A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence.

The central role of gene expression in regulating development has largely been studied by in situ hybridization and antibody staining techniques in fixed material. However, rapid temporal and spatial changes in gene expression are often difficult to correlate with complex morphogenetic movements. A green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, can be used as a real-time reporter for gene expression and could aid analysis of dynamic events during embryogenesis. Here, we describe a transgenic Drosophila line ubiquitously expressing a nuclear GFP fusion protein that highlights morphogenesis, cell movement, and mitosis in living embryos. The fusion protein is highly fluorescent when maternally supplied, but there is a long delay between its zygotic expression and the appearance of fluorescence. GFP is thus an excellent marker for the expression of stable gene products, but a poor reporter for dynamic zygotic gene expression in early Drosophila embryos.

A cell-autonomous, ubiquitous marker for the analysis of Drosophila genetic mosaics.

Mosaic analysis, the study of animals containing cells of two different genotypes, has been used to address a wealth of questions in developmental biology. Up to now, the cell markers used to distinguish cells of the two genotypes have only been applicable to specific experimental situations (e.g., only in adult wings). We have designed a general purpose cell marker for mosaic analysis. It consists of the bacterial LacZ gene expressed under the control of the constitutive promoter of the Drosophila armadillo gene. Transformants carrying this fusion gene express beta-galactosidase in all tissue and at all stages analyzed. Zygotic expression is detectable as early as gastrulation. In mosaics obtained by nuclear transplantation, cells carrying the transgene are easily distinguished from beta-galactosidase-negative host cells. The marker should also be useful for mosaics generated with the "Flp technique."

The use of photoactivatable reagents for the study of cell lineage in Drosophila embryogenesis.

Photoactivatable lineage tracers represent a major advance for clonal analysis in the early embryo and the study of cell movements. Any cell in the blastoderm can be marked, and the nuclear localization of the signal allows excellent resolution in identifying the daughters of individual cells. Although the technique is limited by the availability of the water-soluble caged fluorescein and its derivatives for synthesis of the complete tracer, these may become commercially available in the future. The use of caged rhodamine derivatives or antibody amplification of the signal may greatly extend the developmental period over which marked clones can be identified.

polo encodes a protein kinase homolog required for mitosis in Drosophila.

We show that mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed; normal bipolar spindles with polyploid complements of chromosomes; bipolar spindles in which one pole can be unusually broad; and monopolar spindles. We have cloned the polo gene from a mutant allele carrying a P-element transposon and sequenced cDNAs corresponding to transcripts of the wild-type locus. The sequence shows that polo encodes a 577-amino-acid protein with an amino-terminal domain homologous to a serine-threonine protein kinase. polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos but are specifically concentrated in dividing cells in larval discs and brains. In the cell cycles of both syncytial and cellularized embryos, the polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic.

Chromosome tangling and breakage at anaphase result from mutations in lodestar, a Drosophila gene encoding a putative nucleoside triphosphate-binding protein.

We describe a Drosophila maternal-effect gene, lodestar, mutations in which cause chromatin bridges at anaphase. lodestar maps to cytological position 84D13-14, and we identified the lodestar gene in germ-line transformation experiments by the ability of a genomic fragment to restore fertility to females homozygous for lodestar mutations. lodestar encodes a potential nucleoside triphosphate binding protein, which is a novel member of the D-E-A-H box family of proteins. Antibodies raised against the lodestar gene product detect a protein that undergoes cell cycle-dependent changes in distribution in the embryo. The protein is cytoplasmic at interphase, and rapidly enters the nucleus early in prophase. It is restricted to the region enclosed by the spindle envelope during metaphase and anaphase; but by telophase, the lodestar protein is contained entirely within the reforming nucleus.

Mitosis in Drosophila development.

Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. The pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed.