Pole cell formation in Drosophila melanogaster

DEVELOPMENTAL

BIOLOGY

75, 419-430 (1980)

Pole Cell Formation

in Drosophila

MARGARETMACMORRISSWANSON'AND Division

of Natural

Sciences, Thimann

Laboratories,

University

melanogaster’ CLIFTON A. POODRY

of California,

Santa Crur, California

95064

Received June 12, 1978; accepted in revised form October 11. 1979 The development of the pole cells of Drosophila melanogaster has been investigated in a temperature-sensitive developmental mutant strain, shibire’“. Shibire (shi”) mutants have periods of temperature sensitivity in early embryogenesis; embryos shifted to 29°C during those periods subsequently lack pole cells. The temperature-sensitivity profiles of four independently isolated alleles indicate that the shibire mutation is responsible for this defect in development. Carefully timed heat pulses established that there is a broad range in times of sensitivity extending even to the stage when the pole cells have begun to form. Scanning electron microscopy of the heattreated embryos was compared to that of controls to illustrate the extent of the defects in mutants. Transmission electron microscopy of sectioned embryos lacking pole cells revealed normal stagespecific morphology of polar granules in the posterior end. Speculations are presented on the significance of pole cell formation as a means of segregating germ cell determinants. INTRODUCTION

The development of the germ cells in Drosophila involves a process that extends over the entire period of embryonic and postembryonic development. The cell lineage of the germ cell primordia can be traced, however, to the earliest specializations in the embryo, the pole cells (Hegner, 1908). These distinctive round cells are pinched off from the posterior end of the egg surface immediately after nuclei arrive in that region of the egg cytoplasm. Thus, the pole cells are segregated from the rest of the embryo well before cleavage occurs in the bulk of the egg. They remain outside the blastoderm as it cellularizes and enters gastrulation. Furthermore, pole cells may be a singular example of cytoplasmic localization of morphogenetic determinants in insect embryos (Sander, 1976). Specific injury to the posterior pole of the egg results in loss of subsequent germ cell development (Hegner, 1908; Geigy, 1931; Jazdowska-Zagrod’ Supported by NIH Research Grant GM 20401. ’ Present address: Division of Biological Sciences, Tucker Hall, University of Missouri, Columbia, MO. 65211.

zinska, 1966). Replacement of injured material by cytoplasmic transplantation from a normal posterior polar region restores the embryos’ capacity to form the pole cells (Okada et al., 1974; Warn, 1975). Transplantation of the polar cytoplasm to an ectopic location also produces cells resembling pole cells which are competent in germ-line function (Illmensee and Mahowald, 1974, 1976). The specific transplantable substance in the posterior polar cytoplasm has not been identified, but morphogenetic studies (Counce, 1963; Mahowald, 1962) have revealed distinctive, dark-staining inclusions, the polar granules. There has been speculation that these subcellular organelles are or contain the germ cell determinants (Mahowald, 1971). In some insects such as Wachtliella, the primordial germ cells are distinctive in their nuclear cytology as well as their cellular morphology at an early stage. The primordial germ cells retain a full chromosomal complement while all somatic cells undergo chromosome elimination (GeyerDuszynska, 1959). Clearly, some process occurs at a very early stage which commits these cells to become part of the germ line 419

0012-1606/80/040419-12$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOCIY

and results in chromosome alteration. It is unknown, either in Cecidomyidea or in Drosophila, in which there is no visible effect on the genetic material, whether the physical segregation of the pole cells from the rest of the embryo is a necessary part of germ cell determination. In this paper we report experiments in which the shibire mutation, which has temperature-sensitive pleiotropic effects on development in Drosophila melanogaster (Grigliatti et al., 1973; Poodry et al., 1973), was used to interrupt pole cell formation. Mutant embryos which are heat pulsed during the first 2 hr of development can survive to adulthood but lack germ cells due to a disruption in pole cell formation (Grigliatti, 1974, personal communication). An analysis of the temperature-sensitive periods is described along with an attempt to identify the aspect(s) of the normal process which is disturbed. Polar granule presence, nuclear migration, and membrane formation have been examined with the aim of using the mutation to help identify the important components of the normal process. MATERIALS

AND

METHODS

Flies The allele, shitsl was used as the mutant strain in most of the work presented here and Oregon R-C strain flies were used as wild-type. Allele names are stated in the text whenever shz*‘, shp3, or shitS6 were used. All stocks were raised on standard medium in cornmeal-agar-sugar-yeast half-pint bottles at 22°C.

Egg Collections Five- to ten-day-old, mated female flies were placed in empty half-pint bottles inverted over petri plates of agar spread with a paste of vinegar and yeast. Flies were left to deposit eggs for 0.5 to 2 hr at a time. In cases in which embryos were not subsequently staged, the first collection (prelay) was discarded to eliminate eggs retained in the female and deposited at advanced

VOLUME ?5,1980

stages. In all cases where embryos were separated by developmental stages, they were dechorionated chemically (in 2.6% sodium hypochlorite) .

Heat Pulses High-temperature treatments were given by floating the small plastic collection plates on the surface of a covered Blue M water bath. Temperature was monitored by a YSI temperature recorder responding to a thermistor probe in the incubation plate. Samples for 20- to 30-min pulses were placed in preheated plates. Samples treated for longer periods were shifted up from 22°C plates and took 3-5 min to reach desired temperature.

Whole Mounts Embryos were fixed and prepared as whole mount specimens according to a modification of the method of Zalokar and Erk (1976), using a Feulgen staining procedure (Humason, 1972).

Electron Microscopy Eggs were fixed first in heptane-25% glutaraldehyde for 1 to 2 min and then transferred to an aldehyde fixative containing acrolein, DMSO, and glutaraldehyde (modified Kalt and Tandler, according to Illmensee and Mahowald, 1974). After short incubation in fixative, the vitelline membrane was removed with tungsten needles. In many cases the anterior end of the egg was also removed to allow identification of the posterior pole when embedding and sectioning. After primary fixation for approximately 12 hr at 4°C samples were washed and postfixed in 1% osmium tetroxide. Uranyl acetate (1 hour, 1% aqueous) was used as an additional postfixation. Samples were dehydrated through ethanol and were embedded for transmission electron microscopy (TEM) in Epon/Araldite or Araldite. Thin sections were cut on a Sorvall MT-2 ultramicrotome, stained with uranyl acetate and lead citrate. Sections were ob-

SWANSON AND POODRY

served and photographed 1OOB at 60 kV. Scanning

Electron

with

Pole Cell Formation

a JEOL

Microscopy

From 100% ethanol, samples were taken through an amyl acetate series and dried through the critical point of carbon dioxide. Dried samples were mounted on stubs with sticky copper tape and coated alternately (three to four times) with carbon and gold in a vacuum evaporator (Varian, VE 10). A JEOL JSM-2 scanning electron microscope was used at 10 kV for microscopy of samples. They were transferred from stubs into 100% ethanol and the usual process used for embedding samples for TEM was followed. RESULTS

Sensitivity

of Four shi Alleles

One-hour heat pulses of 29°C given to shi’“’ embryos at prepole cell stages (immediately after oviposition) prevent formation of pole cells. We tested three other shi alleles, shP*, shP3, and shit”“, with 1-hr pulses to prepole cell staged embryos and found that pole cell formation was inhibited in all cases at temperatures below that which disrupted the process in the wildtype strain, Oregon R. The differences in the critical temperature for this phenotype

421

in D. melanogaster

parallel other developmental effects and the differences seen between these four shi alleles (Poodry, unpublished observations). While shit”’ and shits6 require higher temperatures of 31-32°C to disrupt pole cell formation, shit”’ and shit”” are affected by temperatures as low as 27528°C. The wild-type strain Oregon R shows loss of pole cells after treatment at 32-33°C. Timing

of Sensitivity

To establish the bounds of any specific short temperature-sensitive period (tsp) for the effect, 20- and 30-min pulses of 29°C were given to prepole cell .shP’ embryos. The heat-pulsed embryos were fixed at the beginning of gastrulation and scored with the light microscope for the presence of pole cells. The elapsed time from the heat pulse to gastrulation was used to establish the timing of each heat pulse. The data in Table la show that there is not a single short period which is especially sensitive in the mutant. Longer (1 hr) heat pulses are more effective, again at a range of times relative to gastrulation (Table lb). In these experiments there were many cases in which embryos which were still at prepole cell stages at the end of the heat pulse, i.e., showing no sign that nuclear migration had yet occurred, subsequently developed with-

TABLE

1

POLE CELL FORMATION IN shit”’ EMBRYOS HEAT PULSED AT PREPOLE CELL STAGES Time of heat pulse in minutes before gastrulation”

30 min at 29°C

20 min at 29°C

210-240 180-210 150-180 120-150 90-120 60-90 30-60 Control

(shi at 22°C)

(b)

(a)

60 min at 29°C

No PC

PC

*h

No PC

PC

*

4 8 7 10 2

1 24 13 8 0

0 5 2 3 0

28 35 38 15 14

4 7 18 9 6

5 4 3 4 3

0

65

0

No PC

PC

*

28 38 a 2

0 1 0 0

0 0 0 0

’ Incubation of embryos before and after heat pulses was at 22°C. ’ In cases where it was impossible to make a clear distinction between presence (PC) and absence (No PC) of pole cells, the embryos were classified in this third category.

422

DEVELOPMENTALBIOLOC:Y

out pole cells. Apparently, it is unnecessary for the pulse to be given during pole cell formation in order to interfere with the process. The wide range in times of sensitivity indicated that heat pulses given during pole cell formation might also inhibit it. A few embryos in which bulges were visible at the posterior end were shifted to 29°C for 30 min, and in these cases pole cell formation was prevented. In addition, embryos heat pulsed for 1 hr at the stage when pole cells had apparently already formed lacked pole cells in 26 of 50 cases. Thin sections of control embryos at this stage show that the pole cells may not have completely separated at the point when the heat pulse was given. However, we found in sectioned material that some embryos shifted up at later stages, when pole cells definitely had separated, lacked pole cells after the heat pulse (Table 2). This result indicated that in shi mutants apparently resorption of pole cells can occur as well as inhibition of their formation.

Electron Microscopy of Pole Cell Formation As part of the analysis of pole cell formation, shi embryos which were heat pulsed at the sensitive stages for pole cell formation were prepared for scanning electron microscopy. These were compared to 22°C control shi embryos fixed at various stages during normal processes. The shi embryos at the permissive temperature showed no marked differences from the description by Turner and Mahowald (1976) of wild-type morphology. We will briefly review here our observations of control early embryo morphology relevant to pole cell formation as a basis for comparison with the heat-treated shi embryos. The early embryo, prior to nuclear migration, is uniform on all surface areas, with many pits and small, rounded projections visible at high magnification. Just prior to pole cell formation there is extensive differentiation of the surface at the posterior end.

VOLUME 75,198O TABLE

2

POLE CELLFORMATION INS~~~EMBRYOSFIXEDFOR OBSERVATION Preparation No.

1 2 3 4

Stage at time of heat pulse”

cell Early pole stage Prepole cell Early pole cell stage Syncytial blastoderm

No. of specimens No PC

PC

*’

Specimen preparation

26

24

0

WM

20 6

0 4

2 1

Sd s

4

1

1

s

a Heat pulses were at 29°C for one hour; before and after heat pulses embryos were incubated at 22°C. ‘In cases where it was impossible to make a clear distinction between, presence (PC) and absence (No PC) of pole cells, the embryos were classified in this third category. ’ WM, Pole cells were scored in embryos prepared as fured and Feulgen-stained whole mounts. ’ S, Pole cells were scored in embryos, fixed, embedded in Epon/Araldite, and one-micron sectioned.

Numerous round and elongate projections drastically increase the surface area there. The pole cells begin to bulge after the embryonic nuclei migrate to the surface, at which time there is a uniform distribution of surface mounds, as seen in SEM. At this stage too, at the posterior pole there is still blebbing; but in the mounds where cells are forming the surface is smoother with fewer rounded projections (as noted by Turner and Mahowald (1976)). In the early stages when pole cells are visible, they are relatively smooth with some folds and pits (Fig. 1). The surface membrane at the posterior pole, beneath the pole cells, is smooth and punctuated with rounded extrusions. This is in contrast to the lateral portions of the egg where the rounded surfaces over each nucleus are not as smooth and the areas between them more broken with tightly packed clusters of projections. As can be seen in Fig. 1 and as was reported by Turner and Mahowald (1976), seven or eight pole cells arise independently, and increase in number by division.

SWANSON AND POODRY

Pole Cell Formation in D. melanogaster

423

FIGS. 1-13. Scanning electron micrographs are of shi embryos fixed at various stages. Vitelline membranes were removed from each and samples were observed at 10 kV with a JEOL JSM-2. Field widths (FW) are indicated for each micrograph. Figures 1 to 4 are shi embryos at 22°C. FIG. 1. A view of the posterior end of a nuclear migration stage embryo in which eight pole cells are forming. The surface of those cells is smooth (FW 133 pm). FIG. 2. View of the posterior pole of late pole cell stage embryo showing 15 to 20 pole cells. Pole cells elongate, apparently beginning to divide (FW 133 pm). FIGS. 3 AND 4. View of posterior pole of syncytial blastoderm stage embryos. FIG. 3. Pole cells are difficult to distinguish, but are smooth surfaced and packed together closer than outlines of the blastoderm cells (FW 131 am). FIG. 4. Posterior view of cellular blastoderm stage embryo in which the pole cell cluster is well defined. Blastoderm surface no longer has mounds indicating nuclear positions, but has extensive pits and projections (FW 131 pm).

Progressively, the pole cells become more closely packed. After the 10th division of the blastodermal nuclei (Fig. 2), the pole cells are clustered tightly and the underlying cell membrane is no longer visible. The close packing of the cells also modifies their round shapes. After the 11th cleavage, the pole cells become difficult to identify without section-

ing. Some cells at the posterior end appear to be packed more tightly, are rounder or smoother (Fig. 3), or are not dividing synchronously with lateral blastodermal nuclei. We verified that these structures were pole cells by sectioning through plasticembedded SEM samples. As blastoderm nuclei divide and cell membranes form between the blastoderm

424

DEVELOPMENTAL BIOLOGY

nuclei, the surface changes, becoming continuous, with numerous pits and folds. Cell outlines are not obvious on the surface at this stage. The group of pole cells (approximately 30 in Fig. 4) is again well defined. The pole cells have somewhat smooth surfaces with projections interdigitating between the cells. These connections may be an important prelude to the passive migration of pole cells accomplished by gastru-

VOLUME 75,198O

lation. Mutant. Among shi embryos heat pulsed for one hour beginning before any signs of pole cells were evident, surface morphology of the posterior pole was variable. In some cases flattened bumps at the pole resembled abnormal pole cells, but in the majority the area lacked signs of cells entirely (Figs. 5 and 6) or was covered uniformly by mounds resembling blastoderm cells (Fig.

FIGS. 5-8. Posterior views of shi embryos which have been heat pulsed for one hour beginning at prepole cell stage. All lack pole cells. FIG. 5. Fixed 20 min after the end of the heat pulse, embryo resembles a normal syncytial blastoderm or pole cell stage embryo, but lacks pole cells at the posterior end (FW 136 pm). FIG. 6. Fixed 30 min after the end of the heat pulse, the embryo shown here lacks cellular outlines at the posterior pole as does the embryo shown in Fig. 5, but it is at a later stage (noted from the smaller outlines of the blastodermal cells), and the posterior end lacks the many projections seen in Fig. 5 (FW 122 pm). FIG. 7. Fixed 30 min after the end of the heat pulse, the posterior pole is covered with apparently normal blastoderm cell outlines in this embryo (FW 136 pm). FIG. 8. This syncytial blastoderm stage embryo is lacking a complete population of cells at the posterior end. Cells which have formed there resemble the blastoderm cells (FW 136 pm).

SWANSON

AND

POODRY

Pole Cell Formation

7). In a few instances, gaps were present between the surface bulges at the pole (Fig. 8). Phenotypes similar to those for embryos treated earlier were seen when embryos were heat pulsed at 29°C for 30 min at the first signs of pole cell development. Cells covering the pole more or less regularly (Fig. 9) and a flattened area at the pole apparently lacking cells (Fig. 10) are typical. The flattening visible in Figs. 6, 10, and

in D. melanogaster

425

12 may be due to compression of the posterior tips against the vitelline membrane as noted by Turner and Mahowald (1976). Heat pulses of 30 min at 29°C begun at the pole cell stage produced some embryos in which pole cells appeared absolutely normal (Fig. 11). More often, the familiar abnormalities were again seen; e.g., reduced numbers of pole cells (Fig. 12) and blastodermal cells covering the pole with some gaps (Fig. 13). In shi embryos heat pulsed at prepole cell stage and fixed before nuclear migration, in some cases surface membranes resemble those of controls with extensive folds and projections. In others the entire surface, or substantially large regions of it, lack the usual extensions and are smooth. Multivesicular bodies and coated vesicles occur just beneath the smooth membrane in the most extreme cases. Where nuclei were present in the polar region during or after a heat pulse, the characteristic bulges over nuclei do occur (Figs. 14 and 15). However, between nuclei the surface membrane is unusually smooth and lacking in projections (Fig. 15), similar to membrane in lateral areas as reported by Turner and Mahowald (1976).

Polar Granules

FIGS. 9 AND 10. Views of the posterior end of shi embryos heat pulsed for 0.5 hr beginning at the first signs of pole cell formation. Embryos were fixed immediately after the heat treatment (29°C). FIG. 9. A nearly normal layer of blastoderm cell outlines covers the posterior pole. In the center there are a few smaller cells which may be abnormal pole cells (FW 136 pm). FIG. 10. No pole cells occur in this embryo and a smooth area is present at the posterior end. Not only are normal cell outlines lacking, but there are few microprojections of the surface there (FW 136 pm).

Control. The changes in morphology and distribution of polar granules in normal development have been described by Counce (1963) and by Mahowald (1969). Initially in the Drosophila melanogaster oocyte the polar granules are associated with mitochondria and are of an equivalent size (0.5 pm). In the early embryo, the granules appear as aggregates in the form of elongated chains (Figs. 16A and B). As the pole cells form, the granules dissociate from the aggregates and are more often discrete and dispersed with associated polysomes (Figs. 16B and C). After the pole cells have ceased dividing, and the cellular blastoderm is forming, the granules reaggregate into larger, doughnut-shaped structures (Fig. 16D).

FIGS. 11-13. Views of the posterior end of shi embryos heat pulsed at 29“C for 0.5 hr beginning at pole cell stage. Embryos were fiied immediately after the heat treatment. FIG. 11. An apparently normal group of pole cells occur in this late syncytial blastoderm embryo (FW 136 pm). FIG. 12. Approximately four pole cells occur in this embryo which is at the cell blastoderm stage (FW 136 pm). FIG. 13. Pole cells do not occur in this syncytial 426

FIG. 14. TEM of a longitudinal section through the posterior pole of a s/n embryo heat pulsed (29°C) for one hour at prepole cell stage and fLved at the first signs of nuclear migration. Fixation and embedding: K&T (mod), 1% OsO+ Epon/Araldite. Figure shows nucleus (N) associated with polar granules (pg) and surface bulge with adjacent smooth surface. Under the smooth area are large membrane vesicles (V) and multivesicular bodies (mvb). Arrows indicate coated pits in the vesicle membrane. (a) Higher magnification of a portion of Fig. 14. FIG. 15. TEM of longitudinal section through the posterior pole of a shi embryo heat pulsed (20°C) for one hour at the fast signs of pole cell formation and fixed 15 min after the shift down. Nuclei (N) occur beneath surface bulges. Arrow indicates polar granule aggregate. Fixation and embedding; K&T (mod), 1% OsO1, Epon/Araldite. blastoderm embryo and there is a small gap in the covering of blastoderm cells at the posterior pole (FW 136 pm).

SWANSON

AND

POODRY

Pole Cell Formation

in D. melanogaster

427

FIG. 16. Transmission electron micrographs of sectioned embryos showing areas of the posterior polar cytoplasm including polar granules (arrows). Fixation and embedding for E, K&T (mod), Zetterqvist OsO,, Araldite, and for all others, K&T (mod), 1% 0~04, Epon/Araldite. All figures are at approximately the same magnification. Bars indicate one micrometer. (A) Early pole cell stage shi embryo at 22°C. (B) Late pole cellearly syncytial blastoderm stage shi embryo at 22°C. (C) Syncytial blastoderm stage shi embryo at 22°C. (D) Late cellular blastoderm stage OreR embryo after one hour at 29°C beginning at syncytial blastoderm. In (EH) all embryos lack pole cells. (E) Prepole cell stage shi embryo after 1 hour at 29°C. Numerous polar granules are present. (F) Pole cell stage shi embryo, heat pulsed at prepole cell stage for one hour and fixed at the first signs of nuclear migration. (G) Syncytial blastoderm stage shi embryo, shifted to 29°C as pole cells began to form (1 hrl, then fixed 15 min after the shift down. (H) Syncytial-cell blastoderm stage shi embryo shifted to 29°C at early pole cell stage for one hour, incubated for one hour at 22°C after shift down before fixation.

Mutant. In shi embryos heat pulsed for one hour at 29°C beginning at prepole cell stages, pole cells do not form, yet TEM of

such samples reveals polar granules with normal morphology. In such early embryos the polar granules are aggregated into long

428

DEVELOPMENTALBIOLOGY

chains as in the controls at similar stages (Fig. 16E). In the cases where either the heat pulse was given slightly later, so nuclei have arrived in the polar region by the end of the pulse, or the embryo was allowed to develop to this stage after the heat pulse, polar granules are still present but the aggregates have dispersed. Fragmented polar granules are adjacent to nuclei as in normal pole cells as they first form (Fig. 16F). In order to assess the role of the polar granules in germ cell determination, the fate of the posterior polar region was followed in embryos lacking pole cells. After heat pulses in many cases, polar granules were present and adjacent to nuclei but at stages before those nuclei would be included in blastoderm cells. When embryos which had been heat pulsed at prepole cell stages were allowed to develop further at 22°C after the heat pulse, polar granules were found in blastoderm cells at the posterior end. In these embryos, the granules form the characteristic doughnut-shaped masses seen in controls of similar stages (Figs. 16G and H). DISCUSSION

Between fertilization and the syncytial blastoderm stage, heat pulses applied to shi embryos can be effective in deleting pole cells. The affected component(s) of pole cell formation is susceptible to heat treatment for an extended time and there is no specific short period for pole cell lability with more extreme sensitivity. The existence of multiple independently isolated alleles of a temperature-sensitive paralytic mutation which result in altered pole cell formation, in addition to other embryonic defects, leads us to conclude that the disruption of pole cell formation is due to the single gene mutation, shit”. Any putative non-shi mutation responsible for the effects on pole cells would be limited to the region 13F14A on the salivary map (see Lefevre, 1976) since a small duplication (Dp(1; Y) y+shi+B”), which covers the region, pro-

VOLUME 75,198O

tects against all shi phenotypes including temperature sensitivity of pole cell formation. The pleiotropic effects of shi (including eye scars, bristle abnormalities, and paralysis) argue against an effect of any factor specifically or solely responsible for the initiation or general control of pole cell formation. In a normally developing embryo, the beginning of pole cell formation is marked by the slight surface bulges at the posterior end of the embryo which signal the arrival of nuclei there. The transplantation studies of Illmensee and Mahowald (1974, 1976) have aptly demonstrated the necessity for the specific posterior pole cytoplasm and the ability of that cytoplasm to interact with nuclei from other regions to yield cells which are morphologically and functionally similar to pole cells. The only morphologically distinctive cytoplasmic components in the region are the polar granules and it has been suggested (Mahowald, 1971) that these organelles function as germ cell determinants. Studies of mutants are in accord with the requirement for both polar granules and nuclei. The number of polar granules is reduced in two mutant strains which lack normal pole cells, grandchildless of D. subobscura (gs; Fielding, 1967) and female sterile-Nasrat (fs(l)N; Kern, 1975). In gsa7of D. melanogaster, polar granules are morphologically normal, but nuclear arrival in the posterior pole is abnormal (Thierry-Meig et al., 1972). In the case of shP, while disruptions in nuclear migration can occur, even heat pulses given to embryos in which nuclear migration has already progressed normally are effective in preventing pole cell formation. In shi, as in polar granules are morphologically ge normal, and there are cases in which nuclei and polar granules both occur at the posterior pole in apparently normal associations, yet no pole cells form. While stagespecific morphology of the polar granules indicates normality, there could be defects in the polar granules which are unrecog-

SWANSON

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POODR~

Pole Cell Formation

nized in TEM. Until their function is tested, this possibility cannot be eliminated; however, there is no reason to expect that a pleiotropic developmental mutation would have a specific effect on these granules. If the polar granules and nuclei are presumed to be normal, however, another component must be necessary for pole cell formation. An obvious requirement is the membrane which surrounds the pole cells and a mechanism by which that enclosure is accomplished. TEM studies (Swanson, 1977) have revealed microfilaments adjacent to the emerging pole cells which suggest that there is a contractile ring which pinches off the cells. The membrane bulges which occur upon nuclear arrival suggest an interaction between the surface membrane and the nuclei. SEM and TEM studies indicate that the surface membrane, initially distended into many irregular projections and pits in the early embryo, unfolds as contraction pulls it around the nucleus and the polar cytoplasm. In shi mutants apparently some aspect of this process is perturbed. The smooth surface membrane in heattreated samples, and resorption of pole cells in embryos heat treated at later stages, may be indications of a membrane alteration. Such an effect on the membrane could be explained by changes in membrane function, in a nucleus-plasma membrane attachment system, or in a contractile network in contact with the membrane. A change in any of these levels could be brought about by a perturbation of the molecular structure of the membrane. A membrane defect is also consistent with data on the neurophysiological effects of shi paralysis which point to defects in synaptic transmission at the neuromuscular junction (Ikeda et al,, 1976; Siddiqui and Benzer, 1976; Salkoff and Kelly, 1978; Poodry and Edgar, 1979). The early segregation of the pole cells may provide the permissive conditions for expression of the germ-line potential. In shi

in D. melanogaster

429

embryos lacking pole cells, the incorporation of the posterior pole substances into posterior blastoderm cells does not result in functional adult germ cells. Whether these blastoderm cells could potentially function as germ cells has not been determined but might be testable by transplantation. In the absence of such data, however, we are led to conclude that a necessary permissive condition for expression of the germ-line potential is the physical segregation of the posterior pole cytoplasm from the rest of the embryo early in development. This process is what is disturbed by the shibire mutation. REFERENCES BOWNES, M. (1975). A photographic study of development in the living embryo of Drosophila melanogaster. J. Embryol. Exp. Morphol. 33, 789-801. COUNCE, S. J. (1963). Developmental morphology of polar granules in Drosophila including observation on pole cell behavior and distribution during embryogenesis. J. Morphol. 112, 129-445. FIELDING, C. J. (1967). Developmental genetics of the mutant grandchildless of Drosophila .subobscura. J. Embryol. Exp. Morphol. 17, 375-384. GEIGY, R. (1931). Action de l’ultraviolet sur le pole germinal dans l’oeuf de Drosophila melanogaster. Reu. Suisse 38, 187-288. GEYER-DUSZYNSKA, I. (1959). Experimental research on chromosome elimination in Cecidomyidae (Diptera). J. Exp. Zool. 141, 391-448. H., and GRIGLIATTI, T. A., HALL, L. KOSENBLUTH, SUZUKI, D. T. (1973). Temperature-sensitive mutations in Drosophila melanogaster. XV. Selection of immobile adults. Mol. Gen. Genet. 120, 107-114. HEGNER, R. W. (1908). Effects of removing the germ cell determinants from the eggs of some Chysomelid beetles. Biol. Bull. 16, 19-26. HUMASON, G. (1972). “Animal Tissue Techniques,” 3rd ed. Freeman, San Francisco. IKEDA, K., OZAWA, S., and HAGIWARA, S. (1976). Synaptic transmission reversibly conditioned by singlegene mutation in Drosophila melanogaster. Nature (London) ILLMENSEE,

259,489~491.

K., and MAHOWALD, A. I’. (1974). Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. Nat. Acad. Sci. USA 71, 1016-1020. ILLMENSEE, K., and MAHOWALD, A. P. (1976). The autonomous function of germ plasm in a somatic region of the Drosophila egg. Exp. Cell Res. 97, 127. JAZDOWSKA-ZACRO~ZINSKA.

B. (1966). Experimental

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studies on the role of “polar granule” in the segregation of pole cells in Drosophila melanogaster. J. Embryol. Exp. Morphol. 16,391-399. KERN, C. (1975). Cytochemical and ultrastructural observations on the polar plasm of eggs produced by the female-sterility mutant fs(l)N in Drosophila melanogaster. Amer. Zool. 16, 189. LEFEVRE, G., JR. (1976). A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. In “The Genetics and Biology of Drosophila” (M. Ashburner and E. Novitski, eds.), Vol. la, pp. 31-66. Academic Press, London. MAHOWALD, A. P. (1962). Fine structure of pole cells and polar granules in Drosophila melanogaster. J. Exp. Zool. 151,201. MAHOWALD, A. P. (1969). Polar granules of Drosophila. II. Ultrastructural changes during early embryogenesis. J. Exp. Zool. 167, 237-262. MAHOWALD, A. P. (1971). Polar granules of Drosophila. IV. Cytochemical studies showing loss of RNA from polar granules during early stages of embryogenesis. J. Exp. Zool. 176,345-352. OKADA, M., KLEINMAN, I. A., and SCHNIEDERMAN, H. A. (1974). Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm. Develop. Biol. 37,43-54. POODRY, C. A., HALL, L., and SUZUKI, D. T. (1973). Developmental properties of shibire”‘: A pleiotropic mutation affecting larval and adult locomotion and development. Develop. Biol. 32,373-386. POODRY, C. A., and EDGAR, L. (1979). Reversible alterations in the neuromuscular junctions of Dro-

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sophila melanogaster bearing a temperature-sensitive mutation, shibire. J. Cell. Biol. 81, 520-527. SALKOFF, L., and KELLY, L. (1978). Temperature-induced seizure and frequency-dependent neuromuscular block in a ts mutant of Drosophila. Nature (London) 273, 156-158. SANDER, K. (1976). Specification of the basic body pattern in insect embryogenesis. Advan. Insect Physiol. 12, 125-238. SIDDIQUI, O., and BENZER, S. (1976). Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 73, 3253-3257. SWANSON,M. M. (1977). “Embryogenesis in Drosophila melanogaster: A Genetic and Morphological Study.” Ph.D. dissertation, Univ. of California, Santa Cruz. THIERRY-MEIG, D., MASSON, M., and GANS, M. (1972). Mutant de sterilite a effet retarde de Drosophila melanogaster. CR. Acad. Sci. Paris 275, 2751-2754. TURNER, F. R., and MAHOWALD,A. P. (1976). Scanning electron microscopy of Drosophila embryogenesis. I. The Structure of the egg envelopes and the formation of the cellular blastoderm. Develop. Biol. 50,95-108. WARN, R. (1975). Restoration of the capacity to form pole cells in UV-irradiated Drosophila embryos. J. Embryol. Exp. Morphol. 33, 1003-1011. ZALOKAR,M., and ERK, I. (1976). Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J. Microsc. Biol. Cell 25,97106.