InteractiveFly: GeneBrief

elbow B : Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Misexpression | References

BIOLOGICAL OVERVIEW

The elbow B (elB) gene encodes a conserved nuclear protein with a single zinc finger. Expression of ElB is restricted to a specific subset of tracheal cells, namely the dorsal branch and the lateral trunks. Stalled or aberrant migration of these branches is observed in elB mutant embryos. Conversely, ElB misexpression in the trachea gives rise to absence of the visceral branch and an increase in the number of cells forming the dorsal branch. These results imply that the restricted expression of ElB contributes to the specification of distinct branch fates, as reflected in their stereotypic pattern of migration. Since elB loss-of-function tracheal phenotypes are reminiscent of defects in Dpp signaling, the relationship between ElB and the Dpp pathway was examined. By using pMad antibodies that detect the activation pattern of the Dpp pathway, it has been shown that Dpp signaling in the trachea is not impaired in elB mutants. In addition, expression of the Dpp target gene kni is unaltered. The opposite is true as well, because expression of elB is independent of Dpp signaling. ElB thus defines a parallel input, which determines the identity of the lateral trunk and dorsal branch cells. No ocelli (Noc) is the Drosophila protein most similar to ElB. Mutations in noc give rise to a similar tracheal phenotype. Noc is capable of associating with ElB, suggesting that they can function as a heterodimer. ElB also associates with the Groucho protein, indicating that the complex has the capacity to repress transcription of target genes. Indeed, in elB or noc mutants, expanded expression of tracheal branch-specific genes is observed (Dorfman, 2002).

The Drosophila tracheal system is a stereotypical network of interconnected tubes that supplies air to all cells of the organism. Initially, ten tracheal placodes are defined on both sides of the embryo, each consisting of 20 cells. The placodes undergo two rounds of division, giving rise to the final number of tracheal cells. All subsequent events of tracheal morphogenesis and branch migration occur in the absence of any further cell division. The final structure of the tracheal tree is elaborate. Each tracheal pit gives rise to five different branches: dorsal branch (DB), dorsal trunk (DT), visceral branch (VB), lateral trunk anterior (LTa) and lateral posterior/ganglionic branch (LTp/GB). The number of cells allocated to each branch is fixed and the final structure of each branch is stereotyped, reflecting established migration routes. Within each branch, different cell types are formed from an originally equipotent population of tracheal cells. The cells at the termini of the branches differentiate as terminal cells that send long hollow extensions to hypoxic tissues. Another group of specialized cells, termed fusion cells, establishes connections between branches from adjacent segments (Dorfman, 2002 and references therein).

This elaborate tracheal structure is set up by the concerted activity of multiple signaling pathways. The initial assignment of tracheal fates within the population of ectodermal cells is driven by the localized expression of the Trachealess and Drifter transcription factors. Persistent expression of these genes in the trachea provides a 'cell context' for other signals that impinge on the trachea. Prior to the onset of tracheal migration, the precise number of cells must be allocated to each future branch. Several signaling pathways contribute to this decision, and in many cases parallel inputs from different pathways are responsible for the assignment of a particular branch fate. The process of migration is guided by the FGF pathway. All tracheal cells express the FGF receptor, Breathless (Btl). The ligand, Branchless (Bnl), is expressed locally in adjacent ectodermal or mesodermal cells. This restricted ligand presentation is responsible for guided migration. In addition, as the branches elongate, the levels of Btl activation determine the fate of the cells as terminal or fusion cells. Additional accessory guidance systems are present, such as the presence of a mesodermal cell expressing Hunchback (Hb) that assists the migration of the dorsal trunk cells (Dorfman, 2002 and references therein).

Morphogenesis of the tracheal system is determined by highly coordinated signaling events, which are restricted in both space and time. This prompted a search for new genes regulating tracheal development using the EP misexpression screen. The midline- and tracheal-specific btl-Gal4 driver was used, and the collection of EP lines was screened for those that give rise to lethality because of aberrant development of the tracheal system, or other tissues expressing btl-Gal4. Known genes that regulate tracheal patterning, such as dpp, bnl, rhomboid and escargot were scored, validating the specificity of the approach. In addition, this screen identified new genes that were not previously known to be involved in patterning the trachea. ElB defines a pathway acting parallel to Dpp that determines the identity of the lateral trunks and dorsal tracheal branches (Dorfman, 2002).

ElB is a member of a new family of proteins containing a single zinc finger and additional conserved motifs. ElB is a nuclear protein, but it is not yet known whether it binds DNA, or if it functions as a monomer or multimer. The similarity between elB and noc mutant phenotypes, the genetic interactions between them (Davis, 1997), and the ability of ElB and Noc proteins to associate with each other, suggest that ElB/Noc heterodimers are the functional complex (Dorfman, 2002).

ElB overexpression represses expression of genes such as the visceral branch marker and spalt (sal). Conversely, absence of ElB results in expanded expression of Sal to the dorsal branch, and noc mutants display failure to repress Serum response factor (SRF) expression in the fusion cells of the dorsal branch, suggesting that ElB functions as a repressor of gene expression. One piece of evidence strongly indicates that the ElB/Noc complex indeed functions directly to repress the expression of target genes. Both Drosophila proteins, as well as the human homologs, contain the FKPY motif, which is known to be sufficient for interactions with Groucho. Indeed, GST pull-down experiments have demonstrated that ElB can associate with Groucho. It is interesting to note that another Sp1 homolog, Huckebein (Hkb), recruits Groucho through the FRPW motif. The ElB/Noc complex may thus serve to recruit the Groucho protein to specific target sites on the DNA, and repress the expression of distinct genes. Future identification of target genes will determine if ElB/Noc can also facilitate induction of certain genes (Dorfman, 2002).

Expression of ElB is confined to distinct tracheal branches from stage 12, namely the dorsal branch and lateral trunks. This restricted expression is instructive for the future fate and migration pattern of these branches. When misexpressed in other branches, ElB abolishes the migration of the visceral branch and eliminates the expression of a visceral branch marker. It also represses expression of Sal, a protein that defines the dorsal trunk identity. ElB is also able to divert several cells from the dorsal trunk fate into a dorsal branch (Dorfman, 2002).

In the tracheal branches where elB is normally expressed, elB has an essential role. In elB null mutants, fewer cells join the dorsal branch, and these branches migrate abnormally. In addition, the cells normally forming the lateral trunk anterior remain in the transverse connective, and the ganglionic branches are stalled. Attempts can be made to interpret these phenotypes with the repressive activity of the ElB/Noc complex in mind (Dorfman, 2002).

It is possible that in the dorsal branch, the complex represses expression of genes that confer dorsal trunk identity such as sal. A similar paradigm has been shown for the Dpp pathway. kni expression in the dorsal branch is induced by the Dpp pathway. Kni can bind the sal promoter and repress expression of the gene. In elB mutants expression of dorsal trunk genes extends to the dorsal branch and partially converts the identity of these cells to a dorsal trunk fate. Similarly, in the lateral trunk the ElB/Noc complex may be required to repress the expression of genes conferring visceral branch identity. In elB mutants, the expanded expression of visceral branch- and dorsal trunk-specific genes into the lateral trunk may thus abolish or stall the migration of the lateral trunk and ganglionic branches. Subsequently, ElB expression is confined to a specific cell within the dorsal branch, namely the fusion cell. In noc mutants the expression of Srf in the dorsal branch expands to the fusion cell. One way to interpret the expanded expression of Srf is by loss of direct repressive activity of ElB/Noc in the fusion cell (Dorfman, 2002).

Why is the elB mutant tracheal phenotype more severe than that of noc, if the two proteins form a functional complex? Since noc does not have an early maternal RNA, the zygotic noc mutant phenotype should reflect the null situation. ElB can associate not only with Noc, but also with another ElB monomer. It is thus possible that in noc mutant embryos, ElB/ElB homodimers partially substitute for the ElB/Noc heterodimers (Dorfman, 2002).

The tracheal defects observed in elB mutants are reminiscent of tracheal defects in tkv mutant embryos, where signaling of the Dpp pathway is blocked. However, ElB appears to function in parallel to the Dpp pathway. Phosphorylation of Mad and induction of kni expression, both of which mark the activity of the Dpp pathway, are normal in elB mutants. Conversely, ElB expression is independent of Dpp/Tkv activation. The possibility that Dpp signaling directs a post-translational modification of ElB/Noc has not been ruled out. Nevertheless, the available data suggests that generation of the dorsal branch, and migration of the lateral trunk anterior and ganglionic branch, require both the input from Dpp signaling and the expression of ElB/Noc (Dorfman, 2002).

It is not known yet how activation of distinct tracheal cells in the dorsal and ventral region of the tracheal pit by Dpp, as visualized by pMad accumulation, contributes to the capacity of these cells to form the dorsal and lateral branches, respectively. Activation by Dpp induces expression of target genes such as kni in these compartments. Kni in turn was shown to repress expression of dorsal-trunk genes like sal (Dorfman, 2002).

How are the ElB/Noc and Dpp signals integrated in the trachea? One possibility is that they impinge on different target genes. ElB/Noc repress expression of visceral branch or dorsal trunk genes, while the Dpp signal induces the expression of target genes in the same cells. The combined activity of the two pathways will determine the set of branch-specific genes expressed by these cells. The final identity of each branch is likely to be a result of inputs from different pathways, which contribute to the expression of branch-specific genes and to the repression of other genes. This is exemplified most clearly when monitoring kni expression in the dorsal branch of elB mutant embryos. While the correct number of cells are induced by the Dpp pathway and express kni, some of these cells are stalled in the dorsal trunk in the absence of ElB. It is also demonstrated by the fact that only the combined activity of ElB and activated Tkv is capable of inducing an excess of LTa cells. ElB/Noc and Kni also cooperate in the repression of common target genes. Complete repression of sal in the dorsal branch cells requires both complexes, as evidenced by the expanded expression of Sal in elB-mutant embryos (Dorfman, 2002).

Future knowledge regarding the nature of these branch-specific target genes should provide insights into the mechanism that regulate branch-specific fates. These genes may encode adhesion molecules or membrane receptors that allow responses to different sets of external guiding cues. This system could provide further migrational specificity, superimposed on the common Branchless signal guiding the migration of all tracheal branches. Furthermore, it may determine the stereotyped number of cells recruited into each tracheal branch (Dorfman, 2002).

The similarity in the phenotypes of elB and noc mutants, the genetic interaction between the mutants, and the complex formed between the two proteins, strongly suggest that these proteins carry out their biological roles as a complex. However, the two genes are regulated differently in the embryo. noc is broadly expressed and overexpression of the protein does not give rise to a tracheal phenotype, suggesting that spatial and temporal regulation of activity relies on elB expression. The restricted expression of elB is essential, since elB misexpression gives rise to deleterious phenotypes in the trachea. It is not known if additional tiers of regulation, such as inputs from signaling pathways or phosphorylation, also impinge on the complex (Dorfman, 2002).

Expression of elB is initially observed in all tracheal cells, suggesting that it is under the control of Trachealess and Drifter, which confer tracheal identity. However, at stage 12, expression of elB becomes excluded from the central part of the pit. It is possible that the restricted pattern of elB expression is thus a combination of induction by general tracheal transcription factors, and repression of expression in the future dorsal trunk and visceral branch. The signals leading to this repression are not known. The EGF receptor pathway is activated in the central domain of the tracheal placodes. However, when the activity of this pathway is abolished in Star mutants, elB expression remains unchanged (Dorfman, 2002).

It will be interesting to find out the function the ElB/Noc complex in other tissues. In noc mutant embryos, defects in migration of cells from the procephalic lobe are observed (Cheah, 1994). Expression of elB is not restricted to the trachea, and is also observed in the wing imaginal disc and the adult photoreceptors (S. Cohen and C. Desplan, communication to Drofman, 2002). In accordance with the roles of the ElB/Noc complex in the trachea, it is likely that in the above tissues the same complex will be essential for determination of cell fates, by repressing and possibly also inducing critical target genes (Dorfman, 2002).

In conclusion, using a tracheal misexpression screen two proteins have been identified that form a complex and participate in the determination of specific tracheal branch fates. ElB/Noc define a parallel input to Dpp signaling, demonstrating that convergence of several signals contributes to the robust determination of branch-specific cell fates, and to the refinement of these fates (Dorfman, 2002).

REGULATION

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRING) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

Protein Interactions

The similarity between ElB and No ocelli (Noc) proteins prompted an examination of the role of Noc in tracheal development. In situ hybridization shows that noc is expressed from stage 4 in the embryonic termini, and at stage 5/6 in ectodermal stripes. At stage 11, noc is expressed in the invaginating tracheal pits, while from stage 13, noc is expressed ubiquitously (Cheah, 1994). Since noc expression is not spatially restricted, it is not surprising that no tracheal phenotypes were observed after Noc misexpression by btl-Gal4/EP2000. Higher levels of induction of noc expression (using UAS-noc), do not abolish the visceral branches, but their direction of migration is occasionally misrouted. In addition, misexpression of both ElB and Noc in the trachea does not generate a phenotype that is more severe than the one produced by misexpression of ElB alone (Dorfman, 2002).

In order to examine the normal role of Noc in tracheal development, imprecise excisions were generated for the EP(2)2173 element, located 518 bp upstream of the 5'UTR. A lethal excision termed noc^d64 was characterized by Southern blotting and inverse PCR. It represents a deletion of 848 bp, removing all residues between the insertion site of EP2173 and noc transcription start site, as well as 330 bp of the 5'UTR, thus defining a null mutation in noc. Homozygous noc^d64 mutants show a weaker mutant phenotype, but similar to that of elB in terms of the affected tracheal branches. Fewer cells are observed in the lateral anterior trunk and ganglionic branch, and lateral branch fusion is occasionally missing. In addition, while all dorsal branches are formed, some branches have only up to three cells. The basis for partial lack of branch fusion in noc mutants was examined. Normally, in the dorsal branch only one terminal cell expresses SRF, while the preceding cell becomes the fusion cell and expresses specific markers. In noc mutants, repression of SRF expression in the fusion cell fails, giving rise to duplicated terminal cells at the expense of fusion cells (Dorfman, 2002).

In view of the overlapping tracheal expression patterns and the previously reported genetic interactions (Davis, 1997), it is possible that the ElB and Noc proteins form a heterodimeric complex. Since each protein contains only a single zinc finger, a heterodimer may provide the appropriate number of zinc fingers for DNA binding. GST pull-down experiments were carried out using GST fusions to full-length constructs of the ElB protein, as well as to fragments of the ElB protein. Association with in vitro translated full-length ElB or Noc was examined. Indeed, GST-ElB is capable of associating with Noc. In addition, GST-ElB is also capable of associating with ElB, demonstrating that ElB can form homodimers as well as heterodimers with Noc. Truncated constructs of ElB show that its C-terminal region, which contains the Cysteine-rich and zinc-finger domains, is sufficient for these interactions (Dorfman, 2002).

The biological activities of ElB and Noc are consistent with repression of transcription. Overexpression of ElB in the trachea represses the expression of specific genes in the dorsal trunk and visceral branch. Conversely, absence of ElB results in expanded expression of Sal to the dorsal branch, and noc mutants display failure to repress SRF expression in the fusion cells of the dorsal branch. The notion that the complex may posses repressive activity by virtue of its association with known inhibitors of transcription was tested. Two such proteins are known: CtBP and Groucho (Dorfman, 2002).

The Groucho protein is known to mediate long-range transcriptional repression, and to associate with DNA-binding proteins bearing a number of motifs, including FKPY. This sequence is conserved in ElB, Noc and the two human homologs. Thus the capacity of ElB to associate with Groucho was tested. Indeed, specific association was detected. CtBP promotes short-range repression and is known to associate with DNA-binding proteins containing the PxDLSxR/K/H motif. Such a motif is not found in ElB or Noc, and the GST-ElB fusion protein shows only negligible precipitation of labeled CtBP. This result strongly suggests that the ElB/Noc complex represses transcription of target genes directly, by recruiting Groucho to these sites (Dorfman, 2002).

Genetic dissection of photoreceptor subtype specification by the Drosophila melanogaster zinc finger proteins Elbow and No ocelli

The elbow/no ocelli (elb/noc) complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. This study shows that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the 'dorsal rim area' (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. The current data suggest that Elb/Noc genetically interact with Hth, and two conserved domains crucial for this function were identified. Furthermore, Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. It is proposed that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease (Wernet, 2014; PubMed: 24625735).

The transcription factors Homothorax (Hth) and Extradenticle (Exd) have been well characterized as co-factors for Hox genes. Hth/Exd can also act as co-factors for non-Hox transcription factors, like for Engrailed. This study showed that loss of both Elb and Noc phenocopies the loss of Hth at the dorsal rim of the retina. All markers of DRA ommatidia are lost in elb,noc double mutants: Rh3 expression and Sens repression in DRA R8, as well as the DRA-specific inner photoreceptor rhabdomere morphology in DRA R7 and DRA R8. The data shows that Elb/noc act downstream of Hth in the specification of DRA cell fates. Elb and Noc are expressed strongly in DRA R7 and R8. This expression is expanded to all R7 and R8 by ectopic Hth (but never into outer photoreceptors R1-6), while Hth expression is not affected in elb,noc double mutants. One possibility is that Elb/Noc serve as cofactors for Hth/Exd, since Hth loses its potential to induce the DRA fate in a double mutant retina. The vertebrate homologs of Elb and Noc function as repressors of transcription (Nakamura, 2008). Therefore, aspects of the Hth/Exd and Elb/Noc loss-of-function phenotypes could be due to a direct failure of their complex to repress common target genes. For instance, the de-repression of the R8 marker Sens by dominant-negative hth^HM, as well as in elb,noc double mutants could be explained by loss of a repressor complex containing all four proteins. Interestingly, functional antagonism between the Hox/Hth/Exd complex and Sens have been described in the Drosophila embryo. However, in this case the factors were shown to compete for overlapping binding sites in the promoter of the common target gene rhomboid. Gene expression profiling data revealed that the Hox gene Abd-B also directly represses Sens in the embryo using Hth/Exd as cofactors. Elb and Noc might therefore provide a missing link for transcriptional repression of Sens by Hth/Exd (Wernet, 2014).

Much work on NET family proteins has focused on functional characterization of their evolutionarily conserved domains. The C-terminus of NET proteins is required for nuclear localization (Pereira-Castro, 2013; Runko, 2004), as well as for self-association of the zebrafish ortholog Nlz1, although neither self-association nor heterodimerization with Nlz2 was found to be necessary for wild type function (Runko ). 'buttonhead box' , a conserved 7-10 amino acid motif which was not investigated in this study, may be required for transcriptional activation (Athanikar, 1997). Deletion of the 'buttonhead box' in zebrafish Nlz proteins transformed them into dominant-negatives, an effect that was proposed to be due to reduced affinity to co-repressor Groucho and histone de-acetylases. Interestingly, deletion of N-terminal sequences, including the Sp/SPLALLA motif also leads to dominant negative proteins. These data are consistent with findings that a protein with a mutated Sp/SPLALLA motif has a dominant-negative effect on DRA specification. The Sp motif was proposed to mediate transcriptional repression by directly binding to cofactors. It should be noted that both N-terminal Sp/SPLALLA deletion and the VP16 fusions have the same dominant-negative effect for zebrafish Nlz1. While this is consistent with a pure repressor function of the zebrafish protein, the differences between Sp/SPLALLA mutation and VP16-fusion (as well as the observation of a phenotype for the Engrailed fusion) reported in this study hint towards a more complex role of Elb and Noc in transcriptional regulation (Wernet, 2014).

This study has shown that mutation of the conserved zinc finger of Elbow also transforms this protein into a dominant-negative. Usually, multiple zinc fingers are required for DNA binding, suggesting that the NET family zinc finger is a protein-protein interaction domain. Deletion of the zinc finger from zebrafish Nlz proteins leads to a loss of nuclear localization, and the Nlz1 zinc finger is necessary for transcriptional repression. Although the possibility that Elb and Noc bind DNA through their zinc finger cannot be excluded, it is likely that mutation of the zinc finger either leads to an inactive complex by sequestration of another co-repressor, or that such complex could be trapped in the cytoplasm. Given that mutation of either Sp/SPLALLA motif or zinc finger both lead to a dominant-negative effect raises the possibility that protein binding to both motifs could be necessary for in vivo function, possibly through the formation of higher order transcriptional complexes (Wernet, 2014).

Loss of both elb and noc does not result in Rhodopsin phenotypes outside the DRA. However, over-expression of different forms of Elb or Noc recapitulates all Rhodopsin phenotypes observed in otd^UVI mutants. This phenotype might therefore arise from forcing a direct interaction between over-expressed Elb protein and Otd. Little is known about the regulatory relationship between Elb/Noc and Otd. However, the overlapping expression patterns and similar phenotypes for certain alleles of otd named ocelliless, and for no ocelli (noc) at the anterior pole of the fly embryo, as well as their common requirement in the morphogenesis of ocelli suggests that these proteins also interact positively outside of the retina. The antagonism that was observed might therefore be a dominant-negative effect resulting from sequestration of the Otd protein by over-expressed Elb. Alternatively, different combinations of transcriptional cofactors present between tissues (for instance DRA versus non-DRA R8 cells) might decide whether Elb and Noc act in concert with Otd, or as antagonists (Wernet, 2014).

In the retina, Otd acts in a 'coherent feedforward loop' with Spalt to directly activate transcription of rh3 and rh5. As a consequence, Rh3 and Rh5 are lost in otd mutants. Furthermore, Otd activates transcription of the repressor Dve, forming an 'incoherent feedforward loop', resulting in repression of rh3 and rh5 in outer photoreceptors. Since rh6 is activated by a distinct factor, Pph13, loss of Otd leads to a specific de-repression of rh6 into outer photoreceptors. This study shows that different domains of Elb specifically interfere with different aspects of Otd function in these feedforward loops. Mutation of the Groucho-binding motif FKPY only abolishes the ability of over-expressed Elbow protein to antagonize Otd function in repressing rh6 in outer photoreceptors, while mutation of the Sp/SPLALLA motif specifically antagonizes Otd function in activating both rh3 and rh5, without affecting repression of rh6 in outer photoreceptors (mediated by induction of Dve). Furthermore, while the Elb zinc finger is also required for antagonizing the function of Otd in outer photoreceptors, it is also necessary for antagonizing activation of rh3 by Otd, but not rh5. Hence, these two activator functions of Otd could be separated by mutating the zinc finger (Wernet, 2014).

The different Rhodopsin phenotypes caused by loss of Otd can be mapped to different protein domains. The current data therefore reveal specific genetic interactions between the protein domains of Elb/Noc and Otd. Such interactions could be direct or be mediated through additional proteins. For instance, the Otd C-terminus mediates the repression of rh6 in outer photoreceptors, making it a possible interaction domain for Groucho binding to the Elb/Noc FKPY motif. The N-terminus of Otd is necessary for most activation potential on rh3, while activation of rh5 predominantly maps to the C-terminus. This correlates well with the Rhodopsin-specific phenotypes seen after mutation of Sp/SPLALLA (affecting rh3 and rh5), or the zinc finger (affecting rh3 and rh6) motifs. Finally, the results using VP16- and en[R]-fusions of Noc show that potentially direct transcriptional effects on rhodopsin genes can only be induced in R8 cells. Both fusion proteins specifically regulate expression of rh5, while all other rhodopsins remain unaffected. Elb and Noc are both expressed strongly in R8 cells outside of the DRA where they may contribute the repression of Rh5. The absence of a non-DRA R8 rhodopsin phenotype in elb,noc double mutants, as well as the R8-specific action of VP16:noc could therefore be due to the existence of redundant, R8-specific factors required for Elb/Noc function there, but not for DRA specification. These factors remain unknown, since it was found that expression of elb and noc is not altered in homozygous mutants affecting p/y cell fate decisions in R8 cells (melt and wts (Wernet, 2014).

Mutations in the human Elb/Noc homolog ZNF703 promote metastasis (Slorach, 2011). This study has shown that over-expression of both human NET family proteins UAS-ZNF503 and UAS-ZNF703 in the Drosophila retina result in weak co-expression of Rh5 and Rh6, resembling over-expression of a VP16:noc protein. It is therefore possible that the genetic interaction of NET family proteins with Otd/Otx proteins is evolutionarily conserved, especially since a central domain of Otd was previously shown to mediate mutual exclusion of Rh5 and Rh6 (McDonald, 2010). This study presents a new role for Drosophila NET proteins in retinal patterning. Both zebrafish homologs of Elb/Noc, Nlz1 and Nlz2 are also required for optic fissure closure during eye development (Brown, 2009). Furthermore, expression of the Elb/Noc mouse homologue znf503 suggests that NET family genes are involved in the development of mammalian limbs(McGlinn, 2008). Given previous reports from Drosophila on the proximo-distal specification of leg segments, it appears that NET family members act in similar processes across species. This raises the possibility that NET proteins serve as evolutionarily conserved modules that have been re-utilized for analogous processes during evolution. Based on the current data, their conserved domain structure might be crucial for interacting with transcription factor networks involving conserved families of factors like Otx or Meis. Given their medical relevance in breast cancer, a better understanding of the role NET proteins play in the transcriptional control of tissue patterning will be of great importance (Wernet, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

In situ hybridization with an elB RNA probe reveals expression of elB in all tracheal pit cells, starting at stage 11. There is a higher level in the first and the tenth pits. At stage 12, as the primary branches form, elB expression is reduced within the central part of the pit, and becomes restricted to the lateral anterior and posterior branches and to the dorsal branch cells. The elB probe is specific and does not crossreact with noc RNA, which exhibits a different distribution (Dorfman, 2002).

Anti-ElB antibodies are able to detect ectopically expressed protein, but marginally detect the endogenous protein levels. However, a Gal4 element inserted 1260 bp upstream of the 5' UTR provides a sensitive expression pattern marker that correlates with the RNA pattern. A Gal4-responsive UAS-elB element shows that at stage 14 elB expression is detected in the lateral branches and excluded from the dorsal trunk and visceral branch. By stage 16 expression in the transverse connective, spiracular branch, lateral and ganglionic branches is observed. Within the dorsal branch, ElB is specifically expressed at this stage in the fusion cell. Restriction of ElB expression is indeed necessary, since expression of ElB in all branches is sufficient to eliminate the visceral branch, and leads to defects in the dorsal trunk (Dorfman, 2002).

EFFECTS OF MUTATION

In a misexpression screen of the EP collection with the midline- and tracheal-specific btl-Gal4 driver, the EP(2)2039 line gave rise to a lethal phenotype. Examination of the tracheal system of these embryos with antibodies recognizing the tracheal lumen revealed a specific abolishment of the visceral branch. By immunohistochemical analysis using anti-Trh antibody that detects all tracheal nuclei, as well as anti-ElB antibody, which shows a similar pattern, it was observed that cells that normally form the visceral branch remain in the central region of the transverse connective branch. In the dorsal branch, an increase in the number of cells was identified. Quantitation of the number of dorsal branch cells have demonstrated that while in wild-type embryos each branch contains an average of five cells, in ElB misexpression embryos most dorsal branches contain five to eight cells. The phenotypes of ElB tracheal misexpression were verified by crossing the btl-Gal4 line to lines bearing a pUAST construct driving an elB full-length cDNA. Similar phenotypes of reduced visceral branches and enlarged dorsal branches were observed (Dorfman, 2002).

To obtain a finer resolution, molecular markers for the different branches were followed. By using the l(2)01351 enhancer trap line that marks the visceral branch cells, it was shown that this marker is not expressed in the cells remaining in the transverse connective following ElB misexpression. Overexpression indeed abolishes expression of a gene normally marking the visceral branch cell fate. knirps (kni) is normally expressed in the lateral trunks (Lta and LTp), dorsal branch and visceral branch. Uniform expression of ElB in the trachea does not alter the pattern of kni expression in cells that usually form the LT. kni expression is also observed in visceral branch cells that are retained in the transverse connective upon ElB misexpression. Thus, not all visceral branch markers are abolished. Finally, in the dorsal branch more cells express kni due to ectopic migration of cells at the expense of the dorsal trunk (Dorfman, 2002).

In ElB misexpression embryos, several cells normally assigned to the dorsal trunk appear to be migrating into the dorsal branch, thus increasing the cell number in that branch. This suggests that there may also be a defect in specifying the dorsal trunk fate. Therefore, a dorsal trunk marker, Spalt (Sal), was followed in embryos misexpressing ElB. Sal is a transcription factor that is specifically expressed in the dorsal trunk cells and determines their identity. Ectopic ElB expression abolishes all Sal expression in the trachea. Surprisingly, this does not lead to an absence of the dorsal trunk, which is typical of sal mutant embryos, presumably because the btl-Gal4 driver induces the accumulation of ElB and abolishment of Sal expression only after execution of the normal Sal function in the dorsal trunk (Dorfman, 2002).

These experiments suggest that ElB expression must be excluded from the dorsal trunk and visceral branch, in order to specify proper dorsal trunk and visceral identities (Dorfman, 2002).

To determine the role of ElB in the branches where it is normally expressed, analysis of mutations in the elB locus was carried out. Several mutant fly lines in the elB region are available; however, they have not been precisely mapped. In order to create a line with a specific elB mutation, the EP element was excised, using a Delta 2-3 transposase. Only one lethal excision line (termed elB^d47) showed rearrangements in Southern blot analysis in the region of EP2039. Inverse PCR of the rearranged genomic fragment and sequencing provided the following molecular details. The EP2039 element was deleted. All sequences between EP2039 insertion site and elB were intact, while on the other side sequences of the transposable yoyo element were identified. Further Southern analysis indicated a deletion of the genomic sequence in the region upstream of EP2039 insertion. Therefore, it is possible that an inversion coupled to a deletion of sequences upstream of EP2039 has taken place. elB^d47 is lethal over Df(2L)noc10 and over the smaller deficiency Df(2L)fn2, uncovering 35A3-35B2. While elB^d47 does not remove genomic regions containing the elB transcribed region, it is nevertheless allelic to the EMS-induced viable mutation elB¹, which displays the typical smaller wing phenotype. It is thus possible that crucial transcriptional regulatory sequences of elB are removed in elB^d47 (Dorfman, 2002).

Homozygous elB^d47 embryos display defects in tracheal development. In all segments, there is no migration of the lateral trunk anterior, and the ganglionic branches are shorter. The cells that fail to migrate into the lateral trunk remain in the transverse connective. The number of cells that form the dorsal trunk and visceral branch do not change. Dorsal branch migration is also impaired, and failure of dorsal branch fusion is observed at stage 16. Cell counts of dorsal branch cells in the mutant embryos show a considerable number of branches with only three to four cells. In some of the segments, the dorsal branch cells migrate aberrantly, and laterally adjacent branches fuse. The same tracheal phenotype is observed in embryos homozygous for the Df(2L)fn2 deficiency, as well as trans-heterozygotes of elB^d47 over Df(2L)fn2, demonstrating that elB^d47 is a null mutation (Dorfman, 2002).

Since elB^d47 may not affect only the elB gene, it was necessary to confirm that the observed tracheal phenotypes indeed result from loss of elB. Btl-Gal4 was used to drive UAS-elB inserted on the third chromosome, in the background of homozygous elB^d47 embryos. The most pronounced tracheal phenotype of elB^d47 embryos is manifested in stalled anterior lateral trunk migration. Since tracheal misexpression of elB does not give rise to defects in the LTa of wild-type embryos, it was possible to examine rescue of this phenotype. Embryos misexpressing ElB were identified by anti-ElB staining and the characteristic visceral branch defects. While 25% of these embryos are homozygous for elB^d47, no severe LTa migration defects were observed. It was possible to identify specifically some of the rescued homozygous elB^d47 embryos, by virtue of subtle defects in other branches such as the dorsal and ganglionic branch (Dorfman, 2002).

The tracheal phenotype of elB mutants is reminiscent of defects arising when signaling by the Dpp pathway is blocked (e.g. in thickveins mutant embryos). Most notable is the stalled migration of the lateral trunk anterior, the ganglionic branch and the dorsal branch. Dpp is expressed at stage 11 in two ectodermal stripes: the dorsal stripe is restricted to a single row of the dorsal-most ectodermal cells, and is positioned several cells away from the dorsal edge of the tracheal pit. The ventrolateral stripe of Dpp expression abuts the ventral side of the tracheal pit (Dorfman, 2002).

Activation of the Tkv/Punt receptors by Dpp leads to the phosphorylation of the Mad protein, which forms a heterodimer with Medea and translocates to the nucleus to trigger transcription. Antibodies specifically recognizing the C-terminal phosphorylated form of Smad1 also recognize the phosphorylated form of Drosophila Mad. These antibodies were used to follow Dpp signaling in the trachea in wild-type and elB mutant embryos (Dorfman, 2002).

In wild-type embryos, pMad is observed in the tracheal pits beginning at early stage 11, when Dpp is expressed in two stripes in the dorsal and ventrolateral ectoderm. Dpp activation subdivides the tracheal pit into three parts: the activated dorsal and ventral domains and the central region, which is not activated by Dpp. The number of cells in which pMad is generated in the ventral domain of the pit is significantly larger than the number of tracheal cells displaying pMad in the dorsal domain. Since the dorsal stripe of Dpp is positioned several cell diameters above the tracheal pit, the diffusion of Dpp reaches and activates only approx. five tracheal cells. This corresponds to the number of cells that will be recruited to the dorsal branch (Dorfman, 2002).

The ventral Dpp stripe abuts the tracheal pit, and pMad is observed in more cells in the ventral aspect of the pit, in accordance with the larger number of lateral trunk cells influenced by Dpp signaling. It is interesting to note that despite the higher levels of Tkv expression in the tracheal pits, the amount of pMad in the tracheal cells versus the adjacent ectodermal cells is comparable. As migration of the tracheal branches continues, the lateral trunk cells migrate ventrally beyond the stripe of Dpp. Consequently, only the tracheal cells lying under this stripe retain pMad activation (Dorfman, 2002).

The pMad patterns allow a direct determination of whether the reception of Dpp signaling is compromised in elB mutants. elB^d47 embryos were stained with the pMad antibody, and identified by the stalled migration of the lateral trunk anterior. The number of dorsal and ventral cells displaying pMad in the pit at stage 11/12 is comparable with wild-type embryos. ElB is thus not required for normal signaling by the Dpp pathway (Dorfman, 2002).

ElB could be required downstream of pMad, for the induction of Dpp-target genes. Expression of kni, which encodes a zinc-finger transcription factor, is induced by the activated Tkv/Punt receptors. kni-lacZ transgene was used to follow the dependence of kni expression on ElB. Overexpression of ElB does not alter the expression pattern of kni. Likewise, in elB mutants, the expected number of dorsal branch cells retain kni expression. However, while normally the kni-expressing cells segregate completely from the dorsal trunk into the dorsal branch, in the elB mutant several cells are retained within the dorsal trunk. This may be a reflection of failure of the cells destined to form the dorsal branch to lose their identity completely as dorsal trunk cells. Indeed, when expression of the dorsal trunk marker Sal was examined, residual Sal was observed in all the kni-expressing cells that remained within the dorsal trunk, and in some of the cells forming the dorsal branch. Thus, Kni is not sufficient to repress Sal expression, and requires cooperation with ElB for complete repression (Dorfman, 2002).

The normal reception of Dpp signaling and kni expression in elB mutants still leaves open the possibility that ElB is a downstream component of the Dpp pathway. Whether expression of elB is regulated by the Dpp pathway was also tested. Overexpression of activated Tkv with the btl-Gal4 driver does not alter the expression pattern of elB RNA. In addition, ectopic kni or knrl does not alter the elB expression pattern in the trachea, in agreement with the above results with activated Tkv. These results imply that while ElB and the Dpp pathway affect the same tracheal branches, they function in a parallel manner (Dorfman, 2002).

The combined effects of ElB misexpression and uniform activation of Tkv in the trachea could be manifested in cell fate changes leading to excess LTa cells. Following misexpression of only ElB or activated Tkv, the number of LTa cells is unchanged, while the typical visceral branch and dorsal branch abnormalities are observed. However, misexpression of both constructs also leads to changes in LTa cell fates, as extra cells are recruited into this branch (Dorfman, 2002).

The elbow locus is found to be two genes elA and elB, each of which has a distinct phenotype when mutant. Mutations of the elA gene have a strong phenotype where the wing is markedly disrupted. Mutations of elB are weak, mainly affecting the alula and the wing bristles. The two genes are dominant enhancers of each other. Homozygous deletion of the complete elbow region results in lethality. Situated between the elbow genes is the pupal gene and a locus which when deleted causes a crippled leg phenotype. This locus may be a control region for elbow. Immediately adjacent on the proximal side of elA is the no-ocelli locus. The phenotypes of noc alleles vary from extreme, where the ocelli and associated bristles are absent, to weak, where these structures are disrupted. The various noc phenotypes are associated with genetically distinct gene regions, mutations of which act as enhancers of each other. Alleles of el and noc show partial failure of complementation, heterozygotes having weak el or weak noc phenotypes. Alleles of both these genes interact with the antimorphic noc allele Sco (Davis, 1997).

The el-noc complex spans a distance of about 200 kb on chromosome 2L. It consists of three discrete genetic regions el, l(2)35Ba and noc, each of which has a distinct phenotype when mutant. The noc locus itself is complex, including three separate regions. The el locus has been characterized by mapping 30 aberration breakpoints to the DNA. It extends over a distance of about 80 kb. It can be divided into two parts by the aberrations In(2LR)DTD128 and T(Y;2)A80. These break between two sets of el alleles yet are both phenotypically wild type for elbow. The simplest explanation is that el consists of two transcription units elA and elB. The locus pu, which appears to be unrelated to the el-noc complex, is found to map between the two el loci very close to elB (the distal el locus). The loci l(2)35Ba and nocA have been separated by only two l(2)35Ba+nocA- deletions and a nocA- inversion. No l(2)35Ba-nocA+ aberrations have been found. At the molecular level these loci are found to occupy almost the same region, and are probably identical (Davis, 1990).

The phenotype for mutations of the nubbin gene consists of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs from the early stages of larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Mutant clones are approximately 50% smaller than control clones or else they fail to grow in 50% of the cases. Clones located in the proximal region of the wing blade cause an additional nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior-posterior nor the dorsal-ventral boundary but that the selector genes decapentaplegic and engrailed are correctly expressed from early larval development. The phenotypes of nub elbow and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells (Cifuentes, 1997).

A screened was carried out for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, 46 lines were identified, corresponding to insertions in 32 different loci, that elicit a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation (Tseng, 2002).

The lines EP965 and EP2039 have insertions that are 171 bp apart and in the same orientation, ~16.7 kb upstream of the gene elB (CG4220). The EP2039 insertion has been shown to be a weak elB allele, and a putative open reading frame (BG:DS06238.3) was identified by the Berkeley Drosophila Genome Project. This gene is predicted to encode a zinc-finger protein and shows 27% homology to no-ocelli (noc), a gene 100 kb proximal to BG:DS06238.3. The function of elB is unknown. No other transcription units have been identified between the EP insertions and elB. Both EP965 and EP2039 expression increased levels of elB RNA in a GAL4-dependent manner (Tseng, 2002).

The properties of proliferating cells were examined in the wing imaginal disc in the EP2039 line. Compared to the wild-type control, overexpression of the EP2039 line does not have any observable effects on cell size as determined by forward scatter. However, EP2039 overexpression does result in a slight change in cell-cycle phasing. There is a small but reproducible increase in the G2/M population, suggesting that EP2039 overexpression may be able to restrict either entry into or passage through mitosis. The population doubling time of cells overexpressing EP2039 was determined. Overexpression of EP2039 and p35 results in a doubling time of 18.6 hr, which is 26% longer than the doubling time of 14.8 hr calculated for the control cells expressing p35 alone. An increased doubling time with no change in cell size is consistent with a decreased rate of growth (mass accumulation) with a concomitant slowing of the cell cycle (Tseng, 2002).

RNAi was used to reduce the levels of elB in SL2 cells. elB is expressed at extremely low levels in SL2 cells. Expression could not be detected by Northern blotting, but could be detected at low levels using Affymetrix oligonucleotide microarrays. There was no change in the growth properties of the treated SL2 cells, although it is important to point out that the change in the level of elB expression could not be monitored. The possibility that elB and noc might have shared functions was also considered. However, RNAi experiments using dsRNA for both elB and noc showed no effect on cell division (Tseng, 2002).

Proximodistal subdivision of Drosophila legs and wings: the elbow-no ocelli gene complex

Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, attempts were made to identify genes that underlie the specification of all appendage primordia. Evidence is presented that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation (Weihe, 2004).

Evidence suggests that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna, and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do so, when examined in detail (Weihe, 2004).

Dll expression is required for the formation of all leg and antenna elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc. Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh. Thus an essential mediator of the effects of Wg and Dpp was missing. The current results present evidence that El and Noc serve this function, since their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so it is concluded that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc (Weihe, 2004).

The situation differs slightly in the wing. Repression of Tsh is the earliest marker for specification of the distal wing region, preceding the onset of Hth repression or of Nub induction. Loss of Tsh and Hth are required to allow Nub expression. Ectopic expression of Hth and Tsh and loss of Nub is observed in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll). The vestigial gene is also important for wing development and has been proposed to be a wing specifying gene. However, Vestigial is expressed all along the DV boundary of the wing, both in the wing primordium and in the body wall. This led to the suggestion that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages. For this reason analysis of the relationship between El, Noc and Vestigial was not performed in this study (Weihe, 2004).

Is the repression of trunk genes needed to specify appendage, as opposed to body wall, in wing and leg discs? In the wing disc the answer appears to be yes; repression of 'trunk genes' like hth is necessary to make the remaining part of the disc competent to form the appendage. However, in the leg the situation is more complex. Coexpression of Dll and Hth does not disrupt proximal-distal axis formation, but leads to homeotic transformation of leg tissue into antennal tissue. Hth is not repressed and limited to proximal areas in the antenna. However, loss of el and noc activities in the leg disc leads to loss of distal leg tissue without any evident transformation into antennal tissue. Thus, El and Noc may regulate the expression of other 'trunk genes', whose restricted expression is required to make the remaining leg and antenna disc competent to form the appendage (Weihe, 2004).

The regional requirements for El and Noc highlight another interesting difference between leg and wing disc development. el noc double mutant cells are excluded from contributing to the tarsal region of the leg but not from contributing to the femur and tibia. Lineage tracing has shown a considerable net flux of cells from the proximal (Tsh-expressing domain) into femur and tibia. While there is no boundary of lineage restriction separating these domains, cells must be able to change from expressing the proximal marker Hth to expressing the distal marker Dll in order to move from one territory to the other. The wing in contrast does not appear to normally exhibit this large net flux of cells from proximal to distal and the el noc double mutant cells are excluded from contributing to the entire wing region. Clonal analysis has suggested that el noc double mutant cells attempt to sort out toward proximal territory, or if that fails, they can be lost from the disc, apparently by sorting out perpendicular to the epithelium. These observations suggest that El and Noc activity may contribute to the production of proximal-distal differences in cell affinities and thereby may help to maintain segregation of these cell populations during development (Weihe, 2004).

REFERENCES

Search PubMed for articles about Drosophila elbow B

Ashburner, M., Misra, S., Roote, J., Lewis, S. E., Blazej, R., Davis, T., Doyle, C., Galle, R., George, R. and Harris, N. (1999). An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region. Genetics 153: 179-219. 10471707

Athanikar, J. N., Sanchez, H. B. and Osborne, T. F. (1997). Promoter selective transcriptional synergy mediated by sterol regulatory element binding protein and Sp1: a critical role for the Btd domain of Sp1. Mol Cell Biol 17: 5193-5200. PubMed ID: 9271397

Brown, J. D., Dutta, S., Bharti, K., Bonner, R. F., Munson, P. J., Dawid, I. B., Akhtar, A. L., Onojafe, I. F., Alur, R. P., Gross, J. M., Hejtmancik, J. F., Jiao, X., Chan, W. Y. and Brooks, B. P. (2009). Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure. Proc Natl Acad Sci U S A 106: 1462-1467. PubMed ID: 19171890

Cifuentes, F. J. and Garcia-Bellido, A. (1997). Proximo-distal specification in the wing disc of Drosophila by the nubbin gene. Proc. Natl. Acad. Sci. 94(21): 11405-11410. PubMed Citation: 9326622

Cheah, P. Y., Meng, Y. B., Yang, X., Kimbrell, D., Ashburner, M. and Chia, W. (1994). The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures. Mol. Cell. Biol. 14: 1487-1499. 8289824

Davis, T., Trenear, J. and Ashburner, M. (1990). The molecular analysis of the el-noc complex of Drosophila melanogaster. Genetics 126: 105-119. 2121591

Davis, T., Ashburner, M., Johnson, G., Gubb, D. and Roote, J. (1997). Genetic and phenotypic analysis of the genes of the elbow-no-ocelli region of chromosome 2L of Drosophila melanogaster. Hereditas 126: 67-75. 9175495

Dorfman, R., et al. (2002). Elbow and Noc define a family of zinc finger proteins controlling morphogenesis of specific tracheal branches. Development 129: 3585-3596. 12117809

Gutiérrez, L., et al. (2012). The role of the histone H2A ubiquitinase Sce in Polycomb repression. Development 139(1): 117-27. PubMed Citation: 22096074

McDonald, E. C., Xie, B., Workman, M., Charlton-Perkins, M., Terrell, D. A., Reischl, J., Wimmer, E. A., Gebelein, B. A. and Cook, T. A. (2010). Separable transcriptional regulatory domains within Otd control photoreceptor terminal differentiation events. Dev Biol 347: 122-132. PubMed ID: 20732315

McGlinn, E., Richman, J. M., Metzis, V., Town, L., Butterfield, N. C., Wainwright, B. J. and Wicking, C. (2008). Expression of the NET family member Zfp503 is regulated by hedgehog and BMP signaling in the limb. Dev Dyn 237: 1172-1182. PubMed ID: 18351672

Pereira-Castro, I., Costa, A. M., Oliveira, M. J., Barbosa, I., Rocha, A. S., Azevedo, L. and da Costa, L. T. (2013). Characterization of human NLZ1/ZNF703 identifies conserved domains essential for proper subcellular localization and transcriptional repression. J Cell Biochem 114: 120-133. PubMed ID: 22886885

Runko, A. P. and Sagerstrom, C. G. (2004). Isolation of nlz2 and characterization of essential domains in Nlz family proteins. J Biol Chem 279: 11917-11925. PubMed ID: 14709556

Slorach, E. M., Chou, J. and Werb, Z. (2011). Zeppo1 is a novel metastasis promoter that represses E-cadherin expression and regulates p120-catenin isoform expression and localization. Genes Dev 25: 471-484. PubMed ID: 21317240

Tseng, A.-S. K. and Hariharan, I. K. (2002). An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye. Genetics 162: 229-243. 12242236

Weihe, U., et al. (2004). Proximodistal subdivision of Drosophila legs and wings: The elbow-no ocelli gene complex. Development 131: 767-774. 14757638

Wernet, M. F., Meier, K. M., Baumann-Klausener, F., Dorfman, R., Weihe, U., Labhart, T. and Desplan, C. (2014). Genetic dissection of photoreceptor subtype specification by the Drosophila melanogaster zinc finger proteins Elbow and No ocelli. PLoS Genet 10: e1004210. PubMed ID: 24625735