The Journal of Experimental Biology 200, 2007–2015 (1997) 2007

Printed in Great Britain © The Company of Biologists Limited 1997

JEB0867

TISSUE-SPECIFIC VARIATION IN Hsp70 EXPRESSION AND THERMAL DAMAGE IN

DROSOPHILA MELANOGASTER LARVAE
ROBERT A. KREBS1,* AND MARTIN E. FEDER1,2
1Department of Organismal Biology and Anatomy and 2The Committee on Evolutionary Biology, The University of
Chicago, 1027 East 57th Street, Chicago, IL 60637, USA

Accepted 1 May 1997

Summary
All tissues of larval Drosophila melanogaster express
Hsp70, the major heat-shock protein of this species, after
both mild (36 °C) and severe (38.5 °C) heat shock. We used
Hsp70-specific immunofluorescence to compare the rate
and intensity of Hsp70 expression in various tissues after
these two heat-shock treatments, and to compare this with
related differences in the intensity of Trypan Blue staining
shown by the tissues. Trypan Blue is a marker of tissue
damage. Hsp70 was rarely detectable before heat shock.
Brain, salivary glands, imaginal disks and hindgut
expressed Hsp70 within the first hour of heat shock,
whereas gut tissues, fat body and Malpighian tubules did
not express Hsp70 until 4–21 h after heat shock. Differences
in Hsp70 expression between tissues were more
pronounced at the higher heat-shock temperature. Tissues
that expressed Hsp70 slowly stained most intensely with
Trypan Blue. Gut stained especially intensely, which
suggests that its sensitivity to heat shock may limit larval
thermotolerance. These patterns further suggest that some
cells respond primarily to damage caused by heat shock
rather than to elevated temperature per se and/or that
Hsp70 expression is itself damaged by heat and requires
time for recovery in some tissues.
Key words: heat-shock proteins, stress, thermotolerance, vital dyes,
Hsp70, Drosophila melanogaster.

Introduction
In response to heat and other stresses, nearly all organisms
express heat-shock proteins (Hsps), which promote stress
tolerance by functioning as molecular chaperones (Lindquist,
1993). Recent years have witnessed enormous progress both in
the elucidation of chaperone function at the biochemical level
and in the demonstration that heat-shock proteins are
responsible for a large component of organismal
thermotolerance (Morimoto et al. 1994). Progress has not been
as rapid, however, in establishing how the activities of Hsps at
the cellular level enhance the thermotolerance of the individual
(Hartl, 1996). At a more descriptive level, the way in which
tissue-specific expression of Hsps is temporally and
quantitatively related to the thermotolerance of the various
tissues and the patterns of cell damage that ensue during and
after heat shock are poorly understood. Accordingly, we have
examined tissue-specific patterns of Hsp70 expression and cell
damage in Drosophila melanogaster, the fruit fly. In D.
melanogaster, Hsp70 is the primary inducible heat-shock
protein (Lindquist, 1981) and is the product of 10–12 nearly
identical genes at the 87A and 87C loci (Ish-Horowicz et al.
1979a,b). This protein is not expressed before stress and is very
tightly autoregulated as Hsp70 concentrations increase during
stress and/or recovery from stress (Lindquist, 1993).
Although Hsps are best known for their inducibility by heat,
the presence of non-native proteins within cells is sufficient to
induce their expression (Parsell and Lindquist, 1994). Hsp70
expression, which can be detected immunologically, may
therefore be symptomatic both of a direct response to high
temperatures and of damage to cells and tissues by high-
temperature stress (Hofmann and Somero, 1995). To examine
tissue damage directly, we stained with Trypan Blue, a dye that
is excluded from intact cells but is rapidly absorbed by dead
or dying cells. Hsp70 expression and Trypan Blue staining may
pinpoint thermosensitive regions within an organism. We
therefore examined the order in which tissues first expressed
Hsp70 during or after heat shock, and related this to Trypan
Blue staining. We performed this experiment in larvae, the life
stage of D. melanogaster most likely to experience lethal heat
stress in nature (Feder, 1996; Feder et al. 1997).
Much of the extensive literature on Hsps in D. melanogaster
and other insects has little bearing on the tissue specificity of
Hsp expression and heat damage because it combines results
for several different Hsps and/or tissues without distinguishing
among them. Studies of whole D. melanogaster and cells in
culture, which have been the most frequently used subjects of
previous work, are not relevant to tissue-specific variation. All

*e-mail: r-krebs@uchicago.edu.
2008 R. A. KREBS AND M. E. FEDER
or most individual tissues of D. melanogaster (Tissiéres et al.
1974; Mitchell et al. 1979) and other dipterans (Nath and
Lakhotia, 1989; Joplin and Denlinger, 1990; Tiwari et al.
1995) clearly increase their expression of unspecified members
of the major families of Hsps upon heat shock. Seldom,
however, have the individual Hsps been identified [but see
Palter et al. (1986) and Singh and Lakhotia (1995)] or have the
tissues under study been systematically exposed to heat shocks
of graded severity.
Materials and methods
We analyzed Hsp70 expression and tissue damage in the
Chromosome II excision strain of Drosophila melanogaster
(Welte et al. 1993). This strain, which was a control in some
previous analyses of the effects of hsp70 copy number on
thermotolerance, growth and Hsp70 expression (e.g. Krebs and
Feder, 1997), contains a second-chromosome P-element
insertion but expresses Hsp70 normally. Eggs were collected
from, and larvae reared on, yeast–cornmeal–molasses–agar
medium sprinkled with live yeast. Third-instar larvae (6–7
days post-laying) were separated from the medium in 3 mol l−1
NaCl (Ashburner, 1989) and transferred to 5 cm Petri dishes
containing medium. This procedure does not affect Hsp70
concentration.
To characterize the thermal sensitivity of Hsp70 expression
in entire larvae, third-instar larvae were exposed to constant
temperatures between 33 and 40 °C for 1 h, followed by 1 h at
25 °C, as a recovery period. Other larvae were treated at
38.5 °C for 1 h, and then placed at 25 °C for variable periods.
larvae that had not been treated with primary antibody. Then,
Hoechst 33258 dye was added (10 µl ml−1 of a 1 mg ml−1 stock
solution to secondary antibody) for coincident staining of
nuclei. Samples were incubated with secondary antibody for
1 h, after which they were rinsed three times with PBS and
washed for 30 min in PBST after addition of each antibody.
Tissues were transferred to a glass slide and mounted in
several drops of 1 mg ml−1 p-phenylenediamine in 70 %
glycerol. Slides were examined with a Zeiss fluorescent
microscope and photographed (2.5× camera lens) with 100
ASA Ektachrome film, using exposures of 5 s with a 10×
objective and 40 s with a 4× objective. Comparisons of
expression levels were possible for photographs taken with the
same objective. All slides were scanned as Adobe Photoshop
documents (Adobe Systems, Inc.), with assembly and text
additions in PowerPoint (Microsoft Corp.).
Trypan Blue staining
Larvae were dissected as for Hsp70 analysis, a step requiring
less than 10 min, immersed in 0.2 mg ml−1 Trypan Blue in PBS,
and rotated for 30 min at room temperature to bring internal
tissues into contact with dye. Groups of three larvae were then
rinsed three times in PBS, washed for 30 min in PBS, and each
group was immediately scored for Trypan Blue staining of
tissues and cells. Scoring for these groups of larvae was based
100
A
90
80 8
70
Hsp70 expression (as percentage of standard)

Whole-body Hsp70 concentration was then determined by 60 5

enzyme-linked immunosorbent assay (ELISA) and is presented 50
relative to a standard concentration, that produced by 40
Drosophila S2 cells treated for 1 h at 36.5 °C and 1 h at 25 °C 30 8
(Welte et al. 1993; Feder et al. 1996). 20
10 4 8 5
1 1
Fluorescent staining 100
0
Larvae were immersed in PBS (phosphate-buffered saline), 90 32 33 34 35 36 37 38 39 40
and cuticle and muscle tissue were peeled from the body 80 Temperature (°C)
cavity. Larvae were then placed in 4 g l−1 paraformaldehyde 70 B
(Fisher, T-353) in PBS (pH 7.3) and rotated for 30 min at room 60
temperature (23 °C), rinsed three times with PBS, washed with 50 3
rotation in PBST [5 g l−1 bovine serum albumin (Sigma A- 40
6793), 5 g l−1 Triton X-100 (Sigma T-6878) in 1× PBS] for 30 4
30 min to permeabilize cells, and stored overnight at 4 °C in 20
fresh PBST. 10
3
Tissues were incubated for 1 h in 1:1000 anti-Hsp70 rat 0
monoclonal antibody, 7FB, which is specific for the 0 4 8 12 16 20 24
Drosophila melanogaster heat-inducible Hsp70 family Recovery time at 25 °C (h)
member (Velazquez and Lindquist, 1984). The secondary
Fig. 1. Expression of Hsp70 in whole-body lysates of Drosophila
antibody was FITC-conjugated goat anti-rat IgG, affinity- melanogaster larvae relative to a standard, the concentration in
purified F(ab′)2 fragments (Cappel 55747) prepared according Drosophila S2 cells after 1 h at 36.5 °C and 1 h at 25 °C. (A) Hsp70
to the instructions of the manufacturer and diluted 1:300 in level determined by ELISA after 1 h at the indicated temperature
PBST. To remove any nonspecifically binding components of followed by 1 h at 25 °C; (B) Hsp70 concentration after 1 h at 38.5 °C
the secondary antibody, each 1 ml of this dilution was and at the indicated time at 25 °C. Values are means ± S.E.M.; values
incubated at room temperature for 1 h with 3–5 heat-shocked of N are given beside the data points.

on an average composite index per larva: no color, 0; any blue,
1; darkly stained nuclei, 2; large patches of darkly stained cells,
3; or complete staining of most cells in the tissue, 4. As these
data are sequential categories, differences due to treatment
effects (control versus heat shock) and due to recovery time
(immediately after heat shock versus 21 h after heat shock)
were tested by Mann–Whitney U-tests. For presentation,
images were taken with tissues in PBS and recorded on a Wild
microscope with a direct computer feed. Staining for Hsp70
and Trypan Blue was not possible in the same larva, as Trypan
Blue leaches from the sample after fixation.
Results
In whole larvae, Hsp70 was undetectable in the absence of
stress. Hsp70 level varies with temperature in third-instar
larvae (Fig. 1A, F7,32=28.6, P<0.001), and the concentration
of Hsp70 was greater after a 1 h exposure to 36 °C than at any
other temperature tested. After 1 h of heat shock at
temperatures lower than 36 °C, the concentration of Hsp70
declined rapidly (R. Krebs, unpublished results). In contrast, a
1 h exposure to 38.5 °C caused the concentration of Hsp70 to
rise for many hours afterwards (Fig. 1B). The level of Hsp70
4 h after exposure to 38.5 °C for 1 h was significantly greater
A id
fb
gc
pv
sg
mg
gc
br
C mg
mt
hg
Fig. 2. Hsp70 expression in larval tissues after a 1 h exposure to 36 °C and either (A,C) no recovery or (B,D) 4 h of recovery at 25 °C. A and
B are for anterior tissues; C and D are for posterior tissues: sg, salivary glands; fb, fat body; id, imaginal disks, gc, gastric caeca; pv,
proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules. Expression is identified by fluorescent staining with anti-Hsp70
monoclonal antibody. At the presented level of photographic exposure, images of individuals at 25 °C are indistinguishable from background,
representing undetectable expression in all tissues. Scale bars, 500 µm.
Hsp70 expression and tissue damage 2009
than the level immediately after the heat shock, and that after
21 h was significantly higher than that after 4 h (Tukey’s
multiple-comparisons test, P<0.05).
In the immunohistochemical studies, autofluorescence was
limited to a few tissues; for example, at the junction of the
midgut and hindgut, and in parts of the trachea. Incubation with
secondary antibody alone did not increase fluorescence. Non-
heat-shocked samples incubated with both primary and
secondary antibodies exhibited repeatable staining of a few
cells in the base of the right and left lobes of the brain. Staining
in these cells was never observed in the absence of primary
antibody. Male gonadal disks occasionally fluoresced in the
absence of heat treatment, and once quite strikingly, but this
fluorescence was inconsistent.
All tissues expressed Hsp70 after 1 h at 36 °C, but differed
in the intensity of their fluorescent signal (Fig. 2). Larval brain,
salivary glands and imaginal disks stained most strongly
immediately after stress (Fig. 2A), with the hindgut also
showing a pronounced signal (Fig. 2C). When larvae were
allowed to recover at 25 °C for 4 h after exposure to 36 °C for
1 h, the tissues that first showed high Hsp70 levels declined
markedly in fluorescence, while staining in the gut was
uniformly high (Fig. 2B,D). One exception was the
Malpighian tubules, in which staining was initially low but
B
br gc
mg
gc pv
D
fb
mt
hg
mt
mg
2010 R. A. KREBS AND M. E. FEDER
became more pronounced than that of any other tissue by 4 h
after exposure to 36 °C (Fig. 2D). By 24 h after exposure to
36 °C, Hsp70 was undetectable in all tissues, except faintly in
the anterior midgut (not shown).
Immediately after a 38.5 °C heat shock, brain, salivary
glands, imaginal disks and hindgut (the same four tissues
showing pronounced staining at 36 °C) stained more intensely
than all other tissues (Fig. 3), although the signal was relatively
less than that obtained at 36 °C (Fig. 2). Almost no
fluorescence was evident elsewhere. Four hours after the stress
treatment, fluorescence levels increased in most tissues (Fig. 4)
and tended to localize to nuclear regions. Fluorescence was
patchy throughout the gut, and the many small regions of
intense fluorescence (Fig. 4C) appeared to be from expression
in the tiny tracheal cells responsible for aerating the broad cells
of the gut proper. Alternatively, some of this fluorescence may
have been in gut imaginal cells. Of all gut tissues, only in the
gastric caeca was cytoplasmic fluorescence evident by 4 h after
heat shock.
By 21 h of recovery, tissues that stained immediately after
the 38.5 °C heat shock were much less fluorescent than gut and
related tissues, especially caeca (Fig. 5). In contrast, gut
tissues, Malpighian tubules and fat body now stained intensely.
Fat body, which makes up a substantial proportion of the body
mass in late-instar larvae, fluoresced less intensely than most
other tissues after all other treatment conditions. Consequently,
fat body fluorescence more closely paralleled Hsp70 levels in
whole-body lysates than did fluorescence in other tissues. This
general pattern, signal decrease in some tissues with signal
A B
id
br
sg
pv
fb
sg mg
gc
mg
increase in others, is dramatically evident from the faint patch
in Fig. 5D, where the outline of a non-fluorescent female
gonadal disk is clearly discernible amidst fluorescent fat body.
Control larvae, as well as those receiving temperature
treatments, stained with Trypan Blue in the anterior and
posterior remnants of the muscle tissue following dissection,
but otherwise showed minimal staining. This is consistent with
tissue or cell damage in the region of dissection, but little
elsewhere. In the control preparation depicted in Fig. 6A, gut
staining, including caeca, upper midgut and lower gut (the
region labeled hindgut, of which scoring may include damage
in the most posterior part of the midgut) was
uncharacteristically intense by comparison with other controls.
Nevertheless, even in this preparation, the level of staining was
much lower than in treated individuals (Fig. 6B,C). Gut stained
slightly in the controls, probably due to damage from
inadvertent stretching during dissection, but few other cell
types stained with Trypan Blue in larvae not exposed to high
temperature.
In comparison with controls, tissues of heat-shocked larvae
(1 h at 38.5 °C) were more extensively stained with Trypan
Blue, and staining varied among tissues (Fig. 7). Changes from
control levels were relatively larger in caeca, midgut, brain and
salivary glands, where differences were statistically significant
(Mann–Whitney U-tests, P<0.05). The intensity of staining,
however, was strongest in gut tissues (caeca, midgut and
hindgut), but these tissues also showed more staining in
controls than non-gut tissues (Fig. 7). Very little staining after
heat shock occurred in the proventriculus and fat body (data
fgd
mt
mg hg
Fig. 3. Hsp70 expression in larval tissues after 1 h of exposure to
38.5 °C and no recovery at 25 °C. (A) Anterior tissues, (B) posterior
tissues and (C) middle midgut. sg, salivary glands; id, imaginal disks,
gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg,
hindgut; mt, Malpighian tubules; fb, fat body; fgd, female gonadal
disk. Expression is identified by fluorescent staining with anti-Hsp70
monoclonal antibody. Scale bars, 500 µm.
 Hsp70 expression and tissue damage 2011

A sg br B
gc mgd mt
pv fb

id mg
mg

fb gc hg

Fig. 4. Hsp70 expression in larval tissues after 1 h of exposure to

38.5 °C and 4 h of recovery at 25 °C. (A) Anterior tissues, (B)
posterior tissues and (C) middle midgut. sg, salivary glands; fb, fat
body; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br,
mg brain; mg, midgut; hg, hindgut; mt, Malpighian tubules. Expression
is identified by fluorescent staining with anti-Hsp70 monoclonal
antibody. Scale bars, 500 µm.

not presented). By 21 h after the 38.5 °C heat shock, much of and Malpighian tubules stained significantly more at this time
the gut tissue stained with Trypan Blue, as shown by the than immediately after heat shock (Fig. 7), while in all types
extreme individual depicted in Fig. 6C. The midgut, hindgut of tissue, differences between the level of staining in control

A B
fb hg
mg
gc
mg
fb
mg

fb mt

C D
fb

fgd

mg

Fig. 5. Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and 21 h of recovery at 25 °C (A) Anterior tissues, (B) posterior tissues,
(C) middle midgut and (D) a region of the fat body surrounding the female gonadal disk. fb, fat body; gc, gastric caeca; mg, midgut; hg, hindgut;
mt, Malpighian tubules; fgd, female gonadal disk. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale
bars, 500 µm in A,B,C; 250 µm in D.
2012 R. A. KREBS AND M. E. FEDER
A B
pv
gc
mg
fb
mg
hg
Fig. 6. Trypan Blue staining in (A) an untreated larva, (B) a larva exposed to 38.5 °C for 1 h, and (C) a larva exposed to 38.5 °C for 1 h and
then maintained at 25 °C for 21 h: fb, fat body; pv, proventriculus, gc, gastric caeca; mg, midgut; hg, hindgut. Larvae were dissected, immersed
in Trypan Blue, which is excluded from healthy cells, and washed thoroughly. Thus, blue indicates tissue damage. Scale bar, 1 mm.
larvae and in those 21 h after heat shock were significant
(Mann–Whitney U-tests, P<0.05). Large internal necroses also
become evident in some larvae.
Discussion
In whole-body lysates, the pattern of Hsp70 expression
following a potentially lethal heat shock (i.e. 1 h at 38.5 °C)
contrasts with that occurring after a 1 h treatment at 36 °C,
which is seldom lethal. After exposure to the higher
temperature, Hsp70 expression in third-instar larvae is initially
very low, but increases markedly for many hours afterwards.
The milder stress, in contrast, leads to maximal expression in
whole-body extracts after 1 h of treatment, approaching 70 %
of a standard (Feder et al. 1996; Krebs and Feder, 1997), but
concentrations decline rapidly afterwards, and are virtually
undetectable 21 h after the 36 °C exposure. Similar expression
patterns are known for other life stages (Feder et al. 1996) and
for tissue culture cells derived from embryos (Solomon et al.
1991).
C
gc
mg
fb
fb mg
hg
Hsp70 expression in whole larvae is the net result of
differential expression in various tissues, which differ in their
kinetics of Hsp70 expression during stress and recovery from
stress. Fig. 2 exemplifies this finding. Brain, salivary gland,
imaginal disks and hindgut respond quickly during stress and
are responsible for the high levels of Hsp70 that may be found
within minutes of a stress encounter. However, fat body, which
makes up a large portion of larvae late in development, may
account for much of the Hsp70 expressed later after heat shock.
Coincidentally, those tissues showing rapid induction of Hsp70
all have a common embryonic origin in the ectoderm (Martinez
Arias, 1993). Tissues that express Hsp70 more slowly,
however, have disparate embryonic origins that include
mesoderm (fat body), endoderm (caeca and midgut) and
ectoderm (Malpighian tubules) (Skaer, 1993).
The timing of the appearance of Hsp70 varies both within
individual cells and in cell types within tissues. Gut cells show
a fluorescent signal in the cytoplasm, but not in the nuclei, 4 h
after a 36 °C exposure. The transition from nuclear to
cytoplasmic localization of Hsp70 correlates with a resumption
 Hsp70 expression and tissue damage 2013

AA
5 Caeca 12 Brain Control 25 °C

AA 10 Heat shock 38.5 °C

4

AA AA AA
Heat shock 38.5 °C
8 and 21 h recovery
AA AA AA
3
6
2
AA AAAAA AA
AA AA
AAAAA
4
1
AAAAA
2
0 0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

8 Midgut 12 Salivary glands

10
6
AA
Frequency AA AA
8

AA AA
4 6
Fig. 7. Quantification of Trypan Blue staining, an
indicator of tissue damage, among control larvae
AAAA AA 4
AA AAAAAA
AA AA
AA AAAAA
2
(open columns), larvae exposed to 38.5 °C for 1 h 2
(stippled columns) and those exposed to 38.5 °C
for 1 h and thereafter maintained at 25 °C for 21 h
0
AAAA
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0
AA
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
(filled columns). Scoring for each individual was 8 12
Hindgut Malpighian tubules
(0) no color, (1) any blue, (2) darkly stained
10

AA AA
nuclei, (3) large patches of darkly stained cells, 6

AA
and (4) complete staining of most cells in the 8

AAAAAA AA AA
tissue. Each datum was a composite score for 4 6

AA
groups of three larvae that were dissected, stained
4
and visually scored together, with half units 2
AAAAAA
AAAA
representing intermediate degrees of staining after 2
AA AA
averaging within each group. Scoring of hindgut
may have included the most posterior section of
0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0
AA
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
the midgut in some samples. Trypan Blue score

of synthesis of normal cellular proteins (Velazquez and
Lindquist, 1984; Palter et al. 1986). Malpighian tubules
comprise two distinct cell types, of which stellate cells express
Hsp70 first. The more numerous broad cells peak in Hsp70
expression well after most other tissues. This delay in Hsp70
expression may explain the observations of Singh and Lakhotia
(1995), who reported that the broad cells of the Malpighian
tubules do not produce Hsp70.
Extreme heat shock (38.5 °C) produced more extensive
inter-tissue variation in Hsp70 expression than did 36 °C heat
shock (Figs 3–5), although basic patterns followed those
described for Fig. 2. After the more intense heat shock, most
tissues initially exhibited little Hsp70 staining, which is
consistent with the Hsp70 levels reported for whole-larval
lysates. In contrast, brain, salivary glands, imaginal disks and
hindgut fluoresced intensely at this time.
Experimental increases and decreases in Hsp70
concentration affect thermotolerance correspondingly
(Solomon et al. 1991; Welte et al. 1993; Feder et al. 1996).
Our expectation a priori, therefore, was that those tissues
expressing the highest levels of Hsp70, or those expressing
Hsp70 rapidly, should be most resistant to thermal stress. In
general, those tissues of D. melanogaster that produced Hsp70
rapidly in response to high temperatures, particularly brain and
salivary glands, suffered less damage from heat shock than
those expressing Hsp70 slowly, e.g. caeca, and midgut. Trypan
Blue staining, a measure of tissue damage, increased in all
tissues after heat stress. In non-gut tissues, staining was spotty,
suggesting that outright damage or death occurred in a small
proportion of cells. The gut, however, incorporated much more
Trypan Blue, suggesting extensive necrosis. Gut function is not
essential for acute survival, and gut damage may kill only
slowly as it deprives larvae of nutrients and water. Indeed,
deaths from a 1 h heat shock at 38.5 °C occur 24–48 h after the
stress exposure in first- and third-instar larvae, and few larvae
die during the actual stress treatment (Krebs and Feder, 1997).
Rarely do larvae resume feeding, despite crawling along the
food surface and occasionally pupating.
In mammals, Hsp70 expression may vary within specific
tissues, for example, in the central nervous system (CNS)
(Manzerra and Brown, 1992), but whether low levels of
expression correlate with heat susceptibility is not known. In
one test of heat-induced protein denaturation, proteins in a gut
tissue, liver, denatured before those of muscle or eye lens
(Ritchie et al. 1994); the CNS was not examined. Identifying
nerve damage is important, however, because CNS failure
could debilitate the function of an organ, with the cause
attributed incorrectly to the organ itself. Two different
transcription factors, HSF1 and HSF2, which differ in their
response to heat and thus in their affects on Hsp induction,
2014 R. A. KREBS AND M. E. FEDER
contribute to the inter-tissue variation (Marcuccilli et al. 1996;
Brown and Rush, 1996). D. melanogaster possess only one
HSF, but the fact that larval rearing temperatures may affect
Hsp70 expression patterns in some D. melanogaster cell types
(Hochstrasser, 1987) and the increased tissue variability at
higher temperatures found in this study indicate that multiple
factors affect Hsp70 regulation.
Our results make several important contributions. (1)
Although all tissues express Hsp70 in response to heat,
different tissues vary in the timing of their response. (2) Most
Hsp70 expression activated by severe heat shock occurs after
a return to physiologically normal temperatures, suggesting
either that Hsp70 expression responds to the heat-induced
damage rather than to the heat shock itself or that Hsp70
expression is itself damaged by heat. (3) From Trypan Blue
staining after heat shock, gut tissues appear to be especially
sensitive to heat. (4) Covariation among and perhaps even
within tissues for expression of Hsp70 and Trypan Blue
staining affirms the link between Hsp70 expression and tissue
damage, thereby strengthening the evidence for the use of
Hsp70 as a biomarker in environmental monitoring (Ryan and
Hightower, 1996). It is unclear from the present investigation,
however, whether the expression of Hsp70 after heat shock is
more symptomatic of cell damage than of a system for repair.
In contrast, Hsp expression before heat shock clearly enhances
survival (Krebs and Feder, 1997). These results lay the
groundwork for an examination of how differential sensitivity
of the tissues results in whole-organism thermotolerance, and
target the gut for future attention. As gut tissues appear to be
the most sensitive to heat shock and the slowest to produce
Hsp70, we predict that increasing Hsp70 levels experimentally
(Feder et al. 1996) ought to increase the tolerance of these
tissues more than others, and that enhanced thermotolerance of
the gut should enhance whole-organism thermotolerance.
We thank Susan Lindquist and Mark Martindale for use of
microscopes, Julie Feder and Steve Irvine for critically reading
the manuscript and/or assistance with microscopy techniques,
and Pratumtip Boontrakulpoontawee Maddux for
demonstrating her technique for larval dissection. Research
was supported by National Science Foundation grants IBN94-
08216 and BIR94-19545, and the Louis Block Fund of the
University of Chicago.
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