Anthony P. Mahowald

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.

The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster

Abstract Pulse labeling of proteins, in vivo , followed by indirect immunoprecipitation of the vitellogenin polypeptides, has shown that not only the thoracic and abdominal fat bodies but also the ovary devote a significant percentage of their synthetic capacity to vitellogenin (VG) production. These methods have also shown that ovarian stages 9 and 10 contribute the majority of VG synthesized by the ovary and that the follicular epithelium of these stages is the specific site of VG synthesis. In situ hybridization (of a probe containing the coding regions of the two larger polypeptides) to sections of ovaries confirmed that the VG mRNAs are abundant species in the cytoplasm of stage 9 and 10 follicle cells. In addition, two of the three polypeptides (VGP1 and VGP2) are produced at roughly equal levels by the follicle cells, but the smallest polypeptide (VGP3) is produced at one-fourth this level by these cells. Hybridization of cloned genomic probes ( T. Barnett, C. Pachl, J. P. Gergen, and P. C. Wensink, 1980 , Cell 21, 729–738) to RNA bound on nitrocellulose filters has shown that the ovary contributes aproximately 35% of the total amount of the mRNAs coding for VGP1 and VGP2 but only about 10% of the mRNA for VGP3. The same procedure demonstrated that the levels of all three VG mRNAs during follicular development closely parallel VG polypeptide synthesis. Finally, culture of ovaries in males has shown that the mRNA levels accurately reflect the follicle cell contribution to VG synthesis.

Scanning electron microscopy of Drosophila embryogenesis. 1. The structure of the egg envelopes and the formation of the cellular blastoderm.

Abstract As part of a series of detailed observations on embryogenesis in Drosophila , the protective coverings of the egg and surface changes in the embryo prior to gastrulation have been studied with the SEM. Four specializations of the chorion are described: the plastron, micropylar cone, operculum, and the posterior thickening. After removal of the protective coverings the surface changes during development can be observed. During the first eight synchronous nuclear divisions a dense array of thin microprojections covers the whole embryo. After the ninth division between 373 and 408 nuclei reach the surface and become located in cytoplasmic projections. From counts of the number of surface bulges during the syncytial blastema stages, it was established that 13 synchronous divisions take place producing between 5600 and 6500 surface nuclei. During formation of the cellular blastoderm, the location of the prospective cells becomes obscured by a dense pattern of microprojections from each cell. However, with the completion of the blastoderm, the surfaces of the cells become smooth and the cell outlines distinct. The usefulness of the SEM in developmental studies is discussed.

Assembly of the Drosophila germ plasm.

The Drosophila melanogaster germ plasm has become the paradigm for understanding both the assembly of a specific cytoplasmic localization during oogenesis and its function. The posterior ooplasm is necessary and sufficient for the induction of germ cells. For its assembly, localization of gurken mRNA and its translation at the posterior pole of early oogenic stages is essential for establishing the posterior pole of the oocyte. Subsequently, oskar mRNA becomes localized to the posterior pole where its translation leads to the assembly of a functional germ plasm. Many gene products are required for producing the posterior polar plasm, but only oskar, tudor, valois, germcell-less and some noncoding RNAs are required for germ cell formation. A key feature of germ cell formation is the precocious segregation of germ cells, which isolates the primordial germ cells from mRNA turnover, new transcription, and continued cell division. nanos is critical for maintaining the transcription quiescent state and it is required to prevent transcription of Sex-lethal in pole cells. In spite of the large body of information about the formation and function of the Drosophila germ plasm, we still do not know what specifically is required to cause the pole cells to be germ cells. A series of unanswered problems is discussed in this chapter.

Multiple functions of segment polarity genes in Drosophila

l(1)dishevelled (l(1)dsh) is a late zygotic lethal mutation that exhibits a rescuable maternal effect lethal phenotype. l(1)dsh/Y embryos, derived from females possessing a homozygous l(1)dsh germline clone, exhibit a segment polarity embryonic phenotype. Analysis of the development of these embryos indicates: (1) that segmental boundaries do not form although the correct number of tracheal pits is formed; (2) that pockets of cell death occur between the tracheal pits; and (3) that engrailed expression becomes abnormal during germ band shortening. We propose that, in the absence of both maternal and zygotic expression of l(1)dsh+, cells from each posterior compartment die. Subsequently, cells from the anterior compartment must rearrange their positional values to generate the segment polarity phenotype. We have compared the phenotype of five other segment polarity loci: four embryonic lethals [l(1)armadillo, l(2)gooseberry, l(2)wingless, and l(3)hedgehog]; and the late zygotic lethal, l(1)fused. Only l(2)wingless embryos exhibit early segmentation defects similar to those found in l(1)dsh/Y embryos derived from homozygous germline clones. In contrast, segmentation is essentially normal in l(1)armadillo, l(2)gooseberry, l(3)hedgehog, and l(1)fused embryos. The respective maternal and zygotic contribution and the roles of the segment polarity loci for the patterning of the embryo and the adult are discussed.

Scanning electron microscopy of Drosophila melanogaster embryogenesis: II. Gastrulation and segmentation

Abstract The sequence of gastrulation events in Drosophila melanogaster , starting with the cellular blastoderm and culminating in a segmented embryo, have been studied with scanning electron microscopy (SEM). Extensive use is made of dissected embryos to illustrate changes taking place within the embryo during gastrulation. During the first 15 min of gastrulation, the mesodermal portion of the germ band is established by the invagination of approximately 1000 cells through the ventral furrow. The primordia for the proctodeum and hindgut are shown to form during early gastrulation. Detailed examination of the surfaces of invaginating primordia shows similarities to other systems and suggests possible underlying mechanisms. Germ band elongation and the formation of the amnioserosa are described. At the time of segmentation, three pairs of rudimentary cephalic appendages develop posterior to the cephalic furrow. Tracheal pits invaginate on all eight abdominal segments and on the second and third thoracic segments. Modifications of the embryonic fate map are discussed.

The ontogeny of germ plasm during oogenesis in Drosophila.

Abstract Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant? Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12. The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from flies injected with 14C amino acids. Chorion proteins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12--14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979).

Scanning electron microscopy of Drosophila melanogaster embryogenesis: III. Formation of the head and caudal segments

Abstract The formation of both the anterior most and posterior most segments in higher dipteran embryos involves complex movements of primordia which can be best visualized with the scanning electron microscope. During head formation, the gnathocephalic segments partially involute through the stomodeum. The labial segment forms the floor of the mouth, and the fused maxillary and mandibular segments form the lateral sides of the mouth. The involuted clypeolabrum forms the roof of the mouth. Invaginations of cells for segmentally derived sense organs can be found prior to involution on all the gnathocephalic and thoracic segments as well as on the labrum. The antennal sense organ derives from the lateral surface of the procephalic lobe. Following involution of the mouth parts, the dorsal ridge, which arises just anterior to the first thoracic segment, is drawn over the dorsal procephalic lobe producing the deep dorsal sac. The optic lobes of the brain invaginate anterior to the dorsal ridge just prior to the covering over of the head. The formation of the anal segment is similarly complex. Two rudimentary segments are found posterior to the eighth abdominal segment. During shortening of the germ band, the posterior most segment is drawn around the posterior tip of the embryo to lie ventrally. Two large anal pads form lateral to the anus from this segment. The next segment, following dorsal closure, produces a pair of anal sense organs and a central tuft of setae. Finally, the eighth abdominal segment gives rise to the posterior spiracles. Following dorsal closure these three segments fuse to produce the terminal (anal) segment of the larva.

l(1)hopscotch, a larval-pupal zygotic lethal with a specific maternal effect on segmentation in Drosophila

The maternal and zygotic effect phenotypes of mutations at the l(1)hopscotch (l(1)hop) locus are described. l(1)hop is located in 10B6-8 on the salivary gland chromosome map and 17 alleles have been characterized. A complex complementation pattern is observed among the 17 alleles. The lethal phase of null alleles of l(1)hop occurs at the larval-pupal interface associated with a small disc phenotype. Embryos produced from homozygous l(1)hop germline clones show segment specific defects. The extent of these defects depends upon both the strength of the allele and the paternal contribution. In the most extreme case embryos exhibit defects associated with five segments T2, T3, A4, A5, and A8. In the less extreme phenotype defects are only associated with A5. Thus, activity of l(1)hop+ is required both for the maintenance and continued cell division of diploid imaginal precursors and for the establishment of the full array of segments.