BIOLOGICAL OVERVIEW

Closely related in sequence and pattern of expression, the invected and engrailed gene pair lie adjacent to one another in the Drosophila genome (Colemen, 1987). They are transcribed in opposite directions however, their respective start sites at opposite ends of a 50 kb stretch of DNA. invected was cloned on the basis of homology to engrailed.

invected presents an enigma. Mutation of invected has no phenotypic effects. In itself, this phenomenon is not novel. There are several instances (both sloppy paired 1 and 2, and gooseberry distal and proximal) in which the second gene in a closely related pair of genes has a less consequential role than the first. Nature appears to tolerate unequal division of labor. In such cases, a mutation in the second gene demonstrates that the first can do the job alone. Perhaps current techniques are not sensitive enough to detect the role of invected, or maybe the proper environment in which to test its effects has not yet been found.

When both invected and engrailed are missing, things go slightly haywire. Particularly odd is the appearance of double wings (Lawrence, 1994, Simonds, 1995, and Tabata, 1995). When engrailed does not function in the posterior compartment, its normal site of expression, additional posterior compartments can form. On the other side of these additional posterior compartments, a new anterior compartment may also form. The duplicated wing is actually structured in reverse, with the posterior compartment in front. Understanding of the double wing mutation serves to reinforce the fundamental importance of regulatory and structured compartments in the subdivision of the developing organism and the resultant cell specialization within and between segments (Lawrence, 1994).

Involvement of invected in hindgut the development of a hindgut signaling border the only embryonic phenotype known for inv. The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).

At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).

Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).

In addition to expressing Dpp, the li-v domain expresses the Notch ligand Delta (Dl); Dl is also expressed in the anterior of both the rectum and the small intestine. Fringe (Fng), a modulator of Notch signaling, is expressed opposite Dl in the Drosophila wing and eye; in the hindgut, Fng is expressed in li-d and the boundary cell rows, opposite the domain of Dl expression in li-d (Iwaki, 2002).

Interestingly, the Dri- and Crb-expressing boundary cells delimit both AP and DV boundaries in the hindgut. The rings form borders at the anterior and posterior ends of the large intestine, while the rows form borders between the dorsal (li-d) and ventral (li-v) regions of the large intestine. This study focusses primarily on the establishment and characteristics of the boundary cell rows (Iwaki, 2002).

Staining with both anti-Crb and anti-ß_HEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of byn^apro/+ embryos (containing a P-element insert in byn) with anti-ß-Gal antibody reveals that the nuclei of the cells of the boundary rows (identified by strong staining with anti-Crb) are also elongated in the AP axis (Iwaki, 2002).

The dramatically higher level of Crb expression in the boundary cells (both rings and rows) suggests that their apical surface may differ from that of other hindgut epithelial cells, and/or that, in the boundary cells, Crb may be present in cellular compartments in addition to the apical surface. Both of these expectations are borne out by a higher magnification examination of the boundary cells. In cross-sections of the large intestine viewed by electron microscopy, short microvilli on the apical surfaces of two cells on opposite sides of the hindgut lumen were observed; these cells most likely correspond to the boundary cell rows. The microvilli of the presumed boundary cell rows appear more organized and parallel than the irregular protrusions on the surfaces of the other cells of the hindgut epithelium. Because of their apical microvilli, the presumed boundary cell rows have a larger apical membrane surface and are expected to be labeled more strongly with anti-Crb. Consistent with this, cross-sections of anti-Crb-stained embryos viewed by light microscopy reveal two cells on opposite sides of the large intestine lumen with a higher level of Crb on their apical surfaces. In addition to their stronger apical labeling with anti-Crb, these presumed boundary cell rows also display an accumulation of Crb in their cytoplasm; this is strongest apical to the nucleus. The cytoplasmic accumulation of Crb suggests that Crb is produced at a higher level, or is more stable, in the boundary cells (Iwaki, 2002).

In conclusion, differences in gene expression demonstrate that the boundary cells are a separately patterned (fated) group of cells in the large intestine. The unique fate of the boundary cells is manifested both molecularly, in their expression of Dri and high cytoplasmic accumulation of Crb, and morphologically, in their marked AP elongation and development of apical microvilli (Iwaki, 2002).

The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).

In embryos homozygous for a strongly hypomorphic dri allele (dri null mutants lack a discernable hindgut), the hindgut is of roughly normal diameter but only about one-third its normal length. Even in these severely reduced dri hindguts, however, boundary cells can still be observed; this phenotype is similar to that described for embryos lacking both maternal and zygotic dri function. Since reduced hindgut size is observed in embryos that lack zygotic, but retain maternal dri function, it is concluded that zygotic expression of dri (most likely the uniform expression at the blastoderm stage) is required to establish or to maintain the normal-size hindgut primordium. Neither blastoderm expression of dri, nor its later expression in the boundary cells, however, appears to be required to establish the boundary cells (Iwaki, 2002).

In dpp embryos, the large intestine is shorter; this is believed to be due to a requirement for dpp in DNA endoreplication in the large intestine. Although the hindgut is variable and severely deformed in dpp mutant embryos (only rudimentary hindguts are detected in the strongest dpp alleles), boundary cell rows were detectable in the hindguts of embryos carrying several different strongly hypomorphic dpp alleles. Thus even though it is required for normal hindgut development, dpp activity does not appear to be required to establish the boundary cell rows (Iwaki, 2002).

In embryos lacking only en, the boundary cell rows and rings form normally. Similarly, many embryos lacking only inv form boundary cell rows and rings. In a significant number of inv embryos, however, gaps were observed in the posterior of the boundary cell rows. This is the only embryonic phenotype known for inv. When both en and inv are removed [in Df(en^E) embryos], the phenotype is much more dramatic: boundary cell rows and rings are completely absent. Consistent with previous studies demonstrating a functional redundancy of en and inv, it is concluded that en and inv are required largely redundantly to establish the boundary cells. However, while inv can substitute completely for en, there is a requirement for inv that cannot be completely substituted by en. This is likely not due to a difference in protein structure, but rather to the fact that, in the hindgut, inv is expressed earlier and at a higher level than en. As their functions are so closely intertwined, the activities of en and inv, and the highly related proteins that they encode, are referred to as single entities: en/inv and En/Inv (Iwaki, 2002).

Embryos lacking Dl function are extremely deformed and do not always have a recognizable hindgut, indicating that function of Dl early in embryogenesis is required to establish and/or maintain the hindgut. Since Dl encodes a ligand for Notch, embryos lacking the zygotic contribution of Notch were examined. Strikingly, Notch mutant hindguts completely lack both boundary cell rows and rings, revealing that Notch signaling is required to establish the boundary cells. The data demonstrate that formation of the boundary cell rows at the border of Dl expression requires the Notch receptor; however, Fng does not appear to be required for this process (Iwaki, 2002).

To further investigate the required role of Dl in establishing the boundary cells, a dominant-negative form of Dl was expressed throughout the hindgut. bynGal4:UAS-Dl.DN embryos show a complete absence of boundary cell rows and rings; this phenotype closely resembles that seen in Notch loss-of-function embryos. Expression of a dominant negative Notch receptor throughout the hindgut results in a similar absence of boundary cell rows and rings. Furthermore, bynGal4 driven expression of UAS-Hairless, which acts to suppress activity of Su(H) also results in an absence of boundary cells. This last result indicates that the Notch signaling required to establish the boundary cells must act through Su(H). In summary, the above results demonstrate required roles in boundary cell specification of the following Notch pathway components: the ligand Dl, the receptor Notch, and the downstream transcription factor Su(H). It is therefore concluded that the Notch signaling pathway is required for boundary cell induction (Iwaki, 2002).

An intriguing observation, given the demonstrated role of the LIN-12/Notch signaling pathway in generation of left_¯right asymmetry in the Caenorhabditis elegans intestine is that a large portion of 455.2Gal4:UAS_¯Su(H)VP16 hindguts display a reversal of left_¯right looping (Iwaki, 2002).

Ectopic expression experiments, taken together with the loss-of-function experiments, demonstrate that establishment of the boundary cell rows requires the juxtaposition of Dl-expressing and Dl-non-expressing cells and signaling via Notch and Su(H). In addition to Notch and spatially restricted Dl, establishment of the anterior ring requires localized activity of Dpp; the posterior ring requires En/Inv activity (which does not need to be localized) and the localized activity of Dl (Iwaki, 2002).

Since the experiments described in the preceding sections show that both spatially localized En/Inv and a boundary of Dl expression are required to establish the boundary cells, it was asked whether En/Inv might control the boundary of Dl expression. In Df(en^E) embryos, Dl is not restricted to li-v, but rather is uniform in the hindgut circumference, indicating that en/inv is required to repress Dl. In the large intestine, uniform expression of En/Inv results in an absence of Dl expression. Expression of En/Inv in li-d is thus both necessary and sufficient to restrict Dl expression to li-d. While it represses Dl throughout the large intestine, ectopic En/Inv does not affect Dl expression in the rectum. Embryos with ectopic En/Inv not only express Dl at the anterior of the rectum, they also form the posterior boundary cell ring. Thus a boundary of Dl-expressing with Dl-non-expressing cells is required not only to establish the boundary cell rows but also likely to establish the posterior ring; the posterior ring also requires En/Inv activity, but this activity does not need to be localized (Iwaki, 2002).

Consistent with observations that En and Inv are repressors with the same targets, the data presented in this study demonstrate that Dl expression in the large intestine is restricted to the li-v domain by the repressive activity of En/Inv in li-d (Iwaki, 2002).

The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).

In summary, three steps in the establishment of the Drosophila hindgut boundary cell rows are similar to steps characterized in other Notch dependent boundary-forming systems. (1) A homeodomain transcription factor (En/Inv in the case of the boundary cells) is expressed on one side of the forming boundary; (2) this transcription factor defines two domains, one which expresses Dl and one which does not; (3) Notch activation in the Dl-non-expressing cells that confront Dl-expressing cells leads to a unique cell fate (Iwaki, 2002).

Given the essential role of spatially restricted En/Inv expression in establishing the boundary cells, it is of interest to consider how En/Inv expression is restricted to the li-d domain. The activation of en expression in the large intestine at stage 10 requires the T-domain transcription factor brachyenteron (byn), which is expressed uniformly in the hindgut. Since dissection of the en regulatory region has identified fragments that drive reporter expression in all hindgut cells, en expression is likely restricted to li-d by a repressor that remains to be identified (Iwaki, 2002).

Boundary cells could be imagined to provide adhesive differences important for cell rearrangement; alternatively, their AP elongation might provide a mechanical force to drive hindgut elongation. In spite of these tempting scenarios, however, the normal appearance (overall size, diameter, and length) of Notch and Df(en^E) hindguts, which completely lack both boundary cell rows and rings, demonstrates conclusively that the boundary cell rows and rings are not required to establish normal hindgut morphology (Iwaki, 2002).

Rather than playing a required role in hindgut morphogenesis, the boundary cells most likely contribute to the ion and water absorption function of the larval hindgut. In the adult insect, this function is carried out by cells in the rectum that are distinguished by their extensive, mitochondria-rich apical membrane leaflets. In the Drosophila larval hindgut, this characteristic ultrastructure is found not in the rectum, but rather in the cells of li-d, leading to the conclusion that water and ion absorption in the larva occurs in the large intestine. Associated with the absorptive cells of the Dipteran rectum is a distinct cell type referred to as 'junctional cells'; these form a collar surrounding the absorptive cells, have extensive intercellular junctional complexes, and are thought to play an isolating and supportive role. The Drosophila boundary cell rings and rows similarly constitute a collar surrounding the absorptive li-d cells of the larval hindgut and, based on their intensive Crb staining, have unusual membrane characteristics. It is therefore proposed that, like the junctional cells in the adult insect rectum, the boundary cells serve to isolate and support a domain of ion and water absorbing cells in the Drosophila larval hindgut (Iwaki, 2002).

Genomic DNA length - greater than 30 kb (engrailed is 4 kb)

cDNA clone length - 2.3 kb

Bases in 5' UTR - 294

Exons - four

Bases in 3' UTR - 261

PROTEIN STRUCTURE

Amino Acids - 576

Structural Domains

Like Engrailed, Invected is approximately 60 kD and contains a homeobox near its carboxyl terminus. The homeodomain sequence of 117 amino acids in the carboxy-terminal region of both proteins is almost identical (Coleman, 1987).

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for'Homeobox' engrailed-type protein signature.

The mosquito Anopheles gambiae En protein shows significant divergence from the Drosophila protein. The overall sequence identity is only 35% and is confined to 7 domains. Four of these domains, the En/Inv domains are found in both the Drosophila En and Inv proteins and in all En-class proteins, including those of mouse. These include the homeodomain, a region surrounding the first intron of Drosophila En, and a C-terminal region. Two other domains are En specific, including murine En, and are not found in Invected. There is another region, the Dipteran-specific En domain, found in Drosophila and mosquito, but not in Inv or mouse En. An engrailed cDNA from mosquito was expressed from a Drosophila engrailed minimal promoter. The promoter fragment used includes 2.6 kb of regulatory DNA that causes transposons to home to the endogenous Drosophila engrailed gene at high frequencies. This transposon was inserted onto a Drosophila chromosome that produces no functional Engrailed proteins. When this transposon integrates near the engrailed promoter, adult viability is restored to engrailed mutant flies showing that the highly divergent mosquito Engrailed protein can replace the Drosophila Engrailed protein at all stages of development. Insertion of this transposon into the adjacent invected gene, which is transcribed in a pattern similar to engrailed, leads to only embryonic rescue, suggesting an important difference in the regulation of these two genes (Whitley, 1997).

The Engrailed Homology 1 (EH1) motif is a small region, believed to have evolved convergently in homeobox and forkhead containing proteins, that interacts with the Drosophila protein Groucho (C. elegans unc-37, Human Transducin-like Enhancers of Split). The small size of the motif makes its reliable identification by computational means difficult. The predicted proteomes of Drosophila, C. elegans and human have been systematically searched for further instances of the motif. Using motif identification methods and database searching techniques, which homeobox and forkhead domain containing proteins also have likely EH1 motifs was examined. Despite low database search scores, there is a significant association of the motif with transcription factor function. Likely EH1 motifs are found in combination with T-Box, Zinc Finger and Doublesex domains as well as discussing other plausible candidate associations. Strong candidate EH1 motifs have been identified in basal metazoan phyla. Candidate EH1 motifs exist in combination with a variety of transcription factor domains, suggesting that these proteins have repressor functions. The distribution of the EH1 motif is suggestive of convergent evolution, although in many cases, the motif has been conserved throughout bilaterian orthologs. Groucho mediated repression was established prior to the evolution of bilateria (Copley, 2005).

Sequence motifs were sought in homeobox containing transcription factors taken from the proteins of human, Drosophila and C. elegans, by first masking known Pfam domains, and then using the expectation maximization algorithm implemented in the meme program. The first non-subfamily specific motif identified corresponded to previously known examples and new instances of, the EH1 motif, in 100 sites, with an E-value of < 10^-126. The same approach was applied to Forkhead containing transcription factors, identifying 25 sites with a combined E-value of < 10^-31. These motifs also appeared to conform to the consensus of the EH1 motif (Copley, 2005).

To further investigate the significance of this similarity, hidden Markov models (HMM) were constructed of the motif (EH1^hox & EH1^fh) which were then searched against the complete set of predicted proteins from human, D. melanogaster and C. elegans. The highest scoring non homeobox containing domain match of EH1^hox was a Forkhead protein (human FOXL1), and the second highest scoring non-Forkhead containing match of EH1^fh was to a homeobox containing protein (Drosophila Invected). In both cases, nearly all the high scoring hits were to proteins containing domains with transcription factor function. Among the best scoring matches of the EH1^hox searches were several T-box (TBOX), Doublesex Motif (DM), Zinc finger (ZnF_C2H2) and ETS containing proteins (Copley, 2005).

The presence of EH1 motifs within various homeobox, and to a lesser extent, forkhead-containing proteins has been widely reported, although not systematically studied. EH1-like motifs co-occurring with 3 major groupings of homeobox sub-types were found: the extended-hox class, typified by Drosophila Engrailed; the paired class, including Drosophila Goosecoid, and the NK class, including Drosophila Tinman. Related to the paired class homeobox domains, a number of genes containing PAIRED domains only were also found to contain EH1-like motifs. With only a few exceptions, the EH1-like motif occurs N-terminal to the homeobox domain and C-terminal to the PAIRED domain when present. A number of these proteins have been shown to interact with Groucho or its orthologs, e. g., C. elegans cog-1, Drosophila Engrailed and Goosecoid, and in high throughput assays Drosophila Invected and Ladybird late (Copley, 2005).

Promoter Structure

Truncations in the regulatory region of engrailed reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. These mutations affect expression of the linked invected gene, suggesting that engrailed and invected share a complex set of regulatory elements that operate over at least 85 kb (Goldsborough, 1994).

In Drosophila the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmentally crucial regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. The chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. Posterior sexcombs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that all three are members of a common multimeric protein complex. Immunoprecipitation experiments using in vivo cross-linked chromatin indicate that these three Polycomb group proteins are associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. Polycomb, Polyhomeotic, and Posterior sexcombs are, however, differentially distributed on regulatory sequences of the engrailed-related gene invected. High-resolution mapping shows that Pc binding is maximal in a 1.0-kb element, 400 bp upstream of the inv start of transcription. Pc binding sites in en are found in a fragment that contains repetitive elements. The Pc binding sites and the repetitive elements are separable. In fact, Pc associates with two distinct elements, one covering the first intron and the other 1 kb upstream from the start of transcription. Both these regions have been implicated in regulation of en expression during embryogenesis. The binding site upstream of en overlaps with a number of pairing-sensitive elements which have been suggested to mediate PcG repression. Ph and Psc are present at both Pc binding sites in the en upstream region and first intron. The common Pc-Ph-Psc complex does not appear to funcion at inv: no Psc is associated with inv and Ph is associated with a much more restricted element than Pc (Strutt, 1997).

Transcriptional Regulation

invected is under the control of engrailed and hedgehog. engrailed expression has been targeted to different regions of the wing disc. In the anterior compartment, ectopic en expression gives rise to the substitution of anterior structures by posterior ones, thus demonstrating its role in specification of posterior patterns. The en-expressing cells in the anterior compartment also induce high levels of the hedgehog and decapentaplegic gene products. This results in local duplications of anterior patterns. hh is able to activate en and invected in this mutant anterior compartment. In the posterior compartment, elevated levels of en product result in partial inactivation of the endogenous en and inv genes, indicating the existence of a negative autoregulatory mechanism. It is proposed that en has a dual role: a general one for patterning of the appendage, achieved through the activation of secreted proteins like hh and dpp, and a more specific one, determining posterior identity, in which the inv gene may be implicated (Guillen, 1995).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of in at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Like engrailed, invected is expressed in the embryonic and larval cells of the posterior developmental compartments and in the embryonic hindgut, clypeolabrum, and ventral nervous system (Coleman, 1987). invected transcription is delayed compared to that of engrailed. Engrailed appears at the onset of cellularization, while Invected stripes are not present until germ band elongation [Images].

Effects of Mutation or Deletion

Removing engrailed activity causes incomplete morphological transformation from posterior to anterior fate in the wing, and failure to produce an ectopic anterior-posterior organizer. Complete transformation can only be effected by simultaneously eliminating activity of engrailed and its homolog invected. invected functions principally to specify posterior cell fate. Thus establishment of the anterior-posterior organizer and control of compartment identity are genetically distinguishable; invected may perform a discrete subset of functions previously ascribed to engrailed (Simonds, 1995).

Removing engrailed and invected from posterior wing cells created two new compartments: an anterior compartment expressing patched and cubitus interrruptus and a posterior compartment expressing mutant engrailed. patched is expressed ectopically in mutant posterior cells and hedgehog is expressed in the posterior as well. In some cases, these compartments form a complete new wing resulting from a duplication of anterior and posterior compartments. Increasing engrailed activity also affects patterning. Engrailed both directs the posterior compartment pathway and creates the compartment border (Tabata, 1995).

The invected gene of Drosophila melanogaster is a homeobox-containing gene that is closely related to engrailed. A dominant gain of function allele, invected^Dominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invected^Dominant shows that it is associated with the transformation of the anterior compartment of the wing into a posterior fate. This transformation is normally limited to the wing blade itself and does not involve the remaining tissues derived from the wing imaginal disc, including the wing hinge and dorsal thorax of the fly. The ectopic expression of Invected protein associated with invected^Dominant correlates spatially with the normal expression pattern of vestigial in the wing imaginal disc, suggesting that control elements of vestigial are driving ectopic invected expression. This was confirmed by sequence analysis, which has shown that the dominant vestigial activity is eliminated by a deletion that removes the 3' portion of the vestigial coding region. This leaves a gene fusion wherein the vestigial enhancer elements are still juxtaposed immediately 5' to the invected transcriptional start site, but with the vg sequences harboring an additional lesion. Unlike recessive invected alleles, the invected^Dominant allele produces an observable phenotype, and as such should prove useful in determining the role of invected in patterning the wing imaginal disc. Genetic analysis has shown that mutations of polyhomeotic, a gene involved in regulating engrailed expression, cause a reproducible alteration in the invected^Dominant phenotype. Finally, the invected^Dominant allele should prove valuable for identifying and characterizing genes that are activated within the posterior compartment. A screen using various lacZ lines that are asymmetrically expressed in an anterior-posterior manner in the wing imaginal disc has isolated one line that shows posterior-specific expression within the transformed anterior compartment (Simmonds, 1998).

REFERENCES

Coleman, K. G. et al. (1987). The invected gene of Drosophila: sequence analysis and expression studies reveal a close kinship to the engrailed gene. Genes Dev 1: 19-28.

Copley, R. R. (2005). The EH1 motif in metazoan transcription factors. BMC Genomics 6: 169. 16309560

Goldsborough, A. S. and Kornberg, T. B. (1994). Allele-specific quantification of Drosophila engrailed and invected transcripts. Proc Natl Acad Sci 91: 12696-12700

Guillen, I. et al. (1995). The function of engrailed and the specification of Drosophila wing pattern. Development 121: 3447-3456

Iwaki, D. D. and Lengyel, J. A. (2002). A Delta-Notch signaling border regulated by Engrailed/Invected repression specifies boundary cells in the Drosophila hindgut. Mech. Dev. 114: 71-84. 12175491

Lawrence, P.A. and Morata, G. (1994). Homeobox genes: their functionin Drosophila segmentation and pattern formation. Cell 78: 181-189

Simonds, A. J., Brook, W.J., Cohen, S.M. and Bell, J.B. (1995) Distinguishable functions for engrailed and invected in anterior-posterior patterning in the Drosophila wing. Nature 376: 424-427

Simmonds, A. J. and Bell, J. B. (1998). A genetic and molecular analysis of an invected^Dominant mutation in Drosophila melanogaster. Genome 1998 Jun;41(3):381-90.

Strutt, H. and Paro, R. (1997). The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol. Cell. Biol. 17(12): 6773-6783.

Tabata, T. et al. (1995). Creating a Drosophila wing de novo, the role of engrailed, and the compartment border hypothesis. Development 121: 3359-3369

Whiteley, M. and Kassis, J. A. (1997). Rescue of Drosophila engrailed mutants with a highly divergent mosquito engrailed cDNA using a homing, enhancer-trapping transposon. Development 124 (8): 1531-1541.

date revised: 10 May 2006


 

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