Variability of response of early Drosophila embryos to heat shock

Abstract

Several embryonic stages (nuclear multiplication stage up to blastoderm) were incubated at 37°C. The 84,000 dalton heat-shock protein was always present in the heat-shocked embryos at all these stages. This protein was also found in nonheat-shocked controls. Different amounts of two other heat-shock proteins, the 70,000 and 68,000 daltons, were synthesized depending upon the embryonic stage. A massive synthesis of these two later proteins was observed in embryos which were able to recover from the heat-shock.It has been reported that prior to nuclear migration, Drosophila melanogaster embryos incorporate very little uridine (Zalokar, '76) and that at the blastoderm stage the synthesis of RNA is significantly activated (Lamb and Laird, '76; McKnight and Miller, '76; Anderson and Lengyel, '79). These results suggest that during nuclear multiplication the zygotic genome is mainly, if not entirely, silent. This interpretation is also supported by the shortness of interkinesis: 4 minutes a t 24°C (Rabinowitz, '41). During this period the DNA is engaged in replicating 200 million base pairs and presumably there is little time for possible transcription.Transcription and translation of specific messages can be experimentally stimulated by incubation at 37°C in many tissues, cell cultures, and organs (Tissieres et al., '74; McKenzie et al., '75) We used heat-shock as a tool to investigate whether the corresponding genes can be activated, during the earliest stages of embryonic development. MATERIALS AND METHODS Flies maintenance and egg harvestingRoughly 10,000 flies (Oregon-R strain) were kept in a population cage at 24°C. Eggs were collected on food plates left in the cage for L 1 0 minutes. Details on egg harvesting were reported by Graziosi et al. ('79). Labelling of eggs and heat-shock treatmentThe eggs were dechorionated in 50% bleach for 2 minutes, permeabilized, and incubated as described by Limbourg and Zalokar ('73). WMethionine was added to the incubation medium to obtain an activity of 1 mCi/ml (The Radiochemical Centre, Hamersham, United Kingdom, 600 Ciimmol). The heat-shock was carried out in a water bath at 37°C for 15 minutes and the eggs were kept at this temperature further for 45 minutes in radioactive medium. ElectrophoresisThe SDS gels were run according to the method of Laemmli ('70). The O'Farrell ('75) technique was used for the two-dimensional gel electrophoresis. All the two-dimensional gels underwent fluorography as described by Bonner and Laskey ('74). RESULTSSeveral embryonic stages were considered: from early nuclear multiplication (20-to 30-minute average age) up to blastoderm (3 hours). The survival rate of embryos incubated at 37°C for 30 minutes and then transferred to 24°C without any further treatment was recorded. A large proportion of eggs 20 to 30-minUtes-old failed to hatch (about 20% survival rate) while 63-83% of embryos treated at 150 minutes (syncytial blastoderm) hatched as larvae. Treatment at 3 hours did not cause any mortality. A few hundred embryos were obser...

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

“…A similar observation has been reported for very early embryos of Drosophila melanogaster (10,24 (8,10,23). In this report, as Wittig et al (31) have previously reported, we showed that the same result is true for the eight-cell mouse embryo.…”

“…They transiently synthesize high levels of the same set of very conserved polypeptides, the heat shock proteins (HSP). A developmental control of the synthesis of these proteins has been previously demonstrated (8,10,23,24,26) In preblastoderm Drosophila embryos (8,10,24), high levels of some HSP are spontaneously expressed in the absence of stress, whereas the other HSP could not be induced by heat treatment.We have previously shown (2) (20,22,27 Hyperthermia. Cells were grown in tissue culture dishes (diameter, 35 mm).…”

Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo.

“…These authors referred to it as 'the comet'. In early Drosophila embryos (Graziosi et al, 1980;Loyd et al, 1981) the HSP equivalent to the mouse 89-kd and 70-kd have also been found to be spontaneously very abundant. In addition, the heat induction of HSP synthesis is reported to depend heavily upon the embryonic stage in Gel electrophoresis Isoelectrofocusing (O'Farrell, 1975) was performed with 15 cm polyacrylamide gels containing 1.6%7 3.5/10 and 0.4% 4/6 ampholines (LKB): 550 V were applied to the gel tubes for 16 h and then 1000 V for 1 h. Gels were extruded and kept at -20°C until used for the second dimension.…”

Localization of the hsp83 transcript within a 3292 nucleotide sequence from the 63B heat shock locus ofD. melanogaster

“…Fifth, the 63B region is induced to puff in isolated salivary gland nuclei in response to uninduced cellular extracts that do not cause puffing at other heat shock loci (8). Sixth, hsp83 is a common protein in early embryos kept at normal growth temperatures, and is also the only heat shock protein detectable in heat-induced unfertilized eggs (9). Furthermore, of the seven major heat shock genes only the mRNAs encoding hsp83, hsp27, and hsp26 are present during normal development in non-heat shocked adult ovaries and in preblastoderm embryos.…”