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.     

hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein whilst A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. We made clones of cells with altered activities of the engrailed, patched and smoothened genes. Our results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is of A or P type and (2) that Hedgehog signalling, coming from the adjacent P compartments across both A/P and P/A boundaries, organises the pattern of all the A cells. We have uncovered four new aspects of compartments and engrailed in the abdomen. First, we show that engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. Second, we look at the polarity changes induced by the clones, and build a working hypothesis that polarity is organised, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. Third, we show that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from each other. Finally, we find that when engrailed is removed from P cells (of, say, segment A5) they transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6).     

The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary?

Imaginal disks, the primordia of the adult appendages in Drosophila, are divided into anterior and posterior compartments. However, the developmental role of such compartments remains unclear. The expression of decapentaplegic (dpp), a pattern formation gene required for imaginal disk development, has the intriguing property of being expressed in a line at or near the boundary between these compartments. Here, we compare the distribution of dpp-driven reporter gene expression to the pattern of expression of the engrailed (en) gene, known to be required for the maintenance of the compartment boundary. Using confocal microscopy to obtain single cell resolution, we have determined that the majority of the en+ imaginal disk cells expressing the dpp-driven reporter genes about those cells expressing en, while a small percentage of dpp reporter gene expressing cells also express en. In posterior regions of en mutant disks, where compartmentalization is abnormal, we observe ectopic expression of the dpp-driven reporter genes. We conclude that the pattern of dpp expression in imaginal disks is delimited in part through the direct or indirect repression by engrailed. Our results lead us to question the widely held assumption that the anterior edge of en expression demarcates the A/P compartment boundary.     

The state of engrailed expression is not clonally transmitted during early Drosophila development.

In Drosophila embryos, boundaries of lineage restriction separate groups of cells, or compartments. Engrailed is essential for specification of the posterior compartment of each segment, and its expression is thought to mark this compartment. Using a new photo-activatable lineage tracer, we followed the progeny of single embryonic cells marked at the blastoderm stage. No clones straddled the anterior edges of engrailed stripes (the parasegment border). However, posterior cells of each stripe lose engrailed expression, producing mixed clones. We suggest that stable expression of engrailed by cells at the anterior edge of the stripe reflects, not cell-intrinsic mechanisms, but proximity to cells that produce Wingless, an extracellular signal needed for maintenance of engrailed expression. If control of posterior cell fate parallels control of engrailed expression, cell fate is initially responsive to cell environment and cell fate determination is a later event.     

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.     

Compartmental modulation of abdominal Hox expression by engrailed and sloppy-paired patterns the fly ectoderm

In Drosophila, segmentation genes partition the early embryo into reiterative segments along the anterior-posterior axis, while Hox genes assign segments their identities. Each segment is also subdivided into distinct anterior (A) and posterior (P) compartments based on the expression of the engrailed (en) segmentation gene. Differences in Hox expression often correlate with compartmental boundaries, but the genetic basis for these differences is not well understood. In this study, we extend previous results to describe a genetic circuit that controls the differential expression of two Hox genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), within the A and P compartments of the abdominal ectoderm. Consistent with earlier findings, we show that en is essential for high Abd-A levels and low Ubx levels in the P compartment, whereas sloppy-paired (slp) is required for high Ubx levels in the A compartment. Overall, these results demonstrate that the compartmental expression of Ubx and abd-A is established through a repressive regulatory network between en, slp, Ubx and abd-A. We also show that abd-A expression in the P compartment is important for the formation of abdominal-specific cell types, suggesting that en and slp modulation of Hox expression within the A and P compartments is essential for embryonic patterning.     

The hedgehog morphogen and gradients of cell affinity in the abdomen of Drosophila

The adult abdomen of Drosophila is a chain of anterior (A) and posterior (P) compartments. The engrailed gene is active in all P compartments and selects the P state. Hedgehog enters each A compartment across both its anterior and posterior edges; within A its concentration confers positional information. The A compartments are subdivided into an anterior and a posterior domain that each make different cell types in response to Hedgehog. We have studied the relationship between Hedgehog, engrailed and cell affinity. We made twin clones and measured the shape, size and displacement of the experimental clone, relative to its control twin. We varied the perceived level of Hedgehog in the experimental clone and find that, if this level is different from the surround, the clone fails to grow normally, rounds up and sometimes sorts out completely, becoming separated from the epithelium. Also, clones are displaced towards cells that are more like themselves: for example groups of cells in the middle of the A compartment that are persuaded to differentiate as if they were at the posterior limit of A, move posteriorly. Similarly, clones in the anterior domain of the A compartment that are forced to differentiate as if they were at the anterior limit of A, move anteriorly. Quantitation of these measures and the direction of displacement indicate that there is a U-shaped gradient of affinity in the A compartment that correlates with the U-shaped landscape of Hedgehog concentration. Since affinity changes are autonomous to the clone we believe that, normally, each cell's affinity is a direct response to Hedgehog. By removing engrailed in clones we show that A and P cells also differ in affinity from each other, in a manner that appears independent of Hedgehog. Within the P compartment we found some evidence for a U-shaped gradient of affinity, but this cannot be due to Hedgehog which does not act in the P compartment.     

Development of embryonic pattern in D. melanogaster as revealed by accumulation of the nuclear engrailed protein

Engrailed is required to establish and maintain developmental compartments within each segment of the fly. To understand the role of the engrailed protein in this process, we have raised antibodies against engrailed and have visualized an engrailed protein in embryos by indirect immunofluorescence. The protein accumulates in the nucleus, supporting the notion that engrailed is a regulatory factor. The first pattern of expression is in alternating segments followed by expression in every segment, suggesting that engrailed may be responding to pair-rule segmentation gene products. Overall, engrailed protein levels peak in areas undergoing morphogenesis. Finally, the complex final form of the head and terminalia derive from earlier simple subdivision of these areas into developmental fields by engrailed.     

Effects of deficiencies in the engrailed region of Drosophila melanogaster

The engrailed gene of Drosophila melanogaster is believed to be involved in control of determination and differentiation of posterior compartments. en1/en1 causes a partial transformation of the posterior compartment of wing and first leg to mirror-image anterior, which prompted the hypothesis that engrailed + is a "selector gene" required for the posterior pathway decision. The incomplete transformation was thought due to residual en+ activity in en1; a deletion of engrailed (en28) was constructed to determine if a complete transformation can occur. en28 is homozygous lethal and cell lethal. en28/en1 survives to adult stage, but causes a weaker transformation than en1/en1, indicating that en1 is not a simple hypomorph. A more distal deletion, en30, survives over en-lethal alleles. Both en30/en1 and en28/en30 survive to adult stage, but do not cause a stronger posterior to anterior transformation than en1/en1; thus this effect may be allele specific. New abnormalities included (1) transformation of the posterior wing blade to haltere, an effect dependent on the bx+ (but not pbx+) pseudoallele of the bithorax complex; (2) abnormal bristle pattern, tarsal fusion, and degenerate posterior claws of all legs. Although these abnormalities are posterior compartment specific, they are not expected of a "selector gene." Thus the function of engrailed may be more complex than originally believed.     

ceh-16/engrailed patterns the embryonic epidermis of Caenorhabditis elegans

engrailed is a homeobox gene essential for developmental functions such as differentiation of cell populations and the onset of compartment boundaries in arthropods and vertebrates. We present the first functional study on engrailed in an unsegmented animal: the nematode Caenorhabditis elegans. In the developing worm embryo, ceh-16/engrailed is predominantly expressed in one bilateral row of epidermal cells (the seam cells). We show that ceh-16/engrailed primes a specification cascade through three mechanisms: (1) it suppresses fusion between seam cells and other epidermal cells by repressing eff-1/fusogen expression; (2) it triggers the differentiation of the seam cells through different factors, including the GATA factor elt-5; and (3) it segregates the seam cells into a distinct lateral cellular compartment, repressing cell migration toward dorsal and ventral compartments.     

Molecular cloning of engrailed: a gene involved in the development of pattern in Drosophila melanogaster

The engrailed gene acts early in Drosophila embryogenesis and plays an essential role in the processes that establish and maintain the repeating segmental pattern. To begin molecular analysis of the role of the engrailed gene in embryonic pattern formation, we used a chromosomal walk to clone genomic sequences that encompass the locus, and have physically mapped the positions of 15 engrailed mutations. The positions of engrailed rearrangement mutations indicate that the engrailed complementation unit includes a minimum of 70 kb. The locus can be divided into two regions. Rearrangement mutations interrupting the centromere proximal 50 kb of the locus result in embryonic lethality while mutants altered in the distal 20 kb of the locus survive to show morphological abnormalities in several adult segments. It appears that long-range cis interactions play a role in the function of the engrailed gene.     

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.     

Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. We show that the positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively.     

Engrailed gene expression in Drosophila imaginal discs.

Genetic and molecular analyses indicate that the Drosophila engrailed gene is required to distinguish posterior from anterior compartments in each segment of the developing animal. Here, the patterns of engrailed expression in the imaginal discs and ventral ganglion of Drosophila larvae are examined, using an antiserum against the engrailed protein and a novel image processing method to reduce non-specific background. As expected, engrailed expression generally is restricted to cells in the posterior compartment of the discs, and the patterns of expression allow refinements in the fate maps of the discs to be made. More significant is the finding that expression of the gene is highly variable in different regions of posterior compartments, suggesting that engrailed may do more than simply specify 'posteriorness'. In the ventral ganglion engrailed appears to be expressed by a subset of cells, primarily in the posterior regions of each segment. In wing discs from animals that are homozygous for the en1 mutation, the pattern of expression of the gene is altered, as opposed to being simply reduced uniformly in the posterior cells.     

An anterior/posterior communication compartment border in engrailed wing discs: possible implications for Drosophila pattern formation

Our previous studies have suggested that all the known lineage compartment borders in the wing imaginal disc of Drosophila are coincident with boundaries of reduced gap junctional communication (communication compartment borders). Since engrailed discs have a disrupted anterior/posterior (A/P) lineage border (G. Morata and P. A. Lawrence, 1975, Nature (London) 255, 614-617), it was of great interest to determine if their A/P communication restriction boundary is similarly disrupted. Examination of gap-junction-mediated exchange of small fluorescent molecules between cells in the engrailed wing disc revealed a boundary of restricted communication that appeared to be identical to the wild-type A/P communication restriction boundary. This result suggests that lineage compartments are not required for the formation of A/P communication restrictions. Furthermore, we suggest that perhaps communication compartments are the domains within which information is provided for specifying the formation of lineage compartments.     

Barnacle duplicate engrailed genes: divergent expression patterns and evidence for a vestigial abdomen

SUMMARY Cirripedes (barnacles) are crustaceans that are characterized by a very peculiar body plan, in particular by the lack of an abdomen. To study their body plan, we searched for their engrailed gene. We found two engrailed ( en.a/en.b ) genes in cirripedes. The two engrailed genes of the rhizocephalan barnacle Sacculina carcini are expressed in the posterior compartment of developing segments and appendages. When the neuroectoderm differentiates into epidermis and neuroderm the expression patterns of en.a and en.b diverge dramatically. en.a expression fades in segment epidermis whereas it is subsequently detected ventrally in reiterated putative neural cells. At the same time, en.b expression increases in the epidermis, which makes it a very good segmentation marker. Five tiny en.b stripes are observed between the sixth thoracic segment and the telson. We interpret these stripes as the molecular definition of vestigial abdominal segments, being the remnant of an ancestral state in keeping with the bodyplan of maxillopod crustaceans. engrailed expression is the first molecular evidence for a segmented abdomen in barnacles.     

Expression of Patella vulgata orthologs of engrailed and dpp-BMP2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. Here, we show that, in the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. Earlier it was shown that a compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. We conclude that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. We suggest that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains.