Download Drosophila windpipe codes for a leucine

Mechanisms of Development 111 (2002) 173–176
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Gene expression pattern
Drosophila windpipe codes for a leucine-rich repeat protein expressed in
the developing trachea
Janice L. Huff*, Karl L. Kingsley, Jennell M. Miller, Deborah K. Hoshizaki
Department of Biological Sciences, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA
Received 11 September 2001; received in revised form 26 October 2001; accepted 26 October 2001
Abstract
The embryonic tracheal system of Drosophila provides an important model for understanding the process of epithelial branching
morphogenesis. Here we report the sequence and expression analysis of a novel tracheal gene, named windpipe (wdp). wdp is identical
to the predicted gene CG3413 and encodes a transmembrane, leucine-rich repeat family member. wdp transcripts appear abruptly at stage 15
and are restricted to primary tracheal branches that give rise to secondary branches. q 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Drosophila; Trachea; Epithelial branching morphogenesis; Embryonic development; Leucine-rich repeat; Transmembrane glycoprotein
1. Results and discussion
We have characterized a Drosophila cDNA of the windpipe (wdp) gene and document its expression in the developing trachea. wdp corresponds to the predicted gene
CG3413 (chromosomal location: 2R, 58D 2-3; Adams et
al., 2000) and codes for a transmembrane leucine-rich repeat
(LRR) family member (Fig. 1A). The predicted WDP
protein has 677 residues with a potential signal peptide
cleavage sequence located between amino acids 20 and 21
(ANA-TP). Based on hydropathy analysis, residues 451–
472 form a transmembrane domain, followed by an
acidic-rich ‘stop-transfer’ sequence (KRKC). The predicted
extracellular domain contains four putative LRR motifs
spanning amino acid residues 70–92, 93–118, 135–157
and 167–216. LRRs are found in numerous proteins and
function in mediating protein–protein interactions (Kobe
and Deisenhofer, 1994). The hypothetical protein
KIAA0918, which shares structural and low-level amino
acid homology, is predicted to be the human homologue
of WDP (Kotani et al., 1998; Flybase, 1999).
Northern analysis revealed that wdp was constitutively
expressed in all stages of development examined (Fig. 2),
while whole-mount embryo analysis demonstrated that
during embryogenesis wdp transcripts were restricted to
the developing trachea (Fig. 3). The Drosophila trachea is
* Corresponding author. Tel.: 11-702-895-1553; fax: 11-702-895-1656.
E-mail address: [email protected] (J.L. Huff).
a branched tubular network arising at stage 10 from bilateral
ectodermal cell clusters that form tracheal metameres in
segments T2–A8. At stage 12, primary buds form within
each metamere to give rise to primary branches: anterior
and posterior dorsal trunk (DTa and DTp); dorsal branch
(DB); visceral branch (VB); anterior lateral trunk (LTa) and
posterior lateral trunk/ganglionic branch (LTp/GB). The
DTa and DTp connect to form the DT that spans the length
of the embryo, while the VB cells of segments T3–A5
extend to form transverse connections, that through secondary branches, associate with the gut. Secondary branches
also extend from the other primary branches, with the
exception of the DT (Samakovlis et al., 1996; Beitel and
Krasnow, 2000).
wdp transcripts were first detected at early stage 15
(Campos-Ortega and Hartenstein, 1997) in the cells of the
primary branches, excluding the longitudinal DT (Fig. 3).
wdp was simultaneously expressed throughout the length of
the VB, in eight of the DBs, in all of the LTa and LTp/GBs
(Fig. 3A) and in the ventral portion of the transverse connective (TC; Fig. 3E). wdp expression exhibited an anterior–
posterior polarity in the DBs. wdp was detected along the
length (from DT to terminal cells) of DBs within tracheal
hemisegments Tr 1–2 (T2–3), whereas in the more posterior
hemisegments transcripts were gradually restricted to the
distalmost cells. In Tr 7–8, transcripts were limited to the
terminal cells, while in Tr 9–10, expression was absent in
the DB including the terminal cells (Fig. 3F,G). At stage 15,
the primary branches, with the exception of the DT, form
secondary branch outgrowths. wdp expression was absent in
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J.L. Huff et al. / Mechanisms of Development 111 (2002) 173–176
secondary and tertiary branches. By late-stage 15, levels of
wdp transcript regressed to the cells closest to the fork of the
LTa and LTp/GB (Fig. 3H), and to the terminal cells of the
DB within Tr 1–8 (data not shown). By stage 16, transcripts
were limited to the ventral region of the TCs (Fig. 3I,J). The
expression of wdp transcripts in distinct dorsal and ventral
regions (specifically, the DB, VB, ventral TC, LTa and LTp/
GB) suggest that wdp might be a new member of the regional class of tracheal genes identified by enhancer traps
(Samakovlis et al., 1996).
Fig. 2. Northern blot analysis of windpipe expression. Approximately 3.0kb transcript was detected in poly(A 1) mRNA (5 mg/lane) isolated from 0to 24-h embryos, third-instar larvae and male and female y w adults. Equal
loading was confirmed by visualizing the mRNA on the membrane with
EtBr prior to hybridization.
2. Methods
2.1. cDNA library screen, DNA and RNA techniques
Fig. 1. windpipe cDNA sequence and predicted open-reading frame. The
complete coding sequence for windpipe (Genbank accession no. AF395331)
was assembled from overlapping cDNA isolated from an 18-h embryonic
library and from Berkeley Drosophila Genome Project, Expressed Sequence
Tag (BDGP EST) clones GH02310 and LD35218 (Genbank accession nos.
AI062927 and AI518817, respectively). Predicted amino terminal signal
peptide sequence is highlighted in blue (von Heijne, 1986); LRR motifs
are shaded in gray (predicted by Pfam alignment program, Bateman et al.,
2000). Potential N-linked glycosylation consensus sites are boxed and the
hydrophobic transmembrane sequence is highlighted in red. (Sonnhammer
et al., 1998). Base-pairs 222–269 (underlined) in the 5 0 non-translated
regions are deleted in the EST clone SD04444 (Rubin et al., 2000;
AI53219). (B) Schematic diagram of overlapping wdp cDNAs.
windpipe cDNAs were isolated during a screen of the 58D
region for the fat-body gene associated with P[29D] (Hoshizaki et al., 1994; Blackburn, 1994.). lgt10 clones were
isolated from an 18-h embryonic cDNA library (Clontech
Inc., IL1010A) and EST clones were from ResGen. DNA
sequence from both strands of overlapping clones was
assembled using the CAP contig assembly program
(Huang, 1992). Poly (A) 1 RNA was isolated using standard
methods (Chirgwin et al., 1979; Sambrook and Russel, 2001).
2.2. Staining procedures
In situ hybridization to whole-mount embryos was accomplished as described (Hoshizaki et al., 1994). Microscopic
analyses were performed on a Zeiss Axioplan2 microscope
and images taken with a Kodak MDS120 digital camera.
J.L. Huff et al. / Mechanisms of Development 111 (2002) 173–176
175
Fig. 3. Embryonic expression pattern of wdp. wdp transcripts were detected in whole-mount embryos by in situ hybridization of digoxygenin-labeled anti-sense
RNA derived from lgt10 clone 8.2-2. (A–G) wdp expression was first detected at early stage 15. Images (A–G) are of the same embryo. (A) Lateral–dorsal
view, wdp was expressed within the head, the DBs, the VB, the distal portion of the TCs and the LTa, LTp/GB of the developing tracheal system. (B) Higher
magnification and ventral–lateral view of wdp expression in a tracheal branch within the head region. (C) Dorsal view of wdp expression in the VBs. (D) Higher
magnification and a lateral view of wdp expression in the VBs. (E) Higher magnification and a ventral–lateral view of wdp expression in the TC, LTa, LTp/GB
and VB (out of focus). (F) Higher magnification and a lateral view of wdp in DB. (G) Higher magnification and dorsal view of wdp in the terminal cells of the
DBs of Tr 1–8. No wdp activity was detected in the terminal cells of Tr 9–10. (H) Lateral view of a late-stage 15 embryo. (I) Lateral view of stage-16 embryo.
(J) Ventral view of stage-16 embryo. wdp activity (black arrow) was detected in a position that was both internal and posterior to the apodemes, or muscle
attachment sites (open arrow). (K) Schematic drawing representing tracheal metameres, Tr 2–Tr 3, during early stage 15; wdp expression is highlighted in blue;
regions lacking wdp expression (DT and TC) are outlined by black dots.
Acknowledgements
The authors are grateful to Dr George E. Plopper for use of
laboratory space and equipment during the course of this
work. We thank Dr Mark Krasnow for helpful advice on
the expression analysis. This project was supported by a
National Science Foundation POWRE award to J.L.H. and
a generous gift to D.K.H. from Dr and Mrs Shearing of the
North Star Foundation. K.L.K. and J.M.M. were supported
by Barrick fellowships from the U.N.L.V. Graduate College.
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J.L. Huff et al. / Mechanisms of Development 111 (2002) 173–176
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