BIOLOGICAL OVERVIEW

Retained, also known as Dead ringer (Dri), is a founding member of a recently defined ARID family of DNA binding proteins whose members share a conserved DNA binding domain termed the A/T-rich interaction domain. This family includes the B-cell-specific factor Bright and the Drosophila factor Eyelid (Osa). dri is developmentally regulated, being expressed in a restricted set of cells including some neural cells and differentiating cells of the gut and salivary gland ducts. It is unlikely that Dri is a general transcription co-factor or chromatin modifier, as is Eyelid, since transcription of only a small number of the genes are disrupted in dri mutant embryos (Valentine, 1998 and Shandala, 1999).

Dri has been shown to be a sequence-specific DNA binding protein. The in vitro sequence specificity of Dri is strikingly similar to that of many homeodomain proteins: Dri preferentially binds the PuATTAA sequence (Gregory, 1996). It is therefore likely that the phenotypes exhibited by dri mutant embryos result from disruption to the expression of regulatory genes. In terms of specific molecular function, Dri has been shown to act in conjunction with Dorsal to recruit Groucho and repress the zerknullt minimal ventral repression region (VRR) element (Valentine, 1998). Nevertheless there are no major dorsal-ventral patterning defects in dri mutant embryos, although variable gastrulation defects are observed, consistent with some level of disruption to dorsal-ventral patterning (Shandala, 1999).

Terminal development is disrupted in the dri maternal and zygotic mutant embryos. Both head and tail defects are invariably observed in dri maternal and zygotic mutant embryos. One of the most consistent and striking phenotypes is severe disruption of the cephalo-pharyngeal skeleton. Germline and zygotic dri mutant embryos still have a recognizable dorsal bridge, dorsal and ventral arms and mouth hooks, but the H-piece and lateralgraten are missing or severely malformed. In addition, the atypical anterior position of pharyngeal muscles, visualized using anti-muscle myosin immunostaining, indicates that head involution does not proceed properly (Shandala, 1999).

The appearance of these defects prompted an examination of genes that play a role in the formation of terminal structures. Expression of the terminal gene tailless and the genes buttonhead, empty spiracles, orthodenticle and argos was examined. Of these genes, disruption to only argos (aos) and buttonhead (btd) expression was observed. In wild-type embryos, aos is initially expressed at stage 5 in two terminal domains and a domain that flanks the position of the cephalic furrow. In embryos lacking dri maternal and zygotic product, expression of aos in the terminal domains is almost completely eliminated while expression in the region of the cephalic furrow is maintained, both before and after division into two stripes at the time of cephalic furrow formation. Zygotic aos mutant embryos exhibit head defects that are similar to those observed in maternal and zygotic dri mutant embryos, indicating that the dri mutant head defects are likely to be the result of loss of anterior aos expression in the dri mutant embryos. Analysis of btd expression reveals a regulatory relationship that accounts for another consistent dri mutant phenotype, the appearance of ectopic cephalic furrows. btd expression is found to be partially derepressed in the trunk of dri germline and zygotic mutant embryos. The cephalic furrow arises where expression of the head specific gap gene btd overlaps the first stripe of expression of the primary pair rule gene eve. The repetitive appearance of ectopic cephalic furrows is therefore likely to be the result of the coincident ectopic trunk expression of btd with the more posterior eve stripes. The ectopic furrows do not progress, most probably due to the incomplete derepression of btd in this region (Shandala, 1999).

dead ringer is required for proper patterning of the abdomen. To test the basis for defects in patterning, genes required for segment formation in the Drosophila embryo were examined. Expressions of the axis patterning gene, bicoid; the gap genes hunchback, Krüppel, knirps and giant; the primary pair-rule genes even-skipped, hairy and runt, and the segment polarity genes wingless and engrailed were examined in embryos lacking germline and zygotic dri function. Most of these genes are expressed normally with respect to their role in segment formation. The variable disruption to abdominal segment formation correlates with a variable reduction in expression of engrailed and wingless (wg) in stripes 9-14. The most consistent effect on expression of the segmentation genes in the dri maternal and zygotic mutant embryos is a disruption to the expression of even-skipped (eve) stripe 4, observed in nearly all embryos lacking both maternal and zygotic dri product. Specifically, the ventrolateral portion of eve stripe 4, although initiated appropriately is not maintained in dri mutant embryos, leading to the subsequent aberrant appearance of wg stripes 7 and 8 and disruption to the parasegment 4 ventrolateral setal belts (Shandala, 1999).

The simplest interpretation of these results is that Dri can act either as an activator or as a repressor, depending on the context within which it finds itself. These different actions must depend on the combination of regulators acting on the respective position-specific cis-regulatory sequences. The differential regulation of aos and btd domains by dri occurs at a time when Dri is found in all somatic nuclei of the embryo, so that Dri must be acting to permit the proper function of other developmental regulatory factors. However, following gastrulation, dri expression becomes exquisitely tissue and stage specific (Gregory, 1996), raising the possibility that it may specify spatial-specific expression at later stages of development. It is presumed that Dri, like Osa, which is a member of the Trithorax Group of genes implicated in the modification of chromatin structures required for epigenetic regulation (Vazquez, 1999), is acting to establish stable chromatin structures. These structures may favor, but not be essential for, the formation of complexes in which other transcriptional regulators act, so that the absence of factors such as Dri introduces an element of chance into the stable formation of such complexes; consequently, there is an introduction of variability in gene expression and mutant phenotype, a phenomenon repeated observed for Dri (Shandala, 1999).

Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors

Mutation in the Drosophila retained/dead ringer (retn) gene leads to female behavioral defects and alters a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body ß-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. It is posited that some of these retn-expressing cells function in females to repress a male behavioral pathway activated in males by fru^M (Ditch, 2005). retn females show one behavior not shown by dsf, dsx or fru females: male-like courtship of females and males, especially as they age. retn females follow, tap and appear to sing. Although not as robust as male courtship (following is not as sustained, full wing extension and vibration are not seen, and copulatory bending is weak or absent), these behaviors highly resemble courtship. These behaviors vary between and within allelic combinations, but when the behaviors are seen they are striking and continue for hours. retn^z2-428/retn^dri8 females, which show the most consistent behaviors, with maximum penetrance at 3-4 weeks post-eclosion, averaged 42 courtship events per 5-minute observation period, while control females display fewer than three courtship-like events in the same period. Although male behaviors are evident, the fruM-dependent Muscles of Lawrence are not seen in retn females (Ditch, 2005).

Aspects of the retn female behaviors are similar to wild-type female defenses of food and egg-laying resources. One study on Drosophila aggressive behaviors indicated that aggression in wild-type females increases if females are raised individually before pairing for observation. No increase was found in male-like behaviors in females kept separately from eclosion until testing. This suggests that these behaviors are not an exaggerated defense response. Other indications that these behaviors are not based on access to food come from observations of wild-type females starved overnight on moistened filter paper and transferred back onto food. These females showed short head-to-head and head-to-side interactions, but did not show behavior resembling male courtship. Courting retn females, by contrast, primarily show posterior orientation, and will follow other females on and off a food source for minutes at a time (Ditch, 2005).

retn is expressed in the CNS during pupal stages when sexual behavior is hardwired. To map retn expression in the CNS, retn-driven GFP expression was mapped using retn-Gal4 insertions that rescue retn phenotypes with the retn cDNA. These Gal4 enhancer traps, in addition to rescuing retn viability and behaviors, exactly reproduce Retn antibody patterns in embryos and larval eye tissue; therefore, they should represent the later CNS expression to a high degree of accuracy. Expression and projections were monitored using membrane-associated UASCD8::GFP (UAS-mGFP). retn expression in the CNS begins in the embryo, and continues through adulthood, in specific subsets of neurons. Focus was placed on expression of retn in the periods before and during metamorphosis, when adult neurons are born and larval neurons are remodeled into adult-specific forms. Notably, expression is seen in the mushroom bodies, subesophageal ganglion, ventral ganglion and developing photoreceptors. These patterns are essentially the same in both sexes (Ditch, 2005).

In the third instar, MB expression is seen in the Kenyon cell (KC) bodies lying in the dorsoposterior of the central brain, with staining in the calyx, containing KC dendrites, and the pedunculus and lobes, containing KC axons. Between 12 and 18 hours after puparium formation (APF), the calyx retracts, the alpha and ß lobes narrow and what appears to be axonal debris can be seen at the lobe tips. At this stage there are slightly more retn cells in females than in males, perhaps reflecting the greater axon number in female MBs. By 36 hours APF, the adult alpha, alpha', ß, ß', and gamma lobe projections are visible, although retn expression is stronger in alpha/ß projections. Between 24 and 48 hours APF, expression in all lobes except alpha/ß gradually fades, and by 48 hours only the alpha/ß lobes can be seen. This pattern remains through the rest of metamorphosis (Ditch, 2005).

In the larval Subesophageal ganglion (SOG), two central groups of six or seven neurons and two anterior groups of five neurons send projections towards the protocerebrum and ventral nerve cord. Laterally to these neurons are four additional neurons per side. The projections of these neurons form a dense pattern, and individual projections cannot be discerned. Retraction of larval-specific processes can be seen beginning six hours APF; by 36 hours APF, new processes are evident. The number of SOG neurons expressing retn remains constant, but projections become increasingly dense through the pupal period (Ditch, 2005).

retn-Gal4⁸⁹ is expressed posterior to the morphogenetic furrow, in photoreceptor cells R1-R6, which project to the lamina and R8, which projects to the medulla, as is also seen with Retn antibody staining. Beyond 48 hours APF, R8 expression and projections fade, although lamina projections remain. Expression in the eye, MB, SOG and ventral nerve cord is still visible post-eclosion (Ditch, 2005).

MB-specific abnormalities are seen in three different retn mutant genotypes: retn-Gal4⁸⁹/retn^Z2-428 larvae and pupae; retn^dri8/retn^Z2-428, and retn^Ro44/retn^RO44 adults. MB neurons diverge within the nerve tracks and ß-lobe neurons cross the midline and join with the opposite ß-lobe neurons, causing ß-lobe fusion, compared with retn-Gal4⁸⁹/+. This is more common in females than males, but phenotypes of retn; fru males indicate that retn functions in male neurons. Using antibodies to Fas2, which is expressed in MB axons projecting to the alpha- and ß-lobes in retn^dri8/retn^Z2-428 and retn^R044/retn^R044 adults, it was found that in a subset of mutant females, axons in the posterior part of the ß-lobe crossed the midline, resulting in ß-lobe fusion. In addition, in those animals with ß-lobe fusion, there were fewer Fas2-positive axons in the alpha-lobe. These MB fusion phenotypes are similar to the ß-lobe fusion phenotypes reported in other mutants, such as linotte/derailed, Drosophila fragile X mental retardation 1, fused lobes, ciboulot and alpha-lobe absent. Resistance is shown by the vast majority of females of these genotypes, thus MB fusion is unlikely to be causal for resistance (Ditch, 2005).

To determine retn neuronal birth dates and the neural phenotypes of dri-class alleles, the MARCM system, which can simultaneously create homozygous mutant cells and allow them to express Gal4-regulated marker genes, was used. retn-expressing MB neurons are born throughout the larval and pupal stages and eye clones appear at all embryonic and larval stages. The VNC neurons are born only within 48 hours of egg laying, and SOG retn neurons are born in 8-hour-old or younger embryos (Ditch, 2005).

Homozygous retn-Gal4⁸⁹ clones show striking mis-projection phenotypes in SOG neurons. The normal elaboration and symmetry of arbors in mid-pupae is diminished; ventral dendritic branches do not show normal density, and anterior projections wander and fail to extend. Neurons also fail to fasciculate normally. A central SOG midline-crossing tract, visible throughout metamorphosis, contains tightly bundled projections. In mutant clones, projections stray from this tract, apparently losing some adherent ability. Photoreceptor neurons also mis-project. In retn^dri clones, induced in the embryo, R1-R6 cells overshoot the lamina, and a number now target the medulla. Although retn mutations alter neuronal projection patterns, and projection differences are consistent with changes in behavior, retn behavioral functions have not yet been mapped to a particular set of neurons, nor has it been demonstrated that the projection differences, as opposed, for example, to retn-induced reductions in neural activity, are responsible for behavioral changes (Ditch, 2005).

It has been concluded that retn functions in multiple, separable processes during development. It acts in differentiation and control of gene expression along the anterior posterior and dorsal ventral axes in embryos. It also acts in the production of various tube structures such as salivary ducts and gut. Failures in these or other embryonic processes with dri-class (null or near null) alleles lead to embryonic death. retn-class (hypomorphic missense) alleles can perform the embryonic functions but show defects in neural development and projections. Correlating with this are changes in female behavior, including resistance to male courtship and, strikingly, generation of male-like courtship behaviors. Additional functions in development of internal genital ducts and fertility (Ditch, 2005).

retn neural and behavioral phenotypes are substantially different from those of dsf or fru. dsf females, like retn-females, are sterile and resist male courtship. For dsf, sterility results from loss of motor synapses on the circular muscles of the uterus. By contrast, these synapses are intact in retn females. dsf females show no male behaviors, while retn females do. dsf males are bisexual and slow to copulate, owing to inefficient abdominal bending, correlated with abnormal synapses on the muscles of ventral abdominal segment 5. retn males court and mate with normal kinetics and have normal A5 synapses. This suggests that retn and dsf have largely separate functions (Ditch, 2005).

retn and fru also have different phenotypes. In a wild-type background retn behavioral phenotypes are restricted to females. fru behavioral phenotypes are restricted to males and include failure to attempt copulation, bisexual and homosexual courtship, and, in the strongest allelic combinations, complete lack of male courtship. In addition, fru males lack the male-specific muscles of Lawrence in dorsal abdominal segment 5. retn males have normal muscles of Lawrence, and retn females do not have muscles of Lawrence. In addition, the larval and pupal expression patterns of retn and the sex-specific products of the fru P1 promoter, notably the active male-specific fru proteins, show little or no overlap. This all suggests that fru and retn are unlikely to interact intracellularly and would be expected to be involved in different aspects of behavioral control (Ditch, 2005).

The latter conclusion seems to be contradicted by the male-like courtship generated by retn females, since previous work demonstrates that otherwise wild-type males require Fru-M to generate male behavior. It has been operationally and molecularly shown that the male behavior generated by retn females occurs even in the absence of fru P1 transcripts (Ditch, 2005).

A plausible working model has been developed that reconciles the data on the necessity of fruM in males and male-like courtship by retn females. The largely non-overlapping expression patterns of fru and retn suggests that the formal interactions of this model will result from interactions between networks of fru- and retn-influenced neurons rather than by intracellular regulatory interactions involving Fru-M and Retn, although the model can accommodate either situation (Ditch, 2005).

The model posits that in the absence of fruM and retn the nervous system has an inherent tendency to set down some rudiments of neural pathways for male courtship behavior. When retn is wild type and fruM is not expressed, as in wild-type females, retn, or cells expressing retn [perhaps in conjunction or parallel with other factors such as dsxF], act to suppress the basal male courtship pathway. This blocks male courtship behaviors. This is the case in wild-type females (Ditch, 2005).

Finally, in wild-type males, fruM or cells expressing fruM, perhaps along with other factors such as dsxM, act to strengthen the male courtship pathway such that the repressive action of retn-expressing cells is overpowered. This makes fru the switch that results in male behavior and captures both the requirement for fru⁺ in males, and the male-like courtship by retn females (Ditch, 2005).

This model does not rule out involvement of other components. For example, it has been suggested that dsxF can suppress male behaviors in a retn⁺ background. This can be fitted into the model as an additional female-specific block to male behavior. A simple prediction of such a role for dsx is that reduction of dsx expression in a retn mutant background will enhance the retn phenotype. Recent work involving expression of fru RNAi in a subset of fru neurons suggests a role for temporally repression in the sequencing of male behaviors in courtship (Ditch, 2005).

An extensive series of experiments is in progress to test predictions of this model. Experiments are also in progress to determine if dsx participation fits within the context of the model, and to identify the molecules and mechanisms downstream of retn in the control of behavior (Ditch, 2005).

GENE STRUCTURE

cDNA length - 3.7kb

Exons - 12

Bases in 3' UTR - 877

PROTEIN STRUCTURE

Amino Acids - 901

Structural Domains

dead ringer (Gregory, 1996 and Shandala, 1999) is a founding member of a new family of proteins whose members share a conserved DNA binding domain, termed the ARID (A/T Rich Interaction Domain, Herrscher, 1995, Gregory, 1996). Members of this gene family include Drosophila osa (also referred to as eyelid; Treisman, 1997; Vazquez, 1999); yeast SWI1 (O’Hara, 1988); the mammalian jumonji (Motoyama, 1997); Smcx (Agulnik, 1994a); Smcy (Agulnik, 1994b), and the MRF1 and MRF2 (Huang, 1996) genes, as well as genes encoding Retinoblastoma binding proteins RBP1 and RBP2 (Fattaey, 1993). Sequence comparisons show that DRI belongs to a subgroup within this family that exhibits an extended region of similarity either side of the ARID. This motif is referred to as the extended ARID (eARID; Kortschak, 1998). The eARID group, which is poorly characterized, includes DRI, human DRIL1 (Kortschak, 1998), mouse Bright (Herrscher, 1995) and proteins encoded by the C. elegans T23D8.8 and D. rerio dri1 and dri2 genes (Kortschak, 1998). There is some evidence that members of this group are implicated in transcriptional regulatory processes. The mouse dri ortholog Bright (for B-cell regulator of IgH transcription; Herrscher, 1995) encodes a B cell-specific protein that appears to bind the minor groove of a consensus MAR sequence (AT/ATC). Bright acts to displace a conserved human homeoprotein CUX (ortholog of Drosophila Cut) to activate the immunoglobulin heavy chain intronic enhancer, Em, specifically in B cells (Wang, 1999 and Shandala, 1999).

The Dead ringer protein from Drosophila is a transcriptional regulatory protein required for early embryonic development. It is the founding member of a large family of DNA binding proteins that interact with DNA through a highly conserved domain called the AT-rich interaction domain (ARID). The solution structure of the Dead ringer ARID (residues Gly262-Gly398) was determined using NMR spectroscopy. The ARID forms a unique globular structure consisting of eight alpha-helices and a short two-stranded anti-parallel beta-sheet. Amino acid sequence homology indicates that ARID DNA binding proteins are partitioned into three structural classes: (1) minimal ARID proteins that consist of a core domain formed by six alpha-helices; (2) ARID proteins that supplement the core domain with an N-terminal alpha-helix, and (3) extended-ARID proteins, which contain the core domain and additional alpha-helices at their N- and C-termini. Studies of the Dead ringer-DNA complex suggest that the major groove of DNA is recognized by a helix-turn-helix (HTH) motif and the adjacent minor grooves are contacted by an alpha-hairpin and C-terminal alpha-helix. Primary homology suggests that all ARID-containing proteins contact DNA through the HTH and hairpin structures, but only extended-ARID proteins supplement this binding surface with a terminal helix (Iwahara, 1999).

date revised: 30 Sept 99

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