John R. Merriam

Parameters of the wing imaginal disc development ofDrosophila melanogaster

Abstract Using X-ray-induced somatic crossing-over, a clonal analysis of the wing disc development of Drosophila melanogaster was carried out. Individuals carrying y in the X-chromosomes, and sc J 4 ( y + ) jv and mwh in either 3 L chromosome were irradiated at different developmental stages and the induced y;mwh and jv twin clones studied with respect to their frequencies, sizes, and shapes. The results indicate that the wing imaginal disc cells grow exponentially from the beginning of the larval period up to puparium formation. A total of 15.6 divisions is necessary to complete the adult number of cells. The division rate is constant in the intermolt periods, but apparently decreases during the molt periods. The average cell cycle time for the larval period is about 8 hours 30 minutes. The cell sensitivity to SCO is more or less constant during the larval periods (0.2–0.4%). It increases during the pupal period about 4 times for the presumptive hair cells and about 5–10 times for the presumptive trichogen cells. The orientation of the mitotic spindle appears to be a major morphogenetic process. Cell divisions are preponderantly oriented along the proximodistal axis in the wing and alternating transverse and longitudinal or at random in the notum region. Clones grow in indeterminate patterns. At least two determinative events separate cell clones into different prospective developmental pathways. From the beginning of the larval period, dorsal and ventral cells do not mix over the wing margin. Forty hours before puparium formation, induced clones are able to give rise to several bristles or several hair cells but not to both in the same clone. The two differential mitoses giving rise to a bristle organ appear to be equivalent to, and to take place at the same time as, the two divisions give rise to 4 hair cells.

CLONAL PARAMETERS OF TERGITE DEVELOPMENT IN DROSOPHILA.

Abstract A clonal analysis of abdominal development was carried out using chaeta (y, f36a and jv) and trichome (mwh) cell marker mutants. The uncovering of these recessive markers in heterozygous individuals occurred after spontaneous loss of a X ring chromosome (gynandromorphs) or by somatic recombination induced at different developmental stages by 1000 r of X-ray. The minimum spot sizes resulting from preblastodermic gynandromorphs indicate that the least number of primitive analge cells is about 8. This is also the number of imaginal cells per analge present throughout the larval stages of development. Only with the onset of metamorphosis do the imaginal cells start dividing and spreading over the presumptive tergite area, each pair of bilateral anlage giving rise to a complete tergite. All the tergites studied (II–VI) behaved uniformly in the present experiments. Corresponding with the constant number of imaginal cells during the larval development there is a constant frequency of induced spots or a constant cell sensitivity to SCO. This sensitivity varies depending on the chromosome considered, being higher in the X chromosome than in the 3L chromosome arm. The data on gynandromorphs, as well as those of somatic spots, indicate that the adult tergites have an indeterminate pattern of growth. The cell determination to become trichome or micro- or macrochaeta occurs sometime during the pupal expansion of the growing imaginal cell population. Trichome- and chaeta-forming cells may then be separated to construct the adult cuticular pattern.

The zygotic mutant tailless affects the anterior and posterior ectodermal regions of the Drosophila embryo

The recessive zygotic lethal mutation tailless maps to region 100A5,6-B1,2 at the tip of the right arm of chromosome 3, and results in shortened pharyngeal ridges in the head skeleton of the mature embryo and the elimination of the eighth abdominal segment and telson. Although they have a normal body length, tailless embryos have a smaller number of abdominal segments, some of which are larger than normal. The mutant phenotype is seen as early as 8 hr postfertilization, when tailless embryos are observed to have fewer tracheal pits than wildtype. At 9 hr, tailless embryos appear to be missing segments A8, A9, and A10 and have an abnormal clypeolabrum, optic lobes, and procephalic lobe. Segments A4, A5, A6, and A7 appear larger in tailless embryos than wildtype at this stage. The tailless mutation, although affecting anterior and posterior ectodermal structures in the mature embryo, does not affect the formation of pole cells, the posterior midgut, or the proctodeum, which arise from the most posterior region of the embryo. The mutation does result, however, in the failure of Malpighian tubule formation. Consistent with its effect on ectodermal segments, tailless leads to a reduction in the number of segmented, paired ganglia in the ventral nerve cord as well as to an abrupt alteration in the posterior region of the tracheal system. The role the tailless gene may play in the formation of the most anterior and posterior regions of the embryos ectodermal body plan is discussed.

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast

Abstract The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and cbf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophilamelanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast cbf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.

A Novel Ecdysone Receptor Mediates Steroid-Regulated Developmental Events during the Mid-Third Instar of Drosophila

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E—signaling through a nuclear receptor heterodimer consisting of EcR and USP—induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Phototactic and geotactic behavior of countercurrent defective mutants ofDrosophila melanogaster

Ten behavioral mutations, originally isolated in the countercurrent fractionation device, were tested in phototaxis and geotaxis mazes. While none of the mutations caused an altered ERG, they all caused photomaze behavior to differ from that seen in Canton-S controls. Eight of the mutants showed altered geotactic behavior. There was no correlation between the kind of change in phototactic behavior and the geomaze behavior of a given mutant. Certain mutations cause flies to be more photopositive and more geonegative than Canton-S; others result in behavior that is photo- and geopositive. The results suggest that certain mutations may be affecting visual components other than the ERG while other mutations may be more centrally or generally acting.

A model for somatic pairing derived from somatic crossing over with third chromosome rearrangements in Drosophila melanogaster

SummaryX-ray induced crossing-over between the left arms of third chromosome pairs heterozygous for rearrangements was used to measure the somatic pairing of these arms. mwh spots were scored in the wing blades. A significantly reduced number of crossover spots is found in flies heterozygous for inversions or translocations located in the 3L arm. This decrease from control frequencies was experimentally demonstrated to be due to inviability of some crossover spots which are aneuploid as a result of crossing-over within the inverted sequence or between the translocation piece and its centromere. By the same token, some euploid crossover spots are produced by crossing-over outside of the inverted region or between the translocation breakpoint and the mwh locus. Thus, these results are evidence that the euchromatic non-centric portions of the 3L arms are somatically paired in the presence of rearrangements.A model of somatic pairing in which chromosomes are normally found in a “hair pin” configuration is proposed to account for these results and for some previous results with X-chromosomes which indicated that inversion heterozygotes did not pair somatically.

Are dicentric anaphase bridges formed by somatic recombination in X chromosome inversion heterozygotes of Drosophila melanogaster

SummaryMitotic recombination has been induced with X-rays in Drosophila melanogaster larvae and assayed later as twin mosaic spots on the adult tergites. With the use of the In(1)sc4Lsc8R chromosome which lacks the nucleolar organizer and is marked with yellow (y) indirect evidence was obtained that mitotic recombination between ring and rod chromosomes results, in a majority of cases, in XO spots, bearing the rod-X only. This was concluded from the relative scarcity and small sizes of y NO- spots (uncovering the sc4sc8 chromosome), compared to control sisters bearing an extra Y chromosome with its NO locus. Thus, dicentric chromatid bridges formed by mitotic recombination between the ring and rod chromosomes are probably eliminated at the next division.In In(1)sc4sc8/f36a (rod/rod) females, no effect of the Y chromosome on the frequency and sizes of cross-over spots was observed. Either any dicentric chromatid bridges formed by recombination between inverted rod chromosomes fragment at division, with a centromeric piece going to each pole, or such bridges are not usually formed by recombination. The latter case would indicate that somatic pairing of homologues is not accurate in X chromosome inversion heterozygotes and consequently, that recombination yields aneuploid cells.Additional studies are cited which indicate that X chromosome heterozygotes for entire arm inversions may not pair in the typical loop at the time of mitotic recombination.

Drosophila acid DNase is a homolog of mammalian DNase II.

Mammalian DNase II enzymes and the Caenorhabditis elegans homolog NUC-1 have recently been shown to be critically important during engulfment-mediated clearance of DNA. In this report, we describe the cloning and characterization of the gene encoding Drosophila DNase II. Database queries using the C. elegans NUC-1 protein sequence identified a highly homologous open reading frame in Drosophila (CG7780) that could encode a similar enzyme. Analysis of crude protein extracts revealed that wild-type Drosophila contain a potent acid endonuclease activity with cleavage preferences similar to DNase II/NUC1, while the same activity was markedly reduced in an acid DNase hypomorphic mutant line. Furthermore, the pattern of cleavage products generated from an end-labeled substrate by hypomorphic-line extracts was significantly altered in comparison to the pattern generated by wild-type extracts. Sequence analysis of CG7780 DNA and mRNA revealed that the hypomorphic line contains a missense mutation within the coding region of this gene. Additionally, Northern analysis demonstrated that CG7780 expression is normal in the mutant line, which in combination with the lowered/altered enzymatic activity and sequencing data suggested a defect in the CG7780 protein. To conclusively determine if CG7780 encoded the Drosophila equivalent of DNase II/NUC-1, transgenic lines expressing wild-type CG7780 in the mutant background were generated and subsequently shown to complement the mutant phenotype. Our results, therefore, provide compelling evidence that the predicted gene CG7780 encodes Drosophila DNase II (dDNase II), an enzyme related in sequence and activity to mammalian DNase II. Interestingly, overexpression of CG7780 both ubiquitously and in specific tissues failed to elicit any discernable phenotype.

Maximum likelihood estimates for fate map locations of behavior in Drosophila

Abstract Further analysis of the mosaic fly method of Hotta and Benzer shows that the fate map position of behavioral foci affecting all or none of the fly can be estimated in a variety of ways. Maximum likelihood estimation allows one to determine whether or not a given behavioral mutant fits the model of submissive foci or domineering foci or neither.