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Grunge, related to human Atrophin-like proteins, has multiple functions in Drosophila development

Alfrun Erkner^*, Agnès Roure, Bernard Charroux, Michèle Delaage, Nicolas Holway, Nathalie Coré, Christine Vola, Corinne Angelats, Françoise Pagès, Laurent Fasano and Stephen Kerridge

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

View larger version (85K): [in a new window]   Fig. 1. Gug mutations act as dominant suppressors of a tsh reporter gene in the eye. (A,B) Heterozygotes for P(Lac w+)tshJ834 (B is also heterozygous for the GugS2 mutation). (A) Note that the white gene is active in anterior cells of the eye (Sun et al., 1995) as is the tsh gene; (B) expression of these genes is reduced.

View larger version (33K): [in a new window]   Fig. 2. Molecular analysis of the Gug locus. (A) The genomic region of Gug with the two P-element insertions and the deletion Df(3L)Gug35. The restriction enzyme sites of EcoRI, SalI and HindIII are shown. (B) Structure of the cDNA of Gug. The black boxes indicate the coding sequence. (C) Northern analysis: a 9.0 kb zygotic and a 8.0 kb maternal-specific transcript are detected. L, third instar larva; F, female. (D-F) In situ detection of Gug transcripts in a blastoderm (D) and germband extended (E) embryos and leg disc (F) associated with the larval central nervous system (cns, arrow). (D,E) Anterior is towards the left and ventral is towards the bottom. Note that Gug is not detected in the amnioserosa (as) in E and is concentrated in the hemispheres of the brain (arrow, F). In the leg disc, v, d and di indicate the ventral, dorsal and distal regions, respectively.

View larger version (98K): [in a new window]   Fig. 3. Loss of maternal Gug activity affects embryonic segmentation and Tsh expression. (A) Ventral view of a wild-type larva showing the 11 similar trunk segments each with alternating denticled and naked cuticle. (B) Detail of the head region of an embryo homozygous for Gug35. (C-E) Larvae from Gug35 germline clones fertilised by wild-type (C) or Gug35 (D,E) sperm. (C) Note the reduced number of segments compared with wild type. (D) Note the holes in the cuticle or the absence of the ventral cuticle (E). (F) Phenocopy of the Gug segmentation phenotype after injection of antisense Gug mRNA into embryos (compare with C). Expression of the segmentation genes ftz (G), hb (H), Kr (I), kni (J) and the region-specific homeotic protein Tsh (K) in wild type (left) and Gug mutant (right) embryos. Gug/Gug zygotes were distinguished by the absence of ftzlacZ (carried on the TM3 balancer chromosome) expression. Loss of Gug generally increases the number of cells expressing the segmentation genes. Note that Tsh is missing from the ventral parts (arrow) and in stripes in dorsal regions (arrowheads) of the trunk in Gug– embryos (K, right panel), where Tsh is uniformly expressed in the trunk of wild-type embryos (K, left panel).

View larger version (49K): [in a new window]   Fig. 4. The Grunge protein. (A) The predicted amino acid sequence of Grunge protein. The blue box highlights a domain similar to that found in human arginine-glutamic acid dipeptide repeat (RERE) protein (Seki et al., 1997; Yanagisawa et al., 2000) (63% identity, 66% similarity), human Mta1 (25% identity, 33% similarity), Drosophila Mta-1 (27% identity, 36% similarity) and EGL-27 (29% identity, 35% similarity) (see Fig. 4C). The green box corresponds to a domain with weak similarity to human RERE (20% identity, 41% similarity), human Atrophin-related (20% identity, 40% similarity) and mouse atrophin 1 (23% identity, 44% similarity). The purple box is a domain of weak homology found in mouse atrophin 1 (30% identity, 45% similarity) and rat atrophin 1 related (22% identity; 30% similarity). Finally, the orange box shows homology to human RERE (40% identity, 44% similarity), human atrophin-related (28% identity, 45% similarity) and human atrophin 1 (30% identity, 45% similarity; see Fig. 4D). The location of the two poly-glutamine (Q) stretches is indicated in grey and three putative nuclear localisation signal motifs are indicated in yellow. A putative Caspase protease site is present (IEPD, red letters). (B-D) Conserved domains in the Gug protein. Comparison of the structure of Gug and the human arginine-glutamic acid dipeptide repeat (RERE) (Seki et al., 1997; Yanagisawa et al., 2000), Atrophin-1-like, protein. Colour codes are as in A. The black box indicates a GATA-like domain and the yellow box to a brahma adjacent homology (BAH) domain present in RERE but not in Gug. The purple box is a weak domain of homology found in mouse and human atrophin 1 but not human RERE. (C) Alignment of the amino acids from the N-terminal domain of Gug compared with Drosophila Metastasis-associated factor (MTA1), the C. elegans EGL-27 protein and the human RERE, Atrophin-1-like, protein. This domain contains an ELM2 region (dark-blue outline) and a SANT DNA-binding domain (light blue outline) (Solari et al., 1999). (D) Sequence comparison of the C-terminal domains of Gug and the human RERE protein. This region has comparable levels of similarity to vertebrate DRPLA proteins (not shown). Identities are indicated by white letters on black, similarities are indicated by black letters on grey.

View larger version (98K): [in a new window]   Fig. 5. Gug– tissues affect the protein distribution detected by antisera raised against the Gug protein. Anti-Gug antibody stains to embryos at blastoderm (A,C) and early gastrula stages (B,D). Wild-type (A,B) and embryos derived from Gug35 germline clones (C,D). Note that for the lower panels, staining is absent from the nuclei unlike in wild type (arrow, A). The asterisk in B indicates one of several mitotic domains where Gug is lost. Note that staining of embryos with the relevant pre-immune serum when overstained gives similar results to that shown in D. Clones of cells homozygous for GugS2 in imaginal leg discs, detected by the absence of GFP (E), show reduced levels of staining with anti-Gug antibody (F). Clones of cells that lack Gug+ products were induced at 48-72 or 96-120 hours after egg laying. Ventral is towards the bottom.

View larger version (149K): [in a new window]   Fig. 6. Gug– clones affect ventral-specific patterning and morphogenetic events in legs. Clones were induced at 48-72 or 72-96 hours after egg laying and are marked by the yellow bristle marker (highlighted by the broken blue lines). (A,B) Anterodorsal clones in the femur (fe) showing normal morphology (A) and higher density of bristles (B) compared with a wild-type leg. (C) A first leg with a small posterolateral Gug– clone with little effect on leg patterning, although showing a higher density of bristles than normal. Arrowheads indicate the large bristles formed in a wild-type first leg femur, in a ventral position in the posterior compartment. (D) A posterior Gug clone in the ventral region of the femur. The large bristles do not form (arrow; compare with C). (E) Ventral clone in the second tarsal segment of a second leg shows the deletion of a spur bristle (arrow). In wild type, each tarsal segment has two spur bristles at the distal end (arrowheads). (F) First leg carrying a Gug– clone. The transverse rows (arrowhead; compare with the wild type in G) and the sex comb (arrow) in the basitarsus are not formed in the clone. In wild type (G), the sex comb carries 10-12 specific bristles. Here, only four bristles are made, deriving from wild-type tissue. (G) Wild-type basitarsus of a first leg, showing the ventral transverse rows. (H) Antero- and posteroventral Gug– clones in the femur-tibia region lead to fusion of these leg segments.

View larger version (146K): [in a new window]   Fig. 7. Cell autonomous and nonautonomous effects of Gug mosaic clones for proximal identity in the leg. The right panels show higher magnifications of the panels on the left. (A) The proximal region of a second leg showing the region between bracted (arrow) and non-bracted (arrowhead) bristles in the femur. Distal to this border, almost all bristles have bracts. (B) A proximal Gug– M+ clone filling a large region of the anteroproximal part of a second leg. Coxa, trochanter and proximal femur are replaced with unpatterned leg tissue. Bristles show more distal (arrows) identity. Note that bristles change their polarity in a autonomous and non-autonomous (arrowhead) manner. (C) A clone, induced at 120-144 hours, where a secondary leg axis protrudes from the ventral region of the proximal femur and trochanter region. The ectopic leg is incomplete, consisting of mosaic y Gug– and Gug+ cells. Gug+ cells in the ectopic leg have formed large bristles (arrow) typically found in the distal leg. We believe that these bristles have dorsal identity that represents the preapical bristle of the tibia.

View larger version (116K): [in a new window]   Fig. 8. Gug activity is required for regulation of teashirt (tsh). Clones of Gug– cells in imaginal leg discs, detected by the absence of the Myc epitope. (A) Clone, showing a ventral outgrowth in the proximal, ventral region. Overlapping expression of Tsh and Myc is shown in yellow. Tsh is absent in ventral Gug– cells (white line) and in wild-type cells (red line) adjacent to the clones. In dorsal clones, Tsh is expressed normally as shown by the green staining (green line). (B) Tsh (green) and Dll (red) expression in a late third instar disc showing a small secondary axis. Distalisation is indicated by the ectopic Dll expression in this new axis (arrow). Note that this disc is shown at twice the magnification. (C) Distal (ventrally and dorsally) clone marked by the absence of Myc (red); Dll (green) is expressed normally in the clone. (D) Wg-lacZ expression (green and middle panel) is reduced in a small ventral outgrowth (arrow) induced by a Gug– clone (absence of the red staining; right panel). (E) Wg-lacZ expression (green and middle panel) is normal in a distal ventral clone (absence of the red staining and right panel). Note that there must be at least two clones, one in the anterior and another in the posterior compartment. (F) Dpp-lacZ (green and middle panel) is expressed weakly in some Gug– cells in a ventral outgrowth (arrow; right panel).

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