Ecdysteroid-regulated heat-shock gene expression during Drosophila melanogaster development

Peaks in hsp 26, 28, and 83 RNA levels are correlated with peaks in ecdysteroid titers during mid-embryogenesis, pupariation, and mid-pupation, and with a peak in the level of RNA from the 74EF ecdysone puff at pupariation. Inhibition of the ecdysteroid peak at pupariation by temperature shift of the conditionally ecdysteroid-deficient strain ecd-1 was followed by a disappearance of hsp 26 RNA and a decline in hsp 83 RNA level; subsequent addition of exogeneous 20-OH-ecdysone to the temperature-shifted strain resulted in a severalfold increase in hsp 83 RNA level, and a dramatic increase in that of hsp 26. These results are consistent with the induction of the hsp 83, 28, and 26 genes by ecdysteroid at several developmental stages.     

Cloning of heat-shock locus 93D from Drosophila melanogaster.

Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.     

Expression of the polyhomeotic locus in development of Drosophila melanogaster

The polyhomeotic (ph) gene of Drosophila melanogaster encodes a chromatin protein required for negative regulation of homeotic genes, and is a member of the Polycomb group of genes. The distribution of ph mRNA and protein was determined in embryos, imaginal discs, and ovaries. Distribution of ph protein and mRNA coincided, except in early embryogogenesis. During blastoderm, ph mRNA is present in anterior and posterior domains that are themselves subdivided into stripes. During germ band extension, a segmentally repeated striped pattern of mRNA expression is seen. ph protein is first detected as a nuclear protein during cell cycle 10, and is ubiquitously expressed. ph protein stains more heavily in the ectodermal mitotic domains described by Foe (1989). Later, ph mRNA and protein expression is concentrated in the neuronal cell bodies of the central nervous system, and can also be detected in the peripheral nervous system. In imaginal discs, ph expression is non-uniform in metathoracic discs, but appears more regular in other imaginal discs. The ph mRNA is found in the germarium and in stages 1–10 in nurse cells and follicle cells, but we do not detect it in oocytes. These results are discussed with respect to the expression of Polycomb, and with respect to the function of the Polycomb group.     

Deficiency mapping of the 93D heat-shock locus in Drosophila melanogaster

The 93D heat shock locus was mapped relative to an overlapping series of deficiencies of the 93D region by three criteria: the ability of the deleted chromosomes to puff at 93D, the ability of the deleted chromosomes to synthesize RNA from the 93D region after a temperature shift and the presence of heat shock RNA sequences at 93D as assayed by in situ hybridization. The results are essentially the same by all three criteria. Chromosomes with deficiencies that did not extend distal to 93D4 puffed and incorporated ³H-uridine after a temperature shift, and were labelled at 93D following in situ hybridization of heat shock RNA from tissue culture cells. All the other deficiency chromosomes tested failed to puff and to incorporate ³H-uridine following a temperature shift and did not show hybridization in this region after in situ hybridization with heat shock RNA. The heat shock locus was mapped to the overlapping region of Df(3R)e ^Gp4and Df(3R)GC14 just outside the inverted region of In(3R)GC23.     

Expression of the disconnected gene during development of Drosophila melanogaster.

Proper development of the larval visual nerve, Bolwig's nerve, of Drosophila melanogaster requires the wild type function of the disconnected (disco) gene. In disco mutants, the nerve does not make stable connections with its targets in the larval brain. We have begun to explore the role of disco in the formation of the nervous system by examining the distribution of disco mRNA and protein in embryos and third instar larvae using in situ hybridization and antibody staining respectively. No differences between the distribution patterns of the two products are detected; disco is expressed in many tissues including both neural and non-neural cells. Many of the cells which express disco undergo extensive movement during development as they participate in major morphogenetic movements. Antibody staining shows that the protein is found in the cell nucleus. Products of the disco gene are detected in cells near the terminus of the growing Bolwig's nerve. In embryos homozygous for either of two mutant alleles of disco, the disco protein is absent near the nerve terminus, although protein distribution elsewhere is indistinguishable from wild type.     

Localized heat-shock induction in Drosophila melanogaster

We describe a technique for inducing localized expression of genes fused to heat-shock gene promoters. We demonstrate that a localized heat-shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for beta-galactosidase activity. Induced beta-galactosidase activity is confined to the cells in the region of heating; the beta-galactosidase activity can still be detected 48 hr after the heat shock. Given the heat inducibility of Drosophila heat-shock promoters in heterologous systems, we suggest that this technique will be useful for allowing spatially controlled induction of a gene of interest in any organism into which fusion genes can be introduced. Additional uses of the technique for following cell movements during development are discussed.     

Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster

The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and ry² mutants. In vitro complementation experiments demonstrated the presence of the mal⁺ complementation factor in the oocyte, suggesting an explanation for the mal maternal effect. The ry⁺ complementation factor synthesized from paternal template was detected at gastrulation. This is the earliest detection of a paternal enzyme during nonmammalian embryonic development.     

Translational inhibition of heat-shock induced gene expression in picornavirus-infected Drosophila melanogaster cells

Picornavirus infection of Drosophila melanogaster cells inhibited the appearance of heat-shock induced proteins. Examination of intracellular mRNAs revealed that those coding for heat shock proteins were present in a translationally competent form in infected cells. Inhibition of induced gene expression in infected Drosophila cells therefore involves, but is not necessarily solely mediated by, effects at the level of translation.     

Ecdysone titers during postembryonic development of Drosophila melanogaster.

The level of ecdysone in Drosophila melanogaster was determined by a radioimmune assay in organisms selected between the second larval instar and maturity. Maxima in the titer of the hormone were observed at puparium formation and 38 hr later, just prior to the secretion of the adult cuticle. The level of ecdysone was very low in adults of either sex. However, adult females had significantly more ecdysone per organism than did males. The magnitude of this difference could be correlated with ovarian development, suggesting a possible role for ecdysone in ovarian maturation in this organism.     

Expression of cis-regulatory mutations of the white locus in metafemales of Drosophila melanogaster.

At the white eye colour locus, there are a number of alleles that have altered expression between males and females. To test these regulatory mutations of the white eye colour locus for their phenotypic expression in metafemales (3X; 2A) compared to diploid females and males, eleven alleles or transduced copies of white were analysed. Two alleles that exhibit dosage compensation between males and females (apricot, blood) also exhibit dosage compensation in metafemales. White-ivory and white-eosin, which fail to dosage compensate in males compared to females, but that are distinct physical lesions, also show a dosage effect in metafemales. Two alleles with greater expression in males than females (spotted, spotted-55) exhibit even lower expression in metafemales. Lastly, five transduced copies of white carrying three different lengths of the white promoter, but that all exhibit higher expression in males, show reduced expression in metafemales, exhibiting an inverse correlation between the level of expression and the dosage of the X chromosome. Because these alleles of white respond to dosage compensation in metafemales as a continuum of the male and female responses, it is concluded that the same basic mechanism of dosage compensation is involved and that the dosage of the X chromosome conditions the sexually dimorphic expression.     

Fatty-acid preference changes during development in Drosophila melanogaster

Fatty-acids (FAs) are required in the diet of many animals throughout their life. However, the mechanisms involved in the perception of and preferences for dietary saturated and unsaturated FAs (SFAs and UFAs, respectively) remain poorly explored, especially in insects. Using the model species Drosophila melanogaster, we measured the responses of wild-type larvae and adults to pure SFAs (14, 16, and 18 carbons) and UFAs (C18 with 1, 2, or 3 double-bonds). Individual and group behavioral tests revealed different preferences in larvae and adults. Larvae preferred UFAs whereas SFAs tended to induce both a strong aversion and a persistent aggregation behavior. Adults generally preferred SFAs, and laid more eggs and had a longer life span when ingesting these substances as compared to UFAs. Our data suggest that insects can discriminate long-chain dietary FAs. The developmental change in preference shown by this species might reflect functional variation in use of FAs or stage-specific nutritional requirements, and may be fundamental for insect use of these major dietary components.