Laboratoire de Génétique et Physiologie du Développement, UMR 9943 C.N.R.S.-Université, I.B.D.M. CNRS-INSERM-Université de la Méditerranée, Campus de Luminy Case 907, F-13288 Marseille, Cedex 09, France
* Present address: Institut für Molekularbiologie, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland
Author for correspondence (e-mail: kerridge{at}ibdm.univ-mrs.fr)
Accepted 14 December 2001
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SUMMARY |
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Key words: Drosophila, Grunge, Teashirt, Legs, Atrophin-1-like proteins, Metastasis-associated proteins, Segmentation
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INTRODUCTION |
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During the first stages of embryonic development, the tsh expression pattern in the ectoderm is very dynamic and at gastrulation tsh mRNAs are homogeneously distributed in the presumptive trunk region. Genetic evidence suggest that tsh is activated and restricted in the trunk of early embryos by a combination of maternal and segmentation genes (Röder, 1992). Maternal and segmentation genes act either as repressors or activators of tsh transcription, in order to delimit the boundaries of tsh expression domains. The pair-rule gene fushi tarazu (ftz) activates tsh expression directly in even-numbered parasegments in the embryonic ectoderm (Coré et al., 1997
). Later during embryogenesis, Tsh expression is maintained by homeotic genes (Röder, 1992
) and autoregulation (Coré et al., 1997
). Gallet et al. (Gallet et al., 1998
) have shown that Wg signalling is necessary to accumulate a high amount of Tsh protein in the nucleus in order to give a trunk specific output for Wg signalling.
During Drosophila embryogenesis, a group of epithelial cells from each thoracic hemisegment will invaginate to form the primordia of the adult legs. These epithelia proliferate during larval life to give rise to the imaginal discs, which undergo morphogenesis and differentiation during metamorphosis (Bryant, 1978; Cohen, 1993
). In the leg discs, hedgehog (hh) is transcribed in the posterior compartment and its protein is secreted to the anterior part to induce wingless (wg) and decapentaplegic (dpp) transcription in ventral and dorsal domains, respectively. Wg and Dpp proteins, which are homologous to Wnt1 and TGFß in vertebrates, specify the ventral and dorsal cell fates, respectively, and via mutual repression establish the dorsoventral and the proximodistal axes of the leg. These signalling proteins impose progressively restricted patterning decisions on neighbouring cell groups, via independent transduction pathways, to give largely invariant appendages whether in Drosophila or vertebrates (Basler and Struhl, 1994
; Brook and Cohen, 1996
; Diaz-Benjumea and Cohen, 1994
; Ingham and Fietz, 1995
; Jiang and Struhl, 1996
; Klingensmith et al., 1994
; Lecuit and Cohen, 1997
; Massague, 1998
; Penton and Hoffmann, 1996
; Wodarz and Nusse, 1998
; Wolpert, 1969
; Yang and Niswander, 1995
).
Several genes have been isolated that exhibit differential, proximodistal patterns of expression in the imaginal discs (reviewed by Abu-Shaar and Mann, 1998; Couso and Bishop, 1998
; Gonzalez-Crespo et al., 1998
; Wu and Cohen, 1999
). The earliest is the Distal-less (Dll) gene product, which encodes a protein with a homeodomain and is expressed in the leg primordia of embryos before the invagination of the epithelia (Cohen et al., 1989
; Cohen and Jürgens, 1989
). Dll protein is crucial for the formation of specific distal parts of the legs (Cohen, 1990
), as loss of function gives rise to an excess of proximal leg tissue at the expense of distal patterns. The Tsh protein is expressed in a largely complementary way to Dll, in the proximal leg, where it is required for the identity of the coxa and trochanter, and for the formation of a boundary to Dll-expressing cells. In ventral cells, this boundary formation is dependent on Wg signalling (Erkner et al., 1999
).
We have carried out an in vivo screen in order to isolate regulators of tsh, and have identified a new gene called Grunge (Gug). The putative Gug protein shows similarities with human arginine-glutamic acid dipeptide repeat, vertebrate Atrophin-1 and Metastasis-associated-1 (Mta1)-like proteins. Mutations in Gug indicate that it is required for normal segmentation of embryos and patterning of the imaginal discs. In Gug embryos, the expression of segmentation genes and tsh expression is affected. A mosaic analysis of Gug mutations in the leg shows that, despite its ubiquitous pattern of expression, Gug is required for global ventral and proximal patterning of the leg, where it acts as a positive regulator of tsh.
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MATERIALS AND METHODS |
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Cloning and sequencing of the Gug region
DNA was isolated from l(3)PZGug3928/MKRS flies in order to construct a genomic library. Partial Sau3A genomic fragments were cloned into phage (Sambrook et al., 1989
). lacZ DNA probes were employed to isolate genomic DNA from the Gug gene region from this library. Subsequent chromosomal walking gave overlapping phages from the region. Genomic fragments from the walk were used to probe Northern blots in order to identify putative transcription units of the Gug gene. Complementary DNAs were isolated from an embryonic library (Zinn et al., 1988
). The EST clones LD10989 and LD15383 from the Berkeley Drosophila Genome Project (BDGP) were used. Sequencing of the Gug cDNAs was performed by Genome Express (Grenoble, France). The sequence is available with the GenBank Accession Number AF217844. Sequence alignments and calculations of sequence similarity were constructed using the Network Protein Sequence Analysis ClustalW at the Pole Bio-informatique Lyonnais (http://pbil.ibcp.fr/cgbin/npsa_automat.plpage=/NPSA/npsa server.html) with manual editing.
Germline clones
Germline clones homozygous for Gug mutations were induced using the dominant autosomal germline clone technique (Chou and Perrimon, 1996). y w hsFLP/y w hsFLP; GugX P(w+ FRT2A)/TM6C, Sb (where X=S2, 35, 1207D6, this work and R. Finkelstein, unpublished) females were crossed to P{w+mC=ovoD118}3L1 P{w+mC=ovoD118}3L2 P(w+ FRT2A)/TM3 males. Their progeny were heat shocked for 1-2 hours at 36°C in a water bath to induce germline clones homozygous for individual Gug alleles. y w hsFLP/+; GugX P(w+ FRT2A/ P{w+mC=ovoD118}3L1 P{w+mC=ovoD118}3L2 P(w+ FRT2A) females were then crossed to wild type, GugX/TM6C, Sb or, in the case of embryos used for in situ hybridisation, with Gug35/TM3ftzlacZ males.
Sense and antisense injection
Sense and antisense mRNA were synthesised using T3 or T7 RNA polymerase (Sambrook et al., 1989). RNA was injected into preblastoderm embryos and the larval cuticles examined 48 hours later.
Mosaic analyses of Gug mutations in the adult leg
The FRT/FLP technique (Golic, 1991; Xu and Rubin, 1993
) was used to produce clones of Gug (Gug35, Gug1207D6 or GugS2) or Gug+ cells induced by heat shock at 36°C for 1 hour in a water bath at different developmental stages from 24 to 144 hours after egg laying. Clones were induced in larvae of the genotype y w P(hs-FLP, ry+); mwh jv Gug P(w+ FRT2A)/Dp(1;3)scJ4, y+ M(3)i55 P(w+ FRT2A) (FlyBase, 1999
; Lindsley and Zimm, 1992
; Xu and Rubin, 1993
), in order to analyse Gug clones with a growth advantage (Morata and Ripoll, 1975
), and were marked by yellow, multiple wing hairs and javelin.
In discs, Gug cells were detected by absence of the Myc tag or green fluorescent protein (GFP), which are lost after mitotic recombination. All stocks carried balancers with the dominant Tubby mutation (Lindsley and Zimm, 1992), allowing larvae of the correct genotype [y w P(hs-FLP, ry+); Gug35 FRT80/ M(3)i55 P(w+, hs-cMYC) FRT80 or Gug35 FRT2A/ubiGFP FRT2A (Flybase, 1999
)] to be selected for dissection of imaginal discs.
For the analysis of wingless and decapentaplegic expression in Gug clones in leg discs, CyOwgLacZ or dppLacZ chromosomes were incorporated into the crosses described above.
Production of anti-Gug antibodies and immunohistochemical staining
Antibodies were raised in rabbits against the extreme C-terminal peptide (RQSLHDQYFRQRPR) of the putative Gug protein by Neosystem (Strasbourg, France). Mouse anti-Dll (from Stephen Cohen) was used at 1/1000; mouse anti-ß-gal (Promega) was used at 1/500; rat anti-Tsh was used at 1/600 (Gallet et al., 1998); and anti-Gug at 1/250. Anti-Myc (9E10 mouse or rabbit; Santa Cruz Biotechnology) was used at 1/100. Secondary FITC- or TRITC-coupled antibodies (Jackson laboratories) were used at 1/100. Disc fixation and fluorescence labelling was performed as described by Gallet et al. (Gallet et al., 1998
) and Xu and Rubin (Xu and Rubin, 1993
). A Zeiss Confocal Microscope was used for this analysis.
In situ expression analysis
Gug, Kr, hb, kni, ftz and lacZ antisense RNA probes were synthesised. Homozygous Gug embryos were identified by the absence of expression of the ftzlacZ reporter gene carried by TM3.
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RESULTS |
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We cloned the genomic DNA surrounding the insertion point of P(LacZ, ry+)Gug3928 and used these genomic probes to confirm the location of the Gug gene to 66D1-2 on chromosome 3 (not shown). New mutations in the Gug gene were obtained by jump start mutagenesis by mobilisation of the P(LacZ, ry+)Gug3928 element (Materials and Methods). Wild-type revertant chromosomes, presumably with precise excision events, suggest that the P-element is responsible for the Gug mutation. Gug35 corresponds to an imprecise excision of the P-element and a deletion of genomic DNA at the point of insertion (Fig. 2A). All Gug alleles have similar properties, dying as late embryonic zygotic lethals. One exception is P(LacZ w+)Gugj5A3, which dies at the third larval instar stage as a homozygote.
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Wild-type embryos consist of head, trunk and tail segments. In the larvae, the most obvious segments are those of the thorax and abdomen (trunk), each segment consisting of anterior denticle belts and posterior naked cuticle (Fig. 3A). Loss of zygotic Gug activity affects only head morphogenesis (Fig. 3B). In order to test for the maternal contribution of Gug, we have induced germline clones homozygous for different Gug alleles (Materials and Methods). All tested alleles (GugS2, Gug35, P(LacZ, ry+)Gug3928) give essentially similar phenotypes. When fertilised by wild-type sperm, Gug germline clones give rise to embryos with severe segmentation defects (Fig. 3C). In the absence of maternal and zygotic Gug activity, embryos lack ventral pattern elements (Fig. 3D,E) and exhibit holes in the ventral cuticle.
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To understand how loss of Gug activity affects segmentation, we analysed the expression of hunchback (hb), Krüppel (Kr), knirps (kni) and fushi tarazu (ftz) in embryos derived from Gug35 germline clones fertilised by Gug35 sperm. In wild-type embryos, the expression of these segmentation gene products localise to discrete domains in the early embryo (Fig. 3G-J left) (reviewed by Rivera-Pomar and Jackle, 1996). In almost all of the expression domains, loss of Gug activity increases the number of cells expressing these segmentation genes (Fig. 3G-J right) suggesting that Gug plays a role in their repression. Later the expression of ftz displays a more complex defective pattern with some stripes being broader, and others narrower, than wild type (Fig. 3G).
Loss of Gug activity also affects the distribution of the Tsh protein (Fig. 3K). In wild-type embryos at the germ band retraction stage, Tsh is expressed evenly in trunk segments (left) and not the head or tail (Alexandre et al., 1996). In Gug embryos (right), Tsh is expressed in the trunk but is lost from ventral regions (arrow) and is expressed in a striped pattern in the dorsal part of the embryo (arrowheads). These results suggest that Gug is a regulator of the tsh gene during embryogenesis.
Grunge encodes for a protein similar to human RERE
Sequence analysis of two overlapping cDNAs reveals an open reading frame encoding for a putative protein of 1966 amino acids (Fig. 4A; GenBank Accession Number, AF217844). The putative Gug protein has closest similarity to human arginine-glutamic acid dipeptide repeat (RERE) protein, an Atrophin-1-related protein (Seki et al., 1997; Yanagisawa et al., 2000
) (Fig. 4B-D). Distinct domains of this protein also show similarity with vertebrate Atrophin-1-related and with the Metastasis-associated (Mta)-like proteins (Fig. 4C,D). Atrophin-1 and Atrophin-1-related proteins are found in mice, rats and humans. Human Atrophin-1 contains a poly-glutamine repeat, which is expanded in individuals with a dentatorubral-pallidoluysian atrophy (DRPLA), resulting in neuronal apoptosis (reviewed by Kanazawa, 1998
). The normal function of Atrophin-1 is not known. Atrophin-1 and the human Atrophin-1-related (RERE) protein are similar in the C-terminal half of each protein (60% identity), but RERE has no poly-glutamine stretch. Gug contains two poly-glutamine stretches (grey in Fig. 4A) and has a conserved C-terminal box found in Atrophin-1 and Atrophin-1-like proteins (Fig. 4D; orange in Fig. 4A). Human RERE exhibits weaker identity in a second region of Gug, extending from amino acid 334-513 (green box Fig. 4A,B). This domain is also conserved in vertebrate Atrophin-1 proteins but is less extensive (not shown). Another weak region of homology is found between Gug and mouse Atrophin-1 (30% identity, 43% homology) (purple box Fig. 4A,B) and rat Atrophin-1-related (22% identity; 30% similarity); this domain is not found in human RERE.
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Grunge protein is localised in the nucleus
To analyse the cellular localisation of Gug, we raised an antibody to the putative Gug product (see Materials and Methods). During embryogenesis, this antibody recognises epitopes localised in the nucleus in all cells (Fig. 5A,B), although not within the putative mitotic domains (asterisks in Fig. 5B). To verify that the protein corresponds to Gug, we analysed the distribution of this antibody in embryos and in tissues mutant for different Gug alleles. In embryos derived from Gug germline clones, nuclear staining was not detected, as in wild type (Fig. 5C,D). We also induced Gug35 clones (marked by loss of GFP) in the imaginal discs and analysed the expression of Gug. Staining was significantly reduced in the clones (Fig. 5E,F) compared with wild-type GFP control clones (not shown). At present, we do not know if the antibody is specific to Gug but our results show that Gug mutations affect the distribution of a protein detected by the antiserum. Taken as a whole, these results suggest that the Gug alleles correspond to loss-of-function alleles that affect the function of the protein depicted in Fig. 4.
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Differential behaviour of Gug clones is observed along the dorsoventral axis of the legs. Mutant cells located in dorsal or lateral parts of the leg give rise to essentially wild-type patterns (Fig. 6A,C), although they exhibit a slight cell autonomous increase in bristle density, compared with wild type (Fig. 6B).
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Grunge specifies the proximal identity of legs
Large Gug clones located in the coxa, trochanter or proximal femur, irrespective of their provenance in the anterior or posterior compartment, lead to fusion of these segments. Pattern elements, which are associated with clones and in neighbouring cells, were replaced with those found in more distal parts of the legs. That is Gug clones in these proximal parts generally bear bracts (Fig. 7B), as do bristles located more distally (Fig. 7A). Clones situated in dorsal regions do not affect proximal identity (Fig. 6A). However, proximal clones, which occupy a large region of both the dorsal and ventral domains, replace all patterns with more distal identities and cause a reversal of the polarity of bristles (Fig. 7B). These Gug clones have a non-autonomous effect on the polarity of more distally located, ventral bristles (Fig. 7B, arrowheads). Smaller clones affect patterning if they are located ventrally. Such clones lead to outgrowths forming a partial new axis (Fig. 7C). Although bristles in these outgrowths show a distal (bracted) identity, they never form a complete new leg. Outgrowths consist of Gug and Gug+ tissue, suggesting that Gug activity is crucial for normal cell communication.
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DISCUSSION |
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Grunge has motifs with similarity to Atrophin-like and Metastasis-associated protein families
Atrophin-1, which shares homology with Gug, has been implicated in the neurodegenerative disease dentarubral-pallidoluysian atrophy (DRPLA). This pathology results from expansion of glutamine repeats (reviewed in Kanazawa, 1998). Whereas the Gug protein is the only known Drosophila member of the Atrophin family of proteins, vertebrates have at least two Atrophin proteins and several Atrophin-like members. The closest relative to Gug is the Atrophin-1-related protein called human arginine-glutamic acid dipeptide repeat (RERE) protein (Seki et al., 1997
; Yanagisawa et al., 2000
), which has similarity to both the Atrophin-1 and Metastasis-associated (Mta) families of proteins (Fig. 4). Two glutamine repeat regions are found in Gug (Fig. 4A) that are not found in human RERE. Gug, human RERE, human Mta1 and C. elegans EGL-27 proteins possess a homologous ELM2 SANT domain located at the N terminus (Fig. 4B,C). Mta1 is thought to be required for normal chromatin structure, as it associates with histone deacetylase and has nucleosome remodelling activities (Xue et al., 1998
). Additionally, Mta1 is upregulated in metastatic carcinomas. Interestingly, analysis of egl-27 mutations in C. elegans reveals that EGL-27 has a function in common with the Wnt pathway (Herman et al., 1999
), as we describe for Gug. The presence of a SANT-like DNA-binding domain (Fig. 4B,C), three putative nuclear localisation motifs (Fig. 4A) and its nuclear localisation (Fig. 5A,B) suggest that Gug acts as a transcriptional regulator.
Grunge is required for embryonic segmentation
Loss of Gug activity severely affects the process of segmentation and the expression of segmentation genes when missing from the female germ line (Fig. 3). At the blastoderm stage, most of the expression domains of hb, Kr, kni and ftz genes are expanded compared with wild type. These observations indicate that maternal production of Gug is crucial for the repression of these genes to precise domains in the early embryo. Gap proteins, including Hb, Kr and Kni are known to be required to restrict each others domains of expression (Rivera-Pomar and Jackle, 1996). It will be interesting to test if Gug acts with these proteins for these repression activities.
Loss of gap gene products and especially the pair rule product ftz affects the normal regulation of tsh (Coré et al., 1997; Röder, 1992
). Ftz acts as a positive and probably direct regulator of tsh. Loss of Gug activity does not effect the location of Tsh to the trunk segments of the embryo but Tsh expression is affected (Fig. 3K).
Grunge and patterning of the proximal parts of the legs
One striking feature of Gug+ function is its role in the formation of proximal specific patterns of the leg (Fig. 7). Loss of Gug+ activity in proximal ventral cells changes bristle polarity and replaces proximal with more distal cellular identities. Thus, patterns typical of the coxa, trochanter and proximal femur are replaced with leg tissue that partially resembles that found in more distal femur or tibia. These effects resemble those seen in clones of cells lacking Extradenticle or Homothorax activities (Gonzalez-Crespo and Morata, 1996). As Gug+ activity is also crucial for ventral patterning of the leg, the proximal-to-distal change is never complete. Gug mutant clones also affect cell communication in the proximal leg, as they exhibit cellular non autonomy causing neighbouring wild-type tissue to differentiate distal patterns in proximal positions (Fig. 7C-D, Fig. 8B).
The role of Gug in patterning the proximal leg is shown at the molecular level, where tsh requires positive input from the Gug gene specifically in ventral proximal parts of the leg imaginal disc (Fig. 8A). Loss of Gug results in ectopic expression of Dll in this position (Fig. 8B). As Gug is ubiquitously produced in the leg (Fig. 2F, Fig. 5F), proximodistal specificity of Gug function presumably derives from other proteins located in proximoventral parts. Recently, we showed that Dll and possibly Tsh act as mutual repressors only in cells where Wg is signalling (Erkner et al., 1999). Gug may normally be required for this process (Fig. 6, Fig. 7A-D).
Grunge is required for ventral-specific patterning and morphogenesis of the leg
Gug activity is essential for patterning the ventral parts of the leg along the entire proximodistal leg axis (Fig. 6). Loss of Gug in dorsal or lateral parts has no drastic effect on patterning (Fig. 6A-C), although the number of bristles is augmented in Gug mutant cells irrespective of dorsoventral position (Fig. 6B,C).
Ventrally in the femur-tibia region, loss of ventral Gug activity causes the fusion of these leg segments (Fig. 6H). During early pupariation, a sack of cells is known to give rise to the femur and tibia. Ventrally situated cells then migrate to meet and separate the femur and tibia (Fristrom and Fristrom, 1993). If Gug is missing in these migrating groups of cells, the femur and tibia remain attached (Fig. 6D,H). Gug mutant clones also affect the process of segmentation of the tarsus (Fig. 6E-G). Similar defects on the morphogenesis of the femur-tibia and tarsus have been observed in clones lacking components of the Notch signalling pathway (de Celis et al., 1998
). The relationship between Gug and Notch signalling activities will be reported elsewhere.
The normal ventral patterning of the legs is specifically under the control of the Wg signalling cascade of molecules (reviewed by Wodarz and Nusse, 1998); thus, there is a correlation between the domains where Wg signalling occurs and where Gug is active. Furthermore, both Gug and Wg signalling act in domains where wg is transcribed and where Wg is secreted (for example, in the posterior ventral part of the leg) (Fig. 6D-G).
Although Wg and Gug act in the same domains of the leg with similar roles, they exhibit distinct functions. Gug seems to act in a fraction of Wg-dependent developmental events. First loss of Wg signalling induces a novel axis in ventral leg parts, irrespective of proximodistal position. Gug, however, induces bifurcated legs only if its activity is removed from proximal ventral parts of the leg. Contrary to the loss of Wg signalling, Gug mosaics do not distalise bifurcated legs properly, presumably because Gug activity is required for this process (Fig. 6, Fig. 7). Finally, Gug replaces proximal tissue with distal patterns (Fig. 8B, Fig. 7B,C); loss of Wg signalling never produces such homeosis (Brook and Cohen, 1996; Jiang and Struhl, 1996
). These observations suggest that Gug functions are related to those controlled by Wg signalling but are more specialised. This specialisation may reflect the fact that Gug controls the expression of tsh, which is required to modulate Wg signalling activity (Gallet et al., 1998
; Waltzer et al., 2001
).
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ACKNOWLEDGMENTS |
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