Screening a cDNA library generated from poly(A)+RNA of Drosophila cleavage embryos, we selected a cDNA clone (pDE20.6). The cDNA hybridized specifically with a poly(A)+RNA that is capable of restoring embryos from u.v.-caused inability of pole cell formation. The RNA hybrid-selected by pDE20.6 was also able to induce pole cells in the anterior region of embryos, if it was coinjected with u.v.-irradiated polar plasm, although the RNA or irradiated polar plasm alone was not effective. Pole cells thus formed in the anterior or in the u.v.-irradiated posterior region were identified by polar granules and nuclear bodies, morphological markers for normal pole cells. Furthermore, the RNA-induced pole cells were able to migrate into gonadal rudiments. The nucleotide sequence of pDE20.6 cDNA insert was highly homologous with the mitochondrial large rRNA (IrRNA) gene, but not with any nuclear DNA sequences. Using pDE20.6 as a primer, a full-length cDNA of mitochondrial IrRNA was generated and cloned. The RNA transcribed in vitro from the cDNA was able to restore pole cell formation. The cDNA hybridized only with a 1.5 kb poly(A)+RNA on a Northern blot. The 1.5 kb RNA sedimented more with the post-mitochondrial (P3) fraction than with the mitochondrial (P2) fraction, while the majority of transcripts from another mitochondrial gene was detected in the P2 fraction.

Germ plasm or a localized ooplasm is associated with the germ line in many animal groups (Beams and Kessel, 1974, Eddy, 1975). In Drosophila, germ plasm (also called polar plasm) is localized in the posterior polar region of oocytes and cleavage embryos. After the ninth intravitelline nuclear division, nuclei penetrate the polar plasm to form protrusions, each containing a nuclei and part of the polar plasm. The protrusions later become pole cells, which represent the germ line in this animal (Underwood et al. 1980; Technau and Campos-Ortega, 1986).

The polar plasm is able to induce pole cells wherever it is transplanted (Illmensee and Mahowald, 1974,1976; Illmensee et al. 1976), and also is able to restore fertility to u.v.-sterilized embryos (Okada et al. 1974). On the other hand, a subcellular fraction (P3 fraction) sedimented from a homogenate of early cleavage embryos, was shown to restore the ability to form pole cells to u.v.-irradiated embryos (Ueda and Okada, 1982). Furthermore, poly(A)+RNA extracted from P3 fraction can restore pole-cell-forming ability to u.v.-irradiated embryos, whereas no such restoration activities were detectable in poly(A)+RNA prepared from blastoderm embryos (Togashi et al. 1986). Pole cells formed in the embryos that have been u.v.-irradiated and then injected with the poly(A)+RNA are able to migrate into mesodermal gonads to become a cell type morphologically very similar to primordial germ cells (PGCs). However, those PGCs are unable to develop into germ cells. Moreover, the poly(A)+RNA is incapable of inducing pole cells in ectopic sites (Togashi et al. 1986). Based on these observations, we have presumed that the function of poly(A)+RNA from the P3 fraction represents part of the polar plasm function. That is, the poly(A)+RNA takes part in forming pole cells, with the characteristic morphological markers and properties to migrate into gonads; those pole cells, however, need a germ cell determinant proper for their further differentiation into germ cells. Thus the term ‘pole cell’ is henceforth used for a cell with polar granules and nuclear bodies, morphological markers for normal pole cells, and with an ability to migrate into mesodermal gonads to become a morphologically identifiable PGC.

In order to investigate whether the poly(A)+RNA (or its translation products) participates in pole cell formation in normal development, we isolated a cDNA of the poly(A)+RNA that restores ability to form pole cells to u.v.-irradiated embryos. In addition, presuming that polar plasm is a complex of plural factors sharing roles in the developmental process from pole cell formation through germ cell determination, our study of the cDNA would provide us with a useful probe for further analyses of polar plasm function.

In this paper, we report that (1) the isolated cDNA was almost identical to the mitochondrial large ribosomal RNA (IrRNA) gene of Drosophila melanogaster, (2) the transcript from a full-length cDNA of mitochondrial IrRNA was able to restore pole-cell-forming ability to u.v.-irradiated polar plasm, and (3) the remarkable amount of the 1.5 kb RNA hybridizing with the cDNA is sedimented in the P3 fraction, which includes very few mitochondria. In addition, we describe the morphology and developmental fate of pole cells formed in u.v.-irradiated embryos due to the injection of the 1.5 kb RNA.

Drosophila melanogaster strains

Embryos of the wild-type strain Oregon-R were used for preparing subcellular fractions from which poly(A)+RNA and DNA was extracted, and also as recipients for testing for pole-cell-inducing activity in the anterior pole. Embryos from the mutant strain rnwh e11 were used as recipients for microinjection into u.v.-irradiated posterior poles, and also used for histological observation of embryonic gonads.

Poly(A)+RNA and DNA preparation from embryos The procedures for collection of embryos and fractionation of the homogenate of the embryos were described previously (Togashi et al. 1986). Oregon-R female flies were allowed to lay eggs for 40 min on agar plates, the embryos were then collected, washed with distilled water and stocked in liquid nitrogen. The stage of embryos at the egg collection was 20±20min after egg laying (AEL) or at early cleavage. Blastoderm embryos (150±20min AEL) were obtained by incubation of the collected embryos for 130 min at 25 °C.

After thawing in 10 volumes of homogenizing medium, the embryos were gently homogenized. The homogenate was centrifuged at 1000g for 10min, 12000g for 10min, and 27000g for 60min. The pellets were collected separately and designated as Pl, P2 and P3 fractions. Nucleic acid was phenol-extracted from these fractions. RNA was purified from the nucleic acid solution by DNase treatment followed by ethanol precipitation. DNA was extracted from the solution by ethanol precipitation following RNase treatment. The RNA fraction was further separated into poly(A)+RNA and poly(A)-RNA by oligo(dT)-cellulose (Collaborative Research) chromatography.

Colony hybridization

cDNA was synthesized from the poly(A)+RNA extracted from the P3 fraction prepared from cleavage embryos (20±20min after egg laying, AEL) according to the method of Maniatis et al. (1982). A cDNA library was constructed by inserting the cDNA into pBR322 at the PstI site and transforming the plasmid into the DH1 strain of E. coli. The library was plated at a density of about 500 colonies per plate (9 cm in diameter), and 10 plates (5000 colonies) were analyzed by colony hybridization in the presence of competitors. The colonies were transferred to duplicate filters, which had been treated according to Hanahan and Messelson (1983). After a pretreatment for 6h at 42°C in 50% formamide, 5xSSPE, 0.1 % SDS, 20 μg ml-1 polyadenylate, and 100 μg ml-1 salmon sperm DNA, the filters were incubated for hybridization in the same solution containing either poly(A)+RNA from cleavage embryos kinase-labeled with 32P (2.5x106cts min-1 ml-1, 25ngml-1) and cold competitor poly(A)+RNA from blastoderms (150±20min AEL) (10 μg ml-1), or 32P-labeled poly(A)+RNA from blastoderms (2.5x106cts min-1 ml-1, 25ngml-1). Hybridization was continued for 40 h at 42 °C, then filters were washed and exposed for autoradiography at -70°C with an intensifying screen for 1 week. From this competitive colony hybridization procedure, we selected clones that were positive in the filters probed with poly(A)+RNA from cleavage embryos, but negative when probed with poly(A)+RNA from blastoderms.

Hybrid selection

2 μg of the recombinant plasmid per clone was digested by HaeIII, denatured in 0.4N-NaOH and 0.6M-NaCl, and pipetted onto Gene Screen Plus filters (0.5 cm in diameter, NEN). The filters were washed in 96% formamide, 10 mM-Tris-HCl (pH7.5), and 10mM-EDTA, and prehybridized for 2 h at 42°C in 65% formamide, 20mM-Pipes (pH6.4), 1% SDS, 0.4 M-NaCl, 100 μgml-1 yeast tRNA, and ImM-EDTA. Hybridization was carried out in the same solution containing 500pg ml-1 poly(A)+RNA from cleavage embryos for 12h at 42°C. To remove non-specifically bound poly(A)+RNA, the filters were vortexed twice for 1 min each in 10 mM-Tris-HCl (pH7.6), 0.15M-NaCl, ImM-EDTA, and 0.5% SDS at65°C. The poly(A)+RNA hybridizing to the DNA on the filters was eluted by rinsing in 96% formamide, 10 mM-Tris-HCl (pH7.5) and 10mM-EDTA for 30min at 65°C, and was precipitated by ethanol with 8.3 μg of yeast tRNA. The same DNA-holding filters were hybridized and eluted three times, and the eluted poly(A)+RNA was gathered and dissolved in 15 μl of injection buffer (55mM-NaCl, 40mM-KCl, 15 mM-MgSO4, 5mM-CaC12, 10mM-Tricine, 20mM-glucose, and 30mM-sucrose at pH 6.9). The RNA eluted from the filter was injected into u.v.-irradiated embryos.

Nucleotide sequencing

Two Pstl-Ddel fragments of the cDNA from pDE20.6 were inserted between PstI and Hindi sites of the pGEM-3 vector (Promega Biotec). The fragments were sequenced by the dideoxy chain termination method (Sanger et al. 1977) using double-stranded plasmid DNA as a template and synthetic oligonucleotide hybridizing with the T7 promoter region of the vector as a primer.

Southern and Northern blot hybridization

DNA was digested with the indicated restriction enzymes and separated on a 0.8 % agarose gel in TBE buffer. The DNA was transferred to Gene Screen Plus filters (NEN) by a capillary blotting method with 0.4M-NaOH and 0.6M-NaCl.

Poly(A)+RNA was size-fractionated on a 1% agarose gel containing 40mM-Mops (pH7.0), lOmM-sodium acetate, 5 mM-EDTA, and 1 % formaldehyde, and blotted to the filters in 10×SSC.

Hybridization was carried out at 42 °C for 12 h in 65% formamide, 50 mM-Tris-HCl (pH 7.5), 1% SDS, 100 pg ml-1 salmon testis DNA, 5xDenhardt’s solution, 0.4M-NaCl, and 5×105ctsmin-1ml-1 antisense RNA probe transcribed under the control of T7 promoter from pGP6.4, which carries the 101 bp fragment from pDE20.6 (see Fig. 5). Antisense RNA probe hybridizing with ND-1 RNA is transcribed from a plasmid carrying a 157 bp Scal-EcoRI fragment from the 5’ end region of the ND-1 gene (a mitochondrial gene) using SP6 RNA polymerase (Promega Biotec). The filters were washed in 2×SSC at room temperature for 20 min and 2×SSC with 1% SDS at 65°C for 60min. Autoradiography was done for 20 h at -70°C with an intensifying screen.

Measurement of cytochrome C oxidase activity

Each of the P2 and P3 precipitates prepared from 80 mg of cleavage embryos was suspended in 160 μl of the homogenizing buffer. 20 of the suspension and 50 μl of the reduced cytochrome C solution were added to 930 μl of 50 mM-Tris-HCl (pH 7.5) and 5% Triton X-100. Immediately after mixing, A55onmmin-1 was measured. Cytochrome C oxidase activity in each fraction is proportional to the value of Assonmmin-1 in this assay.

Microinjection

Microinjection techniques were principally the same as previously reported (Togashi et al. 1986). Briefly, embryos aged 30±20min AEL were u.v.-irradiated (280nm, 200Jm-2) in the posterior region. The irradiated embryos were injected in the posterior with an RNA sample (0.1 nl per embryo). After injection, the embryos were allowed to develop to the blastodermal stage and examined under a microscope for the presence or absence of pole cells.

For microinjection at the anterior, recipient embryos were dechorionated and placed on a glass slide with double-sided Scotch tape before injection with the RNA solution. For supplementing u.v.-irradiated polar plasm with RNA, polar plasm taken into a glass micropipette from Oregon-R embryos (which had been u.v.-irradiated posteriorly with 280nm at 100 J m-2) was expelled from the pipette into oil as a drop, into which RNA was injected and mixed. The mixture was collected in the pipette for injection into embryos. After injection, the embryos were kept for development to the blastodermal stage and observed for the presence of ‘pole cells’ resting on the outer surface of the anterior blastodermal layer.

Histological observation

Embryos were fixed by the method of Zalokar and Erk (1977) and embedded in epoxy resin according to the method of Spurr (1969) for transmission electron microscopy. Thin sections were cut with an LKB Nova Ultramicrotome and observed under a JEOL 100B electron microscope.

16 h AEL embryos were fixed in an alcohol-formol-acetic acid fixative and processed for paraffin sections. Serial sections were cut and stained with hematoxylin for observation of gonads.

Full-length cDNA of mitochondrial IrRNA

Full-length cDNA of mitochondrial IrRNA was synthesized on poly(A)+RNAs extracted from a P3 fraction prepared from cleavage embryos. A cDNA-synthesizing kit (Boehringer) was used with a denatured 167 bp fragment from the 3’-end region of pDE20.6 cDNA as a primer. The cDNA was inserted into the HindlU-Psil site of a pGEM-3 vector (Promega Biotec). Sense RNA or antisense RNA was transcribed from the cDNA with Sp6 or T7 RNA polymerase, respectively, according to a protocol from Promega Biotec. The transcribed RNA was dissolved in the injection buffer and was microinjected into u.v.-irradiated embryos (0.1 nl per embryo).

Isolation of the cDNA clone of poly(A)+ RNA required for pole cell formation in u. v. -irradiated embryos

5000 clones in a cDNA library generated from poly(A)+RNA extracted from the P3 fraction (from a homogenate of cleavage embryos) were screened in the following two steps. First, cDNA clones of poly(A)*RNA exclusively present in 20min AEL embryos were selected by competitive colony hybridization. This screening plan was based on the fact that pole-cell-forming capacity is restored to u.v.-irradiated embryos by poly(A)+RNA from cleavage embryos, but not by poly(A)+RNA extracted from blastodermal embryos (Togashi et al. 1986). The first screening isolated 100 cDNA clones, which were then screened using the hybrid-selection method to select the cDNA clones whose complementary RNA restored pole-cellforming ability to u.v.-irradiated embryos.

It was confirmed that the poly(A)+RNA hybrid-selected by the 100 clones en masse caused a significant rise in restoration rates (Table 1). 17 of the 100 clones were found to be particularly cleavage-stage-specific with stronger hybridization signals than other clones in the competitive colony hybridization. The poly(A)+RNA selected by these 17 clones had polecell-inducing activity in u.v.-irradiated embryos (Table 1). Southern blot analysis revealed homology between 6 of the 17 clones (data not shown). Poly(A)+RNA selected by the 6 clones, and by a single clone designated pDE20.6, which carried the longest cDNA insert among the 6 clones, exhibited a statistically significant increase in restoration rates (Table 1). In contrast, no significant rise in restoration rate resulted from injection of poly(A)+RNA hybrid-selected by pBR322, or by another set of 8 clones, chosen from the 17 but with no homology to pDE20.6.

Table 1.

Pole-cell-inducing activity of RNA selected by hybridization with the cDNA clones

Pole-cell-inducing activity of RNA selected by hybridization with the cDNA clones
Pole-cell-inducing activity of RNA selected by hybridization with the cDNA clones

To evaluate the accuracy of the hybrid-selection procedure, Northern blots of hybrid-selected poly(A)+RNA were probed with pDE20.6. The pDE20.6 hybridized with a 1.5 kb RNA on the Northern blot of poly(A)+RNA from cleavage embryos (Fig. 1). As expected, the hybridization signal was prominent in RNA hybrid-selected by the pDE20.6, but not detectable in RNA hybrid-selected by the 8 clones with no homology to the pDE20.6.

Fig. 1.

Northern blot hybridization of poly(A)+RNA from P3 fraction prepared from cleavage embryos (lane 1), RNA hybrid-selected by pDE20.6 (lane 2) and by a mixture of 8 clones non-homologous to the pDE20.6 (lane 3), probed with antisense RNA transcribed from pGP6.4 that carries the 101 bp fragment from pDE20.6 (see Fig. 3). Poly(A)+RNA was dissolved into 15/4 of the injection buffer (see Materials and Methods) at 25 μg per 15μl, of which 6 μl was loaded per lane (lane 1). Hybrid-selected RNA sample was dissolved into 15/4 of the injection buffer and 6μl loaded per lane (lane 2 and 3). Arrowhead indicates 1.5 kb.

Fig. 1.

Northern blot hybridization of poly(A)+RNA from P3 fraction prepared from cleavage embryos (lane 1), RNA hybrid-selected by pDE20.6 (lane 2) and by a mixture of 8 clones non-homologous to the pDE20.6 (lane 3), probed with antisense RNA transcribed from pGP6.4 that carries the 101 bp fragment from pDE20.6 (see Fig. 3). Poly(A)+RNA was dissolved into 15/4 of the injection buffer (see Materials and Methods) at 25 μg per 15μl, of which 6 μl was loaded per lane (lane 1). Hybrid-selected RNA sample was dissolved into 15/4 of the injection buffer and 6μl loaded per lane (lane 2 and 3). Arrowhead indicates 1.5 kb.

These results suggest that pDE20.6 contains the cDNA of RNA required for pole cell formation in u.v.-irradiated posterior pole. However, a slight rise in restoration rate was observed even when control RNAs, which are selected by DNA without any homology to pDE20.6, were injected (Table 1). This partial restoration may be due to an unknown ‘surgery’ effect of injection, but not due to specific activities of RNA selected by these clones. In fact, even tRNA, used as a carrier, showed a slight rise in restoration rates in this assay system (Table 1). Statistical tests show that the rise in restoration rates observed in embryos injected with RNA selected by pDE20.6 is significantly higher than the rise caused by injection of these control RNAs (Table 1).

Morphology and developmental fate of the pole cells formed in u. v. -irradiated embryos injected with pDE20.6-selected RNA

Embryos u.v.-irradiated posteriorly and injected with RNA hybrid-selected by pDE20.6 formed round cells located on the outer surface of the blastodermal cell layer in the posterior region (Fig. 2A). Transmission electron microscopy (Fig. 2B-D) demonstrated that those round cells include polar granules and nuclear bodies, which are specific anatomical markers of normal pole cells. These cells thus can be regarded as anatomically very similar to normal pole cells, and also similar to those pole cells induced by injection of either intact polar plasm or total poly(A)+RNA extracted from cleavage embryos (data not shown: cf. Togashi et al. 1986).

Fig. 2.

Light and transmission electron micrographs of sections through the posterior polar region of blastodermal embryos that have been u.v.-irradiated and injected with the 1.5 kb RNA hybrid-selected by the pDE20.6 (A, B, C, D, posterior pole to the top), and the anterior polar region of blastoderms injected with a mixture of the hybrid-selected RNA and u.v.-irradiated polar plasm (E, F, G, H, anterior pole to the top). Arrows in A and E indicate pole cells. Arrows and arrowheads in TEM (C, D, G, H) indicate nuclear bodies and polar granules in the pole cells, respectively. Bars: 10μm (A, E), 5μm (B, F) and 1 μm (C, D, G, H).

Fig. 2.

Light and transmission electron micrographs of sections through the posterior polar region of blastodermal embryos that have been u.v.-irradiated and injected with the 1.5 kb RNA hybrid-selected by the pDE20.6 (A, B, C, D, posterior pole to the top), and the anterior polar region of blastoderms injected with a mixture of the hybrid-selected RNA and u.v.-irradiated polar plasm (E, F, G, H, anterior pole to the top). Arrows in A and E indicate pole cells. Arrows and arrowheads in TEM (C, D, G, H) indicate nuclear bodies and polar granules in the pole cells, respectively. Bars: 10μm (A, E), 5μm (B, F) and 1 μm (C, D, G, H).

There were also similarities in the developmental fate between pole cells induced in u.v.-irradiated embryos by total poly(A)+RNA and those induced by an RNA hybrid-selected by pDE20.6. Serial sections of 16 h embryos developed from blastoderms that had formed pole cells due to the injection of the pDE20.6-selected RNA after u.v.-irradiation were examined for PGCs in their gonads. Gonads in 10 out of 20 observed embryos included cells with nuclei larger than 5.5 μm in diameter. According to our previous work (Togashi et al. 1986), gonadal cells with nuclei larger than 5.5 μm in diameter can be safely judged as PGCs. Thus, some pole cells induced in u.v.-irradiated embryos by RNA hybrid-selected by pDE20.6 are probably able to migrate into mesodermal gonads to become ‘PGCs’, as are pole cells induced by total poly(A)+RNA from P3 or by intact polar plasm.

Pole cell formation in the anterior by coinjection of RNA and u.v.-irradiated polar plasm

When polar plasm is injected into the anterior of cleavage embryos, pole cells are induced there (Illmensee and Mahowald, 1974, 1976; Togashi et al. 1986). In contrast, hybrid-selected 1.5 kb RNA did not induce pole cells in the anterior. However, we found that the 1.5 kb RNA can induce round cells in the anterior, provided the RNA was coinjected with u.v.-irradiated polar plasm (Table 2). The round cells formed in the anterior by this coinjection were very similar to normal pole cells in (1) morphology, (2) resting on the outer surface of the blastoderm cell layer (Fig. 2E), and (3) inclusion of polar granules and nuclear bodies similar to those found in normal pole cells and in RNA-induced pole cells in the u.v.-irradiated posterior region (Fig. 2F,G,H).

Table 2.

Coinjection of RNA and u.v.-irradiated polar plasm into anterior region

Coinjection of RNA and u.v.-irradiated polar plasm into anterior region
Coinjection of RNA and u.v.-irradiated polar plasm into anterior region

Coinjection of irradiated polar plasm and poly(A)+RNA hybrid-selected by clones with no homology to the pDE20.6 never induced pole cells in the anterior. Transplantation of irradiated polar plasm alone did not induce any pole cells in the anterior (Table 2). Combining the results from the coinjection experiments in the anterior and from injection of pDE20.6-selected RNA into the posterior of u.v.-irradiated embryos, it may be deduced that pDE20.6 cDNA codes poly(A)+RNA required for pole cell formation in u.v.-irradiated embryos.

Nucleotide sequencing

The pDE20.6 clone carried about 600 bp of cDNA, of which we sequenced 383 bp from the 3’-terminus. This sequence contained a 25 bp poly(A) tract and a 358 bp unique sequence (Fig. 3A). We searched for homologous sequences in the data base (EMBL) and found that the only sequence showing a good match with the 358 bp in the pDE20.6 was the mitochondrial large ribosomal RNA (IrRNA) gene of Drosophila yakuba (Clary and Wolstenholme, 1985) with a 98% homology (Fig. 3B). The discrepancy of 2% may be due to the difference between D. yakuba and D. melanogaster. Recently, the 3’-terminal region of IrRNA was sequenced in D. melanogaster (Garesse, 1988). The sequenced part of pDE20.6 matched almost completely with the reported sequence (Fig. 3B).

Fig. 3.

Schematic presentation of the cDNA insert in the pDE20.6 clone (A), and nucleotide sequences of the 358 bp fragment from the pDE20.6 insert and of the 3’ terminal region of mitochondrial IrRNA gene of Drosophila yakuba (IrRNA(y)) (Clary and Wolstenholme, 1985) and D. melanogaster (IrRNA(m)) (Garesse, 1988). (B) Dotted line in A represents pBR322. The pDE20.6 insert was approximately 600 bp long, of which we sequenced 383 bp (shown in A by thick line and open box that represents 25 bp of poly(A) tract). Asterisks in B denote positions of match between pDE20.6 and the large mitochondrial IrRNA. The Dde I site indicated in A is boxed in B. The bar under the pDE20.6 sequence in B indicates the sequence (pGP6.4) used for the hybridization probe in experiments shown in Figs 1, 4 and 5.

Fig. 3.

Schematic presentation of the cDNA insert in the pDE20.6 clone (A), and nucleotide sequences of the 358 bp fragment from the pDE20.6 insert and of the 3’ terminal region of mitochondrial IrRNA gene of Drosophila yakuba (IrRNA(y)) (Clary and Wolstenholme, 1985) and D. melanogaster (IrRNA(m)) (Garesse, 1988). (B) Dotted line in A represents pBR322. The pDE20.6 insert was approximately 600 bp long, of which we sequenced 383 bp (shown in A by thick line and open box that represents 25 bp of poly(A) tract). Asterisks in B denote positions of match between pDE20.6 and the large mitochondrial IrRNA. The Dde I site indicated in A is boxed in B. The bar under the pDE20.6 sequence in B indicates the sequence (pGP6.4) used for the hybridization probe in experiments shown in Figs 1, 4 and 5.

Southern blot analysis was performed to examine whether the same sequence is also present in the nuclear genome. Single or double (HindIII and/or EcoRI) digested total DNA from adults was probed with the 101 bp fragment of pDE20.6 (Fig. 3B). Hybridization signals were detected only on the bands expected from a published restriction map of the mitochondrial IrRNA and adjacent genes (Battey et al. 1979; Garesse, 1988) (Fig. 4). This indicates that there is hardly any probability of a sequence homologous with pDE20.6 cDNA being in the nuclear genome. This result is supported by the absence of an in situ hybridization signal in salivary polytene chromosomes probed with a biotinylated sequence from pDE20.6 (data not shown).

Fig. 4.

(A) Southern blot hybridization of total Drosophila DNA digested by EcoRI (1), HindIII and EcoRI (2), or HindIII (3) probed with antisense RNA transcribed from pGP6.4 that carries a 101 bp fragment of pDE20.6 cDNA (barred in Fig. 5). 10 μg of DNA was loaded per lane.

Fig. 4.

(A) Southern blot hybridization of total Drosophila DNA digested by EcoRI (1), HindIII and EcoRI (2), or HindIII (3) probed with antisense RNA transcribed from pGP6.4 that carries a 101 bp fragment of pDE20.6 cDNA (barred in Fig. 5). 10 μg of DNA was loaded per lane.

Northern blot analysis

Since the nucleotide sequence of pDE20.6 was highly homologous with the mitochondrial ribosomal RNA gene, we surveyed the subcellular fractions for the presence of mitochondria and RNA that hybridizes with pDE20.6. The pDE20.6 probe hybridized only with a 1.5 kb poly(A)+RNA, similar in size to IrRNA, on the Northern blot of poly(A)+RNA from the P3 (postmitochondrial) fraction prepared from cleavage embryos (Fig. 5A). The Northern blot analysis also revealed that the hybridization signal was stronger in poly(A)+RNA extracted from the P3 fraction than in poly(A)+RNA extracted from the P2 (mitochondrial) fraction (Fig. 5A). To estimate the mitochondrial content in these subcellular fractions, we measured their cytochrome C oxidase activity, detecting a much higher activity in P2 than in P3 (relative activity of cytochrome C oxidase in the P3 fraction was 7.4, while that in the P2 was 100). In addition, mitochondrial DNA was detected only in the P2 fraction (Fig. 5B).

Fig. 5.

Northern (A, C, D) and Southern (B) blot hybridization probed with antisense RNA transcribed from the 101 bp fragment from pDE20.6 (A, B, D) or antisense RNA from the 157 bp fragment from mitochondrial ND-1 gene (C). Poly(A)+RNA for the Northern (A, C) and DNA for the Southern Blot (B) were extracted from the P2 (lane 1) or P3 (lane 2) fraction, both prepared from cleavage embryos (20+20 min AEL). DNA was digested with HindIII. (D) Northern blot hybridization of poly(A)+RNA from P3 fraction prepared from cleavage embryos (lane 1) and from blastodermal embryos (150±20min AEL) (lane 2). The amount of DNA or poly(A)+RNA loaded per lane were adjusted to the amount contained in each subcellular fraction prepared from 1000 embryos. Arrowheads in A and D and arrow in B indicate the 1.5 kb poly(A)+RNA and mitochondrial DNA fragment both hybridizing with the pDE20.6, respectively. Arrowhead in C shows the 1 kb poly(A)+RNA hybridizing with RNA probe from ND-1 gene.

Fig. 5.

Northern (A, C, D) and Southern (B) blot hybridization probed with antisense RNA transcribed from the 101 bp fragment from pDE20.6 (A, B, D) or antisense RNA from the 157 bp fragment from mitochondrial ND-1 gene (C). Poly(A)+RNA for the Northern (A, C) and DNA for the Southern Blot (B) were extracted from the P2 (lane 1) or P3 (lane 2) fraction, both prepared from cleavage embryos (20+20 min AEL). DNA was digested with HindIII. (D) Northern blot hybridization of poly(A)+RNA from P3 fraction prepared from cleavage embryos (lane 1) and from blastodermal embryos (150±20min AEL) (lane 2). The amount of DNA or poly(A)+RNA loaded per lane were adjusted to the amount contained in each subcellular fraction prepared from 1000 embryos. Arrowheads in A and D and arrow in B indicate the 1.5 kb poly(A)+RNA and mitochondrial DNA fragment both hybridizing with the pDE20.6, respectively. Arrowhead in C shows the 1 kb poly(A)+RNA hybridizing with RNA probe from ND-1 gene.

Since cytochrome C oxidase activities and mitochondrial DNA are associated with membraneous structures of mitochondria, it could be that enrichment of the 1.5 kb IrRNA in the P3 fraction results from leakage of transcripts from mitochondria during preparation of subcellular fractions. However, this possibility seems to be ruled out, because the transcript from the ND-1 gene, which is located immediately downstream of the IrRNA gene in the mitochondrial genome, is enriched in the P2 fraction (Fig. 5C). The above observations indicate that the amount of the 1.5 kb RNA present outside mitochondria is more than can be explained by leakage out of mitochondria during preparation.

The hybridization signal with pDE20.6 in poly(A)+RNA from the P3 fraction was variable depending on the stages of embryos from which the fraction was prepared. When the P3 fraction was prepared from blastodermal embryos, the signal was measured as 10−30% of the level of poly(A)+RNA extracted from the P3 of cleavage embryos (Fig. 5D).

Injection of transcripts from cDNA into u. v. -irradiated embryos

As mentioned above, pDE20.6, a 0.6 kb cDNA, hybridizes only with a 1.5 kb RNA that restores pole-cellforming ability to u.v.-irradiated embryos. Since the pDE20.6 is highly homologous with a portion of mitochondrial IrRNA gene, it is suggested that the 1.5 kb RNA with restoration activities is mitochondrial IrRNA. In order to obtain direct evidence supporting this hypothesis, a full-length cDNA of the 1.5 kb RNA was synthesized using pDE20.6 as a primer. The cDNA sequence obtained was highly homologous with the mitochondrial IrRNA gene Drosophila yakuba (Clary and Wolstenholme, 1985). Detailed description of the complete nucleotide sequence of the obtained cDNA will be published elsewhere.

The full-length cDNA was inserted into the pGEM-3 vector at a downstream site of the SP6 polymerase promoter. Mitochondrial IrRNA was synthesized in vitro on this cDNA using SP6 RNA polymerase, and injected into u.v.-irradiated embryos. Results summarized in Table 3 indicate that the transcript from the cDNA has obvious restoration activities, while the antisense RNA shows no significant restoration.

Table 3.

Pole-cell-inducing activity of RNA transcribed in vitro from the full-length cDNA of mitochondrial IrRNA

Pole-cell-inducing activity of RNA transcribed in vitro from the full-length cDNA of mitochondrial IrRNA
Pole-cell-inducing activity of RNA transcribed in vitro from the full-length cDNA of mitochondrial IrRNA

The strategy we employed for screening the pDE20.6 cDNA clone was principally to select cDNA clones that hybridized with the poly(A)+RNA capable of restoring pole-cell-forming ability to u.v.-irradiated embryos.

The pDE20.6, a 0.6 kb cDNA, hybridized only with a 1.5 kb poly(A)+RNA on a Northern blot of total poly(A)+RNA extracted from the P3 fraction. Northern blot analyses also revealed that the 1.5 kb RNA was detectable in RNA selected for its hybridization with pDE20.6, but not in RNA hybrid-selected by clones with no homology to pDE20.6. Since the condition of hybridization used in the hybrid-selection procedure was as stringent as that used in the Northern blot analysis of total poly(A)+RNA probed with the pDE20.6, it is unlikely that poly(A)+RNA other than the 1.5 kb RNA was selected by pDE20.6 and participated in the restoration of pole cell formation.

The 1.5 kb RNA was 3 to 10 times more abundant in the P3 fraction from cleavage embryos than in the same fraction from blastodermal embryos. The decrease of the 1.5 kb RNA in the P3 fraction during development from cleavage to blastoderm stages suggests a correlation between the 1.5 kb RNA and the restoration activity, which is also reduced during the same developmental stages from total poly(A)+RNA extracted from the P3 (Togashi et al. 1986). Thus pDE20.6, which exclusively hybridizes with the 1.5 kb RNA, probably carries cDNA of poly(A)+RNA required for pole cell formation in u.v.-irradiated embryos.

We showed that pDE20.6 cDNA was almost identical with the mitochondrial large ribosomal RNA (IrRNA) gene of Drosophila. Southern blot analysis and in situ hybridization onto polytene chromosomes using pDE20.6 as a probe indicated that no nuclear gene was homologous to the cDNA. The pDE20.6 cDNA hybridized only with a 1.5 kb RNA that is of the size of mitochondrial IrRNA. These results suggest that this 1.5 kb RNA is of mitochondrial origin. Furthermore, we showed that the transcript from the full-length cDNA to mitochondrial IrRNA had the same restoration activities as the hybrid-selected 1.5 kb RNA. These results support the idea that it is mitochondrial IrRNA that restores pole-cell-forming ability to u.v.-irradiated embryos.

One may argue that the restoration is due to a simple cure of u.v.-damaged mitochondria by the injection of mitochondrial IrRNA. It has been reported that u.v. causes swelling and vacuolation of mitochondria in Smittia (Kalthoff et al. 1975) and Xenopus (Ikenishi et al. 1974). However, in the u.v.-irradiated posterior region of Drosophila embryos, mitochondria did not show any discernible deformity in their fine structures (Yamazaki, unpublished observation). In addition, we have shown that mitochondrial membrane potential, which is essential for the respiratory function, is not lost in the u.v.-irradiated posterior region. The mitochondrial membrane potential can be monitored by the accumulation of a permeant cationic fluorescent dye, Rhodamine 123, in mitochondria (Johnson et al. 1981; Chen et al. 1982). This fluorescent dye stained mitochondria in the posterior region of both normal and u.v.-irradiated Drosophila embryos alike (Akiyama, unpublished observation). Furthermore, cytoplasm either from the anterior or from the lateral region of an embryo, which includes quite a few mitochondria, is unable to restore pole-cell-forming ability to u.v.-irradiated embryos (Okada et al. 1974; and the present observation). These observations support the possibility that the injected mitochondrial IrRNA rescues embryos, in which cellular functions required for pole cell formation have been damaged by u.v., independent of the respiratory function of mitochondria.

Ueda and Okada (1982) showed that the restoration activity is detected mainly in the P3 fraction, and scarcely in the P2 fraction. Our Northern blot analysis revealed that much more 1.5 kb RNA sedimented in the P3 fraction than in the P2 fraction. In contrast, the transcript from a mitochondrial gene located next to IrRNA gene was detected more in the P2 than in the P3 fraction. Furthermore, we showed that most of the mitochondria sedimented in the P2 fraction and very few sedimented in the P3. Those findings suggest that the presence of mitochondrial IrRNA outside mitochondria does not result from leakage during preparation of subcellular fractions. Moreover, the 1.5 kb RNA outside the mitochondria is presumed to be localized in polar plasm, since only the posterior polar plasm shows the restoration activity (Okada et al. 1974). We speculate that IrRNA is transported out of mitochondria into the cytosol and takes part in pole cell formation. U.v.-irradiation may impair the function of the 1.5 kb IrRNA outside mitochondria, and the injected RNA then substitutes for the inactivated RNA resulting in a restoration of pole-cell-forming ability. These speculations need direct evidence to support them. Unfortunately, our attempts to inhibit pole cell formation in normal embryos by injection of antisense mitochondrial IrRNA have so far been unsuccessful.

In the previous report, we presented data that suggest the existence of two u.v.-sensitive cytoplasmic factors, one responsible for formation of morphologically identifiable pole cells, and the other for the determination of those pole cells as germ cells (Togashi et al. 1986). A preliminary result from the present work agrees with this suggestion, because embryos that formed pole cells due to the injection of hybrid-selected IrRNA have so far never developed into fertile adults. The assumption is also supported by the phenotype of mutations, in which pole cell formation is not enough for germ line establishment. For example, mutations at loci such as agametic or ovo cause degeneration of pole cells before their differentiation into germ cells (Engstrom et al. 1982; Oliver et al. 1987).

We have shown that mitochondrial IrRNA can induce pole cells in ectopic sites only when it is coinjected with u.v.-irradiated polar plasm. That may suggest a requirement for an additional factor, which is u.v.-resistant and localized in polar plasm, for pole cell formation.

Recently, it was reported that pole cells formed even if only centrosomes, separated from nuclei, migrated into the periplasm of the posterior region of Drosophila embryos, but somatic cell formation was not initiated by these isolated centrosomes (Raff and Glover, 1989). Reorganization of cortical actin filaments mediated by centrosomes penetrating the posterior periplasm is claimed to be important for pole cell formation. On the other hand, if mitochondrial IrRNA also is responsible for pole cell formation in normal development, it is probable that the function of this RNA is involved in the mechanism underlying reorganization of cortical cytoskeleton.

We thank Drs Norihiro Okada and Osamu Numata for technical advice and Takahiro Akiyama for technical assistance. Our thanks are also due to Dr Robert W. Ridge for critical reading of the manuscript. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan and by a grant from Naito Foundation.

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