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BMC Dev Biol
2007 Jul 05;7:82. doi: 10.1186/1471-213X-7-82.
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echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases.
Copeland JM
,
Bosdet I
,
Freeman JD
,
Guo M
,
Gorski SM
,
Hay BA
.
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BACKGROUND: Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death.
RESULTS: We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss.
CONCLUSION: The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye.
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17612403
???displayArticle.pmcLink???PMC1950886 ???displayArticle.link???BMC Dev Biol ???displayArticle.grants???[+]
Figure 1. echinus gene structure, mutants and surrounding genomic region. Echinus (CG2904) exons are indicated by shaded boxes. Exons are numbered sequentially with respect to the 5' end of the gene. The shaded boxes with diagonal lines represent the conserved USP domain. Three different splice versions identified through cDNA sequencing are illustrated in panels A-C. Exons common to all three splice versions are noted with lines above numbered exons 7,8, and 9. Nearby genes are indicated (open arrows), as are Flybase annotated P element insertions, and ecPlacZ. Open triangles indicate P element insertions that are wildtype with respect to echinus, while filled triangles indicate P element insertion lines with rough eyes that fail to complement echinus. The location of EMS-induced point mutations in echinus, ec56 and ec3c3, and mutations identified in ec1 (a point mutation and a Copia insertion) are indicated by asterisks. The locations of the breakpoints for the echinus deletion alleles ecEPΔ4 and ecΔ9 are indicated by dotted lines at the top. (A) ec-SF1 encodes a version of echinus that contains Cys and His box residues important for USP catalysis, as noted by the highlighted residues. These and surrounding sequences are highly conserved in predicted echinus homologs in other insect species. Highly related sequences are also found in a number of other species. (B) The ec-SF2 transcript initiates at a downstream exon, which contains an initial methionine and coding sequences that lack a Cys box. The 3' exon containing His box sequences (exon 9) is still present. (C) The ec-SF3 transcript initiates at a distinct position further 3', and also contains His box sequences, but lacks Cys box sequences. Sequences highly related to this alternative N-terminus are also found in a number of other species.
Figure 2. Flies with mutations in CG2904 have rough eyes, defects in IOC sorting, an increase in IOC number (A-F) SEM views of adult fly eyes of various genotypes. (G-O) Pupal retinas of various genotypes stained with anti-Dlg. (A, G) Wildtype flies have regularly spaced ommatidia and an invariant number of IOCs. Cell types indicated are bristle (B), 2°, 3°, and asterisk represent extra IOCs. (B,H) ec1 flies obtained from the Bloomington Stock center have rough eyes and a modest number of extra 2° and 3° pigment cells. (C,I) GMR-driven RNAi of CG2904 results in flies with rough eyes and a large increase in IOCs, with many stacked side-by-side in parallel rows. (D,J) Flies homozygous for a deletion in CG2904, ecEPΔ4, have rough eyes, a large increase in IOCs, with many cells stacked side-by-side in parallel rows. (E,K) GMR-dependent expression of ec-SF1 has no effect on the adult eye and does not cause any excess death of IOCs. (F,L) Expression of GMR-ec-SF1 restores normal levels of IOC death to ecEPΔ4 flies. (M,N) Pupal eyes from two independent stocks of ec1 outcrossed for 5 generations. There are increased numbers of IOCs as compared with the original ec1 stock, and many extra cells are aligned side-by-side in parallel rows. (O) Pupal eyes from ec3c3 flies have a modest increase in IOC number and few defects in cell sorting.
Figure 3. The echinus transcript is expressed at low, uniform levels in the pupal eye, and GAL4-driver-dependent expression of ec-SF1 or an ec-silencing microRNA suggests that pigment cells are an important site of ec action. (A,B) Tissue in situ hybridization of an echinus antisense probe complementary to all splice forms in 28 hr APF pupal retinas. (A) Focal plane showing primary pigment cells. (B) Focal plane showing IOCs. (C-E) Pupal retinas from heterozygous ecPlacZ/+ flies showing anti-β galactosidase staining in cone cells (C), primary pigment cells (D), and IOCs (E). Cell types indicated are cones (C), primaries (P), and IOCs (*). (F-F") 24 hr pupal retina from LL54-GAL4; UAS-GFP flies stained with anti-Dlg to outline cell boundaries (F, F") and GFP (F',F") to visualize the LL54-GAL4 expression pattern. (G-G") 29 hr pupal retinas from LL54-GAL4; UAS-GFP flies stained as above. LL54-GAL4 is expressed primarily, if not exclusively in pigment cells, but not bristles or cone cells. (H) 36 hr pupal eye from LL54-GAL4; UAS-CG2904-RNAi stained with anti-Dlg. Extra IOCs and sorting defects are apparent. Arrows indicate bristles.
Figure 4. Echinus does not require deubiquitinating activity to promote normal IOC death. (A-D) SEMs of adult eyes of various genotypes. (E-H) Pupal retinas of various genotypes stained with anti-Dlg. (A,E) GMR-driven expression of a microRNA targeting ec-SF1 results in an echinus phenotype. (B,F) ecEPΔ4 eyes. (C,G) Eyes of genotype ecEPΔ4; GMR-ec-SF2/+. (D,H) Eyes of genotype ecEPΔ4; GMR-ec-SF3/+. Expression of versions of Echinus that lack essential USP catalytic residues rescues the ecEPΔ4 phenotype.
Figure 5. Genetic interactions between echinus and known or potential regulators of cell death in the eye. To the right is a schematic depicting known or suggested interactions between death regulators in the fly. The question mark separating Debcl/Buffy from Ark indicates the uncertainy as to the roles these proteins play in regulating Ark activation or activity. GMR-driven transgenes of the indicated genotype were introduced into the ecEPΔ4 background, or into a wildtype background in the presence of GMR-ec-SF1. For each death regulator tested, similar phenotypes were observed in the presence of GMR-ec-SF2 (data not shown).
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