spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, Y.
Right arrow Articles by Adams, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, Y.
Right arrow Articles by Adams, M. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency

Yoonseong Park1,2, Valery Filippov2, Sarjeet S. Gill2 and Michael E. Adams1,2,*

1 Department of Entomology, 5429 Boyce Hall, University of California, Riverside, CA 92521, USA
2 Department of Cell Biology/Neuroscience, 5429 Boyce Hall, University of California, Riverside, CA 92521, USA



View larger version (64K):

[in a new window]
  		Fig. 1. Timeline depicting the morphological, physiological and behavioral changes preceding ecdysis to the second larval instar in Drosophila. (A) All time points are relative to the double vertical plates (dVP) stage as shown. Broken bars (e.g. A-P) indicate periodically repeated contractions in each stage. Bars depicting a gradient of color indicate gradual changes in morphology. (B) Shedding of the mouthparts and old tracheal tubes (red) during ecdysis. Left: the forward thrust (FT) phase of ecdysis results in deposition of the old mouth hooks and vertical plates onto the substrate (forward blue arrow) and removal of the old tracheal tubes, which are pulled out through spiracular pits, functional only during ecdysis. Right: during the backward thrust (BT) phase of ecdysis, the old spiracles are detached and slide forward (curved red arrow) a maneuver that also extracts the old tracheal trunks (red).

 


View larger version (61K):

[in a new window]
  		Fig. 2. Location of Inka cells shown by ETH immunohistochemistry and EGFP expression in the 2eth3egfp transgenic fly line. Red, blue and green colors are for Cy3, DAPI and EGFP, respectively. Scale bars: (C-J) 10 µm. (A) The larval tracheal system and positions of Inka cells (red). (B) Adult tracheal system and positions of the Inka cells (red). Letters correspond to boxed areas in the diagram shown in C-J. (C) Cy3 staining in the late 1st instar using the DrmETH1 antibody. Old intima (arrow) is already separated from new intima. The Inka cell (arrowhead) is located along the main dorsal tracheal trunk at each branchpoint of the transverse connectives. (D) Expression of eth3-egfp transgene in late 3rd instar. The cell expressing EGFP (arrowhead) at a node (arrow, see Fig. 1B) is shown with low intensity transmitted light. (E) Cy3 staining (arrowhead) in the adult stage using the DrmETH1 antibody. (F) Expression of the eth3-egfp transgene (arrowhead) in the adult stage. (G) The eth deletion mutant eth25b Cy3 (late 1st instar) shows no immunohistochemical staining of the Inka cell. The old intima (arrow) is separated from new intima. Open arrowhead indicates location of the epitracheal gland in wild-type flies. (H) Depletion of ETH immunoreactivity in the Inka cell (arrowhead) of wild-type flies (open arrowhead) immediately after ecdysis to early 2nd instar. (I) Immunoreactive Inka cell in the late 3rd instar using an antibody against MasPETH. (J) Inka cell immunoreactivity (arrowheads) in the adult stage using the MasPETH antiserum.

 


View larger version (48K):

[in a new window]
  		Fig. 3. EGFP co-localized in the Inka cell is depleted during ecdysis to the 2nd instar. (A) Colocalization of ETH and EGFP in an Inka cell in a wandering 3rd instar larva viewed under confocal optics. (B) Depletion of EGFP in a 2eth3egfp transgenic Drosophila Inka cell during ecdysis to the 2nd instar. Sample images of the Inka cell are shown at each time point. Error bars indicate s.e.m. of five to nine individuals for each stage, and more than five cells for each individual.

 


View larger version (21K):

[in a new window]
  		Fig. 4. Molecular map depicting the eth gene, deletions following P-element excision, and structure of the eth-egfp transgene. (A) Molecular map for the 2nd chromosome right arm (60E) region covering eth and adjacent P-element location and direction in the EP(2)1065 line. Boxes are exons for each of the genes, including orc4, eth and reg-5. The primer set used for deletion mutant screening is shown by small arrows. ATGs depict the putative translation initiation sites and directions of the genes. Asterisks indicate the stop codons in each gene. As the transcription initiation site for reg-5 is ambiguous, a thin hatched bar is used to depict the sequence of Van Gelder (Van Gelder and Krasnow, 1996) (GenBank Accession Number, U65105) and a thick hatched bar is used for the EST clones HL04722.5', LP09845.5' and SD04185.5' sequences (GenBank Accession Numbers, AA698461, AI296050 and AI532618, respectively). (B) eth deletion lines generated by imprecise P-element excision from EP(2)1065 line. Three relevant deletion mutant lines are shown. (C) eth gene structure. Boxes show exons, EcRE indicates a putative ecdysteroid responsive element (imperfect repeat of aggtca) (Park et al., 1999), ATG shows the putative translation initiation site for eth, and the star indicates location of the stop codon. ETH1, ETH2 and ETH-AP are depicted with canonical processing sites GKR, GR, KRR and GRR (Park et al., 1999). (D) The transgene pCaSpeR4[eth3egfp]. The ETH chimera with EGFP is constructed after the last canonical processing site GRR. Restriction enzyme sites used for cloning into pCaSpeR4 vector (Thummel and Pirrotta, 1991) are shown as EcoRI and XhoI.

 


View larger version (38K):

[in a new window]
  		Fig. 5. Comparisons of the time lines for ecdysis-related behaviors in wild-type CantonS and in the eth deletion mutant eth25b. See abbreviations in Table 1. (Left) Early DrmETH1 or DrmETH2 injections in CantonS induced premature ecdysis-related behaviors, but ecdysis fails. (Right) The null mutant eth25b lacks pre-ecdysis behaviors anterior-posterior contractions (A-P) and squeezing waves (SW), and exhibits repeated ecdysis-like behavior, and delayed tracheal collapse (TC) and air inflation (AF). Injection of eth25b flies with DrmETH1 or DrmETH2 at the dVP stage rescued the ecdysis deficiency (see text for more details).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002