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.

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

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 beta-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.

Molecular, phenotypic, and expression analysis of vein, a gene required for growth of the Drosophila wing disc.

vein1 (vn1) mutants lack portions of longitudinal wing vein 4 and the anterior crossvein. Stronger alleles, originally called defective dorsal discs, show vn is also required for the growth of the wing and haltere discs, as mutants for these alleles have tiny dorsal discs. vn functions nonautonomously and encodes a secreted EGF-like protein. Here we characterize the role of vn in the imaginal wing disc by describing the expression pattern and correlating this pattern with vn mutant phenotypes and the requirement for vn. vn is expressed in wing discs in a complex and dynamic pattern. In larval wing discs vn is first expressed in the presumptive notum and later in the wing-pouch and hinge regions. There is a striking localization of vn transcripts to intervein regions which begins with a stripe of expression straddling the AP boundary in late larval discs and develops in all intervein regions after puparium formation. We isolated new vn mutations including nulls and hypomorphs. Hypomorphic vn alleles revealed region-specific requirements for vn within the wing disc. We mapped lesions caused by 10 vn mutations and defined a minimum size of 48 kb for the gene. The phenotype and expression analyses show vn has an early role in global proliferation of the wing disc and specific roles in the development of the notum, hinge, longitudinal vein 4, and all intervein regions. The role of vn in the EGF receptor signaling pathway is discussed.

The enzymatic activity of Drosophila AWD/NDP kinase is necessary but not sufficient for its biological function.

The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.

The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function

The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.

Genetic and molecular analysis of hyperplastic discs, a gene whose product is required for regulation of cell proliferation in Drosophila melanogaster imaginal discs and germ cells.

The hyperplastic discs (hyd) gene (formerly called l(3)c43) is located at 85E1-10 on the third chromosome of Drosophila melanogaster. It was originally identified by a temperature-sensitive mutation that causes imaginal disc overgrowth in mutant larvae raised at a restrictive temperature. Twenty new alleles of hyperplastic discs have been recovered in gamma ray, ethyl methanesulfonate and hybrid dysgenesis screens, and the molecular lesions have been identified for several of the alleles. The null phenotype appears to be lethality at or before the second instar. Adults that can be obtained in crosses of temperature-sensitive alleles maintained at permissive temperatures are sterile with one exception and have defects in germ tissue morphology. The hyperplastic discs locus has been cloned by chromosome walking from the alpha-tubulin-2 gene and encodes a 9.5-kb messenger RNA, containing a 2897-amino-acid open reading frame. Sequence analysis of HYD reveals strong similarity to a portion of the C-terminus of poly(A) binding protein and to the RAT 100-kDa PROTEIN, whose function is unknown. Developmental Northern and Western analyses show coincident accumulation of the 9.5-kb transcript and the 280-kDa protein at all stages of development, with high levels at the embryonic and pupal stages. The 280-kDa HYD protein accumulates at decreased levels in mutant alleles and at restrictive temperatures in ts alleles. Examination of relative levels of HYD protein in mutant animals support the idea that less severe mutations are those that result in disc overgrowth, while more severe mutations result in variable disc growth phenotypes.

Molecular genetic analysis of the Drosophila melanogaster gene absent, small or homeotic discs1 (ash1).

The absent, small or homeotic discs1 gene (ash1) is one of the trithorax set of genes. Recessive loss of function mutations in ash1 cause homeotic transformations of imaginal disc derived tissue which resemble phenotypes caused by partial loss or gain of function mutations in genes of the Antennapedia Complex and bithorax Complex. F2 screens were used to isolate P element insertion alleles and EMS-induced alleles of ash1, including one temperature-sensitive allele, and an F1 screen was used to isolate gamma-ray-induced alleles. Analysis of ash1 mutant flies that survive until the adult stage indicates that not only imaginal disc- and histoblast-derived tissues are affected but also that oogenesis requires ash1 function. Mutations in the gene brahma (brm) which also is one of the trithorax set of genes interact with mutations in ash1 such that non-lethal ash1 +/+ brm double heterozygotes have a high penetrance of homeotic transformations in specific imaginal disc- and histoblast-derived tissues. The cytogenetic location of ash1 was determined to be 76B6-11 by the breakpoint of a translocation recovered in the F1 screen. The gene Shal, which is located cytogenetically nearby ash1, was used to initiate an 84-kb genomic walk within which the ash1 gene was identified. The ash1 gene encodes a 7.5-kb transcript that is expressed throughout development but is present at higher levels during the embryonic and pupal stages than during the larval stages. During the larval stages the transcript accumulates primarily in imaginal discs. During oogenesis the transcript accumulates in the nurse cells of developing egg chambers.

Increased type IV collagenase in lgl-induced invasive tumors of Drosophila.

Loss of function mutations in the lethal giant larvae (lgl) gene causes neoplastic brain tumors in Drosophila. We have introduced a lacZ reporter gene into lgl mutant cells and used beta-galactosidase expression as a marker to monitor the growth of such tumors following transplantation into wild-type adult hosts. Whereas normal larval brains do not grow when transplanted, mutant brains can develop into enormous tumors that fill the entire abdominal cavity. To investigate whether these tumors are similar to mammalian tumors at the biochemical level, we examined the accumulation of a specific protein which is differentially expressed in mammalian metastatic tumors and is likely to be involved in the invasive and/or metastatic mechanism. Increased accumulation of a 72 kilodalton (kDa) type IV collagenase has been observed in several metastatic human tumors. Using antibodies directed against this human 72 kDa type IV collagenase, we show for the first time that Drosophila has a cross-reacting 49 kDa protein with gelatinase activity. In brains dissected from lgl mutant larvae, the accumulation of this 49 kDa gelatinase of Drosophila is increased compared to the level in brains dissected from wild-type larvae. In tumors derived from mutant brains, all of the cells express this protein. Moreover, the tumor cells that invade host organs express this protein. These data suggest that the metastasis of Drosophila tumor cells is similar to the metastasis of some human tumors at the biochemical level as well as at the cellular level.

The expression of the Drosophila awd gene during normal development and in neoplastic brain tumors caused by lgl mutations.

The abnormal wing discs (awd) gene of Drosophila is homologous to the nm23 gene of mammals, a gene whose expression is altered in metastatic tumors. Both awd and nm23 encode nucleoside diphosphate kinases (NDP kinases). We have examined the accumulation of AWD/NDP kinase during normal development by assaying enzyme activity in extracts. There is a nearly constant level of activity throughout larval and pupal development. We have examined the tissue-specific transcription of the awd gene by RNA in situ hybridization and by reporter gene expression. In imaginal discs and brains there is no detectable awd gene expression until the beginning of the third larval instar, despite the constant level of enzyme activity measured in extracts of larvae and pupae. The most intense awd gene expression in imaginal discs and brains occurs after the end of larval development. We have also examined awd gene expression in neoplastic brain tumors caused by mutations in the lethal giant larvae (lgl) gene. In lgl mutant brains, as in normal brains, awd gene expression begins during the third larval instar. No tumors form in brains from lgl-; awd- double mutant larva, so awd gene expression is required for tumor formation and/or proliferation. There is more accumulation of AWD/NDP kinase in lgl- mutant brains than there is in normal brains. Using an awd reporter gene, we show that this is a consequence of an increased proportion of awd gene-expressing cells in mutant brains. Using the same awd reporter gene as a marker of donor cells, we have confirmed the invasiveness of lgl-induced neuroblastomas.

Establishment of imaginal discs and histoblast nests in Drosophila.

In Drosophila the homeotic genes of the bithorax-complex (BX-C) and Antennapedia-complex (ANT-C) specify the identity of segments. Adult segment primordia are established in the embryo as the histoblast nests of the abdomen and the imaginal discs of the head, thorax and terminalia. We have used a molecular probe for the limb primordia and in vivo culture to describe the nature of the adult primordia in mutants in which the pattern of homeotic gene expression was altered. The results suggest that the histoblast or disc 'mode' of development is initiated by the extended germ band stage through activity of the BX-C and ANT-C and is relatively inflexible thereafter [corrected].

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.

Imaginal discs can be recovered from cultured embryos mutant for the segment-polarity genes engrailed, naked and patched but not from wingless.

Drosophila embryos homozygous for strong mutations in each of the segment-polarity genes wingless (wg), engrailed (en), naked (nkd) and patched (ptc) form a larval cuticle in which there is a deletion in every segment. The mutant embryos normally fail to hatch but by in vivo culture we were able to show which could produce adult structures. Cultured wg⁻ embryos did not produce any adult structures. Cultured en⁻ embryos produced eye-antennal derivatives and rarely produced partial thoracic structures. nkd⁻ and ptc⁻ embryos produced eye-antennal and thoracic derivatives. The nkd⁻ and ptc⁻ thoracic imaginal discs developed with an abnormal morphology and abnormal pattern of en-expression. Our findings are consistent with the idea that the thoracic imaginal discs derive from two adjacent groups of cells that express wg and en respectively in the embryo.

Analysis of the lethal interaction between the prune and Killer of prune mutations of Drosophila.

The third-chromosome mutation Killer of prune (K-pn) causes no phenotype by itself, but causes lethality in individuals homozygous for the nonlethal X-chromosome mutation prune (pn). We have recovered 12 gamma-ray-induced revertants of Killer of prune. All of the revertants fail to complement a recessive cell lethal mutation in the abnormal wing discs (awd) gene. We present evidence that Killer of prune is a mutation in the awd gene. First, revertant awdKR14 leads to reduced accumulation of the awd gene product, but does not affect flanking genes. Second, when a copy of the awd gene is cloned from Killer of prune homozygous flies and injected into embryos, transformants express the lethal interaction with prune. In individuals of the genotype pn; awdK-pn/awd+ the awd mRNA is present at normal levels but the awd polypeptide fails to accumulate. The absence of the awd gene product in such individuals is the cause of death. Although the awd polypeptide is a subunit of a cytoplasmic protein, its sequence is similar to subunit V of yeast cytochrome oxidase.

Developmental consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis.

In order to recover mutations affecting imaginal discs in a way which would allow the relevant genes to be readily cloned, a hybrid dysgenic screen was performed for mutations causing late larval/early pupal lethality. This paper describes that mutagenesis procedure and the phenotypes caused by the mutations that were recovered. Of 81 late larval/pupal lethal mutations that were recovered, 20 cause imaginal disc defects. These 20 mutations define 12 different genes. This paper also includes a description of the developmental defects caused by a mutation in one of those 12 genes which we have named abnormal wing discs (awd); the following paper (C. Dearolf, N. Tripoulas, J. Biggs, and A. Shearn, 1988, Dev. Biol. 129, 169-178) describes the cloning of the awd gene and an analysis of its pattern of transcription. awdb3 homozygotes develop at a normal rate until the end of the second larval instar, when their rate of development is reduced. After an extended third larval instar, they form puparia and die. Mutant wing discs have an abnormal morphology and extensive cell death. These abnormal wing discs, and also the leg and eye-antenna discs which appear to be morphologically normal, differentiate poorly or not at all when injected into metamorphosing larvae. Analysis of genetic mosaics indicates that the awdb3 mutation is expressed in a cell-autonomous manner in wing, leg, and eye-antenna discs. The larval brain and proventriculus in awdb3 homozygous third-instar larvae appear to be vacuolated due to the accumulation of lipid droplets. Mutant ovaries are unable to develop when injected into wild-type larvae, although mutant germ cells are capable of producing normal eggs.

The defective dorsal discs gene of Drosophila is required for the growth of specific imaginal discs.

The wild-type allele of the gene defective dorsal discs (ddd) is required for the normal development of the dorsal thoracic discs in Drosophila melanogaster. In ddd mutant larvae the dorsal discs (wing, haltere, and humeral) are greatly reduced in size or absent while the ventral discs (leg) are unaffected. We have examined the function of the ddd+ gene in wing development. The ddd+ product is not involved in the initial determination of wing cells but rather is required for their subsequent proliferation during the larval period. Analysis of chimaeras shows that there is a requirement for ddd+ gene expression in wing discs, but it is sufficient for normal development that only some cells in a disc express the gene. We propose that the ddd+ product is involved in the synthesis of a factor which is required for the normal growth of wing discs and which can be transferred between wing disc cells.

Genetic analysis of transdetermination in Drosophila. I. The effects of varying growth parameters using a temperature-sensitive mutation.

The heat-sensitive mutation of Drosophila melanogaster l(3)c4(3)hs1, causes mutant larvae raised at a restrictive temperature to have abnormally large wing discs. The large size of these discs is a disc-autonomous property and results from an increase in the number rather than the size of wing disc cells. We have used wing discs from this mutant to further investigate properties of transdetermination which had previously been investigated with nonmutant discs. Transdetermination can occur in nonmutant discs when the proliferative phase of imaginal disc development is extended by wounding discs and culturing them in vivo. The results indicate that additional proliferation in the absence of wounding does not lead to transdetermination. There is a correlation between the extent of growth of a cultured disc and the probability that it will undergo transdetermination. The results suggest that this correlation does not depend on a differential rate of cell division. Finally, the results indicate that the cells which give rise to transdetermination are at an equivalent developmental stage no later than that characteristic of eye-antenna disc cells before the third larval instar.

Genetic analysis of transdetermination in Drosophila. II. Transdetermination to wing of leg discs from a mutant which lacks wing discs.

Drosophila melanogaster larvae homozygous for lethal mutations at the L6 locus have no wing discs. However, like wild-type leg discs, leg discs from such mutant larvae can transdetermine to wing. Apparently such a transdetermination event bypasses the block to wing disc development caused by mutations at this locus. In order to evaluate the significance of this observation we have examined the cell autonomy of the mutant phenotype and the capacity of mutant larvae to support the growth of normal wing discs. The mutation appears not to be expressed cell autonomously, yet mutant larvae can support the growth of normal wing discs. One way of resolving these paradoxical results is to hypothesize that the normal product of the L6 gene is essential only for an initial step of wing disc development. According to this hypothesis, the fact that mutant leg discs can transdetermine to wing implies that transdetermination does not proceed by recapitulating that step of normal wing disc development.

Multiple Allele Approach to the Study of Genes in DROSOPHILA MELANOGASTER That Are Involved in Imaginal Disc Development.

The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.

Genetic analysis of two allelic temperature-sensitive mutants of Drosophila melanogaster both of which are zygotic and maternal-effect lethals.

After fertilization, the development of a zygote depends upon both gene products synthesized by its maternal parent and gene products synthesized by the zygote itself. To analyze genetically the relative contributions of these two sources of gene products, several laboratories have been isolating two classes of mutants of Drosophila melanogaster: maternal-effect lethals and zygotic lethals. This report concerns the analysis of two temperature-sensitive mutants, OX736hs and PC025hs, which were isolated as alleles of a small-disc mutant, l(3)1902. These alleles are not only zygotic lethals, but also maternal-effect lethals. They have temperature-sensitive periods during larval life and during oogenesis. Mutant larvae exposed continuously to restrictive temperature have small discs. One-or two-day exposures to the restrictive temperature administered during the third larval instar lead to a homeotic transformation of the midlegs and hindlegs to the pattern characteristic of the forelegs. Mutant females exposed to the restrictive temperature during oogenesis produce eggs that can develop until gastrulation, but do not hatch.--The existence of these mutants, and one that was recently described by another group, implies that there may be a class of genes, heretofore unrecognized, whose products are synthesized during oogenesis, are essential for embryogenesis and are also synthesized during larval stages within imaginal disc cells.