Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

The engrailed locus of Drosophila: structural analysis of an embryonic transcript

cDNA clones originating from the engrailed gene of Drosophila have been isolated from recombinant phage libraries that were made using poly(A)+ RNA extracted from early embryos. The DNA sequence of one of these clones includes a homeo box, a 180 bp sequence present in several other Drosophila genes important in formation of body pattern during development. The homeo boxes found in the other Drosophila genes, as well as in cognate sequences from a wide range of segmented animals, including higher vertebrates, are highly conserved. By contrast, the homeo box within the engrailed gene diverges substantially and, unlike the other homeo boxes, is interrupted by an intervening sequence. The engrailed homeo box is located near the 3' end of a 1700 bp open reading frame. If translated, this sequence would produce a protein of unusual composition. We also show that a neighboring gene has a large region with strong homology to engrailed, and that it also contains a homeo box.     

Association of the Drosophila melanogaster engrailed protein with specific soluble nuclear protein complexes.

The Drosophila engrailed protein which contains a homeobox domain and specific DNA binding activity is believed to function in the regulation of gene expression during embryogenesis. Here we show that the engrailed protein interacts stably with specific complexes of soluble nuclear proteins when expressed artificially in a cell line and in the developing embryo. The engrailed complexes have molecular masses between 10(7) and 10(8) which suggests they contain a polymeric protein component. The complex is able to bind reversibly to DNA and a definitive purification shows it to be constituted of 12 distinct protein species, two of which are predominant. Purified, bacterially produced engrailed protein can be reconstituted with both culture cell and embryo nuclear protein fractions to form complexes of the same and related composition respectively. On the basis of these results we propose that protein--protein interactions as well as DNA binding are important for correct engrailed protein function in vivo.     

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'''' Sites within a 1.6-Kb Region

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter.     

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.     

The sequence specificity of homeodomain-DNA interaction

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.     

P-element homing is facilitated by engrailed polycomb-group response elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. We mapped the minimal DNA fragment that could mediate homing to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. We made flies that contain a 1.5 kb deletion of engrailed DNA (en(Δ1.5)) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the en(Δ1. 5) chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. We suggest that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing.     

The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implications for transvection and gene regulation.

The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by-product of normal intragenic zeste action.     

Expression of the homeobox Chick-en gene in chick/quail chimeras with inverted mes-metencephalic grafts

The homeobox gene Chick-en, sharing homologies to the engrailed gene of Drosophila, is expressed, during early steps of development, in a restricted area of the chick embryo including mes-metencephalic neuroepithelia. The expression of the Chick-en gene has been analyzed in chick/quail chimeric embryos in which a portion of the 2-day-old mes-metencephalic neuroepithelium has been transplanted in an inverted position. By means of a monoclonal antibody, "Mab 4D9," recognizing engrailed proteins, it is shown that the expression of the Chick-en gene is regulated in the inverted neuroepithelium according to its new position in the host neural tube. The regulation takes place within 20 hr after transplantation. These results, together with previous data demonstrating that the phenotypic expression of the inverted neuroepithelium depends, also, on its new position in the host neural tube, strongly suggest that the engrailed protein could play an important role in the positional specification of the mes-metencephalic neuroepithelium.     

The zic1 gene is an activator of Wnt signaling

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.     

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system

BACKGROUND: The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS: We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS: The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.     

The beta 3 tubulin gene is a direct target of bagpipe and biniou in the visceral mesoderm of Drosophila

Previous studies have identified the NK homeobox gene bagpipe and the FoxF fork head domain gene biniou as essential regulators of visceral mesoderm development in Drosophila. Here we present additional genetic and molecular information on the functions of these two genes during visceral mesoderm morphogenesis and differentiation. We show that both genes are required for the activation of beta 3Tub60D in the visceral mesoderm, which encodes beta 3 tubulin. We demonstrate that a 254 bp derivative of a previously defined visceral mesoderm-specific enhancer element, vm1, from beta 3Tub60D contains one specific in vitro binding site for Bagpipe and two such sites for Biniou. While the wild-type version of the 254 bp enhancer is able to drive significant levels of reporter gene expression within the entire trunk visceral mesoderm, mutation of either the Bagpipe or the Biniou binding sites within this element results in a severe decrease of enhancer activity. Moreover, mutation of all three binding sites for Bagpipe and Biniou, respectively, results in the complete loss of enhancer activity. Together, these observations suggest that Bagpipe and Biniou serve as direct, partially redundant, and tissue-specific activators of the terminal differentiation gene beta 3Tub60D in the visceral mesoderm.