Allelic interactions at the engrailed locus of Drosophila: engrailed protein expression in imaginal discs

Many embryonic lethal engrailed (enlethal) mutations are known to partially complement the cuticular defects of the original engrailed mutation, en1. To explore the nature of this complementation, the adult phenotypes of several different en1/enlethal transheterozygotes were compared with the corresponding patterns of engrailed protein expression in third larval instar imaginal discs (determined by immunofluorescence). Transheterozygotes of en1 and deletions of the locus (enDf) typically show slight complementation in the adult cuticle. The pattern of engrailed protein expression in some en1/enDf wing discs is indistinguishable from en1 homozygotes, but in others the pattern is nearly normal. en1/enDf leg discs appear to express engrailed protein normally. Transheterozygotes of en1 and EMS-induced, cytologically normal enlethal alleles have almost normal adult cuticle phenotypes and also exhibit normal patterns of engrailed protein expression in all of the thoracic imaginal discs. Surprisingly, the intensity of anti-engrailed staining in these discs is elevated relative to that in wild type. en2 is an unusual lethal allele in that it does not complement either the en1 adult cuticle phenotype or the protein expression pattern in imaginal discs. Moreover, the cytologically normal enlethal alleles also complement en2, at least partially. Both wing and leg imaginal discs from en2/enlethal transheterozygotes show abnormal patterns of engrailed protein expression. These results are discussed in the context of an autoregulatory model for engrailed regulation.     

Compartments in the abdomen of Drosophila and the role of the engrailed locus

A clonal analysis has shown that the dorsal surface of the first abdominal segment of Drosophila melanogaster is subdivided into anterior and posterior compartments. Cells of the posterior compartment grow up to but not beyond the anterior-posterior compartment border within the first abdominal segment and the intersegmental border that defines the boundary between the first and second abdominal segments. Growing within these boundaries, a narrow band of tissue clonally isolated from the adjoining tissue is formed. When these posterior cells are deficient for the engrailed locus, however, neither the compartment nor the segment border is maintained. The implications, that compartmentalization is essential for segmentation, and that all insect segments are subdivided by anterior and posterior compartments, are discussed.     

The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5'' leader.

The Ace locus of Drosophila melanogaster has been mapped at the molecular level. cDNA clones from the locus have been isolated and their sequence determined, confirming that Ace forms the structural gene for acetylcholinesterase (AChE). The cDNAs have a 1950 nucleotide open reading frame from which the complete amino acid sequence of AChE has been deduced. The Drosophila enzyme is found to have extensive homology to the known sequence of Torpedo AChE. Ace cDNAs have an unusual structure with a long 5' leader and several short upstream open reading frames.     

Patterns of engrailed protein in early Drosophila embryos

By the onset of gastrulation during nuclear cycle 14 of Drosophila embryogenesis, the engrailed gene is expressed in fourteen one-cell-wide stripes. Each stripe defines the anlagen of the posterior compartment of a metameric segment. We report here several observations relating to the role and disposition of the engrailed protein during the embryonic stages that precede cellularization. We demonstrate that in embryos mutant for the engrailed gene, there were characteristic morphological abnormalities as early as the 6th cleavage cycle. In addition, the engrailed protein was detected in pre-cycle-9 embryos by Western blot analysis. When localization of engrailed protein begins during cycle 14, engrailed expression was first present in broad anterior and posterior regions before the fourteen-stripe pattern appeared.     

Coordinate embryonic expression of three zebrafish engrailed genes.

We have identified three genes, expressed in zebrafish embryos, that are members of the engrailed gene family. On the basis of sequence comparisons and analyses of their expression patterns, we suggest that two of these genes, eng2 and eng3, are closely related to the En-2 gene of other vertebrates. The third gene, eng1, is probably the zebrafish homolog of En-1. Subsets of cells at the developing junction between the midbrain and hindbrain express three different combinations of these genes, revealing a previously unknown complexity of this region of the CNS. Other cells, for example, jaw and myotomal muscle precursors, express two of the three genes in combinations which, in the myotomal muscles, change during development. Cells in the developing hindbrain and fins express only a single engrailed gene. We propose that the fates and patterning of these cells may be regulated by the coordinate expression of particular combinations of these closely related homeoproteins.     

Drosophila embryonic 'fibroblasts': Extending mutant analysis in vitro

The in vivo analysis of Drosophila using genetics, with almost a hundred year history, has produced an immense body of knowledge about biology. In vitro analysis, while arguably the poor cousin to its in vivo relative, has a utility-in biochemical analyses and in cell-based screening, for example, with RNAi. A major block to the development of in vitro analysis has been the lack of an efficient genetic method to derive cell lines from mutant Drosophila strains. We recently discovered that expression of activated Ras (Ras^V12) provides cells in vitro with both a survival and a proliferative advantage and hence promotes the generation of cell lines.¹ In this addendum, we provide new data describing the genesis of seven cell lines corresponding to a rumi mutant, which demonstrate that the method can be used to derive lines and study genetic mutants in vitro.     

Further studies of the engrailed phenotype in Drosophila.

Although most mutations at the engrailed locus of Drosophila cause embryonic death when homozygous, they are viable in clones of cells. We describe the phenotype of such clones in the eye-antenna, proboscis, humerus, wing, legs, and terminalia. When in anterior compartments the clones are normal, but in most posterior compartments they are abnormal and fail to respect the anteroposterior compartment boundary. We find that the yield of engrailed-lethal clones in posterior compartments is often significantly lower than expected, indicating that these clones are lost during development. Mutant clones are abnormal in the analia and rare in the humerus, suggesting that both structures are of posterior provenance. These results support the hypothesis that the engrailed+ gene is required exclusively in cells of posterior compartments to specify their characteristic cell affinities and pattern.     

The role of Polycomb-group response elements in regulation of engrailed transcription in Drosophila

Polycomb group proteins are required for long-term repression of many genes in Drosophila and all metazoans. In Drosophila, DNA fragments called Polycomb-group response elements (PREs) have been identified that mediate the action of Polycomb-group proteins. Previous studies have shown that a 2 kb fragment located from -2.4 kb to -395 bp upstream of the Drosophila engrailed promoter contains a multipartite PRE that can mediate mini-white silencing and act as a PRE in an Ubx-reporter construct. Here, we study the role of this 2 kb fragment in the regulation of the engrailed gene itself. Our results show that within this 2 kb fragment, there are two subfragments that can act as PREs in embryos. In addition to their role in gene silencing, these two adjacent PRE fragments can facilitate the activation of the engrailed promoter by distant enhancers. The repressive action of the engrailed PRE can also act over a distance. A 181 bp subfragment can act as a PRE and also mediate positive effects in an enhancer-detector construct. Finally, a deletion of 530 bp of the 2 kb PRE fragment within the endogenous engrailed gene causes a loss-of-function phenotype, showing the importance of the positive regulatory effects of this PRE-containing fragment. Our data are consistent with the model that engrailed PREs bring chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments.