Gene name - invected
Synonyms - engrailed related
Cytological map position - 48A2
Function - transcription factor
Keywords - segment polarity
Symbol - inv
Genetic map position - 2-62 next to engrailed
Classification - homeodomain - Engrailed class
Cellular location - nuclear
|Recent literature||Ozsoy, E. D., Yilmaz, M., Patlar, B., Emecen, G., Durmaz, E., Magwire, M. M., Zhou, S., Huang, W., Anholt, R. R. H. and Mackay, T. F. C. (2021). Epistasis for head morphology in Drosophila melanogaster. G3 (Bethesda) 11(10). PubMed ID: 34568933
Epistasis-gene-gene interaction-is common for mutations with large phenotypic effects in humans and model organisms. Epistasis impacts quantitative genetic models of speciation, response to natural and artificial selection, genetic mapping, and personalized medicine. However, the existence and magnitude of epistasis between alleles with small quantitative phenotypic effects are controversial and difficult to assess. This study used the Drosophila melanogaster Genetic Reference Panel of sequenced inbred lines to evaluate the magnitude of naturally occurring epistasis modifying the effects of mutations in jing and inv, two transcription factors that have subtle quantitative effects on head morphology as homozygotes. This study found significant epistasis for both mutations and performed single marker genome-wide association analyses to map candidate modifier variants and loci affecting head morphology. A subset of these loci was significantly enriched for a known genetic interaction network, and mutations of the candidate epistatic modifier loci also affect head morphology (Ozsoy, 2021).
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-ßHEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of bynapro/+ 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(enE) 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(enE) 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(enE) 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
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 (EH1hox & EH1fh) 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 EH1hox was a Forkhead protein (human FOXL1), and the second highest scoring non-Forkhead containing match of EH1fh 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 EH1hox 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).
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).
Enhancers are often located many tens of kilobases away from the promoter they regulate, sometimes residing closer to the promoter of a neighboring gene. How do they know which gene to activate? This study used homing P[en] constructs to study the enhancer-promoter communication at the engrailed locus. engrailed enhancers can act over large distances, even skipping over other transcription units, choosing the engrailed promoter over those of neighboring genes. This specificity is achieved in at least three ways: (1) early acting engrailed stripe enhancers exhibit promoter specificity; (2) a proximal promoter-tethering element is required for the action of the imaginal disc enhancer(s). The data suggest that there are two partially redundant promoter-tethering elements. (3) The long-distance action of engrailed enhancers requires a combination of the engrailed promoter and sequences within or closely linked to the promoter proximal Polycomb-group response elements. These data show that multiple mechanisms ensure proper enhancer-promoter communication at the Drosophila engrailed locus (Kwon, 2009).
engrailed gene exists in a gene complex with the coregulated invected (inv) gene. en and inv are co-expressed in a complex manner throughout development. Early in development, they are required for segmentation, and are expressed in a series of stripes continually throughout embryogenesis. Although the location of En stripes does not change throughout embryonic development, the enhancers and the trans-acting proteins that regulate their expression do change. For example, separate fragments of regulatory DNA act as enhancers for activation by the pair-rule genes, for activation by Wingless signaling and for regulation by the trithorax and Polycomb group genes. en and inv are also expressed in the hindgut, clypeolabrum, central nervous system (CNS), peripheral nervous system (PNS), fat body and the posterior compartments of the imaginal discs. The regulatory sequences of engrailed are distributed throughout a 70 kb region. Interestingly, the en and inv promoters are separated by ~54 kb, yet they appear to be regulated by the same enhancers, suggesting that en/inv enhancers must be able to act over long distances. What ensures they activate only en/inv and not flanking genes? Homing P-transgenes were used to address this question (Kwon, 2009).
Most P-based constructs insert in the genome in a non-selective manner. However, a few pieces of regulatory DNA have been found to alter the insertional specificity of P-constructs, causing the P-construct to insert near the gene that the regulatory DNA came from. This phenomenon is called P-element homing and was first observed with DNA from the en gene. DNA fragments from the bithorax complex, linotte gene and, most recently, even skipped, have also been shown to mediate homing. For en, P-constructs containing a DNA fragment including the engrailed promoter and 2.4 kb of upstream sequences (P[en-lacZ]) cause homing to the en region of the chromosome (Kassis, 1992). Insertions are not site specific, but occur over a region of ~300 kb, including en and inv and flanking genes. P[en-lacZ] has no enhancer activity on its own, but acts as an enhancer detector; that is, its expression is directed by flanking genomic enhancers. It has recently been shown that P[en-lacZ] can be stimulated by en enhancers even when it is inserted into neighboring genes (DeVido, 2008). Furthermore, this long-distance enhancer activity was dependent upon en DNA fragments that also act as Polycomb-group response elements (PREs). PREs are DNA elements that bind and mediate the action of the Polycomb group of transcriptional repressors. It is not known whether the PRE activity can be separated from the enhancer-detection activity of these DNA fragments (Kwon, 2009).
This study shows that, in addition to the PRE fragments, the en promoter is necessary for long-distance interactions with en/inv enhancers. The data suggest that enhancer-promoter specificity at the en locus is complex, using different mechanisms for different enhancers: (1) the early stripe enhancers, which respond to the pair-rule transcriptional activators, exhibit promoter specificity; (2) a promoter-tethering element is required for interactions with the imaginal disc enhancer(s). Finally, both the promoter and the DNA fragment that includes the promoter-proximal PREs are important for the long-range action of en enhancers (Kwon, 2009).
en and inv exist in a gene complex, encode related proteins with redundant functions and share regulatory DNA. Thus, en enhancers must be able to activate both the en and inv promoters, which are separated by 54 kb. What properties do these two promoters share? First, en and inv are both TATA-less promoters. Both en and inv have the initiator promoter element (Inr) and the downstream promoter element (DPE). The inv promoter has a perfect match to the Inr consensus sequence for Drosophila (at nucleotide 7,363,212), and a near match to the DPE 28 bp after the initiating adenine. The en promoter has a near match to the Inr consensus and a perfect match to the DPE located 30 bp downstream of the third nucleotide of the Inr sequence. Second, both promoters have binding sites for the transcription factor GAGA, which are located just upstream of the transcription start site. GAGA-binding sites greatly increase the activity of the en promoter. Third, both have Polycomb response elements (PREs) located very close to the promoters. Finally, the DNA sequences from 600 bp upstream to 400 bp downstream of the inv promoter were compared with the 588 bp en promoter fragment used in this study and a few stretches of sequence identity were found. The longest was a 14/15 bp match located from -57 to -42 upstream of the en transcription start site and from -40 to -25 bp upstream of the inv transcription start site. The functional significance of this is unknown (Kwon, 2009).
The sequences around the presumed transcription start sites for the different transcripts were examined. Strikingly, aside from sprt (well upstream of inv), none of these genes had sequences that matched the TATA, Inr or DPE consensus sequences. Like en and inv, the sprt gene has Inr and DPE elements. Unlike en and inv, no PREs were found at the sprt gene (as judged by the binding of PcG proteins). It is suggested that sprt is not activated by en enhancers because it lacks the PREs (or associated sequences) that are necessary for the long-distance action of the en enhancers (Kwon, 2009).
The minimal heat shock promoter present in P[enHSP] contains sequences -44 to +204 bp of the HSP70 promoter. It contains the TATA element but does not have any of the GAGA sites that are located further upstream. It has been found that a slightly different version of this promoter (from -73 to +70 bp) would not function in a reporter construct with the en stripe enhancer present in the intron, although it was able to function with enhancers that drive expression in the hindgut, fat body and posterior spiracles. Those data, combined with the current results, clearly show that different en enhancers have different promoter requirements. The ability of different types of core promoters to recognize different enhancers has been reported by many other investigators and may be a common mechanism to achieve enhancer specificity in Drosophila (Kwon, 2009).
At least three distinct processes mediate promoter specificity at en. (1) The early stripe enhancers, those activated by the pair-rule transcription activators, require the en promoter; they are not able to stimulate the heat shock promoter. It is suggested that this could be due to the type of core promoter present at en, or to sequences very near the transcription start site. The en allele enJ86 contains a deletion from -412 to -73 bp upstream of the en transcription start site and shows no disruption of early en expression. Thus, sequences within 73 bp of the transcription start site are sufficient for interaction with early stripe enhancers. Caudal, an early acting developmental transcription factor, was recently found to specifically activate DPE-containing promoters. It would be interesting to test whether pair-rule proteins also exhibit promoter specificity (Kwon, 2009).
(2) It is proposed that there are two promoter-tethering elements that mediate interactions with the imaginal disc enhancers. One of them is located in the 181 bp element, PRE2, and another is located between -273 and -73 bp. en joins a growing list of Drosophila genes that have promoter-tethering elements, including the homeotic genes Scr and Abd-B, as well as the white and string genes. It is likely that many other genes with extensive regulatory regions have promoter-tethering elements (Kwon, 2009).
(3) It has been shown that the 2 kb PRE fragment, from -2.4 to -0.4 kb, is required for distantly located transgenes to interact with the en enhancers (DeVido, 2008). The current study shows that the en promoter is also required for long-range enhancer-promoter interactions. It is suggested that both the promoter and the PRE fragment are necessary to form the correct chromatin structure to allow interactions with distant en enhancers. In conclusion, these data suggest that multiple mechanisms exist to ensure that en enhancers activate the correct promoters (Kwon, 2009).
invected (inv) and engrailed (en) form a gene complex that extends about 115kb. These two genes encode highly related homeodomain proteins that are co-regulated in a complex manner throughout development. Dissection of inv/en regulatory DNA shows that most enhancers are spread throughout a 62kb region. Two types of constructs were used to analyze the function of this DNA: P-element based reporter constructs with small pieces of DNA fused to the en promoter driving lacZ expression and large constructs with HA-tagged en and inv inserted in the genome with the phiC31 system. In addition, deletions of inv and en DNA were generated in situ, and their effects on inv/en expression were assayed. The results support and extend knowledge of inv/en regulation. First, inv and en share regulatory DNA, most of which is flanking the en transcription unit. In support of this, a 79-kb HA-en transgene can rescue inv en double mutants to viable, fertile adults. In contrast, an 84-kb HA-inv transgene lacks most of the enhancers for inv/en expression. Second, there are multiple enhancers for inv/en stripes in embryos; some of these may be redundant but others play discrete roles at different stages of embryonic development. Finally, no small reporter construct gave expression in the posterior compartment of imaginal discs, a hallmark of inv/en expression. Robust expression of HA-en in the posterior compartment of imaginal discs is evident from the 79-kb HA-en transgene, while a 45-kb HA-en transgene gives weaker, variable imaginal disc expression. It is suggested that the activity of the imaginal disc enhancer(s) is dependent on the chromatin structure of the inv/en domain (Cheng, 2014).
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).
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].
Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. This study quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. Mechanical tension was found to be approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries (Landsberg, 2009).
A long-standing hypothesis to explain the maintenance of compartment boundaries is based on differential cell adhesion (or cell affinity). Cell adhesion molecules required for the maintenance of compartment boundaries, however, have not been identified. More recently, it has been proposed that actin-myosin-based tension is important for keeping the dorsoventral compartment boundary of the developing Drosophila wing smooth and straight. However, whether a similar mechanism operates at the anteroposterior compartment boundary (A/P boundary) is unclear. Moreover, a physical measurement of differential mechanical tension at compartment boundaries has not been reported. Furthermore, whether and how differential mechanical tension governs cell sorting at compartment boundaries is not well understood (Landsberg, 2009).
To test whether actin-myosin-based tension is increased at the A/P boundary, the levels of Filamentous (F)-actin and nonmuscle Myosin II (Myosin II) were quantified. The A/P boundary in the wing disc epithelium was particularly well defined by the cell bonds located at the level of adherens junctions, indicating that mechanisms maintaining the boundary operate at this cellular level. F-actin and the regulatory light chain of Myosin II (encoded by spaghetti squash, sqh) were increased at these cell bonds along the A/P boundary. Cell bonds displaying elevated levels of Myosin II correlate with decreased levels of Par3 (Bazooka in Drosophila), a protein organizing cortical domains, at the dorsoventral compartment boundary and during germ-band extension in Drosophila embryos. Likewise, Bazooka was decreased at cell bonds along the A/P boundary, indicating a common mechanism of complementary protein distribution of Myosin II and Bazooka. The level of E-cadherin, a component of adherens junctions, was not altered along the A/P boundary (Landsberg, 2009).
To identify signatures of increased tension in the vicinity of the A/P boundary, the morphology of cells were quantitatively analyzed at the level of adherens junctions. Line tension and mechanical properties of cells have been proposed to contribute to cell shape and to influence angles between cell bonds. Line tension associated with adherens junctions, here termed cell bond tension, can be defined as the work, per unit length, performed as a cell bond changes its length. Cell bond tension results from actin-myosin bundles and other structural components at junctional contacts that generate tensile stresses. Wing discs from late-third-instar larvae were stained for E-cadherin and engrailed-lacZ, a marker for the posterior compartment. Cell bonds were identified, and morphological parameters were analyzed. Adjacent anterior and posterior cells (A1 and P1, respectively) displayed a significantly enlarged apical cross-section area compared to cells farther away from the compartment boundary, indicating that apposition of anterior and posterior cells alters specifically the properties of A1 and P1 cells. Angles between adjacent cell bonds along the A/P boundary were larger compared to angles between bonds of the remaining cells and were significantly smaller in mutants for Myosin II heavy chain (encoded by zipper; zip2/zipEbr). Thus, the unique morphology of A1 and P1 cells depends on Myosin II. These data are consistent with an increased Myosin II-based tension of cell bonds located along the A/P boundary (Landsberg, 2009).
Cells on opposite sides of the A/P boundary differ in gene expression. The homeodomain-containing proteins Engrailed and Invected as well as the Hedgehog ligand are only expressed on the posterior side. The Hedgehog signal is transduced exclusively on the anterior side. Hedgehog signal transduction and the presence of Engrailed and Invected are required to maintain this compartment boundary. Whether the altered cell morphology at the A/P boundary could be reproduced by ectopically juxtaposing Hedgehog signaling and non-Hedgehog signaling cells was tested. Clones of cells that expressed Hedgehog from a transgene and that were also mutant for the gene smoothened (encoding an essential transducer of the Hedgehog pathway) were generated. In the P compartment, which is refractory to Hedgehog signal transduction, clones displayed a normal morphology. In the A compartment, a response to Hedgehog that is secreted by the clones is elicited in the surrounding wild-type cells. These clones had a rounder appearance, and at the clone border, but not away from it, apical cross-section area and bond angles were increased. Similarly, juxtaposing cells expressing engrailed and invected with cells that are mutant for these genes resulted in increased apical cross-section area and increased bond angles at the clone border. It is concluded that the morphology that is characteristic of cells at the A/P boundary can be imposed on cells within a compartment by juxtapositioning cells with different activities of Hedgehog signal transduction or Engrailed and Invected (Landsberg, 2009).
Ablating cell bonds generates cell vertex displacements, providing direct evidence for tension on cell bonds. Individual cell bonds were ablated by using a UV laser beam focused in the plane of the adherens junctions. Single-cell bonds were cut, and the displacement of vertices of neighboring cells, visualized by E-cadherin-GFP, was recorded. The P compartment was visualized by expression of GFP-gpi under control of the engrailed gene via the GAL4/UAS system. The increase in distance between the two vertices of the ablated cell bond and the initial velocity of this vertex separation were analyzed. The ratio of initial velocities in response to cell bond ablation is a measure of the tension ratio on these cell bonds. Initial velocity and extent of vertex separation were indistinguishable between anterior (A/A) and posterior (P/P) cell bonds located away from the A/P boundary. This was also the case when specifically cell bonds between the first and second row of anterior cells were ablated. By contrast, ablation of bonds between adjacent anterior and posterior cells (A/P cell bonds) gave rise to a larger vertex separation. This result was not due to the fact that A/P cell bonds have a preferred orientation. Moreover, the initial velocity of ablated A/P bonds was 2.37-fold higher compared to the mean of initial velocities of A/A and P/P bonds. This value provides an estimate of the ratio λ of cell bond tension along the A/P boundary relative to the average tension of cell bonds. In the presence of the Rho-kinase inhibitor Y-27632, the ratio of initial velocity of vertex separation of A/P cell bonds relative to A/A cell bonds was reduced to 1.46. Given that Myosin II is the main effector of Rho-kinase, these results strongly suggest that Myosin II-based tension acting on cell bonds is locally increased along the A/P boundary (Landsberg, 2009).
To quantify λ by an independent method, the displacement field was calculated after laser ablation. Using a vertex model, two populations of adjacent cells were introduced and cell bond ablations were simulated, varying λ between 1 and 4. When λ = 2.5, the vertex displacement, and in particular the anisotropy of displacements, in the simulations closely matched the vertex displacements in the experiment. In the vertex model, λ = 2.5 also resulted in increased bond angles at the interface of the two cell groups, similar to the A/P boundary in the wing disc. Thus, on the basis of two different methods, the data demonstrate that cell bond tension is increased approximately 2.5-fold along the A/P boundary compared to the remaining tissue (Landsberg, 2009).
To test whether a 2.5-fold increase in cell bond tension is sufficient to maintain a compartment boundary, the vertex model was used to simulate the growth of two adjacent cell populations for λ = 1, 2.5, and 4. For λ = 1, the interface between two growing cell populations became increasingly irregular. By contrast, for λ = 2.5 and 4, a well-defined interface was maintained. Moreover, corresponding changes in cell bond tension at borders of simulated clones resulted in the morphology and sorting behavior of cell patches that resembled those of experimental cell clones compromised for Hedgehog signal transduction or Engrailed and Invected activity. The roughness of the interface in the simulations decreased with increasing λ, showing that cell bond tension is sufficient to maintain straight interfaces between growing cell populations. For λ = 2.5, the roughness of the interface was still larger than the roughness of the A/P boundary in wing discs. This suggests that additional mechanisms might contribute to further reduce the roughness of the A/P boundary. Also, because of the uncertainty of the mechanical properties of A1 and P1 cells, which differ from those of the remaining cells, the value of λ, inferred from laser ablation of cell bonds, might be underestimated. Remarkably, the roughness of the A/P boundary could be altered in mutant conditions. In zip2/zipEbr mutant wing discs, the roughness of the compartment boundary was significantly larger than in controls, demonstrating a role for Myosin II in maintaining a sharp and straight A/P boundary (Landsberg, 2009).
In summary, by applying physical approaches and quantitative imaging, this work for the first time demonstrates and quantifies an increase in tension confined to the cell bonds along the A/P boundary. Moreover, simulations show that this increase in tension suffices to maintain a stable interface between two proliferating cell populations. Genetic studies demonstrated that cells of the two compartments differ in their expression profiles and signaling activities. It has therefore been proposed that biophysical properties of cells within the P compartment differ from those within the A compartment, and that such differences could drive cell sorting. When quantifying cell morphology and vertex displacements after laser ablation, no differences were detected in the biophysical properties of cells between the two compartments. However, the two rows of abutting A and P cells show clear differences in biophysical properties from other cells. Most importantly, the cell bond tension along the A/P boundary is increased. Cell divisions in the vicinity of the A/P boundary were randomly oriented in the epithelial plane. Thus, taken together with the simulations, these results suggest a sorting mechanism by which an increased cell bond tension guides the rearrangement of cells after cell division to maintain a straight interface. Increased cell bond tension and the roughness of the A/P boundary depend on Rho kinase activity and Myosin II, indicating a role for actin-myosin-based tension in this process. Because cell bond tension also depends on cell-cell adhesion, differences in the adhesion between A1 and P1 cells as compared to the remaining cells might also contribute to sorting. The heterotypic, but not homotypic, interaction of molecules presented on the surface of A and P cells might trigger the local increase in cell bond tension. Hedgehog signal transduction and the presence of Engrailed and Invected might control the expression of these heterotypically interacting molecules. These data indicate an important role for cell bond tension directing cell sorting during animal development (Landsberg, 2009).
A subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster that express the transcription factors Engrailed (En) and Invected (Inv) form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. Misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. A simple electrophysiological recording paradigm was introduced for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity (Pezier, 2014).
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, invectedDominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invectedDominant 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 invectedDominant 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 invectedDominant 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 invectedDominant phenotype. Finally, the invectedDominant 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).
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