InteractiveFly: GeneBrief

Dorsal interacting protein 3: Biological Overview | References


Gene name - Dorsal interacting protein 3

Synonyms -

Cytological map position - 55B7-55B7

Function - Transcription factor

Keywords - Immune response, eye, Dorsal-ventral patterning

Symbol - Dlip3

FlyBase ID: FBgn0040465

Genetic map position - 2R:14,016,005..14,017,751 [+

Classification - MADF subfamily of SANT domain, and BESS motif

Cellular location - nuclear



NCBI link: EntrezGene

Dlip3 orthologs: Biolitmine
BIOLOGICAL OVERVIEW

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal (DL) Rel homology domain. This study shows that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters (Ratnaparkhi, 2009).

The Drosophila genome encodes three rel homology domain (RHD) containing proteins, Dorsal (Dl), Dorsal-related immunity factor (Dif), and Relish (Rel). The RHD, which is also found in the human NFκB family of transcriptional activators, mediates dimerization and sequence-specific DNA binding. Rel/NFκB family proteins in vertebrates and invertebrates play central roles in the innate immune response by triggering the expression of antimicrobial defense genes in response to signals transduced by Toll and the Immune deficiency (Imd) signal transduction pathways. In Drosophila, Dl also directs dorsoventral (D/V) patterning of the embryo. Specifically, the regulated nuclear localization of maternally expressed Dl in response to Toll signaling in the embryo leads to the formation of a ventral-to-dorsal nuclear concentration gradient of Dl and to the spatially restricted regulation of a large number of genes, including twist (twi), snail (sna), and rhomboid (rho), which are activated by Dl, and zerknullt and decapentaplegic, which are repressed by Dl. This serves to subdivide the embryo into multiple developmental domains along its D/V axis (Ratnaparkhi, 2009).

Unlike Dl, Dif and Rel are not required for D/V patterning. Instead, these two rel-family proteins function along with Dl in the innate immune response. Toll signaling in the immune system leads to the translocation of Dl and Dif to the nucleus and the consequent activation of a subset of anti-microbial defense genes, including drosomycin (drs) and Immune induced molecule 1. Dl and Dif are believed to have redundant roles in this process and thus either one alone is sufficient for the induction of drs. Activation of the Imd signal transduction pathway, leads to proteolytic cleavage of Rel. The N-terminal region of Rel, which contains the RHD, then translocates into the nucleus where it activates expression of anti-bacterial genes, such as diptericin (dipt), cecropin-A1 (cec-A), and attacin-A. Dl, Dif, and Rel homo- and hetero-dimerize to activate different subsets of the anti-microbial defense genes in response to signals from the Toll and Imd pathways (Ratnaparkhi, 2009).

Very little is known about the identity of factors that assist the RHD proteins in the activation of the anti-microbial defense genes. Proteins that modulate expression of these genes include transcription factors such as the GATA factor Serpent (Srp), Hox factors, Helicase89B, and an unknown protein that binds region 1 (R1), a regulatory module in cec-A and other anti-microbial defense genes. In addition, a recent screen identified several POU domain proteins as potential regulators of anti-microbial defense genes (Ratnaparkhi, 2009).

To date, about a dozen proteins that interact directly with Dl and modulate its regulatory functions have been identified by genetic and biochemical means. For example, an interaction between Dl and Twist (Twi) enhances the activation of Dl target genes, while an interaction between Dl and Groucho (Gro) is essential for Dl-mediated repression. A yeast two-hybrid screen to identify Dl interacting proteins yielded, in addition to the well characterized Dl-interactors Twi and Cactus, four novel Dl-interactors (Dip1, Dip2, Dip3, and Dip4/Ubc9; Bhaskar, 2000). Conjugation of SUMO to Dl by Ubc9 was subsequently shown to result in more potent activation by Dl (Ratnaparkhi, 2009).

Dip3 belongs to a family of proteins that contain both MADF (for Myb/SANT-like in ADF) and BESS (for BEAF, Stonewall, SuVar(3)7-like) domains. While MADF-BESS domain proteins are found in both insects and vertebrates, only a few have been characterized and their functions are largely unknown. The Drosophila genome encodes 14 MADF-BESS domain factors. In addition to Dip3, these include Adf-1, which was initially found as an activator of Alcohol dehydrogenase, and Stonewall, which is required for oogenesis (Clark, 1996; England, 1990). The Dip3 MADF domain mediates sequence specific binding to DNA, while the Dip3 BESS domain mediates binding to a subset of TATA binding protein associated factors as well as to the Dl RHD and to Twi (Bhaskar, 2002). In addition to functioning as an activator, Dip3 can function as a coactivator to stimulate synergistic activation by Dl and Twi in S2 cells (Ratnaparkhi, 2009).

This study shows that Dip3 assists RHD proteins during both embryonic development and the innate immune response. By stimulating the expression of antimicrobial defense genes, Dip3 improves survival of both larvae and adults following septic injury. The presence of Dip3 near the promoters of antimicrobial defense genes depends upon Rel family proteins suggesting that Dip3 functions as a coactivator at these promoters (Ratnaparkhi, 2009).

It has been shown that Dip3, which binds both Dl and Twi via its BESS domain, synergistically enhances the activation of a luciferase reporter with multiple Dl and Twi binding sites upstream of the promoter (Bhaskar, 2002). In addition, Dip3 has been implicated as the 'mystery protein' which binds to sites adjacent to Dl and Twi binding sites in a subset of Dl target genes (Erives, 2004). Therefore the ability of Dip3 to enhance the expression of the Dl target promoters twi, sna, and rho in S2 cell transient transfection assays was examined. All three promoters require both Dl and Twi for full activity. Dip3 was found to synergize with Dl and Twi in the activation of the sna and twi promoters, but not in the activation of the rho promoter (Ratnaparkhi, 2009).

A polyclonal antibody against recombinant Dip3 was generated, and used to determine where and when Dip3 is present in the embryo. Maternally expressed Dip3 is observed in all nuclei as early as nuclear cycle 7. It was detected in subsequent nuclear cycles during formation of the Dl nuclear concentration gradient. In interphase embryonic as well as S2 cell nuclei, Dip3 localizes to nuclear speckles of unknown identity. During mitosis Dip3 is enriched on chromosomes. It associates with the centrosome proximal portion of the anaphase chromatids and the inside ring of the polar body rosette suggesting a predominant pericentromeric location at this stage of the cell cycle and hinting at a possible role of Dip3 in centromeric function. Confirming the specificity of the antibodies, the immunoreactivity is absent from Dip31 embryos in which the Dip3 transcriptional and translational start sites as well as a large segment of the Dip3 coding region have been deleted. Weak Dip3 expression is also detected in the larval fat body (Ratnaparkhi, 2009).

Homozygous Dip31 flies are viable and fertile, indicating that Dip3 cannot have an essential role in embryonic D/V pattern formation. However, a small proportion (7±4%) of the embryos fail to hatch and exhibit D/V patterning defects. Embryos produced by females transheterozygous for Dip31 and a deficiency that removes a portion of the second chromosome containing the Dip3 gene (Df(PC4) exhibit similar embryonic lethality (10%) and D/V patterning defects. Also, maternal overexpression of Dip3 using the Gal4-UAS system leads to 54±9 % embryonic lethality with cuticles of the dead embryos showing both anteroposterior and D/V patterning defects, indicating that Dip3 may have a role in embryonic pattern formation (Ratnaparkhi, 2009).

Consistent with a non-essential role for Dip3 in D/V patterning, a Dip3 mutation enhances the temperature sensitive dl haploinsufficieny phenotype. The degree of dorsalization is often quantified by categorizing embryos on a scale from D0 (completely dorsalized, lacking all dorsoventral pattern elements other than dorsal epidermis) to D3 (inviable, but with little or no apparent defect in the cuticular pattern). At 29°, about half the dead embryos produced by dl1/+ females exhibit detectable D/V patterning defects and the majority of these fall into the D2 category (moderately dorsalized, exhibiting mildly expanded ventral denticle belts and a twisted germ band). Removal of maternal Dip3 increases the proportion of dorsalized embryos to about 75% with most of the increase being due to an increase in the number of D2 embryos. The effect seems to be strictly maternal as the paternal genotype does not modulate the dl haploinsufficiency phenotype (Ratnaparkhi, 2009).

Dip3 is present in the fat body, the organ in which RHD factors activate antimicrobial defense genes in response to infection. Since Dip3 binds the Dl RHD, the role of Dip3 in the innate immune response was examined by assessing the sensitivity of Dip31 flies to bacterial and fungal infection. Wild-type and Dip31 adults and larvae were injected with gram positive bacteria (M. luteus), gram negative bacteria (E. coli), and fungi (B. brassiana). For comparison, flies were infected that contained mutations in known components of the Toll (spzrm7) and Imd (RelE20) pathways. Wild-type, RelE20, spzrm7, and Dip31 adults showed little lethality (<15%) 30 days after mock infection. However, the Dip31 adult flies exhibited 55% lethality one month after injection with a 1:1 mixture of M. luteus and E. coli, compared to 10% lethality after 30 days for wild-type flies and 98% after 30 days for RelE20 flies. In contrast, wild-type and Dip31 adults were equally sensitive to fungal infection, both showing 55-70% lethality after 30 days compared to 100% lethality after 22 days for RelE20 adults and 100% lethality after 7 days for spzrm7 adults. Similar results were seen in larvae in which Dip31, RelE20 and spzrm7 mutations resulted in reduced rates of eclosion following septic injury compared to wild-type. The effectiveness of the immune challenge was further verified by an experiment showing that septic injury leads to translocation of Dl into the nucleus (Ratnaparkhi, 2009).

To determine if the sensitivity of Dip31 flies to infection results from reduced induction of antimicrobial peptides, the expression of dipt, drs and cec-A was monitored as a function of time following septic injury. Relative to uninfected flies, the levels of expression of drs and dipt were reduced by the Dip31 mutation, especially at the 2 and 4 hr time points, while the levels of cec-A expression were not significantly altered. Thus, some, but not all, antimicrobial defense genes that are regulated by RHD family proteins exhibit dependence on Dip3. At the 4 hr time point, relative to infected, wild type flies, the spzrm7 mutation reduced drs expression to basal levels while the RelE20 mutation reduced dipt expression ten fold (Ratnaparkhi, 2009).

Dip3 was over expressed in the larvae using the Cg-Gal4 driver to examine the effect of increasing levels of Dip3 on the expression of antimicrobial defense genes in the fat body. Cec-A and drs levels were unaffected, while dipt levels increased two-fold in infected flies. Thus, both loss-of-function and over expression data are consistent with the conclusion that Dip3 makes the immune response more robust by elevating the expression of a subset of antimicrobial defense genes (Ratnaparkhi, 2009).

Radiolabeled Dip3 interacts with FLAG-tagged Dl and Rel immobilized on anti-FLAG beads. Similarly, immobilized FLAG-Dip3 binds Dl (Bhaskar, 2002) and Rel (Residues 1-600). Dip3 binds to DNA via its MADF domain and to the RHD via its BESS domain, and can thus function either as an activator or as a coactivator (Bhaskar, 2002). To determine if Dip3 is present at the promoters of antimicrobial defense genes, ChIP assays were carried out in S2 cells transfected with FLAG-Dip3. FLAG antibody was used to immunoprecipitate Dip3 crosslinked to chromatin. Compared both to mock-transfected cells and to the transcribed region of a ribosomal protein-encoding gene (rp49), Dip3 was highly enriched at the drs, dipt and cecA promoters. As expected, dsRNA directed against Dip3 eliminated the ChIP signal verifying antibody specificity. The association of Dip3 with the promoters of the anti-microbial defense genes depended on Rel family proteins, since knockdown of these proteins by dsRNAi significantly reduced association of Dip3 with the promoters. Similar results were observed with an anti-GFP antibody and cells expressing a Dip3-GFP fusion protein (Ratnaparkhi, 2009).

These results suggest that Dip3 may synergize with RHD proteins in multiple developmental contexts possibly through contact with the Dl rel homology domain. Dip3 is expressed maternally and present in cleavage stage nuclei at the time that Dl is functioning to pattern the D/V axis. Furthermore, Dip3 can potentiate Dl-mediated activation of the twist and snail promoters in S2 cells. These observations suggest that Dip3 might have a role in D/V patterning. Consistent with this possibility, it was found that removal of maternal Dip3 results in occasional D/V patterning defects and significantly enhances the dl haploinsufficiency phenotype suggesting the Dip3 renders D/V patterning more robust perhaps by assisting in Dl-mediated activation (Ratnaparkhi, 2009).

An important aspect of the immune response is activation in the fat body of genes encoding antimicrobial peptides by the Rel family transcription factors Dl, Dif, and Rel. This study found that synergistic killing of flies by a mixture of E.coli and M. luteus is enhanced in Dip31 flies. This suggests roles for Dip3 in the Imd and/or Toll pathways, which mediate the response to microbial infection. In accord with this idea, it was found that activation of the Imd pathway target dipt and the Toll pathway target drs are compromised in Dip3 mutant larvae (Ratnaparkhi, 2009).

To determine if the role of Dip3 at antimicrobial defense gene promoters is direct, ChIP assays were carried out demonstrating that this factor associates directly with the drs, dipt, and cec-A promoters in S2 cells. Since Dip3 contains a DNA binding domain, it is possible that it binds to these promoters through a direct interaction with DNA. However, with one exception in the drs promoter, these promoters lack matches for the consensus Dip3 binding sites. Thus, Dip3 may be acting as a coactivator at these promoters consistent with its ability to bind the rel homology domain. In support of this idea, it was found that simultaneous knockdown of all three rel family proteins significantly reduced recruitment of Dip3 to the promoters (Ratnaparkhi, 2009).

The mechanism of Dip3 co-activation remains unclear. The finding that the Dip3 BESS domain binds TAFs (Bhaskar, 2002) suggests a role for Dip3 in the recruitment of the basal machinery. In addition, the MADF domain is closely related to the SANT domain, which binds histone tails and may have a role in interpreting the histone code. While analysis of RHD targets suggests roles for Dip3 in activation, Dip3 also associates with pericentromeric heterochromatin during mitosis, consistent with a possible role in silencing. Other heterochromatic proteins including a suppressor of position effect variegation (Su(Var)3-7) also contain BESS domains. However, the loss of Dip3 does not appear to modify position effect variegation (Ratnaparkhi, 2009).

In flies, additional roles for RHD-mediated activation have been demonstrated in haematopoesis, neural fate specification, and glutamate receptor expression. Antimicrobial defense genes are also expressed constitutively in barrier epithelia and in the male and female reproductive tracts. It will be interesting to determine if Dip3 is involved in rel protein-dependent and independent gene activation in some or all of these tissues. One tissue in which Dip3 appears to have clear rel-independent functions is in the developing compound eye, where Dip3 overexpression results in conversion of eye to antenna (Duong, 2008a), while Dip3 loss-of-function leads to mispatterning of the retina (Duong, 2008b: Ratnaparkhi, 2009).

Transformation of eye to antenna by misexpression of a single gene

In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dip3, a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc; advantage was taken of this observation to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2009a).

Although previous studies also suggested a link between growth and developmental fate in the eye-antennal disc, the current study adds a number of new insights: (1) Previous studies showed that inhibiting growth of the eye disc leads to overproliferation and duplication of the antennal disc. The current study supports this idea, but also shows that antennal disc overproliferation is not sufficient for duplication, which may also involve communication between the eye and antennal discs. (2) This is first study to suggest a connection between the cell-cycle progression and the eye-antennal decision (Duong, 2009a).

Loss-of-function alleles of Dip3 demonstrate a role for Dip3 in late eye development, but they do not show a role in early fate determination in the eye-antennal field. This may be due to redundancy as there are 14 other homologous MADF/BESS domain transcription factors encoded in the Drosophila genome (Duong, 2009a).

Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2009a).

In this study, it was also found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2009a).

Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments were observed tipped with eye tissue. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2009a).

The arguments presented above support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility cannot be excluded that these two phenotypes are two manifestations of a single mechanism. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2009a).

The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2009a).

Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2009a).

While the results imply that antennal disc overgrowth is required for antennal duplication, it is not believed that overgrowth is sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver was used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2009a).

The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2009a).

Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) Activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation (Duong, 2009a).

Any one of these three conditions by itself is not sufficient: (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2009a).

This study found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between ey and dac. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2009a).

Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2009a).

The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented in this study that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ (Johnston’s organ) associated with the antenna. Small mechanosensory sensilla, such as Johnston’s organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2009a).

Non-cell-autonomous inhibition of photoreceptor development by Dip3

The Drosophila MADF/BESS domain transcription factor Dip3, which is expressed in differentiating photoreceptors, regulates neuronal differentiation in the compound eye. Loss of Dip3 activity in photoreceptors leads to an extra photoreceptor in many ommatidia, while ectopic expression of Dip3 in non-neuronal cells results in photoreceptor loss. These findings are consistent with the idea that Dip3 is required non-cell autonomously to block extra photoreceptor formation. Dip3 may mediate the spatially restricted potentiation of Notch (N) signaling since the Dip3 misexpression phenotype is suppressed by reducing N signaling and misexpression of Dip3 leads to ectopic activity of a N-responsive enhancer. Analysis of mosaic ommatidia suggests that no specific photoreceptor must be mutant to generate the mutant phenotype. Remarkably, however, mosaic pupal ommatidia with three or fewer Dip3+ photoreceptors always differentiate an extra photoreceptor, while those with four or more Dip3+ photoreceptors never differentiate an extra photoreceptor. These findings are consistent with the notion that Dip3 in photoreceptors activates a heretofore unsuspected diffusible ligand that may work in conjunction with the N pathway to prevent a subpopulation of undifferentiated cells from choosing a neuronal fate (Duong, 2009b).

This analysis suggests that expression of Dip3 in one cell population suppresses neuronal differentiation of other cells around it. Loss of Dip3 expression in photoreceptors appears to release the neuronal fate suppression in non-neuronal cells, leading to the differentiation of an extra photoreceptor in each ommatidium. Consistent with this interpretation, when Dip3 is misexpressed in undifferentiated and/or primary pigment cells, which normally do not express Dip3, it inhibits neural precursors from assuming the neuronal fate. However, the possibility cannot be formally ruled out that the loss-of-function phenotype and the gain-of-function phenotype are mechanistically distinct (Duong, 2009b).

Along with inhibiting photoreceptor development, misexpression of Dip3 also leads to ectopic non-neuronal cell specification as reflected by the appearance of extra cone and pigment cells. Whether the extra cone and pigment cells originate from the inhibited neuronal precursors or from undifferentiated cells is unclear. However, some pupal ommatidia contain 8 photoreceptors and 5 cone cells implying that at least some of the extra non-neuronal cells originate from undifferentiated cells as opposed to inhibited neuronal precursors. Furthermore, the Dip3 mutant ommatidia contain the normal number of cone and pigment cells indicating that transformed cone or pigment cells cannot be the source of the extra photoreceptors that result from Dip3 loss-of-function. Therefore, Dip3 must possess at least two independent functions: inhibition of neuronal and promotion of non-neuronal specification (Duong, 2009b).

These two properties are also seen in N signaling. In lateral inhibition, the activation of N in R8 inhibits the surrounding cells from assuming the neuronal fate. Furthermore, in the R7 equivalence group, in which EGFR is active in all cells, the level of N activity determines cell fate. Cells with low N activity differentiate into photoreceptors, while cells with high N activity differentiate into cone cells. Furthermore, restricted activation of Notch in late eye development leads to loss of photoreceptors and extra cone and pigment cells similar to over-expression of Dip3. Thus, there is likely to be an interaction between Dip3 and N signaling. Consistent with this hypothesis, reduction of N signaling suppresses the Dip3 over-expression phenotype, and ectopic expression of Dip3 leads to ectopic expression of a reporter under control of a N-responsive enhancer. Finally, misexpression of Dip3 in the wing, where Dip3 is not normally expressed, inhibits wing vein development, a phenotype similar to the N over-expression phenotype (Duong, 2009b).

While the data suggest that Dip3 potentiates N signaling, they are not consistent with the notion that Dip3 simply triggers the N signaling pathway in an indiscriminate manner. While the N signal is essential and involved in many diverse aspects of development, Dip3 is not an essential gene. Furthermore, N laterally inhibits neuronal development in all cells that surround the signal-emitting cell, but Dip3 normally only suppresses one cell from assuming the neuronal fate. Therefore, Dip3 is apparently responsible for only a subset of N functions. Consistent with this interpretation, misexpression of Dip3 in the eye results in ectopic activation of a N reporter in only a subset of photoreceptors, while misexpression of Dip3 in the wing inhibits formation of only the anterior and posterior cross veins, along with the distal segment of the L5 longitudinal vein. How Dip3 is able to have these spatially restricted effects on N signaling is still unknown. It is likely, however, that the key to this spatial restriction lies in the need for combinatorial interactions between Dip3 and other spatially restricted signaling pathways or transcription factors (Duong, 2009b).

Mosaic analysis shows that no single photoreceptor must be mutant to generate the mutant phenotype. However, the possibility cannot be competely excluded that certain subsets of photoreceptors must be mutant to generate the mutant phenotype. The cells of the R7 equivalence group arise after the second mitotic wave and so may tend to be simultaneously mutant in mosaic ommatidia more often than would be expected if there were no lineage relationships at all between R cells. Thus, one possibility is that it is sufficient for all three photoreceptors of the R7 equivalence group (R1, R6, and R7) to be mutant to generate the extra photoreceptor. However, all the mosaic ommatidia with an extra R cell (all those with nine R cells in total) contain at least six mutant R cells. Thus, it is not sufficient for all the cells of the R7 equivalence group to be mutant to generate the mutant phenotype. Alternatively, it is possible that all the precluster R cells (R2, R3, R4, R5, and R8) must be mutant to generate the mutant phenotype. However, this seems unlikely as it would imply that the five precluster R cells are always mutant together in mosaic ommatidia containing five or more mutant R cells. This would, in turn, suggest a much stronger lineage relationship between R cells than has been previously observed. Thus, it is concluded that it is likely to be the total number of Dip3+ R cells that determines whether or not an extra photoreceptor is recruited to an ommatidium (Duong, 2009b).

The finding that any four Dip3+ photoreceptors are sufficient to prevent the mutant phenotype seems most consistent with the idea of a diffusible factor that must accumulate to a minimum level to inhibit extra photoreceptor development. In theory, this diffusible factor could be a N ligand or coligand. However, given that known N ligands are membrane bound proteins, the notion is favored that a diffusible factor activates a separate pathway that works in parallel with the N pathway to prevent extra photoreceptor development, perhaps by synergistically stimulating N target genes. If it is assumed that this parallel pathway is required for some, but not all, N functions, then this mechanism would explain the specificity of the phenotype (Duong, 2009b).

If Dip3 directly activates transcription of a diffusible factor, misexpression of Dip3 might be expected to produce the same gain-of-function phenotype regardless of where within the developing ommatidium it was misexpressed. Therefore, the observation that misexpression of Dip3 in cone cells does not produce a gain-of-function phenotype, while misexpression in undifferentiated cells does suggests that Dip3 may not directly activate the ligand. Rather it may be required for the modification of a ligand that is absent from cone cells, but present in undifferentiated cells. Alternatively, the lack of a phenotype due to cone cell misexpression of Dip3 could also result if all the photoreceptors are already specified by the time Dip3 is expressed in the cone cells (Duong, 2009b).

The remarkable finding that four Dip3+ cells are always sufficient while three Dip3+ cells are always insufficient to produce a mutant ommatidium suggest that the system is exquisitely tuned to the concentration of the hypothetical diffusible ligand. A number of mechanisms (e.g., zero order ultrasensitivity) have been proposed to explain this kind of extreme sensitivity to the concentration of a ligand (Melen, 2005). Extreme concentration sensitivity would also explain the observation that the non-cell-autonomous effects apparently do not spread between ommatidia (Duong, 2009b).

The MADF-BESS transcription factors each contain a DNA binding domain (the MADF domain) and an activation domain (the BESS domain) and can function as both activators and coactivators (Bhaskar, 2002). To assess the relative importance of the MADF and BESS domains, an unbiased screen was carried out for new Dip3 loss-of-function alleles. All four missense mutations identified map to the MADF domain suggesting that DNA binding is essential for the function of Dip3 in retinal patterning. Although the screen did not identify missense mutations in the BESS domain, the conserved architecture of MADF-BESS domain factors suggests that this domain is nonetheless critical. In support of this speculation, misexpression of a Dip3 deletion lacking the BESS domain with the ey-Gal4 or GMR-Gal4 driver did not produce the lethality or the smooth eye phenotype associated with the expression of full-length Dip3 (Duong, 2009b).

The high degree of conservation of the MADF-BESS domain architecture is surprising. 36 of the 57 known BESS domain-containing proteins also contain a MADF domain, while 36 of the 141 known MADF domain-containing proteins also contain a BESS domain. Although most common in flies, this architecture is conserved across phyla. For example, two of the three recognized MADF domain-containing factors in zebrafish also contain BESS domains. This level of conservation is unusual among sequence-specific transcription factors where it is generally impossible to detect homology across phyla outside the DNA binding domain. The frequency with which the MADF and BESS domains are found together suggests that they interact with one another in the developmental control of gene expression (Duong, 2009b).

The MADF-BESS domain factor Dip3 potentiates synergistic activation by Dorsal and Twist

The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. A novel protein named Dorsal-interacting protein 3 (Dip3) has been identified that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. The possibility is assessed that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators (Bhaskar, 2002).


REFERENCES

Search PubMed for articles about Drosophila Dip3

Bhaskar, V., Valentine, S. A. and Courey. A. J. (2000). A functional interaction between dorsal and components of the Smt3 conjugation machinery. J. Biol. Chem. 275(6): 4033-40. PubMed ID: 10660560

Bhaskar, V. and Courey, A. J. (2002). The MADF-BESS domain factor Dip3 potentiates synergistic activation by Dorsal and Twist. Gene 299(1-2): 173-84. PubMed ID: 12459265

Clark, K. A. and McKearin, D. M. (1996). The Drosophila stonewall gene encodes a putative transcription factor essential for germ cell development. Development 122: 937-950. PubMed ID: 8631271

Duong, H. A., Wang, C. W., Sun, Y. H. and Courey, A. J. (2008a). Transformation of eye to antenna by misexpression of a single gene. Mech. Dev. 125(1-2): 130-41. PubMed ID: 18037276

Duong, H. A., Nagaraj, R., Wang, C. W., Ratnaparkhi, G., Sun, Y. H. and Courey, A. J. (2008b). Non-cell-autonomous inhibition of photoreceptor development by Dip3. Dev. Biol. 323(1): 105-13. PubMed ID: 18761008

England, B. P., Heberlein, U. and Tjian, R. (1990). Purified Drosophila transcription factor, Adh distal factor-1 (Adf-1), binds to sites in several Drosophila promoters and activates transcription. J. Biol. Chem. 265: 5086-5094. PubMed ID: 2318884

Erives, A. and Levine, M. (2004). Coordinate enhancers share common organizational features in the Drosophila genome. Proc. Natl. Acad. Sci. 101: 3851-3856. PubMed ID: 15026577

Melen, G. J., Levy, S., Barkai, N. and Shilo, B. Z. (2005). Threshold responses to morphogen gradients by zero-order ultrasensitivity. Mol. Syst. Biol. 1: 2005.0028. PubMed ID: 16729063

Ratnaparkhi, G. S., Duong, H. A. and Courey, A. J. (2008). Dorsal interacting protein 3 potentiates activation by Drosophila Rel homology domain proteins. Dev. Comp. Immunol. 32(11): 1290-300. PubMed ID: 18538389


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date revised: 20 February 2010

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