MOLECULAR AND CELLULAR BIOLOGY, Oct. 2002, p. 6842–6853
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 19
Control of the Hypoxic Response in Drosophila melanogaster by
the Basic Helix-Loop-Helix PAS Protein Similar
Sofía Lavista-Llanos,1La ´zaro Centanin,1Maximiliano Irisarri,1Daniela M. Russo,1
Jonathan M. Gleadle,2Silvia N. Bocca,1Mariana Muzzopappa,1
Peter J. Ratcliffe,2and Pablo Wappner1*
Instituto de Investigaciones Bioquímicas Fundacio ´n Campomar, Buenos Aires 1405, Argentina,1and The Henry
Wellcome Building of Genomic Medicine, University of Oxford, Oxford OX3 7BN, United Kingdom2
Received 19 February 2002/Returned for modification 10 May 2002/Accepted 12 June 2002
In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)–PAS transcription hypoxia-induc-
ible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous
system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using tran-
scriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in
late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar
(Sima) and Tango (Tgo) function as HIF-? and HIF-? homologues, respectively, and demonstrate a conserved
mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time
course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and
that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic
expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the
nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila
Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima,
indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in
Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.
In multicellular organisms, oxygen homeostasis requires
precise developmental coordination between the growth of
metabolizing tissues and that of systems that supply oxygen.
Recent advances have provided new insights into how this
complex task is achieved. For instance, in mammals it has long
been recognized that local hypoxia is a major stimulus for
angiogenesis (27). More recently, the recognition that specific
angiogenic growth factors such as the vascular endothelial
growth factor are powerfully induced by hypoxia through the
action of a DNA binding complex termed hypoxia-inducible
factor (HIF) has provided mechanistic insights into the process
The HIF DNA-binding complex consists of a heterodimer of
basic-helix-loop-helix–PAS (bHLH-PAS) proteins that binds a
core element A/(G)CGTG within hypoxia response elements
(HREs) (49, 50). Regulation by oxygen involves stabilization of
the ?-subunit in hypoxia, whereas the ?-subunit, a common
partner for several other bHLH-PAS proteins, is constitutively
expressed regardless of oxygen tension (19, 25, 40, 55). Nor-
moxic degradation of HIF-? is mediated via ubiquitination and
subsequent proteolysis, which requires oxygen-dependent in-
teraction with the Von Hippel-Lindau (VHL) tumor suppres-
sor protein (36). This interaction is regulated by hydroxylation
of specific prolyl residues within the HIF-? polypeptides (21,
23, 34), and recent work has identified a series of ?-ketoglu-
tarate-dependent non-heme iron-dependent dioxygenases that
catalyze HIF-? prolyl hydroxylation and thus regulate stability
of the polypeptide in accordance with oxygen availability (4,
15). Other studies of HIF induction in mammalian tissue cul-
ture systems have defined additional regulatory steps that in-
volve oxygen-dependent subcellular localization (2, 26) and
coactivator recruitment (6, 13), although these processes are so
far less well understood.
In addition to angiogenic growth factors, HIF-1 drives the
expression of genes involved in a broad array of systemic and
cellular adaptive responses to hypoxia, suggesting a central
role for HIF-1 as a regulator of oxygen homeostasis (35).
However, although targeted inactivation of different HIF-?
and HIF-? subunits in the mouse is associated with several
severe or lethal phenotypes involving defective vascular devel-
opment (5, 22, 29, 32, 41), few mechanistic studies of the HIF-1
system have been conducted in vivo, and the critical interfaces
between developmental processes and HIF activation remain
To better understand HIF regulation in vivo and the role of
hypoxia in developmental processes, we therefore sought to
define and characterize the system in Drosophila melanogaster.
In insects, air reaches the tissues by passive diffusion through a
specialized tubular network termed the tracheal system (33,
43). Moreover, development of tracheal terminal branches is
oxygen dependent and shares many features with mammalian
oxygen-dependent angiogenesis (37) (52). For instance, oxy-
gen-regulated expression of Drosophila Branchless/FGF guides
tracheal migration during development (47) and also drives
extension of plastic terminal branches in a manner similar to
the function of vascular endothelial growth factor in mamma-
lian angiogenesis (24). The existence of a Drosophila HIF ho-
mologue has been inferred from DNA-binding assays with
* Corresponding author. Mailing address: Instituto de Investigacio-
nes Bioquímicas Fundacio ´n Campomar, Patricias Argentinas 435,
Buenos Aires 1405, Argentina. Phone: (54-11) 4863-4011. Fax: (54-11)
4865-2246. E-mail: email@example.com.
nuclear extracts from normoxic or hypoxic SL2 cells (38), and
transfection studies in mammalian cells have suggested that
the Drosophila bHLH-PAS protein Similar (Sima) (39) might
function as an HIF-? homologue (1). However, the system has
not yet been fully defined or characterized in vivo in the fly.
In the present study, we demonstrate a hypoxia-inducible
transcriptional response in Drosophila that is homologous to
the mammalian HIF system, and we use an HRE reporter
system in transgenic flies to characterize the temporal and
spatial patterns of the activity during development. We confirm
the function of Sima as an HIF-? homologue and demonstrate
a conserved regulatory process that involves dual mechanisms
of protein stabilization and nuclear localization, which are
critically dependent on the function of a Drosophila homo-
logue of the HIF prolyl hydroxylases.
MATERIALS AND METHODS
DNA constructs. The pCaSpeR-LDH-LacZ plasmid was generated by excising
an Xba-BamHI 233-bp fragment containing the murine LDH-A enhancer/pro-
moter from pLDHGH (L1) (16) and inserting the fragment into the same sites
in a CaSpeR-AUG-?gal vector (a gift from Carl Thummel). The LDH-Gal4
plasmid was generated by amplifying a 51-bp fragment from the same enhancer
with a sense oligonucleotide (5?-CGGGATCCCAGCCTACACGTGGG-3?)
bearing a BamHI restriction site, and an antisense oligonucleotide (5?-GAAGA
TCTGCGCTGACGTCAGAGTGG-3?) with a BglII site. After digestion with
BamHI and BglII the fragment was cloned into the BamHI site of a CaSpeR-
Gal4 vector (3), and plasmids bearing a dimerised insert were isolated.
Sima?692-863 deletion was generated by PCR with full-length pNB40-sima as
a template and the divergent primers 5?-GCTTCGGGTACCCATGAGGCCA
ACCAACATGTCAC-3? and 5?-GCTTCGGGTACCCTAACGGAGACGGAT
CTCATGTGG-3? that include KpnI restriction sites at the 5? ends. Amplification
resulted in a 7.7-kb fragment that included the whole pNB40 vector, as well as
the sequence encoding Sima protein, lacking amino acids 692 to 863. The prod-
uct was digested with KpnI and circularized, resulting in pNB40-sima?692-863.
The Sima?692-863 insert was excised with EcoRI and NotI and subcloned into
the same sites of the pCaSpeR-UAS vector.
Fly stocks. Transgenic lines were generated from the above-mentioned con-
structs by standard procedures. Two independent LDH-LacZ lines 33 and 34,
with insertions in the third chromosome, were obtained. Tissue-specific expres-
sion of the reporter was the same in both lines. Line 34 was used in all studies.
Nine independent LDH-Gal4 lines were obtained, and the reporter gene expres-
sion pattern was reproducible in all of them. One insertion in the second chro-
mosome that showed strong expression of a UAS-GFP reporter was selected and
used for generating LDH-Gal4/UAS-nGFP.LacZ or LDH-Gal4/UAS-TAU.GFP
recombinant chromosomes that were used in experiments. The tgo5and simH9
stocks were kindly provided by Steve Crews; btlMZ13was a gift from Benny Shilo;
UAS-nGFP-LacZ stocks were a gift from Shigeo Hayashi and the UAS-TAU.GFP
line was kindly provided by Andrea Brand; lines bearing Df(3L)emc-E12 defi-
ciency and en-Gal4 on the second chromosome were provided by the Blooming-
ton Stock Center. The line bearing UAS-sima on the second chromosome was
previously described (56). For heat shock induction the K25 sev HS-Gal4 line on
the third chromosome was used.
Synchronized collection of embryos, hypoxic treatment, and ?-galactosidase
(?-Gal) assays. To obtain synchronized collections, egg-laying agar plates were
replaced every half an hour, and embryos were kept at 25°C until the desired
Hypoxia was applied by regulating the proportions of oxygen and nitrogen in
a Forma Scientific 3131 incubator at 25°C. ?-Gal activity determinations were
performed as described previously (57). Briefly, embryos were homogenized in
200 to 500 ?l of lysis buffer (50 mM Tris HCl [pH 7.8], 2 mM EDTA, 10%
glycerol, 2 mM dithiothreitol, 1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride) in a Teflon-glass Potter and centrifuged at 2,500 ? g for 3 min at 4°C.
Supernatant was recovered, and the protein concentration was determined. Typ-
ically, 50 ?g of protein extract was used in each assay. Enzymatic reactions were
performed by incubating 20 ?l of extract with 180 ?l of reaction buffer containing
80 mM Na3PO4(pH 7.3), 102 mM ?-mercaptoethanol, 9 mM MgCl2, and 4 mM
Chlorophenol Red ?-D-galactopyranoside (Roche Diagnostics, Mannheim, Ger-
many) at 37°C, and the optical density at 574 nm was read at 10, 30, 60, and 120
min. Rate of color development was linear throughout this time period. Endog-
enous background was substracted by using a sample inactivated at 100°C for 10
min. After checking on the linear progression of the reaction, values at 2 h were
Antibody staining, in situ hybridization, and RNAi. Rabbit anti-?-Gal anti-
bodies (Cappel), rat anti-Sima antibody (1), rat anti-Trh antibody (51), and
mouse monoclonal 2A12 antitracheal lumen antibody (Developmental Studies
Hybridoma Bank, Iowa University) were used. All secondary antibodies (conju-
gated to Cy2, Cy3, or horseradish peroxidase) were from Jackson Laboratories.
Standard procedures for X-Gal (5-bromo-4-chloro-3-indolyl-?-D-galactopyrano-
side) and antibody staining were used. Embryos, larvae, and adults were visual-
ized by fluorescent or Nomarski optics in a BX-60 Olympus microscope or by
confocal microscopy in a Zeiss LSM510 microscope. Homozygous mutant em-
bryos for tgo, trh, sim, and btl genes were identified by absence of balancer
chromosome ?-Gal staining. Sense digoxigenin-labeled RNA probe for CG1114
mRNA in situ hybridization was synthesized with T7 RNA polymerase (Roche)
with pOT2-CG1114 digested with XhoI as a template. For the antisense probe a
PCR product bearing a T7 promoter on the 3? end was used as a template.
CG1114 interference RNA (RNAi) was injected into embryos ca. 30 min after
egg laying. Embryos were then maintained at 18°C for 2 h, transferred to 25°C
until stage 16, and fixed for staining as described above. RNAi was synthesized
by using a PCR product with polymerase T7 promoters at the 5? ends of both
DNA strands. Full-length CG1114 cloned in pOT2 was used as a template for
PCR amplification by using two primers (5-GAATTAATACGACTCACTATA
GGGAGAAGATCTATGATAACCTCCACGACCACGGACTAC-3? and 5?-
TCGCTGCTGGTG-3?) bearing T7 RNA polymerase promoter sequences at the
5? ends. The PCR product was used as a template for generating RNAi with T7
polymerase. Synthesis was performed at 37°C for 3 h. After quantification, RNAi
was divided into aliquots and stored at ?70°C.
Western blot and RNA slot blot. For Western blots, ca. 400 embryos were
rapidly homogenized in 50 ?l of loading buffer in a Kontes 1.5-ml polypropylene
pestle/tube and immediately incubated at 95°C for 3 min. After centrifugation
samples were loaded on a gel and subjected to polyacrylamide gel electrophore-
sis. Blotting and antibody-ECL detection (New England Biolabs) were per-
formed by standard procedures. The anti-?1-tubulin antibody was a gift from
Ricardo Ramos. For RNA slot blotting, RNA was prepared from ca. 400 em-
bryos by the Trizol (Gibco) method. RNA was quantified by absorbance at 260
nm and blotted onto a Zeta-Probe (Bio-Rad) membrane by using a Hybri-Slot
Manifold (BRL). [32P]dCTP DNA probes were prepared by random priming
(Prime-a-Gene; Promega) by using an 842-bp BamHI fragment from the sima
gene, a 1,523-bp EcoRV fragment from ?-Gal gene or the complete rRNA 18S
sequence. Hybridization, washes, and autoradiography were performed by using
A hypoxia-inducible transcriptional response in Drosophila
that is homologous to the mammalian HIF system. Based on
the observation that an HIF homologue present in extracts of
Drosophila SL2 cells can bind a mammalian HRE, we sought to
characterize the transcriptional system predicted by these ex-
periments by the generation of transgenic flies bearing mam-
malian HREs linked to a LacZ reporter gene. We first tested
a transgenic line bearing a pentamer of an 18-bp sequence
from the murine erythropoietin HRE (Epo-LacZ), that is
known to be induced by both Trachealess and Single minded
(56). However, no significant induction of ?-Gal expression
was observed when these embryos were exposed to hypoxia.
Mutational analysis of the erythropoietin HRE in mammalian
cells has indicated that, while this 18-bp sequence is sufficient
for binding to HIF-1, an adjacent site that binds an as-yet-
unknown factor is also critical for the transcription activity (18).
The erythropoietin gene is not conserved in flies, and we rea-
soned that the additional factor(s) binding to this adjacent site
might be missing in flies. Therefore, for the design of a new
HRE-LacZ reporter, we selected the mammalian lactate de-
hydrogenase A (LDH-A) gene, which is hypoxia responsive
VOL. 22, 2002DROSOPHILA TRANSCRIPTIONAL RESPONSE TO HYPOXIA6843
and well conserved in flies. The mammalian LDH-A hypoxic
enhancer includes two HREs separated by an 8-bp spacer and
a cyclic AMP responsive element (CRE) located 16 bp further
downstream. Both HREs and the CRE consensus have been
shown to be necessary for strong hypoxic induction in mam-
malian cells (16). We generated transgenic lines with an LDH-
LacZ reporter based on a 233-bp fragment from the murine
LDH-A enhancer bearing the three relevant boxes described
above (Fig. 1-A). In two independent lines carrying this ele-
ment we observed approximately 10-fold ?-galactosidase in-
duction in hypoxic embryos (Fig. 1B to D). These findings
demonstrate the operation in Drosophila embryos of a con-
served HRE-dependent transcriptional response to hypoxia
and provided an opportunity to characterize the transcriptional
response to hypoxia in developing flies.
Physiological characterization of the transcriptional re-
sponse to hypoxia. First, we studied the manner in which the
overall response to hypoxia is influenced by development. For
this purpose, we used synchronized populations of insects that
were subjected to 5% oxygen for 8 h at different stages of
development and tested their ability to induce the reporter. As
can be seen in Fig. 1E, hypoxia-inducible ?-Gal activity rises
dramatically at late embryogenesis, although a relatively high
induction of the reporter was recorded at all larval stages.
FIG. 1. A hypoxia-inducible transcriptional response in Drosophila. Physiological characterization of the hypoxic response in vivo. (A) Sche-
matic representation of the LDH-LacZ transcriptional reporter: A 233-bp fragment of the murine LDH-A enhancer from bp ?186 to ?47 (relative
to the transcription initiation site) controls ?-Gal expression. The sequences correspond to HRE and CRE elements present in the enhancer. Bases
in bold lettering mark the HRE consensus, and the numbers above indicate the position relative to the transcription initiation site. (B) The
LDH-LacZ reporter is induced in embryos maintained at 5% O2for 8 h (F) compared to normoxia (}). The x axis represents time course of the
?-Gal reaction. (C and D) Stage 17 transgenic embryos carrying the LDH-LacZ reporter maintained in normoxia (C) or hypoxia (8 h at 5% O2)
(D) were stained with X-Gal in a reaction developed overnight. Whereas in embryos exposed to hypoxia the X-Gal staining was ubiquitous, in
normoxic individuals the expression of the reporter was restricted to a small anterior domain. (E) Modulation of the hypoxic response during
development. Individuals bearing the LDH-LacZ reporter were synchronized at different stages of development and were subjected to hypoxia (5%
O2) for 4 h. The hypoxia/normoxia ?-Gal activity ratio is shown. Hypoxic induction of the reporter is maximal at late embryogenesis, although it
is also high at larval stages 1 and 2. P, pupa; A, adult. (F) Effect of oxygen concentration. Stage 16 to 17 embryos bearing the LDH-LacZ reporter
were exposed to different oxygen concentrations for 4 h, the ?-Gal activity was compared with that of normoxic controls, and the ratios of the
activities were calculated. Triplicate determinations of the activity were performed. Induction of the reporter was maximal between 3 and 5%
6844 LAVISTA-LLANOS ET AL.MOL. CELL. BIOL.
Thus, the response is strongly modulated by developmental
parameters, reaching peak levels at late embryogenesis.
Second, we wanted to know the range of ambient oxygen
concentrations over which the reporter is induced in the whole
animal. The response to different oxygen concentrations was
studied in stage 16 to 17 embryos. As depicted in Fig. 1F,
hypoxic induction was found to be maximal between 3 and 5%
oxygen, decreasing gradually as the oxygen concentration rises.
This result indicates that activation by low oxygen is strikingly
concentration dependent although, presumably because of ox-
ygen gradients within the developing flies, the point of maximal
activation is shifted relative to the concentrations of 0.1 to
1.0% oxygen that are maximally active in tissue culture cells.
Remarkably, at 1% oxygen, expression of the reporter was
below that of the normoxic controls, most probably reflecting
general arrest of cell metabolism in very severe hypoxia (10, 11,
Spatially restricted induction of the hypoxic transcriptional
response. Although the first experiments with X-Gal staining
suggested ubiquitous reporter expression (Fig. 1D), this result
was obtained by using long incubation times on stage 17 em-
bryos, where the cuticle is beginning to be secreted, and we
surmised that very high levels of reporter product may have
obscured spatial localization. By using X-Gal staining for 30
min on stage 15 to 16 embryos, a distinct expression pattern
that appeared to correspond to tracheal branches was observed
(Fig. 2A). Indeed, double staining of hypoxic embryos with an
antitracheal lumen antibody revealed hypoxia-inducible ?-Gal
reporter in the tracheal system, with stronger expression in the
lateral trunks and dorsal branches (Fig. 2B and C) (33).
To study the expression of the reporter in more detail and to
assess postembryonic stages, we developed an additional re-
porter construct based on the binary Gal4/UAS system (3) that
would allow more sensitive detection in vivo particularly in the
presence of the developing cuticle. We engineered a 51-bp
fragment from the core of the LDH-A enhancer (16) as a
dimer controlling expression of Gal4 (LDH-Gal4, Fig. 2M)
and generated several transgenic fly lines with single insertions
in all of the three chromosomes. These lines were crossed
with different UAS-green fluorescent protein (GFP) or UAS-
GFPn.LacZ lines (42), and expression of the reporter was
monitored in normoxia and hypoxia (5% oxygen for 4 h).
Unlike the LDH-LacZ lines, these lines showed constitutive
reporter expression in salivary glands. However, hypoxia-in-
ducible expression was remarkably similar between the differ-
ent reporter lines. Double immunofluorescence experiments
with antibodies to ?-Gal and the tracheal marker Trachealess
(53) demonstrated that hypoxia-dependent transcription is in-
deed elicited mostly in tracheal cells but also in scattered
patches on the ectoderm (Fig. 2F). More detailed studies of
developmental regulation showed that, whereas stage 11 em-
bryos showed no induction of the reporter (Fig. 2D), scattered
cells of the tracheal system began to express the reporter dur-
ing stages 12 and 13 (Fig. 2E), with the number of cells ex-
pressing the reporter increasing gradually from this stage on-
ward (Fig. 2F). By the end of embryogenesis and throughout
the larval stages all of the tracheal cells showed a strong re-
sponse to hypoxia (Fig. 2G, lower panel). To answer the ques-
tion of whether enhanced hypoxia responsiveness in the
tracheal system reflects some microenvironmental signal con-
nected with the position or integrity of the developing tracheal
system, we studied hypoxic induction of the reporter in em-
bryos homozygous for a mutation in the gene breathless (btl)
that encodes a Drosophila FGF receptor, which is required for
tracheal cell migration (28). As is shown in Fig. 2H and I,
tracheal cells fail to migrate in btlMZ13mutants, and the tra-
cheal system is not developed. In these mutants, however, the
nonmigrating tracheal cells expressed the hypoxic reporter
normally, clearly indicating that tracheal integrity is not re-
quired for the hypoxic response.
Enhanced hypoxia-inducible transcription in tracheal cells
is of interest since the tracheal system is directly responsible
for oxygen delivery in the fly. Nevertheless, current models of
oxygen-dependent tracheal plasticity involve a receptor-medi-
ated chemotactic outgrowth of terminal branches that is gen-
erated by hypoxia-inducible expression of the Drosophila FGF
homologue Branchless in extratracheal metabolizing tissues
(24). Therefore, it would be predicted that the hypoxic ma-
chinery should operate in nontracheal tissues as well, although
perhaps with different sensitivity. To pursue this, we applied
more severe hypoxic conditions (4% oxygen for 16 h) to em-
bryos bearing the hypoxic reporter and recorded the GFP
expression. Under these conditions, hypoxia-inducible expres-
sion was clearly observed outside the tracheae, with approxi-
mately one-quarter of late embryos or larvae manifesting wide-
spread reporter expression across the ectoderm, esophagus,
gut, fat body, muscles, and gonads (Fig. 2J and K), as well as
the trachea. However, tracheal expression remained dominant,
and responses in extratracheal cells were more patchy in the
majority of embryos. When embryos were placed at an oxygen
concentration of 3% or less, arrest of embryonic development
occurred, and tissue-specific expression of the reporter could
not be recorded. Taken together with the analysis of responses
to graded hypoxia in LDH-LacZ flies, this suggests that most if
not all tissues in the developing fly can respond to this system,
although in many nontracheal tissues this appears to be at a
threshold that is close to that which produces metabolic and
developmental arrest. In contrast, the tracheal system appears
to respond with greater sensitivity. Tracking inducible expres-
sion precisely in adult flies was more difficult because of cuticle
development. Nevertheless, strong hypoxia-inducible expres-
sion was evident in the legs (Fig. 2L).
The bHLH-PAS proteins Similar and Tango mediate the
hypoxic response. The experiments described above estab-
lished that the HIF response is conserved and strongly active in
developing flies, suggesting that genetic studies in Drosophila
may be used to explore the upstream and downstream connec-
tions of the pathway in an in vivo system.
As a first step in understanding the upstream pathway we
wished to define the role of particular Drosophila bHLH-PAS
proteins in the pathway. The Drosophila bHLH-PAS protein
Tango (Tgo) is a partner for several bHLH-PAS proteins and
has been proposed to be homologous to the mammalian pro-
tein ARNT (for aryl hydrocarbon receptor nuclear transloca-
tor) (46, 56) that functions as the HIF-? subunit. To test this
proposed role for Tgo in the hypoxic response, we examined
embryos that were homozygous for a strong tgo mutant allele
(tgo5). These embryos failed to induce the reporter in hypoxia
(Fig. 3A and B), strongly supporting the role of Tgo as the
VOL. 22, 2002 DROSOPHILA TRANSCRIPTIONAL RESPONSE TO HYPOXIA 6845
FIG. 2. Spatially restricted induction of the transcriptional response to hypoxia. The expression pattern of LDH-LacZ (A to C) and LDH-Gal4
(D to L) reporters was examined in transgenic embryos or larvae exposed to hypoxia. (A) X-Gal staining of a stage 15 LDH-LacZ embryo subjected
to 5% O2for 4 h. Some branches of the tracheal system (arrows) are stained. (B) Double immunostaining showing the tracheal lumen in brown
(arrows) and ?-Gal expression in blue (arrowheads). The reporter is expressed at some branches of the tracheal system. (C) In normoxia, no expression
of the reporter is seen. (D to F) An unsynchronized population of embryos was exposed to 5% oxygen for 4 h and induction of the reporter was analyzed.
Double immunofluorescent confocal image shows that the ?-Gal reporter (green) colocalizes with the tracheal marker Trachealess (red). Note that some
extratracheal cells express the reporter as well. Expression of the reporter cannot be detected at embryonic stage 11 (see panel D). By stage 13 (panel
E), scattered tracheal cells (arrowheads) begin to express the reporter, with further cells responding to hypoxia at stage 14 (arrowheads) (F). (G) By the
end of embryogenesis and throughout larval stages, the LDH-Gal4/UAS-TAU.GFP reporter is expressed in the whole tracheal system upon hypoxia
(arrowheads, lower panel), but in normoxia no expression is seen in the tracheae (upper panel), although expression of the reporter can be detected in
salivary glands (arrows). (H and I) In breathlessMZ13homozygous embryos tracheal cells (red) fail to migrate (arrowhead), but the hypoxic reporter
(UAS-nGFP.LacZ) is induced normally (arrows) in hypoxia. (I) Higher magnification of panel H. (J and K) First-instar larvae subjected to 4% O2for
16 h express the UAS-nGFP reporter in nontracheal tissues, as well as in the esophagus (e), gut (g), ectoderm (ec), fat body (fb), and tracheae (t).
Expression in the salivary glands (sg) is constitutive. (L) Hypoxic induction of the UAS-nGFP reporter is seen in the legs of adult flies maintained
at 5% O2for 24 h (lower panel) compared with normoxia (upper panel). (M) Schematic representation of the LDH-Gal4 transcriptional reporter.
A dimerized 51-bp fragment of the murine LDH-A enhancer including the HREs and CRE was constructed as a dimer controlling expression of Gal4.
HIF-? subunit and indicating that, as in mammalian cells, this
protein is absolutely required for the hypoxia response.
We next sought to identify the Drosophila HIF-? homo-
logue(s). Several bHLH-PAS proteins exist in the fruitfly (8, 9),
but the best candidate HIF-? homologue is Similar (Sima)
since it shows the highest amino acid identity (39), and protein
levels are increased at 1% O2in Drosophila SL2 cells (1).
Nevertheless, two other Drosophila bHLH-PAS proteins, Sin-
gle minded (Sim), a regulator of differentiation of the embry-
onic central nervous system midline cells (7), and Trachealess
(Trh), a key regulator of tracheal development (20, 53), show
high identity with HIF-1? in the basic and HLH domains and
were also candidates for mediation of the hypoxic transcrip-
tional response. To examine for a role of Sim or Trh in hy-
poxia-dependent transcription, we crossed the LDH-Gal4/
UAS-GFPn.LacZ reporter into flies bearing a sim strong
mutant allele (simH9) or a chromosomal deficiency that in-
cludes the trh gene and then studied expression of the reporter
in homozygous mutant embryos. Homozygous embryos for the
Df(3L)emc-E12 chromosomal deficiency that includes the trh
gene do not develop a tracheal system and are viable until late
embryogenesis (20). After these embryos were challenged with
severe hypoxia, strong expression of the reporter was observed,
indicating that trh is not required for the hypoxic response (not
shown). Similarly, homozygous simH9embryos subjected to
moderate hypoxia (5% oxygen for 4 h) exhibited normal ex-
pression of the hypoxic reporter, showing that Single minded is
also dispensable for the response (not shown).
FIG. 3. Role of bHLH-PAS proteins in the transcriptional response to hypoxia. (A) Wild-type expression of the ?-Gal reporter in the tracheal
system of LDH-Gal4/UAS-nGFP.LacZ transgenic embryos subjected to hypoxia (arrowheads). (B) Absence of reporter induction in tango5
homozygous embryos subjected to hypoxia. Only constitutive reporter expression is seen in the salivary glands (sg) and cells of the gut (arrow).
Typical tracheal defects are observed in tango mutants (B, arrowheads). (C and D) Sima cannot be detected in normoxic wild-type embryos (C) but
is induced upon hypoxia (5% O2for 14 h) in a pattern that follows the tracheal tree (D). (E) Western blot analysis of lysates of embryos bearing
the LDH-Gal4/UAS-GFP.LacZ reporter subjected to hypoxia (5% O2for 14 h) or maintained in normoxia showing hypoxic induction of both Sima
and LDH-Gal4/UAS-GFP.LacZ. (F) Slot blot analysis of RNA extracted from hypoxic or normoxic embryos treated as in panel E. ?-Gal mRNA
exhibited strong hypoxic induction (9.7- to 10.4-fold), whereas Sima mRNA was induced only slightly (1.3- to 1.5-fold).
VOL. 22, 2002 DROSOPHILA TRANSCRIPTIONAL RESPONSE TO HYPOXIA 6847
To investigate the role of Sima in the transcriptional re-
sponse to hypoxia, we performed immunofluorescent detection
of Sima in embryos subjected to hypoxia (5% oxygen for 14 h).
By performing the fixation immediately (0.5 to 1 min) after
opening the hypoxic chamber, strong Sima staining could be
detected in stage 15 to 16 embryos subjected to hypoxia, but
not in normoxic controls, in a pattern of expression corre-
sponding to the tracheae that strikingly mimics expression of
the reporter in hypoxia (Fig. 3C and D). To pursue this further,
synchronized stage 8 wild-type embryos bearing the LDH-
Gal4/UAS-GFP.LacZ reporter were kept in hypoxia as de-
scribed above, and protein extracts were analyzed by Western
blotting with anti-Sima and anti-?-Gal antisera (1). As shown
in Fig. 3E, Sima protein levels are increased in hypoxia, par-
alleling induction of the ?-Gal reporter. However, Sima
mRNA increased only slightly in hypoxia (1.3- to 1.5-fold),
whereas ?-Gal mRNA was upregulated by 9.7- to 10.4-fold
(Fig. 3F). These results indicate that Sima is controlled post-
transcriptionally, most probably at the level of protein degra-
dation. To test this, we overexpressed the protein in hs-Gal4/
UAS-Sima embryos by giving a 37°C heat shock for 20 min and
compared the decay of Sima protein levels in hypoxia and
normoxia. As depicted in Fig. 4B and E, Sima levels 4 h after
heat shock are much higher in hypoxia than in normoxia. At
16 h after heat shock, expression of Sima can still be visualized
in patches of cells in hypoxic embryos (Fig. 4F) but is unde-
tectable in embryos kept in normoxia (Fig. 4C), thus confirm-
ing that Sima is stabilized in hypoxia.
Proteolytic control of mammalian HIF-? is dependent on a
central oxygen-dependent degradation domain (ODDD) (19,
40) that contains two sites of oxygen-dependent prolyl hydroxy-
lation (34). Although overall conservation between HIF-1?
and Sima outside the basic and HLH domains is low (1, 39),
sequence comparison indicates that these prolyl residues are
both conserved (Sima Pro747 and Pro850), suggesting that
they might define a Sima ODDD. To test this, we generated a
deletion of Sima between amino acids 692 and 863 (Sima?692-
863) and expressed the deleted Sima in UAS transgenic lines
through a heat shock-Gal4 driver as described above for full-
length Sima. As shown in Fig. 4G to I, this deletion stabilized
Sima in normoxia with no further changes in stability when
heat-shocked embryos were incubated in hypoxia (Fig. 4J to
L). These findings demonstrated that Sima is regulated by
oxygen-dependent proteolysis in a manner similar to mamma-
FIG. 4. Sima protein is stabilized in hypoxia: identification of the ODDD. Ectopic Sima was expressed by using a heat shock-Gal4 driver (A
to F) and detected with an anti-Sima antibody after a 20-min period of heat shock in embryos kept in normoxia (N) or hypoxia (5% O2) (H). At
2 h after heat shock, moderate levels of Sima are detected, but there are no differences between normoxia (A) and hypoxia (D). At 4 h after heat
shock, Sima can no longer be detected in normoxia (B), but levels in hypoxia are high (E). At 16 h after heat shock the protein can still be detected
in patches of cells in the hypoxic embryos (F, arrows) but not in the normoxic controls (C). (G to L) The ?692-863 deletion of Sima generated
a stable protein. At 2 (G and J), 4 (H and K), or 16 h (I and L) after heat shock, Sima?692-863 was detected both in normoxic (G to I) and hypoxic
(J to L) embryos, showing that the instability of Sima in normoxia depends on the deleted sequences.
6848 LAVISTA-LLANOS ET AL.MOL. CELL. BIOL.
Oxygen-dependent subcellular localization of Sima. To
override the rate of Sima degradation and shed light on an
additional mode of regulation, we sought to overexpress the
protein by using an engrailed-Gal4 (en-Gal4) driver. The en-
Gal4 element was recombined in a chromosome bearing a
UAS-nGFP-LacZ element (with a nuclear localization signal)
(42) that was used to mark the nuclei. Sima protein, expressed
in the characteristic striped engrailed pattern, was easily de-
tected by immunofluorescence in stages 11 to 15 normoxic
embryos. Surprisingly, however, we found that the protein was
almost exclusively localized in the cytoplasm (Fig. 5A to C). In
another experiment, Sima was expressed in embryos bearing
the direct LDH-LacZ hypoxic reporter (lacking the UAS-
nGFP-LacZ element). In normoxia, the reporter was induced
only at very low levels (Fig. 5D). In contrast, when the same
experiments were performed with embryos exposed to 5%
FIG. 5. Regulation of Sima subcellular localization by hypoxia. Sima (A and E) or Sima?692-863 (I and M) were expressed ectopically through
an en-Gal4 driver, and subcellular localization was detected with an anti-Sima antibody. (B, F, J, and N) The embryos also contained a
UAS-nGFP.LacZ reporter (bearing a nuclear localization signal) detected with an anti-?-Gal antibody that was used to mark the nuclei. In
normoxia, Sima was localized exclusively in the cytoplasm (A to C) and became nuclear in hypoxia (5% O2for 8 h) (E to G). Sima?692-863 was
constitutively detected in the nuclei irrespective of oxygen levels (I to K and M to O). (D, H, L, and P) Sima or Sima?692-863 ectopic expression
driven by en-Gal4 was also performed in embryos bearing the direct LDH-LacZ hypoxic reporter and lacking the UAS-nGFP.LacZ element. Upon
ectopic expression of Sima, the LDH-LacZ reporter was strongly induced in hypoxia (H) but only weakly in normoxia (D), a finding consistent with
the subcellular localization of Sima. Expression of Sima?692-863 produced strong expression of the reporter irrespective of oxygen levels (L and
VOL. 22, 2002DROSOPHILA TRANSCRIPTIONAL RESPONSE TO HYPOXIA6849
oxygen, Sima was detected predominantly in the nucleus (Fig.
5E to G), and embryos exhibited strong expression of the re-
porter in the expected engrailed pattern (Fig. 5H). In contrast,
expression of Trachealess or a chimeric protein exhibiting
specificity for Single minded target genes (56) under en-Gal4
did not activate the reporter in either normoxia or hypoxia
(data not shown).
This confirmed that Sima mediates activity of the HRE re-
porter but also indicated the existence of an additional oxygen-
regulated nuclear localization mechanism, as has been pro-
posed in mammalian cells (26, 31). Studies of mammalian
HIF-? have demonstrated that stabilization by deletion of the
ODDD is associated with constitutive upregulation of tran-
scriptional activity (12, 19). Therefore, we reasoned that the
deletion might also affect subcellular localization. The Dro-
sophila system provided an opportunity to compare the local-
ization of Sima?692-863 with full-length Sima under similar
expression conditions. Accordingly, Sima?692-863 was ex-
pressed similarly in embryos through the en-Gal4 driver. As
depicted in Fig. 5I to K and M to O, deletion of the ODDD
caused constitutive localization of Sima in the nucleus irrespec-
tive of oxygen levels. Consistent with this, the LDH-LacZ tran-
scriptional reporter showed strong constitutive expression in
the expected engrailed pattern after crossing into embryos
expressing Sima?692-863 (Fig. 5L and P). These results indi-
cate that the Sima ODDD also mediates signals critically re-
quired for cytoplasmic localization of Sima in normoxia (see
Role of the prolyl 4-hydroxylase homologue CG1114 in the
hypoxic response in vivo. A Drosophila sequence homologous
to the HIF prolyl hydroxylases was recently reported (gene
CG1114) (4, 48). These enzymes are known to promote pro-
teasomal destruction of HIF-? through the hydroxylation of
key prolyl residues in the ODDD. To assess whether CG1114
regulates Sima levels and the transcriptional response to hyp-
oxia in vivo, we abrogated CG1114 expression by injecting
double-stranded RNA into early embryos bearing the LDH-
Gal4/UAS-nGFP.LacZ reporter gene. As depicted in Fig. 6A,
Sima protein was strongly upregulated in normoxic embryos
injected with CG1114 RNAi but not in individuals injected
with an unrelated double-stranded RNA (Fig. 6B). Sima up-
regulation (Fig. 6A and C) induced expression of the LDH-
Gal4/UAS-nGFP.LacZ nuclear hypoxic reporter (Fig. 6D).
Furthermore, complete overlap between Sima protein and the
nuclear reporter product demonstrated that Sima was exclu-
sively localized in the nucleus (Fig. 6C to E). To further assess
the role of CG1114 in the regulation of Sima, we examined
flies that bore mutations predicted to inactivate this gene com-
pletely. A lethal P element insertional mutation [l(3)02255],
mapping 336 nucleotides upstream of the initiation codon of
the CG1114 gene, is available from Drosophila public stock
collections. By crossing l(3)02255 heterozygous mutant flies
with a strain carrying the Df(3R)3-4 chromosomal deficiency
that covers CG1114 gene, we verified that the insertion was
indeed causing the lethal phenotype. We analyzed Sima levels
and induction of the transcriptional response to hypoxia in
l(3)02255 embryos bearing the LDH-Gal4/UAS-nGFP.LacZ
reporter. As depicted in Fig. 6F, Sima is ubiquitously upregu-
lated in homozygous mutant embryos in normoxia, and high
levels of Sima result in concomitant strong induction of the
LDH-Gal4/UAS-nGFP.LacZ reporter in all embryonic tissues
Ectopic stabilization of Sima could potentially exert a dom-
inant-negative effect on Trachealess or other bHLH-PAS pro-
teins. To investigate a possible effect on Trachealess, the em-
bryonic tracheal system was studied with a specific antitracheal
lumen antibody, but no obvious defects were observed (not
shown). Taken together, these results imply a general and
nonredundant role of CG1114 in the regulation of both sub-
cellular localization and protein stability of Sima. In support of
FIG. 6. Role of the prolyl 4-hydroxylase homologue CG1114 in the
hypoxic response in vivo. (A and B) CG1114 expression was abrogated
by injecting double-stranded RNA; Sima protein detected with a spe-
cific antiserum was strongly upregulated (A) but not when injected
with an unrelated double-stranded RNA (B). (C and D) Higher mag-
nification of an embryo injected with CG1114 RNAi revealing Sima
stabilization (C) and concomitant expression of the nuclear hypoxic
reporter LDH-Gal4/UAS-nGFP.LacZ detected with an anti-?Gal an-
tibody (D). (E) The merged confocal image shows that Sima is local-
ized in the nucleus. (F) Sima is upregulated ubiquitously in homozy-
gous mutant l(3)02255 embryos and is localized in the nuclei. (G) These
high levels of Sima result in strong, widespread induction of the LDH-
Gal4/UAS-nGFP.LacZ reporter. (H) Expression of CG1114 mRNA in
embryos in normoxia. (I) Control antisense probe. (J) CG1114 gene
expression was strongly upregulated in hypoxia (compare with panel
H). (K) Ectopic expression of Sima under control of en-Gal4 induces
CG1114 mRNA expression in the typical engrailed pattern.
6850 LAVISTA-LLANOS ET AL.MOL. CELL. BIOL.
this, we found that CG1114 mRNA is ubiquitously expressed
throughout embryogenesis (Fig. 6H and I).
Mammalian HIF prolyl hydroxylase genes are themselves
induced by hypoxia (12), suggesting the existence of a feedback
response that limits HIF induction in hypoxia. To test whether
this aspect of regulation is also conserved in Drosophila and
does indeed represent a feedback response dependent on ac-
tivity in the transcriptional pathway itself, we tested for induc-
tion by both hypoxia and Sima overexpression. As shown in
Fig. 6J, CG1114 gene expression was strongly upregulated in
hypoxia (compare with Fig. 6H) and was strongly induced in
the typical engrailed pattern by overexpression of Sima by
using an en-Gal4 driver (Fig. 6K). In contrast, the branchless/
FGF gene was not induced in embryos exposed to hypoxia or
overexpressing Sima (not shown), suggesting that the hypoxic
regulation of branchless (24) starts at larval stages.
In this work we have used transgenic flies to demonstrate
and characterize in vivo the operation of a hypoxia inducible
transcription response in Drosophila that is homologous to
mammalian HIF. The work confirms the candidacy of Sima
and Tgo as the Drosophila homologues of mammalian HIF-?
and HIF-?, respectively, defines a conserved multistep mode
of regulation for Sima and provides new insights into the
mechanisms regulating HIF proteins, as well as into the spatial
and temporal operation of the hypoxia-responsive system dur-
ing Drosophila development.
By tracking reporter gene activation in developing flies, we
analyzed the oxygen concentration dependence, developmen-
tal regulation, and spatial distribution of the transcriptional
response. Serial studies of the hypoxia response during devel-
opment indicated that induction by hypoxia is modest in early
embryogenesis and mid-embryogenesis and then rises sharply
to peak levels at the end of embryogenesis, thereafter remain-
ing relatively high throughout the larval stages. This develop-
mentally restricted capacity fits well with the adaptive require-
ments of Drosophila larvae. After eclosion larvae usually dig
into the substrate, while feeding actively, and are probably
subjected to major variations in environmental oxygen tension
so that enhanced activity of the HIF system is likely to be of
critical importance at this stage.
Interestingly and somewhat unexpectedly, analysis of re-
porter expression patterns in developing flies showed en-
hanced hypoxia-inducible activity in the cells of the tracheal
system. Although experiments using severe hypoxia and ge-
netic inactivation of Sima proteolysis demonstrated a wide-
spread potential for transcriptional activation by this system,
exposure to more moderate hypoxia clearly demonstrated en-
hanced activity in tracheal cells. This was reflected both in
higher expression levels of Sima and in higher activity of dif-
ferent HRE-linked reporter genes and, moreover, was shown
to be a cell autonomous function that was preserved in cells of
tracheal fate even in the face of mutations that disrupt tracheal
The existence of enhanced responses to hypoxia in cells
composing the organ of oxygen delivery is clearly of interest
and raises questions as to its function, particularly since cur-
rent models indicate that the regulation of tracheal develop-
ment by oxygen is guided by signals arising in the metabolizing
tissues outside the tracheae (24). Interestingly, some of the
branches of the tracheal system run alongside the Drosophila
nervous system (14), and one possibility is that the tracheae
function as sensory organs for hypoxia, as the carotid body
does in mammals. A hypoxia pathway affecting behavioral re-
sponses has been described in flies (54), and it will be inter-
esting to determine whether hypoxia-induced behavioral re-
sponses share a regulatory mechanism with the HIF system.
In the current work we also utilized the HRE transgenic
reporter system to define upstream control mechanisms oper-
ating on the Drosophila HIF system. Our studies identify Sima
as the regulatory Drosophila HIF subunit and demonstrate a
major mode of regulation through oxygen-dependent proteol-
ysis that involves a central ODDD. Interestingly, both of the
sites of prolyl hydroxylation that operate in mammalian HIF-?
subunit ODDD (34) appear to be conserved in Sima. Further-
more, genetic ablation of the Drosophila HIF prolyl hydroxy-
lase homologue CG1114 results in striking upregulation of
both Sima and reporter gene activity in vivo. This strongly
supports a conserved mode of proteolytic regulation of Sima
following prolyl hydroxylation at one or both of these sites.
In contrast with the mammalian system, where HIF prolyl
hydroxylase activity is represented by the three PHD isoforms
(4, 15), survey of the Drosophila genome revealed only one
homologue (48), raising questions about the potential of this
activity to regulate precisely tuned physiological responses.
Interestingly, however, we found that the CG1114 gene is itself
a Sima target, demonstrating the operation of a conserved
feedback control with the potential to contribute to the com-
plex demands of physiological oxygen homeostasis.
Studies of Sima regulation also demonstrated an additional
regulatory step. Transgenic overexpression of Sima in nor-
moxic embryos resulted in cytoplasmic accumulation of the
protein and little transcriptional activity. In contrast, similar
levels of overexpression in hypoxia resulted in nuclear accu-
mulation and a strong transcriptional response, demonstrating
the presence of a second oxygen-regulated mechanism control-
ling Sima subcellular localization. An oxygen-regulated nuclear
localization step has previously been demonstrated for mam-
malian HIF-? (2, 26, 31), although not in every study. Our
demonstration of conservation of this mode of regulation in
Drosophila Sima, however, provides strong support for the
physiological relevance of this process. Our findings suggest
that Sima subcellular localization is controlled by an active
mechanism that maintains the protein in the cytoplasm in
normoxia as opposed to an hypoxia-dependent machinery that
mediates nuclear import. Although the strong transcriptional
activity of mammalian HIF-? that is observed after deletion of
the ODDD (12, 19), mutation of the VHL binding sites (34), or
inactivation of VHL (36) is consistent with a role for this
domain in cytoplasmic localization in normoxia, this has not
been tested in studies of mammalian HIF-? that have exam-
ined subcellular localization directly. Moreover, although in-
duction of nuclear localization by iron chelators and cobaltous
ions (26) suggests a similar mode of regulation to proteolytic
regulation, neither the source of the oxygen-sensitive signal
nor the mechanism of transduction have been defined. Our in
vivo studies in flies show induction of nuclear Sima after inac-
tivation of CG1114 either by RNAi or by mutation, thus clearly
VOL. 22, 2002 DROSOPHILA TRANSCRIPTIONAL RESPONSE TO HYPOXIA6851
implicating this gene product in the process of cytoplasmic
localization in normoxia. Moreover, Sima nuclear localization
was also observed in flies bearing the Sima?692-863 transgene,
indicating that this sequence is absolutely required for cyto-
Very recently nonproteolytic regulation of mammalian
HIF-? subunits involving the C-terminal transactivation do-
mains has been shown to be regulated by hydroxylation of a
specific asparaginyl residue by an enzymatic activity that, like
the prolyl hydroxylases involved in HIF proteolysis, demon-
strates the properties of an ?-ketoglutarate-dependent dioxy-
genase (30). Thus, regulatory hydroxylation of HIF-? residues
by this class of enzyme appears to extend to both specific
asparaginyl and prolyl residues. Currently, the precise sub-
strate requirements of the CG1114 gene product are not de-
fined, and it is not clear whether effects on nuclear localization
are mediated through the conserved prolyl residues, possibly
reflecting additional functions of the VHL ubiquitylation com-
plex, or whether other sequences within the Sima ODDD me-
diate this process. Further biochemical and genetic studies
should clarify these new insights into the HIF system.
Overall, the high degree of conservation in the Drosophila
system indicates that genetic studies in this organism should be
highly informative in analyses of both the upstream pathways
regulating the HIF system, and the downstream physiological
effects in an intact organism.
We thank Andrea Brand, Steve Crews, Shigeo Hayashi, Luis Que-
sada-Allue ´, Ricardo Ramos, Benny Shilo, Carl Thummel, the Bloom-
ington Stocks Center, and the Developmental Studies Hybridoma
Bank for plasmids, antibodies, and fly stocks. We thank Benny Shilo
and Osvaldo Podhajcer for critical reading of the manuscript.
This work was supported by the Wellcome Trust grant HHBR6 and
Fundacio ´n Antorchas grant A-13740/1-119 to P.W. and P.J.R. S.L.-L.
and D.M.R. are fellows of the Argentinean National Council of Sci-
entific Research (CONICET), S.N.B. received a postdoctoral fellow-
ship from The Wellcome Trust, L.C. is a fellow of the FONCyT, M.I.
and M.M. have FOMEC fellowships, and P.W. is a career investigator
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