lozenge

Promoter

An enhancer element located within the second intron of the lozenge gene is responsible for its eye-specific expression. The existence of three independently isolated eye-specific alleles harboring deletions of intron II strongly supports the hypothesis that an eye-specific enhancer lies within this intron. In addition to the eye phenotype, lz null mutants have antenna and tarsal claw defects and defects in leg discs. In the eye-specific alleles, which are deleted for most of intron II, Lz continues to be expressed at wild-type levels in the antenna and leg discs. Furthermore, the antenna and tarsal claw phenotypes are not rescued in lz r1 by any of the transformation constructs mentioned above. Thus, the intron II enhancer is eye specific and is required solely to restrict Lz expression to the pool of undifferentiated cells posterior to the morphogenetic furrow in the eye disc, thereby allowing Lz to properly regulate the expression of multiple cell-specific transcription factors in the developing eye (Flores, 1998).

Lozenge is not itself a cell-specific transcription factor, rather it prepatterns the eye disc by positioning cell-specific factors in their appropriate locations. The developmental events in the eye disc can be separated into two stages of patterning. The first occurs within the morphogenetic furrow and leads to the formation of the 5-cell precluster, while the second occurs in the undifferentiated cells posterior to the furrow, which give rise to the remainder of the cells of the mature ommatidium. The first prepatterning event is controlled by transcription factors such as Atonal and Rough. Lz plays no role in this process since it is not expressed in the 5-cell precluster and lz mutants show no disruption in the patterning of these cells. In fact, the results presented here show that misexpression of Lz at the 5-cell stage leads to a re-programming of cell fates within the precluster. In contrast, proper expression of Lz is crucial for the second phase of prepatterning that completes the ommatidium by adding the last three photoreceptor cells and the non-neuronal cell types to the precluster. Other transcription factors that play a role in this process include the zinc-finger protein Tramtrack and the Ets domain proteins Yan and Pointed. The activity of these three proteins is modulated by the EGFR and Sevenless receptor tyrosine kinase signaling pathways. It seems likely that Lz may function combinatorially with these transcription factors in order to differentially regulate its target genes in different cells. It is interesting to note that the mammalian homolog of Pointed, Ets-1, directly binds to AML1; together, they cooperatively activate transcription of the T cell receptor (Flores, 1998 and references).

In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prospero enhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable Yan^ACT cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. The eye-specific enhancer of lozenge has been sequenced in three Drosophila species spanning 17 million years of evolution and complete conservation of three ETS consensus binding sites was found. lozenge expression increases as cells differentiate, and Yan^ACT blocks this upregulation at the level of transcription. Expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of Yan^ACT. These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. It is proposed that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development (Behan, 2002).

A genetic approach was used to examine Yan/Lz interactions; Yan is shown to temper lz expression. The degree of regulation is dependent upon the presence of the eye-specific enhancer. The complete conservation of three Ets binding sites across 17 million years of evolution is strong supporting evidence that this regulation is direct. At this time, however, the possibility that this regulation is indirect cannot be ruled out (Behan, 2002).

Separate inputs by Ets factors and Lz have been shown to be required for regulation of prospero and D-Pax2. The current data must be interpreted in this context. The lz^gal4 reporter system was used to show that hyperstable Yan^ACT is able to block lz expression at the level of transcription. The GMR and Sev ectopic expression systems have been used to tease out Yan control of lz apart from control of other genes (Behan, 2002).

A model is here proposed for prospero regulation by Prospero and Yan, with the added information that lz is also a target of Yan. Yan tempers Lz expression. In the undifferentiated cell, Yan represses prospero by directly binding to Ets sites. The transcriptional activator Pointed competes for the same DNA but with much less affinity. Lozenge transcription is tempered by Yan, but not entirely repressed. Upon activation of Egfr and Sevenless by their respective ligands Spitz and BOSS, Ras1 is stimulated. Ultimately, Yan and Pointed are phosphorylated, downstream of Ras1 but with opposite effects. Phosphorylated Yan is targeted for degradation. Phosphorylated Pointed binds DNA with a higher affinity. Yan repression of Lz is alleviated, and becomes upregulated by some other mechanism. Upregulated Lz binds with Pointed to mediate prospero transcription (Behan, 2002).

Transcriptional Regulation

Two major classes of cells observed within the Drosophila hematopoietic repertoire are plasmatocytes/macrophages and crystal cells. The transcription factor Lz (Lozenge), which resembles human AML1 (acute myeloid leukemia- 1) protein, is necessary for the development of crystal cells during embryonic and larval hematopoiesis. Another transcription factor, Gcm (glial cells missing), is required for plasmatocyte development. Misexpression of Gcm causes crystal cells to be transformed into plasmatocytes. The Drosophila GATA protein Srp (Serpent) is required for both Lz and Gcm expression and is necessary for the development of both classes of hemocytes, whereas Lz and Gcm are required in a lineage-specific manner. Given the similarities of Srp and Lz to mammalian GATA and AML1 proteins, observations in Drosophila are likely to have broad implications for understanding mammalian hematopoiesis and leukemias (Lebestky, 2000).

Hemocytes of the Drosophila embryo are derived from the head mesoderm. The hemocyte precursors express the GATA factor Srp and give rise to two classes of cells: plasmatocytes and crystal cells. Plasmatocytes spread throughout the endolymph and act as macrophages, whereas crystal cells contain crystalline inclusions and are involved in the melanization of pathogenic material in the hemolymph. These cells can be first recognized in the late embryo, where they form a cluster around the proventriculus. Crystal cells are made clearly visible by the Black cell (Bc) mutation, which causes premature melanization of the crystalline inclusions (Lebestky, 2000 and refereces therein).

In larval stages, hemocytes are produced from a separate organ called the lymph gland. Precursors of this gland first appear during embryogenesis in the dorsal mesoderm of the thoracic segments. Later, these precursors migrate dorsally, forming a tight cluster adjacent to the dorsal vessel, the larval circulatory organ. The larval lymph glands form a bilateral chain of cell clusters ('lobes') flanking the dorsal vessel. In the temperature-sensitive allele lz^ts1, crystal cells develop normally at 25°C. However, crystal cell development is completely blocked at 29°C. Consistent with earlier genetic analysis, crystal cells are missing in lz null mutant alleles. Plasmatocytes develop normally in number and pattern in lz null embryos. Temperature shifts of lz^ts1;Bc flies show that Lz function during stages 10 to 14 of embryogenesis is essential for crystal cell development. Crystal cells formed in the embryo do not persist into late larvae, and Lz function is continuously required during the late larval stages for further crystal cell development. The time scale for de novo crystal cell development in the larva is about 4.5 hours (Lebestky, 2000).

Lz is first detected in a small cluster of cells within the embryonic head mesoderm in a bilaterally symmetric pattern. Lz expression remains localized in bilateral clusters of 20 to 30 cells within the head mesoderm. At later stages, these crystal cell precursors (CCPs) form a loose cluster around the proventriculus. These cells have smooth, round morphology with large nuclei. The CCPs form a subset of the Srp-expressing hemocyte precursors (Lebestky, 2000).

Colocalization with a mitotic marker suggests that Lz-expressing cells can divide. Interestingly, not all of the daughter cells from these divisions will become crystal cells. This is inferred from the observation that lz-lacZ expression is also seen in a group of plasmatocytes that do not express lz mRNA or Lz protein. The expression of lz-lacZ in these cells is interpreted to be due to the long half-life of beta-galactosidase protein that is left over from the parent cell. This is also observed with additional, independent lz promoter fusions to lacZ. Thus, Lz is expressed in a small subset of hemocyte precursors that may undergo cell division. All crystal cells resulting from these precursors maintain Lz expression. The few daughter cells that will differentiate into plasmatocytes do not express Lz protein (Lebestky, 2000).

In the larval lymph gland, Lz expression is initiated in a small number of cells during the second larval instar. The number of cells expressing Lz steadily increases during the third larval instar, reaching 50 to 100 cells per lobe. Lz-expressing cells are scattered uniformly throughout the large, primary lobe of the lymph gland, whereas the smaller secondary lobes do not express Lz. Similar to the embryonic head mesoderm, all lymph gland cells express Srp, but only a small subset of them express Lz. Interestingly, the Lz-expressing cells appear to down-regulate Srp when compared to the surrounding non-Lz-expressing hemocyte precursors (Lebestky, 2000).

Immunolocalization studies of circulating hemocytes in third-instar larvae suggest that the expression of Lz protein is maintained in circulating crystal cells. Given that crystal cells are missing in lz mutants, this demonstrates an autonomous requirement for Lz in crystal cell development. As observed for embryonic hemocytes, Lz-expressing precursors give rise to all crystal cells and a small subset of plasmatocytes, as evidenced by morphology as well as expression of the plasmatocyte marker Croquemort. However, Lz protein is not observed in any circulating larval plasmatocytes (Lebestky, 2000).

An allele of srp (srp^neo45) specifically abolishes Srp expression in embryonic hemocytes. Because this allele also eliminates lz mRNA expression, Srp function is required for the expression of Lz. This finding establishes that srp functions upstream of lz during embryonic hematopoiesis. The lethality of srp precludes the analysis of Lz expression in larval lymph glands of srp mutants. However, as in the embryo, Srp is expressed earlier than Lz in the larval hemocyte precursors, which suggests that srp acts upstream of lz during both developmental stages (Lebestky, 2000).

The transcription factor Gcm promotes glial cell fate, and it also functions downstream of Srp in plasmatocyte differentiation. Lz expression is unaffected in gcm mutants. Gcm expression is initiated in a number of Srp-expressing hemocyte precursors, but Gcm is excluded from the CCPs. Consistent with their cell fate, the small subset of plasmatocytes derived from Lz-expressing progenitors do initiate Gcm expression. Gcm was misexpressed in the CCPs to assess whether exclusion of Gcm from these cells is essential for proper fate determination. This results in the transformation of CCPs into plasmatocytes. The converted cells exhibit morphological characteristics of plasmatocytes and express Croquemort. Moreover, in third-instar larvae, misexpression of Gcm in CCPs prevents the development of all crystal cells. These results suggest that the restricted expression of Gcm is required for the developmental program of embryonic plasmatocytes, and that its misexpression can override Lz-mediated crystal cell differentiation during both embryonic and larval hematopoiesis. The converse experiment of Lz misexpression in the entire hemocyte pool under the control of a heat shock promoter does not convert plasmatocytes into crystal cells. Vertebrate homologs of Gcm have been identified, but any role in hematopoiesis has not been investigated (Lebestky, 2000).

A model of Drosophila hematopoiesis is presented in which a pool of Srp-positive hemocyte precursors gives rise to a large population of Gcm-positive cells and a smaller subpopulation of Lz-positive cells. These results support a genetic hierarchy in which Srp, a Drosophila GATA factor, acts upstream of both Gcm and Lz, two mutually exclusive, lineage-specific transcription factors in hematopoiesis. Although the description of this hierarchy is incomplete in terms of the breadth of molecules involved, it does provide a theoretical framework for understanding how early hematopoietic progenitors in the embryo can differentiate and assume distinct cell fates (Lebestky, 2000).

Hairless induces cell death by downregulation of EGFR signalling activity

Overexpression of the Notch antagonist Hairless (H) during imaginal development in Drosophila is correlated with tissue loss and cell death. Together with the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP), H assembles a repression complex on Notch target genes, thereby downregulating Notch signalling activity. This study investigated the mechanisms underlying H-mediated cell death in S2 cell culture and in vivo during imaginal development in Drosophila. First, the domains within the H protein that are required for apoptosis induction in cell culture were mapped. These include the binding sites for the co-repressors, both of which are essential for H-mediated cell death during fly development. Hence, the underlying cause of H-mediated apoptosis seems to be a transcriptional downregulation of Notch target genes involved in cell survival. In a search for potential targets, transcriptional downregulation of rho-lacZ and EGFR signalling output were noted. Moreover, the EGFR antagonists lozenge, klumpfuss and argos were all activated upon H overexpression. This result conforms to the proapoptotic activity of H, as these factors are known to be involved in apoptosis induction. Together, the results indicate that H induces apoptosis by downregulation of EGFR signalling activity. This highlights the importance of a coordinated interplay of Notch and EGFR signalling pathways for cell survival during Drosophila development (Protzer, 2008).

This work allows two important conclusions: that overexpression of H induces cell-autonomous apoptosis, and that H requires the co-repressors Gro and CtBP for its proapoptotic activity. It is known that H assembles a repression complex together with the two co-repressors, resulting in transcriptional downregulation of Notch target genes. Hence, the ability of H to induce cell death is most likely a consequence of the repression of Notch target genes that are involved in cell survival. It is noted, however, that not every cell that receives an overdose of H dies. One simple explanation for this observation is that the only cells that die are those in which the relevant Notch target genes are normally active, as these cells require a Notch signal for survival. As H results in a repression of Notch activity, these cells would be driven into cell death, whereas those cells that do not depend on higher Notch levels for survival would be resistant to an H overdose. How is this effect of H realised at the molecular level? So far, it has not been possible to narrow down the analyses towards one target gene, the repression of which by the H repressor complex induces apoptosis. The most straightforward idea, repression of the anti-apoptotic protein Diap1, is not supported by the data. Instead, it was found that EGFR signalling activity is downregulated as a consequence of the upregulation of several negative regulators of EGFR (Protzer, 2008).

The existence of a densely woven network of genetic interactions between the EGFR and Notch signalling pathways is well established. This intensive cross-talk harmonises many developmental processes, such as proliferation, differentiation, cell fate specification, morphogenesis and programmed cell death. Still, the molecular basis of this genetic interplay remains largely obscure. So far, few molecular intersections between the Notch and EGFR pathways have been revealed. For example, EGFR signalling causes phosphorylation of the co-repressor Gro, thereby negatively modulating the transcriptional outputs of Notch signalling via the Enhancer of split [E(spl)] genes. Conversely, a myc-Gro complex was shown to inhibit EGFR signalling during neural development in the Drosophila embryo. Although mutual antagonism is probably the most prominent relationship in EGFR-Notch interactions, in some developmental situations both pathways cooperate to potentiate each other's signalling activities. One such example with regard to cell survival has been described in the retina of rugose mutant flies, where cell type-specific cell death could be reversed by an increase in Notch or EGFR signalling activity, indicating that both pathways adopt an anti-apoptotic function in this developmental context. Also, R7 photoreceptor cell specification requires the combined input of both Notch and EGFR signals. Moreover, Notch defines the scope of rho expression in the Drosophila embryo, thereby activating the EGFR pathway required for early ectodermal patterning. Also, during the development of mouse embryonic fibroblast, the Notch receptor-processing γ-secretase presenilin acts as a positive regulator of ERK basal level activity (Protzer, 2008).

A significant decrease was observed in the levels of activated MAPK (diP-ERK), which provides a good assessment of EGFR pathway activation, upon induction of H. Activated MAPK directly phosphorylates two transcription factors, Aop (Yan) and Pointed (Pntp2). Phosphorylation inactivates Aop, which in the unmodified state, represses EGFR targets. At the same time, phosphorylation activates Pointed, which then causes EGFR target gene transcription. As H is a well-defined transcriptional repressor of Notch target genes, it is most unlikely that it impedes EGFR activity at the level of phosphorylation. Moreover, it is not thought that H acts at the level of transcriptional regulation of EGFR target genes, even though combinatorial and antagonistic activities of the nuclear effectors of the EGFR and Notch signalling pathways have been described during eye development. Instead, the hypothesis is favored that H represses the transcription of EGFR activators, or might indirectly provoke the activation of EGFR repressors that affect, for example, the production of EGFR ligands or signal transduction (Protzer, 2008).

Rho activity is required for a timely and spatially regulated release of EGFR ligands. Accordingly, the expression of rho is highly dynamic during Drosophila development, and precedes the appearance of EGFR-induced activated MAPK. Hence, downregulation of rho by H would eventually result in lower levels of activated MAPK (diP-Erk). In contrast to other components of the EGFR signalling pathway, ectopic expression of rho results in EGFR activation in a wide range of tissues, indicating that Rho is an essential and limiting factor. So far, transcriptional control is the only known means of rho regulation. The complex array of enhancers regulating rho expression reflects the dynamic pattern of EGFR activation throughout Drosophila development (Protzer, 2008).

Interestingly, a transcriptional repression of rho-lacZ was observed in H gain-of-function clones that was dependent on the co-repressors Gro and CtBP. This effect might very well be direct, because it was shown previously that rho transcription is regulated by Su(H) in the neuroectoderm as well as in the gut of the Drosophila embryo. As mentioned above, Notch signalling has also been shown to regulate rho expression in the embryonic ectoderm. Moreover, during egg development, a band of Notch activity establishes the boundary between the two dorsal appendage tube cell types, whereby Notch levels are high in rho-expressing cells. In accordance with this, potential Su(H)-binding sites are present in the regulatory regions of rho1 and rho3, making a direct regulation of rho during eye development via the Notch-Su(H)-H complex very likely. It is noted, however, that the downregulation of rho-lacZ and of activated MAPK were focussed at the morphogenetic furrow, where primary photoreceptor cells are specified and ommatidia are founded. Regulation of rho by H would then be expected to interfere with photoreceptor formation rather than with cell survival, which is in agreement with the disturbed cellular architecture of H gain-of-function flies (Protzer, 2008).

Most interestingly, upon H overexpression, ectopic induction of lz, klu and aos was observed. All three genes are known to be involved in cell death induction during pupal eye development. There it was shown that the Runx protein Lz binds to the regulatory regions of klu and aos, resulting in the direct transcriptional activation of these target genes. Therefore, one might speculate that H executes its effect on klu and aos activity via the activation of lz. Moreover, as klu and aos are well-known inhibitors of EGFR signalling activity, this in itself suggests that H impedes EGFR signalling activity via these factors. This interpretation helps to explain why aos expression is induced in H gain-of-function clones, although it is well known that aos is triggered by EGFR signalling, thereby forming an inhibitory loop that acts on EGFR activity. The high levels of Lz still activate aos in H gain-of-function clones, keeping activity of the EGFR pathway low. Alternatively, aos and klu levels might be increased as a consequence of the downregulation, by H, of an as yet unknown repressor. Since H behaves as a kind of 'multi-adaptor protein', which not only recruits the transcriptional silencers Gro and CtBP to Notch targets but also binds other proteins such as Pros26.4, it is also possible that H interacts with positive regulators of lz, klu and aos (Protzer, 2008).

However, a model is favored whereby H influences EGFR signalling activity on two levels. On the one hand, through transcriptional repression of rho, H causes a loss of EGFR signalling output that interferes with cell specification. On the other hand, by interfering with their repressor(s), H relieves the restriction on lz, klu and aos expression, causing their accumulation. In consequence, the survival/death balance is tipped towards apoptosis in those cells that are susceptible to the effects of a lowered EGFR signal. Those cells that do not depend on high Notch and EGFR activity levels for survival would be resistant to an H overdose (Protzer, 2008).

Finally, one can envisage that a downregulation of Notch and EGFR signalling activities, resulting from the overexpression of H, might leave a cell in a state of 'uncertainty' that does not allow any further differentiation towards a certain cell type, but leaves the cell vulnerable to the apoptotic programme (Protzer, 2008).

The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila.

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).

A genome-wide RNA interference screen identifies a differential role of the mediator CDK8 module subunits for GATA/ RUNX-activated transcription in Drosophila

Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. This study used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory "CDK8 module," composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, the data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. It is thus proposed that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, it is suggested that the set of conserved factors identified in this study may regulate GATA/RUNX activity in mammals (Gobert, 2010).

During development, a combination of general and lineage-specific transcription factors integrate different regulatory inputs at the transcriptional levels to unfold the proper gene expression program. The identification of the complete panel of genes that participate in the regulation of the activity of these transcription factors is critical to understand the fine-tuning of transcription that underlies cellular differentiation. In this study, a genome-wide RNAi screen was conducted to uncover regulators of the activity of the GATA/RUNX complex Srp/Lz. This approach highlighted the function of the Mediator complex in Srp/Lz-induced transcriptional activation. Moreover, it was found that, within the Mediator CDK8 module, Med12 and Med13 act independently of CycC and Cdk8 to promote Srp/Lz-dependent transactivation and blood cell differentiation (Gobert, 2010).

The activity of GATA and RUNX transcription factors has been shown to be regulated by interaction with several factors, such as the coactivator CBP/p300 or the corepressors HDAC and Sin3A. However, proper transcriptional regulation relies on the coordinated action of several transcription factors binding a particular cis-regulatory element. Notably, GATA and RUNX factor have been shown to cooperate in both mammals and Drosophila to regulate the expression of specific target genes. Hence, this study used Srp/Lz cooperation as a paradigm to identify putative coregulators of GATA/RUNX activity. Among the genes that were identified, five (CKD9, SIN3A, MED1, enok homolog MYST3/MOZ, and pnt homolog ETS1) have been linked previously to GATA and/or RUNX activity in mammals, and four (Pcf11, CtBP, med13, and Sin3A) have been linked to crystal cell development in flies. This brings strong support to the conclusion that the cell-based assay is suitable to identify genuine modulators of Srp/Lz activity and, more generally, of GATA/RUNX factors. However, further work will be required to discriminate between factors affecting GATA/RUNX interplay specifically or impinging also on either GATA or RUNX activity. Along this line, the results suggest that the three MED core modules but not the CDK8 regulatory module participate in Srp-induced transactivation. Importantly, too, 117 (90%) of the genes identified in the screen have well-conserved human homologs, suggesting they may regulate GATA/RUNX activity in humans. Actually, this sharp bias toward conserved genes underscores the fact that cell-based assays in Drosophila can serve as a powerful system to identify and characterize genes that may play similar roles in humans. Moreover, some homologs of Srp/Lz modifiers that were identified have been implicated in human diseases. These notably include MLF1, which is translocated in t(3;5)(q25.1;q34)-associated AML and whose Drosophila homolog is a target of Srp/Lz expressed in the crystal cells, as well as DDX10, which is translocated in inv(11)(p15q22)-associated AML. Whether these genes participate in GATA and/or RUNX function in normal or pathological situations in humans remains to be determined (Gobert, 2010).

The data show that the Mediator complex plays a central role in Srp/Lz-induced transactivation. Studies of yeast and metazoa highlighted the critical role of Mediator in both transcriptional activation and repression and showed that different Mediator subunits are required for the regulation of specific sets of genes or developmental processes. In addition, different transcription factors interact directly with specific Mediator subunits. Hence, the prevailing view is that different transcription factors depend on particular target proteins of the Mediator complex to regulate transcription. However, this study found that 20 of the 30 Mediator subunits were implicated as positive coregulators of Srp/Lz. Although some Mediator subunits, notably in the head module, play a global role in transcription, a general defect in transcription is unlikely to account for the observed decrease in Srp/Lz activity under the RNAi conditions, since no significant changes were observed in srp and Lz expression levels, except with Med19, a component of the head module, whose depletion decreased Lz levels. It is thus proposed that the integration of Srp/Lz transcriptional output requires the coordinated action of the different Mediator modules. However, it cannot be exclude that some of the Mediator subunits that were not identified in the screen may actually be dispensable for Srp/Lz activity (Gobert, 2010).

Remarkably, the CDK8 module, which is generally considered an accessory repressor module, was also required as a coactivator of Srp/Lz. Furthermore, in line with recent results revealing that all the functions of the CDK8 module do not rely on the CycC/Cdk8 pair, strong evidence is provided that only Med12/Med13 are required for the activation of Srp/Lz target genes in cell culture and in vivo. While different molecular mechanisms of repression by Cdk8/CycC and Med12/Med13 have been described, how Med12/Med13 may promote transcription remains elusive. These subunits may serve as an anchor to recruit the Mediator complex, as they have been shown to bind to Pygopus or ß-catenin to promote Wnt signaling and to Gli3 to inhibit Shh signaling. Accordingly, it was found that Srp and Lz interact with Med12 and Med13. However, this interaction could be due to another Mediator subunit required for Srp/Lz-induced transactivation. Alternatively, Med12/Med13 may be required for the proper folding of the Mediator complex to promote its interaction either with Srp/Lz or with downstream components of the transcriptional initiation machinery (Gobert, 2010).

In vivo, analysis of CDK8 module subunits shows that, reminiscent of what has been observed in larval imaginal discs, CycC/Cdk8 and Med12/Med13 have both common and specific functions during crystal cell development. Indeed, in the embryo, mutations in any of the four CDK8 module components resulted in a similar reduction in the absolute number of Lz⁺ blood cells and, concomitantly, of differentiated crystal cells, indicating that the whole CDK8 module controls the emergence of the crystal cell lineage. While the signaling that induces lz expression in the prohemocytes remains unknown, it was shown that the transcription factor Glial cell missing (Gcm) and the Friend of GATA corepressor U-shaped (Ush) oppose crystal cell fate choice. Both factors are expressed in the prohemocytes and interfere with lz expression to limit the number of crystal cells. Thus, loss of CDK8 module activity may impair crystal cell lineage emergence either by decreasing lz induction or by enhancing gcm or ush function (Gobert, 2010).

Similarly, it was found that targeted downregulation of Med12, CycC, or Cdk8 in the crystal cell lineage by RNAi after the onset of lz expression induced a cell-autonomous decrease in the absolute number of Lz⁺ larval blood cells. Hence, it is likely that the whole CDK8 module also controls the maintenance or the proliferation of the Lz⁺ cells during larval life. Recently, Wg signaling was shown to promote Lz⁺ larval blood cell proliferation. Interestingly, the CDK8 module participates in Wnt signaling. However, its coactivating function seemed to rely only on Med12/Med13 in Drosophila, whereas it depended on Cdk8/CycC in humans. Whether the CDK8 module regulates Lz⁺ cell number in response to Wg signaling or to another unknown pathway remains to be determined (Gobert, 2010).

In addition, the observation that only Med12 or Med13 downregulation caused a drop in the proportion of differentiated Lz⁺ cells in the larva strongly suggest that Med12/Med13 participates in crystal cell differentiation independently of the CycC/Cdk8 pair. In light of the results in cell culture, the in vivo data support an essential and direct function for Med12 and Med13 in the activation of the crystal cell differentiation program by Srp/Lz independently of CycC and Cdk8. All together, these data underline the functional flexibility of the CDK8 module, which appears to be reiteratively and specifically used at different stages of crystal cell development (Gobert, 2010).

In conclusion, it is anticipated that the results presented in this study lay the foundation for future investigations aiming at understanding the different levels of regulation of GATA and RUNX transcription factor activity not only in Drosophila but also in other species (Gobert, 2010).

The Hippo pathway regulates hematopoiesis in Drosophila melanogaster

The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. This study defines the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion (Milton, 2014).

Targets of Activity

Lozenge was initially identified by mutation caused by a P-element insertion in the X chromosome. Because the P-element contained two copies of the sevenless enhancer, DNA adjacent to the site of insertion was expressed in cells eye disc precursor cells normally expressing sevenless (R7, the R3/R4 pair and cone cell precursors). The P-element caused a dominant mutant phenotype resembling loss-of-function mutations of seven-up. Consequently the dominant mutant was called Sprite. In Sprite/+ heterozygotes, 72% of the ommatidia show transformation of R4 into an R7 cell, and in 10% of ommatidia, both R3 and R4 become converted. The phenotype is more extreme in Sprite mutant homozygotes. One explanation for the mutant phenotype caused by the insertion is that the adjacent DNA codes for a protein that represses seven-up. A null mutation was used to test whether lozenge regulates seven-up. Whereas svp is normally expressed in the R1/R6 pair and the R3/R4 pair, in lozenge mutant flies, svp is expressed in R7 and the four cone cell precursors as well. It has been concluded that LZ negatively regulates svp in R7 cells, and in cone cells. In the absence of lozenge each of these cells develop an R7 fate. This transformation is partially dependent on the functioning of sevenless (Daga, 1996).

Lozenge is implicated in the regulation of Bar proteins, specifically required to specify R1/R6 cell fate. The expression of Bar in R1/R6 cells is dramatically reduced but not completely eliminated in lz mutants. The antibody used to detect Bar is raised against BarH1. Upon lz overexpression, Bar expression is no longer restricted to R1/R6, but ectopically staining cells are consistently detected in the developing cluster (Daga, 1996).

lozenge mutants do not express the two Bar genes, and the enhancer-trap O32 (associated with an unknown gene specific to cells R3/4 and R7) is expressed in too many cells. Thus the defective recruitment that occurs in lozenge mutants can be attributed to abnormalities in the expression of genes like Bar, the gene marked by O32, and seven-up, which are essential for establishing the correct cell fate for the final three photoreceptor cells, R1, R6 and R7. seven-up is derepressed in R7 cells in lozenge mutants. The derepression of seven-up is reminiscent of the derepression of svp in rough mutants. rough normally represses svp in R3/R4. Thus Lozenge both actively represses some genes and activates others (Crew, 1997).

A new Drosophila Pax gene, sparkling (spa), implicated in eye development, has been isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system, and in cone, primary pigment, and bristle cells of larval and pupal eye discs. Transcripts are expressed in the posterior portion of the eye disc, with the anterior boundary of expression lagging clearly behind the morphogenetic furrow. In spa(pol) mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Because Spa is not expressed in R7 cells, its expression in newly recruited cone cells distinguishes their fate from that of R7 cells. Lozenge may be the transcription factor whose synthesis would have to precede that of Spa, which is required for the specification of the R7 equivalence group, including R1/R6, R7 and the cone cells. Lozenge helps define the R7 equivalence group by repressing seven-up (Fu, 1997).

Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. Thus Spa exerts at least part of its control of primary pigment cell development through its regulation of Bar expression. Bar is also expressed in R1 and R6 precuror cells, where Lozenge rather than Spa is one of its activators. It is suggested that close functional analogies exist between Spa and Pax2 in the development of the insect and vertebrate eye. In the absence in Pax2, the optic stalk epithelium develops into pigmented retina and fails to proliferate and differentiate into glial cells, which populate the optic nerve and are essential for guidance of the retinal axons. Thus the cone cell in Drosophila might be considered as a kind of neuronal support, or glial --a cell that may have evolved from a more primitive ancestral glial cell. In favor of such a hypothesis, it is observed that spa is expressed in glial cells in the developing PNS (Fu, 1997).

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Together with the transcription factor Lozenge, the nuclear effectors of the Egfr and Notch signaling pathways directly regulate D-Pax2 (shaven) transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of shaven expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. It is expected that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication (Flores, 2000).

shaven is the Drosophila homolog of the vertebrate Pax2 gene. This locus is represented by at least two classes of mutant alleles: shaven (sv) and sparkling (spa). spa mutants show cone cell defects resulting from mutations in the fourth intron of the gene, which have led to the identification of a 926 bp SpeI fragment within this intron that includes the eye-specific enhancer (Flores, 2000).

spa alleles give an enhancement of lz eye phenotypes. Two new spa alleles were isolated as enhancers of the temperature-sensitive lz allele, lz^ts1. The strongest eye-specific allele of shaven, spa^pol, which is not transcribed in cone cell precursors, also enhances lz^ts1. Shaven is not expressed in cone cell precursors of lz mutants, which suggests that Lz regulates shaven expression. There are three Lz/Runt domain (RD) binding sites (5'-RACCRCA-3', where R = purine) in the shaven eye-specific enhancer (RDI-RDIII). To determine whether these sites are required for proper shaven expression, a series of smaller enhancer fragments derived from the SpeI fragment was combined with the shaven promoter and the transcribed region from which introns 1-8 had been removed. This combination was tested as transgenes for the ability to rescue spa^pol mutants. There is no loss in rescue efficiency if the truncation does not eliminate any of the three RD binding sites. However, if RDI is deleted, the rescue efficiency and Shaven expression in cone cell precursors are considerably reduced, and rescue cannot be improved by two copies of the transgene. Similarly, when both RDII and RDIII are removed, the rescue efficiency and expression in cone cell precursors are clearly reduced, but rescue to wild type is achieved with two copies of the transgene. These experiments suggest that the RD binding sites are essential for the control of shaven transcription and that omission of RDI has more severe effects than that of RDII and RDIII (Flores, 2000).

Electrophoretic mobility-shift assays (EMSA) demonstrate that in vitro translated Lz can bind specifically to each of the RD binding sites in the minimal eye specific enhancer (SME). As an in vivo correlate to these experiments, the three RD sites were mutated in the context of a transgenic shaven rescue construct. Mutation of all three RD binding sites (mRDx3) causes a failure to rescue the spa^pol eye phenotype and Shaven expression in cone cell precursors. Together, the in vitro and in vivo data demonstrate that Lz directly regulates shaven transcription through the RD binding sites in the SME (Flores, 2000).

The results described so far suggest that shaven expression is limited to cells which (1) express Lz; (2) receive a sufficiently strong Egfr signal to both alleviate Yan-imposed repression and stimulate PntP2 activation, and (3) receive a N signal able to stimulate Su(H) activation. The tripartite control of shaven expression in the cone cell precursors requires that they receive all three inputs at the proper time in their development. Lz expression in cone cell precursors has been documented. Consistent with their reception of the Egfr signal, activated MAPK is detected in cone cell precursors at the time when they initiate Shaven expression. Dl is expressed in developing photoreceptor clusters at the time when the cone cell precursors express Shaven. Thus, the neuronal clusters signal through an inductive Dl/N pathway to activate shaven expression in the neighboring cone cell precursors. These results suggest that, in addition to expressing Lz, the cone cell precursors receive the Egfr and N signals at the time of fate acquisition and Shaven expression. Presumably, at least one of these three activation mechanisms is lacking in cells that do not express shaven. This hypothesis was tested through genetic manipulation of the system (Flores, 2000).

Undifferentiated cells immediately posterior to the furrow receive the N signal and express Lz, but they do not express Shaven. It is hypothesized that the absence of Shaven expression in these cells is caused by a lack of the Egfr signal. This hypothesis is consistent with the observation that Egfr signaling causes these cells to differentiate. Indeed, Shaven is ectopically expressed in undifferentiated cells that express an activated form of Egfr. Loss-of-function yan^e2D/yan^pokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SME^mETSx6-lacZ, in which the six ETS sites in the SME are mutated, ß-galactosidase is also expressed in undifferentiated cells. Presumably, relief of Yan repression is sufficient to activate some shaven in undifferentiated cells. In SME^mETS(1,6)-lacZ,where the Pnt binding sites are eliminated but two of the Yan binding sites are still intact, there is no expression of ß-galactosidase in the undifferentiated cells. These results suggest that while the undifferentiated cells posterior to the furrow express Lz and receive the N signal, they fail to express Shaven because they do not receive the Egfr signal and are therefore unable to relieve the Yan-imposed repression of shaven (Flores, 2000).

The R7 precursors express Lz and receive RTK signals, yet they do not express Shaven. It is hypothesized that this is due to the lack of the N signal at the time of R7 determination. Indeed, expression of an activated form of N (N^act), leads to ectopic Shaven expression in R7 precursors, which suggests that Shaven is not normally expressed in R7 because this cell does not receive the N signal. These results are consistent with the previous observation that the R7 cell loses its neuronal characteristics upon expression of N^act (Flores, 2000).

Thus far, this study has focused on cells that express Lz. However, the regulation of shaven expression can also be tested in cells that lack Lz, such as the R3/R4 precursors. These cells receive the Egfr signal but receive the N signal after their initial fate specification, during ommatidial rotation. Ectopic expression of either Lz or N^act in the R3/R4 precursors fails to activate shaven expression in these cells. However, when Lz and N^act are coexpressed in the R3/R4 precursors, Shaven is expressed in these cells. These results demonstrate that the lack of both N signaling and Lz during the proper time window prevents R3/R4 cells from expressing shaven (Flores, 2000).

lozenge (lz) functions in eye, antennal, and tarsal claw development. In leg and antennal discs, the pattern of lz expression strongly resembles that of absent MD neurons and olfactory sensilla (amos), although amos expression begins later than lz. These observations suggest that lz might regulate amos expression during the process leading to the formation of basiconic and trichoid SOPs in the antenna and perhaps in the tarsal claw. Therefore, changes in the expression pattern of amos in lz mutants were sought. Strong lz alleles (including lz¹, lz³, and lz³⁴) almost completely lack sensilla basiconica and exhibit up to a 50% reduction in sensilla trichodea. The number of sensilla coeloconica is reported to be unaffected. In these strong alleles, AMOS mRNA is absent from the middle of all three antennal bands. For band 3, the affected region corresponds to the area fated to form sensilla basiconica SOPs. The correlation between this loss of amos expression and the loss of sensilla basiconica is therefore consistent with a requirement for amos in sensillum basiconica formation. In addition, it may be deduced that the middle regions of the other two bands give rise to those sensilla trichodea that are missing in strong lz mutants. Conversely, lz-independent sensilla trichodea may arise from the lz-independent tips of the amos-expressing bands. Topologically, SOPs from the band tips will end up on the lateral edge of the antenna after metamorphosis, which is where the sensilla trichodea are concentrated. Interestingly, comparison with the ato expression pattern suggests that amos is also expressed in regions of sensillum coeloconica formation in bands 1 and 2. Since these SOPs are not lost in lz mutants, the loss of amos expression from the middle of these regions provides evidence that amos is not required at least for many sensilla coeloconica (Goulding, 2000).

In weaker lz alleles (such as lz^g), amos expression appears patchy but spatially normal, suggesting that SOP selection itself is not strongly altered. This would be consistent with observation that the major phenotype of weak lz alleles is one of subtype transformation from basiconic to trichoid fate rather than sensillum loss. This is postulated to result from a role of lz in subtype specification, such that higher levels are required for SOPs to take on basiconic fate while lower levels are sufficient for trichoid fate. It was determined in a complementary experiment whether ectopic lz expression could induce ectopic amos expression. When ubiquitous lz expression is activated in pupae containing a heatshock-inducible lz construct (hs-lz). lz misexpression also results in ectopic amos expression in pupal wings and legs. These experiments show that lz is both necessary for much of amos's expression pattern and also sufficient to drive ectopic amos activation in many other locations (Goulding, 2000).

To investigate further the relationship between lz and amos, it was determined whether amos gene dosage reduction would modify the number of sensilla formed in lz mutants. In the intermediate allele, lz^g, the number of basiconica is reduced to 28% of wild-type. Removing one copy of the chromosomal region containing amos results in a further 70% reduction in this number. The number of sensilla trichodea is unaltered, probably because these are not affected in this intermediate lz allele. To gauge the effect on sensilla trichodea, amos's modification of a strong lz allele, lz³, was examined. In addition to a total lack of sensilla basiconica, lz³ exhibits a strong reduction of sensilla trichodea. In the absence of one copy of amos, sensilla trichodea are reduced by a further 54% in lz³, to 24% of wild-type (Goulding, 2000).

From the genetic and expression analyses, it has been concluded that amos transcription is partly downstream of lz and that its loss of expression may explain the loss of sensilla basiconica and trichodea in lz mutants. It was therefore tested whether experimentally induced amos expression could rescue the loss of sensilla basiconica in strong lz mutants. Using hsGal4 as a driver, UAS-amos was misexpressed in lz³ pupal antennae. Such misexpression results in a significant recovery of sensilla basiconica when compared with lz³ alone. This rescue is still far short of wild-type levels, perhaps because amos is not optimally expressed using hsGal4. Alternatively, lz might need to activate other genes required for basiconic fate in addition to amos (i.e., amos alone cannot replace all the functions of lz). Significantly, ato is unable to direct any rescue under the same conditions, even though the number of sensilla coeloconica is increased. Therefore, amos, but not ato, can partially bypass the requirement for lz. Interestingly, many of the rescued basiconica were located in the lateral region of the antenna (Goulding, 2000).

Since the expression of ato overlaps with the inner two bands of amos expression, it is possible that one gene may be dependent on the other. However, no defect in amos expression was observed in ato mutant antennal discs. Furthermore, ato expression is not dependent on lz, and therefore by inference ato does not depend on amos, at least not in the medial antennal region. It is concluded that the two olfactory proneural genes, ato and amos, are largely independent of each other. Furthermore, ato shows no interaction with lz. Thus, lz³; ato¹/ato¹ double mutants exhibit a complete absence of sensilla basiconica and coeloconica, as expected from the loss of lz and ato functions, respectively. However, the number of sensilla trichodea is not reduced below that observed in lz³ mutant flies. This suggests that there is no redundancy between lz and ato in formation of the remaining sensilla trichodea, which are instead likely to require the lz-independent part of amos's expression (Goulding, 2000).

According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus, indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).

In contrast to ato broad expression just anterior to the furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential for uncommitted cells to acquire ommatidial cell fate or the ability to express ommatidial marker genes. Consistent with this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).

This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow. Uncommitted cells just posterior to the morphogenetic furrow are presumed to acquire ro expression potential at the earliest stage of the model (stage 1). In stage 2, R3/R4 precursors expressing ro acquire svp expression potential. svp expression in wild type R3/R4 precursors along with Ras1 activation-dependent svp misexpression in uncommitted cells is assumed to be not only positively regulated by the concerted action of Ras1 signaling and Dac and Eya but also negatively regulated by the protein product of the prepattern gene, lz. R1/R6 photoreceptors are recruited into ommatidia between stages 2 and 3. R1/R6 fate is previously shown specified by dual Bar homeobox genes, BarH1 and BarH2, whose expression is positively regulated by the cell-autonomous function of lz and svp. Consistent with this, in the putative R1/R6 arising area (around row 6), considerable svp expression occurs even in the presence of Lz. svp expression is regulated by Dac and Eya, so that normal Bar expression or R1/R6 fate eventually comes under the control of putative eye prepattern genes Lz, Dac, and Eya.

In stage 3, which may correspond to R7 and cone cell formation stages, pros is positively regulated through the concerted action of Ras1 signaling and prepattern gene lz. Lz and Pnt both been shown to directly bind to the pros promoter/enhancer region and pros expression occurs only when Pnt and Lz have bound simultaneously to the pros enhancer/promoter. In wild type, Lz is expressed prior to pros expression in rows 4-7; subsequent to Ras1 ubiquitous activation, pros expression takes place in this region. Thus, in all wild-type progenitors situated in rows 4-7, Lz may bind to the pros enhancer/promoter so as to impart progenitor cells with pros expression potential. In wild type, pros expression first becomes apparent in R7 precursors at row 8. The absence of pros expression in rows 4-7 in wild type may then be accounted for by the possible absence of Ras1 signal activity. This possibly may be an oversimplification since, for instance, this does not explain why pros is repressed in R1/R6 photoreceptors which also arise from Lz-positive progenitor cells, or why pros is not induced efficiently on Ras1 activation in row 10 and more posterior regions (Hayashi, 2001).

The former might be caused by the absence of the strong N signal in R1/R6 precursors, but another unknown mechanism may be required to explain the latter. Therefore, more remains to be discovered about pros regulation, but what is known is nonetheless an excellent model for understanding the manner in which cooperative action of prepattern genes and differentiation signals give rise to specific cell fates from common progenitors (Hayashi, 2001).

In the developing Drosophila eye, differentiation of undetermined cells is triggered by Ras1 activation but their ultimate fate is determined by individual developmental potential. Presently available data suggest that developmental potential is important in the neurogenesis of vertebrates and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along the dorso-ventral axis in response to graded Sonic Hedgehog signals and this presages their future fates. Subdivision of originally equivalent neural progenitors through the action of prepattern genes may accordingly be a general strategy by which diversified cell types are produced through neurogenesis (Hayashi, 2001).

Why are both Lozenge and Pointed required for prospero regulation? It is hypothesized that Lz, functioning as a transcription factor, is involved directly in the Ets developmental switch, acting as a cofactor to enhance the ability of Pointed to compete with Yan for Ets sites. What follows supports this argument. The developmental potential of R7 in the presence of one dose of sev-yan^ACT has been analyzed. In this mutant background, Pointed is phosphorylated normally by MAP kinase, but is unable to compete with the mutant hyperstable Yan. The result is that Prospero expression is never upregulated: Runt expression is not turned on, and R7 differentiation fails. Coexpression of GMR-[lz-c3.5] rescues Prospero expression. Furthermore, the ectopic R7 cells that develop in the GMR-[lz-c3.5] background follow their normal developmental pathway. Unlike the native R7 precursors, these ectopic R7 precursors express both upregulated Prospero and Runt. This may indicate a difference in the level of expression of the sev-yan^ACT transgene between the two cell types, or some other cell-specific factors involved in regulation. The results in both the endogenous and ectopic R7 cells indicate that the presence of Lz effects a change in the dynamic between Yan and Pointed (Behan, 2002).

Although this could be accomplished by a number of mechanisms, the hypothesis is favored that Lz induces a change in the ability of Pointed to bind to DNA. This is based on a mammalian paradigm. The mammalian homologs of Lz and Pointed are RUNX1 and Ets-1. Lz is 71% identical to RUNX1 in its homologous domains. Pointed is 95% identical to Ets-1 in the Ets DNA binding domain; Pointed and Ets-1 proteins are functionally homologous and can replace each other in vitro and in vivo. It has been shown that RUNX1 and Ets-1 bind cooperatively to separate, but nearby, DNA sites on the T cell receptor, and that this cooperativity can exist even when these sites are as far apart as 33 base pairs. Notably, RUNX1 and Ets-1 can not substitute for each other. Both inputs are required for stable ternary complex development. The presence of RUNX1 enhances Ets-1 DNA binding affinity by as much as 20 times in vitro. Regions of RUNX1 and Ets-1 outside of the DNA binding domains that are necessary for cooperative DNA binding, and similar regions exist in both Lz and Pointed proteins. Furthermore, it has been speculated that this type of cooperativity may exist in Drosophila eye development (Behan, 2002 and references therein).

Strikingly, in the prospero enhancer one Ets binding site is only 7 base pairs away from a Lz binding site. The genetic results reported in this study are consistent with Lz and Pointed acting cooperatively on the prospero enhancer. The double input of Lz and Pointed effectively competes with Yan^ACT. Clearly a dynamic exists between the three factors Lz, Yan and Pointed. Work in flies and mammals has shown that this dynamic is influenced by phosphorylation, competition, transcriptional regulation and cofactor availability (Behan, 2002).

In vivo analysis of a developmental circuit for direct transcriptional activation and repression in the same cell by a Runx protein

Runx proteins have been implicated in acute myeloid leukemia, cleidocranial dysplasia, and stomach cancer. These proteins control key developmental processes in which they function as both transcriptional activators and repressors. How these opposing regulatory modes can be accomplished in the in vivo context of a cell has not been clear. The developing cone cell in the Drosophila visual system was used to elucidate the mechanism of positive and negative regulation by the Runx protein Lozenge (Lz). A regulatory circuit is described in which Lz causes transcriptional activation of the homeodomain protein Cut, which can then stabilize a Lz repressor complex in the same cell. Whether a gene is activated or repressed is determined by whether the Lz activator or the repressor complex binds to its upstream sequence. This study provides a mechanistic basis for the dual function of Runx proteins that is likely to be conserved in mammalian systems (Canon, 2003).

To understand negative regulation by the Lz protein, regulation of the deadpan (dpn) gene was investigated. In wild-type eyes, Dpn is expressed in photoreceptors R3/R4 and R7. In lz mutants, dpn is also ectopically activated in cone cells, suggesting that Lz either directly or indirectly represses dpn in these cells. Dpn was therefore used as a marker to investigate negative regulation by Lz (Canon, 2003).

The presence of two perfect consensus Runx protein-binding sites (5'-RACCRCA-3') upstream of the dpn-coding region suggested possible direct negative regulation by Lz. Gel-shift experiments showed that Lz specifically binds to both sites. To determine whether these sequences are required for proper dpn regulation, lacZ reporter constructs were made driven by dpn upstream and intronic fragments, and these were transformed into flies. A 4667-bp upstream fragment plus intron I (227 bp) caused expression of lacZ in R3/R4 and R7 faithfully recapitulating the pattern of wild-type dpn expression in the eye. This site is therefore referred to as the dpn eye enhancer (DEE). When the two Lz-binding sites (LBS) in the DEE were mutated (to 5'-RAAARCA-3'; DEE-MutLBS), lacZ expression was also seen in cone cells. Therefore, lack of Lz binding to this enhancer will cause its derepression in cone cells, establishing that Lz directly represses transcription of dpn in cone cells (Canon, 2003).

Like all Runx proteins, Lz contains the conserved C-terminal pentapeptide motif VWRPY, which binds the global corepressor Groucho (Gro). Gro does not bind DNA on its own, but functions as a repressor for sequence-specific DNA-binding factors. Gro is expressed ubiquitously and has early pleiotropic roles in eye development, such as mediating repression by bHLH proteins, making it difficult to study possible involvement of Gro in cone cell development in loss-of-function mutant clones in the eye. Therefore the Gro-interaction domain at the C terminus of Lz was altered from VWRPY to VWEAA, a change that abrogates Gro binding to bHLH proteins. Lz-EAA protein was then expressed under the control of the endogenous eye-specific lz enhancer and its ability to repress dpn was tested in vivo. Whereas a wild-type lz⁺ transgene efficiently represses dpn in cone cells, Lz-EAA was unable to keep dpn off in these same cells. Neuronal differentiation occurs normally in both cases as determined by the neural marker Elav. This shows that the C terminus of Lz, a known Gro-interaction domain, is required for Lz-mediated repression of dpn. The activation function of Lz-EAA, as determined by its ability to activate D-Pax2 expression, remains intact. Therefore, Gro mediates repression by Lz as it does for other Runx proteins. It still remained unclear, however, why in the same cell Lz represses dpn transcription while it directly activates D-Pax2. Clearly, the presence of Gro alone does not cause Lz to become a dedicated repressor in the cone cell (Canon, 2003).

Hairy-related proteins constitutively bind Gro through the conserved sequence WRPW, and function as dedicated repressors. To further address the significance of the C terminus of Lz, the C-terminal amino acids of Lz were changed from WRPY to WRPW to resemble Hairy-related repressors. As a correlate, a Lz-VP16 fusion was made, with the potent activation domain of VP16 fused onto the C terminus of Lz. The ability of Lz-WRPW and Lz-VP16 to regulate Lz targets was tested in vivo. Lz-WRPW efficiently represses dpn in cone cells like the wild-type Lz⁺ but was unable to activate expression of D-Pax2. In contrast, Lz-VP16 failed to repress dpn in cone cells but effectively activates D-Pax2 in cone cells. Therefore, Lz-WRPW functions as a dedicated repressor, and Lz-VP16 as a constitutive activator. These results suggest that Runx-Gro interactions are regulated, because wild-type Runx proteins function as both activators and repressors (Canon, 2003).

The Lz-binding sites in the dpn and D-Pax2 enhancers were compared and distinct differences were found in the neighboring sequences. In the dpn enhancer, each Lz-binding site is followed by AT-rich sequences that are similar to each other (5'-AATCTTT-3' and 5'-TAATCTT-3'). In contrast, sequences near the three Lz-binding sites in the D-Pax2 enhancer, a positively regulated enhancer, are dissimilar and are not as rich in AT sequences. To determine if the difference in these sequences influences the mode of Lz regulation, both AT-rich sequences in the DEE were replaced with the corresponding sequence (GCTG) from the D-Pax2 enhancer. When transformed into flies, the resulting DEE-MutAT enhancer could not support repression of the reporter gene in cone cells. This was the same phenotype that was seen when the Lz-binding sites were mutated in the DEE. In this case, however, alteration of the AT-rich sites had no effect on Lz binding. Therefore, disruption of the AT-rich sequences in the DEE prevents repression of this enhancer by a mechanism that is independent of Lz binding. It is concluded that a cofactor binds to the AT-rich regions next to the Lz-binding sites and is essential for mediating repression of dpn in cone cells (Canon, 2003).

Next, whether it was possible for the dpn enhancer to be repressed independently of the AT-rich sequences was investigated. Lz-WRPW has been shown to function as a dedicated repressor in cone cells. The ability of Lz-WRPW to regulate DEE-MutAT was tested. Significantly, although wild-type Lz failed to repress DEE-MutAT, Lz-WRPW was effective in repressing this enhancer in cone cells. Therefore, Lz-WRPW is able to repress transcription of the DEE without a requirement for the nearby AT-rich sites (Canon, 2003).

The homeodomain protein Cut is expressed specifically in the four cone cells in the eye and has been shown previously to bind AT-rich sequences. The ability of Cut to bind the AT-rich sequences next to the Lz sites in the DEE was tested. Electromobility-shift assays were conducted using probes containing the Lz-binding sites and adjacent AT sequences from the dpn enhancer. Nuclear extracts of cells transfected with a Cut-expressing vector bind the two AT-rich sequences, and this binding is specific as established by competition assays. Further, extracts from cells transfected with both lz and cut cause a supershifted band, indicating that Lz and Cut can bind together to the same probe (Canon, 2003).

To address the in vivo relevance of these results, FLP/FRT-mediated clones were made in the eye that were mutant for the cut locus. Strikingly, Dpn was ectopically expressed in cone cells in the absence of Cut. This provides genetic proof that, in vivo, Cut represses dpn expression in cone cells. Cut is therefore required along with Lz for repression of dpn in these cells (Canon, 2003).

Interestingly, D-Pax2, which is directly activated by Lz, is needed to activate cut in cone cells. Therefore, although indirectly, Lz positively regulates cut. This presents an interesting developmental circuit in which Lz, acting as a transcriptional activator, causes expression of a cofactor that then binds with Lz to convert it into a direct repressor of transcription. Both the presence of the cofactor and binding sites for this cofactor in the controlling regions of an Lz target gene are required for Lz-mediated repression (Canon, 2003).

This model was then tested in R7 cells where both Dpn and Lz are coexpressed. Here, Lz does not repress dpn, presumably because Cut is absent from R7. Consistent with this notion, mis-expression of Cut in R7 cells using lz-Gal4 causes repression of dpn in these cells. This is not a secondary result of a change in cell fate because the expression of the R7 cell-specific marker Prospero remains unchanged in this genetic background (Canon, 2003).

These results add another level of complexity to recent studies demonstrating a combinatorial code whereby a relatively small number of signaling pathways and activated transcription factors work together to generate unique cell fates. In cone cells, the Notch and EGFR pathways are required along with Lz to activate D-Pax2, and therefore cut. In contrast, the combination of these few inputs is not right for activation of cut in the R7 neurons, and therefore dpn is not repressed. The circuit described here demonstrates a higher order of sophistication necessary for a cell to choose between a neuronal and nonneuronal fate using a very limited number of inputs. Using a self-regulated circuit and just two signaling pathways, a single Runx protein is capable of causing opposing effects on different enhancers in the same cell, resulting in a unique fate (Canon, 2003).

These observations suggest that Gro binds proteins with a WRPW motif in a stable manner and causes constitutive repression as seen for both Lz-WRPW and Hairy-related proteins that contain the WRPW motif. In contrast, Gro interaction with the WRPY motif in Runx proteins requires a cofactor, such as Cut, for stabilization. Therefore, repression is regulated as Runx forms a functional repressor complex with Gro only in the presence of the cofactor Cut. This hypothesis was tested in immunoprecipitation (IP) experiments. On its own, Lz weakly interacts with Gro. In the presence of Cut, however, the Lz-Gro interaction is dramatically increased. As expected, Lz-WEAA did not coimmunoprecipitate with Gro, with or without Cut, and Lz-WRPW interacted strongly with Gro, in both the presence and absence of Cut. These results are entirely consistent with all of the in vivo observations: (1) Lz functions as a repressor only in the cells that express the Cut protein; (2) Lz-WRPW, which functions as a constitutive repressor, can repress DEE-MutAT, in spite of the mutant AT-sites and absence of Cut binding; (3) wild-type Lz does not repress DEE-MutAT because Cut cannot bind, and therefore the Lz-Gro complex is not stabilized (Canon, 2003).

Runx proteins have been shown to act as positive and negative regulators. This study, however, is the first to demonstrate that a Runx protein can act as both a direct transcriptional activator and repressor in vivo in the same cell, and that the repressive role requires involvement of the cofactor Cut. The mechanism unraveled here for a Runx protein is similar to that described for a Rel protein, suggesting a common strategy adopted by transcription factors that switch between positive and negative regulation. Furthermore, Cut is conserved in mammals (called CDP or Cux) and has been implicated in the repression of several genes, including osteocalcin (OC). Interestingly, the OC gene is positively regulated by Runx2. These in vitro studies did not investigate a relationship between Runx and Cux. This analysis of dpn repression by Lz and Cut raises the possibility that mammalian Runx proteins may also switch from activation to repression modes through involvement of Cux proteins. If confirmed, such correlations will prove to be important as the mammalian Runx protein AML-1 (Acute Myeloid Leukemia-1) is the most frequent site of translocations that cause leukemia, and human CutL1 is located in a chromosomal region that is often rearranged in cancers, including myeloid leukemia (Canon, 2003 and references therein).

Lozenge directly activates argos and klumpfuss to regulate programmed cell death

Reducing the activity of the Drosophila Runx protein Lozenge (Lz) during pupal development causes a decrease in cell death in the eye. Lz-binding sites were identified in introns of argos (aos) and klumpfuss (klu); these genes were shown to be directly activated targets of Lz. Loss of either aos or klu reduces cell death, suggesting that Lz promotes apoptosis at least in part by regulating aos and klu. These results provide novel insights into the control of programmed cell death (PCD) by Lz during Drosophila eye development (Wildonger, 2005a).

These findings, together with what is known about aos and klu, support the following model: Lz induces aos expression in cone cells, wherefrom Aos diffuses to antagonize EGFR activity in the surrounding 2° and 3° cells. The expression pattern of aos⁹²³-lacZ indicates that Lz also regulates aos expression in 2° and 3° cells, suggesting that these cells may also send antisurvival signals. The data further suggest that within the 2° and 3° cells, Lz activates klu, which antagonizes EGFR signaling downstream of the receptor. Lz also activates klu expression in cone and 1° cells, but it is unclear what function klu has in these cells. Although two phases of PCD during retinal development have been proposed, these experiments support a role for Lz in promoting only the EGFR-dependent phase. An alternative possibility is that the decrease in cell death in lz mutant retinas is due to an increase in 2° and 3° cell differentiation stimulated by an increase in EGFR signaling. However, given the large body of evidence demonstrating that lz normally functions to promote differentiation, a model in which lz acts to suppress differentiation is not favored (Wildonger, 2005a).

The mammalian homolog of Lz, Runx1 (also known as AML1), is also a transcriptional regulator. In humans, translocations that affect Runx1 are associated with acute myelogenous leukemia (AML), which is characterized by the proliferation of undifferentiated hematopoietic cells. Effects on cell cycle regulators have been implicated in contributing to this overproliferation, but it is likely that PCD also plays a role . Changes in the amount of the apoptotic regulator Wilms Tumor 1 (WT1) are often found in AML patients. lz promotes cell death in the Drosophila eye in part by activating the expression of klu, the Drosophila homolog of WT1. It is suggested that these findings may be relevant to how Runx1 chimeras lead to the development of AML in humans. Furthermore, they suggest that WT1 may be a direct target of Runx1 (Wildonger, 2005a).

Transcriptional regulation of odorant receptors; Mechanisms of odor receptor gene choice in Drosophila

A remarkable problem in neurobiology is how olfactory receptor neurons (ORNs) select, from among a large odor receptor repertoire, which receptors to express. Computational algorithms and mutational analysis were used to define positive and negative regulatory elements that are required for selection of odor receptor (Or) genes in the proper olfactory organ of Drosophila, and an element was identified that is essential for selection in one ORN class. Two odor receptors are coexpressed by virtue of the alternative splicing of a single gene, and dicistronic mRNAs were identified that each encode two receptors. Systematic analysis reveals no evidence for negative feedback regulation, but provides evidence that the choices made by neighboring ORNs of a sensillum are coordinated via the asymmetric segregation of regulatory factors from a common progenitor. Receptor gene choice in Drosophila also depends on a combinatorial code of transcription factors to generate the receptor-to-neuron map (Ray, 2007).

Transcription factors were investigated whose expression had been reported in at least one olfactory organ and whose mutations had been shown to cause olfactory defects. One such protein, the Runx domain-containing transcription factor Lozenge, was found had predicted binding sites (RACCRCA, R = purine) adjacent to four maxillary palp Or genes. Specifically, it was found that two maxillary palp Or genes, Or59c and Or85d, had two Lz binding sites, and two genes, Or71a and Or85e, had one Lz binding site, within 1 kb upstream or downstream of the coding region. Lz is required for the specification of cell fate in the eye and for normal numbers of olfactory sensilla in the antenna. In the maxillary palp the numbers of sensilla are normal, but electropalpogram recordings showed large reductions in odor responses (Ray, 2007).

To investigate the possibility that Lz is required for normal receptor gene expression, it was first asked whether it is expressed in ORNs of the maxillary palp. Lz is coexpressed with Elav, indicating that it is expressed in the nuclei of all maxillary palp ORNs. Then the expression of six maxillary palp Or genes was examined, one from each ORN class, in lz³, a strong hypomorphic mutant. The four genes that are flanked by predicted Lz binding sites all showed reduced levels of expression; the two genes that contain two Lz binding sites, Or59c and Or85d, showed particularly severe reductions (of 47% and 87%, respectively) in the number of labeled cells. The mildest reduction, 18%, was observed for Or85e; consistent with this result, a 14% reduction was observed when DNA including the predicted Lz binding site was removed from an Or85e-GAL4 driver (the construct containing 3 kb of upstream DNA labeled 13.4 ± 0.4 cells, whereas the construct containing 0.45 kb labeled 11.5 ± 0.3 cells; n = 12). The two genes that did not contain Lz binding sites did not show a reduction in labeling in lz³. These results demonstrate that lz is required for the expression of a subset of Or genes in the maxillary palp (Ray, 2007).

Next a weaker, temperature-sensitive allele, lz^ts1, was used to investigate the possibility that levels of Or gene expression are susceptible to modulation during the adult stage. It was found that Or85d is expressed in 18% fewer cells (p < 0.05) when lz^ts1 flies are raised at the restrictive temperature (29°) than when raised at the permissive temperature (18°). When flies were raised at the restrictive temperature and then shifted to the permissive temperature for 24 hr, 1 week after eclosion, the number of Or85d-expressing cells showed an increase of 19%, to a level indistinguishable from that of flies that had been cultured continuously at the permissive temperature. These results confirm the finding of a functional role for lz in Or expression, provide direct evidence that levels of Or expression can be altered after eclosion, and invite investigation of epigenetic modulation of odor receptor expression in Drosophila (Ray, 2007).

Only one other transcription factor, the POU domain protein Acj6, has previously been demonstrated to be required for odor receptor expression in Drosophila. Specifically, expression of Or33c, Or42a, Or46a, Or59c, and Or85e was severely reduced by the null allele acj6⁶, whereas expression of Or71a and Or85d was unaffected. It has been shown in this study that expression of Or59c, Or71a, Or85e, and Or85d was reduced by lz³, but expression of Or42a and Or46a was not. Thus, the maxillary palp Or genes can be divided into three classes based on their sensitivity to these mutations: those sensitive to both acj6⁶ and lz³ (Or59c and Or85e), to acj6⁶ alone (Or42a and Or46a), or to lz³ alone (Or71a and Or85d). These results support a model in which Or gene expression depends not only on a combinatorial code of regulatory elements but also on a combinatorial code of transcription factors (Ray, 2007).

In summary, in mammals, it is thought that transcriptional regulatory mechanisms direct expression of OR genes in specific zones of the olfactory epithelium, but that within a zone, OR gene choice is based on a stochastic selection mechanism. A third mechanism, negative feedback, could then operate to limit the number of OR genes expressed in individual neurons (Ray, 2007).

In Drosophila, the process of receptor gene choice achieves a conceptually simple end: it produces a highly stereotyped receptor-to-neuron map. However, the large number of receptors and neurons presents a regulatory problem of great complexity. To achieve such a precise and highly ordered organization, Drosophila has evolved a sophisticated suite of regulatory mechanisms. This study has documented organ-specific and neuron-specific levels of transcriptional control, including both positive and negative mechanisms. A posttranscriptional mechanism, alternative splicing, was identified and the system has even evolved a relatively rare innovation, dicistronic mRNAs (Ray, 2007).

The worm Caenorhabditis elegans has a much larger repertoire of odor receptor genes than Drosophila, but the number of ORNs to which it allocates them is very limited. Thus the number of receptor genes per neuron is increased, but the complexity of the regulatory problem is decreased. In vertebrates, however, the repertoire is very large and the number of receptor genes expressed per neuron is very low. Perhaps as the receptor gene repertoire expanded in vertebrate evolution, the complexity of the regulatory problem eventually exceeded the ability of the system to execute a deterministic plan with sufficient fidelity, and deterministic mechanisms were replaced by a stochastic mechanism and a negative feedback mechanism. In any case, the ultimate result of receptor gene choice in Drosophila is the same as in vertebrates: a spectacular diversity of ORNs that underlie the detection and discrimination of odors (Ray, 2007).

Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer of sparkling

Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? This study examined in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparkling's regulatory sites converts it into a robust photoreceptor-specific enhancer. These results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer's organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others (Swanson, 2010).

The goal of this study was to use a well-characterized, signal-regulated developmental enhancer to examine, in fine detail, the regulatory interactions and structural rules governing transcriptional activation in vivo. This study used functional in vivo assays to test the power of the proposed combinatorial code of 'Notch/Su(H) + Lz + MAPK/Ets' to explain the activity and cell type specificity of the spa cone cell enhancer of dPax2. In the course of this work, several surprising properties of spa were discovered that are not accounted for in current models of enhancer function (Swanson, 2010).

The spa enhancer for fine-scale analysis because (1) the known direct regulators and their binding sites are well defined, (2) they could, in theory, constitute the sum total of the patterning information received by the enhancer, and (3) the enhancer, at 362 bp, is relatively small, simplifying mutational analyses. Surprisingly, a large proportion of the previously uncharacterized sequence within spa is vital for normal enhancer activity in vivo, and of that subset, a large proportion directly influences cell type specificity (Swanson, 2010).

In addition to necessary inputs from Lz, Pnt, and Su(H), three segments of spa were identified, regions 4, 5, and 6, that make essential contributions to gene expression in cone cells. In addition, region 2 makes a relatively minor contribution. (Region 1, another essential domain, will be discussed separately.) Fine-scale mutagenesis reveals that within regions 4, 5, and 6, very little DNA is dispensable for cone cell activation. The previously uncharacterized regulatory sites in spa are very likely bound by factors other than Lz/Pnt/Su(H), for the following reasons: no sequences resembling Lz/Pnt/Su(H)-binding sites reside in these regions; mutations in the newly mapped sites have different effects than removing the defined TFBSs or the proteins that bind them; doubling the known TFBSs fails to compensate for the loss of the newly mapped sequences; and, most importantly, mutating the newly mapped regulatory regions does not significantly affect binding of the known activators to nearby binding sites in vitro. It is not known whether the proposed novel regulators are cone cell-specific, eye-specific, or ubiquitous in their expression. It is known that the newly mapped sites are necessary both for normal cone cell expression and ectopic PR expression. Cut, Prospero, and Tramtrack are expressed in cone cells, but are thought to act as transcriptional repressors. The transcription factor Hindsight is required for dPax2 expression and cone cell induction, but acts indirectly, activating Delta in R1/R6 to induce Notch signaling in cone cells (Swanson, 2010).

Unsurprisingly, placing the enhancer closer to the promoter boosts expression of spa(wt), as well as some of the impaired mutants. The spa enhancer is located at +7 kb in its native locus, and nearly all mutational studies place the enhancer immediately upstream of the promoter. If the entire analysis had been performed at −121 bp, the functional significance of several critical regulatory sequences would have been underrated, and region 1 would have been dismissed as nonregulatory DNA. Other well-characterized enhancers, which have been analyzed in a promoter-proximal position only, may therefore contain more critical regulatory sites than is currently realized (Swanson, 2010).

Like many transcriptional activators, all three known direct activators of spa (or their orthologs) recruit p300/CBP histone acetyltransferase coactivator complexes. Doubling the number of binding sites for these transcription factors (to 6 Lz, 8 Ets, and 10 Su(H) sites) does not suffice to drive cone cell expression in the absence of the newly mapped regulatory regions. It may be, then, that factors recruited to the newly mapped regulatory sites within spa employ mechanisms that are distinct from those of the known activators. The remote activity of spa, mediated by region 1, appears to be an example of such a mechanism (Swanson, 2010).

It was possible to convert spa into a R1/R6-specific enhancer in three ways: (1) by moving the defined TFBSs to one side of the enhancer in a tight cluster; (2) by placing Lz and Ets sites next to regions 1, 4, and 6a; and (3) by mutating regions 2, 3, 5, and 6b within spa while maintaining the native spacing of all other sites. From these experiments, it is concluded that spa contains short-range repressor sites that prevent ectopic activation in PRs by Lz + Pnt + regions 4 + 6a. spa contains at least two redundant repressor sites, because both region 5 and regions 2, 3, and 6b must be mutated to attain ectopic R1/R6 expression (Swanson, 2010).

klumpfuss, which encodes a putative transcriptional repressor, is directly activated by Lz in R1/R6/R7, but is also present in cone cells, making it an unlikely repressor of spa. seven-up, another known transcriptional repressor, is expressed in R3/R4/R1/R6 and could therefore act to repress spa in PRs. However, no putative Seven-up-binding sites were identified within spa. Phyllopod, an E3 ubiquitin ligase component, represses dPax2 and the cone cell fate in R1/R6/R7, but the transcription factor mediating this effect is not yet known (Shi, 2009). Perhaps the best candidate for a PR-specific direct repressor of spa is Bar, which encodes the closely related and redundant homeodomain transcription factors BarH1 and BarH2. Bar expression is activated by Lz in R1/R6 and is required for R1/R6 cell fates. Furthermore, misexpression of BarH1 in presumptive cone cells can transform them into PRs. It is unclear whether Bar-family proteins act as repressors, activators, or both. BarH1/2 can bind sequences containing the homeodomain-binding core consensus TAAT, and region 5 of spa contains two TAAT motifs. Future studies will explore the possibility that Bar directly represses spa in PRs (Swanson, 2010).

The combinatorial code of spa, then, requires multiple inputs in addition to Lz, MAPK/Ets, and Notch/Su(H). Indeed, the data suggest that the known regulators can contribute to expression in multiple cell types, depending on context. The newly mapped control elements identified within spa are necessary not only to facilitate transcriptional activation, but also to steer the Lz + Ets + Su(H) code toward cone cell-specific gene expression (Swanson, 2010).

Enhancers are often located many kilobases from the promoters they regulate. Enhancer-promoter interactions over such distances are very likely to require active facilitation. Even so, few studies have focused specifically on transcriptional activation at a distance, and the majority of this work involves locus control regions (LCRs) and/or complex multigenic loci, which are not part of the regulatory environment of most genes and enhancers. Like spa, many developmental enhancers act at a distance in their normal genomic context, yet can autonomously drive a heterologous promoter in the proper expression pattern, without requiring an LCR or other large-scale genomic regulatory apparatus. However, in nearly all assays of enhancer function, the element to be studied is placed immediately upstream of the promoter. In such cases, regulatory sites specifically mediating remote interactions cannot be identified. Because the initial mutational analysis of spa was performed on enhancers placed at a moderate distance from the promoter (−846 bp), it was possible to screen for sequences required only at a distance, by moving crippled enhancers to a promoter-proximal position. Only one segment of spa, region 1, was absolutely essential at a distance but completely dispensable near the promoter. This region, which contains the only block of extended sequence conservation within spa, plays no apparent role in patterning, or in basic activation at close range. Therefore this segment of spa is termed a 'remote control' element (RCE) (Swanson, 2010).

The remote enhancer regulatory activity described in this study differs from previously reported long-range regulatory mechanisms in two important ways. First, the remote function of spa does not require any sequences in or near the dPax2 promoter. This functionally distinguishes spa from enhancers in the Drosophila Hox complexes that require promoter-proximal 'tethering elements' and/or function by overcoming insulators. This distal activation mechanism also likely differs from enhancer-promoter interactions mediated by proteins that bind at both the enhancer and the promoter, as occurs in looping mediated by ER, AR, and Sp1. Second, studies of distant enhancers of the cut and Ultrabithorax genes have revealed a role for the cohesin-associated factor Nipped-B, especially with respect to bypassing insulators, but it has not been demonstrated that Nipped-B, or any other enhancer-binding regulator, is required only when the enhancer is remote (Swanson, 2010).

The spa RCE is the first enhancer subelement demonstrated to be essential for enhancer-promoter interactions at a distance, but unnecessary for proximal enhancer function and cell type specificity. However, the present work contains only a limited examination of this activity, as part of a broader study of enhancer function. These functional studies, testing for potential promoter preferences and distance limitations, and the identities of factors binding to the RCE are being persued(Swanson, 2010).

As discussed above, it is fairly easy to switch spa from cone cell expression to R1/R6 expression (though, curiously, a construct that is active in both cell types has yet to be constructed). The results show that multiple regions of spa mediate a repression activity in R1/R6, but not in cone cells. It is further concluded that these spa-binding repressors act in a short-range manner; that is, they must be located very near to relevant activator-binding sites, because moving Lz and Pnt sites to one side of spa, without removing the repressor sites (KO+synth^CS), abolishes repression. Despite this failure of repression, synergistic interactions among Lz and Ets sites and the newly mapped sites still occur in this reorganized enhancer -- at least in R1/R6 cells. Cone cell-specific expression is lost, however, revealing (along with other experiments) that transcriptional activation in cone cells is highly sensitive to the organization of regulatory sites within spa. Slightly wider spacing of regulatory sites (KO+synth^NS) kills the enhancer altogether, suggesting that synergistic positive interactions within spa, though apparently longer in range than repressive interactions, are severely limited in their range. The structural organization of spa, then, appears to be constrained by a complex network of short-range positive and negative interactions. Activator sites must be spaced closely enough to trigger synergistic activation in cone cells; at the same time, repressor sites must be positioned to disrupt this synergy in noncone cells, preventing ectopic activation (Swanson, 2010).

Recent work has shown that changes to enhancer organization can 'fine-tune' the output of a combinatorial code, subtly changing the sensitivity of the enhancer to a morphogen. Given the importance of the structure of the spa enhancer for its proper function, it is proposed that any combinatorial code model, no matter how complex, is insufficient to describe the regulation of spa, because the same components can be rearranged to produce drastically different patterns (Swanson, 2010).

One might expect that the regulatory and organizational complexity of the spa enhancer, and its extreme sensitivity to mutation, would be reflected in strict evolutionary constraints upon enhancer sequence and structure. Yet, very poor conservation of spa sequence was observed, both in the known TFBSs and in most of the newly mapped essential regulatory elements. The reduced presence of Lz/Ets/Su(H) sites in D. pseudoobscura could potentially be attributed to redundancy of those sites in D. melanogaster, or to compensatory gain of binding sites for alternate factors in the D. pse enhancer. Perhaps more difficult to understand is the apparent loss of critical regulatory sequences in regions 4, 5, and 6a in D. pse; the experiments in D. mel suggest that the absence of those inputs would result in loss of cone cell expression and/or ectopic activation. It remains possible that many of these inputs are in fact conserved, but that conservation is not obvious due to binding site degeneracy and/or rearrangement of elements within the enhancer. Fine-scale comparative studies are ongoing (Swanson, 2010).

spa is by no means the first example of an enhancer that is functionally maintained despite a lack of sequence conservation. The most thoroughly characterized example of this phenomenon is the eve stripe 2 enhancer; its function is conserved despite changes in binding site composition and organization. Note, however, that spa has undergone much more rapid sequence divergence than eve stripe 2, with no apparent change in function. In general, the ability of an enhancer to maintain its function in the face of rapid sequence evolution suggests that enhancer structure must be quite flexible. These observations support the 'billboard' model of enhancer structure, which proposes that as long as individual regulatory units within an enhancer remain intact, the organization of those units within the enhancer is flexible. Yet, the findings concerning the importance of local interactions among densely clustered, precisely positioned transcription factors are more consistent with the tightly structured 'enhanceosome' model. Further structure-function analysis will be necessary to fully understand the players and rules governing this regulatory element (Swanson, 2010).

Rapid evolutionary rewiring of a structurally constrained eye enhancer

Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as - in some cases - the arrangement and spacing of binding sites for these factors. This study examined evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye. Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. The data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that were observe. Furthermore, stereotypical spatial relationships between certain binding sites ('grammar elements') can be identified in all sparkling orthologs - although the sites themselves are often recently derived. It was also found that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in non-cone cells. It is concluded that rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. These findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale (Swanson, 2011).

Because of spa's rapid structural evolution and binding-site turnover, multispecies sequence alignments do not reveal many conserved features. Only the extreme 5' end of spa is unequivocally alignable across 12 Drosophila genomes. Given spa's complex regulatory circuitry and structure, its unusually rapid sequence divergence between D. mel and D. pse was surprising, especially because both orthologs of spa have identical cell-type specificities (Swanson, 2011).

This study demonstrated that even an enhancer that is subject to structural constraints can be evolutionarily flexible; therefore, an apparent lack of conserved cis-regulatory structure does not imply an absence of organizational rules within an enhancer (Swanson, 2011).

A model for the structural divergence of spa between the melanogaster and obscura groups is proposed, based on sequence analyses and experimental data. Although the remote control element (RCE) and its flanking Lz1-Ets1 pair are relatively stable, many other essential regulatory sites have been relocated. Within regions 4, 5, and 6a, putative novel regulatory motifs, essential for full-strength activation of both spa orthologs, have been identified whose movements are consistent with experimental data on spa's evolutionary restructuring (Swanson, 2011).

Important changes to the Lz/Ets/Su(H) inputs have also occurred: D. pse has fewer Su(H) and Lz sites, relative to the melanogaster group -- which can be compensated by newly acquired, functionally significant 5' Ets and epsilon (AGCCAG) sites. Meanwhile, the melanogaster group has gained a new Lz site and also has a relative abundance of Su(H) sites, which may compensate for relatively few epsilon and Ets sites (Swanson, 2011).

By tracking the reorganization of Su(H), Lz, Ets, and epsilon motifs across multiple species, a speculative phylogeny of the spa enhancer within the genus Drosophila is proposed and the cis-regulatory content of the last common ancestors (LCAs) of several species groups is predicted by reconstructing the gain and loss of sites, and the changing strengths of transregulatory inputs, in specific lineages. The main conclusions to be drawn from this evolutionary view of spa, informed by functional experiments, are: (1) significant enhancer rewiring has occurred since the divergence of the mel and pse lineages; (2) this rewiring involves the loss and gain of individual regulatory motifs, as well as compensatory changes in the overall strength of several trans-regulatory inputs through changes in binding-site number, position, and possibly affinity; (3) despite very rapid site turnover, characteristic configurations of sites ('grammar elements') can be identified; (4) these grammar elements can be relocated within the enhancer, suggesting that a specific arrangement of sites can be more ancient than the individual sites that compose it. These last two points, taken together, may explain how spa can continue to obey structural rules while being significantly reconfigured (Swanson, 2011).

A large proportion of the grammar elements that have been identified involve Lz/Runx and Ets motifs. Unlike the case of linked sites for Dorsal, Twist, and other factors in insect neurogenic enhancers, there is no single, clearly preferred arrangement of Lz and Ets sites within spa: seven distinct types of Lz/Ets grammar element were identified that are at least as ancient as the LCA of the melanogaster group (Swanson, 2011).

Perhaps Runx and Ets factors, which are known to directly interact and to cooperatively activate transcription in flies and vertebrates, can synergize productively in several different spatial configurations. This is consistent with mapped Runx and Ets sites in vertebrate genomes, which are frequently associated with one another in target enhancers, but not with a single rigid arrangement or spacing (Swanson, 2011).

A nonstructural constraint on the sequence of spa was discovered: a requirement for nonconsensus, low-affinity Su(H) sites for proper cone-specific patterning. Because ectopic dPax2 expression in photoreceptor precursors causes faulty cell fate specification and differentiation, resulting in defective eye morphology, it is reasonable to suppose that the expression pattern of spa[Su(H)-HiAff] would have negative fitness consequences for the fly. Taken together with previous work, the data presented in this study suggest that spa requires input from Notch/Su(H) but also requires that input to be attenuated at the cis-regulatory level, in order to generate the proper levels and cell-type specificity of dPax2 expression in a tissue with widespread Notch signaling. Like Notch/Su(H), EGFR/Ets signaling and Lz are also used to specify multiple cell types in the retina, which presents a challenge for combinatorial gene regulation: enhancers must be able to make fine qualitative distinctions in regulatory inputs and often must translate this information into relatively sharp on/off decisions. These pressures could result in a cis-regulatory logic for genes like dPax2 in which many weak inputs are independently tuned (and spatially arranged) to maximize activation in the proper cell type, while minimizing ectopic activation. Previous studies of spa present a picture of an enhancer operating just above a functional threshold, such that the loss of a single regulatory site, or a loss of proper grammar, can result in transcriptional failure in cone cells. One of the main conclusions from this study is that, over a relatively short evolutionary timescale, a cis-regulatory module can find multiple solutions to this complex computational problem (Swanson, 2011).

The presence of weak, nonconsensus binding sites for signal-regulated TFs is a common, but little remarked upon, feature of developmental enhancers. Low-affinity TF binding sites have well-documented functions in shaping a stripe of gene expression across a morphogen gradient and in determining temporal responses to developmental regulators. This study provides direct evidence supporting a role for weak signal response elements in preventing ectopic transcriptional responses to highly pleiotropic signaling pathways such as Notch (Swanson, 2011).

There is one striking question not addressed by this study: why is this enhancer evolving at an unusually high rate, given that its expression pattern is stable? Two plausible explanations are given for which supporting data exist. First, dPax2 is on chromosome 4, the 'dot' chromosome of Drosophila, which has a severely reduced recombination rate, resulting in inefficient selection and relaxed sequence constraint. No other cis-regulatory module on the fourth chromosome has been subjected to an extensive evolutionary analysis, nor are any as well-mapped as sparkling, but enhancers of the fourth-chromosome genes eyeless and toy contain fairly large blocks of sequence conservation, compared to spa. An alternative explanation for the rapid turnover observed within spa involves the presence of nonconsensus, predicted low-affinity sites for Su(H) and, in some cases, Lz and PntP2. For a typical TF, there are many more possible low-affinity binding sites than high-affinity sites: for example, the highest-affinity Su(H) consensus YGTGDGAAM encompasses only 12 variants (TGTGGGAAA, etc.), whereas the lower-affinity consensus of the same length nRTGDGWDn, which accommodates all of the known Su(H) sites within spa, contains 576 possible sequences. Accordingly, it is much more likely that an enhancer will acquire a low-affinity binding site via a single mutational event than a high-affinity site. Thus, an enhancer that does not require high-affinity binding sites for given trans-regulators may rapidly sample a variety of configurations of weak sites and may thereby undergo considerable sequence turnover without losing the input from that regulator. In other words, an enhancer such as spa, which must maintain a weak regulatory linkage with Notch/Su(H), may be less constrained than a high-affinity target with respect to the sequence, number, and position of its Su(H) binding sites. Whatever the reason for the rapid sequence divergence of spa, it provides an opportunity to examine in detail the evolutionary mechanisms by which a complex cis-regulatory module can be significantly reorganized, while still conforming to specific constraints of combinatorial logic and grammar (Swanson, 2011). >

Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference

Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, this study performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, direct TF-DNA interactions were derived using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, network motifs, cis-regulatory codes, and regulators of eye development were found. The predicted target regions of Grainyhead were validated by ChIP-seq and this factor was identified as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions (Potier, 2014).

The development of the Drosophila eye is a classical model system to study neuronal differentiation and patterning. The TFs that represent the core of the retinal determination network are Eyeless (Ey), Twin of Eyeless (Toy), Dachsund (Dac), Sine Oculis (So), and Eyes Absent (Eya). Although many regulatory interactions are known between these TFs, as they intensively cross-regulate each other, knowledge about interactions with downstream target genes and of other TFs involved in the eye-antennal gene regulatory network (GRN) is sparse. This study aimed at combining classical reverse genetics-starting from a mutant allele and analyze its (molecular) phenotype-with genomics. Doing so, attempts were made to unveil genetic regulatory interactions in an unbiased way, and many regulators of the eye and antennal developmental programs were identified; most of these did not require or use any mutation or direct perturbation (Potier, 2014).

The mapping approach began by systematically perturbing the developmental system. Attempts were made to include multiple perturbations into one data matrix to obtain a broad spectrum of expression profile changes. These perturbations included TF mutants, TF overexpression, TF knockdown, and cell sorting (Potier, 2014).

Eye-antennal discs were dissected at the stage where in the WT discs about half of the eye disc contains pluripotent cells that are dividing asynchronously, while the other half contains differentiating PR neurons, in consecutive stages of differentiation. Simultaneously, the antennal disc contains neuronal precursors that are undergoing specification. The expression changes induced by the perturbations often result from a shift in proportion of cell types. This is trivial for the cell-sorting experiments; for example, the GMR>GFP-positive cells show, as expected, a very strong enrichment of genes related to PR differentiation. TF mutants and TF perturbations can also result in cell type shifts; for example, overexpression of Atonal yields more R8 PRs, and the glass mutant results in fewer differentiated PRs. Other TF perturbations cause changes in gene expression downstream of the TF without changing the cell type composition, such as Retained, which disturbs axonal projection. The key technique that was applied, however, was not to compare each TF perturbation with WT discs to identify differentially expressed genes. Rather, linear and nonlinear correlations of gene expression profiles were used across the entire vector of 72 gene expression measurements. This TF-gene coexpression network contains both direct and indirect edges, and although this network is informative, a second layer of predicted TF-DNA interactions was added, thus making this a direct GRN. To increase the sensitivity, a very large collection was used of TF motifs, also including position weight matrices derived for yeast and vertebrate TFs and including computationally derived motifs (e.g., highly conserved words). Using motif-motif similarity measures and TF-TF orthology relationships, each motif was linked to a candidate binding factor. This yielded a large network with 335 TFs and their predicted direct targets. The only functional network of comparable size and comparable directedness to this in vivo network is the TH17 GRN that was derived in vitro in a recent study (Yosef, 2013). That study used a microarray time course of naive CD4+T cells differentiating into TH17. From these gene expression data, they derived TF-gene interactions by clustering and filtered those with TF-DNA interactions obtained by ChIP-seq data, TF perturbations, and cis-regulatory sequence analysis (Potier, 2014).

The predicted direct and functional eye-antennal GRN includes many previously reported interactions, such as known target genes for Eyeless and Sine Oculis. Target genes in the network were also found for late factors (e.g., Glass, Onecut) and very late factors (e.g., Pph13). The fact that information was captured at different time points during development is because several cell populations were sorted that are loosely correlated with the temporal axis of development, consisting of undifferentiated pluripotent cells anterior to the furrow, all PR cells undergoing differentiation posterior to the MF, R8 PR cells, and late populations of chp-positive cells. However, the temporal information encoded in the network is limited to these broad domains, and a more detailed reconstruction of the time axis would require higher resolution cell sorting or microdissection experiments. Although the perturbed TFs were mainly chosen for their development of the retina, master regulators of antennal development, such as aristaless were also identified (Potier, 2014).

Interestingly, general factors like Grainyhead were found that were ubiquitously expressed. Grh was found as one of the TFs with the largest number of target enhancers and its binding correlates with open chromatin. Previous studies have shown that Grainyhead may interact with Polycomb and Trithorax proteins to regulate (both activate and repress) target gene expression. It is speculated that this observed correlation can be explained by the fact that Grh is present ubiquitously in the eye disc, thus yielding many sequence fragments from bound and nucleosome-free enhancers by FAIRE-seq (Potier, 2014).

It is well known that network motifs such as FFLs play an important role in biological networks. One such network motif was examined in more detail, namely the TF pair Glass-Lozenge, and their common targets. These TFs constitute a double-feedback loop (Glass regulates Lozenge, Lozenge regulates Glass, and they together regulate 36 targets). For this network motif, it was found that Glass and Lozenge motifs co-occur at the same enhancer, where they furthermore overlap; this may indicate competition for binding between Glass and Lozenge. Given that Lozenge, an important regulator of cone cell differentiation, could be a repressor and Glass, an important regulator of PR differentiation, could be an activator, such a competition at the CRM level could indeed be a plausible mechanism for their regulatory action (Potier, 2014).

Another interesting feature that can be derived from a GRN is the proportion of autoregulatory TFs (108 autoregulatory TFs in the eye network) and the proportion of activating versus repressive TFs. A recent large-scale study in yeast found a small majority of yeast TFs to have a repressive role. In that study, each individual TF was perturbed, thereby providing information on positive versus negative edges from the TF to its direct predicted targets, whereby TF-DNA information was used from ChIP-chip data. Since the eye GRN was started from a coexpression TF-gene network, the correlations between TFs and their candidate targets were revisited, and 151 TFs were found that have their motif enriched in the positively correlated target genes, but not in the negatively correlated targets, and 127 TFs showing the opposite; 62 TFs show enrichment in both. This finding agrees, to some extent, with the results in yeast concerning the high amounts of gene-specific repressors. On the other hand, the eye network suggests relative more TFs with a dual activator/repressor function, while the yeast study found only a few such cases (Potier, 2014).

In conclusion, starting from an expression matrix derived from large-scale perturbations and combining TF-gene coexpression with TF-DNA interactions based on motif inference enabled drawing an extensive eye-antennal GRN. All predicted regulatory interactions, target genes, and candidate regulatory regions are stored in a Neo4J database and can be queried from a laboratory website. The database can also be accessed directly from Cytoscape using the CyNeo4j plugin or can be queried programmatically using the Neo4j query language Cypher. Although many known regulators and cis-regulatory elements were uncovered and several other ones were revealed, a large part of the predicted network, including how the dynamics of the developmental program are encoded in the cis-regulatory regions and in the topology of the network, remains to be explored (Potier, 2014).

Protein Interactions

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBFb, which interacts with the mammalian CBFa Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. Functional interactions between Brother proteins and Runt domain proteins have been demonstrated in Drosophila. A specific point mutation in Runt has been shown to disrupt interaction with Brother proteins but does not affect DNA binding activity. The point mutation was introduced into Runt by a PCR based site-directed mutagenesis. The mutant is dysfunctional in several in vivo assays. Interestingly, this mutant protein acts dominantly to interfere with the Runt-dependent activation of Sex-lethal transcription. To investigate further the requirements for Brother proteins in Drosophila development, an examination was carried out of the effects of expression of a Brother fusion protein homologous to the dominant negative CBFb::SMMHC fusion protein that is associated with leukemia in humans. This Bro::SMMHC fusion protein interferes with the activity of Runt and a second Runt domain protein, Lozenge. The effects of lozenge mutations on eye development are suppressed by expression of wild-type Brother proteins, suggesting that Brother/Big brother dosage is limiting in this developmental context. Results obtained when Runt is expressed in developing eye discs further support this hypothesis. These results firmly establish the importance of the Brother and Big brother proteins for the biological activities of Runt and Lozenge, and further suggest that Brother protein function is not restricted to enhancing DNA-binding (Li, 1999).

The Core Binding Factor is a heterodimeric transcription factor complex in vertebrates that is composed of a DNA binding alpha-subunit and a non-DNA binding ß-subunit. The alpha-subunit is encoded by members of the Runt Domain family of proteins and the ß-subunit is encoded by the CBFß gene. In Drosophila, two genes encoding alpha-subunits, runt and lozenge, and two genes encoding ß-subunits, Big brother and Brother, have been identified. A sensitized genetic screen was used to isolate mutant alleles of the Big brother gene. Expression studies show that Big brother is a nuclear protein that co-localizes with both Lozenge and Runt in the eye imaginal disc. The nuclear localization and stability of Big brother protein is mediated through the formation of heterodimeric complexes between Big brother and either Lozenge or Runt. Big brother functions with Lozenge during cell fate specification in the eye, and is also required for the development of the embryonic PNS. ds-RNA-mediated genetic interference experiments show that Brother and Big brother are redundant and function together with Runt during segmentation of the embryo. These studies highlight a mechanism for transcriptional control by a Runt Domain protein and a redundant pair of partners in the specification of cell fate during development (Kaminker, 2001).

Sensitized genetic screens have proved to be powerful tools in identifying interacting proteins that participate in many different developmental pathways. A particularly impressive use of this technique in the Drosophila eye has led to the identification of the mutations in the components of the RTK pathway. Such a screening technique was used to generate mutations in genes that function with lz during eye development. The identification of mutations in a direct transcriptional target of Lz, D-Pax2, and the gene encoding a binding partner of Lz, Bgb, suggests that this screen is able to detect proteins whose function is directly related to that of Lz (Kaminker, 2001).

In this screen, two alleles of hsp83 were isolated as dominant enhancers of lz^ts1. Drosophila Hsp83 is a chaperone protein that has been shown to physically interact with Raf. Mutations in hsp83 were identified as downstream modifiers of the sevenless and EGFR RTK pathways. Recent studies have indicated an extensive collaboration between RTK pathways and Lz in the regulation of direct target genes such as D-Pax2 and pros. It is therefore likely that hsp83 strengthens the RTK signal transduction cascade that functions with Lz in the regulation of target genes. In addition, HSP90, the mammalian homolog of hsp83, has been shown to associate with a variety of different transcription factors and has also been proposed to function in nuclear transport. An analysis of the relationship between Hsp83 and Lz/Bgb might provide insight into the mechanism by which this transcription factor complex is translocated to the nucleus (Kaminker, 2001).

The screen also uncovered two alleles of osa/eld, a member of the brahma (brm) complex, involved in chromatin remodeling. The identification of osa as a dominant enhancer suggests that Lz may have a function related to chromatin remodeling. This is not surprising since other Runx family members are thought to function in this manner. For example, Runx2 binding has been implicated in the remodeling of the rat osteocalcin promoter. Additionally, during myeloid differentiation, Runx1 has been shown to interact with p300/CBP, a protein involved in histone acetylation. Further, Drosophila Run has been shown to bend DNA and is likely involved in modifying the architecture of target enhancers. In the eye, Lz is essential for pre-patterning an undifferentiated population of cells and preparing them to activate different target genes in response to signal transduction cascades. It is possible that this process involves remodeling of the individual enhancers through the mediation of an Osa/Lz complex. The identification of osa as a genetic modifier of lz suggests the need for future biochemical experiments to establish if such protein complexes are indeed formed during development (Kaminker, 2001).

This paper focused on the function of the partner proteins since mutations in Bgb were identified as modifiers of lz. The similarity in the phenotype of lz^ts1; Bgb^D/Df(3L)Bgb^K4 mutants to the null allele of lz suggests an absolute functional requirement of the partner protein during eye development. Similarly, ds-RNA interference results suggest that both partner proteins are able to function with Run during embryonic pattern formation (Kaminker, 2001).

It remains to be proven whether the disorganization seen in the PNS of Bgb can be attributed to Bgb function with the known Runt domain proteins. Similar PNS defects are seen in run mutants, but these phenotypes are difficult to interpret because of the additional segmentation phenotypes that could indirectly affect PNS development. It remains possible that Bgb functions with an as yet uncharacterized RD protein in the PNS. Consistent with this explanation, a survey of the sequence of the Drosophila genome reveals two additional runt domain proteins (Kaminker, 2001).

S2 cell expression data show that Bgb is translocated to the nucleus only in the presence of Lz. Although Bgb has a nuclear localization signal (NLS), these data suggest an additional requirement of Lz binding for its transport to the nucleus. Similar regulation of nuclear transport has been reported with Single-minded (Sim) and Tango (Tgo) heterodimers as well as with Homothorax (Htx) and Extradenticle (Exd) heterodimers. In these examples, the localization to the nucleus of either Tgo or Exd, depends on the presence of Sim or Hth, respectively. Recent work has shown that Hth binding allows nuclear transport of Exd by simultaneously inhibiting its nuclear export signal (NES) while activating its NLS. Bgb does not have a leucine-rich sequence typically associated with an NES; co-localization into the nucleus in this case is likely to involve an unmasking of the NLS causing its exposure to the transport machinery. Obviously, nuclear localization of both the alpha- and the ß-subunit is a prerequisite for activation of transcription. In fact, in human AML caused by Inv(16), the CBFß fusion protein is exclusively retained within the cytoplasm (Kaminker, 2001).

The Lz/Bgb complex provides an interesting example of post-translational stabilization of proteins through the formation of heterodimeric complexes. While the possibility that low levels of Bgb protein remain in the cytoplasm of the cell in a lz mutant background cannot be ruled out, the likely explanation for the Bgb protein not being detectable in the absence of Lz or Run is that the ß-subunit is degraded in the absence of the alpha-partner. Similar mechanisms involving degradation of a subunit operate in creating stable Exd/Hth and Sim/Tgo complexes. Tissue lacking Hth or Sim will cause degradation of Exd and Tgo, respectively. As an interesting contrast to these results, in mammalian systems it is the alpha-subunit, Runx1, that is stabilized by CBFß. In this case, the absence of the ß-partner causes a proteosome-mediated degradation of the alpha-subunit (Kaminker, 2001).

The initial cloning of Bro and Bgb raised the possibility that these genes might function redundantly during development. Although there is a stretch of 156 amino acids at the N terminus of Bgb that is not present in Bro, these proteins are 59% identical throughout the remainder of their sequence. Furthermore, Bro and Bgb have overlapping expression domains during embryogenesis. ds-RNA-mediated genetic interference experiments used in this study clearly show that Bro and Bgb function redundantly during development as heterodimeric partners of Run. A loss-of-function phenotype equivalent to a complete run null allele is revealed only in the absence of both Bro and Bgb (Kaminker, 2001).

The two partner proteins do not function redundantly in all tissues. This is highlighted by the fact that Bgb mutants have a PNS defect on their own. Thus, at least in this tissue, Bgb function is not redundant with that of Bro. This is different from redundant gene pairs such as BarH1 and BarH2 which are co-regulated in all tissues and always function together. It is also interesting to note that injection of ds-Bro generates a fairly strong segmentation phenotype, while injection of ds-Bgb does not affect segmentation patterning at all. Therefore, it is possible that in the wild-type fly, when both partners are present, Run preferentially functions with Bro. However, only in the absence of Bro, can compensation of Run function be achieved through its binding Bgb. A comparable situation exists in mice. The paralogs Hoxa3 and Hoxd3 are expressed in the same tissue, but clearly have distinct functional requirements. Yet, a compensating mechanism can be created in a background when one of the two genes is eliminated (Kaminker, 2001).

Detection of mutations in genes that function redundantly poses a difficult challenge to genetic analysis. The data show that at least for the case in study, dosage-sensitive screens involving sensitized genetic backgrounds can be used for the purpose of identifying redundant genes. Bro and Bgb together can be considered to contribute 4 copies of the partner gene. Loss of 1 out of these 4 copies in a sensitized background (lz^ts1; Bgb^- Bro⁺/Bgb⁺ Bro⁺) gives rise to a detectable eye phenotype. Yet, loss of 2 copies in a wild-type background (lz⁺; Bgb^- Bro⁺/Bgb^- Bro⁺) does not generate a mutant phenotype. This remarkable sensitivity to dosage suggests that properly sensitized genetic screens could be used in the detection of redundant gene function (Kaminker, 2001).

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, the role of the Drosophila MLF homolog was investigated during blood cell development. mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, these data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO-induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, it was shown that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO-dependent proliferation. Thus, it is proposed that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development (Bras, 2012).

Development of the hematopoietic system relies on the activity of several signaling pathways and transcription factors that have been conserved from mammals to fly. Drosophila has emerged as a model organism to gain insight into the gene regulatory networks controlling hematopoiesis. Deregulation of these networks lies at the origin of several diseases in humans, including leukemia and lymphoma. Interestingly, these hematologic malignancies are frequently associated with recurring chromosomal abnormalities. Along with their clinical prognostic value, these rearrangements have led to the identification of many genes involved in the etiology of cancer; thus, characterizing the function of these genes in normal and pathological blood cell development is of particular interest (Bras, 2012).

One notorious gene identified by cloning translocation breakpoints is RUNX1/AML1, which is affected by the t(8;21) (q22;q22) translocation found in ~15% of all cases of acute myeloid leukemia (AML). RUNX1 belongs to the RUNX transcription factor family, which is characterized by the presence of a highly conserved DNA binding domain. RUNX proteins activate or repress transcription in a context-dependent manner, thereby regulating cell proliferation and differentiation in a variety of metazoans. In particular, RUNX genes have been shown to regulate hematopoiesis in both vertebrates and Drosophila. For instance, RUNX1 plays a prominent role in definitive hematopoietic stem cell emergence, as well as in megakaryocyte and lymphocyte differentiation in mammals, and mutations affecting RUNX1 are among the most frequent genetic abnormalities associated with blood cell malignancies in humans. These alterations promote leukemia by altering RUNX1 dosage or by producing mutant proteins that act as dominant negative and/or display neomorphic activities, as for RUNX1-ETO, the product of the t(8;21)(q22;q22) translocation, which comprises the RUNX1 DNA-binding domain fused to the transcriptional corepressor ETO. In Drosophila, the RUNX factor Lozenge (LZ) is specifically expressed in and required for development of one of the two main blood cell types: the crystal cells, a megakaryocyte- like lineage that participates in clotting. In fact, LZ interacts and cooperates with the pan-hematopoietic GATA transcription factor Serpent (SRP) to activate the crystal cell differentiation program. This cooperation is conserved in mammals, where it controls megakaryopoiesis and hematopoietic stem cell development. Moreover, reminiscent of the situation in humans, RUNX1-ETO expression in the Drosophila LZ+ blood cell lineage induces a preleukemic phenotype characterized by a switch of cell fate from differentiation to self-renewal. Thus, Drosophila provides a valuable model for studying the normal and oncogenic functions of RUNX factors during hematopoiesis (Bras, 2012).

The myeloid leukemia factor (MLF) family comprises a small group of evolutionarily conserved genes whose founding member was first identified as the target of the t(3;5)(q25;q35) translocation associated with myelodysplastic syndrome (MDS) and AML. This translocation generates a fusion protein between the N-terminal region of nucleophosmin (NPM; a multifunctional nucleolar protein) and most of MLF1. Whereas NPM, which is involved in other translocations, seems to act by providing a dimerization domain and a nucleolar targeting sequence, little is known about MLF1 activity. Significantly, however, increased MLF1 expression correlates with poor prognosis in AML and with malignant progression in MDS, and MLF1 ectopic expression affects the myeloerythroid lineage switch and cell cycle progression in cell cultures. Nonetheless, MLF1 function in hematopoiesis remains poorly defined. Only one MLF gene is present in Drosophila, whereas there are two MLF paralogs in vertebrates. As in mammals, MLF encodes a nucleocytoplasmic shuttling protein with no recognizable structural domain apart from a 14-3-3 binding motif. However, its in vivo function remains unclear, given that mlf mutant flies are subviable and do not exhibit any obvious developmental defect (Bras, 2012 and references therein).

Given the conservation between MLF proteins and the parallels between mammalian and fly blood cell development, this study assessed whether MLF controls hematopoiesis in Drosophila. In particular, it was found that mlf expression is activated in the crystal cells by SRP/LZ, and that mlf regulates the expansion of this lineage. At the molecular level, the data indicate that MLF controls the number of crystal cells by protecting LZ from proteasome- mediated degradation. Interestingly, human MLF1 is able to substitute for mlf function in the crystal cell lineage. Furthermore, mlf is required for the activity and stable expression of the human leukemogenic protein RUNX1-ETO in Drosophila, whereas human MLF1 depletion causes a decrease in RUNX1- ETO protein and impairs RUNX1-ETO-dependent proliferation in human leukemic blood cells. Thus, it is proposed that the control of RUNX levels is a conserved function of MLF proteins that play an important role in the control of blood cell homeostasis (Bras, 2012).

Although deregulation of MLF1 has been linked to AML, the physiological role of MLF family members in hematopoiesis remains largely unknown. Focusing analysis on Drosophila hematopoietic development, this study has demonstrated that MLF controls blood cell homeostasis. In particular, strong evidence is provided that MLF is required to stabilize the RUNX factor LZ during crystal cell development. In addition, the findings suggest that the regulation of RUNX activity by MLF is conserved in humans, where it could play an important role in leukemogenesis. These findings reveal MLF's regulatory function in the control of crystal cell production. Actually mlf, which exhibits a rather ubiquitous expression pattern, is highly expressed in these blood cells, and mlf expression in this lineage is activated by SRP/LZ. MLF controls crystal cell number in a cell-autonomous manner, chiefly by impinging on LZ levels. It is proposed that the induction of mlf expression by SRP/LZ contributes to crystal cell development by stabilizing LZ. As such, this gene regulatory network forms a two-component positive feedback loop that drives development forward by stabilizing the expression of lineage-specific regulators. These findings also show that MLF controls lymph gland homeostasis, where it seems to promote hematopoietic progenitor maintenance. Although some of the factors controlling lymph gland development have been identified, no RUNX factor has been implicated in prohemocyte maintenance, suggesting that MLF has other partners in these cells. Of interest, MLF has been shown to bind Su(fu) and to possibly antagonize its function (Fouix, 2003). Because Su(fu) negatively regulates both Hh and Wnt pathways, which are required for prohemocyte maintenance, their premature differentiation in mlf mutants could result from increased Su(fu) activity. It is anticipated that deciphering MLF's mode of action in the lymph gland will provide valuable insight into the regulation of blood cell progenitor fate (Bras, 2012).

Along with revealing the role of MLF in hematopoiesis, these findings shed light on the function and regulation of LZ. It was found that decreased LZ levels in mlf mutant larvae resulted in an increased number of circulating LZ+ cells, but did not block these cells' differentiation, suggesting that low levels of LZ are sufficient to induce crystal cell differentiation. In addition, expansion of the pool of LZ+ cells might reflect a slowdown in the cells' rate of differentiation or a direct function of LZ in controlling blood cell proliferation or apoptosis. Along this line, LZ was shown to promote cell death in the eye, notably by regulating the expression of the Drosophila homolog of the Wilms' tumor gene 1 (WT1). Alternatively, decreased LZ levels may make crystal cell progenitors more susceptible to proliferative cues from the Notch pathway, which regulates larval crystal cell numbers. In mammals, RUNX1 acts mostly as a brake on blood cell progenitor proliferation, and decreased RUNX1 dosage, as well as MDS/AML-associated mutations or translocations affecting RUNX1, tend to promote aberrant self-renewal. Interestingly, RUNX1-ETO also promotes hematopoietic progenitor cell expansion in Drosophila, and both WT1 overexpression and activation of the Notch signaling pathway have been linked to RUNX1-ETO-induced AML. Given these similarities, characterizing the function of LZ in the control of crystal cell number may have broader implications (Bras, 2012).

Notwithstanding the evolutionary distance between human and fly, the human MLF1 protein rescued mlf-associated crystal cell defects, including LZ down-regulation, whereas mlf and MLF1 were required for the stable expression of RUNX1-ETO in Drosophila and human leukemia cells, respectively. Thus, the regulation of RUNX turnover seems to be a conserved function of MLF family members. The proteasome was found to regulate RUNX1-ETO as well as other RUNX proteins in human cells, and the current data indicate that LZ is degraded in a proteasome-dependent manner in the absence of mlf. An important area of future inquiry will be to determine more precisely how RUNX stability is regulated by MLF. This is of particular interest given that altered RUNX levels are associated with several diseases in humans, including familial platelet disorders and AML for RUNX1 and cleidocranial displasia for RUNX2. Actually, the slightly reduced LZ levels that were observed in mlf mutant eye discs suggests that the regulation of RUNX stability by MLF is not restricted to the hematopoietic system. Moreover, few genes required for RUNX1-ETO-induced AML have been identified so far, and the data suggest that MLF1 is a critical component for RUNX1-ETO leukemogenic activity. Indeed, along with the MDS/AML-associated t(3;5) translocation that generates the NPM-MLF1 fusion protein, MLF1 overexpression was correlated with malignant progression. The mechanisms underlying the oncogenic activity of NPM-MLF1 or MLF1 remain largely unknown, however. Similarly, the function of MLF1 in mammals remains poorly characterized. Exploring the relationship between MLF and RUNX factors could shed further light on MLF's role and mode of action (Bras, 2012).

Blood cell development is controlled by an intricate network of genes, the activity of which must be tightly controlled to ensure proper cell lineage choice, proliferation, and differentiation. The current findings show that the dynamic and coordinated control of gene expression through positive feedback loops participates in the fine-tuning of hematopoiesis, and provides a framework for future investigations of the cross-regulatory interactions that control blood cell fate. Finally, the results open up new avenues of research into the mode of action of MLF family members as conserved regulators of RUNX protein stability, and it is envisioned that Drosophila will provide a powerful model for deciphering MLF's function in hematopoiesis and leukemia (Bras, 2012).

Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis

In Drosophila, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). This study further characterized MLF's mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. The results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, dnaj-1 loss causes an increase in Lz+ blood cell number and size similarly as in mlf mutant larvae. Moreover dnaj-1 was found to genetically interact with mlf to control Lz level and Lz+ blood cell development in vivo. In addition, mlf and dnaj-1 loss was shown to alter Lz+ cell differentiation, and the increase in Lz+ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, high levels of Lz were shown to be required to repress Notch transcription and signaling. These data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of Notch expression and signaling to prevent aberrant blood cell development. Thus these findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo (Miller, 2017).

Proper blood cell development requires the finely tuned regulation of transcription factors and signaling pathways activity. Consequently mutations affecting key regulators of hematopoiesis such as members of the RUNX transcription factor family or components of the Notch signaling pathway are associated with several blood cell disorders including leukemia. Also, leukemic cells often present recurrent chromosomal rearrangements that participate in malignant transformation by altering the function of these factors. The functional characterization of these genes is thus of importance not only to uncover the molecular basis of leukemogenesis but also to decipher the regulatory mechanisms controlling normal blood cell development. Myeloid Leukemia Factor 1 (MLF1) was identified as a target of the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) more than 20 years ago. Further findings suggested that MLF1 could act as an oncogene or a tumor suppressor depending on the cell context and it was shown that MLF1 overexpression either impairs cell cycle exit and differentiation, promotes apoptosis, or inhibits proliferation in different cultured cell lines. Yet, its function and mechanism of action remain largely unknown (Miller, 2017).

MLF1 is the founding member of a small evolutionarily conserved family of nucleo-cytoplasmic proteins present in all metazoans but lacking recognizable domains that could help define their biochemical activity . Whereas vertebrates have two closely related MLF paralogs, Drosophila has a single mlf gene encoding a protein that displays around 50% identity with human MLF in the central conserved domain. In the fly, MLF was identified as a partner of the transcription factor DREF (DNA replication-related element-binding factor), for which it acts a co-activator to stimulate the JNK pathway and cell death in the wing disc. MLF has been shown to bind chromatin, as does its mouse homolog, and it can either activate or repress gene expression by a still unknown mechanism. MLF also interacts with Suppressor of Fused, a negative regulator of the Hedgehog signaling pathway, and, like its mammalian counterpart, with Csn3, a component of the COP9 signalosome, but the functional consequences of these interactions remain elusive. Interestingly the overexpression of Drosophila MLF or that of its mammalian counterparts can suppress polyglutamine-induced cytotoxicity in fly and in cellular models of neurodegenerative diseases. Moreover phenotypic defects associated with MLF loss in Drosophila can be rescued by human MLF1. Thus MLF function seems conserved during evolution and Drosophila appears to be a genuine model organism to characterize MLF proteins (Miller, 2017).

Along this line, the role of MLF during Drosophila hematopoiesis has been studied. Indeed, a number of proteins regulating blood cell development in human, such as RUNX and Notch, also control Drosophila blood cell development. In Drosophila, the RUNX factor Lozenge (Lz) is specifically expressed in crystal cells and it is absolutely required for the development of this blood cell lineage. Crystal cells account for ±4% of the circulating larval blood cells; they are implicated in melanization, a defense response related to clotting, and they release their enzymatic content in the hemolymph by bursting. The Notch pathway also controls the development of this lineage: it is required for the induction of Lz expression and it contributes to Lz⁺ cell differentiation as well as to their survival by preventing their rupture. Interestingly, the previous analysis revealed a functional and conserved link between MLF and RUNX factors. In particular, MLF was shown to control Lz activity and prevent its degradation in cell culture, and the regulation of Lz level by MLF is critical to control crystal cell number in vivo. Intriguingly, although Lz is required for crystal cell development, mlf mutation causes a decrease in Lz expression but an increase in crystal cell number. In human, the deregulation of RUNX protein level is associated with several pathologies. For instance haploinsufficient mutations in RUNX1 are linked to MDS/AML in the case of somatic mutations, and to familial platelet disorders associated with myeloid malignancy for germline mutations. In the opposite, RUNX1 overexpression can promote lymphoid leukemia. Understanding how the level of RUNX protein is regulated and how this affects specific developmental processes is thus of particular importance (Miller, 2017).

To better characterize the function and mode of action of MLF in Drosophila blood cells, this study used proteomic, transcriptomic and genetic approaches. In line with recent findings, MLF was found to bind DnaJ-1, a HSP40 co-chaperone, as well as the HSP70 chaperone Hsc70-4, and that both of these proteins are required to stabilize Lz. It was further shown that MLF and DnaJ-1 interact together but also with Lz via conserved domains and that they regulate Lz-induced transactivation in a Hsc70-dependent manner in cell culture. In addition, using a null allele of dnaj-1, it was shown to control Lz⁺ blood cell number and differentiation as well as Lz activity in vivo in conjunction with mlf. Notably, w mlf or dnaj-1 loss leads to an increase in Lz⁺ cell number and size due to the over-activation of the Notch signaling pathway. Interestingly, these results indicate that high levels of Lz are required to repress Notch expression and signaling. A model is proposed whereby MLF and DnaJ-1 control Lz⁺ blood cell growth and number by promoting Lz accumulation, which ultimately turndowns Notch signaling. These findings thus establish a functional link between the MLF/Dna-J1 chaperone complex and the regulation of a RUNX-Notch axis required for blood cell homeostasis in vivo (Miller, 2017).

Members of the RUNX and MLF families have been implicated in the control of blood cell development in mammals and Drosophila and deregulation of their expression is associated with human hemopathies including leukemia. The current results establish the first link between the MLF/DnaJ-1 complex and the regulation of a RUNX transcription factor in vivo. In addition, these data show that the stabilization of Lz by the MLF/DnaJ-1 complex is critical to control Notch expression and signaling and thereby blood cell growth and survival. These findings pinpoint the specific function of the Hsp40 chaperone DnaJ-1 in hematopoiesis, reveal a potentially conserved mechanism of regulation of RUNX activity and highlight a new layer of control of Notch signaling at the transcriptional level (Miller, 2017).

MLF binds DnaJ-1 and Hsc70-4, and these two proteins, like MLF, are required for Lz stable expression in Kc167 cells. In addition, these data show that MLF and DnaJ-1 bind to each other via evolutionarily conserved domains and also interact with Lz, suggesting that Lz is a direct target of a chaperone complex formed by MLF, DnaJ-1 and Hsc70-4. Of note, a systematic characterization of Hsp70 chaperone complexes in human cells identified MLF1 and MLF2 as potential partners of DnaJ-1 homologs, DNAJB1, B4 and B6, a finding corroborated by Dyer (2017). Therefore, the MLF/DnaJ-1/Hsc70 complex could play a conserved role in mammals, notably in the regulation of the stability of RUNX transcription factors. How MLF acts within this chaperone complex remains to be determined. In vivo, this study demonstrated that dnaj-1 mutations lead to defects in crystal cell development strikingly similar to those observed in mlf mutant larvae, and these two genes were shown to act together to control Lz⁺ cells development by impinging on Lz activity. The data suggest that in the absence of DnaJ-1, high levels of MLF lead to the accumulation of defective Lz protein whereas lower levels of MLF allow its degradation. Thus it is proposed that MLF stabilizes Lz and, together with DnaJ-1, promotes its proper folding/conformation. In humans, DnaJB4 stabilizes wild-type E-cadherin but induces the degradation of mutant E-cadherin variants associated with hereditary diffuse gastric cancer. Thus the fate of DnaJ client proteins is controlled at different levels and MLF might be an important regulator in this process (Miller, 2017).

This work presents the first null mutant for a gene of the DnaJB family in metazoans and the results demonstrate that a DnaJ protein is required in vivo to control hematopoiesis. There are 16 DnaJB and in total 49 DnaJ encoding genes in mammals and the expansion of this family has likely played an important role in the diversification of their functions. DnaJB9 overexpression was found to increase hematopoietic stem cell repopulation capacity and Hsp70 inhibitors have anti-leukemic activity, but the participation of other DnaJ proteins in hematopoiesis or leukemia has not been explored. Actually DnaJ's molecular mechanism of action has been fairly well studied but there are only limited insights as to their role in vivo. Interestingly though, both DnaJ-1 and MLF suppress polyglutamine protein aggregation and cytotoxicity in Drosophila models of neurodegenerative diseases, and this function is conserved in mammals. It is tempting to speculate that MLF and DnaJB proteins act together in this process as well as in leukemogenesis. Thus a better characterization of their mechanism of action may help develop new therapeutic approaches for these diseases (Miller, 2017).

As shown in this study, mlf or dnaj-1 mutant larvae harbor more crystal cells than wild-type larvae. This rise in Lz⁺ cell number is not due to an increased induction of crystal cell fate as we could rescue this defect by re-expressing DnaJ-1 or Lz with the lz-GAL4 driver, which turns on after crystal cell induction, and it was also observed in lz hypomorph mutants, which again suggests a post-lz / cell fate choice process. Moreover mlf or dnaj-1 mutant larvae display a higher fraction of the largest lz>GFP⁺ cell population, which could correspond to the more mature crystal cells. It is thus tempting to speculate that mlf or dnaj-1 loss promotes the survival of fully differentiated crystal cells. RNAseq data demonstrate that mlf is critical for expression of crystal cell associated genes, but both up-regulation and down-regulation of crystal cell differentiation markers were observed in mlf or dnaj-1 mutant Lz⁺ cells. Also these changes did not appear to correlate with crystal cell maturation status since alterations were found in gene expression in the mutants both in small and large Lz⁺ cells. In addition the transcriptome did not reveal a particular bias toward decreased expression for 'plasmatocyte' markers in Lz⁺ cells from mlf^- mutant larvae. Thus, it appears that MLF and DnaJ-1 loss leads to the accumulation of mis-differentiated crystal cells (Miller, 2017).

The data support a model whereby MLF and DnaJ-1 act together to promote Lz accumulation, which in turn represses Notch transcription and signaling pathway to control crystal cell size and number. Indeed, an abnormal maintenance of Notch expression was observed in the larger Lz⁺ cells as well as an over-activation of the Notch pathway in the crystal cell lineage of mlf and dnaj-1 mutants or when Lz activity was interfered with. Moreover the data as well as previously published experiments show that Notch activation promotes crystal cell growth and survival. Importantly too the increase in Lz⁺ cell number and size observed in mlf or dnaJ-1 mutant is suppressed when Notch dosage is decreased. Yet, some of the mis-differentiation phenotypes in the mlf or dnaj-1 mutants might be independent of Notch since changes in crystal cell markers expression seem to appear before alterations in Notch are apparent. At the molecular level, the results suggest that Lz directly represses Notch transcription as this study identified a Lz-responsive Notch cis-regulatory element that contains conserved RUNX binding sites. The activation of the Notch pathway in circulating Lz⁺ cells is ligand-independent and mediated through stabilization of the Notch receptor in endocytic vesicles. Hence a tight control of Notch expression is of particular importance to keep in check the Notch pathway and prevent the abnormal development of the Lz⁺ blood cell lineage. Notably, Notch transcription was shown to be directly activated by Notch signaling. Such an auto-activation loop might rapidly go awry in a context in which Notch pathway activation is independent of ligand binding. By promoting the accumulation of Lz during crystal cell maturation, MLF and DnaJ-1 thus provide an effective cell-autonomous mechanism to inhibit Notch signaling. Further experiments will now be required to establish how Lz represses Notch transcription. RUNX factors can act as transcriptional repressors by recruiting co-repressor such as members of the Groucho family. Whether MLF and DnaJ-1 directly contribute to Lz-induced-repression in addition to regulating its stability is an open question. MLF and DnaJ-1 were recently found to bind and regulate a common set of genes in cell culture. They may thus provide a favorable chromatin environment for Lz binding or be recruited with Lz and/or favor a conformation change in Lz that allows its interaction with co-repressors. The scarcity of lz>GFP⁺ cells precludes a biochemical characterization of Lz, MLF and DnaJ-1 mode of action notably at the chromatin level but further genetic studies should help decipher their mode of action. While the post-translational control of Notch has been extensively studied, its transcriptional regulation seems largely overlooked. The current findings indicate that this is nonetheless an alternative entry point to control the activity of this pathway. Given the importance of RUNX transcription factor and Notch signaling in hematopoiesis and blood cell malignancies, it will be of particular interest to further study whether RUNX factors can regulate Notch expression and signaling during these processes in mammals (Miller, 2017).

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