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doi: 10.1242/10.1242/dev.00168

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Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

¹ Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer Center, Houston, TX 77030, USA
² Program in Genes and Development, M. D. Anderson Cancer Center, Houston, TX 77030, USA
³ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
⁴ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
⁵ Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (96K): [in a new window]   Fig. 1. shar-pei mutant clones result in outgrowths on head, thorax and halteres. Wild-type (left column) and mutant (right column) adult structures imaged by light and scanning electron microscopy (SEM). (A,B) Dorsal views of a normal sized fly (A) and a fly with a shrp mutant head (B). Both flies are genetic mosaics. We used the eyFLP transgene to induce recombination in most cells of the eye-antennal disc (Newsome et al., 2000). To increase the number of clone cells, a cell-lethal mutation on the homologous chromosome was used to eliminate homozygous twin clone cells (Newsome et al., 2000). In the normal sized fly, 80% of cells are white but otherwise wild type. In the mutant fly, white cells are also homozygous mutant for shrp. These mutant cells make up virtually the entire eye. The body is wild type and serves as a reference for comparison of head sizes, because mitotic recombination was specifically induced in the developing head by using eyFLP. The genotypes are (A) y w eyFLP; FRT82B/FRT82B cell lethal p[w+] and (B) y w eyFLP; FRT82B shrp1/FRT82B cell lethal p[w+]. (C,D) SEM images of a wild-type fly and a fly with a shrp3 mutant head produced by eyFLP induced mitotic recombination as for (B). (E,F) Higher magnifications of C,D. The mutant tissue is severely overgrown and folds up. Ocelli (arrows), bristles and hairs differentiated normally. (G,H) Wild-type thorax and a thorax with shrp3 mutant clones. The clones result in overgrown tissue (arrow). (I,J) Wild-type haltere (I) and haltere with shrp3 mutant clones (J). The mutant haltere is much larger than normal.

View larger version (131K): [in a new window]   Fig. 2. shar-pei mutant clones in the eye show excess interommatidial cells, resistance to apoptosis and normal patterning. (A) Plastic thin section through an adult eye that is mosaic for shrp5 mutant cells. Mutant tissue lacks dark pigment granules in pigment and photoreceptor cells. Mutant ommatidial clusters have the normal complement of seven rhabdomeres in the correct trapezoidal arrangement. The spaces between the photoreceptor clusters, however, are significantly larger in mutant tissue than in wild-type tissue (arrowhead). (B,C) Mid pupal stage retinas with shrp4 mutant clones (B) and wild type (C) stained with anti-Dlg antibody to detect cell outlines. (D,E) Confocal section of the basal side of a 38 hours after puparium formation (APF) pupal retina mosaic for shrp3. Mutant cells are marked by the absence of GFP expression (shown in red). The retina was stained with antibodies against activated Drice to detect apoptotic cells (green in D). All apoptotic cells are wild-type and express GFP (arrowheads). (F) Cell outlines in a retina expressing p35 under GMR control revealed by Dlg expression. (G-L) shrp mutant clones in third instar eye discs marked by the absence of GFP (grayscale in G,J and blue in I,L). Discs are stained for Sens (green) and Elav (red) expression. (G-I) A shrp6 mutant clone spanning the morphogenetic furrow (arrowhead) that shows normal patterns of Sens and Elav expression. (J-L) A shrp1 clone at the posterior edge shows normal patterning but increased spacing between ommatidial clusters (arrowhead). Anterior is towards the left in G-L.

View larger version (109K): [in a new window]   Fig. 3. shar-pei mutant cells display ectopic cell proliferation. All panels show imaginal discs stained to detect S phases by BrdU incorporation (green). (A) Wild-type and (B) eyFLP induced mosaic eye disc nearly entirely mutant for shrp1. In wild-type, cells arrest in G1 phase in the morphogenetic furrow (arrow) and non-differentiating cells go through one synchronous S phase in the second mitotic wave (SMW, arrowhead). (B) shrp1 mutant cells also arrest in G1 and go through a synchronous SMW, but cells then continue to proliferate posterior to the SMW (asterisk). (C,D) BrdU incorporation (green) in shrp1 mutant clones marked by the absence of GFP (red). shrp1 mutant cells behind the SMW (arrowhead) continue to proliferate (arrows). This effect of shrp is cell autonomous. (E) Apical and (F) basal focal plane of an eye disc with a posterior shrp1 mutant clone stained for Elav (purple) and BrdU (green). Mutant cells were marked by the absence of GFP expression (not shown). The clone boundary is indicated by a white line in (E). BrdU-incorporating cells are located basally (F) and none of the Elav-positive cells incorporated BrdU. S phases in the SMW are marked by an arrowhead in F. Anterior is towards the left for all discs.

View larger version (69K): [in a new window]   Fig. 4. shar-pei mutant cells upregulate Cyclin E levels. (A-C) shrp1 mutant clones in the eye disc marked by the absence of GFP expression (red) stained for Cyclin E (green). (C) merged channels. Cyclin E is upregulated in cells of shrp mutant clones (arrows), in particular posterior to the SMW (arrowhead).

View larger version (20K): [in a new window]   Fig. 5. shar-pei mutant retinas contain more photoreceptor clusters than wild type. The numbers of photoreceptor clusters in 18 wild-type (wt) and 18 eyFLP induced shrp1 mosaic retinas were counted in whole mid-pupal retinas. Photoreceptor clusters were visualized by Elav-GAL4 driven GFP expression. Each square/triangle represents one retina.

View larger version (41K): [in a new window]   Fig. 6. shar-pei mutant cells proliferate faster, but show normal cell cycle profiles and cell size. (A) Cell numbers in 50 shrp3 mutant clones (gray bars) and (B) 50 control clones of the isogenized wild-type FRT chromosome on which the shrp mutations were induced, compared with their twin clones (red bars). Twin clones were homozygous for an isogenized FRT 82B ubi-GFPNLS chromosome. Cell numbers were counted in wandering third instar wing disc clones. (C) DNA profiles and (D) forward scatter distributions (FSC) of third instar wing disc cells measured by flow cytometry (FACS). shrp4 mutant clones were induced at 24-48 hours after egg laying (AEL) and wing discs dissected 72 hours after clone induction. Blue trace represents shrp mutant cells, red trace wild-type cells. Mutant and wild-type cells were sorted by GFP expression. The mutant cell population had a similar distribution of cell cycle profiles and cell sizes when compared with the wild-type cells. (E-G) shrp3 mutant clone in the presumptive wing pouch of a third instar wing disc marked by the absence of GFP expression (red). The disc is stained for Dlg to reveal cell outlines (green) and DNA to label nuclei (blue). Mutant cells have the same sized outlines as wild-type cells. Large cell outlines are from dividing cells showing apical mitotic figures (blue). (G) Merge of the three channels.

View larger version (49K): [in a new window]   Fig. 7. Identification and sequence analysis of shar-pei. (A) Mapping of shrp relative to five P elements inserted in the 94A-96A region on 3R (horizontal line). Triangles show P elements with their names and genomic position in kb. Recombination distances between shrp (vertical line with star) and each P element are given in centiMorgan (cM, double arrows). (B) The genomic region of shrp determined by recombination mapping. Known and predicted ORFs are shown by arrowed boxes and genomic positions are given in kb above the DNA. The five gray boxed genes and shrp (black box) were sequenced. (C) Schematic representation of the protein structures of the fly, human and nematode Shrp homologs. Numbered arrows indicate the positions of the mutations in the six shrp alleles. The mutations in alleles 1-5 result in premature STOP codons, allele six has a +2 frameshift that results in the addition of 76 amino acids not related to any other protein in GenBank. (D) Sequence alignment of Drosophila Shrp (Dm), human WW45 (Hs) and C. elegans T10H10.3 (Ce). Identical residues are on black background, similar residues are shaded. The two WW domains are outlined by dark boxes and the shrp-specific domain is boxed by a broken line. Asterisks indicate the residues, the codons of which are mutated to STOP codons in the respective alleles, the position of the frame-shift in shrp6 is identified by an arrow. (E) Expression of shrp RNA in a wild-type eye-antennal disc. (F) The sense control shows no staining.