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.

Allocation of the thoracic imaginal primordia in the Drosophila embryo.

The primordia of the thoracic imaginal discs of the Drosophila embryo originate as groups of cells spanning the parasegment boundary. We present evidence that the thoracic imaginal primordia are allocated in response to signals from the wingless (wg) and decapentaplegic (dpp) gene products. Rows of cells that express wg intersect rows of cells that express dpp to form a ladder-like pattern in the ectoderm of the germ band extended embryo. The imaginal primordia originate as groups of cells which lie near these intersection points. We have used a molecular probe derived from the Distal-less (Dll) gene to show that this population contains progenitor cells for both the dorsal (i.e. wing) and ventral (i.e. leg) discs. Although we show that Dll function is not required for allocation of imaginal cells, activation of an early Dll enhancer may serve as a molecular marker for allocation. A group of cells, which includes the imaginal progenitors, activate this enhancer in response to intercellular signals from wg and perhaps from dpp. We have used a conditional allele of wg to show that wg function is transiently required for both allocation of the imaginal primordia and for initiation of Dll expression in these cells during the brief interval when wg and dpp form the ladder-like pattern. Allocation of the imaginal primordium and activation of Dll expression appear to be parallel responses to a single set of positional cues.

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].

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.

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.

A deletion of the 3' end of the Drosophila melanogaster hsp70 gene increases stability of mutant mRNA during recovery from heat shock.

hsp40, an X-ray-induced deletion mutant of the major Drosophila melanogaster heat shock protein gene hsp70, was shown to be incorrectly regulated at the translational level. hsp40 protein synthesis persisted at a high level after the release from heat shock, whereas hsp70 protein production was rapidly repressed. This result was observed both in flies heterozygous for the hsp40 gene and in tissue culture cells transfected with the truncated gene. Analysis of the transcription of the hsp40 gene indicated that its mRNA, unlike hsp70 mRNA, was not actively destabilized after a return to control temperatures, permitting prolonged production of the mutant protein.

Establishment and characterization of continuous cell lines derived from temperature-sensitive mutants of Drosophila melanogaster.

Embryos of 21 temperature-sensitive Drosophila mutants were used to set up 232 primary cultures. From these, 57 cell lines are being subcultured, and we consider 33 of them to be continuous. Nineteen of the 21 mutants are represented in these 57 lines. Four characteristics of each of the continuous lines are described: morphology, ecdysterone response, karyotype, and response to a restrictive temperature. We discuss the potential uses of such cell lines.

When does determination occur in Drosophila embryos?

Small groups of blastoderm cells were transplanted from wild-type donor embryos into genetically marked host embryos of the same age. Donor cells were injected either into an homologous or an ectopic region of the recipient, and both donor and recipient embryos were allowed to develop. Donor flies were examined for defects in external structures. Recipients were scored for patches of donor-type marked tissue derived from the injected cells. After ectopic transfer, the donor cells recovered in chimaeric recipients differentiated structures consistent with the donor site of cell removal. No apparent fate change was observed. In the rare cases when both individuals of a donor/host pair survived, a direct correspondence could be made between the deleted region in the donor and the chimaeric patch in the host. The results show that blastoderm cells are stably determined to within a segment.