TERATOLOGY 44:77-89 (1991)
Developmental Regulation of Heat Shock Protein Synthesis and HSP 70 RNA Accumulation During Postimplantation Rat Embryogenesis PHILIP E. MIRKES, RICHARD H. GRACE, AND SALLY A. L I m L E Department of Pediatrics, Division of Embryology, Teratology and Congenital Defects (P.E.M., R.H.G., S.A.L.),Child Development and Mental Retardation Centre (P.E.M.), University of Washington, Seattle, Washington 98195
ABSTRACT Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to a n in vitro heat shock of 43°C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage a t the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit a n increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43°C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages). In utero exDosures to elevated temDerature (hypertgermia) are known to dause congenital defects in a variety of animals including chickens (Alsop, ’19, Dela Cruz et al., ’661, mice (Pennycuik, ’65), rats (Kreshover and Clough, ’53; Skreb and Frank, ’63; Edwards, ’68), hamsters (Kilham and Ferm, ’761, guinea pigs (Edwards, ’67), rabbits (Brinsmade and Rubsaamen, ’57), nonhuman primates (Poswillo et al., 1974; Hendrickx et al., 1979). Although unproved, hyperthermia is likely to prove teratogenic in humans as well (Smith et al., ’78; Warkany, ’86). In addition, more recent experiments in rats (Webster et al., ’85) indicate t h a t the critical period during which acute hyperthermia induces malformations observable at term spans the interval from 8.5 to 10.5 days of gestation. Before and subsequent to this critical period, acute hyperthermia produced a very low percentage of malformed fetuses (18%and 5%, reQ 1991 WILEY-LISS, INC.
spectively) compared with the critical period (93%). These data of Webster et al. (’85) clearly demonstrate a “window of sensitivity” during early postimplantation development during which elevated temperature induces subsequent abnormal embryogenesis, a window bracketed by periods of relative “resistance” to hyperthermia-induced abnormal embryogenesis. In addition to the teratogenic effects of hyperthermia, it is now well documented that elevated temperatures can also induce a heat shock response in organisms varying from primitive bacteria to humans (see Schlessinger et al., ’82; and Lindquist, ’86 for reviews). This heat shock response is
Received July 9, 1990; accepted January 22, 1991. Address reprint requests to Dr. Philip E. Mirkes, Department of Pediatrics, University of Washington, Seattle, WA 98195.
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characterized by the induction of a set of taining embryos were placed in a 37°C inheat shock genes and the synthesis of a set cubator at 12 noon and rotated for 1 hr at of heat shock proteins, or HSPs. In addition, 20-40 rpm to equilibrate culture medium a variety of stresses can induce the synthe- and embryos to 37°C. After this equilibrasis of stress proteins and thus this response tion phase, the culture bottles were removed should more properly be called the stress from the 37°C incubator and placed upright in a 43°C waterbath (Haake FK2) for 15,30, response (Lindquist, '86). The heat shock response has been exten- or 60 min. Culture medium temperature sively studied in a variety of systems reached 43°C within 2-3 min. Culture me(Lindquist, '86); however, relatively little is dium temperatures were monitored with a known about this process in mammalian digital thermometer (VWR model 500) and embryos. Several reports have appeared temperatures were maintained within documenting that heat shock protein syn- 0.2"C of the reported temperature. Bottles thesis cannot be induced by elevated tem- containing control embryos were also mainperatures until the morula/blastocyst stage tained upright during this period. Lack of (Wittig et al., '83; Muller et al., '85; Heikilla rotation for 5 4 h r had no measurable effect et al., '86). At this stage, heat and other on embryo growth and development stresses induce the synthesis of a 70-kDa (Mirkes, '85a). After the hyperthermia exheat shock protein. At a later postimplanta- posure, bottles were placed briefly on ice to tion stage, Mirkes ('87) has demonstrated cool the culture medium to 37"C, returned that day 10 rat embryos respond to elevated to the 37°C incubator and incubated for a n temperature (43°C) by synthesizing a set of additional hour. After this 1-hr incubation, eight HSPs, the most prominent having a embryos were either cultured in dialyzed molecular weight of 68-70 kDa. rat serum supplemented with glucose and Given this background, we initiated stud- pyruvate (Gunberg, '76) containing leucine, (sp. act. 140.8 Ci/mmole, 50 ies to determine (1)whether the ability of ~-(3,4,5-3H(N)) postimplantation embryos (presomite to kCi/ml, New England Nuclear) for 1 h r a t somite stage) to mount a heat shock re- 37°C or immediately homogenized in prepsponse was developmentally regulated, and aration for RNA isolation. (2) the relationship between the ability of Two-dimensional gel electrophoresis: heat to induce a heat shock response and the ability of heat to induce abnormal embryo- Analysis of rat embryo heat shock proteins Embryos were heat shocked as described genesis. previously. For these experiments a 43°C MATERIALS AND METHODS heat shock for 30 min was chosen because I n uitro embryo culture this was the heat shock protocol used to Time-mated Sprague-Dawley rats were characterize the rat embryo heat shock repurchased from Tyler Labs (Bellevue, WA) sponse in previous studies (Mirkes, '87), Afand maintained in the animal facility of the ter a 1-hr labeling period, embryos were reCentral Laboratory for Human Embryology moved from culture, and washed through with access to food (Purina Lab Chow) and three changes of 0.8% NaC1, dissected free water ad libitum. Pregnant animals were of accompanying membranes, and frozen a t maintained on a 14-hr light/lO-hr dark cy- -20°C. The next day embryos were thawed cle and a t a temperature of 21°C. The morn- briefly and then sonicated (two 5-sec bursts ing following copulation was considered day at setting 2 of Heat System Ultrasonics) in 0. On the mornings of day 9 (presomite 300-400 p1 of lysis buffer (9.5 M urea, 2% stage), day 10 (6-10 somite embryos), day NP-40, 5% 2-mercaptoethanol). The soni11 (21-25 somite embryos), and day 12 (31- cate was then centrifuged for 10 min a t 35 somite embryos) embryos were removed 7,OOOg. Duplicate 10-kl aliquots of the sufrom the uterus and cultured in vitro ac- pernatant were taken for the determination cording to the method of New ('78) as mod- of total TCA-precipitable radioactivity (dpm) and the remaining supernatant was ified in our laboratory (Mirkes et al., '84). stored at -80°C. Heat shock induction Before electrophoresis, homogenates were In order to demonstrate the induction of slowly thawed a t room temperature. Equivheat shock proteins or HSP 70 RNA, the alent amounts of acid precipitable tritium following protocol was used. Bottles con- from heat shocked and control embryos
79
RAT EMBRYO HEAT SHOCK RESPONSE
were analyzed by the nonequilibrium pH gradient electrophoresis (NEPHGE) method of O'Farrell et al. ('77) in order to resolve both acidic and basic proteins. NEPHGE first-dimension gels were then electrophoresed in the second dimension using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gels of 5% stacking gel and 10% resolving gel. Second dimension gels were fixed in 10% acetic and 30% methanol and then impregnated with Enhance (New England Nuclear), dried on filter paper, and exposed to preflashed Xray film (Xomat R) for 5 2 weeks. Identification of HSPs a-h, originally described for day 10 embryos by Mirkes ('87) on autoradiographs made a t different developmental stages (days 9, 11, and 12) was accomplished in the following manner. Several HSPs, i.e., HSPs b, e, f, g, and h , were identified in reference to surrounding radiographic spots which were present a t al! developmental stages analyzed. Thus, HSP b was localized with reference to spots 1 and 2, HSP e with reference to Actin (AC), HSP f with reference to spots 3 and 4 and HSPs g, h with reference to spot 5. Identification of HSPs a, c, and d was difficult to make on day 11 and 12 autoradiograms because neighboring landmark spots changed with developmental stage. Thus a n assessment relative to the presence or absence of these HSPs on days 11 and 12 is tentative. Isolation of RNA and Northern blot analyses Embryos were homogenized in a Dounce homogenizer in a buffer containing 10 mM Tris (pH 7.9), 10 mM EDTA, 1% Sarkosyl, and cesium trifluoroacetate (CsTFA) at 1.3 glml. Homogenates were then incubated at 50°C for 1 hr, layered on CsTFA step gradients, and centrifuged for 14-16 h r at 36,000 rpm in a SW 50.1 rotor (Mirkes, '85b). RNA (banded on CsTFA) a t 1.8 g/ml was removed from the gradient with a syringe and precipitated with ethanol. RNA was then collected by centrifugation, dissolved in a small volume of distilled water, and absorbance of a n aliquot was measured at A,,,. Equivalent amounts of RNA were then electrophoresed in 1% agarose gels after denaturation with formaldehyde as described by Maniatis et al. ('82). After electrophoresis, RNA was transferred overnight to Gene Screen Plus (DuPont NEN) with 20 x SSPE (3.6M NaC1, 0.2 M NaH,PO,, 0.2 M EDTA,
pH 7.4). To measure HSP 70 RNA, we used a 4.3-kb segment of mouse DNA selected from a A phage library and subcloned into pBR322 a t the BamHl site (gift of Dr. R Morimoto, Northwestern University). This clone encodes a portion of the 5' end of a mouse 70-kDa HSP gene. To measure actin mRNA, we used a Drosophila genomic probe (DMA2) subcloned in pBR322 (Fryberg et al., '80; gift of Dr. Gilbert Schultz, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada). Plasmid DNA containing the HSP insert or the actin insert were labeled by nick translation with deoxycytidine 5' [32Pltriphosphate (3,000 Ci/mmole, New England Nuclear) to a specific activity of -2 x loBdpm/ ug. The 32P-labeled cDNA was hybridized to the RNA blot using the following protocol: The RNA filter was prehybridized in a solution containing 0.75M NaC1, 50 mM Tris (pH 7 . 3 , 1%SDS, 0.1% Na pyrophosphate, 10% dextran sulfate, and 5 x Denharts. After prehybridization, the nick translated probe was boiled for 10 min, placed on ice, and added to the bag containing hybridization medium. Hybridization occurred overnight a t 70°C. After hybridization, the filter was removed from the bag and washed in 2 x SSC, 1% SDS a t 70°C for 30 min (two washes) and then further washed in 0 . 2 ~ SSC, 1% SDS a t 70°C for 15 min (two washes). The filter was then exposed to Kodak X-omat R film. RESULTS
Embryos heat shocked (43"C, 30') on day 10 of gestation consistently respond by synthesizing a set of heat shock proteins previously designated HSPs a-h (Fig. 1).One of these HSPs, HSP f, has a n isoelectric point and molecular weight characteristic of the typical mammalian HSP 70. In addition, HSP f is recognized by a n antibody directed against the Drosophila HSP 70 (data not shown). The induction of this putative ra t embryo HSP 70 by heat on days 9, 11, and 12 of gestation is depicted in Figures 2-4. On day 9 of gestation a 43"C, 30' heat shock leads to the induction of HSP f synthesis although the level of synthesis, as judged by autoradiographic spot size and intensity, is considerably less than seen on day 10. On day 11, when neural tube closure and embryo rotation are complete, a 43"C, 30-min heat shock also induces the synthesis of HSP f. The level of HSP f synthesis on day
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Fig. 1. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 10 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 1.5 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
RAT EMBRYO HEAT SHOCK RESPONSE
Fig. 2. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 9 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
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tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 3.2 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
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Fig. 3. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 11 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 3.2 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
RAT EMBRYO HEAT SHOCK RESPONSE
Fig. 4. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 12 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
83
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 2.7 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
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Dav 9 10
TABLE 1 . Relatiue levels o f HSP synthesis as a function of uostimulantation deueloumental stage'," a b C d e f p. h i ND ++ ++ ND +++ + + ++++ + ++++ +++ ++ +++ ++++ ++ ++ ND ND ++ ND ND + -+ + 2 ND ND ND ND ND
11 12
'Relative levels of individual HSP synthesis (indicated by of spot size and density. 'ND, not determined.
~
~
2
through
11 is, again, considerably less than on day 10. On day 12, no detectable HSP f synthesis is observed even when the autoradiograph is overexposed. Identical results are observed when embryos are given a n embryo lethal heat shock (43"C, 60'), suggesting that the attenuated response observed on day 11 and 12 is unrelated to a n insufficient heat shock (data not shown). These results indicate that the ability of the postimplantation r a t embryo to respond to a n acute heat shock by the synthesis of HSP f (HSP 70) is developmentally regulated. In addition to HSP f (HSP 70) synthesis, we also compared the effects of acute heat shock on the synthesis of other HSPs observed on day 10 with those detected on days 9, 11, and 12. This analysis is summarized in Table 1. On day 9, clearly detectable synthesis of HSPs b, c, and e is seen in heat shocked embryos but not in control embryos. Also, synthesis of HSP h is detectable in heat shocked embryos but not control embryos although the level of synthesis is less than for HSPs b, c, and e. The synthesis of HSPs a and d could not be ascertained because unambiguous reference spots were unavailable, while HSP g synthesis could not be detected. Finally, two autoradiographic spots (HSP i, j) are prominent in day 9 heat shocked but not control embryos. Whether these two putative HSPs are induced a t later stages of development is difficult to determine because the two-dimensional gel patterns become increasingly complex and show significant developmental changes between days 9 and 12. On day 11, heat induction of HSPs b and e synthesis is clearly detected. No obvious synthesis of HSPs g or h is detected, although low levels of synthesis cannot be ruled out. Synthesis of HSPs a, c, and d could not be ascertained because unambiguous reference spots were unavailable. On day 12, no detectable synthesis of HSPs a-h are observed, although unambiguous identification of HSPs a, c, and d could not be made.
1
++ ND ND ND
+ + + + ) were determined from autoradiographs on the basis
In order to extend our analysis of the developmental regulation of HSP 70 synthesis, we also assessed the accumulation of HSP 70 RNA in embryos exposed to a temperature of 43°C for 30 or 60 min on gestational days 9-12. Figure 5a depicts the ethidium bromide staining patterns for RNAs isolated from embryos (control and heat shock) of developmental stages 9-12. The prominent 28 and 18s rRNA bands and the 2 : l ratio of 28s rRNA/18s rRNA indicate that the RNA samples are undegraded. Northern analysis of these RNA samples (Fig. 5b) reveals the following. First, no detectable HSP 70 RNA is found in the control embryos at days 9-12 of gestation, Second, HSP 70 RNA is detectable in day 9 embryos heat shocked at 43°C for 30 min. Under this same heat shock schedule, HSP 70 mRNA levels are increased in day 10 embryos compared with day 9 embryos, HSP 70 RNA levels in day 11 embryos are reduced compared with either day 9 or day 10 embryos, and barely detectable in day 12 heat shocked embryos. Third, a t all stages examined a more severe heat shock of 43°C for 60 min (embryo lethal) results in a n attenuated accumulation of HSP 70 RNA. Identical RNA samples were also probed for actin RNA (Fig. 5c). Actin RNA levels are somewhat lower in day 9 embryos compared with later stages; however, no dramatic developmental regulation of actin RNA levels compared with HSP 70 RNA is seen. In addition, heat shock has no dramatic effect on actin RNA levels. Because we observed such a dramatic diminution of HSP 70 RNA accumulation in day 12 embryos exposed to a temperature of 43°C for 30' or 60', we repeated our Northern analysis but added a less severe heat shock (i.e., 43"C, 15') to determine whether HSP 70 RNA could be induced to levels seen a t earlier gestational ages. Results depicted in Figure 6 indicate the following. First, a 43"C, 15' heat shock does lead to the accumulation of HSP 70 RNA in day 12 em-
RAT EMBRYO HEAT SHOCK RESPONSE
85
Fig, 5. Northern analysis of HSP 70 (B) and actin ( C )RNA in embryos cultured at 37°C (lanes 1,4,7, and lo), 4TC, 30' (lanes 2,5,8, and 111, or 43"C,60' (lanes 3, 6,9, and 12). RNAs were isolated from day 9 (lanes l a ) , day 10 (lanes a), day 11(lanes 7-9), and day 12 embryos (lanes 10-12) (see under Materials and Meth-
ods). For each stage and exposure condition, 8 pg of total RNA was electrophoresed, transferred to Gene Screen Plus, and sequentially probed for HSP 70 and actin RNAs. A. Ethidium bromide staining patterns of total RNA samples subsequently probed for HSP 70 (B) or actin RNA (0.
bryos. Second, the relative amount of HSP 70 RNA accumulated in day 12 embryos exposed to a 43"C, 15' heat shock is similar t o the amounts of HSP 70 accumulated at earlier gestational stages exposed to this same heat shock. Third, developmental changes
in the accumulation of HSP 70 RNA become manifest only after embryos are exposed to more severe heat shocks, i.e., 43"C, 30' or 60' (Fig. 7). As development progresses from day 9 to day 12, embryos exhibit the highest level of HSP 70 RNA accumulation after a
P.E. MIRKES ET AL.
86
Fig. 6 . Northern analysis of HSP 70 and actin RNA in embryos cultured at 37% (lanes 4,8,12,and 16),at 43°C for 15' (lanes 1, 5, 9, and 13), at 43°C for 30 (lanes 2, 6, 10, and 14), or at 43°C for 60' (lanes 3, 7, 11, and 15). Total RNA was isolated from day 9 (lanes
1 4 , day 10 (lanes 549, day 11 (lanes 9-12),or day 12 (lanes 13-16) embryos. For each stage and exposure condition, 8 pg of total RNA was electrophoresed, transferred to Gene Screen Plus, and sequentially probed for HSP 70 and actin RNAs.
&
0
15
30
45
60
day9 day 10 day 11 day 12
75
TIME AT 43°C (MINUTES) Fig. 7. Relative levels of HSP 70 RNA induced in rat embryos exposed in vitro to a temperature of 43°C for 15', 30', or 60' on day 9 ( +I, 10 ( -t ), 11 ( +), or 12 ( -u-) of gestation. Individual lanes in the autoradio-
graphic film of HSP 70 depicted in Figure 6 were scanned with a Helena densitometer. Integrated areas for each band were plotted as the relative amount of HSP 70 RNA.
60' heat shock on day 9, a 30' heat shock on day 10 and a 15' heat shock on day 11 and 12. Fourth, as seen in Figure 6, heat has little effect on the levels of actin RNA.
bryos of a variety of species are incapable of mounting a heat shock response. The ability to mount a heat shock response is achieved at the cellular blastoderm stage in Drosophila (Graziosi et al., '80; Dura, '81; Bergh and Arking, '84),at the hatched blastula stage in the sea urchin (Roccheri et al., '81; Howlett et al., '831, at the mid- to late-blas-
DISCUSSION
Previous work from several laboratories has shown that the oocytes and early em-
RAT EMBRYO HEAT SHOCK RESPONSE
tula stage in Xenopus (Bienz, '84; Heikkila et al., '85, '86), and at the blastocyst stage in mouse and rabbit (Wittig et al., '83; Heikkila and Schultz, '84; Heikkila et al., '85, '86). Results presented in this manuscript extend these observations to the early to midpostimplantation stages of the mammalian embryo. Like the mouse blastocyst, the day 9 rat embryo head-fold stage is also capable of mounting a heat shock response. Unlike the mouse blastocyst in which heat induced primarily the level of HSP 70 (Heikkila et al., '85), in the rat embryo heat induced a set of HSPs, one of which (HSP f) is probably HSP 70. The heat shock response is prominent in day 10 embryos when a set of 8 HSPs are induced. Subsequent stages, days 11 and 12, exhibit attenuation of the embryos ability to respond to an acute heat shock culminating in the absence of a response on day 12 when measured by the synthesis of HSP f, as well as other HSPs. In addition, our results indicate that the ability of rat embryos to accumulate RNA for one of the HSPs, i.e., HSP 70, in response t o an acute heat shock is also developmentally regulated. This developmental regulation is seen as an attenuation of HSP 70 RNA accumulation as a function of the length of the heat shock. Day 9 embryos are able to mount the most sustained heat shock response when viewed as the ability to accumulate HSP 70 RNA after an acute heat shock (43"C, 60'). As development proceeds from day 10 to day 12, the embryo becomes increasingly less able to accumulate HSP 70 RNA when exposed to a 43°C heat shock of increasing duration. Moreover, this attenuation of the ability of the embryo to accumulate HSP 70 RNA after acute heat shocks parallels the attenuation of HSP 70 synthesis as revealed by two-dimensional gel electrophoresis. What is the mechanism(s) controlling this stage dependent expression of a heat shock response in mammalian embryos? Little definitive data are available but several possibilities have been suggested. Krone and Heikkila ('88) have shown that heatinduced HSP 30 gene expression does not occur until the tailbud stage in Xenopus, unlike HSP 70, which is induced by heat at the late blastula stage. These authors suggest that heat-induced expression of HSP 30 might be controlled by an inhibitor-HSP 30 gene interaction. Another possibility is that
87
heat shock transcription factors (HSTF) or accessory transcription factors necessary for the induction of heat shock genes are absent or sequestered at different stages of development. Likewise, the fact that not all heat shock proteins are expressed in embryos capable of mounting a response, i.e., blastocyst through day 11, may reflect the fact that different heat shock genes require different transcription factors or that different heat shock genes differentially bind a limited pool of HSTF. Another possibility, suggested by the work of Di Domenico et al. ('82), is that heat shock genes are inducible at all times, but a t certain stages the HSP transcripts are unstable, rapidly turned over, and therefore not translated into HSPs. Our data from day 12 embryos, which do not synthesize any detectable HSP f in response to heat, also accumulate a very low level of HSP 70 mRNA after prolonged heating (43°C' 30-60'). Still another possibility is that HSP transcripts are induced by heat at all developmental stages; however, the translational machinery is incapable of translating some or all of these transcripts at different developmental stages. Although other regulatory mechanisms are possible, it is of great interest t o understand the mechanism used by embryos to regulate their response to heat and other stresses known t o be capable of inducing the so called stress response. Perhaps more interesting is our finding that there is a close relationship between the ability of the postimplantation embryo to mount a heat shock response and the induction of abnormal development as a consequence of an exposure to elevated temperature. Thus, one period of rat development during which hyperthermia can induce abnormal embryogenesis, i.e., days 8.5-10.0, is also the period of development during which hyperthermia can induce a detectable heat shock response. This relationship between hyperthermia-induced teratogenesis and hyperthermia-induced heat shock response has also been studied in Drosophila embryos where hyperthermia induces so called phenocopies (Capdevila and GarciaBellido, '74; Milkman, '66; Mitchell and Peterson, '82; Eberlein, '86; Peterson and Mitchell, '87). In addition, a variety of agents that are teratogenic in mammals are also teratogenic in Drosophila and induce a heat shock response (Buzin and BourniasVardiabasis, '84).
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P.E. MIRKES ET AL
Given this relationship between the heat shock response and abnormal embryogenesis, the obvious question is what role, if any, does the heat shock response play in stressinduced teratogenesis? Heat-induced multihair phenocopies in Drosophila are associated with disruption of protein synthesis and subsequently with discoordinate gene expression (Mitchell and Peterson, '81) and a similar disruption of gene activity in the mammalian embryo could be expected to have profound effects. This is especially true in the postimplantation mammalian rodent embryos undergoing the complex process of organogenesis with its demands of coordinated gene activation and inactivation (German, '84). Alternatively, one or more of the HSPs may be directly involved in inducing abnormal development. Perhaps the inappropriate activation of specific HSP genes that are now known to be constitutively produced at certain developmental stages (Bensaude and Morange, '83; Bensaude et al., '83; Zimmerman et al., '83; Bienz, '84; Kothary et al., '87) plays a role in subsequent pathogenesis induced by heat or other stresses. Furthermore, recent evidence suggests that the heat shock response may play a role in protecting the mammalian embryo from the deleterious effects of heat (Whittig et al., '83; Heikkila et al., '85; Mirkes, '87; Walsh et al., '87). Perhaps the heat shock response may play a dual role, on the one hand protecting the embryo and on the other disrupting embryonic development. Which outcome is observed would depend on developmental stage of the embryo at the time of insult, the severity of the stress, and other factors yet to be determined. ACKNOWLEDGMENTS
This work was supported by NIH grants HD 16287 and HD 22095, and in part by HD 00836. I thank Liz Walker for technical assistance and Rajan John Puthenpurackal for secretarial assistance. LITERATURE CITED Alsop, F.M. (1919) The effects of abnormal temperatures upon the developing nervous system in the chicken embryo. Anat. Rec., 15:307-332. Bensaude, O., and M. Morange (1983) Spontaneous high expression of heat shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo. EMBO J.,2:173-177. Bensuade, O., C. Babinet, M. Morange, and F. Jacob (1983) Heat shock proteins, first major products of zy-
gotic gene activity in mouse embryos. Nature, 305: 331-333. Bergh, S., and R. Arking (1984) Developmental profile of the heat shock response in early embryos of Drosophzla. J. Exp. Zool., 231:379-391. Bienz, M. (1984) Developmental control of the heat shock response in Xenopus. Proc. Natl. Acad. Sci. USA, 81:3138-3142. Brinsmade, A.B., and H. Rubsaamen (1987) Zur teratogenetischen wirkung von unspeczitischon fieber aut der sick entwick endren kaninchen embryo. Beitr. Pathol. Anat. Allg. Pathol., 11 7:154-164. Buzin, C.H., and N. Bournais-Vardiabasis (19841 Teratogens induce a subset of small heat shock proteins in Drosophzla primary embryonic cell cultures. Proc. Natl. Acad. Sci. USA, 81:4075-4079. Capdevila, M.P., and A. Garan-Bellido (1974) Development and genetic analysis of bithorax phenocopies in Drosophzla. Nature, 250:500-502. Dela Cruz, M.W., C. Campillo-Sainz, and S. MunrozArmas (1966) Congenital heart defects in chick embryos subjected to temperature variations. Cell Res., 18t257-262. DiDomenico, B.J., G.E. Bugaisky, and S. Lindquist (1982) The heat shock response is self-regulated at both the transcriptional and post-transcriptional levels. Cell, 31.593-603. Dura, J.M. (1981) Stage-dependent synthesis of heat shock induced proteins in early embryos of Drosophzla melanogaster. Mol. Gen. Genet., 184:381-385. Emberlein, S. (1986) Stage specific embryonic defects following heat shock in Drosophila. Dev. Gen., 6:179197. Edwards, M.J. (1967)Congenital defects in guinea pigs. Arch. Pathol., 843:42-48. Edwards, M.J. (1968) Congenital defects in the rat following induced hyperthermia during gestation. Teratology, 1:173-175. Fryberg, E.A., K.L. Kindle, and N. Davidson (1980) The actin genes of Drosophila a dispersed multigene family. Cell, 19:365-378. German, J . (1984) The embryonic stress hypothesis of teratogenesis. Am. J . Med., 76:293-301. Graziosi, G., F. Micali, F. Marzari, F. DeChristini, and A. Savoini (1980) Variability of response of early Drosophila embryos to heat shock. J. Exp. Zool., 214: 141-145. Gunberg, D.L. (1976) In vitro development of postimplantation rat embryos cultured in dialyzed rat serum. Teratology, 14:65-70. Hendrickx, A.G., G.W. Stone, R.V. Hendrickson, and K. Matayoshi (1979) Teratogenic effects of hyperthermia in the bonnet monkey ( M ~ c ~ radiata). cQ Teratology, 19: 177-182. Heikkila, J.J., and G. Schultz (1984) Different environmental stresses can activate the expression of a heat shock gene in rabbit blastocysts. Gamete Res., 10.4546. Heikkila, J.J., M. Kloc, J. Burg, G.A. Schultz, and L.W. Browder (1985) Acquisition of the heat shock response and thermotolerance during early development of Xenopus laeuis. Dev. Biol., 107:483-489. Heikkilla, J.J., L.W. Browder, L. Gedamu, R.W. Nickells, and G.A. Schultz (1986) Heat shock expression in animal embryonic systems. Can. J . Genet. Cytol., 28: 1093-1 105. Howlett, S.J., J. Miller, and G.A. Schultz (1983) Induction of heat shock proteins in early embryos of Arbacia punctulata. Biol. Bull., 165,500. Kilham, L., and V.H. Ferm (1976) Exencepahly in fetal
RAT EMBRYO HEAT SHOCK RESPONSE hamsters following exposure to hyperthermia. Teratology, 14t323-326. Kothary, R., M.D. Perry, L.A. Moran, and J. Rossant (1987) Cell-lineage-specific expression of the mouse HSP 68 gene during embryogenesis. Dev. Biol., 12: 342-348. Kreshover, S.J., and O.W. Clough (1953) Prenatal influences on tooth development 11. Artificially induced fever in rats. J. Dent. Res., 325.565-577. Krone, P.H., and J.J. Heikkilla (1988) Analysis of HSP 30, HSP 70 and ubiguitin gene expression in Xenopus laeuis tadpoles. Development, 103t59-67. Lindquist, S. (1986) The heat shock response. Annu. Rev. Biochem., 55:1151-1 191. Maniatis, T., E.F. Fritsch, and J . Sambrook (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Milkman, R. (1966) Analysis of some temperature effects on Drosophila pupae. Biol. Bull., 131 :331-345. Mirkes, P.E., J.C. Greenaway, J.G. Rogers, and R.B. Brundrett (1984) Role of acrolein in cyclophosphamide teratogenicity in rat embryos in vitro. Toxicol. Appl. Pharmacol., 72:281-291. Mirkes, P.E. (1985a) Effects of acute exposure to elevated temperatures on rat embryo growth and development in vitro. Teratology, 32t259-266. Mirkes, P.E. (198513) Simultaneous banding of rat embryo DNA, RNA and protein in cesium trifluoroacetate gradients. Anal. Biochem., 148:376-383. Mirkes, P.E. (1987) Hyperthermia-induced heat shock responses and thermotolerance in post-implantation rat embryos. Dev. Biol., 119t115-122. Mitchell, H.K., and N.S. Peterson (1981) Rapid changes in gene expression in differentiating tissues of Drosophila. Dev. Biol., 85t233-242. Mitchell, H.K., and N.S. Peterson (1982) Developmental abnormalities in Drosophila induced by heat shock. Dev. Genet., 3:91-102. Muller, W.U., G.C. Li, and L.S. Goldstein (1985) Heat does not induce synthesis of heat shock proteins or thermotolerance in the earliest stages of mouse embryo development. Int. J. Hyperthermia, 1 :97-102. New, D.A.T. (1978) Whole embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev., 53%-122.
89
OFarrell, P.Z., H.M. Goodman, and P.H. O'Farrell (1977) High resolution two dimensional electrophoresis of basic as well as acidic proteins. Cell, 12t11331142. Pennycuik, P.R. (1965) The effects of acute exposure to high temperature on prenatal development in the mouse with particular reference to secondary vibrissae. Aust. J . Biol. Sci., 18:97-113. Peterson, N.W., and H.K. Mitchell (1987)The induction of a multiple wing hair phenocopy by heat shock in mutant heterozygotes. Dev. Biol., 121:335-341. Poswillo, D., H. Nunnerly, D. Sopher, and J . Keith (1974) Hyperthermia as a teratogenic agent. Annu. R. Coll. Surg. Engl., 55t171-174. Roccheri, M.C., M.G. Di Bernardo, and G. Giudice (1981) Synthesis of heat shock proteins in developing sea urchins. Dev. Biol., 83:173-177. Schlesinger, M.J., M.A. Ashburner, and A. Tissieres (1982) Heat Shock: From Bacteria to Man. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Skreb, N., and Z. Frank (1963) Developmental abnormalities in the rat induced by heat shock. J. Embryol. Exp. Morphol., 11:445-457. Smith, D.W., S.K. Clarren, and M.A. Sedgwick-Harvey (1978) Hyperthermia as a possible teratogenic agent. J. Pediatr., 92:878-883. Walsh, D.A., N.W. Klein, L.E. Hightower, and M.J. Edwards (1987) Heat shock and thermotolerance during early rat embryo development. Teratology, 36: 181-191. Warkany, J . (1986) Teratogen update: Hyperthermia. Teratology, 33:365 -37 1. Webster, W.S., M.A. Germain, and M.J. Edwards (1985) The induction of microphthalmia, encephaloceles, and other head defects following hyperthermia during gastrulation process in the rat. Teratology, 31:73-82. Wittig, S., S. Hensse, C. Keitel, C. Elmer, and B. Wittig (1983) Heat shock gene expression is regulated during teratocarcinoma cell differentiation and early embryonic development. Dev. Biol., 96507-514. Zimmerman, J.L., W. Petri, and M. Meselson (1983) Accumulation of a specific subset of D . rnelanogaster heat shock mRNAs in normal development without heat shock. Cell, 32t1161-1170.
Developmental Regulation of Heat Shock Protein Synthesis and HSP 70 RNA Accumulation During Postimplantation Rat Embryogenesis PHILIP E. MIRKES, RICHARD H. GRACE, AND SALLY A. L I m L E Department of Pediatrics, Division of Embryology, Teratology and Congenital Defects (P.E.M., R.H.G., S.A.L.),Child Development and Mental Retardation Centre (P.E.M.), University of Washington, Seattle, Washington 98195
ABSTRACT Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to a n in vitro heat shock of 43°C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage a t the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit a n increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43°C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages). In utero exDosures to elevated temDerature (hypertgermia) are known to dause congenital defects in a variety of animals including chickens (Alsop, ’19, Dela Cruz et al., ’661, mice (Pennycuik, ’65), rats (Kreshover and Clough, ’53; Skreb and Frank, ’63; Edwards, ’68), hamsters (Kilham and Ferm, ’761, guinea pigs (Edwards, ’67), rabbits (Brinsmade and Rubsaamen, ’57), nonhuman primates (Poswillo et al., 1974; Hendrickx et al., 1979). Although unproved, hyperthermia is likely to prove teratogenic in humans as well (Smith et al., ’78; Warkany, ’86). In addition, more recent experiments in rats (Webster et al., ’85) indicate t h a t the critical period during which acute hyperthermia induces malformations observable at term spans the interval from 8.5 to 10.5 days of gestation. Before and subsequent to this critical period, acute hyperthermia produced a very low percentage of malformed fetuses (18%and 5%, reQ 1991 WILEY-LISS, INC.
spectively) compared with the critical period (93%). These data of Webster et al. (’85) clearly demonstrate a “window of sensitivity” during early postimplantation development during which elevated temperature induces subsequent abnormal embryogenesis, a window bracketed by periods of relative “resistance” to hyperthermia-induced abnormal embryogenesis. In addition to the teratogenic effects of hyperthermia, it is now well documented that elevated temperatures can also induce a heat shock response in organisms varying from primitive bacteria to humans (see Schlessinger et al., ’82; and Lindquist, ’86 for reviews). This heat shock response is
Received July 9, 1990; accepted January 22, 1991. Address reprint requests to Dr. Philip E. Mirkes, Department of Pediatrics, University of Washington, Seattle, WA 98195.
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P.E. MIRKES ET AL.
characterized by the induction of a set of taining embryos were placed in a 37°C inheat shock genes and the synthesis of a set cubator at 12 noon and rotated for 1 hr at of heat shock proteins, or HSPs. In addition, 20-40 rpm to equilibrate culture medium a variety of stresses can induce the synthe- and embryos to 37°C. After this equilibrasis of stress proteins and thus this response tion phase, the culture bottles were removed should more properly be called the stress from the 37°C incubator and placed upright in a 43°C waterbath (Haake FK2) for 15,30, response (Lindquist, '86). The heat shock response has been exten- or 60 min. Culture medium temperature sively studied in a variety of systems reached 43°C within 2-3 min. Culture me(Lindquist, '86); however, relatively little is dium temperatures were monitored with a known about this process in mammalian digital thermometer (VWR model 500) and embryos. Several reports have appeared temperatures were maintained within documenting that heat shock protein syn- 0.2"C of the reported temperature. Bottles thesis cannot be induced by elevated tem- containing control embryos were also mainperatures until the morula/blastocyst stage tained upright during this period. Lack of (Wittig et al., '83; Muller et al., '85; Heikilla rotation for 5 4 h r had no measurable effect et al., '86). At this stage, heat and other on embryo growth and development stresses induce the synthesis of a 70-kDa (Mirkes, '85a). After the hyperthermia exheat shock protein. At a later postimplanta- posure, bottles were placed briefly on ice to tion stage, Mirkes ('87) has demonstrated cool the culture medium to 37"C, returned that day 10 rat embryos respond to elevated to the 37°C incubator and incubated for a n temperature (43°C) by synthesizing a set of additional hour. After this 1-hr incubation, eight HSPs, the most prominent having a embryos were either cultured in dialyzed molecular weight of 68-70 kDa. rat serum supplemented with glucose and Given this background, we initiated stud- pyruvate (Gunberg, '76) containing leucine, (sp. act. 140.8 Ci/mmole, 50 ies to determine (1)whether the ability of ~-(3,4,5-3H(N)) postimplantation embryos (presomite to kCi/ml, New England Nuclear) for 1 h r a t somite stage) to mount a heat shock re- 37°C or immediately homogenized in prepsponse was developmentally regulated, and aration for RNA isolation. (2) the relationship between the ability of Two-dimensional gel electrophoresis: heat to induce a heat shock response and the ability of heat to induce abnormal embryo- Analysis of rat embryo heat shock proteins Embryos were heat shocked as described genesis. previously. For these experiments a 43°C MATERIALS AND METHODS heat shock for 30 min was chosen because I n uitro embryo culture this was the heat shock protocol used to Time-mated Sprague-Dawley rats were characterize the rat embryo heat shock repurchased from Tyler Labs (Bellevue, WA) sponse in previous studies (Mirkes, '87), Afand maintained in the animal facility of the ter a 1-hr labeling period, embryos were reCentral Laboratory for Human Embryology moved from culture, and washed through with access to food (Purina Lab Chow) and three changes of 0.8% NaC1, dissected free water ad libitum. Pregnant animals were of accompanying membranes, and frozen a t maintained on a 14-hr light/lO-hr dark cy- -20°C. The next day embryos were thawed cle and a t a temperature of 21°C. The morn- briefly and then sonicated (two 5-sec bursts ing following copulation was considered day at setting 2 of Heat System Ultrasonics) in 0. On the mornings of day 9 (presomite 300-400 p1 of lysis buffer (9.5 M urea, 2% stage), day 10 (6-10 somite embryos), day NP-40, 5% 2-mercaptoethanol). The soni11 (21-25 somite embryos), and day 12 (31- cate was then centrifuged for 10 min a t 35 somite embryos) embryos were removed 7,OOOg. Duplicate 10-kl aliquots of the sufrom the uterus and cultured in vitro ac- pernatant were taken for the determination cording to the method of New ('78) as mod- of total TCA-precipitable radioactivity (dpm) and the remaining supernatant was ified in our laboratory (Mirkes et al., '84). stored at -80°C. Heat shock induction Before electrophoresis, homogenates were In order to demonstrate the induction of slowly thawed a t room temperature. Equivheat shock proteins or HSP 70 RNA, the alent amounts of acid precipitable tritium following protocol was used. Bottles con- from heat shocked and control embryos
79
RAT EMBRYO HEAT SHOCK RESPONSE
were analyzed by the nonequilibrium pH gradient electrophoresis (NEPHGE) method of O'Farrell et al. ('77) in order to resolve both acidic and basic proteins. NEPHGE first-dimension gels were then electrophoresed in the second dimension using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gels of 5% stacking gel and 10% resolving gel. Second dimension gels were fixed in 10% acetic and 30% methanol and then impregnated with Enhance (New England Nuclear), dried on filter paper, and exposed to preflashed Xray film (Xomat R) for 5 2 weeks. Identification of HSPs a-h, originally described for day 10 embryos by Mirkes ('87) on autoradiographs made a t different developmental stages (days 9, 11, and 12) was accomplished in the following manner. Several HSPs, i.e., HSPs b, e, f, g, and h , were identified in reference to surrounding radiographic spots which were present a t al! developmental stages analyzed. Thus, HSP b was localized with reference to spots 1 and 2, HSP e with reference to Actin (AC), HSP f with reference to spots 3 and 4 and HSPs g, h with reference to spot 5. Identification of HSPs a, c, and d was difficult to make on day 11 and 12 autoradiograms because neighboring landmark spots changed with developmental stage. Thus a n assessment relative to the presence or absence of these HSPs on days 11 and 12 is tentative. Isolation of RNA and Northern blot analyses Embryos were homogenized in a Dounce homogenizer in a buffer containing 10 mM Tris (pH 7.9), 10 mM EDTA, 1% Sarkosyl, and cesium trifluoroacetate (CsTFA) at 1.3 glml. Homogenates were then incubated at 50°C for 1 hr, layered on CsTFA step gradients, and centrifuged for 14-16 h r at 36,000 rpm in a SW 50.1 rotor (Mirkes, '85b). RNA (banded on CsTFA) a t 1.8 g/ml was removed from the gradient with a syringe and precipitated with ethanol. RNA was then collected by centrifugation, dissolved in a small volume of distilled water, and absorbance of a n aliquot was measured at A,,,. Equivalent amounts of RNA were then electrophoresed in 1% agarose gels after denaturation with formaldehyde as described by Maniatis et al. ('82). After electrophoresis, RNA was transferred overnight to Gene Screen Plus (DuPont NEN) with 20 x SSPE (3.6M NaC1, 0.2 M NaH,PO,, 0.2 M EDTA,
pH 7.4). To measure HSP 70 RNA, we used a 4.3-kb segment of mouse DNA selected from a A phage library and subcloned into pBR322 a t the BamHl site (gift of Dr. R Morimoto, Northwestern University). This clone encodes a portion of the 5' end of a mouse 70-kDa HSP gene. To measure actin mRNA, we used a Drosophila genomic probe (DMA2) subcloned in pBR322 (Fryberg et al., '80; gift of Dr. Gilbert Schultz, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada). Plasmid DNA containing the HSP insert or the actin insert were labeled by nick translation with deoxycytidine 5' [32Pltriphosphate (3,000 Ci/mmole, New England Nuclear) to a specific activity of -2 x loBdpm/ ug. The 32P-labeled cDNA was hybridized to the RNA blot using the following protocol: The RNA filter was prehybridized in a solution containing 0.75M NaC1, 50 mM Tris (pH 7 . 3 , 1%SDS, 0.1% Na pyrophosphate, 10% dextran sulfate, and 5 x Denharts. After prehybridization, the nick translated probe was boiled for 10 min, placed on ice, and added to the bag containing hybridization medium. Hybridization occurred overnight a t 70°C. After hybridization, the filter was removed from the bag and washed in 2 x SSC, 1% SDS a t 70°C for 30 min (two washes) and then further washed in 0 . 2 ~ SSC, 1% SDS a t 70°C for 15 min (two washes). The filter was then exposed to Kodak X-omat R film. RESULTS
Embryos heat shocked (43"C, 30') on day 10 of gestation consistently respond by synthesizing a set of heat shock proteins previously designated HSPs a-h (Fig. 1).One of these HSPs, HSP f, has a n isoelectric point and molecular weight characteristic of the typical mammalian HSP 70. In addition, HSP f is recognized by a n antibody directed against the Drosophila HSP 70 (data not shown). The induction of this putative ra t embryo HSP 70 by heat on days 9, 11, and 12 of gestation is depicted in Figures 2-4. On day 9 of gestation a 43"C, 30' heat shock leads to the induction of HSP f synthesis although the level of synthesis, as judged by autoradiographic spot size and intensity, is considerably less than seen on day 10. On day 11, when neural tube closure and embryo rotation are complete, a 43"C, 30-min heat shock also induces the synthesis of HSP f. The level of HSP f synthesis on day
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P.E. MIRKES ET AL.
Fig. 1. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 10 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 1.5 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
RAT EMBRYO HEAT SHOCK RESPONSE
Fig. 2. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 9 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
81
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 3.2 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
82
P.E. MIRKES ET AL.
Fig. 3. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 11 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 3.2 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
RAT EMBRYO HEAT SHOCK RESPONSE
Fig. 4. Fluorographs of second-dimension polyacrylamide gels comparing the profile of proteins synthesized by day 12 rat embryos cultured at 37°C or exposed to a heat shock, 43°C for 30 min. Lowercase letters designate positions of rat embryo HSPs. AC designates ac-
83
tin and numbers 1-5 are reference points for HSPs b, f, g, and h (see under Materials and Methods). Approximately 2.7 x lo5 dpm of acid-precipitable radioactivity was loaded on the first-dimension isoelectric focusing gels.
84
P.E. MIRKES ET AL.
Dav 9 10
TABLE 1 . Relatiue levels o f HSP synthesis as a function of uostimulantation deueloumental stage'," a b C d e f p. h i ND ++ ++ ND +++ + + ++++ + ++++ +++ ++ +++ ++++ ++ ++ ND ND ++ ND ND + -+ + 2 ND ND ND ND ND
11 12
'Relative levels of individual HSP synthesis (indicated by of spot size and density. 'ND, not determined.
~
~
2
through
11 is, again, considerably less than on day 10. On day 12, no detectable HSP f synthesis is observed even when the autoradiograph is overexposed. Identical results are observed when embryos are given a n embryo lethal heat shock (43"C, 60'), suggesting that the attenuated response observed on day 11 and 12 is unrelated to a n insufficient heat shock (data not shown). These results indicate that the ability of the postimplantation r a t embryo to respond to a n acute heat shock by the synthesis of HSP f (HSP 70) is developmentally regulated. In addition to HSP f (HSP 70) synthesis, we also compared the effects of acute heat shock on the synthesis of other HSPs observed on day 10 with those detected on days 9, 11, and 12. This analysis is summarized in Table 1. On day 9, clearly detectable synthesis of HSPs b, c, and e is seen in heat shocked embryos but not in control embryos. Also, synthesis of HSP h is detectable in heat shocked embryos but not control embryos although the level of synthesis is less than for HSPs b, c, and e. The synthesis of HSPs a and d could not be ascertained because unambiguous reference spots were unavailable, while HSP g synthesis could not be detected. Finally, two autoradiographic spots (HSP i, j) are prominent in day 9 heat shocked but not control embryos. Whether these two putative HSPs are induced a t later stages of development is difficult to determine because the two-dimensional gel patterns become increasingly complex and show significant developmental changes between days 9 and 12. On day 11, heat induction of HSPs b and e synthesis is clearly detected. No obvious synthesis of HSPs g or h is detected, although low levels of synthesis cannot be ruled out. Synthesis of HSPs a, c, and d could not be ascertained because unambiguous reference spots were unavailable. On day 12, no detectable synthesis of HSPs a-h are observed, although unambiguous identification of HSPs a, c, and d could not be made.
1
++ ND ND ND
+ + + + ) were determined from autoradiographs on the basis
In order to extend our analysis of the developmental regulation of HSP 70 synthesis, we also assessed the accumulation of HSP 70 RNA in embryos exposed to a temperature of 43°C for 30 or 60 min on gestational days 9-12. Figure 5a depicts the ethidium bromide staining patterns for RNAs isolated from embryos (control and heat shock) of developmental stages 9-12. The prominent 28 and 18s rRNA bands and the 2 : l ratio of 28s rRNA/18s rRNA indicate that the RNA samples are undegraded. Northern analysis of these RNA samples (Fig. 5b) reveals the following. First, no detectable HSP 70 RNA is found in the control embryos at days 9-12 of gestation, Second, HSP 70 RNA is detectable in day 9 embryos heat shocked at 43°C for 30 min. Under this same heat shock schedule, HSP 70 mRNA levels are increased in day 10 embryos compared with day 9 embryos, HSP 70 RNA levels in day 11 embryos are reduced compared with either day 9 or day 10 embryos, and barely detectable in day 12 heat shocked embryos. Third, a t all stages examined a more severe heat shock of 43°C for 60 min (embryo lethal) results in a n attenuated accumulation of HSP 70 RNA. Identical RNA samples were also probed for actin RNA (Fig. 5c). Actin RNA levels are somewhat lower in day 9 embryos compared with later stages; however, no dramatic developmental regulation of actin RNA levels compared with HSP 70 RNA is seen. In addition, heat shock has no dramatic effect on actin RNA levels. Because we observed such a dramatic diminution of HSP 70 RNA accumulation in day 12 embryos exposed to a temperature of 43°C for 30' or 60', we repeated our Northern analysis but added a less severe heat shock (i.e., 43"C, 15') to determine whether HSP 70 RNA could be induced to levels seen a t earlier gestational ages. Results depicted in Figure 6 indicate the following. First, a 43"C, 15' heat shock does lead to the accumulation of HSP 70 RNA in day 12 em-
RAT EMBRYO HEAT SHOCK RESPONSE
85
Fig, 5. Northern analysis of HSP 70 (B) and actin ( C )RNA in embryos cultured at 37°C (lanes 1,4,7, and lo), 4TC, 30' (lanes 2,5,8, and 111, or 43"C,60' (lanes 3, 6,9, and 12). RNAs were isolated from day 9 (lanes l a ) , day 10 (lanes a), day 11(lanes 7-9), and day 12 embryos (lanes 10-12) (see under Materials and Meth-
ods). For each stage and exposure condition, 8 pg of total RNA was electrophoresed, transferred to Gene Screen Plus, and sequentially probed for HSP 70 and actin RNAs. A. Ethidium bromide staining patterns of total RNA samples subsequently probed for HSP 70 (B) or actin RNA (0.
bryos. Second, the relative amount of HSP 70 RNA accumulated in day 12 embryos exposed to a 43"C, 15' heat shock is similar t o the amounts of HSP 70 accumulated at earlier gestational stages exposed to this same heat shock. Third, developmental changes
in the accumulation of HSP 70 RNA become manifest only after embryos are exposed to more severe heat shocks, i.e., 43"C, 30' or 60' (Fig. 7). As development progresses from day 9 to day 12, embryos exhibit the highest level of HSP 70 RNA accumulation after a
P.E. MIRKES ET AL.
86
Fig. 6 . Northern analysis of HSP 70 and actin RNA in embryos cultured at 37% (lanes 4,8,12,and 16),at 43°C for 15' (lanes 1, 5, 9, and 13), at 43°C for 30 (lanes 2, 6, 10, and 14), or at 43°C for 60' (lanes 3, 7, 11, and 15). Total RNA was isolated from day 9 (lanes
1 4 , day 10 (lanes 549, day 11 (lanes 9-12),or day 12 (lanes 13-16) embryos. For each stage and exposure condition, 8 pg of total RNA was electrophoresed, transferred to Gene Screen Plus, and sequentially probed for HSP 70 and actin RNAs.
&
0
15
30
45
60
day9 day 10 day 11 day 12
75
TIME AT 43°C (MINUTES) Fig. 7. Relative levels of HSP 70 RNA induced in rat embryos exposed in vitro to a temperature of 43°C for 15', 30', or 60' on day 9 ( +I, 10 ( -t ), 11 ( +), or 12 ( -u-) of gestation. Individual lanes in the autoradio-
graphic film of HSP 70 depicted in Figure 6 were scanned with a Helena densitometer. Integrated areas for each band were plotted as the relative amount of HSP 70 RNA.
60' heat shock on day 9, a 30' heat shock on day 10 and a 15' heat shock on day 11 and 12. Fourth, as seen in Figure 6, heat has little effect on the levels of actin RNA.
bryos of a variety of species are incapable of mounting a heat shock response. The ability to mount a heat shock response is achieved at the cellular blastoderm stage in Drosophila (Graziosi et al., '80; Dura, '81; Bergh and Arking, '84),at the hatched blastula stage in the sea urchin (Roccheri et al., '81; Howlett et al., '831, at the mid- to late-blas-
DISCUSSION
Previous work from several laboratories has shown that the oocytes and early em-
RAT EMBRYO HEAT SHOCK RESPONSE
tula stage in Xenopus (Bienz, '84; Heikkila et al., '85, '86), and at the blastocyst stage in mouse and rabbit (Wittig et al., '83; Heikkila and Schultz, '84; Heikkila et al., '85, '86). Results presented in this manuscript extend these observations to the early to midpostimplantation stages of the mammalian embryo. Like the mouse blastocyst, the day 9 rat embryo head-fold stage is also capable of mounting a heat shock response. Unlike the mouse blastocyst in which heat induced primarily the level of HSP 70 (Heikkila et al., '85), in the rat embryo heat induced a set of HSPs, one of which (HSP f) is probably HSP 70. The heat shock response is prominent in day 10 embryos when a set of 8 HSPs are induced. Subsequent stages, days 11 and 12, exhibit attenuation of the embryos ability to respond to an acute heat shock culminating in the absence of a response on day 12 when measured by the synthesis of HSP f, as well as other HSPs. In addition, our results indicate that the ability of rat embryos to accumulate RNA for one of the HSPs, i.e., HSP 70, in response t o an acute heat shock is also developmentally regulated. This developmental regulation is seen as an attenuation of HSP 70 RNA accumulation as a function of the length of the heat shock. Day 9 embryos are able to mount the most sustained heat shock response when viewed as the ability to accumulate HSP 70 RNA after an acute heat shock (43"C, 60'). As development proceeds from day 10 to day 12, the embryo becomes increasingly less able to accumulate HSP 70 RNA when exposed to a 43°C heat shock of increasing duration. Moreover, this attenuation of the ability of the embryo to accumulate HSP 70 RNA after acute heat shocks parallels the attenuation of HSP 70 synthesis as revealed by two-dimensional gel electrophoresis. What is the mechanism(s) controlling this stage dependent expression of a heat shock response in mammalian embryos? Little definitive data are available but several possibilities have been suggested. Krone and Heikkila ('88) have shown that heatinduced HSP 30 gene expression does not occur until the tailbud stage in Xenopus, unlike HSP 70, which is induced by heat at the late blastula stage. These authors suggest that heat-induced expression of HSP 30 might be controlled by an inhibitor-HSP 30 gene interaction. Another possibility is that
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heat shock transcription factors (HSTF) or accessory transcription factors necessary for the induction of heat shock genes are absent or sequestered at different stages of development. Likewise, the fact that not all heat shock proteins are expressed in embryos capable of mounting a response, i.e., blastocyst through day 11, may reflect the fact that different heat shock genes require different transcription factors or that different heat shock genes differentially bind a limited pool of HSTF. Another possibility, suggested by the work of Di Domenico et al. ('82), is that heat shock genes are inducible at all times, but a t certain stages the HSP transcripts are unstable, rapidly turned over, and therefore not translated into HSPs. Our data from day 12 embryos, which do not synthesize any detectable HSP f in response to heat, also accumulate a very low level of HSP 70 mRNA after prolonged heating (43°C' 30-60'). Still another possibility is that HSP transcripts are induced by heat at all developmental stages; however, the translational machinery is incapable of translating some or all of these transcripts at different developmental stages. Although other regulatory mechanisms are possible, it is of great interest t o understand the mechanism used by embryos to regulate their response to heat and other stresses known t o be capable of inducing the so called stress response. Perhaps more interesting is our finding that there is a close relationship between the ability of the postimplantation embryo to mount a heat shock response and the induction of abnormal development as a consequence of an exposure to elevated temperature. Thus, one period of rat development during which hyperthermia can induce abnormal embryogenesis, i.e., days 8.5-10.0, is also the period of development during which hyperthermia can induce a detectable heat shock response. This relationship between hyperthermia-induced teratogenesis and hyperthermia-induced heat shock response has also been studied in Drosophila embryos where hyperthermia induces so called phenocopies (Capdevila and GarciaBellido, '74; Milkman, '66; Mitchell and Peterson, '82; Eberlein, '86; Peterson and Mitchell, '87). In addition, a variety of agents that are teratogenic in mammals are also teratogenic in Drosophila and induce a heat shock response (Buzin and BourniasVardiabasis, '84).
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P.E. MIRKES ET AL
Given this relationship between the heat shock response and abnormal embryogenesis, the obvious question is what role, if any, does the heat shock response play in stressinduced teratogenesis? Heat-induced multihair phenocopies in Drosophila are associated with disruption of protein synthesis and subsequently with discoordinate gene expression (Mitchell and Peterson, '81) and a similar disruption of gene activity in the mammalian embryo could be expected to have profound effects. This is especially true in the postimplantation mammalian rodent embryos undergoing the complex process of organogenesis with its demands of coordinated gene activation and inactivation (German, '84). Alternatively, one or more of the HSPs may be directly involved in inducing abnormal development. Perhaps the inappropriate activation of specific HSP genes that are now known to be constitutively produced at certain developmental stages (Bensaude and Morange, '83; Bensaude et al., '83; Zimmerman et al., '83; Bienz, '84; Kothary et al., '87) plays a role in subsequent pathogenesis induced by heat or other stresses. Furthermore, recent evidence suggests that the heat shock response may play a role in protecting the mammalian embryo from the deleterious effects of heat (Whittig et al., '83; Heikkila et al., '85; Mirkes, '87; Walsh et al., '87). Perhaps the heat shock response may play a dual role, on the one hand protecting the embryo and on the other disrupting embryonic development. Which outcome is observed would depend on developmental stage of the embryo at the time of insult, the severity of the stress, and other factors yet to be determined. ACKNOWLEDGMENTS
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