Article

A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
PLoS Genetics (Impact Factor: 7.53). 07/2011; 7(7):e1002087. DOI: 10.1371/journal.pgen.1002087
Source: PubMed

ABSTRACT

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general "house-keeping" genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system.

Full-text

Available from: Amin S Ghabrial, Jul 07, 2014
A Systematic Screen for Tube Morphogenesis and
Branching Genes in the
Drosophila
Tracheal System
Amin S. Ghabrial
1,2
*, Boaz P. Levi
, Mark A. Krasnow
1
*
1 Department of Biochemistry and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, United States of America, 2 Department
of Cell and Developmental Biology, University Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America
Abstract
Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of
the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in
many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or
discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third
chromosome genes (,40% of the genome) that function in development of the tracheal system, a tubular respiratory organ
that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the
screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary
screen to exclude mutations in general ‘‘house-keeping’’ genes. From a collection including more than 5,000 lethal
mutations, we identified 133 mutations representing ,70 or more genes that subdivide the tracheal terminal branching
program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis
and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the
70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal
analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching
morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through
cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can
elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build
even a relatively simple organ such as the Drosophila tracheal system.
Citation: Ghabrial AS, Levi BP, Krasnow MA (2011) A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System. PLoS
Genet 7(7): e1002087. doi:10.1371/journal.pgen.1002087
Editor: Eric Rulifson, University of California San Francisco, United States of America
Received March 28, 2011; Accepted April 5, 2011; Published July 7, 2011
Copyright: ß 2011 Ghabrial et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the National Institutes of Health (http://grants.nih.gov/grants/funding/r01.htm; NIH R01 GM47735 ‘‘Cellular
Communication in Morphogenesis’’) and by the Howard Hughes Medical Institute (www.HHMI.org). ASG was an NRSA postdoctoral fellow, BPL was an American
Heart Association predoctoral fellow (0415030Y), and MAK is an investigator of the Howard Hughes Medical Institute. ASG gratefully acknowledges current
support from the University of Pennsylvania, a March of Dimes Basil O’Connor Award, and the NIH (1R01GM089782-01A1). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ghabrial@mail.med.upenn.edu (ASG); krasnow@stanford.edu (MAK)
¤ Current address: University of Michigan, Ann Arbor, Michigan, United States of America
Introduction
Elucidating the genetic programs of organ formation and
maintenance is a central goal of developmental biology and
medicine. Many organogenesis genes have been isolated in
systematic genetic screens in model organisms, and many others
have been identified by their organ-selective expression patterns
and by candidate gene analysis. These approaches have been very
successful at discovering the signaling pathways and transcription
factors that induce and pattern organs and specify cell fates, but
they have been much less successful at identifying the downstream
effectors that execute morphogenesis programs, what we call
morphogenesis genes [1,2,3,4]. A similar abundance of signaling
and transcription factor genes and dearth of morphogenesis genes
has obtained from the pioneering genetic dissection of Drosophila
body axis formation and other early developmental events [5]. We
reasoned that many morphogenesis genes would function in
multiple organs and developmental processes, so mutations in
these genes would be pleiotropic and hence discarded in most
genetic screens. We therefore designed systematic, saturation
screens for genes required for Drosophila tracheal system organo-
genesis that included clonal analysis of gene function in the
tracheal system, to identify all tracheal genes including those with
pleiotropic phenotypes. The results of a screen of the third
chromosome, representing ,40% of the Drosophila genome [6], are
described here, and the results of a first (X) chromosome screen
initiated earlier will be described elsewhere ([7]; M. Metzstein and
M.A.K., unpublished data).
The Drosophila tracheal (respiratory) system is a branched
tubular network that transports oxygen throughout the body [8].
It is one of the most intensively studied and best understood
organogenesis programs [9,10], and it has emerged over the past
decade as a paradigm of branching morphogenesis, the develop-
mental process that gives rise to many organs including the lung,
vascular system, kidney, and pancreas. Understanding how
branching networks are patterned and how cellular tubes are
made, shaped, and maintained is of fundamental importance in
cell and developmental biology, and in medicine for understanding
PLoS Genetics | www.plosgenetics.org 1 July 2011 | Volume 7 | Issue 7 | e1002087
Page 1
and treating tubular diseases such as aneurysms and polycystic
kidney disease.
The tracheal system develops from 10 pairs of tracheal sacs that
arise by invagination of the embryonic ectoderm [8,11]. Each sac
is an epithelial monolayer composed of ,80 cells. Primary
tracheal branches are formed by groups of 3–20 cells that bud
from the sacs in different directions and successively sprout
secondary and terminal branches. Some specialized primary and
secondary branches grow towards and fuse with branches from
neighboring sacs to interconnect the tracheal network [12]. The
transformation of the simple epithelial sacs into an extensively
branched tubular network occurs without cell proliferation, and is
mediated by cell migration, rearrangement, and dramatic changes
in cell shape [11,13,14,15]. During embryogenesis, the lumens of
the developing tracheal branches are filled with a complex and
changing matrix, which is cleared and replaced with gas just
before the embryo hatches and the tubes become functional in
respiration [8,16]. In the larva, terminal cells ramify extensively to
form many new terminal branches (tracheoles), long cytoplasmic
extensions that grow toward oxygen-starved cells and then form a
cytoplasmic, membrane-bound lumen, creating tiny tubes (,1um
diameter) that supply the targets with oxygen (Figure 1B) [14,17].
Unlike primary (multicellular) and secondary (unicellular) branch-
es, tubes sealed by intercellular and autocellular junctions
(Figure 1B), terminal branches lack cell junctions and resemble
the ‘‘seamless’’ endothelial tubes of the mammalian microvascu-
lature [18,19,20] and C. elegans excretory system [21].
The first important tracheal gene identified was trachealess ,
isolated in the classical screens for embryonic patterning mutants
by the complete and selective absence of the tracheal system [22]
and later shown to encode a bHLH-PAS transcription factor, the
earliest expressed tracheal-specific gene and a master regulator of
tracheal identity [23,24]. Ten years after the discovery of
trachealess,aDrosophila homolog of mammalian FGFRs was isolated
and named Breathless because it is selectively expressed in the
developing tracheal system and required for branching [25,26,27].
Around this time the first systematic screens for tracheal mutants
were conducted, screens of P[lacZ] insertions that identified about
50 tracheal genes that subdivided embryonic tracheal develop-
ment into genetically distinct processes including primary,
secondary, and terminal branching, branch fusion, and tube size
control [11,28]. Mapping and molecular characterization of these
genes identified many components and modulators of the
Breathless FGFR signaling pathway. These include Branchless
FGF, which activates Breathless FGFR and plays a central role in
controlling and coupling each of these processes by guiding
outgrowth of primary branches and inducing expression of key
genes encoding transcription factors such as pointed, blistered/pruned,
and escargot required, respectively, for secondary and terminal
branching and branch fusion [12,14,26,29]. Many other impor-
tant genes have been identified by their tracheal expression
patterns, analysis of candidate genes, and serendipitous discovery
of a tracheal function for genes initially studied in other contexts
[30,31,32,33]. And, over the past several years, several screens of
chemically-induced mutations for tracheal morphogenesis defects
in embryos and larval tracheal and air sac primordium clones have
been conducted [34,35,36] along with more targeted genetic and
genomic screens for genes that are expressed or function
downstream of some of the key early signaling pathways (branchless,
breathless) and transcription factors (trachealess, ribbon) [37,38,39,40].
Together these approaches have implicated ,100 genes in
tracheal development, most of which encode transcription factors
or components of signaling pathways (FGF, TGFa/EGF, TGFb,
Wnt, Notch, Slit/Robo, Jak/Stat, and Hedgehog) [1,2,4,9] (Table
S1). However, the downstream targets of the signaling pathways
and transcription factors, the morphogenesis and maturation
genes that create, shape, and stabilize the tubes, have only recently
begun to be identified. And, although expected to be a large class,
they are substantially under-represented among characterized
tracheal genes (Table S1) [4].
We conducted a large-scale screen of chemically-induced
mutations to assess the function of nearly all Drosophila third
chromosome genes, including early essential genes and genes with
pleiotropic phenotypes. We sought to identify most or all of the
genetically separable steps in tracheal development; to identify
new tracheal genes associated with each step; and to provide an
estimate of the total number of genes required to build an organ.
We were especially interested in identifying tracheal morphogen-
esis genes. Our approach involved clonal analysis in the tracheal
system of all chemically-induced mutations that did not survive
late enough in development as homozygotes to assess their
tracheal function, and a secondary screen to exclude general
‘‘house-keeping’’ genes. We isolated mutations representing ,70
genes, 14 of which we identified molecularly, implicating most of
the genes as morphogenesis genes and revealing new cell biological
pathways in tracheal development. Many of the mutations affect
terminal branch morphogenesis, genetically subdividing this
poorly understood process into five major morphogenetic steps
including an integral cell growth step.
Results
Screen design
To identify tracheal morphogenesis genes, we screened the third
chromosome for EMS-induced mutations that affect larval
tracheal morphology. Approximately 4,300 mutagenized third
chromosomes were generated, and balanced lines were established
for each. Three-quarters (73%) of the lines were homozygous
lethal. Assuming a Poisson distribution, there were ,1.3 lethal
mutations per mutagenized third chromosome and a total of 5600
lethal mutations screened. Because there are ,3600 essential
Drosophila genes [6], with roughly 40% (,1370) on the third
Author Summary
Elucidating the genetic programs that control formation
and maintenance of body organs is a central goal of
developmental biology, and understanding how these
programs go awry in disease has important implications
for medicine. Many such organogenesis genes have been
identified, but most are early-acting ‘‘patterning genes’’
encoding signaling proteins and gene regulators that
control expression of a poorly characterized set of
downstream ‘‘morphogenesis genes,’’ which encode pro-
teins that generate the remarkable organ forms and
structures of the constituent cells. We screened ,40% of
the fruit fly Drosophila genome for mutations that affect
tracheal (respiratory) system development. We included
steps to bypass complexities from mutant effects on other
tissues and steps to exclude mutations in general cell
‘‘housekeeping genes.’’ We isolated mutations in ,70
genes that identify major steps in the organogenesis
program including an integral cell growth control step.
Many of the new tracheal genes are ‘‘morphogenesis
genes’’ that encode proteins involved in cell structure or
intracellular transport. The results suggest that genetic
screens can elucidate a full organogenesis program and
that over 200 patterning and morphogenesis genes are
required to build even a relatively simple organ.
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 2 July 2011 | Volume 7 | Issue 7 | e1002087
Page 2
chromosome [41], we expected to obtain an average of about four
(5600/1370) mutations per gene, with at least one mutation in
97% of all third chromosome genes.
The mutants were screened for tracheal defects in two steps. First,
homozygous third instar larvae of the F3 generation (Figure 1E),
which carried btl-GAL4 and UAS-GFP transgenes (abbreviated
btl.GFP) to label tracheal cells, were scored for tracheal defects by
fluorescence microscopy. The balancer chromosome carried a Tub-
GAL80 transgene that inhibits Gal4 and blocks expression of UAS-
GFP so only homozygous mutant animals expressed GFP,
facilitating screening (Figure 1C). For 40% of lines, GFP
+
F3 third
instar larvae were not recovered, presumably because the
homozygous mutations caused early lethality.
These pre-pupal lethal lines were analyzed in a second step of
the screen, using a genetic mosaic strategy in which we examined
clones of homozygous mutant tracheal cells in otherwise
heterozygous larvae (Figure 1F). We devised a variant of the
MARCM clone marking strategy [42] employing a UAS-
GFP(RNAi) transgene on the homologous chromosome, in trans
to the mutation of interest, which allowed us to label all tracheal
cells with btl .DsRed and homozygous mutant tracheal cells
(lacking UAS-GFP(RNAi)) with btl.GFP (Figure 1D). This
facilitated comparison of homozygous mutant cells (DsRed
+
,
GFP
+
) with surrounding wild type tracheal cells (DsRed
+
, GFP
-
),
enhancing the sensitivity of the screen and detection of cell non-
autonomous effects in the tracheal system.
Overview of screen results
Over 600 mutants with highly penetrant and expressive tracheal
defects were identified. However, the vast majority were lethal
Figure 1. Design of tracheal mutant screen. (A) Diagram of Drosophila tracheal system in third instar larva (dorsal view, anterior up unless noted
otherwise). A close up of two hemisegments (Tr4 and Tr5) are shown at right, with some primary branches indicated. DT, dorsal trunk; DB, dorsal
branch; LT, lateral trunk. (B) Schematic showing cellular structure of dorsal trunk and dorsal branch. Dashed lines indicate plane of section of cross-
sections shown. DT is a multicellular tube with multiple cells and intercellular junctions seen in cross-section. DB stalk is an autocellular tube, a single
cell wrapped around the luminal space and sealed by an autocellular junction. DB terminal cell (TC) forms multiple terminal branches, each of which
is a ‘‘seamless’’ tube lacking junctions. The base of the terminal cell (*), from its junction with a stalk cell to the nucleus, is an autocellular tube. The
fusion joint (FJ) is the position where two fusion cells, each of which forms a seamless tube, connect contralateral tracheal hemisegments. Lum,
tracheal lumen (black); Jxn, intercellular junctions (red); Nuc, cell nuclei (black). (C) Fluorescence micrograph of two sibling F3 larvae from the F3
screen diagrammed in panel E. The GFP
-
larva at left is heterozygous for the mutagenized third chromosome; it is nearly invisible because it contains,
in trans to the mutagenized chromosome, a Gal80-expressing balancer chromosome that prevents expression of btl-Gal4, UAS-GFP (btl.GFP). The
GFP
+
larva at right is homozygous for a mutagenized third chromosome; it lacks the Gal80 chromosome, so expresses GFP throughout the tracheal
system. (D) Fluorescence micrograph of a segment (Tr5) of the tracheal system from a third instar larva generated by the genetic mosaic strategy
shown in panel F. All tracheal cells express btl.DsRED (red); homozygous clones lack the UAS-GFP(RNAi), so express in addition btl.GFP (green).
Dorsal trunk (DT), dorsal branch (DB) and terminal cell (TC) clones are marked (arrowheads). Dorsal branch fusion joint (FJ) connecting the left and
right hemisegments is indicated. (E) Genetic scheme of F3 screen. EMS, ethyl methanesulfonate; Pr, Prickly; P[hs-hid], heat shock inducible hid
transgene; TM3, third chromosome balancer; P[Gal80], transgene with ubiquitous tubulin promoter driving expression of Gal80, a Gal4 inhibitor;
2FRT, two Flp Recombinase Target (FRT) site transgenes (FRT2A on 3L and FRT82B on 3R) flanking the third chromosome centromere; Sb, Stubble;
*, mutagenized chromosome. (F) Genetic scheme of the mosaic screen. hs-FLP, heat-inducible FLP recombinase transgene; UAS-GFP(RNAi), Gal4-
inducible (Gal4 upstream activating sequence) GFP RNAi transgene.
doi:10.1371/journal.pgen.1002087.g001
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 3 July 2011 | Volume 7 | Issue 7 | e1002087
Page 3
mutations in genes required cell autonomously for tracheal cell
growth and survival, and later found in a secondary screen (see
below) to be presumptive housekeeping genes and discarded. 133
tracheal mutations were saved (Table 1 and Table S3), 18 from the
F3 screen (alleles with two letter prefix, e.g. PC213) and 115 from
the genetic mosaic screen (no prefix).
Most of the mutations could be placed into one of five
phenotypic classes (Table 1 and Table S3): (1) cell selection/
specification; (2) cell size; (3) branch number, pattern size and
shape; (4) tube formation, number, position, and shape; and (5)
lumen clearance/gas filling. Within each broad class, phenotypic
subgroups were defined and representative mutations in each
subgroup were selected and subjected to detailed phenotypic
characterization and genetic mapping as detailed below. Some
mutations had more than one defect and were placed into more
than one subgroup or class. Only a few mutants with cell non-
autonomous effects were recovered from the clonal screen (see
below), implying that such tracheal mutations are rare.
Genetic complementation groups
Complementation tests allowed assignment of 68 mutations to
24 loci (Table 1). In addition, four mutations that were mapped to
specific chromosomal deficiencies were found to be new alleles of
extant genes in the mapped intervals (see below). In addition to
these 72 definitively assigned mutations in 28 loci, we also
characterized and named 30 other mutations with interesting
tracheal phenotypes (Table 1). The rest of the saved mutations
(Table S3) were not extensively characterized; 12 of these are
associated with mapped lethal mutations that complement extant
tracheal mutations in the mapped interval, so may represent
additional essential tracheal genes.
It is difficult to estimate the number of mutations we obtained in
previously known tracheal genes because the mosaic loss of
function phenotype is not known for most tracheal genes, and the
number of complementation tests necessary to determine this
number directly is prohibitive. However, the apparent absence of
mutations in two known tracheal genes (stumps and trachealess)
whose mosaic phenotype we determined, and the lower than
expected allele frequencies (mean 2.6) obtained for the 28
definitively identified loci, indicate that the screen did not achieve
the degree of saturation predicted by a Poisson distribution.
Nevertheless, the screen was extensive so we think it is likely it
identified mutations in most processes and molecular pathways
involved in tracheal tube morphogenesis.
Below, we describe each of the major phenotypic classes and
subcategories, and representative mutations in each. Most of the
mutations are homozygous lethal and all caused highly penetrant
and expressive tracheal phenotypes. For ease in presentation, we
treat the strongest phenotype in each complementation group as
the null phenotype; however, we do not know for most if they truly
represent the null condition because it is not readily possible to
generate hemizygous (mutant/deficiency) clones for comparison or
to exclude partial masking of phenotypes due to perdurance of
wild type protein in mutant cells.
Class 1: cell selection/specification mutants
These mutations eliminated specific tracheal cell types or
blocked their differentiation.
No mutant terminal cells (1A). Two complementation
groups (no terminal cell clones-3L and no terminal cell clones-3R) gave
normal numbers of mutant clones in the mosaic screen but the
mutant cells rarely if ever included terminal cells (Figure 2A, 2B).
This novel phenotype lead to new insights into the terminal cell
selection process (see Discussion).
Region-specific terminal cell loss (1B). Two lines lacked
terminal cells in specific body regions of homozygous mutant
animals. In missing parts (AG33) mutants, dorsal branch, fat body,
and CNS terminal cells were frequently missing, but terminal cells
in other positions were unaffected (Figure 2C, 2D). steeple (AI87)
mutants frequently lacked dorsal branch terminal cells (Figure 2E,
2F), although all other terminal cells were present. These
phenotypes demonstrate that there are region- or branch-specific
modulators of terminal cell selection, specification, or survival, the
existence of which had been suggested by marker expression
patterns [11].
Failed branch fusions (1C). Four lines showed frequent
branch fusion failures in homozygous larvae. These included failed
fusions (PA14, AZ63) and missing parts (Figure 2D), which showed
dorsal branch and lateral trunk fusion defects as well as the
terminal cell defects noted above. loose caboose (AB56) mutants had
frequent defects in fusion of the most posterior dorsal branches
(Figure 2G, 2H).
Class 2: cell size mutants
These mutants had their most profound affects on tracheal cell
size. For nearly all mutations, the effect on terminal cell size
correlated with branch number: larger cells had more terminal
branches and smaller cells had fewer. One exceptional mutant,
sprout, is presented below.
General tracheal cell overgrowth (2A). Two comple-
mentation groups showed tracheal cell overgrowth phenotypes in
the mosaic screen, and the enlarged terminal cells had more
branches (see below). miracle-gro (338,878,1483,1489) mutant cells
were several times larger than normal (Figure 3A, 3B), and the
lumens of the mutant cells had larger bores and pursued a more
tortuous path through the cytoplasm. The latter phenotype was
fully penetrant in the seamless tubes of terminal cells and partially
penetrant in larger tracheal tubes. jolly green giant (1149) caused a
more subtle increase in cell size, most readily detected in terminal
cells, and no obvious alteration in lumen morphology. Both
miracle-gro and jolly green giant mutations also cause overgrowth of
cells outside the tracheal system, because eyes derived from miracle-
gro or jolly green giant clones in the EGUF/hid assay [43] described
below were enlarged.
General tracheal cell undergrowth (2B). In roughly 500
lines, homozygous mutant cells in mosaic animals were small or
absent, and mutant terminal cells when present were small with
few or no branches (Figure 3C). Most of these lines presumably
carried mutations in general cell growth (‘‘house-keeping’’) genes,
but we suspected a subset might carry mutations in genes
specifically required for tracheal cell growth. Indeed, we found
that clones mutant for the tracheal master regulator trachealess,
resembled clones homozygous mutant for the housekeeping gene
glutamyl-prolyl-tRNA synthetase (Figure S1). To distinguish tracheal-
specific from more general cell growth mutations, we tested the
,500 tracheal undergrowth mutations in the developing eye. We
used the EGUF/hid system [43] in which forced expression of a
cell death gene in the eye imaginal disc eliminates wild type cells,
so adult eyes develop almost entirely from clones of homozygous
mutant cells. Nearly all (.99%) of the tracheal cell undergrowth
mutations failed to rescue eye development, resulting in adults
lacking eyes or with grossly undersized eyes. This substantiated
that these mutations affected more general cell growth genes, and
the lines were discarded. However, two tracheal undergrowth
mutants, lotus (312) (Figure 3C) and etiolated (1736), formed eyes of
normal size and shape, demonstrating that they are tracheal-
selective growth mutations. Other aspects of the lotus phenotype
are detailed below.
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 4 July 2011 | Volume 7 | Issue 7 | e1002087
Page 4
Table 1. Tracheal morphogenesis mutant collection.
Name (alleles
1
) Tracheal Phenotype (category) [other affects] Map Position (method
2
)
appaloosa (771) TC gas-filling defect, variable penetrance (5A1). [Abundance of epidermal clones; ectopic bristles
in trans to Df35]
87F9; 87F12 (Df 1,2)
asthmatic (742, 1530) TC gas-filling defect (5A1). Incompletely penetrant autocellular gas-filling defect (5B). 3R (MA)
balloon (1448) DT cell clones with dilated lumen (4C1). Variable TC shape defect, mild pruning, rare multi-lumen defect. 70C; D (Df 11,12)
black hole (538) Large cytoplasmic vacuoles in TC (4A5). Variable TC pruning and sporadic odd positioning of nucleus. 64C; 65C (Df 3)
braided (1615) Variable TC multi-convoluted lumen defect (4A3). Moderate pruning. 3L (MA)
bulgy (636) Dorsal trunk clones with lumen dilation (4C1). TC pruned with gas-filling defect. 83C;D (Df 4)
burs (942, 1139) Selective TC pruning (3C1). 942 allele with TC gas-filling defect. 73D1 (Df 5)
carbuncle (804) Large GFP-excluding bodies in all mutant tracheal cells (4A5). TC lumen forma tion is variably
discontinuous (4A1).
66B; 66C (Df 6,7)
cincher (773) Dorsal trunk cell clones are tiny (2C) 83B7; 83C2 (Df 8)
conjoined (356) Thick and severely pruned TC (3C2). Defective intercalation/autocellular tube forma tion (4B). Auto/
subcellular tube gas-filling defect (5A3). DT cells rounded and contribute minimally to multicellular lumen.
3R (MA)
constricted (960) Dorsal trunk lumen constriction (4C2) 70C; D (Df 11,12)
creeper (153) Variable TC multi-convoluted lumen defect (3A). Mild to moderate TC pruning (3A). 96A (Df 9, 10)
corset (897) Dorsal trunk cell clones are tiny (2C) 3L-4 lethals (Df 14-18)
curlicue (1629) TC tips with variable multilumen defect (4A3). TCs moderately pruned. 3L (MA)
cystic lumens (1243) Dramatic TC lumen dilation defect (4A4). Moderate TC pruning. 63F6; 64C15 (Df 19)
dark matter (1417) Cytoplasmic vacuoles (4A5). Moderate TC pruning and lumens slightly convoluted. 84B;D (Df 20)
denuded (PC213, 1520) Selective TC pruning (3C1). Lumens in remaining branches have small bore. cu-sr (RM)
Disjointed (169) TC autocellular-seamless tube junction gas-filling defect (5A3). Mild to moderate pruning. 3R (MA)
dyspneic (1348, 1359) Strong TC gas-filling defect (5A1) and autocellular tube gas-filling defect (5B). DT cells show mild lumen
constriction (4C2).
3R (MA)
etiolated (1537, 1736) Tracheal-specific growth defect (2B). [Normal size eyes in EGUF/hid assay] 73A; 74F (Df 21)
failed fusions (PA14,
AZ63)
Dorsal branch and sporadic lateral trunk fusion defects (1C) 3R (MA)
flash flood (AP67) Lateral trunk fusion and clearance defect in mutant larvae (5B) Ch 3
ichorous (206) TC gas-filling defect with lumGFP accumulation (5A1) 85A2; 85C1-2 (Df 22 )
impatent (1472, 1490,
1757)
TC lumen formation defective with lumGFP accumulating in puncta (4A1) 61 (Df 23)
ivy (1781) TC moderately to severely pruned with multiple convoluted lumens (4A3) 65A,B; 66B,C & 70E;71F (Df
6,7,15,16, 24)
jolly green giant (1149) All mutant tracheal cells overgrown with most dramatic effect on TCs (2A). [Distal hairy-wing
in trans to Df9]
95D; 95F & 98E; 99A (Df 25,26)
liquid-filled (725) TC defective for gas-filling (5A1) 82F (Df 27)
3
littoral (762) Variable TC gas-filling defect, with tips most often affected (5A2) 3R (MA)
loose caboose (AB56) DB10 fusion defect with posterior spiracles often misaligned (1C) Ch 3
lopped (784) Moderate to strong TC pruning (3A) 82F (Df 27)
lotus (312) TC pruned and sometimes appears fragmented (as if degenerating) and other tracheal cells are small (2B).
TC seamless/autocellular tube connection is defective (5A3). TC rounded and contributes minimally to
lumen. [Normal size eyes in EGUF/hid assay]
3R (MA)
miracle-gro (338, 878,
1483, 1489)
All mutant cells overgrown with most dramatic effect on TC (2A) 99F; 100B (Df 28)
missing parts (AG33) Region-specific TC loss and fusion defect (1B). Ch 3
moon cheese (1524) All cells accumulate GFP-excluding vacuoles (4A5). TC pruned with variable lumenal discontinuities (4A1). 64C;D (Df 29)
no tc clones-L (602,
724, 788, 1118, 1187,
1476, 1684, BN40)
Mutant cells never occupy TC position (1A) h-th (RM)
no tc clones-R (198,
1318)
Mutant cells never occupy TC position (1A). When clones present near branch tip, TC often missing. 94D; 95A (Df 30)
oak gall (696) Thick and severely pruned TC (3C2). Defective intercalation/autocellular tube formation (4B). Auto/
subcellular tube gas-filling defect (5A3). DT cells rounded and contribute minimally to multicellular lumen.
3R (MA)
paltry (1181, 1803) Severe TC pruning (3A), sometimes appear to be degenerating. Varia ble gas-filling defect. 63F; 64C & 68A; 69A (Df 19, 31)
panting (1318, 1584) Gas-filling defect at TC branch tips (5A2). Possible mild TC pruning. 77B-C; 77F-78A (Df 32)
piddling (1002, 1834) Mild to moderate TC pruning (3A) with variable gas-filling defect 69C; F (Df 33)
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 5 July 2011 | Volume 7 | Issue 7 | e1002087
Page 5
sprout (574) caused a general reduction of tracheal cell size, as
well as a growth defect in the EGUF/Hid assay. However, it was
distinguished from all the general undergrowth mutations
described above by the ability of the small mutant terminal cells
to branch extensively, like a bonsai plant (Figure 3D).
Selective tracheal cell undergrowth (2C). Two mutations,
cincher (773) and corset (897), caused a growth defect selective for
dorsal trunk cells: homozygous mutant dorsal trunk cells were less
than half normal size, whereas mutant terminal cell clones were
unaffected (Figure 3E). This selectivity contrasts with that of the
,500 house-keeping mutants, many of which caused their most
pronounced effects on terminal cells, presumably because of the
dramatic growth and branching these cells undergo during larval
life. Thus, cincher and corset are growth genes required in dorsal
trunk but dispensable in terminal cells.
Class 3: branch number, pattern, size, and shape mutants
These mutations affected the number of terminal branches, and
in some cases also the position at which new branches bud from
the parental branches. Many also affected the shape of the buds
and mature branches. Mutations caused different but character-
istic spectrums of defects so that, for example, mutations that
reduced terminal cell branching to a similar extent could
reproducibly give rise to terminal cells of very different
morphology, such as short and thick versus elongate and wispy.
Terminal branch pruning (3A). We identified ,40 mutants
in which terminal cell clones had fewer branches than normal but
were distinct from the general cell growth genes described above
because mutant dorsal trunk cells were of normal size and
morphology. This phenotype is similar to that of blistered (pruned),
the canonical terminal branching gene [14,44]. winded alleles
(613,1227,1375,1508) showed severe pruning defects, like blistered
null alleles: mutant terminal cells had few branches, and any
residual branches were typically thin and wispy and lacked
subcellular tubes (Figure 4A, 4B). Other severe pruning mutants
were paltry and topiary. Mutations in most other genes of this class
showed more modest pruning defects (e.g. lopped, truncated),
comparable to blistered partial loss of function alleles.
Excess branching (3B). Although many mutations that
reduced terminal branching were identified, mutations that
Name (alleles
1
) Tracheal Phenotype (category) [other affects] Map Position (method
2
)
scrub (659) Severe TC pruning (3A). Gas-filling defect with no visible lumGFP (5). 64C; 65C & 76B; 77B (Df 42,51)
short of breath (360,
404, 483, 791, 1705)
Strong TC gas-filling defect (5A1). Autocellular tubes also show gas-filling defect (5B). DT cells show
mild lumen constriction (4C2).
82F (Df 27)
short round (1103,
1695)
Moderate to severe TC pruning with incomplete gas filling (3A). DT cells are small and rounded. 87B; 87D (Df 34,35)
small potatoes (1113,
1166, 1694)
Moderate TC pruning (3A) and incomplete gas filling. DT lumen bulges outward (4C1). 95A; D, 96A; B, & 97A; 98A (Df
36,37,38)
spikes (735) Mild TC pruning defect but branches show excess filopodia (3C) 3R (MA)
sprout (574) All tracheal cells are tiny, but TC are nevertheless robustly branched (2B) 3R (MA)
steeple (A187) Dorsal branch TC missing with high penetrance in homozygous larvae (1B) Ch 3
stertorous (1290, 1321) All seamless tubes defective for gas-filling (5A1) 65F3; 66B10 (Df 16)
tendrils (666, 1308,
1469, 1539)
TC pruning with multiple convoluted lumens (4A3) ru-h (RM)
tiny tubes (630, 1309) Mild to moderate TC pruning with narrow bore lumens (3A) 83A6; 83B6 (Df 39,40)
topiary (700, 1019) Moderate (1019) to severe (700) TC pruning (3A) 61A; D3 & 64C; 65C (Df 41,42)
truncated (533, 1659) Moderate TC pruning (3A) & incomplete gas-filling 69A2-3 (Df 31,43)
vine (512) TC with multiple convoluted lumens (4A3) and moderate pruning 89E (Df 44,45)
wavy lumens (894) TC with tortuous lumens (4A2) and moderate pruning 3R (MA)
whacked (PC24, 220) Variable TC pruning and lumen formation defect, including prematurely truncated tubes ending in local
dilations and discontinuous tubes (4A1, 4A4).
86 E14; 86E17 (Df 47)
wheezy (770) Mild TC pruning (3A) and air filling defect. DT clones show darker cuticle over apical membrane.
[Cross-veinless wing defect in trans to Df 13]
89E11; 90A7 (Df 46,49)
winded (613, 1227,
1375, 1508)
Strong TC pruning (3A) and air filling or seamless tube formation defect 65F3; 66B10 (Df 16)
wobbly lumens (BG13) Tortuous lumens in TC branches of third instar larvae (4A2) Ch 3
1
Allele names beginning with two letters (e.g. PC213) were identified in the F3 screen. All others were identified in the genetic mosaic analysis.
2
Methods used for mapping: MA, mosaic analysis; RM, meiotic recombination mapping with recessive markers; Df, deficiency mapping with failure to complement
deficiencies indicated; Ch 3, unmapped mutation on the third chromosome. Chromosomal deficiencies used: (1) Df(3R)126c, (2) Df(3R)Urd, (3) Df(3L)ZN47, (4)
Df(3R)EXEL7284, (5) Df(3L)EXEL9002, (6) Df(3L)ZP1, (7) Df(3L)66C-G28, (8) Df(3R)EXEL7283, (9) Df(3R)crb87-5, (10) Df(3R)XS, (11) Df(3L)fz-GF3b, (12) Df(3L)fz-CAL5, (13)
Df(3R)MAP11, (14) Df(3L)ru-22, (15) Df(3L)RM5-2, (16) Df(3L)pbl-X1, (17) Df(3L)AC1, (18) Df(3L)ED230, (19) Df(3L)GN24, (20) Df(3R)Antp17, (21) Df(3L)81k19, (22) Df(3R)p-
XT103, (23) Df(3L)bab-PG, (24) Df(3L)Brd6, (25) Df(3R)crb-F89-4, (26) Df(3R)3450, (27) Df(3R)3-4, (28) Df(3R)tll-g, (29) Df(3L)EXEL6105, (30) Df(3R)M95A, (31) Df(3L)vin5,
(32) Df(3l)ri-79c, (33) Df(3L)ED4486, (34) Df(3R)KarD2, (35) Df(3R)ry615, (36) Df(3R)mbc-R1, (37) Df(3R)96B, (38) Df(3R)Tl-P, (39) Df(3R)Dr-rvl, (40) Df(3R)01215, (41)
Df(3L)emc-E12, (42) Df(3L)ZN47, (43) Df(3L)F10, (44) Df(3R)Spf, (45) Df(3R)EXEL6270, (46) Df(3R)C4, (47) Df(3R)EXEL6276, (48) Df(3R)tll-e, (49) Df(3R)ED5780, (50)
Df(3R)EXEL6274, (51) Df(3L)XS533, (52) Df(3L )GN34, (53) Df(3R)Win11, (54) Df(3R)ry27, (55) Df(3R)XF3, (56) Df(3L)AC1, (57) Df(3R)DG4 , (58) Df(3R)Cha7.
3
Fails to complement l(3)82Fa.
Other abbreviations: TC, terminal cell; DT, dorsal trunk.
doi:10.1371/journal.pgen.1002087.t001
Table 1. Cont.
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 6 July 2011 | Volume 7 | Issue 7 | e1002087
Page 6
increased branching were rare. Indeed, the only mutations that
increased branching were the miracle-gro alleles described above,
which also increased cell size. Although some extra branches
observed in miracle-gro terminal cells might simply be terminal
branches that are normally present but bigger than normal and
hence easier to detect, there clearly were also extra branches not
present in wild type, such as those in proximal positions of miracle-
gro terminal cells. In many cases, these extra seamless tubes
coursed through the soma of the terminal cell, giving it a striking
multiple lumen phenotype (Figure 4C9, inset).
Branch pattern and shape alterations (3C). Eight
mutations affected terminal cell branch pattern or shape and
included mutations that caused selective pruning (category 3C1,
e.g. burs), thick and severely pruned branches (3C2, e.g. lotus), and
excess protrusions (3C3, e.g. spikes), as detailed below.
burs (942, 1139) mutant terminal cells ramified much less
extensively than wild type, but the branching defect appears
selective for side branches and possibly other later rounds of
terminal branching (Figure 4D). Likewise, in the complementation
group denuded (PC213, 1520) the first terminal branches appeared
largely normal, although sometimes thickened, but subsequent
branches were absent or severely compromised: small, un-
branched, and with a narrow bore tube. These mutants
demonstrate genetic differences between early and later rounds
of terminal branching.
Terminal cells mutant for lotus (312), conjoined (356), and oak gall
(696) were severely pruned, in the most extreme cases with just a
single significant branch, and the remaining branches were
unusually thick (Figure 4E). However, lumens within the thickened
branches were of normal diameter.
spikes (735) mutant terminal cells had variable numbers of
cytoplasmic protrusions emanating along the length of terminal
branches (Figure 4F, inset), protrusions that in wild type are
typically restricted to the growing tip and sites of lateral sprouting
Figure 2. Tracheal cell selection/specification mutants. (A, B) Lateral views (anterior left) of a portion of the lateral tracheal trunk (between two
transverse connectives) of genetic mosaic third instar larvae with control wild-type clones (A) and homozygous no terminal cell clones-3L clones (B).
All tracheal cells express DsRed (red) and tracheal clones also express GFP (green) so appear yellow. Terminal cell clones (arrowheads) are present inA
but absent in B. (C–H) Portions of the tracheal system of wild type control and mutant third instar larvae homozygous for the mutations indicated. (C,
D) Lateral views (anterior left) of wild type (C) and missing parts mutant (D). Tracheae are labeled with GFP (white). Positions of two normal terminal
cells (arrows) and a lateral trunk (LT) fusion joint (arrowhead) are indicated in C. In D, the corresponding terminal cells and LT fusion joint are missing
(*), with broken ends of LT indicated by white dots. (E, F) Dorsal views of distal ends of a pair of dorsal branches labeled with GFP (white) in wild type
(E) and steeple mutant (F). Note terminal cells (arrowheads in E) are missing (*) in steeple mutant (F). (G, H) Dorsal view of posterior of wild-type (G)
and loose caboose mutant (H) with tracheae labeled with GFP (white). Arrowhead, position where contralateral dorsal branches (Tr10) connect to form
the DB10 fusion joint (G). DB10 fusion joint is missing (*) in H; in the absence of the fusion joint, the positions of the disconnected parts of the
tracheal system are more variable. Open circles, posterior spiracles.
doi:10.1371/journal.pgen.1002087.g002
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 7 July 2011 | Volume 7 | Issue 7 | e1002087
Page 7
Figure 4. Terminal cell branching mutants. Fluorescence (A–F) and brightfield (A’–F’) images of homozygous terminal cell clones (DsRED
+
, GFP
+
so appear yellow in A–F) of the mutations indicated, with schematics of the phenotypes shown below. Open boxes, area enlarged in insets. (A, A’)
Control wild type clone. There are dozens of terminal branches (A), and each mature branch contains a single, continuous gas-filled lumen (A’). New
terminal branches arise from filopodial growth cones (A, inset). (B, B’) winded
1508
clone. Note absence of terminal branches. (C, C’) miracle-gro
1483
clone. Note enlarged branches and multiple convoluted seamless tubes in enlarged soma (C’, inset). (D, D’) burs
1139
clone. Note presence of first
generation terminal branches but absence of most second and all subsequent generations. (E, E’) oak gall
696
clone. Note all but one terminal branch is
missing, and remaining branch is short and stout (arrowheads). Another phenotype is the tiny gap in the gas-filled lumen at or near the position
where autocellular and subcellular tubes connect in terminal cell (E’, inset; compare to inset in A’). (F, F’) spikes
773
clone. Note excess filopodia arising
from terminal branches (F, inset) but normal or slightly reduced numbers of mature terminal branches (F’). Bar, 20
mm.
doi:10.1371/journal.pgen.1002087.g004
Figure 3. Tracheal cell size mutants. Micrographs (top panels) and schematics (lower panels) of genetic mosaic third instar larva showing
terminal cell (TC, A–E) and dorsal trunk (DT, A’–E’) clones (GFP
+
, green; at right) of control wild type (A, A’), miracle-gro
338
(B, B’), lotus
312
(C, C’),
sprout
574
(D, D’), and cincher
773
(E, E’) cells. In A–E, a contralateral control heterozygous terminal cell (DsRED
+
, red; at left) is included for comparison.
The maximal soma cross-sectional area of miracle-gro
338
terminal cell clones (0.8760.05 units in Image J (mean6SEM), n = 10 clones) was four-fold
greater than that of wild type control terminal cell clones (0.2260.03 units). Extra branches in the miraclo-gro clone are highlighted in Figure 4C/4C’.
Bar, 50
mm (A–E), 10 mm (A’–E’).
doi:10.1371/journal.pgen.1002087.g003
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 8 July 2011 | Volume 7 | Issue 7 | e1002087
Page 8
(Figure 4A, inset). Most of the excess protrusions, however, were
short and nonproductive as spikes mutant terminal cells had slightly
fewer mature terminal branches than normal.
Class 4: tube formation, number, position, and shape
mutants
These mutations prevented lumen formation, or altered the
number, placement, or shape of the lumens that formed. Most of
these mutations did not affect all tracheal tubes, but rather
structurally distinct subsets of tubes. We start with a description of
mutations that affect the seamless tubes of terminal branches (see
Figure 1B; Figure 5A–5F; Figure 6B).
Seamless tube defects (4A). These mutations caused defects
in seamless tubes including missing and discontinuous lumens
(category 4A1, e.g. impatent), convoluted lumens (4A2, e.g. wavy
lumens), multiple convoluted lumens (4A3, e.g. tendrils), lumen
dilation and other irregularities (4A4, e.g. cystic lumens ), and large
vacuoles (4A5, e.g. black hole), as detailed below.
We recovered ,25 lines in which gas-filled lumens were not
detected in terminal cell clones by brightfield microscopy. Some of
Figure 5. Tubulogenesis mutants. Fluorescence photomicrographs of control wild type (A, G, I) and homozygous mutant (B–F, H, J, K) clones in
seamless, autocellular, and multicellular tracheal tubes in third instar larvae. Schematics of the phenotypes are diagrammed below. Clones are
marked with GFP (white in A–F, green in G–K) and all tracheal cells with DsRED (red in G–K); brightfield images in I’–K’ show air-filled lumens of
multicellular tubes. (A) Wild type control clone in seamless tube. (B) whacked
220
clone. Note most of the lumen is missing and the terminus of the
residual lumen (arrowhead) is dilated and irregularly shaped. (C) moon cheese
1524
clone. (D) wavy lumens
894
clone. (E) cystic lumens
1243
clone. (F) black
hole
538
clone. The regions where the lumen appears to be dilated (e.g., boxed area, upper inset) are actually regions in which a vacuole, which can be
distinguished from the lumen by its accumulation of lumGFP (not shown), intimately surrounds a lumen of normal diameter (lower inset, brightfield
view of boxed area). The vacuole is outlined in red in schematic. (G) Wild type control clone in autocellular tube. The single marked cell (GFP
+
, green)
surrounds the lumen, sealed by an autocellular junction. (H) conjoined
356
clone. The mutant cell (GFP
+
, green) does not form an autocellular junction
but instead forms the lumen by making intercellular junctions with a heterozygous cell (DsRED
+
, red). (I) Wild type control clone in dorsal trunk, a
multicellular tube. (J) bulgy
636
clone. Lumen bulges outward into mutant cell, forming a local dilatation. (K) constricted
960
clone. Lumen constricts
inward at site of mutant cell by ,7% relative to the neighboring, fully wild type dorsal trunk segments. Bar, 5
mm (A–F), 10 mm (G,H), 10 mm (I–K).
doi:10.1371/journal.pgen.1002087.g005
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 9 July 2011 | Volume 7 | Issue 7 | e1002087
Page 9
these mutants are defective in lumen formation as assessed by a
secreted GFP reporter (lum-GFP), whereas others form lumens
that are difficult to detect by brightfield microscopy because they
remain filled with matrix (see below). The three alleles of impatent
(1472, 1490, 1757) lacked or had seriously compromised seamless
tubes, whereas other aspects of terminal branches appeared
normal and lumens of other branches were unaffected. In impatent
mutant terminal cells, the lumGFP reporter accumulated in large
puncta, up to 1–2 um in diameter, located mostly in the cell body
(Figure 6B). These were probably enlarged vesicles representing
trapped or aberrant intermediates in lumen formation.
Some of the other mutations in this class displayed a more
complex spectrum of defects. whacked mutations (PC24, 220)
eliminated the distal portions of seamless tubes, with the lumen
typically terminating prematurely in an irregularly-shaped local
dilatation (Figure 5B). moon cheese (1524) and carbuncle (804) caused
a variable discontinuous lumen phenotype in which regions along
the terminal branch were missing lumen but flanked by regions
containing blind-ended and irregularly-shaped lumen (Figure 5C).
These mutations also caused a fully penetrant large vacuole
phenotype (see below).
In wavy lumens (894) and wobbly lumens (BG13), terminal cell
lumens formed but followed a convoluted path through the
cytoplasm (Figure 5D). Similar convoluted lumens were seen in
miracle-gro mutant terminal cells and cells exposed to hypoxia [45].
However, the convoluted lumens of wavy lumens and wobbly lumens
were not associated with excessive terminal cell growth and
branching as in miracle-gro mutants and under hypoxia: wobbly
lumens terminal cells were of normal size and wavy lumens terminal
cells were mildly pruned.
miracle-gro mutations caused multiple convoluted lumens in the
soma of mutant terminal cells, along with the extra growth and
branching described above. Eight additional mutants were
identified that had multiple, disorganized lumens but fewer
branches than normal. Four compose the complementation group
tendrils [46], and vine (512) defines another locus whose phenotype
was remarkably similar to tendrils except that it began to manifest a
day or so earlier in development. creeper, braided and ivy had similar
but less penetrant phenotypes.
Figure 6. Lumen clearance and gas-filling mutants. Fluorescence (A–G) and bright field micrographs (A’–G’) of control wild type (A, F) and
homozygous mutant clones (B–E, G) in seamless and autocellular tracheal tubes as indicated. Clones are labeled with cytoplasmic DsRed (red) and
also express lumGFP (green), a secreted form of GFP; the fluorescence micrographs (A–G) are DsRed/lumGFP merged images, except for E, which
shows only the lumGFP channel (white). Lumen defects are diagrammed below, with air-filled lumens in white and matrix-filled lumens and tracheal
cell cytoplasm in grey. (A, A’) Wild type control terminal cell. lumGFP has been cleared from the mature, gas-filled lumen (A’). The only lum-GFP visible
is small puncta in the cytoplasm at the tip (A, arrowhead). (B, B’) impatent
1757
clone. This is a mutant, like those described in Figure 5, in which the
seamless lumen is missing (B’): lumGFP is detected only in puncta (B, arrowheads), presumably aberrant intermediates in lumen formation,
distributed in the soma and along the lumenless terminal branch. (C, C’) ichorous
206
clone. Although no mature, gas-filled lumen is detected by
brightfield optics (C’) as in impatent mutant cells, a lumen has formed–just not cleared–as shown by luminal lumGFP staining (C). (D, D’) littoral
762
clone. A specialized clearance defect: the central terminal branch forms a normal gas-filled lumen but the tips of growing side branches (brackets)
contain a lumen that has not cleared (D’) and remains loaded with lumGFP (green, D). (E, E’) lotus
312
clone. Another specialized clearance defect,
restricted to the junction between (arrowheads) the base of the branch (connection with stalk cell) and the seamless tube. The fluorescence signal in
E above and below the arrowheads is autofluorescence of the cuticle, not lumGFP. (F, F’) Control wild type autocellular tube. (H, H’) asthmatic
1530
clone. Lumen is difficult to detect (G’) because it remains filled with luminal matrix and lumGFP (green, G). Bar, 5 mm (in C, A–E), 10 m m (F,G).
doi:10.1371/journal.pgen.1002087.g006
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 10 July 2011 | Volume 7 | Issue 7 | e1002087
Page 10
Several mutants had irregular but gas-filled tubes. As described
above, mutations in whacked had irregularly shaped, and often
prematurely terminated tubes. cystic lumens (1243) caused areas of
lumen dilation and constriction (Figure 5E) and occasional gas-
filling defects. The tube morphology defects in these mutants most
closely resemble those previously reported for larger tubes (dorsal
trunk) in which the secretion or modification of chitin is affected
[47], and more recently by mutation of receptor tyrosine
phosphatase activity [48].
Other mutations caused large cytoplasmic vesicles that excluded
cytoplasmic GFP in mutant tracheal cells. In black hole (538) mutant
cells, the vacuoles were centered over gas-filled lumens, suggesting
that transport into the lumenal space is defective (Figure 5F). black
hole and dark matter (1417) caused vacuole accumulation specifically
in tracheal terminal cells, whereas moon cheese (1524) and carbuncle
(804) caused vacuole accumulation in all tracheal cells. For moon
cheese and carbuncle, a secreted form of GFP (lum-GFP) accumu-
lated to high levels within mutant cells, particularly within the
vacuoles, suggesting a defect in trafficking of secreted proteins.
moon cheese and carbuncle also caused a variable discontinuous tube
defect described above.
Autocellular tube defects (4B). lotus (312), conjoined (356), oak
gall (696) interfered variably with the formation of tubes sealed by
autocellular junctions (Figure 1B): mutant dorsal branch cells
appeared either to completely avoid contributing to autocellular
tubes, forming exclusively intercellular rather than autocellular
junctions (Figure 5G, 5H), or contributed only minimally with
most of the cell body protruding basally away from an otherwise
smooth epithelial tube. Dorsal trunk clones also protruded basally,
although still formed intercellular junctions (not shown). These
mutations demonstrate that autocellular junctions and tubes are
genetically distinct from intercellular junctions and multicellular
tubes (Figure 1B).
Multicellular tube defects (4C). These mutations caused
defects in multicellular tubes, including lumen dilation (category
4C1, e.g. bulgy) and lumen constriction (4C2, e.g. short of breath), as
detailed below.
small potatoes (1113, 1166, 1694), bulgy (636), and balloon (1448)
caused subtle outward bulges in the lumen of dorsal trunk tubes
(Figure 5I, 5J). Bulges occurred at the sites of clones, even single
cell clones, hence these mutations identify cell autonomous
regulators of multicellular tube diameter.
Eight mutations caused the opposite phenotype: dorsal trunk
cells mutant for short of breath (5 alleles), dyspneic (1348, 1359), and
constricted (960) caused lumenal constrictions at the sites of mutant
cells (Figure 5K), reminiscent of the recently described mutant
stenosis [49].
Class 5: lumen clearance/gas-filling mutants
We expected that some mutants from the screen that appeared
to lack lumens would instead be lumen clearance and gas-filling
mutants in which the lumen was present but difficult to detect by
brightfield optics because it remained filled with matrix, which has
a similar refractive index as the surrounding cytoplasm. To
identify such mutants, lines from the screen that appeared under
brightfield optics to lack lumens were subjected to a secondary
screen using a transgene expressing a fusion protein containing the
signal peptide of p23 [50] linked to GFP. The fusion protein
(lumenal-GFP or lumGFP) was designed to transit the secretory
pathway, as it does in mammalian cells [50], entering the tracheal
lumen and remaining there to mark the lumen of mutants that
affect lumenal clearance, such as ichorous and asthmatic (Figure 6C).
By contrast, no lumenal accumulation of lumGFP was observed in
mutants such as impatent described above that lack or have
seriously compromised lumens (Figure 6B), or in wild type control
clones because lumGFP is cleared from the lumen during the
normal lumenal maturation process (Figure 6A). The only
lumGFP that remained in impatent mutant clones was the large
puncta already described (Section 4A1), and the only lumGFP
detected in control clones was the rare puncta near branch tips or
the junctions between branches (Figure 6A). In mutations such as
scrub (659), lumGFP was not detected in either matrix-filled lumens
or cytoplasmic puncta (data not shown); these mutations might
alter lumenal targeting such that lumGFP is secreted from other
positions in the cell so does not accumulate intracellularly.
Seamless tube clearance defects (5A). These mutations
caused liquid clearance defects in all seamless tubes (category 5A1,
e.g. asthmatic), in the tips of seamless tubes (5A2, e.g. littoral), or at
the junction between seamless and autocellular tubes (5A3, e.g.
lotus), as detailed below.
Mutants such as asthmatic and ichorous (Figure 6C) described
above, and stertorous and liquid-filled, were defective in matrix
clearance: mutant terminal cells had normal morphology and
formed seamless tubes, but the tubes failed to clear and gas-fill.
These cells cannot supply oxygen to their targets, and neighboring
wild type cells were frequently found invading the region normally
supplied by the mutant cell; under normal conditions, terminal cell
domains do not overlap [45], similar to neuronal tiling [51].
Seven mutants (littoral, burs, ivy, panting, 826, 928, 1809) had gas-
filling defects that typically affected only new or distal portions of
subcellular tubes (Figure 6D). The tip-clearance defects were
variable, with some mutant terminal cells more severely affected
than others, even within the same mosaic animal. These mutants
suggest that the tips of terminal branches have specialized
requirements for clearance and gas filling, or that these regions
are particularly sensitive to defects in the general machinery.
lotus, oak gall, conjoined, and disjointed mutants showed an
exquisitely specific clearance and gas-filling defect: in mutant
terminal cells, no gas-filled lumen could be detected connecting
the secondary branch tube, which has an autocellular tube, to the
terminal branch seamless tubes, which lack junctions and extend
throughout the rest of the terminal cell and gas-filled normally
(Figure 4E9, Figure 6E and 6E9).
Other branches (5B). Nine mutants representing four
complementation groups showed dramatic defects in liquid
clearance/gas-filling of tracheal tubes containing autocellular
junctions. Two of the complementation groups, short of breath and
dyspneic, were described above, because they also cause defects in
dorsal trunk and terminal cells (sections 4C2 and 5A1). asthmatic
mutations, which caused a strong terminal cell gas-filling defect as
noted above, also caused a partially penetrant autocellular tube
gas-filling defect (Figure 6F, 6G). flashflood (AP67) selectively
blocked clearance of lateral tracheal trunks, as detected in
homozygous third instar larvae.
Mutation mapping and gene identification reveal new
cell biological pathways in tracheal development
To begin to define the molecular functions of tracheal genes
identified in the screen, we mapped representative mutations and
molecularly identified 14 of the genes (Table 2). Six of the
identified genes (no tc clones-L, no tc clones-R, short of breath, dyspneic,
lopped and failed fusions) were previously implicated in tracheal
development. However, new functions were revealed for each by
our clonal analysis. Two are allelic to canonical tracheal genes in
the branchless/breathless FGF pathway, the breathless FGFR itself [26]
and pointed [11,59], which we showed are differentially required for
competition during tip cell selection [13]. Two others, short of breath
and dyspneic, which our results implicate in lumen clearance and
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 11 July 2011 | Volume 7 | Issue 7 | e1002087
Page 11
gas-filling and as cell autonomous promoters of tube expansion,
are allelic to krotzkopf verkehrt (kkv) and knickkopf (knk), chitin synthesis
pathway genes previously shown to coordinate the behavior of
cells in the tracheal epithelium during tube expansion
[53,54,60,61]. Our results demonstrate that chitin synthesis genes
also have an unexpected, cell autonomous function in lumen
clearance and gas filling of autocellular and seamless tubes.
lopped
784
is allelic to fatiga that encodes Drosophila Hif1 prolyl
hydroxylase, and appears to be required in terminal cells for
normal branching. Previous studies with hypomorphic fatiga alleles
Table 2. Molecular identification of tracheal genes.
Comp.
Group
1
Alleles Map Position
Comp.
Test
2
Mutation [strength
3
]
Flybase
Name (gene
loc’n) Protein Function
Extant Tracheal
Function
4
Reference P/M
5
burs 942 1139 73D1 TSG101
D9
TSG101
D10
TSG101
D18
CCC.TCC (P86L)
CCT.CTT (P113L)
TSG101
(73D1)
Homolog of TSG101/
VPS23, part of ESCRTI
complex in endosome
sorting
None [52] M
dyspneic 1348 1359 3R knk
1
ND knickkopf
(85F13)
Chitin synthesis protein,
dopamine b-
monooxygenase motif
Size and shape
of dorsal trunk
lumen
[28,53,54] M
failed
fusions
AZ63 PA14 3R pyd
C5
ND polychaetoid
(85B2-7)
Homolog of ZO-1
junctional MAGUK
Cell intercalation [37] M
jolly green
giant
1149 95D; 95F & 98E;
99A
TSC1
9834
ND TSC1 (95E1) Homolog of tumor
suppressor TSC1; putative
vesicular transport role
None [55] M
or P
lopped 784 82F l(3)82Fe
dHph
02255
ND Hph (82F7-
82F8)
Homolog of Hif1 prolyl
hydroxylase, regulator of
Hif1a transcription factor
Inhibition of
terminal cell
branching
[56] P
miracle-gro 338 878
1483 1489
99F; 100B lats
XI
wts
MGH1
ND warts
(100A5)
MD kinase homolog in
hippo growth control
pathway
None [57,58] M
or P
moon cheese 1524 64C;D CAG.TAG (Q85stop) membrin
(61C13-
64C14)
Homolog of membrin, an
ER-Golgi t-snare
None This study M
no terminal
cell clones-L
602 724
788 1118
1187 1476
1684 BN40
h-th btl
LG18
ND TGG.TGA(W275stop)
CAG.TAG (Q296stop)
CGC.CAC (R863H)
TCG.TTG (S912L) [m]
CCA.TCA (P487S)
CGA.TGA (R402stop)
GAG.AAG (E796K) [w]
(Ref 10)
breathless
(70D2)
FGFR, receptor for
Branchless-FGF
Primary,
secondary
and terminal
branching
[13,26] P
no terminal
cell clones-R
198 1313 94D; 95A pnt
D88
ND pointed
(94E10-
94E13)
Ets-box transcription
factor
Secondary
and terminal
branching
13,59 P
short of
breath
360 404
483 761
791 1705
82F kkv
1
ND krotzkopf
verkehrt
(83A1)
Chitin synthase Size and shape
of dorsal trunk
lumen
[28,53,60,61] M
tendrils 666 1308
1469 1539
ru-h rhea
79A
CAA.TAA (Q1250stop)
AG.AA (splice site) [a]
CAG.TAG (Q934stop)
CAG.TAG (Q2051stop)
[w] (Ref 16)
rhea (66D6-
66D7)
Homolog of Talin, an
integrin/actin cross-linker
None [46,62] M
vine 512 89E GGT.GAT (G297D) cctg (89D6) Homolog of cct-c,
component of cct/TriC
chaperonin
None This study M
whacked PC24 220 86 E14; 86E17 (Schottenfeld and
Ghabrial, unpublished
data)
whacked
(86E11)
Putative RabGAP None This study M
winded 613 227
1375 1508
65F3; 66B10 cdsA
1
cdsA
7
ND CTC.TTC (L227F),
TGG.AGG (W238R) ND
TGG.TAG (W83stop)
cdsA (66B7) Homolog of CDP
diglyceride synthetase in
PI biosynthesis
None [63] M
or P
1
Complementation group name.
2
Mutations in known genes that failed to complement tested mutations in complementation group.
3
The relative strengths of the sequenced alleles of each gene were similar unless noted in brackets next to an allele that it was weak [w] or moderate [m] c ompared to
the other, presumed null allele(s), or stronger than the presumed null and likely antimorphic [a].
4
Previously known tracheal function.
5
P, presumptive patterning gene; M, presumptive morphogenesis gene.
doi:10.1371/journal.pgen.1002087.t002
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 12 July 2011 | Volume 7 | Issue 7 | e1002087
Page 12
gave opposite results [56], although new studies indicate that early
exposure to hypoxia (mimicked by loss of fatiga) result in stunted
tracheal development while later exposure stimulates branching
[64]. failed fusions is allelic to polychaetoid, which has been implicated
in branch fusion and in tracheal cell intercalation, but our mosaic
analysis, along with other new data from our lab, suggest another
function for polychaetoid in tip cell selection (E. Chao, A.S.G. and
M.A.K., unpublished data).
The other eight molecularly identified genes had not been
previously implicated in tracheal development; indeed, three (vine,
moon cheese, and whacked) had not been genetically defined (Table 2).
All eight identify new cell biological pathways in tracheal
development. jolly green giant, which encodes the Drosophila ortholog
of TSC1 [55,65], and miracle-gro (see below) [57,58] implicate
general growth control pathways in tracheal growth and
branching. tendrils, which is allelic to rhea and encodes talin [46],
and vine, which encodes the Drosophila ortholog of CCTgamma,
show that talin-dependent integrin adhesion and a component of
the TriChaperonin complex are required for maintenance of
terminal branches and lumenal organization.
The four other genes implicate membrane and vesicle
trafficking genes in tracheal development. Such genes have been
speculated to function in tube morphogenesis but few have been
genetically identified. winded, essential for terminal branching,
encodes the Drosophila homolog of CdsA [63], an enzyme that
converts phosphatidic acid to cds-diacyl glycerol in the production
of the membrane lipid, phosphatidyl inositol. moon cheese, another
terminal branching gene also implicated in lumen continuity, and
burs, a terminal branching gene selectively required for side
branches, encode the Drosophila homolog of the ER-Golgi t-
SNARE membrin, and TSG101/erupted, a component of the
ESCRTI complex that sorts endocytic vesicles to the multivesic-
ular body, respectively [52,66,67]. whacked, which promotes the
growth and proper shape of terminal cell lumens, encodes a
putative RabGAP (A.S.G. and M.A.K., unpublished data). The
identification of membrane lipid and vesicle trafficking genes in
terminal branching supports the idea that outgrowth of cellular
processes and lumen formation require targeting of apical and
basolateral membrane components at a distance from the cell
soma. It will be important to determine the number of trafficking
pathways involved, how the pathways are activated at the
appropriate times and places, and how the identified t-SNARE,
Rab-GAP, and ESCRTI component function in the pathways.
Thus, all 14 molecularly characterized genes from the screen
reveal new cell biological pathways in tracheal development or
new functions for established pathways.
Most of the identified genes are morphogenesis genes
The identities of the molecularly characterized genes allowed us
to assess the success of the screen in identifying morphogenesis
effectors. Although it was not possible to unambiguously classify all
14 genes in this way from their sequence alone, eight very likely
function as morphogenesis effectors: the vesicle trafficking genes
moon cheese/membrin, burs/TSG101, and whacked/RabGAP; the cell
junction and cytoskeletal genes failed fusions/polychaetoid/ZO-1, rhea/
tendrils/talin, vine/cctc; and the chitin synthesis genes short of breath/
kkv/chitin synthase and dyspneic/knk. Three others are established
patterning genes: the receptor btl/no-terminal cell clones-L, the
transcription factor pnt/no terminal cell clones-R, and the transcrip-
tion factor regulator lopped/fatiga/Hif prolyl hydroxylase. The
remaining three are more difficult to categorize because they
encode enzymes that likely couple patterning signals to cytoplas-
mic outgrowth (winded/(CdsA) and cell growth (Tsc1/jolly green giant
and miracle-gro), as discussed below. Thus, over three-quarters of
the identified genes (11 of 14, 79%) appear to be downstream
effectors/morphogenesis genes (8 of 14, 57%) or genes that couple
patterning signals to morphogenesis (3 of 14, 21%), supporting our
hypothesis that systematic clonal analysis is an effective way of
identifying such genes.
A cell growth regulator that also regulates lumen
morphogenesis
In addition to identifying new tracheal genes and pathways, the
screen suggested new functional connections between pathways.
One example came from characterization of the miracle-gro cell
overgrowth mutations (Section 2A). In terminal cells, not only was
the soma enlarged but there were many ectopic seamless tubes
coursing through it (Figure 4C, 4C9 and Figure 7B). This
phenotype is nearly unique: it is seen otherwise only upon
hyperactivation of the Breathless FGFR pathway (Figure 7C).
However, miracle-gro mutations did not map near breathless or any
other extant loci in the pathway. Mapping and complementation
Figure 7. Genetic analysis of terminal cell growth control pathway. (A–D) Close-ups of the soma of third instar larva terminal cell clones of
the indicated genotypes. Terminal cell cytoplasm is marked with GFP (green) and nuclei in A–C are marked with nuclear DsRed2 (red). Note that the
cell body and nucleus of the miracle-gro(warts)
388/388
clone (B) and the clone expressing l-Breathless (C), a constitutively active form of Breathless
FGFR, are enlarged with ectopic lumens coursing through the soma. By contrast, the soma of the miracle-gro(warts)
388/388
clone in a larva
homozygous for blistered
l(2)3267
, a downstream transcription factor in the Breathless pathway (D), is smaller and there are no ectopic lumens (black
asterisk). However, the single, truncated lumen of the clone is dilated compared to the truncated lumen of the contralateral control terminal cell
(white asterisk). (E) Genetic pathway of terminal cell growth control. Bar, 20
mm.
doi:10.1371/journal.pgen.1002087.g007
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 13 July 2011 | Volume 7 | Issue 7 | e1002087
Page 13
tests (Table 2) demonstrated that miracle-gro is allelic to warts/lats-1
[57,58], which encodes a kinase that suppresses cell growth in a
well-established general growth control pathway. Thus, loss of
a key growth regulator in terminal cells leads not only to
excessive cell growth but excessive lumen formation, revealing
an unexpected coupling between cell growth control and
tubulogenesis.
The striking similarity of the warts/miracle-gro loss of function
phenotype and the btl pathway gain-of-function phenotype
suggested that FGF signaling might stimulate terminal cell growth
and tubulogenesis by inactivating Warts function. To test this, we
sought to define the genetic epistasis relationship between warts/
miracle-gro and breathless-FGFR pathway mutations. Because
mutations that disrupt FGF signaling abrogate terminal cell
specification [11,29], it is not possible to generate terminal cells
doubly mutant for warts and breathless, so we examined terminal
cells doubly mutant for warts/miracle-gro and blistered/pruned/SRF,
the downstream transcription factor in the breathless FGFR
pathway required for terminal cell growth and branching. Doubly
mutant cells were unbranched and small, similar or slightly bigger
than blistered mutant terminal cells, and with a single lumen in the
soma (Figure 7D). However, the lumen diameter was larger than
normal and similar in size to those in warts/miracle-gro mutant
terminal cells. Thus, the cell growth and excessive lumen
formation seen in warts/miracle-gro mutant terminal cells are
dependent on blistered, whereas lumen diameter can be modulated
independently of blistered. This supports a model in which Warts/
Miracle-gro functions downstream of Breathless FGFR but
upstream of Blistered/SRF in the regulation of terminal cell size
and lumen number, and upstream of another, as yet unidentified,
transcription factor that controls lumen diameter (Figure 7E).
Discussion
Our systematic screen for tracheal mutations on the third
chromosome identified new tracheal phenotypes and scores of new
tracheal genes as well as new functions for established tracheal
genes. Molecular identification of 14 of the genes indicates that
most of the isolated genes are downstream effectors/morphogen-
esis genes, an important category of genes substantially underrep-
resented in previous screens. Several of the identified genes encode
proteins involved in vesicle trafficking, implying that such genes
are a major class of morphogenesis genes, at least for terminal
branching.
Figure 8. Genetic dissection of terminal branch morphogenesis. The major, genetically separable processes in the terminal branching
program are illustrated, in the order in which they occur, along with representative mutations that disrupt them. There is an initial patterning step
(Selection/Specification) that selects and specifies the terminal cell, followed by five morphogenesis (Branching, Growth, Tubulogenesis) and
maturation (Clearance/Gas-Filling, Maintenance) steps. The steps can be functionally subdivided further by the more specific phenotypes of the
mutants shown. Where the molecular identities of the genes are known, the protein products are given (in parentheses) to indicate some of the
molecular functions involved in each step. The SRF transcription factor Blistered (Pruned), a key regulator of terminal branching and the last gene in
the Selection/Specification step, presumably controls expression of at least some of the downstream morphogenesis and maturation genes including
ones involved in growth and tubulogenesis (Figure 7E).
doi:10.1371/journal.pgen.1002087.g008
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 14 July 2011 | Volume 7 | Issue 7 | e1002087
Page 14
Our screen succeeded in identifying morphogenesis genes
because (i) it was systematic and extensive, surveying most third
chromosome genes, nearly 40% of the genome; (ii) it included a
clonal analysis of mutations using a new cell marking method that
allowed facile identification of tracheal functions of pleiotropic
mutations such as vesicle trafficking and cytoskeletal genes; and (iii)
it employed a secondary screen in another tissue (eye) to exclude
mutations in general housekeeping genes. Housekeeping genes are
a huge class of genes that would have dominated the results of our
clonal screen, as they have in previous clonal screens [35].
Exclusion of housekeeping genes also allowed the identification of
tracheal-selective growth regulators, which contributed to our
discovery of cell growth control as an important new step in
branching (see below).
Many of the mutations we identified, particularly in the clonal
analysis, affect terminal branching, a morphogenesis process for
which little was known beyond the key signaling pathway and
transcription factors that control terminal cell selection. The
systematic nature of our screen and the distinct phenotypes of
terminal branching mutations we identified provide a comprehen-
sive genetic outline of this morphogenetic process (Figure 8). We
propose that terminal branching involves an initial cell selection and
specification step plus five major morphogenesis processes:
branching, growth, tubulogenesis, gas filling, and maintenance.
Each of these processes is associated with one or two defining genes
that are required quite generally for the process plus additional
genes, mutations in which further subdivide each process into
distinct morphogenetic steps or reveal additional levels of
regulation. Below we discuss each of these processes and the
associated genes, highlighting cell growth regulation because its
critical role in branching morphogenesis had not been recognized.
We return at the end to discuss implications of the results for the
corresponding processes in primary and secondary branching.
Cell selection and specification
The earliest step of cell selection and specification in terminal
branching is a well-characterized process that previous genetic
studies have shown is controlled by the Branchless FGF pathway
(Bnl/Btl/RAS/Pointed) that induces expression of the Blistered/
Pruned SRF transcription factor that selects cells at the ends of
budding branches for a terminal branching fate [9,10]. Re-
expression of Bnl FGF later in hypoxic tissues is proposed to
initiate terminal branch budding, at least in part by activation of
the blistered SRF transcription complex and its downstream effector
genes [14,45].
Our screen identified third chromosome genes previously
implicated in the selection process, but revealed an interesting
new aspect of the process because of the novel ‘‘no terminal cell
clones’’ phenotype. These turned out to be mutations in breathless
FGFR and pointed [13], and lead to the discovery there is
specialization among cells in a budding branch and only the
leaders need to receive the Branchless FGF signal. Cells mutant for
Breathless FGFR cannot receive the signal, and are relegated to
trailing positions, never to be specified a terminal cell.
We also discovered genes (steeple, missing parts) required for
specification of a subset of terminal cells in specific regions or
branches. These may encode region-specific enhancers of the Bnl-
Btl pathway because sporadic failure of terminal cell formation is
seen in animals in which this signaling pathway is partially
compromised [11,13,34].
Branch budding and extension
Although the Branchless pathway and Blistered SRF transcrip-
tion complex are key regulators of branch budding and outgrowth
[14,29], little is known of the signal transduction pathway that
connects them or of the downstream effectors. winded/cdsA
mutations caused a severe, cell autonomous terminal branch
pruning phenotype similar to that of blistered/SRF null alleles.
winded/cdsA encodes an enzyme (CDP-diacylglycerol synthase)
required for phosphoinositide (PI) synthesis, suggesting that a PI-
dependent signaling process, presumably like those involved in
other receptor tyrosine kinase (RTK) signaling pathways [68],
functions downstream of the Btl RTK in the control of Blistered/
SRF and branch budding. Some of the other genes with pruned
phenotypes (e.g., topiary, paltry, truncated) might encode additional
signal transduction components or targets of the SRF transcription
factor required for polarized cell growth (see below).
We propose that other branching genes regulate bud site
selection and the pattern of branching. spikes encodes a negative
regulator of bud site selection because small ectopic buds form in
mutant terminal cells. One appealing idea is that spikes restricts the
normal budding response of terminal cells to the sites of maximal
induction by Branchless FGF. TSG101/erupted/burs and denuded
regulate branch pattern by promoting lateral and late rounds of
terminal branching, perhaps by catalyzing the local disassembly or
reorganization of the cytoskeleton within a maturing terminal
branch.
One set of genes ( lotus, oak gall, conjoined) affected branch number
but also dramatically altered the size and shape of the remaining
branches (see below). These were difficult to categorize purely as
branching genes or growth regulators, so we place them in a
special class at the boundary between those categories because
they share features of both. They may function as integrators of
branch outgrowth and size control signals.
Growth regulation
A new aspect of the tracheal developmental program highlight-
ed by the mutants is cell size and growth regulation. Outgrowth of
terminal branches requires not only chemoattractant signaling to
induce and guide migration, but synthesis of cellular and
membrane components to support cytoplasmic outgrowth.
Terminal cells mutant for glutamyl-prolyl-tRNA synthetase or hun-
dreds of other presumptive house keeping genes failed to form and
extend terminal branch buds. Many such growth-promoting genes
were presumably among those identified in a previous clonal
screen for tracheal mutations [35], but because there are many
such mutations and their phenotypes are non-specific (small, sick,
or missing cells) and difficult to distinguish from genes simply
required for cell viability, it was hard to evaluate their
developmental significance.
Three types of data argue that growth control is an integral part
of the tracheal developmental program. First, clonal analysis of the
master regulator trachealess in terminal cells gave a similar
phenotype (Figure S1), implying that terminal cell growth is a
process actively regulated by Trachealess. etiolated is a particularly
interesting mutant of this class because it resembled trachealess not
just in its clonal phenotype in terminal cells, but in its tracheal
specificity. Second, we obtained mutations in two canonical
growth suppressor genes, warts/miracle-gro and Tsc1/jolly green giant,
which gave the opposite phenotype: terminal branches and
terminal cells were overgrown with particularly large somas that
in warts/miracle-gro mutant cells contained multiple seamless tubes
passing through them. This shows that a general growth regulator
controls not only cell size but tubulogenesis, an essential step in the
tracheal developmental program. Third, the phenotype of warts/
miracle-gro mutant terminal cells is very similar to that of activated
Btl, and genetic epistasis experiments suggest that warts/miracle-gro
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 15 July 2011 | Volume 7 | Issue 7 | e1002087
Page 15
functions downstream of, and is negatively regulated by, btl FGFR
but upstream of blistered SRF (Figure 7E).
sprout is the most intriguing undergrowth mutant because it was
the only one that formed small but normally patterned terminal
branches. sprout cleanly decouples branch size from branch
budding and outgrowth, so we propose it is a key gene in branch
size control.
Three other genes, lotus, oak gall, and conjoined, also function in
branch size control, but in a different way. In mutant cells,
branches were much thicker and more variable than normal, but
the diameter of the seamless tubes that form within them were
normal. We propose that these genes function in the size control
pathway by regulating the distribution of plasma membrane or
other cell constituents among branches. When this process fails,
branches become thicker and fewer in number.
Most of the undergrowth mutations and all of the overgrowth
mutations affected not only terminal cells but other tracheal cell
types and cells outside the tracheal system, implying that the
affected genes encode general growth regulators. However, many
undergrowth mutations had their most extreme effects on terminal
cells, presumably because they are larger and grow more than
other cells. But two mutations, cincher and corset, affected the growth
of dorsal trunk cells and spared terminal cells. Thus, the growth
control programs of these tracheal cell types are genetically
separable. Because terminal cell growth appears to be controlled
primarily or exclusively by Bnl-Btl signaling and operates
selectively under hypoxic conditions, conditions that arrest the
growth of most other cell types, cincher and corset might identify
specific regulators or components of aerobic growth pathways or
other general growth processes dispensable in terminal cells.
Tubulogenesis genes
The striking phenotype of impatent mutant terminal branches,
branches that superficially appear normal but lack air-filled tubes
and hence are nonfunctional, leads us to propose that impatent
encodes a key regulator or component of terminal branch lumen
formation. We further propose that lumen shape is governed by
cystic lumens, perhaps in conjunction with whacked, mutations in
which result in irregularly-shaped lumens, and that lumen length
and position are controlled by wobbly lumens and wavy lumens,
mutations that cause long and convoluted lumens. Long and
convoluted lumens are also seen in terminal cells under hypoxic
conditions and other conditions that cause excessive branchless
FGF pathway activity and/or terminal cell growth (e.g. warts/
miracle-gro), so an appealing model is that these conditions and this
signaling pathway inhibit the activity of wobbly lumens and wavy
lumens, which themselves function to restrict the length or the
position of terminal branch lumens.
Lumen continuity requires carbuncle and membrin/moon cheese,
suggesting that lumens of seamless tubes are made piecemeal and
these genes promote their connection. We also identified four
genes (disjoined, lotus, conjoined, and oak gall) required to make
functional connections between seamless tubes and the adjacent,
architecturally distinct autocellular tubes that form by wrapping.
The short lumenal gap in these mutant terminal cells may result
from a failure to connect the tubes or a structural defect that
prevents clearance of the connection.
Lumen clearance and gas filling genes
For tracheal branches to become functional, the lumenal matrix
must be cleared and replaced by gas, the molecular composition of
which is unknown. ichorous and asthmatic are required for clearance
and gas filling of most or all terminal branches, and littoral and
panting and others are required to clear the tips of terminal
branches. Recent studies highlight the importance of secretion into
the lumen and subsequent endocytosis of lumenal matrix and
liquid during tube expansion and air-filling of large multicellular
tracheal tubes [16,69]; the genes identified in our screen may
mediate related processes in seamless tubes.
Maintenance genes
These genes maintain the elaborate shape and structure of
terminal branches under mechanical stress such as muscle
contraction. In the mutant rhea/tendrils/talin [46], terminal branches
begin to form normally but branches break down and their lumens
retract as the larva begins to move and the developing branches are
subjected to the stress of stretch. The phenotypes of cctgamma/vine,
creeper, braided, and ivy are similar to tendrils, suggesting that they
function in the same integrin/talin cell adhesion and cytoskeletal
support system. For example, the cctgamma/vine chaperonin may
facilitate the folding or assembly of talin or some other component
in the support system, an appealing hypothesis given that CCT
chaperonins have been shown in other systems to mediate the
folding of cytoskeletal proteins [70].
These maintenance genes emphasize the importance of analyzing
the onset and evolution of a mutant phenotype when elucidating
gene function, because similar phenotypes can arise from early
developmental aberrations or later defects in maintenance. Other
elaborate cell types and organs likely also require maintenance
genes. For example, mutations in mouse Dlg5 perturb delivery of
adherens complex proteins to the plasma membrane of brain and
kidney epithelial tubes, resulting in cyst formation [71]. Many such
structural maintenance genes are expected to function late in life so
would be missed in typical developmental screens. A major effort
should be aimed at their identification and isolation because of their
importance in medicine and disease.
Coordination and coupling of morphogenesis processes
How are the genetically separable terminal branch morpho-
genesis processes described above coordinated and controlled in
time and space? An important part of this control and
coordination almost certainly involves the Branchless FGF
pathway. Expression of both the ligand branchless and the receptor
breathless are induced by hypoxia during larval life, and terminal
cell outgrowth and branching are stimulated and directed to
hypoxic cells by local production of Branchless FGF [56]. One
way Bnl-Btl signaling stimulates branching is likely through
transcriptional induction of morphogenesis genes via modification
and activation of the Blistered /SRF transcription complex. In
other systems, the SRF transcription complex has been shown to
be regulated by the actin cytoskeleton and to regulate cytoskeletal
genes [72], and such genes are almost certainly required for
growth of the actin-rich terminal branch buds. It will be important
to determine which of the identified morphogenesis genes are
regulated by Branchless signaling and SRF, and to identify the full
set of downstream targets by transcriptional profiling.
Because Branchless functions as a chemoattractant, it must also
provide a spatial cue that guides terminal branch outgrowth. One
appealing idea is that the ligand-bound Breathless FGFR at the
surface of the terminal cell generates a spatial cue that directs
polarized growth of cytoplasmic extensions toward hypoxic, FGF-
secreting cells. Such a spatial cue could be used to direct vesicular
traffic to the growing ends of terminal cell extensions, both for
polarized growth of the new branch and construction of a lumen
within it. The spatial cue might be a modified membrane PI
because PI signaling functions downstream in many RTK
signaling pathways [68] and can regulate vesicle trafficking [73]
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 16 July 2011 | Volume 7 | Issue 7 | e1002087
Page 16
and tubulogenesis [74], and mutations in the PI synthesis gene
cdsA/winded severely abrogated terminal branching.
Although Branchless-Btl signaling likely controls and coordi-
nates many of the events in terminal branch morphogenesis, it is
unlikely to be the sole control and coordination mechanism
because not all of the morphogenesis events occur at the same time
and place. Lumen formation (tubulogenesis) occurs after cytoplas-
mic outgrowth, and lumen maturation including clearance and gas
filling occur even later, in some cases days after the lumen has
formed. Likewise, branch and lumen maintenance are late steps in
the process. Although there may be delays built into some of the
effector pathways downstream of Breathless to stagger its effects,
other factors likely also contribute to the timing and spatial
organization of the events. For example, ecdysone signaling may
gate the timing of lumen clearance and gas filling, and there are
presumably cell intrinsic cues that direct transport vesicles carrying
integrins and other basolateral markers to the plasma membrane
of growing buds, and vesicles carrying apical markers and lumenal
components to internal positions.
One surprising finding of our clonal analysis of known tracheal
genes was that terminal cell clones of the tracheal master regulator
trachealess gave a pruned phenotype. This implies that trachealess is
required not only for its well established role in the initiation of
tracheal development [23,24], but also for much later steps in the
developmental program such as terminal cell growth and
branching. Perhaps it functions in conjunction with SRF and
other cell type and stage-specific transcription factors in the
program to impart tracheal specificity in the control of
downstream effector genes, as shown for the C. elegans pharyngeal
master regulator pha-4 [75].
Primary and secondary branch morphogenesis genes
We identified a number of genes required for proper formation
of the larger branches of the tracheal system that form earlier than
terminal branches and from which they arise. For example, we
identified mutants required for proper shape of multicellular tubes,
including mutations that cause tracheal dilatations (small potatoes,
bulgy, balloon) and others that cause local constriction of
multicellular and autocellular tubes (kkv/short of breath, knk/dyspneic,
constricted). An especially intriguing set of genes (lotus, conjoined, oak
gall) are those required to form autocellular junctions and lumens.
These may encode specialized components or regulators of
autocellular junctions and tubes, such as proteins required for a
cell to wrap on or seal to itself.
Although we identified some primary and secondary branch
morphogenesis genes, there was a surprising paucity of such genes
relative to the large number of terminal cell branching genes
identified; a similarly skewed distribution obtained in a second
chromosome screen, if the large number of putative housekeeping
genes is excluded [35]). Although it is possible that morphogenesis
of these larger branches requires fewer genes, more likely such
genes were just not as efficiently identified in our screen. One
reason is that some such genes only show a tube phenotype when
most or all cells in the branch are mutant, as with breathless
(Figure 2B) and grainyhead mutations [76]. Another reason is that
perdurance of maternally expressed gene products likely obscures
early functions of some genes. Finally, terminal cells have an
elaborate structure that may make them more sensitive to
mutations and makes phenotypes easier to detect.
The number of genes required to build the tracheal
system
Our systematic genetic dissection of an organogenesis process,
including a clonal analysis to identify tracheal genes with
pleiotropic functions, allows an estimate of the number of genes
required to build an organ–an important question not just for
developmental biology but for medicine and tissue engineering.
Because we identified ,70 tracheal genes on the third chromo-
some (Table 1 and Table S3), which represents ,40% of the
genome, the full genome likely contains roughly two hundred
genes required to construct the larval tracheal system. This almost
certainly represents a lower limit because our screen did not
achieve full saturation and, as described above, the screen would
miss essential embryonic genes required non-cell autonomously
and genes with a significant maternal contribution.
Genomic profiling of developing and mature organs indicates
that there are hundreds of differentially expressed genes among
different organs, and genetic profiling to identify downstream genes
of organ master regulators such as the C. elegans pharyngeal
regulator pha-4 [77], the mouse pancreas regulator Pdx1 [78,79],
and the Drosophila tracheal regulator Trachealess (E. Chao and
M.A.K. unpublished data), suggests that there are 110–240 genes
dependent on the master regulator for expression, at least at certain
stages of development. Although it is not known how many of these
downstream genes are required for organ morphogenesis, or what
fraction of organ morphogenesis genes are both selectively
expressed and downstream targets of organ master regulators,
these genomic results are in line with the estimate from our genetic
studies that organ morphogenesis programs, even ones for relatively
simple organs like the Drosophila tracheal system, are likely to involve
several hundred genes. The approach used here, involving a clonal
analysis in the tracheal system of all mutations that do not survive
late enough in development as homozygotes to assess their tracheal
function, has begun to be extended to the other major chromosomes
to identify the rest of the tracheal morphogenesis program [7,35]
(Metzstein M. and M.A.K., unpublished data); most of the identified
mutations fit with the genetic scheme described here, with the
exception of a novel set of mutations on the second chromosome
that compromise terminal branch mutual avoidance and spacing
[35]. The clonal approach could easily be adapted to other organs
to systematically dissect additional organogenesis programs.
Materials and Methods
Drosophila strains
D. melanogaster strains used in the screen and meiotic mapping
experiments were: (1) btl-GAL4, UAS-GFP; Pr, Hs-hid/TM3Sb,
Tub-GAL80, (2) a newly isogenized btl-GAL4, UAS-GFP;
FRT2A,FRT82B, (3) ywFLP
122
; btl-GAL4, UAS-DsRED;
FRT82B cu UASi-GFPhp/TM6B, (4) ywFLP
122
; btl-GAL4,
UAS-DsRED; UASi-GFPhp th st FRT2A/MKRS (5) ywey-FLP;
cell-lethal, GMR-hid FRT2A/MKRS, (6) ywey-FLP; FRT82B,
cell-lethal, GMR-hid/MKRS, (7) y w FLP122; breathless-GAL4,
UAS-lumGFP, UAS-DsRED; FRT82B TubGal80, (8) y w
FLP122; breathless-GAL4, UAS-lumGFP, UAS-DsRED; Tub-
Gal80 FRT2A; (9) a newly isogenized ru h th st cu sr e ca; (10) ru
h th st cu sr e Pr ca/TM6B. Other strains were: FRT2A, FRT82B
(from Trudi Schu¨pbach); Hs-hid (on chromosome III; from Ruth
Lehman), onto which Pr was recombined; TM3Sb, Tub-GAL80
(from Stefan Luschnig); and strains used in complementation tests
and deficiency mapping experiments (see Table S3). All other
strains, except the mutants isolated here, have been described
(http://flybase.bio.indiana.edu) and are available from http://
flystocks.bio.indiana.edu.
Vector and transgene construction
UAS-DsRed. This Gal4-dependent DsRed transgene was
constructed by inserting a 0.7 kb Kpn I-Xba I restriction fragment
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 17 July 2011 | Volume 7 | Issue 7 | e1002087
Page 17
containing the DsRed coding sequences from pDsRed (Clontech)
between the corresponding sites of the vector pUAST [80]. The
resultant plasmid, pUAST-DsRed, was used to establish transgenic
lines on the X, second, and third chromosome by P element
mediated transformation of w
1118
embryos. The second chromo-
some insertion (line 5A) was recombined with breathless-GAL4 and
used here.
pUASTi. This P element vector for generating RNAi transgenes
was constructed by PCR amplification (primers Xho+trh-intron F,
Kpn+trh-intron B; see Table S2 for primer sequences) and TA
cloning of the 221 bp third intron from the trachealess gene into the
vector, pCRII-TOPO (Invitrogen). The intron fragment was then
excised with Xho I and Kpn I and inserted at those sites in
pUAST. Note that the trachealess intron contains an Eco RI site,
leaving Bgl II, Not I and Xho I as the only unique restriction sites
59 of the trachealess intron, and Kpn I and Xba I as the only unique
sites 39 of the trachealess intron. To generate RNAi constructs, a
,500 bp fragment from the gene of interest is inserted in the
forward orientation just upstream of the intron, and in the reverse
orientation downstream of the intron, as described below for UAS-
GFP(RNAi). Gal4-driven expression of this transgene results in
tissue specific transcription of the self-complementary RNA, which
is predicted to form a double-stranded ‘‘hairpin’’ conformation
that initiates the RNAi response.
UAS-GFP(RNAi). This Gal4-dependent GFP(RNAi) transgene
was created by PCR amplification (primers Not-GFP-F, Xho-
GFP-R) and insertion of an ,500 bp fragment of GFP (in the
forward, sense orientation) between the Not I and Xho I sites
upstream of the intron in pUASTi, and amplification of the same
fragment (primers Xba-GFP-F, Kpn-GFP-R) and insertion (in the
reverse orientation) between the Kpn I and Xba I sites
downstream of the intron, to create plasmid pUASTi-GFP(RNAi).
Transgenic flies were generated as above, and insertions were
identified on all major chromosomes. For this study, an insertion
on 3L (insertion B) was recombined onto ru h th st FRT2A (from
Stefan Luschnig) to generate the UASi-GFPhp th st FRT2A
chromosome, and an insertion on 3R (insertion 4A) was
recombined onto FRT82B cu sr e ca (from S. Luschnig) to generate
the FRT82B cu UASi-GFPhp chromosome.
UAS-lumGFP. This Gal4-dependent transgene expressing
secreted (lumenal or ‘‘lum’’) GFP was constructed by inserting
an Nhe I-Kpn I restriction fragment with the GFP coding
sequence from plum-GFP [50] between the Xba I and Kpn I sites
of pBS-KS (Stratagene), and then subcloning the Not I/Kpn I
lum-GFP fragment into those same sites in the pUAST vector.
Transgenic flies were generated as above, and second and third
chromosome insertions were recovered. A second chromosome
insertion was recombined onto a breathless-GAL4 bearing second
chromosome for use here.
Mutagenesis
A standard F3 EMS mutagenesis screen was performed
(Figure 1C). Strains used were homozygous for breathless-GAL4
[81] and UAS-GFP transgenes on chromosome II. Males
homozygous for an isogenized FRT2A, FRT82B chromosome
III were fed 25 mM EMS as described [82] and mass mated to
breathless-GAL4, UAS-GFP; Pr, Hs-hid/TM3, Sb, Tub-GAL80
virgin females. F1 males were each mated to two virgins of the
genotype used in the P cross. After five days, parents were
removed from the F1 cross and on days five and six F2 larvae were
heated to 38uC for 1.5 hours to induce Hs-hid and eliminate
animals carrying that chromosome. In the few cases (,2–3%)
where animals carrying Pr, Hs-hid survived, virgins of the
appropriate genotype were selected to generate a stock of FRT2A,
FRT82B*/TM3, Sb, Tub-GAL80 (*, newly induced mutation). If
animals homozygous for the treated third chromosome (non-Sb)
were not detected in the F3 or subsequent generations, a lethal
mutation was assumed to be present. Two hundred mutagenized
chromosomes were assayed in three small-scale pilot screens, and
4100 mutagenized chromosomes were assayed in a final large-
scale screen.
F3 screen
Sibling F2 flies (described above) were allowed to mate and were
brooded to produce two clutches of F3 individuals, one-quarter of
which should be homozygous for the mutagenized third
chromosome. The first F3 brood (after five days) was used to
maintain the stock and assess for presence of a lethal mutation; the
second F3 brood (at 12 days) was screened for tracheal phenotypes
(see below). If less than four pairs of flies were obtained in the F2
generation, screening was postponed for a generation. For tracheal
phenotype screening, F3 larvae were washed out of their food vials
with distilled water and examined under an M2 Zeiss or a Leica
fluorescence stereomicroscope. Homozygous third instar larvae
were identified by tracheal expression of GFP, and tracheal
morphology was analyzed; at least three homozygous larvae were
examined for each mutant line. Animals that appeared to have
tracheal defects were heat-killed (70–75uC for 3–5 s), mounted in
50% glycerol and examined under a Zeiss Axioplan 2 compound
fluorescence microscope. Lines in which a tracheal defect was
detected were retested to confirm the phenotype. If no
reproducible phenotype was found, the line was discarded.
Mutant lines that did not give rise to viable homozygous third
instar animals were analyzed in genetic mosaics as follows.
Genetic mosaic screen
Males from the mutant stock established in the F3 screen were
crossed to y, w, FLP
122
; breathless-GAL4, UAS-DsRED; UASi-
GFPhp, th, st, FRT2A/MKRS virgin females, and to y, w, FLP
122
;
breathless-GAL4, UAS-DsRED;FRT82B, cu, UASi-GFPhp/ TM6B
virgin females, to test mutants on 3L or 3R, respectively. For each
arm (3L and 3R) of every mutant stock, a cross with 20–40 pair
matings was done. Embryos (0–4 hr old) were heat-treated as
above for 0.75–1 hr to induce FLP-mediated recombination;
animals 2 hrs old or less at the time of heat treatment typically do
not survive. Crosses were maintained at 25uC for five days and
then mosaic animals were examined under a fluorescence
stereomicroscope. All tracheal cells are marked by expression of
UAS-DsRED; mutant tracheal cells also express GFP. This GFP
marking of mutant cells was achieved by inducing recombination
between a chromosome arm carrying the mutation of interest and
the homologous chromosome arm carrying UASi-GFPhp (see
above). Daughter cells homozygous for the mutagenized chromo-
some arm lack the GFP(RNAi) transgene and thus express GFP.
Animals of the correct genotype were selected, heat killed, and
analyzed for tracheal defects as above. Under these clone
induction conditions, ,6068 (mean +/- SEM) tracheal clones
were generated per animal (n = 5 animals). Among dorsal branch
clones (n = 127 clones), 50% appeared to be composed of a single
cell, 37% of two cells, 10% of three cells, and 3% of four or five
cells.
EGUF/HID secondary screen
Mutations that caused undergrowth defects in tracheal clones
were tested for growth defects in eye development using the
EGUF/HID technique that generates eye imaginal discs com-
posed exclusively of mitotic clones of a single genotype [43]. Males
from mutant lines were crossed to virgin females of genotype y, w,
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 18 July 2011 | Volume 7 | Issue 7 | e1002087
Page 18
ey-FLP; *, GMR-hid FRT2A/MKRS, or y, w, ey-FLP; FRT82B,
*, GMR-hid/MKRS, where * is the undergrowth mutation. Adult
progeny lacking Sb (carried on both of the balancer chromosomes
used) were scored for eye size. Mutations unable to support normal
eye development were presumed to affect general cell growth and
viability genes (‘‘housekeeping’’ genes) and were discarded.
Lumenal GFP secondary screen
Mutants with no detectable lumen under brightfield optics were
tested for presence of liquid-filled or discontinuous lumens using
the lumGFP transgene described above, which expresses GFP with
a signal peptide that we found accumulates in liquid-filled tracheal
lumens but is not detectable in normal, gas-filled lumens. Males
from the mutant stocks were crossed to ywFLP
122
; breathless-
GAL4, UAS-lumGFP; TubGal80 FRT2A/MKRS or ywFLP
122
;
breathless-GAL4, UAS-lumGFP; FRT82B TubGal80/MKRS vir-
gin females, and mosaic analysis was carried out as described
above in the Genetic Mosaic Screen.
Quantitative analysis of phenotypes
Effect of miracle-gro
338
on cell size was determined using ImageJ
software to measure the maximal cross-sectional area in a stack of
2D optical sections through the soma of mutant and wild type
control terminal cell clones (see Figure 3). Effect on dorsal trunk
lumen diameter was determined by comparing lumen diameter
between a section of tube containing a single mutant cell and the
average diameter of the immediately anterior and posterior
regions containing no mutant cells.
Mutation mapping and gene identification
Initial mapping was carried out by complementation tests
against a panel of chromosomal deficiencies spanning the third
chromosome, and by meiotic recombination mapping using visible
recessive markers ru h th st cu sr e ca. Fine scale mapping was carried
out using available single nucleotide polymorphisms (SNPs)
[83,84] and new ones specifically identified in this study, in
conjunction with complementation tests with chromosomes
carrying small, molecularly characterized deletions. The affected
gene in the mapped interval was then identified by sequencing
candidate genes and comparing their sequences to those in the
isogenized parental chromosome to reveal new EMS-induced
nonsense mutations or other mutations predicted to compromise
gene function, and by complementation tests with extant
mutations in the interval.
miracle gro/warts epistasis analysis
To compare the miracle gro/warts terminal cell phenotype to that
of activated Breathless FGFR, the MARCM system [42] was used
to generate marked (GFP
+
) clones of tracheal cells expressing l-
Breathless [85], a constitutively dimerized form of the protein, and
marked cells at terminal cell positions were examined using a
compound fluorescence microscope. To determine the genetic
epistasis relationship between miracle-gro/warts and blistered/pruned/
SRF, a downstream transcription factor in the Breathless pathway,
virgins of the genotype y, w, hsFLP122; bs
l(2)3267
, btl-Gal4, UAS-
GFP/CyO; FRT82B cu, UAS-GFP(RNAi) were crossed to males
of the genotype bs
l(2)3267
, btl-Gal4, UAS-GFP/CyO; FRT82B
miracle gro/warts
338
/MKRS. Mutant animals homozygous for
bs
l(2)3267
were identified by the strong pruned phenotype of
unmarked (GFP
-
) terminal cells, and marked (GFP
+
) wts
338
mutant
terminal cell clones in these animals were examined and scored as
above.
Supporting Information
Figure S1 Tracheal terminal cell clones mutant for trachealess
resemble those for the house keeping gene Aats-gln. Fluorescence
(A–D) and brightfield (A’,D’) images of larval dorsal branch
terminal cell (A,D) or dorsal trunk (B,C) clones (DsRED
+
, GFP
+
;
yellow) homozygous mutant for the house keeping gene glutamine
aminoacyl tRNA synthetase (Aats-gln
05461
; A, C) or the tracheal
master regulator trachealess (trh
10512
; D). Mutant DB terminal cells
are small and lack terminal branches (asterisks in A,D) and air-
filled lumens (dashed ovals in A’,D’). Control DB terminal cells
(DsRED
+
, GFP
-
; red) are shown at left in the images. Homozygous
Aats-gln
05461
mutant dorsal trunk tracheal cells (yellow cells in B)
are smaller than control wild-type dorsal trunk cells (yellow cells in
C). Bars in A (for A,D) and C (for B,C), 50
mm.
(TIF)
Table S1 Molecularly identified tracheal genes. Previously
identified Drosophila genes with defined tracheal phenotypes.
Each of the genes has been assigned as either a presumptive
tracheal patterning (P) or morphogenesis (M) gene.
(DOC)
Table S2 Polymerase chain reaction (PCR) primers. Primers
were used to generate the modified pUAST vector, pUASTi, and
the GFP(RNAi) construct, pUASTi-GFPhp.
(DOC)
Table S3 Additional tracheal morphogenesis mutants. Genes
that were identified in the screen but represented by a single allele,
and for which the molecular identity of the gene remains
unknown. Abbreviations and gene mapping methods are given
in the footnotes of Table 1. The estimate of 70 tracheal genes
identified in our screen includes the 58 named loci in Table 1 plus
the 12 loci in this table (PC146, 137, 198, 826, 889, 928, 1055,
1106, 1631, 1663, 1801) in which a mapped lethal mutation was
identified by deficiency mapping (see ‘‘Map Position’’). However,
these mapped lethal mutations may not be in all cases the
mutation responsible for the tracheal phenotype.
(DOC)
Acknowledgments
We thank Dr. Mark Metzstein for sharing his optimized tracheal clone
induction protocol and reagents and the results of his X chromosome
screen, Dr. Vikram Sudarsan for his observations about tracheal cell
growth control, and these and other members of the Krasnow and
Ghabrial labs for valuable discussions and input.
Author Contributions
Conceived and designed the experiments: ASG BPL MAK. Performed the
experiments: ASG BPL. Analyzed the data: ASG BPL MAK. Contributed
reagents/materials/analysis tools: ASG BPL MAK. Wrote the paper: ASG
BPL MAK.
References
1. Affolter M, Bellusci S, Itoh N, Shilo B, Thiery JP, et al. (2003) Tube or not tube:
remodeling epithelial tissues by branching morphogenesis. Dev Cell 4: 11–18.
2. Hogan BL, Kolodziej PA (2002) Organogenesis: molecular mechanisms of
tubulogenesis. Nat Rev Genet 3: 513–523.
3. Lubarsky B, Krasnow MA (2003) Tube morphogenesis: making and shaping
biological tubes. Cell 112: 19–28.
4. Nelson WJ (2003) Tube morphogenesis: closure, but many openings remain.
Trends Cell Biol 13: 615–621.
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 19 July 2011 | Volume 7 | Issue 7 | e1002087
Page 19
5. Wieschaus EF (1996) From molecular patterns to morphogenesis-The lessons
from studies on the fruit fly Drosophila (Nobel Lecture). Angewandte Chemie
International Edition in English 35: 2188–2194.
6. Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, et al. (1999) The
Berkeley Drosophila Genome Project gene disruption project: Single P-element
insertions mutating 25% of vital Drosophila genes. Genetics 153: 135–177.
7. Metzstein MM, Krasnow MA (2006) Functions of the nonsense-mediated
mRNA decay pathway in Drosophila development. PLoS Genet 2: e180.
8. Manning G, Krasnow MA (1993) Development of the Drosophila tracheal
system. In: Bate M, Martinez Arias A, eds. The Development of Drosophila
melanogaster. Cold Spring HarborNY: Cold Spring Harbor Press. pp 609–685.
9. Ghabrial A, Luschnig S, Metzstein MM, Krasnow MA (2003) Branching
morphogenesis of the Drosophila tracheal system. Annu Rev Cell Dev Biol 19:
623–647.
10. Cabernard C, Neumann M, Affolter M (2004) Cellular and molecular
mechanisms involved in branching morphogenesis of the Drosophila tracheal
system. J Appl Physiol 97: 2347–2353.
11. Samakovlis C, Hacohen N, Manning G, Sutherland DC, Guillemin K, et al.
(1996) Development of the Drosophila tracheal system occurs by a series of
morphologically distinct but genetically coupled branching events. Development
122: 1395–1407.
12. Samakovlis C, Manning G, Steneberg P, Hacohen N, Cantera R, et al. (1996)
Genetic control of epithelial tube fusion during Drosophila tracheal develop-
ment. Development 122: 3531–3536.
13. Ghabrial AS, Krasnow MA (2006) Social interactions among epithelial cells
during tracheal branching morphogenesis. Nature 441: 746–749.
14. Guillemin K, Groppe J, Ducker K, Treisman R, Hafen E, et al. (1996) The
pruned gene encodes the Drosophila serum response factor and regulates
cytoplasmic outgrowth during terminal branching of the tracheal system.
Development 122: 1353–1362.
15. Ribeiro C, Neumann M, Affolter M (2004) Genetic control of cell intercalation
during tracheal morphogenesis in Drosophila . Curr Biol 14: 2197–2207.
16. Tsarouhas V, Senti KA, Jayaram SA, Tiklova K, Hemphala J, et al. (2007)
Sequential pulses of apical epithelial secretion and endocytosis drive airway
maturation in Drosophila. Dev Cell 13: 214–225.
17. Wigglesworth VB (1983) The physiology of insect tracheoles. Adv Insect Physiol
17: 88–148.
18. Bar T, Guldner FH, Wolff JR (1984) ‘‘Seamless’’ endothelial cells of blood
capillaries. Cell Tissue Res 235: 99–106.
19. Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, et al. (2006) Endothelial
tubes assemble from intracellular vacuoles in vivo. Na ture 442: 453–456.
20. Yoshida Y, Yamada M, Wakabayashi K, Ikuta F, Kumanishi T (1989)
Endothelial basement membrane and seamless-type endothelium in the repair
process of cerebral infarction in rats. Virchows Arch A Pathol Anat Histopathol
414: 385–392.
21. Berry KL, Bulow HE, Hall DH, Hobert O (2003) A C. elegans CLIC-like
protein required for intracellular tube formation and maintenance. Science 302:
2134–2137.
22. Jurgens G, Kluding G, Nusslein-Volhard C, Wieschaus E (1984) Mutations
affecting the pattern of the larval cuticle in Drosophila melanogaster. II. Zygotic
loci on the third chromosome. Roux’s Archives of Developmental Biology 193:
283–295.
23. Isaac DD, Andrew DJ (1996) Tubulogenesis in Drosophila: a requirement for
the trachealess gene product. Genes Dev 10: 103–117.
24. Wilk R, Weizman I, Shilo BZ (1996) trachealess encodes a bHLH-PAS protein
that is an inducer of tracheal cell fates in Drosophila. Genes Dev 10: 93–102.
25. Glazer L, Shilo BZ (1991) The Drosophila FGF-R homolog is expressed in the
embryonic tracheal system and appears to be required for directed tracheal cell
extension. Genes Dev 5: 697–705.
26. Klambt C, Glazer L, Shilo BZ (1992) breathless, a Drosophila FGF receptor
homolog, is essential for migration of tracheal and specific midline glial cells.
Genes Dev 6: 1668–1678.
27. Shishido E, Higashijima S, Emori Y, Saigo K (1993) Two FGF-receptor
homologues of Drosophila: one is expressed in mesodermal primordium in early
embryos. Development 117: 751–761.
28. Beitel GJ, Krasnow MA (2000) Genetic control of epithelial tube size in the
Drosophila tracheal system. Development 127: 3271–3282.
29. Sutherland D, Samakovlis C, Krasnow MA (1996) branchless encodes a
Drosophila FGF homolog that controls tracheal cell migration and the pattern of
branching. Cell 87: 1091–1101.
30. Ikeya T, Hayashi S (1999) Interplay of Notch and FGF signaling restricts cell fate
and MAPK activation in the Drosophila trachea. Development 126: 4455–4463.
31. Li n X, Buff EM, Perrimon N, Michelson AM (1999) Heparan sulfate
proteoglycans are essential for FGF receptor signaling during Drosophila
embryonic development. Development 126: 3715–3723 .
32. Chihara T, Hayashi S (2000) Control of tracheal tubulogenesis by Wingless
signaling. Development 127: 4433–4442.
33. Llimargas M (2000) Wingless and its signal ling pathway have common and
separable functions during tracheal development. Development 127:
4407–4417.
34. Myat MM, Lightfoot H, Wang P, Andrew DJ (2005) A molecular link between
FGF and Dpp signaling in branch-specific migration of the Drosophila trachea.
Dev Biol 281: 38–52.
35. Baer MM, Bilstein, A. , Leptin, M (2007) A clonal genetic screen for mutants
causing defects in larval tracheal morphogenesis in Drosophila. Genetics 176:
2279–2291.
36. Chanut-Delalande H, Jung AC, Lin L, Baer MM, Bilstein A, et al. (2007) A
genetic mosaic analysis with a repressible cell marker screen to identify genes
involved in tracheal cell migration during Drosophila air sac morphogenesis.
Genetics 176: 2177–2187.
37. Jung AC, Ribeiro C, Michaut L, Certa U, Affolter M (2006) Polychaetoid/ZO-1
is required for cell specification and rearrangement during Drosophila tracheal
morphogenesis. Curr Biol 16: 1224–1231.
38. Luschnig S, Batz T, Armbruster K, Krasnow MA (2006) serpentine and
vermiform encode matrix proteins with chitin binding and deacetylation
domains that limit tracheal tube length in Drosophila. Curr Biol 16: 186–194.
39. Stahl M, Schuh R, Adryan B (2007) Identification of FGF-de pendent genes in
the Drosophila tracheal system. Gene Expr Patterns 7: 202–209.
40. Zhu MY, Wilson R, Leptin M (2005) A screen for genes that influence fibroblast
growth factor signal transduction in Drosophila. Genetics 170: 767–777.
41. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, et al. (2000) The
genome sequence of Drosophila melanogaster. Science 287: 2185–2195.
42. Lee T, Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM)
for Drosophila neural development. Trends Neurosci 24: 251–254.
43. Stowers RS, Schwarz TL (1999) A genetic method for generating Drosophila
eyes composed exclusively of mitotic clones of a single genotype. Genetics 152:
1631–1639.
44. Affolter M, Nellen D, Nussbaumer U, Basler K (1994) Multiple requirements for
the receptor serine/threonine kinase thick veins reveal novel functions of TGF
beta homologs during Drosophila embryogenesis. Develop ment 120:
3105–3117.
45. Jarecki J, Johnson E, Krasnow MA (1999) Oxygen regulation of airway
branching in Drosophila is mediated by branchless FGF. Cell 99: 211–220.
46. Levi BP, Ghabrial AS, Krasnow MA (2006) Drosophila talin and integrin genes
are required for maintenance of tracheal terminal branches and luminal
organization. Development 133: 2383–2393.
47. Swanson LE, Beitel GJ (2006) Tubulogenesis: an inside job. Curr Biol 16:
R51–53.
48. Jeon M, Zinn K (2009) Receptor tyrosine phosphatases control tracheal tube
geometries through negative regulation of Egfr signaling. Development 136:
3121–3129.
49. Forster D, Armbruster K, Luschnig S (2009) Sec24-dependent secretion drives
cell-autonomous expansion of tracheal tubes in Drosophila. Curr Biol 20: 62–68.
50. Blum R, Stephen s DJ, Schulz I (2000) Lumenal targeted GFP, used as a marker
of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular
network. J Cell Sci 113 3151-3159.
51. Grueber WB, Ye B, Moore AW, Jan LY, Jan YN (2003) Dendrites of distinct
classes of Drosophila sensory neurons show different capacities for homotypic
repulsion. Curr Biol 13: 618–626.
52. Moberg KH, Schelble S, Burdick SK, Hariharan IK (2005) Mutations in
erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101,
elicit non-cell-autonomous overgrowth. Dev Cell 9: 699–710.
53. Devine WP, Lubarsky B, Shaw K, Luschnig S, Messina L, et al. (2005)
Requirement for chitin biosynthesis in epithelial tube morphogenesis. Proc Natl
Acad Sci U S A 102: 17014–17019.
54. Moussian B, Tang E, Tonning A, Helms S, Schw arz H, et al. (2006) Drosophila
Knickkopf and Retroactive are needed for epithelial tube growth and cuticle
differentiation through their specific requirement for chitin filament organiza-
tion. Development 133: 163–171.
55. Tapon N, Ito N, Dickson BJ, Treisman JE, Hariharan IK (2001) The Drosophila
tuberous sclerosis complex gene homologs restrict cell growth and cell
proliferation. Cell 105: 345–355.
56. Centanin L, Dekanty A, Romero N, Irisarri M, Gorr TA, et al. (2008) Cell
autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce
terminal branch sprouting. Dev Cell 14: 547–558.
57. Justice RW, Zilian O, Woods DF, Noll M, Bryant PJ (1995) The Drosophila
tumor suppressor gene warts encodes a homolog of human myotonic dystrophy
kinase and is required for the control of cell shape and proliferation. Genes Dev
9: 534–546.
58. Xu T, Wang W, Zhang S, Stewart RA, Yu W (1995) Identifying tumor
suppressors in genetic mosaics: the Drosophila lats gene encodes a putative
protein kinase. Development 121: 1053–1063.
59. Klambt C (1993) The Drosophila gene pointed encodes two ETS-like proteins
which are involved in the development of the midline glial cells. Development
117: 163–176.
60. Araujo SJ, Aslam H, Tear G, Casanova J (2005) mummy/cystic encodes an
enzyme required for chitin and glycan synthesis, involved in trachea, embryonic
cuticle and CNS development--analysis of its role in Drosophila tracheal
morphogenesis. Dev Biol 288: 179–193.
61. Tonning A, Hemphala J, Tang E, Nannmark U, Samakovlis C, et al. (2005) A
transient luminal chitinous matrix is required to model epithelial tube diameter
in the Drosophila trachea. Dev Cell 9: 423–430.
62. Brown NH, Gregory SL, Rickoll WL, Fessler LI, Prout M, et al. (2002) Talin is
essential for integrin function in Drosophila. Dev Cell 3: 569–579.
63. Wu L, Niemeyer B, Colley N, Socolich M, Zuker CS (1995) Regulation of PLC-
mediated signalling in vivo by CDP-diacylglycerol synthase. Nature 373:
216–222.
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 20 July 2011 | Volume 7 | Issue 7 | e1002087
Page 20
64. Mortimer NT, Moberg KH (2009) Regulation of Drosophila embryonic
tracheogenesis by dVHL and hypoxia. Dev Biol 329: 294–305.
65. Potter CJ, Huang H, Xu T (2001) Drosophila Tsc1 functions with Tsc2 to
antagonize insulin signaling in regulating cell growth, cell proliferation, and
organ size. Cell 105: 357–368.
66. Lowe SL, Peter F, Subramaniam VN, Wong SH, Hong W (1997) A SNARE
involved in protein transport through the Golgi apparatus. Nature 389:
881–884.
67. Hay JC, Chao DS, Kuo CS, Scheller RH (1997) Protein interactions regulating
vesicle transport between the endoplasmic reticulum and Golgi apparatus in
mammalian cells. Cell 89: 149–158.
68. Gips SJ, Kandzari DE, Goldschmidt-Clermont PJ (1994) Growth factor
receptors, phospholipases, phospholipid kinases and actin reorganization. Semin
Cell Biol 5: 201–208.
69. Jayaram SA, Senti KA, Tiklova K, Tsarouhas V, Hemphala J, et al. (2008)
COPI vesicle transport is a common requirement for tube expansion in
Drosophila. PLoS One 3: e1964.
70. Sternlicht H, Farr GW, Sternlicht ML, Driscoll JK, Willison K, et al. (1993) The
t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo.
Proc Natl Acad Sci U S A 90: 9422–9426.
71. Nechiporuk T, Fernandez TE, Vasioukhin V (2007) Failure of epithelial tube
maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice. Dev Cell 13:
338–350.
72. Geneste O, Copeland JW, Treisman R (2002) LIM kinase and Diaphanous
cooperate to regulate serum response factor and actin dynamics. J Cell Biol 157:
831–838.
73. Haucke V (2005) Phosphoinositide regulation of clathrin-mediated endocytosis.
Biochem Soc Trans 33: 1285–1289.
74. Martin-Belmonte F, Gassama A, Datta A, Yu W, Rescher U, et al. (2007)
PTEN-mediated apical segregation of phosphoinositides controls epithelial
morphogenesis through Cdc42. Cell 128: 383–397.
75. Mango SE (2009) The molecular basis of organ formation: insights from the C.
elegans foregut. Annu Rev Cell Dev Biol 25: 597–628.
76. Hemphala J, Uv A, Cantera R, Bray S, Samakovlis C (2003) Grainy head
controls apical membrane growth and tube elongation in response to
Branchless/FGF signalling. Development 130: 249–258.
77. Gaudet J, Muttumu S, Horner M, Mango SE (2004) Whole-genome analysis of
temporal gene expression during foregut development. PLoS Biol 2: e352.
78. Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulin-promoter-factor 1 is
required for pancreas development in mice. Nature 371: 606–609.
79. Svensson P, Williams C, Lundeberg J, Ryden P, Bergqvist I, et al. (2007) Gene
array identification of Ipf1/Pdx1-/- regul ated genes in pancreatic progenitor
cells. BMC Dev Biol 7: 129.
80. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering
cell fates and generating dominant phenotypes. Development 118: 401–415.
81. Shiga Y, Tanaka-Matakatsu M, Hayashi S (1996) A nuclear GFP/b-galactosidase
fusion protein as a marker for morphogenesis in living Drosophila. Dev Growth
Differ 38: 99–106.
82. Ashburner M (1989) Drosophila: A laborat ory handbook. Cold Spring
HarborNY: Cold Spring Harbor Laboratory Press.
83. Berger J, Suzuki T, Senti KA, Stubbs J, Schaffner G, et al. (2001) Genetic
mapping with SNP markers in Drosophila. Nat Genet 29: 475–481.
84. Martin SG, Dobi KC, St Johnston D (2001) A rapid method to map mutati ons
in Drosophila. Genome Biol 2: RESEARCH0036.
85. Lee T, Hacohen N, Krasnow M, Montell DJ (1996) Regulated Breathless
receptor tyrosine kinase activity required to pattern cell migration and branching
in the Drosophila tracheal system. Genes Dev 10: 2912–2921.
Screen for Tube Morphogenesis and Branching Genes
PLoS Genetics | www.plosgenetics.org 21 July 2011 | Volume 7 | Issue 7 | e1002087
Page 21
  • Source
    • "In addition, terminal cell branching is readily quantifiable. Assessment of the effects of genetic mutations on terminal cell development has revealed terminal-cell-autonomous and nonautonomous requirements for oxygen (Ghabrial et al., 2011). Drosophila models have also been used to test for genes associated with congenital lung disease such as asthma (e.g. "
    [Show abstract] [Hide abstract] ABSTRACT: Fly models that faithfully recapitulate various aspects of human disease and human health-related biology are being used for research into disease diagnosis and prevention. Established and new genetic strategies in Drosophila have yielded numerous substantial successes in modeling congenital disorders or inborn errors of human development, as well as neurodegenerative disease and cancer. Moreover, although our ability to generate sequence datasets continues to outpace our ability to analyze these datasets, the development of high-throughput analysis platforms in Drosophila has provided access through the bottleneck in the identification of disease gene candidates. In this Review, we describe both the traditional and newer methods that are facilitating the incorporation of Drosophila into the human disease discovery process, with a focus on the models that have enhanced our understanding of human developmental disorders and congenital disease. Enviable features of the Drosophila experimental system, which make it particularly useful in facilitating the much anticipated move from genotype to phenotype (understanding and predicting phenotypes directly from the primary DNA sequence), include its genetic tractability, the low cost for high-throughput discovery, and a genome and underlying biology that are highly evolutionarily conserved. In embracing the fly in the human disease-gene discovery process, we can expect to speed up and reduce the cost of this process, allowing experimental scales that are not feasible and/or would be too costly in higher eukaryotes.
    Full-text · Article · Mar 2016 · Disease Models and Mechanisms
  • Source
    • "Many identified genes related to terminal branching were found in genetic screens with their tracheal expression pattern during embryogenesis. More recently, several studies on the direct identification of genes involved in larval tracheal branching have started to reveal more about the genetic control cascades on the branching mechanism [10,11]. To gain further insight into the regulation of the formation of tracheal terminal branches during larval stages, we carried out a genetic screen on the Kiss collection of P-element enhancer trap mutants with tracheal terminal branching defects, taking advantage of the P-element insertion into genes, which can be identified and cloned relatively easily [12,13] . "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Endothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, the terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, we searched for mutants with very few terminal branches using the Kiss enhancer-trap line collection. Results: In this analysis, we identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in the terminal cells increased cellular branching and the Cropped protein levels in vivo. Conclusions: We find that the branching morphogenesis of the terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of the terminal cells.
    Full-text · Article · Dec 2015 · BMC Developmental Biology
  • Source
    • "Diametric expansion is followed by axial growth. A series of studies of tube elongation defective mutants [8,21] have shown that axial growth requires the septate junc- tion2223242526272829, subapical protein complex [20,30,31], planar cell polarity proteins [32], and Src kinase [33 ,34 ]. With the exception of Src kinases, all known tube length mutants show over-elongation phenotypes. "
    [Show abstract] [Hide abstract] ABSTRACT: The shape of biological tubes is optimized for supporting efficient circulation of liquid and gas and to maintain organismal homeostasis. Maintaining a constant tube diameter and fitting tube length to body size are two requirements for proper tube function. The tracheal system of the Drosophila embryo is established through branching of ectodermal epithelia in the absence of environmental air, and the branching pattern and geometry of this system are genetically specified. Recent studies identified apical extracellular matrix (aECM) as a crucial regulator of tube expansion and elongation. Evidence suggests that aECM coordinates apical membrane growth and cell contractility to control tube growth at the tissue level. In the present review, we will discuss the physical mechanisms underlying this interaction. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jun 2015 · Current opinion in genetics & development
Show more