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Ectopic expression of Drosophila ELAV and human HuD in Drosophila wing disc cells reveals functional distinctions and similarities

Gakuta Toba, Jan Qui, Sandhya P. Koushika^* and Kalpana White

Department of Biology and Center for Complex Systems, MS 008, Brandeis University, Waltham Massachusetts 02454, USA
^* Present address: Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (21K): [in a new window]   Fig. 1. Structure of GFP reporter transgenes. Boxes represent exons, and solid bars represent introns. Unfilled and filled circles represent translation start and stop codons, respectively. Arrows show transcription start sites. All the transgenes are driven by Ubi-p63E promoter and have Ubi-p63E 5'UTR with an intron (intron not shown) which is 5' to the open reading frame (ORF) starting at ATG. The ORF encodes a Myc-tag fusion GFP. In UGA and UGH, the ORF is not interrupted by an intron, whereas in UgGA and UgGH, a generic intron GI (Mottes and Iverson, 1995) is inserted within the ORF. In UnGA and UnGH, an alternatively spliced intron from nrg (nASI) is inserted at the same position as GI, necessitating neuronspecific splicing of the intron for GFP expression. UGA, UgGA, and UnGA have Act5C 3'UTR, while UGH, UgGH, and UnGH have Hsp70Ab 3'UTR. This figure is not drawn to scale.

View larger version (62K): [in a new window]   Fig. 2. The 3'UTR sequences used in this study. (A) The Hsp70Ab 3'UTR sequence (UGH, UgGH, and UnGH) contains four AUUUA motifs that are often found in AREs. The AU content of the sequence is 76%. The sequence was obtained from the pP{CaSpeR-hs} sequence (Pirrotta, 1988). The 3' end of the sequence is an expected approximate polyadenylation site at 220 bases downstream to the Hsp70Ab stop codon (Dellavalle et al., 1994). (B) The Act5C 3'UTR sequence (UGA, UgGA, and UnGA). The sequence has a single AUUUA motif and 58% AU content. The sequence was obtained from the pP{CaSpeR-hs/act} sequence (Pirrotta, 1988) and is terminated at an expected approximate polyadenylation site (655 bases downstream to the Act5C stop codon) (Bond and Davidson, 1986). The AUUUA motifs are boxed. The underlined sequences are protein coding in the original genes, but constitute 3'UTR in our constructs. All the transgenes have a common 77-base sequence between the GFP stop codon and the sequences shown here.

View larger version (64K): [in a new window]   Fig. 3. Expression of GFP reporter transgenes in third instar larvae. (A) UgGA larva shows broad GFP signal in most tissues implying ubiquitous transcription from the Ubi-p63E promoter. (B) In the UgGH larva, the GFP signal is weaker in most tissues compared with the UgGA larva. However, strong expression is observed in the CNS and salivary glands, and a very strong expression is seen in the male gonads. (C) In the UnGA larva, GFP signal is primarily in the CNS. However, a very low level of expression is also found in other tissues including the male gonads. (D) In the UnGH larva, GFP signal is similar to UnGA, but strong signal is seen also in the male gonads. Arrow, CNS; asterisk, salivary gland; arrowhead, male gonad.

View larger version (71K): [in a new window]   Fig. 4. Effects of HuD and ELAV ectopic expression on GFP reporter expression in the third instar larval wing disc. The ectopic expression is achieved by expressing either UAS-HuD or UAS-elav using dpp-GAL4 as a driver. (A) Wild-type (Canton-S) wing disc shows background level of fluorescence. (B) The expression pattern of dpp-GAL4 driver in the wing disc is visualized using UAS-GFP. (C-N) Effect of ectopic HuD (D,G,J,M) and ELAV (E,H,K,N) are examined for UnGA (D,E), UnGH (G,H), UgGA (J,K) and UgGH (M,N) expression. Basal GFP signal of UnGA (C), UnGH (F), UgGA (I) and UgGH (L) is shown as control. Ectopically expressed HuD has no discernable effect on either UnGA (D) or UnGH (G) expression, while ectopic ELAV expression leads to strong increase in GFP signal along dpp-GAL4 expression pattern for both reporters (E,H). On UgGA expression, neither HuD nor ELAV have any obvious effect (J,K). In contrast, ectopic expression of either HuD or ELAV strongly enhances UgGH expression (M,N). For the genotype of each wing disc, see Materials and Methods.

View larger version (130K): [in a new window]   Fig. 5. Effect of HuD and ELAV ectopic expression on UgGA and UgGH reporter expression. UgGA and UgGH expression was examined at the RNA level by in situ hybridization in the third instar larval wing discs. No apparent change in UgGA mRNA level is observed when either UAS-HuD (A) or UAS-elav (B) are ectopically expressed using dpp-GAL4. However, in UgGH-expressing discs, the mRNA level is increased along the dpp-GAL4 expression pattern by ectopic expression of either UAS-HuD (C) or UAS-elav (D).

View larger version (88K): [in a new window]   Fig. 6. Localization of ectopic HuD and ELAV in the wing disc cells. Third instar larval wing disc cells ectopically expressing HuD or ELAV under the control of dpp-GAL4 driver were dissected and immunoprocessed with anti-HuD and anti-ELAV antibodies. Propidium iodide was used to visualize nuclei. Confocal images were collected for wing pouch cells. Immuno-signal in green (A,D); propidium iodide signal in red (B,E); merged images (C,F). ELAV shows both nuclear and cytoplasmic localization (A,B,C). In contrast, HuD shows mainly cytoplasmic localization (D,E,F). For details see Materials and Methods.