Allen Shearn

A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase

The product of the abnormal wing discs (awd) gene of Drosophila is 78% identical to the product of the nm23 gene of mammals, which is differentially expressed in certain metastatic tumors. We present evidence that the awd gene codes for a nucleoside diphosphate kinase (NDP kinase) and that this Awd/NDP kinase is microtubule associated. Neuroblasts in Drosophila larvae homozygous for a null mutation in the awd gene are arrested in metaphase, indicating that microtubule-associated Awd/NDP kinase plays a critical role in spindle microtubule polymerization.

Molecular consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis☆

The abnormal wing disc locus, which is at salivary gland chromosome position 100C-D of the Drosophila melanogaster genome, has been identified by a recessive lethal mutation, awdb3, induced by hybrid dysgenesis. When homozygous, this mutation causes abnormal development of the brain, the ovaries, and the larger imaginal discs as described in the preceding paper (C.R. Dearolf, E. Hersperger, and A. Shearn, 1988, Dev. Biol. 129, 159-168). The DNA corresponding to this locus was isolated from a genomic library prepared from awdb3 heterozygotes by screening with a P-element probe. The awdb3 allele resulted from the insertion of a P-element fragment into a gene that encodes an 0.8-kb poly(A)+ RNA. In mutant larvae, that 0.8-kb transcript is replaced by two chimeric transcripts that are 0.7 and 1.3 kb in length, both of which contain P-element and awd sequences. The wild-type awd+ gene transcript is most abundant during the second and third larval instars but is found at a lower level during every developmental stage as well as in continuous cell lines. Thus the awd+ gene transcript can be detected in normal larvae at a developmental stage long before defects are expressed in mutant larvae. Moreover, some tissues, for example the salivary gland of nonmutant, third-instar larvae, contain high levels of this transcript, even though these tissues appear to develop normally in mutant larvae.

The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. The two genes are independently regulated by hyperplastic discs; decapentaplegic can still be misexpressed in cells mutant for both hyperplastic discs and hedgehog, and a repressor form of the transcription factor Cubitus interruptus can block decapentaplegic misexpression but not hedgehog misexpression. Loss of hyperplastic discs causes the accumulation of full-length Cubitus interruptus protein, but not of Smoothened, in both the eye and wing discs. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis.

Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in specific tumor suppressor genes

Abstract More than 50 genes have been identified in Drosophila by loss-of-function mutations that lead to overgrowth of specific tissues. Loss-of-function mutations in the lethal giant larvae, discs large, or brain tumor genes cause neoplastic overgrowth of larval brains and imaginal discs. In the present study, the growth and metastatic potential of tumors resulting from mutations in these genes were quantified. Overgrown brains and imaginal discs were transplanted into adults and β-galactosidase accumulation was used as a marker to identify donor cells. Mutations in these three genes generated tumors with similar metastatic patterns. For brain tumors, the metastatic index (a measure we defined as the fraction of hosts that acquired secondary tumors normalized for the amount of primary tumor growth) of each of the three mutants was similar. Analysis of cell proliferation in mutant brains suggests that the tumors arise from a population of several hundred cells which represent only 1–2% of the cells in third instar larval brains. For imaginal disc tumors from lethal giant larvae and brain tumor mutants, it is shown for the first time that they can be metastatic and invasive. Primary imaginal disc tumors from lethal giant larvae and brain tumor mutants formed secondary tumors in 43 and 53% of the hosts, respectively, although the secondary tumors were, in general, smaller than the secondary tumors derived from primary brain tumors.

Developmental regulation of Drosophila imaginal disc proteins: Synthesis of a heat shock protein under non-heat-shock conditions☆

A low-molecular-weight heat-shock protein is reported here to be synthesized in Drosophila imaginal discs under non-heat-shock conditions. This protein has been identified as hsp 23 by its mobility in SDS-gel electrophoresis, by its isoelectric point, and by its partial proteolytic digest pattern. This protein is shown to be synthesized in tissue from late-third-instar larvae using both in vitro and in vivo radiolabeling techniques. Tissues from late-third-instar larvae synthesize hsp 23 at a level much higher than tissues from mid-third-instar larvae. The increased synthesis of this protein at the end of third instar coincides with the rise in ecdysone titer that occurs at this time of development. The developmental regulation of this protein suggests that hsp 23 plays a role in normal development as well as in the heat-shock response.

minidiscs encodes a putative amino acid transporter subunit required non-autonomously for imaginal cell proliferation.

Drosophila minidiscs mutant larvae have smaller imaginal discs than wild-type larvae. However, transplantation experiments have revealed that minidiscs mutant imaginal discs can grow if cultured in non-mutant hosts. These data suggest that minidiscs is required in one or more non-imaginal tissues for synthesis and/or secretion of a diffusible factor that stimulates imaginal cell proliferation. The 2. 3 kb minidiscs transcript accumulates in the larval fat body and encodes a protein containing 12 putative membrane spanning domains that is similar in sequence to amino acid transporter subunits from other eukaryotes, including humans. We propose that in response to amino acid uptake by the transporter encoded by minidiscs, the fat body secretes a diffusible factor required for imaginal disc proliferation.

Trans-regulation of thoracic homeotic selector genes of the Antennapedia and bithorax complexes by the trithorax group genes: absent, small, and homeotic discs 1 and 2

Genes of the trithorax group appear to be required for the maintenance of expression of the homeotic selector genes of the Antennapedia and bithorax complexes. According to genetic criteria, the Drosophila melanogaster genes absent, small, or homeotic discs 1 and 2 (ash1 and ash2) are members of the trithorax group. In this paper we examine the consequences of ash1 and ash2 mutations on the expression of homeotic selector genes in imaginal discs. The results of these experiments demonstrates that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed. Complete loss of ash2 activity causes the loss of expression of Sex combs reduced in first leg imaginal discs, loss of expression of Ultrabithorax in third leg discs, and a late-patterned loss of expression of Ultrabithorax within haltere discs, yet has no effect on engrailed or Antennapedia expression. These results suggest that the range and action of trithorax group genes is varied and complex and argue against any model in which all of the products of the trithorax group act together in a single mechanism or complex.

Studies of l(3)c43hs1 a polyphasic, temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc defects

Abstract The heat-sensitive, lethal mutation l (3) c 43 hs 1 (3–49.0) produces wide variety of defects in the imaginal discs of Drosophila melanogaster . At permissive temperatures (20°C or lower), homozygotes are viable, but sterile. At 22°C, lethality occurs during the late pupal stage, and at 25°C or higher, lethality occurs during the third larval instar. The imaginal-disc abnormalities observed after exposure to restrictive temperatures include: deficiencies of head structures, duplications and deficiencies of the antenna, a homeotic transformation of the arista to tarsus, duplications and deficiencies of wing and haltere structures, differentiation of amorphous cuticular material in the wing blade, an increase in the number of sex-comb teeth, and disruption of the normal segmentation of the tarsus. Exposure to 27°C for 24 hr at different times in the life cycle revealed that each of these defects has a characteristic temperature-sensitive period (TSP) during the larval stages. Injection of wing discs before and after their TSP showed that the mutation is expressed autonomously. These results are discussed in relation to the role that the l (3) c 43 + gene plays in the development of imaginal discs.

Green fluorescent protein/beta-galactosidase double reporters for visualizing Drosophila gene expression patterns.

We characterized 120 novel yeast Ga14-targeted enhancer trap lines in Drosophila using upstream activating sequence (UAS) reporter plasmids incorporating newly constructed fusions of Aequorea victoria green fluorescent protein (GFP) and Escherichia coli beta-galactosidase genes. Direct comparisons of GFP epifluorescence and beta-galactosidase staining revealed that both proteins function comparably to their unconjugated counterparts within a wide variety of Drosophila tissues. Generally, both reporters accumulated in similar patterns within individual lines, but in some tissues, e.g., brain, GFP staining was more reliable than that of beta-galactosidase, whereas in other tissues, most notably tests and ovaries, the converse was true. In cases of weak enhancers, we occasionally could detect beta-galactosidase staining in the absence of discernible GFP fluorescence. This shortcoming of GFP can, in most cases, be alleviated by using the more efficient S65T GFP derivative. The GFP/beta-gal reporter fusion protein facilitated monitoring several aspects of protein accumulation. In particular, the ability to visualize GFP fluorescence enhances recognition of global static and dynamic patterns in live animals, whereas beta-galactosidase histochemistry affords sensitive high resolution protein localization. We present a catalog of Ga 14-expressing strains that will be useful for investigating several aspects of Drosophila melanogaster cell and developmental biology.

Role of AWD/nucleoside diphosphate kinase in Drosophila development.

The abnormal wing discs gene of Drosophila encodes a soluble protein with nucleosidediphosphate kinase activity. This enzymic activity is necessary for the biological function ofthe abnormal wing discs gene product. Complete loss of function, i.e., null, mutations causelethality after the larval stage. Most larval organs in such null mutant larvae appear to benormal, but the imaginal discs are small and incapable of normal differentiation.Killer-of-prune is a neomorphic mutation in the abnormal wing discs gene. It causes dominant lethalityin larvae that lack prune gene activity. The Killer-of-prune mutant protein may have alteredsubstrate specificity. Null mutant larvae have a low level of nucleoside diphosphate kinaseactivity. This suggests that there may be additional Drosophila genes that encode proteinswith nucleoside dipthosphate kinase activity. Candidate genes have been found in theDrosophila genome.