
intron 12/13, as well as wild-type mRNAs. Translation into intron 12/13 would
truncate Rdx at the end of the BTB domain. rdx5 included
two missense mutations: Q193H in sequences unique to RdxE, and D653V in a
highly conserved residue of the BTB domain. X-ray structure of a BTB domain
predicts that the side chain of D653 hydrogen bonds with the peptide backbone
(Ahmad et al., 1998), and
disruption of this hydrogen bond could destabilize the BTB domain.
All six rdx alleles, in all combinations, caused recessive lethality. rdx1 or rdx4 homozygotes hatched, but the sluggish, slow-growing larvae seldom survived beyond the first instar. Fewer rdx5 larvae hatched, and none survived beyond first instar. rdx6 embryos seldom hatched. Homozygotes for Df(3R)red1, a deficiency that includes many genes in addition to rdx, never hatched. These data defined the allelic series Df(3R)red1>rdx5>rdx6>rdx4=rdx1> wild type. This is consistent with the molecular lesions: rdx1 and rdx4 should affect only the E form and thus be partial loss of function; rdx6 should affect all forms but make both wild-type and mutant product; and rdx5 should disrupt the protein structure for all forms. In the rest of this work, we use rdx5 to define rdx loss-of-function phenotypes.
|
) to generate embryos lacking both maternal and zygotic
rdx. GLC of the weak loss-of function alleles
rdx1 or rdx4 yielded embryos that
developed and hatched normally. No eggs were recovered from
rdx5 GLC, suggesting that rdx is essential for
oogenesis. Embryos derived from rdx6 GLC displayed a
spectrum of developmental defects ranging from normal patterning (2%), through
a complete failure of cellularization (75%). An intermediate class (23%)
included errors in segmental patterning. It also included embryos that
developed at one end, but that slumped at the other end into an amorphous
mass, a phenotype most accurately described as roadkill
(Fig. 3C). The failure of
cellularization in most rdx6 GLC embryos reflected severe
defects during the early mitotic cycles
(Fig. 3D). Abnormalities
included mitotic asynchrony, uneven nuclear spacing, failed anaphase
resolution and multipolar mitotic figures. The requirement for rdx
function in early mitoses is strictly maternal, as wild-type sperm did not
rescue the defects of rdx GLC embryos (not shown). As
rdx5 or rdx6 homozygous clones were
the same size as their wild-type counterparts in eye and wing imaginal discs
(Figs 4 and
5), we conclude that the
mitotic requirement for rdx is limited to the earliest mitoses in
embryos. As there is no requirement for Hh signaling in the female germline,
the requirement for rdx in the meiotic/mitotic transition must
represent a Hh-independent function.
MEL-26, the C. elegans of the MATH/BTB protein family, promotes
the meiotic/mitotic transition through degradation of the MEI-1/katanin
(Mains et al., 1990;
Pintard et al., 2003;
Xu et al., 2003). Maternal
loss of MEL-26 leads to a mitotic catastrophe similar to that observed for
rdx. This suggests that a phylogenetically conserved function for the
Rdx/MEL-26 family may be to assist in the meiotic/mitotic transition by
targeting Katanin/MEI-1 for degradation.
A role for rdx in Ci regulation
As the mitotic failure precluded studies of rdx during embryonic
segmentation, we turned to imaginal discs to investigate the connection
between rdx and Hh. In the developing wing, Hh is made in the
posterior compartment while anterior compartment cells respond to Hh because
they express Ptc and Ci (schematized in
Fig. 4N). Deep in the anterior
compartment, cells see no Hh; Ci155 is processed to CiR, resulting in low
levels of Ci155 and repression of target genes. Immediately adjacent to the
compartment border, high levels of Hh activate Ci and block its processing to
CiR; target genes with the highest threshold for Hh are expressed, and levels
of Ci155 are low. In between are moderate levels of Hh; Ci155 accumulates but
is not activated, CiR may be reduced and target genes with lower thresholds
for Hh are induced.
rdx expression was detected in a strip of cells just anterior to the AP border (Fig. 4A,B). Its posterior edge coincided with that of Ci155, at the compartment border. It extended slightly anterior to the En expression that marks the highest responses to Hh, and overlapped almost exactly with the decreased Ci155 immediately adjacent to the compartment border. This rdx expression is most similar to the domain where ptc is induced by Hh. Moreover, rdx expression in the wing is Hh dependent: it was expanded with overexpression of Hh and disappeared when Hh response was blocked by overexpression of Ptc (Fig. 4C,D).
In rdx loss-of-function clones, we found increased levels of
Ci155, but only when those clones were within 10 cells of the compartment
border (Fig. 4F). Ci155 also
accumulated in rdx6 clones near the border, though levels
were somewhat lower (Fig. 4E).
As Ci155 levels were only affected in cells that lost rdx function
(e.g. Fig. 4F), the effect of
rdx on Ci is cell-autonomous. We found no change ci
transcription in clones lacking rdx (using ß-gal from
ci-lacZ as a reporter, not shown), indicating that the effects of
rdx on Ci155 are post-transcriptional. Taken together, these data
suggest that rdx acts as part of a negative feedback loop that
downregulates levels of Ci155 in response to Hh.
Wings filled with unmarked rdx clones (28 of 40 wings) showed a
mild elongation of the anterior compartment
(Fig. 4G). However,
rdx loss-of-function clones had no discernible effect on expression
of the Hh target genes En, Ptc, Collier or dpp. This is consistent
with previous observations that forced overexpression of Ci155 has little
phenotypic consequence if it is limited to the domain immediately adjacent to
the compartment border (Dominguez et al.,
1996; Hepker et al.,
1997; Wang et al.,
1999). The molecular basis for the altered wing morphology could
not be determined.
To ask whether rdx could affect Ci155 beyond the range of Hh, we
overexpressed RdxA in the wing pouch (red in
Fig. 4J). This reduced Ci155
levels both at the compartment border and deep in the anterior compartment
(Fig. 4L). Ptc and En induction
was reduced or blocked (Fig.
4J,K), while dpp expression broadened
(Fig. 4K). The adult wings were
smaller than normal, with ectopic veins anterior to vein 3
(Fig. 4I). This loss of some
targets and activation others is most consistent with reduction in both CiR
and CiA, in all Ci activity (Methot and
Basler, 1999). In western blots of wing imaginal discs, we found
that overexpression of Rdx reduced levels of both Ci155 and Ci75
(Fig. 4M; the modest reduction
is all that could be expected, given that Rdx was overexpressed only in the
wing pouch and not in the remainder of the disc). Taken together, these data
show that rdx attenuates levels of all forms of Ci in response to Hh.
This pathway for Ci regulation plays a minor role in wing development but may
be more significant in other tissues.
rdx regulates levels of Ci155 in the eye
In the eye imaginal disc, a wave of Hh signaling initiates differentiation
as it propagates across the disc (Fig.
5A). Between undifferentiated and differentiating tissue lies the
morphogenetic furrow (MF), a contraction of the columnar epithelium. In front
of (anterior to) the MF, CiR maintains the undifferentiated state by
repressing photoreceptor specification
(Heberlein et al., 1993;
Hsiung and Moses, 2002;
Ma et al., 1993). Behind
(posterior to) the MF, differentiating photoreceptors make Hh
(Lee et al., 1992). Within the
MF, undifferentiated cells fall under the influence of Hh from adjacent
photoreceptors; CiR production is blocked, and a new row of photoreceptors is
initiated. Thus, the MF/differentiation moves from posterior to anterior
across the eye imaginal disc. Hh signaling, as measured by continuing
expression of ptc, remains active behind the MF
(Shyamala and Bhat, 2002). A
developmental role for this Hh signaling has not yet been identified.
In the eye, Hh regulates two distinct pathways that effect Ci155 turnover
(Fig. 5A,C,D). In front of the
MF, Ci155 is processed to CiR via phosphorylation and the Slimb/Cul1-based
ubiquitin E3 ligase. Within the MF, Hh blocks that pathway and Ci155
accumulates. Behind the MF, Ci155 is depleted by a Cul3-dependent pathway that
involves neither PKA nor Slimb (Ou et al.,
2002). That pathway requires Hh signaling, as Ci155 accumulates in
smo clones behind the MF. As Cul3 is present throughout the eye disc
(Ou et al., 2002), it is not
known why Cul3 depletes Ci155 only behind the MF.
rdx is expressed in all cells posterior to the MF, with highest
levels in photoreceptor clusters and cone cells
(Fig. 5B,C, and not shown).
rdx expression in the eye is Hh dependent, as it is in wing and
embryonic ectoderm; it fills the eye when Hh is overexpressed, and disappears
when Ptc is overexpressed (not shown). In loss-of-function
rdx5 clones, Ci155 levels increased behind (posterior to)
the MF but not in front of (anterior to) the MF
(Fig. 5E). This accumulation of
Ci155 was cell-autonomous, limited to the cells lacking rdx. Thus,
rdx, like cul3 (Ou et
al., 2002), acts to reduce levels of Ci155 behind the MF but has
little effect on Ci155 within or in front of the MF. To determine whether Rdx
could affect Ci levels throughout the eye, we misexpressed RdxA in clones
(Fig. 5F). We found that
ectopic Rdx could destabilize Ci155 within and anterior to the MF. Thus, Rdx
expression is both necessary and sufficient for degradation of Ci155
throughout the eye.
|
|
0.2%). We conclude that in vivo, Rdx binds Ci155 better than Ci75. Taken
together, these data suggest that Rdx regulates degradation of Ci155 by acting
as a specificity factor bringing Ci155 to Cul3. However, we cannot eliminate
the possibility that Rdx may act through a Cul3-independent mechanism.
rdx reveals a role for Hh in the eye after photoreceptor recruitment
What might be the role of Hh-dependent depletion of Ci155 in the eye? Small
rdx clones lacked ommatidial bristles, but produced adult eyes with
otherwise normal external morphology (not shown). In eyes that mostly
comprised rdx mutant cells, the ommatidia were uneven in size,
misaligned and often lacked or duplicated ommatidial bristles
(Fig. 6B). MF movement,
photoreceptor and cone cell specification (monitored by morphology, Senseless
and Elav expression) were normal in these rdx mutant eyes (not
shown). Sections through mutant ommatidia revealed the correct number of
photoreceptors, but the photoreceptor clusters were loose, imperfectly aligned
and irregularly spaced (Fig.
6B). Thus, rdx has a role in retinal patterning after
specification of photoreceptor cells and during the rectification of the
ommatidial lattice.
To investigate the cellular basis for the rdx phenotype, we
examined retinas at 36-42 hours of pupation, when the non-neural cells (cone,
pigment and bristle cells) are moving into their final patterns. The spatial
relationships of these non-neuronal cells are shown in
Fig. 6D.
rdx5 pupal ommatidia
(Fig. 6E) showed misaligned
1° cells (40% of ommatidia) that sometimes failed to enclose cone cells
(6%), as well as reduced numbers of cone cells (4%), inappropriate numbers of
lattice cells (56%), ommatidial fusions (2%) and misplaced bristles (19%). As
cone and 1° cells organize lattice cells and prevent their apoptosis
(Cagan and Ready, 1989;
Cordero et al., 2004;
Miller and Cagan, 1998;
Rusconi et al., 2000;
Wolff and Ready, 1991), the
ommatidial fusions and lattice cell defects are likely to be secondary to the
problems with cone and 1° cells. We conclude that loss of rdx
function interferes with patterning in the pupal retina, probably at the level
of the cone and 1° cells.
The dramatic effect of rdx on Ci155 levels suggested that
rdx is acting in retinal patterning via Ci and the Hh pathway.
Indeed, hh– or smo– clones
generate disorganized ommatidia similar to those in rdx mutant eyes,
though this has been attributed to a distortion of the MF, rather than to a
direct effect of Hh (Dominguez,
1999; Heberlein et al.,
1993; Ma et al.,
1993). Moreover, hyperactivation of Hh responses (via insufficient
ptc) causes a variety of morphological defects in the differentiating
eye (Thomas and Ingham, 2003).
To determine whether Hh and Ci affect ommatidial organization after the MF has
passed, we used Lozenge:Gal4 (Lz:G4) to drive
UAS:transgene expression behind the MF
(Flores et al., 1998). The
effects of Hh overexpression were limited to the anterior margin, with some
reduction and with mildly disordered bristles and ommatidial packing
(Fig. 7B). The pupal retinas
showed mild defects near one edge (Fig.
7F), with occasional lost or excess cone cells (4%), mis-oriented
or incompletely wrapped 1° cells (44%) and excess lattice cells (55%).
Expression of Ci5M, a Ci whose mutated PKA sites stabilize it and prevent its
processing to a transcriptional repressor
(Price and Kalderon, 1999),
caused loss of ommatidial bristles (40-50%) and mildly disordered ommatidial
packing (Fig. 7C). The
corresponding pupal ommatidia (Fig.
7G) showed occasional loss of cone cells (2%), failure of the
1° cells to wrap around the equatorial cone cells (12%) and excess lattice
cells (38%). As these defects were not seen with Lz:G4 alone
(Fig. 7E), we conclude that
excess Ci can interfere with 1° and lattice cell patterning. To maximally
stimulate Hh signaling, we used a Smo transgene whose high level of expression
makes it a potent activator (Hooper,
2003). Lz:G4; UAS:Smo generated rough eyes, with defects
most pronounced in the posterior (Fig.
7D). At 24 hours, pupal retinas were relatively normal (not
shown), although 1° cells were often misaligned (27%). By 30 hours,
pupal ommatidia had reduced numbers of cone cells (16%) and of lattice cells
(75%), with occasional cone cells adopting a rounded morphology characteristic
apoptotic cells (Fig. 7H).
Apparently, Smo overexpression causes cone cell death in the pupal retina.
Photoreceptor differentiation, assessed by counting rhabdomeres in pupal
retinas, was unaffected in these experiments (not shown).
|
|
Summary and future prospects
rdx encodes a protein belonging to a phylogenetically conserved
protein family of substrate-specific adaptors for Cullin3-based ubiquitin E3
ligases (Pintard et al., 2003;
van den Heuvel, 2004;
Xu et al., 2003). rdx
loss-of-function and gain-of-function studies suggest that rdx has at
least two substrates – a regulator of early embryonic mitoses and the Hh
regulated transcription factor Ci155. Our data support a model where Rdx
regulates the Hh-dependent degradation of Ci by acting as the adaptor that
presents Ci to the Cul3-based E3 ubiquitin ligase. Because rdx is
expressed in response to Hh, rdx is involved in a novel regulatory
loop that attenuates Hh responses through reducing levels of Ci. In the wing,
this feedback regulation of Ci by rdx plays a minor role, but in the
eye it is essential for proper packing of ommatidia into a hexagonal
array.
Hh is key regulator in human health
(Beachy et al., 2004;
McMahon et al., 2003;
Ruiz i Altaba et al., 2002).
The haploinsufficiency of Ptc in humans
(Johnson et al., 1996) and its
activity as a morphogen in the spinal column
(Jessell, 2000) argue that the
level of Hh response is often crucial. Although there are differences in the
Hh pathway between flies and vertebrates, many regulatory mechanisms are
conserved (Hooper and Scott,
2005; Huangfu and Anderson,
2006; Lum and Beachy,
2004). In particular, Gli2 and Gli3 are regulated much like Ci,
becoming repressors or activators, depending on levels of Hh. The Rdx ortholog
SPOP lies in 17q21.33, a chromosomal region that has been linked with ovarian
cancer and cervical immature teratoma
(Bernardini et al., 2005;
Miliaras et al., 2005). Future
studies will determine whether the Rdx orthologs SPOP or LOC339745 modulate
Gli levels and Hh-mediated responses, and even contribute to cancer.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Ahmad, K. F., Engel, C. K. and Prive, G. G.
(1998). Crystal structure of the BTB domain from PLZF.
Proc. Natl. Acad. Sci. USA
95,12123
-12128.
Alcedo, J., Ayzenzon, M., Von Ohlen, T., Noll, M. and Hooper, J. E. (1996). The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell 86,221 -232.[CrossRef][Medline]
Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. P., Schwartz, C. and Kornberg, T. B. (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89,1043 -1053.[CrossRef][Medline]
Beachy, P. A., Karhadkar, S. S. and Berman, D. M. (2004). Tissue repair and stem cell renewal in carcinogenesis. Nature 432,324 -331.[CrossRef][Medline]
Bentrop, J., Schwab, K., Pak, W. L. and Paulsen, R. (1997). Site-directed mutagenesis of highly conserved amino acids in the first cytoplasmic loop of Drosophila Rh1 opsin blocks rhodopsin synthesis in the nascent state. EMBO J. 16,1600 -1609.[CrossRef][Medline]
Bernardini, M., Lee, C. H., Beheshti, B., Prasad, M., Albert, M., Marrano, P., Begley, H., Shaw, P., Covens, A., Murphy, J. et al. (2005). High-resolution mapping of genomic imbalance and identification of gene expression profiles associated with differential chemotherapy response in serous epithelial ovarian cancer. Neoplasia 7,603 -613.[CrossRef][Medline]
Bier, E., Vassin, H., Shepard, K., Lee, K., McCall, K., Barbel, L., Ackerman, R., Carreto, T., Vemura, E., Grell, L. et al. (1993). Searching for pattern and mutation in the Drosophila genome with a P–LacZ vector. Genes Dev. 3,1273 -1287.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Cagan, R. L. and Ready, D. F. (1989). The emergence of order in the Drosophila pupal retina. Dev. Biol. 136,346 -362.[CrossRef][Medline]
Chou, T. B. and Perrimon, N. (1996). The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144,1673 -1679.[Abstract]
Cordero, J., Jassim, O., Bao, S. and Cagan, R. (2004). A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye. Mech. Dev. 121,1523 -1530.[CrossRef][Medline]
Dai, P., Akimaru, H. and Ishii, S. (2003). A hedgehog-responsive region in the Drosophila wing disc is defined by debra-mediated ubiquitination and lysosomal degradation of Ci. Dev. Cell 4,917 -928.[CrossRef][Medline]
Dominguez, M. (1999). Dual role for Hedgehog in the regulation of the proneural gene atonal during ommatidia development. Development 126,2345 -2353.[Abstract]
Dominguez, M., Brunner, M., Hafen, E. and Basler, K. (1996). Sending and receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus. Science 272,1621 -1625.[Abstract]
Flores, G. V., Daga, A., Kalhor, H. R. and Banerjee, U. (1998). Lozenge is expressed in pluripotent precursor cells and patterns multiple cell types in the Drosophila eye through the control of cell-specific transcription factors. Development 125,3681 -3687.[Abstract]
Heberlein, U., Wolff, T. and Rubin, G. M. (1993). The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina. Cell 75,913 -926.[CrossRef][Medline]
Heemskerk, J. and DiNardo, S. (1994). Drosophila hedgehog acts as a morphogen in cellular patterning. Cell 76,449 -460.[CrossRef][Medline]
Hepker, J., Wang, Q. T., Motzny, C. K., Holmgren, R. and Orenic, T. V. (1997). Drosophila cubitus interruptus forms a negative feedback loop with patched and regulates expression of Hedgehog target genes. Development 124,549 -558.[Abstract]
Hooper, J. E. (2003). Smoothened translates
Hedgehog levels into distinct responses. Development
130,3951
-3963.
Hooper, J. E. and Scott, M. P. (2005). Communicating with Hedgehogs. Nat. Rev. Mol. Cell Biol. 6,306 -317.[CrossRef][Medline]
Hsiung, F. and Moses, K. (2002). Retinal
development in Drosophila: specifying the first neuron. Hum. Mol.
Genet. 11,1207
-1214.
Huangfu, D. and Anderson, K. V. (2006).
Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways
from Drosophila to vertebrates. Development
133, 3-14.
Ingham, P. W. and Fietz, M. J. (1995). Quantitative effects of hedgehog and decapentaplegic activity on the patterning of the Drosophila wing. Curr. Biol. 5, 432-440.[CrossRef][Medline]
Ingham, P. W., Taylor, A. M. and Nakano, Y. (1991). Role of the Drosophila patched gene in positional signalling. Nature 353,184 -187.[CrossRef][Medline]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D. (1997). The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124,761 -771.[Abstract]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Jia, J., Amanai, K., Wang, G., Tang, J., Wang, B. and Jiang, J. (2002). Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus. Nature 416,548 -552.[CrossRef][Medline]
Jiang, J. and Struhl, G. (1998). Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb. Nature 391,493 -496.[CrossRef][Medline]
Jiang, J., Hoey, T. and Levine, M. (1991).
Autoregulation of a segmentation gene in Drosophila: combinatorial interaction
of the even-skipped homeo box protein with a distal enhancer element.
Genes Dev. 5,265
-277.
Johnson, R. L., Grenier, J. K. and Scott, M. P. (1995). patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development 121,4161 -4170.[Abstract]
Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas, J. M., Quinn, A. G., Myers, R. M., Cox, D. R., Epstein, E. H., Jr et al. (1996). Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272,1668 -1671.[Abstract]
Laird, P. W., Zijderveld, A., Linders, K., Rudnicki, M. A.,
Jaenisch, R. and Berns, A. (1991). Simplified mammalian DNA
isolation procedure. Nucleic Acids Res.
19, 4293.
Lee, J. D., Amanai, K., Shearn, A. and Treisman, J. E. (2002). The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms. Development 129,5697 -5706.[Medline]
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell 71,33 -50.[CrossRef][Medline]
Lum, L. and Beachy, P. A. (2004). The Hedgehog
response network: sensors, switches, and routers.
Science 304,1755
-1759.
Ma, C., Zhou, Y., Beachy, P. A. and Moses, K. (1993). The segment polarity gene hedgehog is required for progression of the morphogenetic furrow in the developing Drosophila eye. Cell 75,927 -938.[CrossRef][Medline]
Mains, P. E., Kemphues, K. J., Sprunger, S. A., Sulston, I. A. and Wood, W. B. (1990). Mutations affecting the meiotic and mitotic divisions of the early Caenorhabditis elegans embryo. Genetics 126,593 -605.[Abstract]
Maniatis, T. and Sambrook, J. (1989).Molecular Cloning: A Laboratory Manual (2nd edn) . Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
McMahon, A. P., Ingham, P. W. and Tabin, C. J. (2003). Developmental roles and clinical significance of hedgehog signaling. Curr. Top. Dev. Biol. 53, 1-114.[Medline]
Methot, N. and Basler, K. (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96,819 -831.[CrossRef][Medline]
Miliaras, D., Grimbizis, G., Conroy, J., Psarra, N., Miliaras, S., Nowak, N. and Bontis, J. (2005). Novel karyotypic changes detected by comparative genomic hybridization in a case of congenital cervical immature teratoma. Birth Defects Res. Part A Clin. Mol. Teratol. 73,572 -576.[Medline]
Miller, D. T. and Cagan, R. L. (1998). Local induction of patterning and programmed cell death in the developing Drosophila retina. Development 125,2327 -2335.[Abstract]
Mistry, H., Wilson, B. A., Roberts, I. J., O'Kane, C. J. and Skeath, J. B. (2004). Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development. Mech. Dev. 121,1495 -1507.[CrossRef][Medline]
Mortin, M. A., Zuerner, R., Berger, S. and Hamilton, B. J. (1992). Mutations in the second-largest subunit of Drosophila RNA polymerase II interact with Ubx. Genetics 131,895 -903.[Abstract]
Moses, K., Ellis, M. C. and Rubin, G. M. (1989). The glass gene encodes a zinc-finger protein required by Drosophila photoreceptor cells. Nature 340,531 -536.[CrossRef][Medline]
Muller, B. and Basler, K. (2000). The repressor and activator forms of Cubitus interruptus control Hedgehog target genes through common generic gli-binding sites. Development 127,2999 -3007.[Abstract]
Nagai, Y., Kojima, T., Muro, Y., Hachiya, T., Nishizawa, Y., Wakabayashi, T. and Hagiwara, M. (1997). Identification of a novel nuclear speckle-type protein, SPOP. FEBS Lett. 418, 23-26.[CrossRef][Medline]
Ogden, S. K., Ascano, M., Jr, Stegman, M. A., Suber, L. M., Hooper, J. E. and Robbins, D. J. (2003). Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2. Curr. Biol. 13,1998 -2003.[CrossRef][Medline]
Ohlmeyer, J. T. and Kalderon, D. (1998). Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396,749 -753.[CrossRef][Medline]
Ou, C. Y., Lin, Y. F., Chen, Y. J. and Chien, C. T.
(2002). Distinct protein degradation mechanisms mediated by Cul1
and Cul3 controlling Ci stability in Drosophila eye development.
Genes Dev. 16,2403
-2414.
Pintard, L., Willis, J. H., Willems, A., Johnson, J. L., Srayko, M., Kurz, T., Glaser, S., Mains, P. E., Tyers, M., Bowerman, B. et al. (2003). The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425,311 -316.[CrossRef][Medline]
Price, M. A. and Kalderon, D. (1999). Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by protein kinase A. Development 126,4331 -4339.[Abstract]
Price, M. A. and Kalderon, D. (2002). Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell 108,823 -835.[CrossRef][Medline]
Ruiz i Altaba, A., Sanchez, P. and Dahmane, N. (2002). Gli and hedgehog in cancer: tumours, embryos and stem cells. Nat. Rev. Cancer 2, 361-372.[CrossRef][Medline]
Rusconi, J. C., Hays, R. and Cagan, R. L. (2000). Programmed cell death and patterning in Drosophila. Cell Death Differ. 7,1063 -1070.[CrossRef][Medline]
Shyamala, B. V. and Bhat, K. M. (2002). A positive role for patched-smoothened signaling in promoting cell proliferation during normal head development in Drosophila. Development 129,1839 -1847.[Medline]
Sisson, J. C., Ho, K. S., Suyama, K. and Scott, M. P. (1997). Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell 90,235 -245.[CrossRef][Medline]
Spradling, A. C. and Rubin, G. M. (1982).
Transposition of cloned P elements into Drosophila germ line chromosomes.
Science 218,341
-347.
Stegman, M. A., Vallance, J. E., Elangovan, G., Sosinski, J.,
Cheng, Y. and Robbins, D. J. (2000). Identification of a
tetrameric hedgehog signaling complex. J. Biol. Chem.
275,21809
-21812.
Struhl, G. (1989). Differing strategies for organizing anterior and posterior body pattern in Drosophila embryos. Nature 338,741 -744.[CrossRef][Medline]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[CrossRef][Medline]
Thomas, C. and Ingham, P. W. (2003). Hedgehog signaling in the Drosophila eye and head: an
