BIOLOGICAL OVERVIEW

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis (Lee, 2002).

Communication is essential for multicellular development. Intercellular signals regulate the timing and pattern of cellular events such as growth, division, movement, and differentiation, allowing large groups of cells to behave in a coordinated manner during events such as organogenesis. Cell-cell signaling can regulate the activity of cells at many different levels, including gene expression, cell division and motility. Within the receiving cell, signal transduction cascades transform the reception of ligand at the cell surface into the proper cellular response. This may involve regulation of protein activity by post-translational modification or by destruction or proteolytic processing. Ubiquitination, the covalent addition of a multimeric chain of the 76-amino-acid Ubiquitin (Ub) protein, is the most common intracellular signal for proteolysis. Ubiquitination is a multi-step process that begins with the activation of a Ub molecule by an E1 or Ub-activating enzyme. The activated Ub is transferred to an E2 enzyme, which is then responsible, either directly or indirectly, for attaching the Ub to a substrate protein. Specificity of the ubiquitination reaction is achieved at the level of the E3 ubiquitin ligase, which is thought to directly bind the substrate. Many such ligases exist and have been classified into families based on the structure of the ubiquitination domain. HECT-domain E3 ligases directly attach Ub to a substrate, while RING domain E3s direct specific substrate ubiquitination by the E2 (Lee, 2002).

A small number of signaling pathways appear to direct most developmental processes. In Drosophila, the BMP family member Decapentaplegic (Dpp) and the founding member of the Hedgehog family (Hh) are used repeatedly throughout development. One function of Hh and Dpp is to direct the progressive differentiation of the eye imaginal disc. In the second instar eye disc, hh is expressed in a complex pattern in both the disc proper and the peripodial membrane, before being refined to a small domain centered on the dorsoventral midline of the disc's posterior margin. Hh signals more anterior cells to express dpp and atonal (ato), which encodes the bHLH transcription factor required for the formation of the R8 'founder' photoreceptor in each cluster. These cells then differentiate as photoreceptors. These cells also express hh, allowing the cycle to propagate toward the anterior of the disc. dpp is expressed in the morphogenetic furrow, an indentation at the front of differentiation, where it is responsible for coordinating the timing of differentiation through synchronization of the cell cycle. Loss of either Hh or Dpp blocks the initiation of differentiation, while loss of both blocks progression. While it is known that Hh activates dpp expression in the furrow, it is not known how hh expression is controlled, nor how dpp is turned off in cells leaving the furrow (Lee, 2002).

To identify novel genes contributing to pattern formation in the Drosophila eye disc, a mosaic genetic screen was carried out in which homozygous mutant clones of cells were generated specifically in the eye disc. In this screen, four alleles were recovered of hyd, which encodes a large protein containing a HECT family E3 ubiquitin ligase domain (Callaghan, 1998; Mansfield, 1994). hyd was initially isolated in a screen for mutations causing imaginal disc overgrowth (Martin, 1977). Adult eyes containing hyd mutant clones show extensive overgrowth of the eye tissue surrounding a hyd clone, although the clones themselves did not persist to adulthood. In the third instar eye disc, premature photoreceptor differentiation is observed, visualized by expression of the markers Elav and Neuroglian, in hyd mutant clones in a region just anterior to the morphogenetic furrow. Very rarely, differentiation is seen in clones lying near the anterior margin. The spatial restriction of this phenotype is consistent with work demonstrating the existence of a preproneural zone anterior to the furrow. Ectopic differentiation spreads beyond the borders of the hyd mutant clones into the surrounding wild-type tissue. The proneural transcription factor Atonal (Ato) is also misexpressed within and surrounding hyd clones anterior to the furrow. The overgrowth of wild-type tissue in adult eyes is visible in third-instar imaginal discs as folding and distortion of the disc epithelium surrounding hyd clones. This effect is more widespread than the ectopic differentiation phenotype, appearing in more anterior regions of the disc (Lee, 2002).

Growth and differentiation of cells in the eye disc both depend on Hh and Dpp secreted by more posterior cells. The non autonomous differentiation and overgrowth caused by hyd clones suggested that the clones might be producing one or both of these molecules; a similar phenotype is produced by ectopic expression of hh or activation of the hh pathway ahead of the furrow. Therefore hh expression was examined in hyd clones using both an enhancer trap line and antibody staining. hyd mutant clones anterior to the furrow indeed expressed hh-lacZ and Hh protein earlier than surrounding wild-type cells. When hyd clones were made in a Minute background, thereby reversing the growth disadvantage of hyd mutant tissue, hh-lacZ expression was observed throughout the eye disc. The widespread hh expression in hyd/Minute clones may explain the accelerated differentiation and small size of these eye discs (Lee, 2002).

The ectopic hh expression observed in hyd mutant clones could be a consequence, rather than a cause, of their premature differentiation. To rule out this possibility, hyd;ato double mutant clones were generated. Clones mutant for ato cannot form the R8 photoreceptor, which is itself required for the recruitment of photoreceptors R1-R7. Thus ato mutant clones do not differentiate, unless a cell at the margin of a clone is recruited by a neighboring wild-type R8. It was confirmed that ato single mutant clones do not express hh. While hyd;ato mutant clones do not differentiate, they nevertheless expressed hh-lacZ and are capable of directing ectopic differentiation in surrounding wild-type tissue. This demonstrates that loss of hyd function has a direct effect on hh expression that is not simply due to differentiation (Lee, 2002).

As an E3 ubiquitin ligase, Hyd is likely to promote the degradation of one or more proteins. Based on the ectopic expression of hh in hyd mutant clones, it was hypothesized that Hyd was acting in the anterior of the third instar eye disc to prevent premature expression of hh. hyd is expressed in proliferating tissues in the embryo and larva, but its expression in the eye disc had not been described in detail (Mansfield, 1994). Using in situ hybridization, it was found that hyd RNA is highly expressed in the anterior of the eye imaginal disc, especially around the dorsoventral midline. hyd is expressed at lower levels towards the dorsal and ventral margins but was still restricted to the anterior. This expression pattern is consistent with a role for hyd in preventing the premature expression of hh (Lee, 2002).

If the ectopic differentiation and overgrowth associated with hyd clones is due to ectopic hh expression, it should be possible to rescue this phenotype by removing hh function from the clones. The phenotype of hyd;hh double mutant clones was determined. Similar results were obtained with three hh alleles, hh^rJ413, hh^ts2 grown at 29°C, and the null allele hh^AC. While hh mutant clones appear wild type unless located on the margin of the eye disc, hyd;hh double mutant clones show a partial suppression of the hyd phenotype. Ectopic photoreceptors are no longer present in or around hyd;hh clones, and hyd;hh double mutant clones generated in a Minute background have only a few photoreceptors associated with the remaining wild-type tissue. Thus hyd mutant tissue requires Hh in order to differentiate. However, some hyd;hh mutant clones are still able to stimulate proliferation of surrounding tissue, leading to overgrowth of the adult eye (Lee, 2002).

dpp has also been shown to stimulate proliferation in the eye disc. The remaining non autonomous overgrowth induced by hyd;hh double mutant clones might therefore be due to ectopic expression of dpp. Using a dpp-lacZ reporter construct, ectopic dpp expression was observed in and around hyd mutant clones anterior to the furrow. Ectopic dpp expression appears more widespread than ectopic hh expression and occurs in more anterior regions of the disc, raising the possibility that dpp misexpression is not merely induced by ectopic Hh in the clones but is an independent consequence of the loss of hyd. Indeed, dpp is still expressed in some hyd;hh mutant clones, although its expression is limited to clones close to the morphogenetic furrow. Thus in the absence of hyd function, dpp expression is no longer strictly regulated by Hh signaling (Lee, 2002).

The persistence of dpp expression in hyd;hh double mutant clones has suggested that dpp regulation by Hyd is at least partially independent of hh. One molecule that is known to transcriptionally regulate both hh and dpp is Ci. In the wing imaginal disc it has been shown that Hh controls dpp expression both by suppressing the production of Ci₇₅, which inhibits dpp expression, and by activating Ci₁₅₅, which activates dpp expression. In addition, Ci₇₅ inhibits hh expression in the anterior compartment of the wing disc; ci transcription is repressed by en in the posterior compartment, allowing hh to be expressed there. To test whether hyd acts through Ci to affect hh and dpp expression in the eye disc, the expression of a truncated constitutive repressor form of Ci (Ci₇₆) was driven specifically in hyd mutant clones using the MARCM system. dpp-lacZ is no longer expressed in hyd clones expressing UAS-ci₇₆ in or anterior to the furrow. However, Ci₇₆ does not prevent the ectopic expression of hh in hyd mutant clones. Continued hh expression in these clones sometimes led to ectopic differentiation in tissue surrounding the clone. Thus Ci₇₆ is sufficient to block dpp but not hh expression in hyd clones, suggesting that Hyd regulates hh and dpp expression through at least partially independent mechanisms (Lee, 2002).

If the overgrowth phenotype induced by hyd;hh double mutant clones is indeed due to their misexpression of dpp, it should be blocked by introducing Ci₇₆ into the mutant cells. Indeed, when hyd;hh mutant clones expressing UAS-ci₇₆ were generated, the adult eyes no longer exhibited any overgrowth. Thus all the ectopic growth and differentiation caused by loss of hyd in the eye can be attributed to independent effects on hh expression and activation of the Hh pathway (Lee, 2002).

If hyd regulates dpp expression by altering Ci activity, loss of hyd should lead to upregulation of full-length, active Ci. Increased levels of full-length Ci are indeed observed in hyd mutant clones in the anterior of the eye disc. However, this could be due to misexpression of hh in the same clones. To determine whether hyd has a direct effect on Ci, hyd;hh double mutant clones anterior to the morphogenetic furrow were examined. High levels of full-length Ci accumulated in these clones, confirming that Hyd normally reduces Ci levels independently of Hh activity (Lee, 2002).

Hyd might act directly on Ci to promote its proteolytic cleavage or degradation. An alternative possible target for Hyd activity is Smoothened (Smo). Smo is a transmembrane protein that acts positively in Hh signaling. Smo levels are kept low by the receptor protein Patched (Ptc) in the absence of Hh, but Smo is stabilized and localized to the membrane when Hh binds to Ptc. To test whether Hyd normally contributes to Smo degradation, eye discs containing hyd;hh clones were stained with Smo antibody. No Smo accumulation is apparent in the clones. Thus loss of hyd leads to accumulation of full-length Ci without altering the level of Smo (Lee, 2002).

Since hyd is expressed in the wing disc and is required for its normal growth (Mansfield, 1994), whether its effects on wing development might also be mediated by alterations in hh expression and Ci levels was examined. In wild-type wing discs, hh is expressed uniformly throughout the posterior compartment of the wing pouch, while dpp is expressed in the anterior compartment in a stripe along the AP border. Ci₁₅₅ is present at high levels in a similar stripe at the AP border and at lower levels elsewhere in the anterior compartment. Expression of hh, dpp and Ci₁₅₅ in hyd clones remains restricted to the correct compartment. However, some hyd mutant clones in the posterior compartment express elevated levels of hh-lacZ. This misexpression of hh is correlated with a rounded shape and apparent overgrowth of the clones. The only known regulator of hh expression in the wing disc is Ci, which is restricted to the anterior compartment by En-mediated repression; Ci₇₆ represses hh there. These results suggest that a Ci-independent activator of hh expression must be present in the posterior compartment and kept in check by Hyd activity (Lee, 2002).

In addition, Ci₁₅₅ is upregulated in anterior hyd mutant clones. In contrast to the eye disc, no hh misexpression was observed in anterior hyd clones; thus Ci upregulation in hyd clones must be independent of hh. This is consistent with findings that hyd regulates Ci and hh independently in the eye disc. Smo levels were not significantly increased in hyd mutant clones in the anterior compartment of the wing disc, suggesting that as in the eye disc, hyd affects Ci independently of Smo (Lee, 2002).

Thus hyd acts as a negative regulator of both hh and dpp expression in the anterior of the Drosophila eye disc. Loss of hyd function leads to the ectopic expression of both genes, resulting in non autonomous overgrowth of the disc and premature photoreceptor differentiation that propagates into the surrounding tissue. The ability to suppress this overgrowth by preventing both expression of hh and activation of the Hh pathway indicates that the hyperplastic effects of hyd in the eye are entirely mediated by Hh signaling. This is probably not the case in the wing disc; in this tissue hh and dpp expression are also blocked by hyd, but hyd clones in the posterior compartment can autonomously overgrow without expressing Ci, the transcription factor required for the response to Hh. hyd may thus have an independent effect on the cell cycle in wing disc cells. The human homolog of hyd, EDD, is located in a chromosomal region that is disrupted in a variety of cancers (Callaghan, 1998). It will be of interest to determine whether loss of hyd is responsible for any of these syndromes, and if so, whether the tumorous growth can be attributed to misregulation of hh or dpp homologs (Lee, 2002).

Little is known about the control of hh expression in the eye disc. In the embryo and other imaginal discs hh expression is controlled by en, which defines the posterior compartment in a lineage-dependent manner. The eye disc has no anterior-posterior compartment boundary, and loss of en function in the eye has no effect. Since hh expression is repressed by Ci₇₆ in the anterior wing disc, it seemed possible that this was also the case in the eye disc; Hh could then activate its own expression in more anterior cells by blocking Ci cleavage. However, it was not possible to prevent hh expression by providing Ci₇₆ to hyd mutant cells in the eye disc, although this does suffice to repress hh target genes such as dpp. In agreement with this result, ci mutant clones anterior to the furrow do not induce ectopic differentiation, indicating that loss of the repressor form of Ci is not sufficient to allow hh transcription in the eye disc. hyd must therefore be a component of the Ci-independent mechanism that restricts hh expression (Lee, 2002).

Regulation of hh by hyd is also clearly independent of Ci in the wing disc, since loss of hyd leads to hh upregulation in the posterior compartment, where Ci is not present, and not in the anterior compartment. The Groucho (Gro) corepressor has been proposed to contribute to a Ci-independent mechanism of hh repression in cells close to the compartment boundary. However, the effects of loss of hyd differ from those of loss of gro, which affects only the anterior compartment of the wing disc and promotes excessive photoreceptor differentiation only posterior to the furrow in the eye disc, suggesting that a third mechanism of hh regulation may exist. hh expression may not be merely a default state resulting from the absence of the Ci repressor and Gro, but may require another activator, the levels of which are normally kept in check by Hyd (Lee, 2002).

Control of dpp expression by hyd, in contrast, appears to be mediated by Ci. Ci₁₅₅ is upregulated in hyd mutant cells in the eye disc in a hh-independent manner, and ectopic dpp expression in these cells can be blocked by Ci₇₆. In the wing disc, dpp misexpression is limited to the ci-expressing anterior compartment, and is again associated with upregulation of Ci₁₅₅. Thus hyd acts on Hh signaling as well as hh expression, preventing full activation of the Hh pathway in anterior cells (Lee, 2002).

Hyd independently regulates hh and dpp expression, suggesting either that Hyd has multiple substrates or that its substrate has multiple functions. dpp expression in hyd clones is blocked by Ci₇₆, placing the effect of Hyd on dpp upstream or at the level of Ci activity. Ci₁₅₅ but not Smo accumulates to high levels in hyd;hh mutant cells in the eye disc and in anterior cells in the wing disc; thus Hyd may act on Ci itself, on a component of the Hh pathway between Smo and Ci, or on a nuclear cofactor that stabilizes Ci. Consistent with an effect on Ci or a cofactor, Hyd protein has been detected in both the cytoplasmic and nuclear compartments (Mansfield, 1994). The human Hyd homolog EDD appears to be predominantly localized in the nucleus, where it interacts with the progesterone receptor and DNA topoisomerase II-binding protein (Henderson, 2002; Honda, 2002). Ubiquitination can function to enhance the potency of transcriptional activation domains; however, the ectopic gene expression observed in hyd mutant clones would be difficult to explain by this mechanism (Lee, 2002).

The SCF ubiquitin ligase complex containing the F-box protein Slmb and the RING finger protein Roc1 has been implicated in the ubiquitination of Ci that mediates its processing to Ci₇₅. There are several possible explanations for the apparent overlap between Slmb and Hyd functions. Slmb may directly ubiquitinate Ci, while Hyd acts on another substrate; the more dramatic effect of slmb than hyd clones on Ci accumulation argues for this possibility. However, it is also possible that Hyd, rather than Slmb, is the direct ubiquitin ligase for Ci. The consensus sequence for Slmb is not present in Ci, although it has been suggested that several weakly matching sequences might suffice for its recognition. In addition, two groups have obtained inconsistent genetic evidence as to whether slmb acts upstream or downstream of smo and protein kinase A (PKA). It is unlikely that Hyd and Slmb carry out the same process in different cells, since hyd is expressed throughout the wing pouch (Mansfield, 1994), and slmb is clearly active in the same region, although its expression has not been examined. Finally, Hyd and Slmb could both act on Ci, either additively or with different outcomes. For example, ubiquitination by Slmb promotes processing of Ci to Ci₇₅, while ubiquitination by Hyd might promote complete degradation of the Ci protein. Unfortunately, it was not possible to obtain large enough quantities of hyd;hh mutant tissue to test this possibility directly by Western blotting. The lack of hh misexpression in hyd clones in the anterior compartment of the wing disc suggests that these cells still contain Ci₇₅ as well as Ci₁₅₅; loss of hyd may therefore stabilize both forms of the Ci protein rather than altering their ratio. However, this is not a definitive test of hyd function, as loss of PKA does not lead to ectopic hh-lacZ expression despite its effect on Ci processing. Hyd-mediated degradation of both forms of Ci, or its redundancy with Slmb for Ci cleavage, would explain the limited effect on dpp expression in hyd;hh double mutant clones in the eye disc and in hyd mutant clones in the wing disc; dpp misexpression in these cases is restricted to a region in which endogenous Hh may contribute to altering the ratio of the two forms of Ci (Lee, 2002 and references therein).

Ubiquitination is a mechanism commonly used to regulate protein activity by targeting proteins for degradation or processing, during the cell cycle and in a number of signaling pathways (reviewed in Ciechanover, 2000). The Slmb-containing SCF complex is also required for the degradation of Armadillo (Arm)/ß-catenin, allowing Wnt signaling, as well as for the degradation of IkappaB. Hyd is unlikely to act on Arm in the eye disc, since Arm accumulation would prevent the ectopic photoreceptor differentiation seen in hyd mutant clones; this may indicate another difference in the substrate specificity of Hyd and Slmb. However, it is possible that hyd affects Wg signaling in the wing disc, since hyd mutant clones can induce ectopic expression of the Wg target gene scute (Kazuhito Amanai, unpublished data cited in Lee, 2002). The HECT domain ligases Smurf1 and Smurf2 are important antagonists of BMP signaling, promoting downregulation of both Smads and receptors. Itch/Suppressor of deltex, another HECT domain ligase, ubiquitinates Notch. In addition, nuclear Notch is degraded by Sel-10, while the ligand Delta is the target of ubiquitination by Neuralized. Placement of hyd within the Hh pathway and upstream of hh expression expands this growing list of cases in which signaling pathways are regulated by ubiquitination (Lee, 2002).

GENE STRUCTURE

cDNA clone length - 9053

Exons - 19

Bases in 3' UTR - 237

PROTEIN STRUCTURE

Amino Acids - 2895

Structural Domains

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. Hyd is likely to act as an E3 ubiquitin ligase; its human homologue has been shown to ubiquitinate at least one substrate in vitro (Honda, 2002). The substrate specificity of HECT domain E3 ubiquitin ligases appears to reside within their unique N-terminal domains (Ciechanover, 2000), making it difficult to predict the sequence or structure recognized by Hyd. The only potential clue is that Hyd contains a peptide-binding domain homologous to the C-terminus of poly(A)-binding protein (PABP) (Callaghan, 1998; Kozlov, 2001); the human HYD protein can interact with Paip1, a binding partner of PABP (Craig, 1998; Deo, 2001; Lee, 2002 and references therein).

date revised: 22 November 2002

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