InteractiveFly: GeneBrief

roadkill: Biological Overview | References


Gene name - roadkill

Synonyms -

Cytological map position - 88A3-88A4

Function - signal transduction

Keywords - eye, segment polarity, substrate adaptor for Cullin3-mediated ubiquitylation

Symbol - rdx

FlyBase ID: FBgn0264493

Genetic map position - 3R:9,793,674..9,857,777 [-]

Classification - MATH_SPOP, Speckle-type POZ protein (SPOP) family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The final step in Hedgehog (Hh) signal transduction is post-translational regulation of the transcription factor, Cubitus interruptus (Ci). Ci resides in the cytoplasm in a latent form, where Hh regulates its processing into a transcriptional repressor or its nuclear access as a transcriptional activator. Levels of latent Ci are controlled by degradation, with different pathways activated in response to different levels of Hh. The roadkill (rdx) gene is expressed in response to Hh. The Rdx protein belongs to a conserved family of proteins that serve as substrate adaptors for Cullin3-mediated ubiquitylation. Overexpression of rdx reduces Ci levels and decreases both transcriptional activation and repression mediated by Ci. Loss of rdx allows excessive accumulation of Ci. rdx manipulation in the eye revealed a novel role for Hh in the organization and survival of pigment and cone cells. These studies identify rdx as a limiting factor in a feedback loop that attenuates Hh responses through reducing levels of Ci. The existence of human orthologs for Rdx raises the possibility that this novel feedback loop also modulates Hh responses in humans (Kent, 2006; full text of article).

Hedgehog (Hh) signaling is a phylogenetically conserved pathway that is crucial to the growth and development of metazoans. Misregulation in the Hh pathway results in numerous pathologies ranging from birth defects to cancers. Hh itself is a secreted protein that acts over a short range, up to about 20 cell diameters. It often acts as a morphogen, where differences in Hh levels across a field of cells generate distinct developmental outputs (Kent, 2006)

Hh signaling is a tightly regulated cascade that acts through Gli-family transcription factors to alter gene expression and reprogram cell fate. 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, 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 (Kent, 2006)

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)]. 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. The resulting CiR, freed from the regulatory complex, translocates to the nucleus where it represses transcription of Hh target genes (Kent, 2006)

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. 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, 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 (Kent, 2006)

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. 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 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. 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. 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 (Kent, 2006).

This study describes 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 (Kent, 2006).

The rdx locus was identified by an enhancer trap with embryonic expression in a pattern suggesting Hh-regulation. When genomic DNA flanking the insertion was used to screen a Drosophila embryonic cDNA library, cDNAs were obtained that initiated near the enhancer trap insertion and spliced into a cluster of seven downstream exons. These cDNAs represented the predicted gene CG10235 spliced into the predicted gene CG9924. ESTs recovered by the BDGP identified four additional isoforms (CG9924 A-D), which differ in their 5' ends but which share the cluster of seven downstream exons with rdxE, and are designated rdxA-rdxD (Kent, 2006).

The A, C/D and E forms are predicted to encode proteins with unique and novel amino termini fused to a common C terminus. The B form lacks unique coding sequence and is predicted to initiate translation within exon 7. The 398 C-terminal residues encoded by exons 7-13 contains two conserved domains: a MATH (Meprin and TRAF homology) domain and a BTB (Broad/Tramtrack/Bric-a-brac) domain. These two protein interaction domains are found together in an evolutionarily conserved protein family where the BTB domain binds to Cul3, while the MATH domain recruits specific substrates to the Cul3-based E3 ubiquitin ligase complex for ubiquitylation and subsequent degradation (Kent, 2006).

rdxA, rdxE and the initial enhancer trap produced expression patterns that were indistinguishable from those of a probe common to all rdx forms. Maternally deposited rdx transcripts were detected in early embryos, but disappeared during mid-cleavage stages. The first zygotic transcripts appeared in pole cells. During cellularization of the blastoderm, rdx transcripts appeared in two broad stripes in the head, in seven narrower stripes along the segment primordium, and in a ring surrounding the pole cells. Seven additional stripes appeared during germ band extension, so that by stage 8, rdx was expressed in 14 evenly spaced ectodermal stripes characteristic of segment polarity genes. At this time, strong expression was seen in the anterior and posterior midgut primordia. During stage 9/10, expression appeared in a subset of neuroblasts. During stage 10 each segmental stripe split so that by stage 11, ectodermal expression consisted of two thin stripes corresponding to the anterior and posterior margins of the former stripe. At this time, strong expression was seen in the mesoderm. As germ band retraction began, expression faded from most of the ectoderm, but was retained in the salivary glands and in abdominal segment 9. After stage 14, rdx expression was detected only in the clypeolabrum, anal plate and salivary glands (Kent, 2006).

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, it was found that the segmentally repeated stripes of rdx were centered around Hh during stage 8/9, while the double stripes of rdx during stage 10/11 flanked cells expressing Hh. Thus, rdx is expressed in cells known to be responding to Hh. To test whether expression of rdx requires Hh signaling, rdx expression was examined in ciCe mutant embryos. This allele produces a truncated Ci protein that mimics CiR and constitutively represses Hh target genes. 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. To test whether Hh signaling is sufficient to activate rdx expression, ptc mutant embryos were used to activate Hh signaling throughout the anterior compartment. In ptc mutants, rdx expression filled the anterior compartment in the ectoderm, and was also extensively activated in the mesoderm. 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 (Kent, 2006)

Five additional alleles of rdx were identified by failure to complement the enhancer trap allele rdx1. The molecular lesions are mapped onto rdxE. A LacW insertion near the start of exon 5 is responsible for rdx1, as precise excision of the P-element restored viability. rdx2 and rdx3 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. rdx4 is a complex rearrangement that was generated by imprecise excision of rdx1. rdx5 and rdx6 derived from an EMS mutagenesis. 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, and disruption of this hydrogen bond could destabilize the BTB domain (Kent, 2006)

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, rdx5 was used to define rdx loss-of-function phenotypes (Kent, 2006)

Since Hh regulates rdx transcription during segmentation, a role was expected for rdx in Hh signaling or segmental patterning. However, segmentation, as assessed by cuticle pattern, was normal in rdx mutant embryos. To ask whether maternally deposited rdx mRNA might mask a role for rdx in early patterning, clones were used 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. The failure of cellularization in most rdx6 GLC embryos reflected severe defects during the early mitotic cycles. 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. Since rdx5 or rdx6 homozygous clones were the same size as their wild-type counterparts in eye and wing imaginal discs, it is concluded that the mitotic requirement for rdx is limited to the earliest mitoses in embryos. Since 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 (Kent, 2006)

MEL-26, the C. elegans of the MATH/BTB protein family, promotes the meiotic/mitotic transition through degradation of the MEI-1/katanin (Mains, 1990; Pintard, 2003; Xu, 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 (Kent, 2006)

Since the mitotic failure precluded studies of rdx during embryonic segmentation, the connection between rdx and Hh was investigated in imaginal discs. In the developing wing, Hh is made in the posterior compartment while anterior compartment cells respond to Hh because they express Ptc and Ci. 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 (Kent, 2006)

rdx expression was detected in a strip of cells just anterior to the AP border. 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 (Kent, 2006)

In rdx loss-of-function clones, increased levels of Ci155 were found, but only when those clones were within ~10 cells of the compartment border. Ci155 also accumulated in rdx6 clones near the border, though levels were somewhat lower. Since Ci155 levels were only affected in cells that lost rdx function, the effect of rdx on Ci is cell-autonomous. No change was found in ci transcription in clones lacking rdx (using ß-gal from ci-lacZ as a reporter), 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 (Kent, 2006)

Wings filled with unmarked rdx clones (28 of 40 wings) showed a mild elongation of the anterior compartment. 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. The molecular basis for the altered wing morphology could not be determined (Kent, 2006)

To ask whether rdx could affect Ci155 beyond the range of Hh, RdxA was expressed in the wing pouch. This reduced Ci155 levels both at the compartment border and deep in the anterior compartment. Ptc and En induction was reduced or blocked, while dpp expression broadened. The adult wings were smaller than normal, with ectopic veins anterior to vein 3. This loss of some targets and activation others is most consistent with reduction in both CiR and CiA, in all Ci activity. In western blots of wing imaginal discs, it was found that overexpression of Rdx reduced levels of both Ci155 and Ci75 (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 (Kent, 2006)

In the eye imaginal disc, a wave of Hh signaling initiates differentiation as it propagates across the disc. 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. Behind (posterior to) the MF, differentiating photoreceptors make Hh. 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. A developmental role for this Hh signaling has not yet been identified (Kent, 2006)

In the eye, Hh regulates two distinct pathways that effect Ci155 turnover. 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). That pathway requires Hh signaling, as evidenced by accumulation of Ci155 in smo clones behind the MF. As Cul3 is present throughout the eye disc, it is not known why Cul3 depletes Ci155 only behind the MF (Kent, 2006)

rdx is expressed in all cells posterior to the MF, with highest levels in photoreceptor clusters and cone cells. 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. In loss-of-function rdx5 clones, Ci155 levels increased behind (posterior to) the MF but not in front of (anterior to) the MF. This accumulation of Ci155 was cell-autonomous, limited to the cells lacking rdx. Thus, rdx, like cul3, 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, RdxA was missexpressed in clones. Ectopic Rdx destabilizes Ci155 within and anterior to the MF. Thus, Rdx expression is both necessary and sufficient for degradation of Ci155 throughout the eye (Kent, 2006)

The similar effects of rdx and cul3 on Ci155 raise the possibility that Rdx and Cul3 act in the same pathway. Indeed, its paired MATH and BTB domains suggest that Rdx acts as a substrate-specific adaptor, bringing Ci155 to a Cul3-based ubiquitin E3 ligase. In this scenario, ubiquitously expressed Cul3 depletes Ci155 only behind the MF, because that is where rdx is expressed; when Rdx is ectopically expressed, Ci155 is ectopically degraded. If Rdx acts as an adaptor bringing Ci to Cul3, then a small fraction of Ci should be associated with Rdx in cells. To test for association of Rdx and Ci, Myc-tagged Rdx was overexpressed in embryos, and then Myc immunoprecipitates were probed for Ci. A small fraction of Ci155 (significantly greater than 0.2%) was found in MycRdx immunoprecipitates, while Ci75 was not detected (threshold of detection, ~0.2%). It is concluded 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, the possibility cannot be eliminates that Rdx may act through a Cul3-independent mechanism (Kent, 2006)

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. In eyes that mostly comprised rdx mutant cells, the ommatidia were uneven in size, misaligned and often lacked or possessed duplicated ommatidial bristles. MF movement, photoreceptor and cone cell specification (monitored by morphology, Senseless and Elav expression) were normal in these rdx mutant eyes. Sections through mutant ommatidia revealed the correct number of photoreceptors, but the photoreceptor clusters were loose, imperfectly aligned and irregularly spaced. Thus, rdx has a role in retinal patterning after specification of photoreceptor cells and during the rectification of the ommatidial lattice (Kent, 2006)

To investigate the cellular basis for the rdx phenotype, retinas at 36-42 hours of pupation were examined, when the non-neural cells (cone, pigment and bristle cells) are moving into their final patterns. rdx5 pupal ommatidia show misaligned 1° cells (40% of ommatidia) that sometimes fail 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%). Since cone and 1° cells organize lattice cells and prevent their apoptosis, the ommatidial fusions and lattice cell defects are likely to be secondary to the problems with cone and 1° cells. It is concluded that loss of rdx function interferes with patterning in the pupal retina, probably at the level of the cone and 1° cells (Kent, 2006)

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. Moreover, hyperactivation of Hh responses (via insufficient ptc) causes a variety of morphological defects in the differentiating eye. To determine whether Hh and Ci affect ommatidial organization after the MF has passed, Lozenge:Gal4 (Lz:G4) was used to drive UAS:transgene expression behind the MF. The effects of Hh overexpression were limited to the anterior margin, with some reduction and with mildly disordered bristles and ommatidial packing. The pupal retinas showed mild defects near one edge, 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, caused loss of ommatidial bristles (40-50%) and mildly disordered ommatidial packing. The corresponding pupal ommatidia 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%). Since these defects were not seen with Lz:G4 alone, it is concluded that excess Ci can interfere with 1° and lattice cell patterning. To maximally stimulate Hh signaling, a Smo transgene was used whose high level of expression makes it a potent activator. Lz:G4; UAS:Smo generated rough eyes, with defects most pronounced in the posterior. At ~24 hours, pupal retinas were relatively normal, 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. 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 (Kent, 2006)

Taken together, these data show that overstimulation of the Hh pathway interferes with behavior of the non-neuronal cells in the pupal retina. These effects are subsequent to and independent of the role of Hh in the MF. The similarity of the phenotypes resulting from Hh overstimulation, from Ci overexpression and from rdx loss of function suggests that rdx affects retinal patterning via Ci. Without this fine-tuning by rdx, excess Ci generates excess Hh response, which ultimately interferes with retinal patterning (Kent, 2006)

Thus, rdx encodes a protein belonging to a phylogenetically conserved protein family of substrate-specific adaptors for Cullin3-based ubiquitin E3 ligases. 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. The 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 (Kent, 2006).

Hh is key regulator in human health. The haploinsufficiency of Ptc in humans and its activity as a morphogen in the spinal column 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. 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. Future studies will determine whether the Rdx orthologs SPOP or LOC339745 modulate Gli levels and Hh-mediated responses, and even contribute to cancer (Kent, 2006).

The Ci/Gli family of transcription factors mediates Hedgehog (Hh) signaling in many key developmental processes. This study identified a Hh-induced MATH and BTB domain containing protein (HIB) as a negative regulator of the Hh pathway. Overexpressing HIB down regulates Ci and blocks Hh signaling, whereas inactivating HIB results in Ci accumulation and enhanced pathway activity. HIB binds the N- and C-terminal regions of Ci, both of which mediate Ci degradation. HIB forms a complex with Cul3, a scaffold for modular ubiquitin ligases, and promotes Ci ubiquitination and degradation through Cul3. Furthermore, HIB-mediated Ci degradation is stimulated by Hh and inhibited by Suppressor of Fused (Sufu). The mammalian homolog of HIB, SPOP, can functionally substitute for HIB, and Gli proteins are degraded by HIB/SPOP in Drosophila. Evidence is provided that HIB prevents aberrant Hh signaling posterior to the morphogenic furrow, which is essential for normal eye development (Zhang, 2006).

This study demonstrates that Ci is regulated by dual ubiquitination systems that act in two distinct signaling states. In the absence of Hh, the Slimb-Cul1-based ubiquitin ligase targets CiFL for proteolytic processing to generate CiRep, and this process requires prior phosphorylation of CiFL by PKA, GSK3, and CKI. In the presence Hh, Ci phosphorylation is inhibited and Slimb-Cul1-mediated Ci processing is blocked. Hh further induces nuclear translocation and maturation of CiFL into CiAct, which activates Hh target genes and concomitantly induces the production of HIB. The HIB-Cul3-based E3 ligase then binds and targets CiAct for ubiquitination and degradation. The subcellular distribution of HIB is consistent with its acting in a nuclear E3 complex that targets nuclear Ci for degradation in Hh responding cells (Zhang, 2006).

Although these observations support the idea that HIB targets CiAct to downregulate Hh responses, it is not suggested HIB-Cul3 exclusively regulates CiAct. As a matter of fact, t Ci703, which is similar to CiRep, can be degraded by HIB, raising the possibility that HIB/SPOP could also modulate Hh signaling in certain development contexts, such as vertebrate limb development, that are regulated only by the repressor form of Ci/Gli. Nevertheless, these results do suggest that CiAct is a better substrate for HIB because (1) it is nuclear and (2) it may dissociate from Sufu or bind Sufu less tightly than latent forms of CiFL (Zhang, 2006).

Neither do this study rule out the possibility that a partially redundant mechanism(s) may exist that targets Ci for degradation in Hh-responding cells. It was noticed that HIB expression is downregulated near the D/V boundary and that hib mutant cells near the junction between the A/P and D/V boundaries failed to accumulate CiFL. Although the possibility cannot be ruled out that residual HIB in hib mutant cells could be responsible for degrading Ci, these observations raise the possibility that a HIB-independent mechanism may exist to degrade CiAct in response to high thresholds of Hh (Zhang, 2006).

The HIB-Cul3-based regulatory mechanism may provide a means to regulate the strength and/or duration of Hh pathway activity in a spatially or temporally regulated manner. It may also provide a mechanism to terminate Hh signaling in certain developmental contexts, as appears to be the case in Drosophila eye development. During Drosophila eye development, Hh signaling is required for the initiation and progression of the MF. Although Hh is expressed in differentiating cells posterior to the MF, Hh target genes are not expressed in differentiating cells. Evidence is provided that Hh signaling is downregulated through HIB-mediated degradation of Ci posterior to the MF. Furthermore, it was demonstrated that termination of Hh signaling posterior to the MF is essential for normal eye development. Indeed, misexpression of truncated forms of Ci that are resistant to HIB-mediated degradation or removal of HIB resulted in rough eye phenotypes. Hence, during eye development, differentiating cells turn on HIB to prevent aberrant Hh signaling by degrading Ci, which is essential for normal eye development (Zhang, 2006).

The HIB-Cul3-based regulatory mechanism identified in this study is likely to be evolutionarily conserved and may play a more general role in fine-tuning Hh responses in various developmental contexts. Indeed, it was found that both Gli2 and Gli3, the two primary transcription factors for the vertebrate Hh signaling pathway, are degraded by HIB in a fashion similar to Ci. There are multiple HIB homologs in vertebrates. For example, human and mouse each have two whereas zebrafish has three. This study demonstrates that mouse SPOP can functionally replace HIB in degrading Ci. It would be interesting to determine whether the other mouse HIB homolog, LOC76857, which shares with SPOP 94% and 83% identical amino acids in their MATH and BTB domains, respectively, can also functionally substitute for HIB, and whether the vertebrate homologs of HIB play any roles in modulating Hh responses in vertebrate development (Zhang, 2006).

A recent study demonstrated that Gli proteins possess dual degradation signals, one regulated by Slimb/β-TRCP-Cu11 E3 ligase and the other by an unknown mechanism (Huntzicker, 2006). Moreover, perturbation of Gli degradation mechanisms appears to greatly potentiate the ability of Gli proteins to induce tumor formation in transgenic animals. Similarly, this study found that hib mutation greatly enhances the ability of an active form of Ci to induce disc overgrowth. The finding that HIB/SPOP also regulates Gli stability raises the exciting possibility that SPOP and its paralogs may act as tumor suppressors whose loss of function could synergize with aberrant Gli activation to induce tumor formation (Zhang, 2006).

It is also noted that a recent study demonstrated that the Wnt pathway induces a Cul3-based ubiquitin ligase to attenuate pathway activity through degradation of the key pathway component Dishevelled (Angers, 2006). The finding that the Hh pathway also induces a Cul3-based ubiquitin ligase to attenuate pathway activity extends the many interesting parallels between these two pathways and suggests that similar mechanisms could be employed by other signaling pathways. Given the large number of BTB proteins present in the genomes of various species, it should be fruitful to explore the potential roles of BTB-Cul3 E3 ligases in various signaling pathways (Zhang, 2006).

Regulation of Hh signal transduction as Drosophila eye differentiation progresses

Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change (Baker, 2009).

As the morphogenetic furrow crosses the eye disc, Ci155 accumulates most highly just anterior to the morphogenetic furrow, even though Hh is secreted posterior to the morphogenetic furrow. The sharp reduction in Ci155 as the furrow passes is associated with a switch from Cul1-dependent processing to Cul3-dependent degradation (Ou, 2002). The posterior eye expresses rdx, encoding a BTB protein that couples Ci 155 to the Cul3 pathway (Kent, 2006; Zhang, 2006). This study identified the signals that induce rdx and that process Ci155 in the posterior eye (Baker, 2009).

The induction of rdx transcription couples Ci155 processing to Cul3 (Kent, 2006; Zhang, 2006). rdx transcription is regulated by both Hh signaling and Ras signaling, and there were distinctions between cell types. The smo mosaic and hhts2 experiments show that Hh signaling is continuously required for rdx transcription in unspecified cells with basal nuclei. In the absence of smo, EGFR-dependent rdx transcription occurs in differentiating photoreceptor cells only, not in unspecified cells. The egfr mosaics show that EGFR is essential for rdx transcription in all cells except the R8 photoreceptor class. Thus, EGFR-dependent differentiation was sufficient to induce rdx in photoreceptors even without Hh signaling, but Hh was not sufficient to induce rdx anywhere without EGFR signaling, except for the R8 cells. Undifferentiated cells might require both the Ras and Hh signaling pathways to induce rdx because the level of Ras signaling is lower in unspecified cells than in differentiating cells of the ommatidia. Alternatively, there may be a combinatorial requirement for both pathways in unspecified cells (Baker, 2009).

There has been some discussion of whether proteolysis of Ci155 by Cul-3 is regulated directly by Hh, as is Cul-1 dependent Ci processing. The current studies provide no support for this idea. In all the genotypes examined, Ci proteolysis correlates with the expression of rdx, and the simplest explanation is that the only effect of Hh on the Cul-3 pathway is through rdx transcription, directly in unspecified cells, and indirectly via EGFR-mediated differentiation in most specified cells (Baker, 2009).

Two mechanisms, acting in different cells, appear to reduce Hh responses through Ci155 after the furrow passes. One also occurs in wing development, where rdx is transcribed only by cells experiencing high Hh signaling levels close to the source of Hh. In wing development, rdx and the Cul3-pathway modulate the amount of Ci155 available for Cul1-dependent processing, lowering the maximum level of Ci155 activity at high Hh levels. Rdx could lower Ci155 levels in unspecified eye cells posterior to the furrow by this mechanism, in which an equilibrium between Hh-dependent induction of rdx, and rdx- and Cul3-dependent degradation of Ci155, leads to a lower level of Ci155 protein than anterior to the furrow. By contrast, in the specified, differentiating eye cells, rdx transcription becomes independent of Hh signaling, and Ci155 is degraded more completely (Baker, 2009).

If there is Hh signaling posterior to the furrow, as these studies find maintains rdx transcription in unspecified retinal cells, why are genes such as atonal that are induced by Hh signaling ahead of the furrow not also expressed posterior to the furrow? There are at least three possible explanations. First, rdx may dampen Ci155 accumulation in unspecified cells such that the threshold necessary for ato expression is not achieved posterior to the furrow. This is unlikely to be the sole explanation, since mutating rdx or cul3 permits Ci155 accumulation but does not lead to ectopic R8 specification, but it could contribute in conjunction with other mechanisms. Secondly, other genes may interfere posterior to the furrow. This could include egfr induction of Bar gene expression, since Bar genes antagonize ato expression. There seem to be multiple respects in which EGFR-dependent differentiation renders cells unable to continue anterior responses to Hh, and it is also envisaged that egfr might play a role in further mechanisms that modulate the response to Dpp signaling posterior to the furrow, should such mechanisms exist. Finally, recent evidence suggests that induction of ato by Hh is not so simple as the induction of a target gene above a threshold in a morphogen gradient, but depends indirectly on Hh repressing Eyeless and activating Sine Oculis, so that these transcription factors are coexpressed and turn on ato only in a domain ahead of the furrow. In this case, persistent Hh signaling would not be expected to activate ato expression once Ey had been repressed (Baker, 2009).

Recently, Hh has been discovered to induce compensatory proliferation in response to eye disc cell death, a further example of post-furrow Hh function. The current results now suggest the model that loss of EGFR-dependent rdx expression elevates Ci155 locally to permit Hh responses when photoreceptor cells that secrete EGFR ligands are lost. Consistent with this idea, loss of rdx or cul3 also result in proliferation of eye disc cells (Baker, 2009).

The regulation of rdx expression and thus degradation of Ci by Cullin-3 may not be sufficient to explain Ci regulation posterior to the furrow. In order for Ci155 to be stable, as observed in cul3 mutant clones and egfr mutant clones, Ci155 must escape processing to Ci75 by Cul-1. Ahead of the furrow, and in most other tissues, rdx is not expressed, Ci is not coupled to Cul3, and Ci155 is stabilized wherever Hh inhibits Smo and the Cul1 pathway. The observation that Ci155 is stable in cul3 clones, or in the genotypes where rdx is not expressed, shows that Ci155 escapes processing by the Cul1 pathway in the posterior eye as well, but this is not due to Hh. Ci155 accumulates in smo egfr mutant clones that do not express rdx and cannot respond to Hh (Baker, 2009).

One model would be that once rdx is induced, Ci155 is sequestered and not available to be processed by Cul1. This model cannot explain why Ci155 accumulates in egfr clones that lack rdx expression, where Ci155 should be available for Cul1. Therefore Ci155 must escape Cul1-mediated processing in the posterior eye by a distinct mechanism. This could be explained by the induction of a component distinct from Rdx that inhibits the processing of Ci155 by Cul1, or sequesters Ci155. It is equally possible that a component essential for processing of Ci155 by Cul1 is repressed posterior to the morphogenetic furrow (Baker, 2009).

Previous studies show that Ci155 never accumulates in smo tkv clones that are unable to respond to either Hh or Dpp signaling. Clones of cells unable to respond to Dpp, but able to respond to Hh and Ras, show only a subtle change in Ci155 labeling. These previously published observations suggest that Ci155 remains a target of Cul1 in the absence of both Dpp and Hh signaling, perhaps through failure to transcribe or repress transcription of a gene that modulates Ci155 proteolysis by Cul1 posterior to the furrow (Baker, 2009).

It is now possible to account for why smo clones affect Ci155 levels differently from cul3 clones, a previously puzzling observation. In cul3 clones, or egfr clones that do not express rdx, the Cul3 pathway cannot degrade Ci155 and the Cul1 pathway is inactivated posterior to the furrow exactly as in wild type discs, so Ci155 accumulates. In smo clones, Ci155 transiently accumulates in those cells in which processing by Cul1 has been lost but rdx not yet induced. In such cells, Ci155 is not coupled to any cullin, and is stable. Eventually, differentiation spreads into the posterior of smo clones, leading to rdx expression, and Cul3-dependent Ci degradation. If differentiation and rdx expression are prevented, as in smo egfr clones, then Ci155 remains stable. Because there is a delay in expressing rdx in smo clones compared to wildtype, Ci155 is not subject to Cul3-mediated processing as soon as in wild type, and there is a period when Ci155 has been uncoupled from Cul1-processing but not yet coupled to the Cul3 pathway. It is during this period that Ci155 accumulates in smo mutant cells (Baker, 2009).

These findings help explain how a wave of differentiation moves across the eye disc uni-directionally. Hh, secreted from differentiating photoreceptor cells, must be present at highest concentrations posterior to the furrow. Indeed, ahead of the furrow Ci155 is stabilized in a decreasing posterior-to-anterior gradient, consistent with a gradient of Hh protein coming from a source posterior to the furrow. Yet, the cell-autonomous responses to Hh signaling that are seen ahead of the furrow, such as cell cycle arrest and atonal expression, do not occur posterior to the furrow, where Ci is rendered unstable by Rdx and Cul3, induced both directly by Hh itself, and indirectly by the photoreceptor differentiation that is largely induced by EGFR posterior to the furrow (Baker, 2009).

There are other examples where Hh-secreting tissues are not the targets of Hh signaling. For, example, in Drosophila wing development, anterior compartments respond to Hh secreted by posterior compartments, but posterior compartment cells do not respond because ci transcription is repressed by the posterior-specific protein Engrailed. In vertebrate development, notochord cells express Shh but the responses seen in the nearby spinal cord are not seen in notochord. Such segregation of Hh-producing cells from fields competent to respond to Hh makes sense, if the purpose of Hh signaling in development is to pattern new body regions. Hh signaling is also deregulated in many tumors. Whether any of these tumors activate Hh signaling by affecting GLI protein stability, or other normal down-regulatory mechanisms, remains to be seen. In any case, mechanisms that render cells unresponsive to Hh by coupling Ci155 to the proteosome might prove useful in the treatment of cancers that depend on Hh signaling (Baker, 2009).


REFERENCES

Search PubMed for articles about Drosophila Roadkill

Angers, S., et al. (3006). The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Nat. Cell Biol. 8(4): 348-57. PubMed ID: 16547521

Baker, N. E., Bhattacharya, A. and Firth, L. C. (2009). Regulation of Hh signal transduction as Drosophila eye differentiation progresses. Dev. Biol. 335(2): 356-66. PubMed ID: 19761763

Dai P, Akimaru H, 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(6): 917-28. PubMed ID: 12791275

Huntzicker, E. G., et al. (2006). Dual degradation signals control Gli protein stability and tumor formation. Genes Dev. 20(3): 276-81. PubMed ID: 16421275

Kent, D., Bush, E. W. and Hooper, J. E. (2006). Roadkill attenuates Hedgehog responses through degradation of Cubitus interruptus. Development 133: 2001-2010. PubMed ID: 16651542

Mains, P. E., et al. (1990). Mutations affecting the meiotic and mitotic divisions of the early Caenorhabditis elegans embryo. Genetics 126(3): 593-605. PubMed ID: 2249759

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. PubMed ID: 12231629

Pintard, L., et al. (2003). The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425(6955): 311-6. PubMed ID: 13679921

Xu, L., et al. (2003). BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425(6955): 316-21. PubMed ID: 13679922

Zhang, Q., et al. (2006). A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor. Dev. Cell 10: 719-729. PubMed ID: 16740475


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date revised: 10 February 2010

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