InteractiveFly: GeneBrief

spineless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Originally reported by Bridges in 1914, spineless plays a central role in defining the distal regions of both the antenna and leg. spineless encodes the closest known homolog of the mammalian dioxin receptor, a transcription factor of the bHLH-PAS family. Loss-of-function alleles of spineless cause three major phenotypes: transformation of distal antenna to leg, deletion of distal leg (tarsal) structures, and reduction in size of most bristles. Consistent with these phenotypes, spineless is expressed in the distal portion of the antennal imaginal disc, the tarsal region of each leg disc, and in bristle precursor cells (Duncan, 1998).

Is there a connection between the antennal and leg transformation, that is some evolutionary or developmental logic to this phenomenon? The following essay explores this question. The deeper question of the relationship between the function of spineless in insect morphogenesis and its relation to the function of the dioxin receptor in vertebrates, is not be dealt with here.

Whenspineless was ectopically expressed in Drosophila, a few flies survived to the pharate adult stage. In this way, the effects of ectopic spineless expression on adult structures have been studied. The results indicate that spineless is a primary determinant of distal antennal identity. Ectopic expression causes transformation of the maxillary palp and distal leg to distal antenna. Transformation of the maxillary palps varies from essentially no effect to an almost complete transformation to the third antennal segment and arista (bristle like structures at the tip of the antenna). Palps are also often deleted. Surprisingly, ectopic spineless induces ectopic antennal structures in the rostral membrane between the maxillary palp and the normal antenna. These range from small patches of third antennal segment and arista to entire ectopic antennae, and are always arranged in mirror symmetry to the normal antenna. Ectopic spineless expression also causes transformation of the distal leg to arista. In some cases, aristal-claw intermediatesare present, indicating that aristae can arise by transformation of claws. More proximal antennal structures are never present in the leg. Ectopic expression of spineless also causes the deletion of medial leg structures. More distal tarsal segments are usually unaffected, with the exception of transformation of claw to arista. Ectopic spineless expression also causes deletion of the central part of the wing, and induces a zone of polarity reversal in the abdominal tergites. A few scattered bristles are also induced in the wing blade (Duncan, 1998).

Ectopic expression of the Antennapedia gene can cause a complete transformation of distal antenna to second leg. It was suspected that the loss of function spineless mutation that caused antennal transformation to leg might have resulted from the ectopic activation of Antp+. To test this, dual mutant Antp;spineless clones were studied in the distal antenna. Surprisingly, these are indistinguishable from Antp+;spineless clones, and are transformed to second leg tarsus. This demonstrates that the mutant spineless antennal transformation is independent of Antp+, and leads to the belief that spineless controls distal antennal identity directly (Duncan, 1998).

In addition to specifying the distal antenna, spineless also specifies the tarsus. The tarsus appears to develop without input from the Antennapedia complex and bithorax complex genes. spineless is first expressed in the tarsal region in the late second instar. Staining increases in this region through the early third instar, and then gradually declines. As far as is known, spineless is the only disc-patterning gene that shows such transient expression. Forcing the tarsal segment to undergo premature metamorphosis results in an unsegmented tarsal region. Thus, it would appear that tarsal development occurs in two phases: first, a uniform tarsal region is established, and then this region is subdivided into specialized segments. It has been suggested that spineless is responsible for the first of these phases, whereas downstream genes are responsible for the second. The timing of maximal spineless expression, a temperature-sensitive period for spineless function, and the finding that spineless expression is not segmentally modulated within the tarsal ring are all consistent with this view (Duncan, 1998).

It is generally considered that the arthropod antenna evolved from a leg-like locomotory appendage, a view that has received support from work on Drosophila homeotic and segmentation genes. It is also generally accepted that unsegmented tarsi are ancestral in the hexapods, because simple tarsi resembling those present in spineless mutant Drosophila occur in crustaceans and primitive hexapods. Thus, both the transformation of antenna to leg and the tarsal deletions caused by spineless mutations appear to be atavistic (representing an ancestoral form), suggesting that spineless played an important role in the evolution of distal limb structures in the arthropods. Because antennal specialization occurred very early in arthropod evolution, it is likely that the first function of spineless was antennal specification, and that spineless was recruited into tarsal development much later, during the evolution of the hexapods. Antennae are often elongated appendages used for probing the environment, and it is plausible that as part of antennal specification, spineless came to have an appendage elongation function. Transient expression of this function could then have served to extend other limbs, including the legs. This evolutionary sequence predicts that spineless homologs will prove to be expressed in the antenna, but not the legs, of crustaceans and primitive hexapods, but in both locations in the insects (Duncan, 1998).

Temporally dynamic antagonism between transcription and chromatin compaction controls stochastic photoreceptor specification in flies

Stochastic mechanisms diversify cell fates during development. How cells randomly choose between two or more fates remains poorly understood. In the Drosophila eye, the random mosaic of two R7 photoreceptor subtypes is determined by expression of the transcription factor Spineless (Ss). This study investigated how cis-regulatory elements and trans factors regulate nascent transcriptional activity and chromatin compaction at the ss gene locus during R7 development. The ss locus is in a compact state in undifferentiated cells. An early enhancer drives transcription in all R7 precursors, and the locus opens. In differentiating cells, transcription ceases and the ss locus stochastically remains open or compacts. In Ss(ON) R7s, ss is open and competent for activation by a late enhancer, whereas in Ss(OFF) R7s, ss is compact, and repression prevents expression. These results suggest that a temporally dynamic antagonism, in which transcription drives large-scale decompaction and then compaction represses transcription, controls stochastic fate specification (Voortman, 2022).

Cell fate specification is controlled by lineage, signaling, and stochastic regulatory inputs, leading to highly precise developmental outcomes. Stochastic mechanisms promote diversity in populations of photoreceptors (PRs), olfactory neurons, motor neurons, and immune cells. Despite the importance of stochastic cell fate specification, how cells randomly choose between fates is poorly understood (Voortman, 2022).

Stochastic cell fate specification is best understood in prokaryotes. One well-characterized example is the bet-hedging mechanism utilized by Bacillus subtilis. To minimize losses in a changing environment, populations of genetically identical bacteria maintain a subpopulation of cells that are competent for DNA uptake. The transient and random transition into the competent fate is controlled by expression of the transcriptional regulator ComK. Though most cells maintain low expression of ComK, a subset will experience a pulse of ComK expression that exceeds a threshold and induces a transition to the competent fate. A similar mechanism occurs in the HIV life cycle, where transcription of the regulatory factor trans-activator of transcription (Tat) determines the switch from proviral latency to active replication. Thus, stochastic cell fate specification often requires a pulse of expression of a critical regulator that determines a fate decision (Voortman, 2022).

In addition to transcriptional dynamics, chromatin-mediated repression is a key mechanism mediating stochastic fate specification. In mice, each olfactory sensory neuron (OSN) expresses only one olfactory receptor (OR) gene from a battery of ∼1,300 possibilities. Despite residing in numerous clusters across many chromosomes, all ∼1,300 OR genes are repressed and coalesce into heterochromatic foci within the nucleus prior to OR selection. In mutants that impact chromatin modifications and nuclear organization, co-expression of multiple ORs is observed. While the mechanism of selection remains elusive, a single OR allele escapes the repressive heterochromatic environment and is expressed in each OSN. Thus, chromatin-mediated silencing and selective de-silencing are paramount for the stochastic expression of a single OR gene (Voortman, 2022).

The random mosaic of R7 PRs in the fly eye provides a paradigm to study the integration of transcription and chromatin-mediated repression in stochastic cell fate specification. In the fly eye, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) establishes the random pattern of two R7 subtypes across the retina. SsON R7s express Rhodopsin 4 (Rh4), while SsOFF R7s express Rhodopsin 3 (Rh3). In wild-type flies, each R7 has a 67% chance of adopting the SsON R7 fate and a 33% chance of assuming the SsOFF R7 fate, yielding a consistent ratio yet unique, random pattern of R7 subtypes across eyes. In ss protein null mutants, all R7s express Rh3. The stochastic ON/OFF ss expression is controlled by an enhancer (late enhancer, LE) that drives expression in all R7s and silencers that limit expression to a subset of R7s (Voortman, 2022).

This study describes a mechanism that controls stochastic R7 subtype specification. Initially, the ss locus is compact in all undifferentiated cells. An early enhancer (EE) drives ss expression and the ss locus opens in all R7 precursors during larval development. Expression ceases and the ss locus randomly compacts or remains open. In R7s in which ss remains open, the LE drives ss expression and SsON R7 fate. In R7s with compact chromatin, repression prevents expression driven by the LE, yielding the SsOFF R7 fate. The data suggest that stochastic fate specification is controlled by the dynamic, intertwined relationship of transcription and chromatin: transcription opens chromatin then chromatin compaction represses transcription. Transcription was found to be a source of stochasticity as modulating early transcription in precursors alters the proportions of alternative R7 fates (Voortman, 2022).

This study investigated how regulation of transcription and chromatin compaction at the ss locus controls stochastic R7 patterning in the fly eye. ss is initially in a compact, repressed state in undifferentiated cells. This compacted state is similar in other SsOFF cell types including peripodial cells and SsOFF R7s. As the eye develops, ss is transcribed in precursors and chromatin is opened. ss transcription and large-scale decompaction are lost in mutants deleting the EE or the promoter, suggesting that ss transcription drives the opening of the ss locus in precursors early (Voortman, 2022).

As the cells mature, ss expression ceases, and the ss locus compacts during the transition from the precursor to the differentiating cell phase. Our observations that (1) the ss locus is open in SsON R7s and compact in SsOFF R7s and (2) the similarity of median compaction in differentiating cells and all R7s (including SsON R7s and SsOFF R7s) suggest that the ss locus assumes either an open or compact state in differentiating cells that is maintained until terminal R7 subtype specification. Our data are consistent with stable compaction states in differentiating cells, but they cannot rule out changes during this phase of R7 development (Voortman, 2022).

ss expression and compaction during the transition from precursor to differentiating cell phases are critical processes that determine the stochastic R7 fate choice. Decreasing EE activity reduced ss expression in precursors and the proportion of SsON R7s. Extending ss transcription into the differentiating cell phase increased the proportion of SsON R7s. It is proposed that variable activation and duration of transcription in each precursor determines the probability of recompaction, which ultimately dictates the SsON or SsOFF expression state in R7s (Voortman, 2022).

In the last stage of R7 subtype specification, ss expression driven by the LE is repressed in a subset of R7s. The repression reporter strategy showed that repression at the ss locus limits expression to a subset of R7s. The chromatin compaction assays showed that the ss locus is open in SsON R7s and compact in SsOFF R7s. Deletion of the LE ablated expression but did not alter compaction in R7s. Thus, the chromatin state is set and maintained independent of expression at this stage of R7 maturation. Further, open chromatin is not sufficient to activate ss expression. Together, these data suggest that open chromatin allows activation by the LE whereas compact chromatin represses ss expression (Voortman, 2022).

Based on these findings, a mechanism is proposed that controls stochastic R7 subtype specification. The ss locus is in a compact state in undifferentiated cells. The EE drives transcription and opens the ss locus in precursors. Early expression ceases and the ss locus randomly assumes an open or compact state in differentiating cells. R7s with open chromatin at the ss locus reactivate ss and take on the SsON R7 fate, whereas R7s with compact chromatin at the ss locus repress ss and take on the SsOFF R7 fate (Voortman, 2022).

A key aspect of this mechanism is the initial 'priming' or opening of the ss locus during the early expression in precursors. Transcription-based priming plays important roles in several stereotyped developmental programs. A well-understood example has been described in C. elegans, where the bilateral pair of ASE gustatory neurons display asymmetric gene expression and function. Stereotyped specification of the left neuron ASEL is dependent upon the asymmetric expression of the microRNA lsy-6, achieved by a 'prime and boost' mechanism. Several cell divisions prior to the birth of the terminal ASEL neuron, a pulse of lsy-6 expression in the precursor cell promotes decompaction of the lsy-6 locus. This decompacted state is maintained in the ASEL lineage throughout development, allowing for reactivation of lsy-6 in the terminal ASEL neuron. In the ASER lineage that never experiences the early pulse of lsy-6 expression, the locus remains in a repressed, compacted state, preventing later activation by transcription factors that are expressed in both ASE neurons. Thus, early transcription of a key regulator (lsy-6) promotes one cell fate (ASEL) by antagonizing chromatin-mediated repression important for the specification of the alternative fate (ASER) (Voortman, 2022).

The transcription-based prime and boost mechanism controlling ASEL/R sensory neuron specification in C. elegans has many similarities to the mechanism that this study has identified for R7 subtype specification. In both systems, early expression of a key regulator in precursor cells opens a locus (prime) so that it can be reactivated later upon terminal specification (boost). A major difference is that the ASEL/R decision requires priming in only the ASEL lineage to reproducibly generate the ASEL fate, whereas the R7 subtype decision utilizes priming in all precursors, which opens the chromatin followed by variable chromatin compaction and repression that ultimately determines the SsON or SsOFF R7 fate (Voortman, 2022).

Both the ASEL/R and R7 subtype decisions also exhibit a window of inactivity between the early and late expression phases. However, this window appears to play two very different roles. In the ASEL/R decision in worms, the early priming of the lsy-6 locus occurs several cell divisions prior to terminal differentiation. The time between the prime and boost is an obstacle that must be overcome to remember the early developmental event. In contrast, the window between the early and late stages of ss expression appears to enable chromatin compaction and repression that determine the SsON or SsOFF expression states in R7s (Voortman, 2022).

Though stochastic fate specification is an important feature of many cell fate programs, general features of these mechanisms have not been identified. In the bacterium Bacillus subtilus, transcriptional regulation is critical, as ComK transcription drives a stochastic cell fate switch to the 'competent' fate. In both the competence decision in bacteria and R7 subtype specification in flies, all 'precursor' cells express the key regulator, yet only a subset undergo the cell fate switch (Voortman, 2022).

Stochastic R7 subtype specification in flies also shares mechanistic features with OR selection in mice, particularly in the repression of alternative fates. In the olfactory system, OR genes are found in a compact heterochromatic region in the nucleus, with one gene that escapes repression and activates. Similarly, chromatin compaction and repression play key roles in determining ssON and ssOFF R7 fates. The current studies in flies bridge the roles of transcription in bacteria and chromatin in mice for stochastic cell fate specification (Voortman, 2022).

Understanding of the relationship between transcription and chromatin is often a chicken and egg problem: it is unclear whether transcription state dictates large-scale chromatin state or vice versa. This study provides evidence that clearly identifies these cause-effect relationships and show how they change during development. The EE drives transcription to open chromatin in precursors. In differentiating cells, the EE ceases to function and transcription stops. Chromatin remains open or closes, marking the stochastic step. Finally, the LE turns on in mature R7s. In cells where the locus is open, transcription reinitiates, while in cells where the locus is closed, transcription is repressed. Thus, initially, transcription state regulates chromatin state and later, chromatin state controls transcription state. Our studies not only outline this simple mechanism, but also identify how the stochastic step is regulated. The stochastic step occurs as cells cease ss transcription in the precursor phase and assume the open or compact chromatin state in differentiating cells. Decreasing or extending early transcription alters the probability of chromatin closing and ultimately, the proportion of R7 subtypes. Thus, variability in the duration of early transcription is likely a key input that determines the stochastic decision. These findings provide an important step in understanding how transcription and large-scale chromatin states regulate one another to control how cells randomly assume fates (Voortman, 2022).

The data suggest that transcription drives large-scale chromatin decompaction and then compaction represses transcription, which controls stochastic cell fate specification. This study focused on large-scale chromatin remodeling. It is hypothesized that local changes of histone modifications at the enhancer and promoter likely precede transcription in precursors. The heterogeneity of the tissue limited testing this hypothesis in a cell-type-specific manner in intact tissue. Moreover, it is concluded that chromatin compaction represses ss expression in a subset of R7s. The repression reporter experiments showed that the chromatin context at the ss locus is sufficient to repress expression in a subset of R7s. The DNA FISH experiments showed that the ss locus is open in ssON R7s and compact in ssOFF R7s. These experiments were limited as it was not possible to identify conditions to artificially induce compaction and show sufficiency of compaction to repress ss. Additionally, these studies were conducted in fixed tissue, limiting the observation of the rapid temporal interplay between transcription and chromatin. Future studies could address this challenge with live imaging, enabling assessment of the transcriptional and chromatin dynamics of the ss gene throughout the maturation of individual R7s during subtype specification (Voortman, 2022).

A common mechanism for antenna-to-leg transformation in Drosophila: suppression of homothorax transcription by four HOM-C genes

Mutations in the clustered homeotic genes (HOM-C genes) can cause specific homeotic transformation, suggesting that the HOM-C genes determine segmental identities by acting on different target genes. However, misexpression of several HOM-C genes in the antenna disc causes similar antenna-to-leg transformations. No HOM-C genes are normally expressed in the eye-antenna disc proper. It has been considered that Antp, when ectopically expressed in the eye-antenna disc, suppresses an antenna-determining gene. This study shows that Scr, Antp, Ubx, and abd-A HOM-C genes all exert their effects through a common mechanism: suppression of the transcription of the homothorax (hth) homeobox gene, thereby preventing the nuclear localization of the Extradenticle homeodomain protein. If hth is a key effector suppressed by these four HOM-C genes, addition of hth should reverse the antennal transformations. Coexpression of the hth and HOM-C genes completely or partially reverts the transformation phenotype. It is noted, however, that suppression of hth is probably not the only effect of HOM-C expression in the antenna disc, since Scr, Antp, and Ubx each induce the antenna to transform into leg, showing different segmental characters (i.e., thoracic 1, thoracic 2 and thoracic 3 legs, respectively). Ectopic hth expression can cause duplication of the proximodistal axis of the antenna, suggesting that it is involved in proximodistal development of the antenna (Yao, 1999).

A possible mechanism for the suppression of hth by different HOM-C proteins assumes that the HOM-C proteins compete with a factor required for hth transcription. One candidate protein that fits all of these criteria is Hth itself. The gene spineless exhibits a similar antenna-determining function. It is possible that hth and spineless represent separate pathways specifying antennal identity. Since hth and ss are expressed in the leg discs as well as in the antenna discs, it is not their simple presence that determines antennal identity. What then distinguishes the antenna vs. the leg? One possibility is that the detailed spatial and temporal expression pattern makes the difference. The broader expression pattern of hth in the antenna disc may distinguish the antenna from the leg. It is also possible that the level of spineless makes a difference: high levels of ss correlate with antennal identity and low levels of ss correlate with leg identity. The duplication in the antenna caused by ectopic hth could be explained by the creation of a new proximodistal interface in the distal portion region of the disc. In both antenna and leg discs, Distal-less is expressed in the distal regions and is required for distal development. The roughly complementary expression of hth/nuclear Exd vs. Dll, defines the proximal and distal domains of appendages, respectively. The combined action of Wg and Dpp signaling defines the two domains by activating Dll and repressing hth in the distal domain. Antennal duplication due to ectopic hth could be explained by the juxtaposition of distal (Dll expressing) and proximal (hth expressing) cells (Yao, 1999 and references).

Control of the spineless antennal enhancer: direct repression of antennal target genes by Antennapedia

It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, a 62bp enhancer was isolated from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. It is concluded that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring (Duncan, 2010).

This report examines the regulation of an enhancer from the antennal gene ss that drives expression specifically in the third antennal segment (A3). The work provides the first look at how the homeodomain proteins Dll, Hth, and Exd function in the antenna to activate antennal target genes. These proteins form a trimeric Dll/Hth/Exd complex on the enhancer, suggesting that Dll acts much like a Hox protein in antennal specification. This work also reveals how the Hox protein Antp functions in the leg to repress antennal development. The conventional view has been that the primary function of Antp is to repress hth in the distal leg, which then prevents the activation of all downstream antennal genes. However, this study found that Antp represses the ss A3 enhancer directly. This repression is essential within a proximal ring in the leg that coexpresses the antennal gene activators Dll, Hth, and Exd. Antp competes with Dll for binding to the enhancer, and this competition is part of a molecular switch that allows the ss A3 element to be activated in the antenna, but represses its activation in the leg. The results suggest that repression of antenna-specific genes in the proximal ring is the sole function of Antp in the leg imaginal disc (Duncan, 2010).

At 62 bp, the ss A3 enhancer (called D4) is one of the smallest enhancers to be identified in Drosophila, and yet it is quite strong; only a single copy is required to drive robust expression of lacZ reporters. The enhancer is also very specific, driving expression in A3 and nowhere else in imaginal discs. It has been proposed that antennal identity in Drosophila is determined by the combined action of Dll, Hth, and Exd. Consistent with this proposal, all three of these factors were found to be required for D4 expression. Although these activators are coexpressed in both A2 and A3, D4/lacZ expression is restricted to A3 by Cut, which represses the enhancer in A2. Like ss itself, D4/lacZ is also repressed by ectopically expressed Antp (Duncan, 2010).

A previous report (Emmons, 2007) showed that the antennal expression pattern of ss is reproduced by lacZ reporters containing a 522 bp fragment from the ss 5' region. This fragment contains five conserved (41%-90% identity) domains, each of which was deleted and tested for effect on expression in vivo. Expression in the arista and the third antennal segment (A3) prove to be under separate control; expression in the arista requires domains 1, 3 and 5, whereas expression in A3 is lost only when domain 4 is deleted. Moreover, reporters containing domain 4 alone show expression in A3 and nowhere else in imaginal discs. Thus, domain 4 is both necessary and sufficient for A3-specific expression. Domain 4 (D4) is 62 bp in length and is highly conserved, being invariant at 50/62 base pairs in the 12 Drosophila species sequenced (Duncan, 2010).

Surprisingly, Dll, Hth, Exd, Cut, and Antp all act directly upon D4. The activators Hth and Exd bind with strong cooperativity to directly adjacent sites. Their joint binding site matches the optimum site for in vitro binding of the mammalian homologs of Hth and Exd (Meis and Prep), consistent with the robust activity of the enhancer in vivo. Mutation of either of these sites abolishes activity of the enhancer. The coactivator Dll binds three sites in D4; one of these sites (Dlla) is required for almost all activity of the enhancer. Dll shows strong cooperativity with Hth and Exd for binding to D4, indicating that Dll interacts physically with these proteins. This interaction requires DNA binding, as Dll protein containing a missense change that blocks DNA binding (a change of asn51 to ala in the homeodomain) shows no ability to associate with D4-bound Hth and Exd. A curious feature of the cooperativity seen in the binding studies is that although Hth and Exd increase the affinity of Dll for D4, Dll appears to have little effect on the affinity of Hth and Exd for the enhancer. Since Hth and Exd already bind cooperatively with one another, it may be that additional cooperative interactions with Dll have little effect. Alternatively, it may be that Hth and Exd interact with Dll only after binding DNA. If so, Hth and Exd would be expected to increase Dll binding to D4, but Dll would have little effect on the binding of Hth and Exd, as observed. Interactions between Dll and Hth in the absence of DNA have been reported in immunoprecipitation experiments. However, this study was unable to repeat these observations. Moreover, the finding that the asn51 mutant of Dll fails to associate with D4-bound Hth and Exd argues strongly against such interactions (Duncan, 2010).

The repressor Cut also acts directly upon D4. Binding of Cut requires two sites, one overlapping Dlla and the other overlapping the joint Hth/Exd site. These binding sites suggest that D4 is controlled by Cut in much the same way that a structurally similar Abdominal-A (Abd-A) regulated enhancer from the rhomboid gene is controlled by the repressor Senseless (Sens). In the rhomboid enhancer, adjacent Hth and Exd sites are also present, and these create a binding site for Sens. Activity of the rhomboid enhancer is controlled by a competition between binding of the Sens repressor and binding of the activators Abd-A, Hth, and Exd. It seems likely that D4 is controlled similarly, with the repressor Cut competing for binding with the activators Dll, Hth, and Exd. It will be of interest to determine whether enhancers similar to D4 are used more widely to control Cut targets involved in its role as an external sense organ determinant (Duncan, 2010).

A key finding in this work is that Antp represses D4 by direct interaction. Antp binds a single site in D4, which overlaps or is identical to the Dlla binding site. Like Dll, Antp binds cooperatively with Hth and Exd. Using purified proteins, it was showm that binding of Dll and Antp to the Dlla site is mutually exclusive. This indicates that Antp represses the enhancer at least in part by competing with Dll for binding. Similar competition may occur at other enhancers; when Antp expression is driven artificially in the distal leg, variable deletions of the tarsal segments occur. These defects might arise because Antp competes with Dll for binding to its target genes in the distal leg. In most other contexts examined, Antp is an activator of transcription; why it fails to activate D4 is not clear. The similar behavior of Dll and Antp in binding to D4 supports the idea that Dll behaves like a Hox protein in activating D4 (Duncan, 2010).

Although the initial focus of this study was on the antenna, the finding that Antp interacts directly with D4 led to an examination of D4 regulation in the leg, where Antp is normally expressed. In second leg imaginal discs, Antp is required only in a proximal ring of cells that coexpresses Dll and Hth. This ring appears in the early third instar, and is of uncertain function. Large Antp⁻ clones in T2 leg discs that do not enter this ring appear to develop completely normally, regardless of whether they are located distal or proximal to the ring. However, clones that overlap the ring show activation of D4/lacZ within the ring cells. Importantly, such clones have no effect on the expression of Dll or Hth within the ring. By examining Antp⁻ clones of increasing age the following sequence of events is inferred. First, D4/lacZ is activated in cells of the ring that are included within Antp⁻ clones. Second, many such clones begin expressing the antennal markers Ss and Cut, indicating a transformation to antenna, and round up as if they have lost affinity for neighboring cells. Third, such clones appear to extend and move distally in the disc (Duncan, 2010).

The events described for Antp⁻ clones in the leg make sense of several previously enigmatic observations. It has been noted that many Antp⁻ clones in the leg do not transform to antenna and appear to develop normally. The finding that only clones that overlap the proximal ring undergo transformation accounts for this observation. Antp⁻ clones that do contain transformations usually show apparent nonautonomy in that not all cells in the clone are transformed to antenna. The current results account for this observation as well, since within an Antp⁻ leg clone only those cells located in the proximal ring undergo transformation to antenna; cells located elsewhere in the clone retain normal leg identity. Most importantly, these observations provide an explanation for why ss is controlled directly by Antp. Antp^- clones have no effect on hth or Dll expression in the proximal ring. Therefore, Antp must function in the ring at the target gene level to repress antennal genes that would otherwise be activated by combined Hth and Dll (and Exd). Since several such targets are known, it seems likely that several, perhaps many, antennal genes in addition to ss are repressed directly by Antp (Duncan, 2010).

Transformed Antp⁻ clones in the leg often show ectopic hth expression in distal locations. If hth is not directly controlled by Antp in the leg, as this study suggests, then why is hth ectopically expressed within such clones? A likely explanation is that downstream antennal genes that have become activated in such clones feed back to activate hth. This interpretation is strongly supported by the finding that ectopic expression of the antennal genes ss, dan, or danr in the distal leg causes ectopic activation of hth. Thus, the distal expression of hth seen in Antp⁻ leg clones is likely a consequence rather than a cause of the transformation to antenna. Whether repression of hth in the antenna by ectopic Antp is also indirect is not clear. Dll is also expressed ectopically in transformed Antp⁻ leg clones, suggesting that it is also subject to feedback activation by downstream antennal genes (Duncan, 2010).

The function of the proximal Dll- and Hth-expressing ring in the proximal leg is not well understood. The ring is highly conserved among the insects, and may serve as a boundary between the proximal and distal portions of the legs. In the context of this work, a striking feature of the ring is that it contains a microcosm of gene expression domains corresponding to the three major antennal segments. Thus, proceeding from proximal to distal through the ring, cells express hth alone, hth + Dll, and hth + Dll + strong dachshund. These expression combinations are characteristic of the A1, A2, and A3 antennal segments, respectively. Looked at in this way, the ring would appear to resemble a repressed antennal primordium within the leg (Duncan, 2010).

It has been known for almost thirty years that Antp is required in the leg to repress antennal identity. However, an understanding of how this repression occurs has been lacking. The current results indicate that Antp functions within the proximal ring to directly repress antennal genes that would otherwise be activated by combined expression of Dll, Hth, and Exd. This appears to be the only function of Antp in the leg, at least during the third instar larval stage. The results are entirely consistent with the idea that second leg is the 'ground state' ventral appendage (the limb type that develops in the absence of identity specification) and that the role of Antp in the leg is to preserve this ground state by repressing the activation of 'head-determining' genes (Duncan, 2010).

REGULATION

Promoter Structure

spineless alleles broken in the upstream region of the gene have no effect on spineless expression in the leg, but alter expression in the antenna to resemble that normally seen in the leg. The effects of a translocation broken at 4 kb to 5 kb (ssD114.3) are described. spineless is expressed in a transient ring in the antennal and leg discs in this mutant. This indicates that the antennal expression pattern of spineless is controlled by the region upstream of 4 kb to 5 kb, whereas the tarsal pattern of expression is likely controlled by a downstream, perhaps intronic, region. Consistent with their effects on spineless expression, mutants located in the upstream region cause transformation of the distal antenna to tarsus. However, only the distal part of the third antennal segment and the arista are affected. Detailed examination of one upstream mutant (ssD114.7) indicates that the antennal tarsus produced has second leg identity (Duncan, 1998).

Regulation of the Drosophila distal antennal determinant spineless

The transformation of antenna to leg is a classical model for understanding segmental fate decisions in Drosophila. The spineless (ss) gene encodes a bHLH-PAS transcription factor that plays a key role in specifying the identity of distal antennal segments. This report identifies the antennal disc enhancer of ss and then uses enhancer-lacZ reporters to work out how ss antennal expression is regulated. The antennal determinants Distal-less (Dll) and homothorax (hth) are key activators of the antennal enhancer. Dll is required continuously and, when present at elevated levels, can activate the enhancer in regions devoid of hth expression. In contrast, homothorax (hth) is required only transiently both for activation of the enhancer and for specification of the aristal portion of the antenna. The antennal enhancer is repressed by cut, which determines its proximal limit of expression, and by ectopic Antennapedia (Antp). Repression by Antp is not mediated by hth, suggesting that ss may be a direct target of Antp. ss⁺ is not a purely passive target of its regulators: ss⁺ partially represses hth in the third antennal segment and lies upstream of Dll in the development of the maxillary palp primordia (Emmons, 2007; full text of article).

This study used lacZ reporters to identify the enhancers responsible for most aspects of ss expression during embryonic and imaginal development. Antennal expression is driven by two large fragments from the ss 5' region, B6.9 and EX8.2. Both of these fragments drive expression in the antennal segment of the embryo and in the distal portion of the pupal antenna. B6.9 is also expressed in the antennal disc through most or all larval development. Dissection of B6.9 allowed localization of the larval antennal enhancer to a fragment of 522 bp. The B6.9 and 522 reporters were used as a proxy for ss expression in experiments to determine the effects of potential upstream regulators of ss. This strategy has its strengths and weaknesses, but has been made necessary by an inability to generate antisera against Ss. A major strength of the approach is that it was possible to assess the effects of regulators on individual enhancers. It is likely that monitoring endogenous ss expression would give results that are less clear cut since both the antennal and tarsal enhancers of ss are active within the antenna. A potential weakness is that the reporters may not faithfully reproduce the normal expression of ss. However, as far as is possible to tell, the antennal reporters reproduce ss expression very well. The expression of B6.9 and EX8.2 in the embryonic antennal segment and the pupal antenna corresponds very closely to that of endogenous ss. Expression of B6.9 and 522 in the larval antennal disc appears very similar or identical to that of ss⁺, and the transient requirement for hth⁺ in the activation of these reporters corresponds well to the transient requirement for hth⁺ in aristal specification. The tarsal enhancer P732 likely also reproduces the spatial pattern of ss⁺ expression as its tarsal expression domain corresponds well to the region deleted in ss⁻ mutants (Emmons, 2007).

The results of this dissection of the B6.9 fragment were surprising. Removal of the left-hand 2 kb of B6.9 to produce S4.9 resulted in the loss of antennal specificity; S4.9 reporters are expressed in both antennal and leg discs. The E2.0 subfragment of S4.9 shows a similar expression pattern, and expression of this fragment in both leg and antennal discs is independent of Hth, but requires Dll continuously. On further subdivision of the E2.0 fragment, it was found that antennal and leg expression are separable; the 522 fragment is largely specific for the antenna, whereas the 531 fragment drives expression primarily in leg discs. To summarize, antennal specificity is present in B6.9, lost in S4.9 and E2.0 and regained in 522. How can sense be made of this? The region deleted from B6.9 to produce S4.9 clearly plays an important role in enforcing antennal specificity. Since this region contains a PRE, one might suspect that it functions in larval stages to maintain repression of the enhancer outside of the antennal segment. However, that the E2.0 fragment has lost the requirement for Hth in both the antenna and leg (S4.9 has not been tested) suggests that the PRE-containing region might function in both locations. One possibility is that this region represses the enhancer in both antennal and leg discs. In the antenna, this repression can be overcome by the combined action of Hth and Dll, while in the leg Dll alone is not sufficient for activation. When the PRE-containing region is deleted, repression is absent or reduced, so that Dll can activate the enhancer without assistance from Hth, and expression is seen in both antennal and leg discs. Why then is antennal specificity restored in the 522 subfragment? Perhaps this fragment is lacking a subset of Dll interaction sites so that it can no longer be activated by Dll alone, but requires combined activation by Hth and Dll. Although this model is consistent with many of the results, it does not provide a ready explanation for the leg specificity of the 531 fragment (Emmons, 2007).

In addition to activation by combined Hth and Dll, the ss antennal disc enhancer is repressed by Cut and by ectopic Antp. Each of these regulators will be discussed separately. It was found that hth⁺ is required only transiently for activation of the B6.9 reporter. hth⁻ clones induced in the embryo or first instar lose expression of B6.9 autonomously in both A3 and the aristal primordia. However, some time in the second of early third instar. Regulatory instar expression of B6.9 becomes independent of hth. Consistent with this transient requirement, it is shown that hth⁺ is required only early in larval development for specification of the arista. hth⁻ clones induced in the first and second instars show a transformation of the entire antenna to a leg-like appendage. However, clones induced after this time show normal aristal development. These temporal requirements are reflected in the expression pattern of hth: hth is expressed throughout the antennal primordium early in development, but in the second or early third instar is repressed in the central domain, which will produce the arista (Emmons, 2007).

The stable activation of B6.9 by Hth suggests that this fragment contains a 'cellular memory module'. The presence of a PRE within B6.9 is consistent with this idea. The ss locus binds Polycomb protein in salivary gland chromosomes and was recently shown to contain PREs by chromatin immunoprecipitation. In the latter work, ss PREs were localized to within the E1.6 subfragment of B6.9 as well as the EX8.2 fragment, both of which showed pairing dependent suppression in this work. PREs are generally thought of as functioning to stably repress genes. However, PREs can also be associated with activating elements to form memory modules that mediate stable activation. It seems likely that B6.9 contains such a module that responds to Hth. Like a memory module from the hedgehog gene, activity of the ss module is set sometime around the second instar. Surprisingly, it was found that activation of the 522 reporter by Hth can also be persistent, although not as stable as for B6.9. The 522 fragment does not appear to contain a PRE, suggesting that Hth may directly recruit factors to the 522 element that cause semi-stable transcriptional activation (Emmons, 2007).

ss is not a completely passive target of hth; ss partially represses hth in antennal discs, which causes hth to be expressed at a lower level in A3 than in A2. This repression appears to be important for normal development as ectopic expression of Hth can delete A3. Moreover, clones ectopically expressing Hth are largely blocked from entering A3 from the proximal (A2) side, suggesting that the different levels of Hth present in A2 and A3 cause a difference in cell affinities between these segments. Hth-expressing clones are similarly restricted to the two most proximal segments in leg discs, although here there is no endogenous expression of hth more distally (Emmons, 2007).

In contrast to hth, Dll is required continuously for expression of both B6.9 and 522 as Dll⁻ clones induced even very late in development lose expression of these reporters. This continuous requirement for Dll indicates that stable activation of the B6.9 memory module by Hth does not by itself commit the reporter to expression; rather, activation by Hth appears to render B6.9 open to interaction with Dll and perhaps other positive factors (Emmons, 2007).

Three lines of evidence suggest that Dll is the primary activator of the ss antennal enhancer. (1) It was found that expression of B6.9 and 522 is sensitive to the dosage of Dll⁺. Expression of both reporters is reduced in animals carrying only one dose of Dll⁺, and for 522, expression is enhanced in clones having extra doses of Dll⁺. This dose sensitivity suggests that ss is a direct target of Dll. (2) It was found that expression of both reporters is often induced within clones expressing ectopic Dll, even in the apparent absence of Hth expression. Such activation is seen in clones in the distal leg, wing and elsewhere. (3) It was found that the embryonic antennal enhancer carried by B6.9 is absolutely dependent upon Dll⁺, but independent of hth. Taken together, these observations suggest that Dll is a primary activator of the ss antennal enhancers. Hth may provide antennal specificity by boosting the level of activation by Dll in the antennal disc (Emmons, 2007).

Surprisingly, it was found that the regulatory relationship between ss and Dll is reversed in the maxillary palp. Here, ss is expressed prior to Dll and is required for the normal initiation of Dll expression. Although some Dll expression ultimately takes place in the palp primordium in ss⁻ animals, this expression is weak and occurs in only a few cells. It has not been worked out how ss is activated in the palp. However, it seems likely that dpp plays a role as the 531 subfragment of B6.9 drives expression in a stripe in the region of the palp that roughly coincides with a stripe of dpp expression. The positioning of ss upstream of Dll in the palp may explain why the region ventral to the antenna is so sensitive to ectopic expression of Ss. Strong activation of Dll here by ectopic Ss combined with endogenous expression of hth might be expected to cause frequent induction of ectopic antennae, as is observed. Since ss is normally expressed in the palp, why should earlier ectopic Ss cause the palp primordium to develop as antenna? It seems likely that timing is key, but level of Ss expression could also be important (Emmons, 2007).

The reciprocal regulatory roles of ss and Dll in the antenna and palp suggest a particularly close relationship between these genes. This relationship is reinforced by the finding that ss is required for the development of bracts in the femur, as is Dll (Emmons, 2007).

The finding that Dll and Hth are both activators of the ss antennal reporters is consistent with the proposal that antennal identity is defined by the combined activity of these regulators. However, the results indicate that this model is an oversimplification. Examination of clones expressing Dll, Hth, or both proteins together revealed little correlation between activation of the B6.9 and 522 antennal reporters and combined expression of Dll and Hth. Strikingly, Dll-expressing clones often activate the reporters ectopically without any apparent concomitant expression of Hth, and clones expressing both proteins usually do not activate the reporters. These experiments also reveal strong context dependence. Examples include the leg, where Dll-expressing clones can activate the reporters distally, but not proximally (where endogenous hth expression occurs) and the wing disc, where clones expressing Dll or both Dll and Hth activate the reporters in the wing pouch, but not at all in the notum. The level of expression of both proteins also appears to be key as high levels of Dll can activate the reporters in the leg in the absence of Hth and elevated levels of Hth can repress expression in the normal antennal domain. Previous results have shown that antennal structures can be induced by ectopic expression of Dll in the wing hinge region or proximal leg (which express hth endogenously) or by combined expression of Dll and Hth elsewhere. While this is true, the results indicate highly variable effects in such ectopic expression experiments and fail to detect the strongly synergistic activation of antennal identity by combined Hth and Dll implied by the model. The results indicate that Dll is the primary activator of the ss antennal reporters, that Hth serves to promote this activity and that activation by Dll and Hth is highly context-dependent (Emmons, 2007).

Consistent with direct control of the antennal reporters by Dll and Hth, two highly conserved regions within the 522 fragment contain apparent binding sites for Dll, Hth, and the Hth dimerization partner Extradenticle. The functional importance of these binding sites is currently being tested (Emmons, 2007).

This study has show that the proximal boundary of B6.9 and 522 expression is defined by repression by cut. This repression likely explains why ectopic Cut causes a transformation of arista to tarsus. cut has been shown to define the proximal expression limit of distal antenna (dan) and distal antenna related (danr); since ss lies upstream of these genes , it seems very likely that their regulation by cut is indirect. The mechanism of action of Cut is not well understood, since only one direct target has been characterized in Drosophila (Emmons, 2007).

Ectopic expression of Antp in the antenna represses the B6.9 and 522 reporters. This finding was expected, since it is well known that expression of Antp or other Hox genes in the antenna causes a transformation to leg. The conventional view is that this transformation results from the repression of hth by ectopic Hox proteins. Repression of hth early in development would be expected to lead secondarily to loss of ss expression and loss of distal antennal identity. However, it was found that clones expressing Antp repress the B6.9 and 522 reporters even when these clones are induced very late in development, long after the requirement for activation by hth has passed. Late repression of the antennal reporters by Antp must therefore occur independently of hth and could be direct. One possibility, currently being tested, is that Antp might compete with Dll for binding to the 522 enhancer. Late repression of the ss antennal enhancer by Antp is consistent with the effects of Antp-expressing clones on antennal identity: such clones induced in the mid to late third instar cause transformations of distal antenna to leg (Emmons, 2007).

Clones induced late that ectopically express Antp in a sustained fashion were examined. In contrast, previous work studied the effects of pulses of Antp expression induced by one-hour heat shocks in a heat shock/Antp line. It had been found that transformations of arista to tarsus were induced by such pulses only when they were administered at the end of the second instar. Why do pulses of Antp at this time cause a stable, heritable transformation of the distal antenna? The current results suggest an explanation. The period sensitive to Antp pulses coincides roughly with when the ss antennal enhancer becomes independent of hth. This correlation suggests that pulses of Antp in the second instar cause heritable transformations by interfering with the stable activation of ss by Hth. Recently, it has been reported that ectopic Antp does not repress hth in the antenna early in larval development. This observation suggests that Antp might act directly on the ss antennal enhancer to prevent its stable activation by Hth (Emmons, 2007).

The regulation of ss by ectopic Antp suggests that Antp may normally play a significant role in repressing ss antennal enhancer activity in the legs. Although this idea has not been tested directly, it seems unlikely that Antp is primarily responsible for keeping the ss antennal enhancers inactive in the leg. Antp null clones do cause activation of the ss target gene dan in leg discs, implying ectopic activation of ss. However, this activation occurs only proximally, with the distal leg appearing to develop independently of Antp. Expression of Antp in the proximal leg may account for why Dll-expressing clones fail to activate B6.9 or 522 in this location. Ectopic activation of the ss antennal enhancers in the leg primordia of the embryo is not seen in an Antp null mutant (Emmons, 2007).

These studies suggest that antennal structures are specified in a combinatorial fashion by Hth, Dll, Ss and probably other factors. In A3, all three proteins are required for normal antennal identity. In ss⁻ antennae, hth continues to be expressed in A3 (although at elevated levels), as does Dll. Despite this continued expression of hth and Dll, A3 develops without antennal characteristics and produces only naked cuticle. Thus, Hth and Dll are unable to specify A3 characters in the absence of Ss. Conversely, assuming that ss is stably activated in the antenna by Hth, as is B6.9, then hth⁻ clones induced late would show persistent expression of both ss and Dll in A3. Such clones are transformed to leg, implying that Ss and Dll have no ability to direct A3 identity in the absence of Hth. Taken together, these observations suggest that Hth, Dll and Ss must act together to specify A3 identity. This requirement for combined action accounts for why ectopic expression of Ss does not induce A3 tissue in the medial leg, since hth is not normally expressed here. The view of combinatorial control suggests that many A3-specific target enhancers might be identifiable in genome searches as regions that contain clustered binding sites for Hth, Dll and Ss; tests of this prediction will be presented elsewhere (Emmons, 2007).

In contrast to A3, the aristal primordium appears to be specified by ss and Dll acting together in the absence of hth expression. hth is expressed in the aristal region early in development, where it functions to establish ss expression, but it is soon repressed here. Therefore, for most of development, the arista is specified by Ss and Dll acting without input from Hth. Consistent with this picture, the arista adopts leg identity in ss null mutants, and ectopic expression of ss causes the distal tip of the leg to develop as arista (Emmons, 2007).

In ss⁻ mutants, the distal antenna is terminated by a single tarsal segment (the fifth). In contrast, in ss mutants that lack only antennal enhancer activity (e.g. the breakpoint mutations ss^D114.3 and ss^D114.7, the distal antenna develops with a near complete set of tarsal segments. This difference likely reflects the activity of the tarsal enhancer in the antenna. In support of this view, the ss tarsal enhancer drives expression in the segmented base of the arista, a region known as the basal cylinder. This region transforms to tarsal segments 2-4 in Antp-induced transformations of antenna to leg. However, the question arises as to why normal antennal expression of ss causes the proximal arista to develop as basal cylinder, whereas ss expression driven by the tarsal enhancer alone causes this same region to develop as tarsal segments. Likely, the key difference is that expression driven by the tarsal enhancer is transient, whereas expression driven by the antennal enhancer is sustained. Perhaps transient expression of ss allows growth and subsegmentation to produce a full set of tarsal segments, whereas sustained expression inhibits growth, producing the basal cylinder. Consistent with this idea, sustained expression of ss driven by the GAL4 method can cause deletion of tarsi in the legs. The levels of expression driven by the tarsal and antennal enhancers may also be important as flies having only one dose of ss show a partial transformation of the basal cylinder to tarsus. The ss tarsal enhancer drives weak expression in A3 as well as in the basal cylinder, likely accounting for the presence of some specialization of A3 in ss mutants lacking the antennal enhancers (Emmons, 2007).

The view that antennal identity is specified by the combined action of Hth, Dll and Ss contradicts the now prevalent view that antennal identity is determined solely by hth. The major evidence supporting the latter view is that early hth⁻ clones transform the entire antenna to leg, and ectopic expression of Hth can induce ectopic antennal structures in the anal plates. Moreover, Dll shows little antennal specificity, being expressed in the distal portions of all of the ventral appendages, and ss expression in the antenna is dependent upon hth⁺. Should hth be viewed as the antennal 'selector' gene? hth does not seem to be a selector in the same sense as the Hox genes; it is expressed very broadly in the embryo and in other imaginal discs and plays no role in activating ss in the antennal segment of the embryo. Moreover, the ability of ectopic Hth to induce antennal structures is very limited: transformations of anal plate to distal antenna have been reported following ectopic expression of Hth or Meis1, a mammalian homolog. However, others have been unable to reproduce this effect by ectopic expression of Hth, matching the results of this study. That anal plates are susceptible to transformation at all is likely due to the fact that Dll and ss are coexpressed here in normal development. A further dissimilarity is that hth acts only as an establishment regulator of ss in the antennal disc, unlike the continuous requirements usually seen for the Hox genes. Ultimately, assessment of the importance of hth will depend on whether its function in the antenna is conserved. The expression pattern of hth in the antenna does appear to be conserved in the milkweed bug Oncopeltus. However, localization of nuclear Exd (a proxy for Hth expression) indicates that Hth is not differentially expressed in the antenna and leg of the cricket. Expression of hth in the crustacean Porcellio also appears to be identical in the second antenna and the legs. Characterization of hth, Dll and ss expression and function in additional arthropods will be required to assess properly the importance of these genes in antennal specification (Emmons, 2007).

Transcriptional Regulation

In both the antenna and leg, spineless expression is shown to depend on Distal-less (Dll), a master regulator of ventral appendage formation The Dll gene is required for the development of all leg segments distal to the coxa. To test whether spineless lies downstream of Dll in limb development, spineless expression was examined in a weak Dll loss-of-function mutant, DllPK. This allele survives to the pharate adult stage when heterozygous with Dll null alleles such as DllB, and causes the deletion of distal limb structures. spineless expression is almost completely eliminated in the tarsus, antenna, and maxillary palp of DllPK/DllB heterozygotes. Thus, spineless lies downstream of Dll in all three of these appendages. In the antenna, spineless expression is reduced in animals that carry only one dose of Dll+. This presumably accounts for the weak transformation of distal antenna to leg seen in most Dll mutant heterozygotes (Duncan, 1998).

To monitor Dll expression in relation to spineless, a monoclonal antibody was isolated against Dll protein. Dll is expressed uniformly in the central portions of the leg and antennal imaginal discs. In the early third instar, when spineless is first expressed in the leg, the outer edge of the spineless tarsal ring coincides precisely with the proximal limit of Dll expression. As the leg disc grows, the boundary of Dll expression expands beyond the spineless tarsal ring, so that a proximal zone of cells that express Dll, but not spineless, is created. In the antenna, Dll expression extends more proximally than spineless at all stages examined (Duncan, 1998).

Hox genes regulate the same character by different strategies in each segment

Hox genes control regional identity along the anterior-posterior axis in various animals. Each region contains morphological characteristics specific to that region as well as some that are shared by several different regions. The mechanism by which one Hox gene regulates region-specific characteristics has been extensively analyzed. However, little attention has been paid to the mechanism by which different Hox genes regulate the same characteristics in different regions. This study shows that two Hox genes in Drosophila, Sex combs reduced and Ultrabithorax, employ different mechanisms to achieve the same out-put, the absence of sternopleural bristles, in the prothorax and metathorax, respectively. Sternopleural bristles are characteristics of the mesothorax, and it was found that spineless is involved in their development. Analysis of the regulatory relationship between Hox genes and spineless indicated that ss expression is repressed by Sex combs reduced in the prothorax. Since sole misexpression of ss could induce ectopic sternopleural bristle formation in the prothorax irrespective of the expression of Sex combs reduced, spineless repression appears to be critical for inhibition of sternopleural bristles by Sex combs reduced. In contrast, spineless is expressed in the metathorax independently of Ultrabithorax activity, indicating that Ultrabithorax blocks sternopleural bristle formation through mechanisms other than spineless repression. This finding indicates that the same characteristics can be achieved in different segments by different Hox genes acting in different ways (Tsubota, 2008).

This study found that three genes, Antp, ss and al, are involved in sternopleural bristle formation. In the al mutant, no appreciable Ac expression in the T2 leg disc is detected and sternopleural bristles are not formed, indicating that the requirement of al is absolute. In contrast, Ac expression is detectable in the ss mutant T2 leg disc and in the Antp mutant clones, indicating that the requirement of both ss and Antp for ac expression is not absolute. However, sternopleural bristles were never found in the ss mutant, despite the fact that Antp expression was unaffected in the ss mutant clone in the T2 leg disc. In contrast, Antp mutant cells, in which ss is expressed normally, formed sternopleural bristles. In addition, sole misexpression of ss in the T1 segment produces sternopleural bristles ectopically, while that of Antp did not. Therefore, ss appears to be necessary and sufficient for sternopleural bristle formation, while Antp appears to be insufficient and not necessarily required. Moreover, Ac expression is ectopically induced in the T1 leg disc by misexpression of ss but not of Antp and in the ss mutant T2 leg disc is very weak, highly restricted, and only transient. This indicates that ss but not Antp appears to be one of the major activators of ac expression. Taken together, ss appears to be much more fundamental for sternopleural bristle formation than Antp (Tsubota, 2008).

The initiation of ac expression coincides with the initiation of ss expression. Since al and Antp are already expressed before ac induction in the early third instar stage, the timing of ac induction may be determined by the regulation of ss expression. Interestingly, the residual Ac expression seen in the ss mutant leg disc is first observed in the mid third instar as in the wild-type leg disc. This implies that at least one additional gene (referred to as X hereafter), whose expression or function is activated at the same stage as the initiation of ss expression, may be involved in ac induction. One possibility may be a gene functioning in hormonal regulation. Nonetheless, the ability of the sole misexpression of ss to induce ectopic ac expression and sternopleural bristle formation strongly indicates that ss is much more fundamental than X (Tsubota, 2008).

The restriction of ac expression to the overlap between the ss and al expression domains indicates the importance of determining the distal limit of ss expression and the proximal limit of al expression. Analysis of clones lacking ss activity or misexpressing ss indicates that ss has a repressive activity on al expression. How can al be expressed in the overlap domain? In the overlap domain, ss represses al expression when misexpressed at high levels but does not when misexpressed at approximately endogenous levels. The level of ectopic Al expression in the ss mutant clone located in a region proximal to the normal al expression domain is lower than that of endogenous Al expression. Moreover, Al expression in the wild-type leg disc gradually decays at its proximal edges. Considering all of these observations, the following hypothesis is suggested: al expression is activated according to the proximodistal information and the proximal limit of the al expression domain may be determined by a balance between activation according to the proximodistal information and repression by ss. The activation force may dominate the repressive activity of ss in the overlapping region but may gradually decay towards the proximal edges of the al expression domain. In contrast, ss expression does not appear to be regulated by al. As with the case of al activation, it may be possible that ss is repressed according to the proximodistal information (Tsubota, 2008).

The morphological identities of the T1 and T3 segments, including the absence of sternopleural bristles, are determined by Scr and Ubx, respectively. Analyses of the T1 leg disc with Scr mutant clones and the T2 leg disc with ectopic Scr activity indicate that both ss and Antp are repressed by Scr in the T1 leg disc. In addition, there is a possibility that the expression or function of gene X is repressed by Scr. Weak Ac expression is transiently observed in the ss mutant T2 leg disc, indicating that ac expression can be weakly activated without ss activity in the presence of gene X and Antp activity. In addition, Scr does not appear to repress ac expression directly, since ectopic induction of ac by ss misexpression in the T1 leg disc was not associated with an alteration in Scr expression. If gene X is active in the T1 leg disc, sole misexpression of Antp is expected to activate ac expression at least weakly and transiently. However, no ectopic Ac expression was found upon sole misexpression of Antp. Therefore, the activity of gene X is likely to be repressed in the T1 leg disc. For evaluating the significance of these three genes on Scr-dependent inhibition of sternopleural bristle formation, the ability of ss misexpression to induce ectopic ac expression and sternopleural bristle formation without affecting Scr expression is of crucial importance. At present, whether ac expression and sternopleural bristle formation can be induced solely by ss or only in a combination of ss and Antp and/or gene X is unclear. However, ss misexpression induced Antp expression and, thus, at least ss and Antp were coexpressed upon sole misexpression of ss. As for gene X, if it is not activated by ss misexpression, the results indicate that ac expression and sternopleural bristle formation can be induced without gene X activity at least in the presence of both ss and Antp expression. In contrast, if ac expression and sternopleural bristle formation require gene X activity, ss misexpression must activate gene X. After all, the results indicate that sole misexpression of ss can fulfill at least a minimum requirement for ac expression and sternopleural bristle formation. In other words, if Scr could not repress ss expression, ac expression would be activated and sternopleural bristles would be formed irrespective of the expression and function of Antp and gene X. Therefore, Scr must repress ss expression and this appears to be a key step to block sternopleural bristle formation in the T1 segment (Tsubota, 2008).

In contrast to the T1 leg disc, strong Ss expression was observed in the wild-type T3 leg disc and it is unaltered in Ubx mutant clones. Therefore, Ubx appears to act through a mechanism unrelated to ss expression. How does Ubx function? Simultaneous expression of both ss and Antp seemed insufficient for ac expression and sternopleural bristle formation in the T3 segment, since Antp misexpression failed to induce Ac expression in the T3 leg disc, in which ss is prominently expressed. It may be possible that Ubx represses ac expression directly. Alternatively, Ubx may compromise the function of the Ss protein directly or indirectly through regulation of its downstream gene products. Another possibility is that Ubx acts through repression of gene X activity. These possibilities are not mutually exclusive with each other (Tsubota, 2008).

The occurrence of ac expression and sternopleural bristle formation in the absence of Antp activity indicates that the absence of sternopleural bristles is not the ground state. However, the number of sternopleural bristles is variable in that condition, indicating that the complete formation of sternopleural bristles is not also the ground state. Since ss misexpression experiment suggests that sternopleural bristles can be formed as long as ss is expressed, one possible aspect of the ground state may be the expression of ss and the production of at least some kind of bristles. Antp may have acquired the ability to modify this state to produce the current-type of sternopleural bristles. On the other hand, Scr may have evolved the ability to block sternopleural bristle formation by acquiring the activity to repress ss expression and Ubx by acquiring another, yet unknown function. Taken together, the current state of sternopleural bristles in all three thoracic segments appears to be the derived state (Tsubota, 2008).

Targets of Activity

The transient early expression of spineless in the leg suggests that spineless plays a role in the establishment of the tarsal region. Support for such a role is provided by the finding that bric à brac (bab) lies downstream of spineless. In wild type, bab expression is initiated in the tarsal region in the mid-third instar; at disc eversion, bab expression can be seen to extend from the middle of the first tarsal segment through the fifth segment. In spineless null mutants, bab expression is abolished in the leg (Duncan, 1998).

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).

To test whether hern and fer are sufficient to induce eye or antennal development, they were expressed ectopically using the GAL4/UAS system. When either the hern or the fer genes are misexpressed in the leg discs with dpp-GAL4 or Dll-GAL4 (EM212) drivers, distal legs are transformed to aristae. These transformations are accompanied by the ectopic expression of hth, sal, and ss, three genes expressed in the antennal primordium but not in the distal region of mature wild-type leg disc. Clones expressing either the hern or the fer genes in the leg or wing disc have smooth borders and frequently activate the sal and hth genes cell-autonomously. In dpp-GAL4/UAS-fer or ptc-GAL4/UAS-hern leg (or wing) discs, the expression of ss is also activated. Curiously, although ss is downstream of hth in the antenna and leg, ectopic ss in the leg disc can also activate hth in a few cells (Suzanne, 2003).

The hth or ss genes, together with Dll, are sufficient to develop ectopic distal antennae when expressed in different regions of the adult. The hern or fer genes are also able to elicit this transformation in the leg and they activate hth and ss. Conversely, when high levels of the Hth or Ss products are induced in the leg discs, ectopic expression of the hern and fer genes is found. To study the interactions between these genes in normal development, the relationship between Dll, hth, ss, and hern/fer in the antennal primordium was examined. A reduction of Hth activity using a dominant negative form of hth (UAS-EN-HTH^1-430) results in a decreased activity of the MD634 and AC116 GAL4 lines, which reveal hern and fer expression, respectively. Similarly, in antennal discs of a Dll strong hypomorph or a ss null mutation, the expression of hern and fer disappears. These results suggests that hth, Dll, and ss are required to maintain hern and fer expression in the antenna. By contrast, high levels of hern or fer may reduce hth expression. In dpp-GAL4/UAS-fer or dpp-GAL4/UAS-hern larvae, the expression of hth (and sal) in the third antennal segment is eliminated or strongly reduced dorsally (where levels of hern and fer are high) and does not change or is ectopically activated ventrally (where levels of hern and fer are low). Similarly, fer-expressing clones are able to downregulate hth expression in the antennal primordium. These results suggest that levels of hern and fer expression may be important for a normal antennal development (Suzanne, 2003).

The differentiation of legs or antennae depends on the activity of the hth and Antp genes. The ss gene, however, is also able to transform distal leg (and also maxillary palp and rostral membrane) into distal antenna, and the absence of ss, like that of hth, transforms antenna into leg. Although ss seems to be downstream of Dll and hth in antenna specification, ectopic ss can activate hth in some cells of the leg disc. Similarly, misexpression of ss in the rostral membrane induces Dll expression. It seems, therefore, that ss can trigger an antennal genetic program when misexpressed in certain places (Suzanne, 2003).

The fer and hern genes are both required and sufficient to make part of the distal antenna. Four different genes, hth, ss, hern, and fer, are able to form distal antenna, together with Dll, when ectopically expressed. Their mutual regulation seems to differ when misexpressed in the leg disc or when normally expressed in the antennal primordium. In the leg disc, hern or fer activates hth and ss and, reciprocally, hth and ss induce hern and fer expression. Moreover, even ss can promote hth transcription, although just in a few cells. Taken together, these results suggest that the four genes can form distal antenna by activating each other's transcription when ectopically expressed (Suzanne, 2003).

In the third antennal segment, Dll, hth, and ss are required to activate hern/fer expression. Since ss is downstream of Dll and hth in the antenna, the activation of hern/fer by Dll and hth could be mediated by ss. It is noted, however, that the levels of hern and fer may modulate hth expression. Moderately increased levels of fer can activate hth in dpp-GAL4/UAS-fer discs but, when the levels of hern or fer in the antenna are highly increased, the transcription of hth is prevented. These results suggest that the total amount of hern and fer expression may be regulated in the antennal primordium. Accordingly, in clones mutant for danr (hern), the expression of dan (fer) is upregulated. Also supporting the conclusion that levels of hern and fer have to be regulated, it was found that, in ey-GAL4/UAS-hern or ey-GAL4/UAS-fer flies, where levels of either hern or fer are highly increased in the eye–antennal disc, both the eye and the antenna disappear (Suzanne, 2003).

The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site. One such element is the xenobiotic response element to xanthotoxin (XRE-Xan) that lies upstream of consensus XRE-AhR (xenobiotic response element to the aryl hydrocarbon receptor) and OCT-1 (octamer-1 binding site) element known to be utilized in mammalian aryl hydrocarbon response cascades. Two-plasmid transfections conducted in Sf9 cells have indicated that XRE-Xan, XRE-AhR and a number of other proximal elements, but not OCT-1, are critical for basal as well as xanthotoxin- and benzo[alpha]pyrene-induced transcription of the CYP6B1 promoter. Four-plasmid transfections with vectors co-expressing the Spineless (Ss) and Tango (Tgo) proteins, the Drosophila melanogaster homologues of mammalian AhR and ARNT, have indicated that these proteins enhance basal expression of the CYP6B1 promoter but not the magnitude of its xanthotoxin and benzo[alpha]pyrene induction. Based on these results, it is proposed that these Drosophila transcription factors modulate basal expression of this promoter in a ligand-independent manner and attenuate its subsequent responses to planar aryl hydrocarbons (benzo[alpha]pyrene) and allelochemicals (xanthotoxin) (Brown, 2005).

Protein Interactions

The Drosophila spineless gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). The ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss minus mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus (Emmons, 1999).

Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. In the embryo, ss is expressed in the antennal segment, the gnathal segments, the leg anlage, and the peripheral nervous system. Strong nuclear accumulation of Tgo is seen in the antennal segment, which expresses the highest level of ss. Nuclear accumulation of Tgo is also observed in the gnathal segments (mandibular, maxillary, and labial), but the intensity of staining is relatively weak compared to the antennal segment. This correlates with the relatively weak expression of ss in the gnathal segments, when compared to the antennal segment. Nuclear localization of Tgo in the antennal and gnathal segments is dependent on ss, since it is not seen in a ss null mutant. The expression of ss in the appendage primordia and the peripheral nervous system also correlates with Tgo nuclear accumulation. Sensory cells that express ss are in close proximity to the tracheal cells that express trh. To distinguish these, embryos were labeled with anti-Trh and anti-Tgo. Non-tracheal cells that show nuclear Tgo are observed in the location of ss-expressing sensory cells. This non-tracheal Tgo nuclear accumulation is absent in ss mutant embryos. These results indicate that Tgo accumulates in the nuclei of ss-expressing antennal, gnathal and sensory cells, consistent with the formation and nuclear accumulation of Ss:Tgo heterodimers in vivo. Surprisingly, no significant Tgo nuclear accumulation is seen in the limb primordia, even though ss is expressed in these cells. This may reflect regulatory events idiosyncratic to the limb primordia, or a lack of sensitivity of the immunostaining, since the limb primordia express ss at considerably lower levels than the antennal segment. Tgo nuclear accumulation is also observed in the cells of the dorsal vessel. Since, sim, ss and trh are not expressed in the dorsal vessel, an additional bHLH-PAS protein may function in combination with Tgo in controlling the development or physiology of these cells, which comprise the Drosophila circulatory system. When ectopic expression of a UAS-ss transgene is driven by en-Gal4, Tgo is found to accumulate in nuclei in circumferential ectodermal en stripes. Similarly, expression of ss in mesodermal cells (driven by twi-GAL4) causes nuclear accumulation of Tgo in the mesoderm. These experiments support the conclusion that Ss and Tgo interact in vivo, and suggest that their interaction and nuclear accumulation does not depend on additional, spatially-restricted, factors. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved (Emmons, 1999).

How did Ss and Ahr come to have such different functions in vertebrates and arthropods? One possibility is that Ahr functioned as some type of chemosensory protein in an ancestral organism. In vertebrates, this function became utilized by all cells to sense aryl hydrocarbon toxins, whereas in arthropods it became intimately associated with the specification of a major chemosensory organ, the antenna. It is hoped that studies of organisms from other lineages will shed light on how Ss and Ahr came to adopt such different roles (Emmons, 1999).

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. The distal antenna (dan) and distal antenna-related (danr) genes encode novel 'pipsqueak' motif nuclear proteins that probably function as DNA binding proteins serving as sequence-specific transcription factors but may serve instead as more general chromatin modification factors. dan and danr are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore it is suggested that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. It is proposed that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures (Emerald, 2003).

Dan plays an important role in specification of antennal identity downstream of spineless (ss), but rescue of the spineless mutation by Dan suggests that there may be additional genes acting downstream from ss to specify antennal identity. spineless mutants lead to ectopic expression of Antennapedia and concomitant loss of Dan/Danr expression and cause a strong phenotypic transformation of distal A3 and arista to tarsus. To determine whether morphological transformation depends on loss of Dan/Danr, use was made of Gal4 to direct Dan expression in the ss mutant discs. ptc^Gal4 directed expression of Dan causes strong suppression of the arista-to-tarsus transformation in the ss mutant antenna. ptc^Gal4 is expressed in a stripe of cells adjacent to the AP boundary in the antenna region of the disc. Dan expression does not repress ectopic expression of Antp in the ptc^Gal4 stripe of the mutant discs. This suggests that Dan can direct antennal differentiation in the presence of Antp, and overcome the ability of Antp to cause transformation to tarsus. Remarkably, this transformation can affect the entire distal arista, even though ectopic Dan is expressed in only a subset of Antp-expressing cells. These observations suggest that Dan plays an important role in specification of antennal identity (Emerald, 2003).

An additional line of evidence to indicate that both genes contribute to distal antenna identity comes from examining genetic interactions with spineless^aristapedia. ss^a mutants lose Dan/Danr expression and express Antennapedia ectopically in the antenna disc. Restoring Dan expression is able to partially suppress the transformation to antenna, implicating Dan as an effector of ss^a function. The consequences of removing one copy each of Dan and Danr was examined in a ss mutant background. The spineless^114.4 allele shows a mild transformation of the basal capsule of the arista when heterozygous, suggesting that the reduced level of ss activity in this allele is not sufficient to support normal development. Removing one copy of danr using the danr^ex35 deletion in this background causes a modest increase in the size of the basal capsule and in the number of ectopic bristles. The dan danr^ex56 deletion causes a stronger phenotype, with the basal capsule adopting a two-segment structure with multiple bracted bristles and obvious tarsal morphology. Flies heterozygous for the dan danr^ex56 deletion are morphologically normal. Thus, reduction of both Dan and Danr gene dose leads to a more severe phenotype under conditions where ss activity is limiting. Even more extreme arista transformation phenotypes are observed when one copy of ss is removed in animals homozygous for the dan danr^ex56 deletion (Emerald, 2003).

Loss of Hth activity has been shown to cause transformation of arista to tarsus, presumably because of loss of ss. It has been suggested that uniform expression of Hth in second and early third instar antennae might be responsible for its role in specification of distal antenna identity. However, the results of this study indicate that Hth can have a non-autonomous effect on the expression of Dan in the antenna. Hth-expressing cells sort out from the distal part of the leg. Nonetheless they are able to induce Dan expression in cells that remain integrated in the distal leg. This observation is best explained by a non-autonomous induction of Dan in response to a signal from Hth-expressing cells. Responsiveness to this signal apparently requires Dll, which limits it to the distal region. These effects are presumably mediated by regulation of ss, which is required for Dan and Danr expression. These observations provide an explanation for the apparently non-autonomous role of Hth together with Dll in the distal antenna (Emerald, 2003).

ss is also required to induce Dan and Danr and to repress Antp expression. Repression of Antennapedia may be mediated in part by repression of Cut. The findings described above implicate Dan and Danr as downstream effectors of ss that promote development of distal antennal structures. Remarkably, expression of Dan or Danr under Gal4 control can restore antenna development and prevent transformation of antenna to leg in the ss mutant, even when Antp is present. A striking feature of these results is that there appears to be non-autonomous activity. Transformation is blocked in cells expressing Dan and Danr, as well as in nearby cells that do not express these proteins. The identity of the genes responsible for these non-autonomous effects in antenna specification remains to be determined. In view of recent reports of non-autonomous effects of vein/EGFR signaling in development of distal leg pattern, it will be of interest to learn if there is a link to this pathway in the non-autonomous effects of Dan and Danr (Emerald, 2003).

DEVELOPMENTAL BIOLOGY

Embryonic

Spineless mRNA staining first appears at embryonic stage 8 in a crescent just anterior to the cephalic furrow. This staining develops rapidly into an intense patch. As germ-band extension continues, staining develops in the maxillary, labial, and mandibular segments, followed by expression in a ventral patch in all three thoracic segments. These patches have been identified as the leg anlage by double-labeling for transcripts from Spineless and Aristaless. Spineless mRNA staining also appears in cells of the peripheral nervous system in each abdominal segment. The staining pattern is maintained through germ-band retraction and continues until the deposition of larval cuticle makes it difficult to follow further. To position the Spineless head patch, embryos were stained for both Spineless transcript and Engrailed protein. The posterior boundary of the Spineless antennal patch coincides precisely with the posterior edge of the antennal En stripe. There is a one-to-one correspondence between Spineless- and En-expressing cells for some distance along this stripe, although the En antennal stripe extends ventrally several cells beyond the Spineless patch. The anterior limit of Spineless expression lies just posterior to the En head spot, which delimits the posterior border of the ocular segment. Thus, Spineless mRNA is expressed throughout most or all of the embryonic antennal segment, and is expressed in a segmental, not parasegmental, register (Duncan, 1998).

The expression of spineless in embryos is unexpected, as no embryonic defects have been described for spineless mutants. However, examination of null mutant larvae reveals that the antennal sense organ is misshapen and often sclerotized. In addition, the dorso-medial papilla of the maxillary sense organ, which is thought to be derived from the antennal segment, fails to migrate completely. Examination of spineless mutant embryos stained with the monoclonal antibody 22C10 fails to reveal any defects in the peripheral nervous system (Duncan, 1998).

Pupal

Spineless mRNA is expressed in the distal portion of the antennal imaginal disc, the tarsal region of each leg disc, and in bristle precursor cells. In leg discs, Spineless mRNA staining is first seen in the late second instar in a central ring that corresponds to the presumptive tarsal region. This ring is transient, and fades out by the late third instar. The Spineless tarsal ring likely corresponds to the tarsal structures deleted in spineless mutants, which include the distal part of the first tarsal segment and the second through fourth tarsal segments. After the tarsal ring fades out, Spineless mRNA becomes expressed in a patch in the anterior-proximal portion of the disc in a region that will give rise to structures of the thorax proper. spineless null alleles show no defects in these structures (Duncan, 1998). Spineless mRNA staining in the antennal disc is first detected during the late second instar. At all times after this, intense staining is seen in an oval patch in the central (distal) portion of the antennal disc. After disc eversion, the limits of intense Spineless mRNA expression coincide precisely with the boundary between the second (AII) and third (AIII) antennal segments. In spineless null mutants, the antenna shows both transformation to leg and tarsal deletion. In this case, the entire AIII segment and arista are affected. Strikingly, the AIII segment in spineless null mutants is unlike any normal appendage segment in that it completely lacks bristles or cuticular hairs. This segment is followed distally by most or all of a fifth tarsal segment terminated by claws (Duncan, 1998).

In the late third instar, spineless is expressed in a small patch in the antennal disc that corresponds to the maxillary palp anlage. Consistent with this, the maxillary palps of null mutants are truncated. This suggests a general requirement for spineless in the development of distal structures in ventral appendages (Duncan, 1998).

In the wing disc, spineless is expressed in the presumptive notum and wing hinge region, and in a ventral stripe. Perhaps related to this expression, the wings of null mutants are held perpendicular to the body and curve ventrally. The haltere disc stains in a similar pattern. Expression is also detected in the genital and labial discs, and in the morphogenetic furrow of the eye disc. Genital, labial, and eye development appear normal in spineless mutants (Duncan, 1998).

At pupariation and disc eversion, stereotyped patterns of single large intensely stained cells are seen in most discs, including the leg. The pattern of labeled cells is identical to that shown by the neuralized enhancer trap A101, indicating that these late spineless expressing cells are sensory organ precursors. At later stages, intense staining is seen in developing bristle cells, but not in the associated socket cells (Duncan, 1998).

EFFECTS OF MUTATION

Mutations in the spineless-aristapedia (ssa) gene of Drosophila are pleiotropic; their classical manifestations include a reduction in size of all bristles (spineless phenotype), transformation of distal parts of antennae into tarsal segments of the mesothoracic leg (aristapedia phenotype), and, in extreme alleles, fusion of tarsal segments on all six legs and the transformed aristaes. A new allele has been isolated, which is a severe loss-of-function mutation and, in addition to the above-mentioned features, is characterized by amplification of sex combs on the first leg. This phenotype can be caused by a change in the expression of the Sex combs reduced (Scr) gene of the Antp-C. Identification of this phenotype, together with observed variations in the extent of the fusion of tarsal segments in the legs of different segments, raises the possibility that ssa interacts with homeotic genes controlling the identity of segments. This possibility was tested in genetic experiments using flies with loss-of-function mutations in several homeotic genes and flies transformed by heat shock-driven homeotic genes. Analysis of adult phenotypes of different ssa alleles in the background of under-, over-, or ectopic expression of some genes of Bx-C and Ant-C suggests that the ssa product is required to prevent the effect of the homeotic gene products in the distal segments of the appendages (Kuzin, 1997).

The transformation of antenna to leg in Drosophila was carried out using ectopically expressed transgenes with heat shock promoters: heat shock Antennapedia, heat shock Ultrabithorax, and heat shock mouse Hox A5. The frequency of transformation of several leg markers was determined in response to Antennapedia protein delivered by heat shock at different times and doses. Stage-specific responses to the transgene, heat shock mouse Hox A5, were also studied. Each marker has its own stage and dose-specific pattern of response. The same marker can pass through a period of high-dose inhibition followed by a dose-independent response and then a positive dose-dependent phase. The heat shock-induced transgenes and spineless aristapedia transform the apterous enhancer trap antenna disc expression pattern toward the pattern found in leg discs. These results are considered in relation to developmental competence: the ability of developing tissue to respond to internal or external influences. The results suggest that all genes tested interact with the same competence system and that at least two classes of mechanisms are associated with antenna to leg transformation: one comprises global mechanisms that permit transformation over approximately 24 hr; the second class of mechanisms act very locally and are responsible for changes in dose response on the order of 4-8 hr (Larsen, 1996).

The Drosophila gene stubarista (sta) encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. The basis of the sta1 visible phenotype which consists of shortened antennae and bristles has been analyzed. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. Genetic evidence is provided that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein (Melnick, 1993).

The homeotic mutation spineless-aristapedia (ssa) transforms the aristae into second tarsi. Flies with an ssa phenotype also show extremely positive geotaxis as measured in a Hirsch-type geotaxis maze. Other antennal mutants and flies with their aristae amputated do not show such extreme positive geotaxis. Deletion analysis has co-mapped the geotaxis effect with ssa in band 89C on the third chromosome. A biometrical analysis has detected additional genes on the X chromosome that also affect geotaxis (McMillan, 1992).

Loss-of-function mutations in the spineless-aristapedia gene of Drosophila (ssa mutants) cause transformations of the distal antenna to distal second leg, deletions or fusions of the tarsi from all three legs, a general reduction in bristle size, and sterility. Because ssa mutants are pleiotropic, it has been suggested that ss+ has some rather general function and that the ssa antennal transformation is an indirect consequence of perturbations in the expression of other genes that more directly control antennal or second leg identity. A test has been made of whether the ssa transformation results from aberrant expression of Antennapedia (Antp), a homeotic gene thought to specify directly the identity of the second thoracic segment. Antp-ssa mitotic recombination clones in the distal antenna behave identically to Antp+ ssa clones, and are transformed to second leg. This demonstrates that the ssa antennal transformation is independent of Antp+, and suggests that ss+ may itself directly define distal antennal identity. The results also reveal that Antp+ is not required for the development of distal second leg structures, as these develop apparently normally in Antp- ssa antennal clones. It is suggested that ss+ and Antp+ may play similar, but complementary, roles in the distal and proximal portions of appendages, respectively, because Antp- mutations cause deletions or transformations that are restricted to proximal structures, whereas ssa alleles cause similar defects that are distally restricted (Burgess, 1990).

A two-step screen for isolating null mutations of the spineless-aristapedia locus has been performed, and several amorphic mutations, as well as a small deficiency, have been obtained. With the exception of the deficiency, which deletes genes required for viability on either side of the spineless-aristapedia locus, these mutations result in a transformation of only the distal antenna into distal leg, thereby indicating that the spineless-aristapedia gene is required for specifying antennal, as opposed to leg development, in only the distal portion of the antenna. Because this distal region does not appear to be a developmental compartment, it is probable that the spineless-aristapedia gene, unlike several other homeotic genes, is required for maintaining the correct determined state in a population of cells defined by their relative position, not by their ancestry (Struhl, 1992).

The development of the sensory neuron pattern in the antennal disc of Drosophila melanogaster was studied with a neuron-specific monoclonal antibody (22C10). In the wild type, the earliest neurons become visible 3 h after pupariation, much later than in other imaginal discs. They lie in the center of the disc and correspond to the neurons of the adult aristal sensillum. Their axons join the larval antennal nerve and seem to establish the first connection towards the brain. Later on, three clusters of neurons appear in the periphery of the disc. Two of them most likely give rise to the Johnston's organ in the second antennal segment. Neurons of the olfactory third antennal segment are formed only after eversion of the antennal disc (clusters t1-t3). The adult pattern of antennal neurons is established at about 27% of metamorphosis. In the mutant lozenge3 (lz3), which lacks basiconic antennal sensilla, cluster t3 fails to develop. This indicates that, in the wild type, a homogeneous group of basiconic sensilla is formed by cluster t3. The possible role of the lozenge gene in sensillar determination is discussed. The homeotic mutant spineless-aristapedia (ssa) transforms the arista into a leg-like tarsus. Unlike leg discs, neurons are missing in the larval antennal disc of ssa. However, the first neurons differentiate earlier than in normal antennal discs. Despite these changes, the pattern of afferents in the ectopic tarsus appears leg specific, whereas in the non-transformed antennal segments a normal antennal pattern is formed. This suggests that neither larval leg neurons nor early aristal neurons are essential for the outgrowth of subsequent afferents (Lienhard, 1991).

Drosophila melanogaster females expressing the homoeotic mutation, spineless-aristapedia (ssa), were tested for their ability to hear the song of courting males. Since courtship song increases a females' receptivity to copulation, the frequency of mating within a short observation period was used as a measure of the ability of mutant females to distinguish between singing males and males that were unable to sing. These results show that ssa females, although lacking aristae, can distinguish between the two types of males in that they mated more readily with males that sang. Furthermore, the homoeotic legs of ssa females are not required to be present for the detection of courtship song, since females whose homoeotic legs were removed could still distinguish between singing and non-singing males (McRobert, 1991).

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).

As with Dll and hth loss-of-function mutants, loss of spineless (ss) also results in antenna to leg transformations. The genetic relationship among these genes was investigated. The expression of both Dll and hth appears relatively normal in the ss null antennal disc. It is therefore concluded that ss is not required for either the activation or the maintenance of Dll or hth expression in the antenna. It has been reported that Dll is required for the antennal expression of ss. To test whether Hth is also required to activate antennal ss expression, the effect of ectopic hth was examined. Ectopic Hth where Dll is expressed, for example in the wing pouch and leg disc, can activate ss expression. Conversely, loss of hth in the antenna results in loss of ss expression. Taken together, these results indicate that ss functions downstream of both Dll and hth in the antenna (Dong, 2002).

ss is expressed in a circular pattern in the antenna covering the presumptive a2 through the arista. In the leg disc, ss is transiently expressed in a ring pattern in the presumptive tarsal region and subsequently becomes restricted to leg bristle precursors. Consistent with the ss expression domain, cuticular defects in ss null mutants can be found from a2 through the arista. These include the elongation of a2, loss of olfactory sensilla from a3, and transformation of a4, a5, and arista to tarsal segments (Dong, 2002).

The large differences in the expression patterns of these genes between the antenna and the leg begs the question of whether these differences are due to differential regulation by antenna-determining genes such as Dll and hth. To test whether Dll or hth are responsible for the antenna-specific expression patterns of these genes, the effects on their patterns were examined in Dll and hth loss-of-function mutants. Whether Dll and hth are regulating their antenna-specific targets via ss was tested by examining their expression in ss null antennal discs (Dong, 2002).

The antennal dac expression domain expands in Dll hypomorphs and in hth null clones. This expansion of dac expression in Dll and hth mutant antennae resembles the leg pattern of dac expression. In contrast, in the ss null antenna, there appears to be neither expansion nor reduction of dac expression. The only detectable difference in the ss null antennal disc is overgrowth in the central (distal) area such that the ring of dac expression has a larger radius. This correlates with the transformation phenotype of the ss null arista into a tarsus, which is a larger structure. Since the expression of dac relative to other genes appears normal in ss null antennae, ss is not thought to regulate dac (Dong, 2002).

The expression of ato is required for the formation of the JO. The JO is a structure unique to the antenna and is required to sense sound vibrations transmitted from the arista. ato function is generally associated with neuronal differentiation, so it is interesting that cuticular defects are associated with ato null antennae. It may be that formation of the JO is required for the normal morphology of the a2/a3 joint. The circular outline of the a2/a3 joint is lost in hth and Dll loss-of-function mutants, but is present in ss null mutants. Consistent with this, the antennal expression of ato is lost in hth null clones and in Dll hypomorphs, but persists in the ss null antenna discs. Thus although ss null mutants exhibit cuticular defects in a2 and a3, the a2/a3 joint to which the JO is attached is present. It is noted that the Dll hypomorphic combination used, Dll^GAL4/Dll³, does not lead to loss of a2. Thus the absence of ato expression in these antennae is not due to death of the cells that would normally express it (Dong, 2002).

Dll and hth are required for the expression of sal in the antenna. sal expression does not appear to be affected in ss null antenna. The fact that Ss is not required for the expression of either ato or sal in a2 is consistent with the observation that the a2/a3 joint is still present in the ss null antenna (Dong, 2002).

This study serves to initiate an understanding of the different roles that these homeotic genes are playing in antenna specification. During imaginal disc development, the expression of Dll and ss is found from a2, a3, a4, a5 and arista. Expression of hth is dynamic and retracts from the distal-most segments by late third instar, but hth is expressed and cell-autonomously required throughout the antenna from a1 through to the arista (Dong, 2002).

Since ss is not required to activate antenna-specific expression of genes such as sal/salr and ato that are involved in antenna differentiation, the question arises as to what ss does do in the antenna. ss represses tarsus and tarsal claw organ formation in the antenna. Since loss of ss also leads to loss of olfactory sensillae on a3, ss probably potentiates the formation of these sensillae, either cooperating with or mediating Dll and hth activities in a3. Similarly, since ectopic expression of ss elsewhere in the body can lead to the formation of ectopic aristae, ss may also cooperate with or mediate Dll and hth activities in arista differentiation (Dong, 2002).

sal and salr, like ato, are required for normal auditory functions. Since both Dll and hth are required for the antennal expression of ato and sal, Dll and hth mutant antennae are also hearing defective. In contrast, ss null antennae exhibit normal expression of both ato and sal and normal morphology of the a2/a3 joint, leading to the idea that ss mutants are likely to be functional in audition (Dong, 2002).

Stochastic spineless expression creates the retinal mosaic for colour vision

Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called 'pale' and 'yellow', defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. The Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless (Wernet, 2006).

The ability to discriminate between colours has evolved independently in vertebrates and invertebrates. However, despite the obvious differences in eye development and design, both flies and humans have developed retinal mosaics where classes of photoreceptor cells (PRs) with different spectral sensitivity are randomly distributed. The compound eye of Drosophila consists of ~800 optical units (ommatidia), each containing eight PRs in addition to accessory cells. In each ommatidium, the six 'outer PRs' (R1-R6) function like the vertebrate rod cells, as they are required for motion detection in dim light. These cells express the broad-spectrum rhodopsin, Rh1. The 'inner PRs' (R7 and R8) may be viewed as the equivalent of the colour-sensitive vertebrate cone cells, which express a range of different rhodopsin molecules (Wernet, 2006).

The general rule of sensory receptor exclusion also applies to Drosophila ommatidia, where only one rhodopsin gene is expressed by a given PR. The expression of inner PR rhodopsins can be used to distinguish three ommatidial subtypes. Two of the subtypes are distributed randomly throughout the retina: ~30% of ommatidia express ultraviolet-sensitive Rh3 in R7 cells and blue-sensitive Rh5 in R8 cells, and therefore are specialized in the detection of short wavelengths ('pale' ommatidia). The remaining ~70% express another ultraviolet-sensitive opsin (Rh4) in R7 and green-sensitive Rh6 in R8, making them more responsive to longer wavelengths ('yellow' ommatidia). The coupled expression of Rh3/Rh5 or Rh4/Rh6 within the same ommatidium results from communication between R7 and R8. In the dorsal rim area (DRA), a third type of ommatidia exists in which both R7 and R8 express ultraviolet-sensitive Rh3. These ommatidia are used to detect the e-vector of polarized sunlight for orientation. Spatially localized polarized light detectors and stochastically distributed colour-sensitive ommatidia therefore reflect two fundamentally different specification strategies that shape the retinal mosaic of Drosophila (Wernet, 2006).

The current model for specifying colour-sensitive ommatidia combines stochastic and instructive steps. First, a subset of R7 (pale R7, pR7) stochastically chooses Rh3 expression over the 'R7 default', Rh4. Second, these cells then impose the p fate (Rh5) onto R8 (pale R8, pR8) of the same ommatidium (Wernet, 2006).

This study reports the identification of spineless (ss) as a key regulatory gene for establishing the retinal mosaic. ss encodes the Drosophila homologue of the human arylhydrocarbon ('dioxin') receptor, a member of the bHLH-PAS (basic helix-loop-helix- Period-Arnt-Single-minded) family of transcription factors. At mid-pupation, ss is stochastically expressed in a majority of R7 that seem to correspond to the y subtype. ss is both necessary and sufficient to specify the yellow R7 (yR7) fate and subsequently the entire y ommatidia; pR7 cells are thus specified by default, and stochastic expression of ss represents the key regulatory event defining the retinal mosaic required for fly colour vision (Wernet, 2006).

homothorax (hth) has been identified as the key regulatory gene necessary and sufficient for the specification of DRA ommatidia. ss and hth cause similar homeotic phenotypes: that is, complete (hth) or partial (ss, 'aristapedia') transformation of antennae into legs. Therefore, a potential role of ss in ommatidial subtype specification was tested by generating whole-mutant eyes, as well as mitotic clones, lacking ss function using the null allele ss^D115.7 and the ey-FLP/FRT technique. Owing to ey-FLP expression in the antennal imaginal disc, ss mutant flies showed a strong aristapedia phenotyp, but lacked any obvious morphological eye phenotype. However, expression of rhodopsin genes was severely affected. In wild-type eyes, Rh3 is found in ~30% of R7 cells, as well as in both R7 and R8 of DRA-ommatidia, whereas the remaining ~70% of R7 contain Rh4. In ss mutant eyes, Rh4 was completely absent, whereas Rh3 was expanded into all R7 cells. The total number of ommatidia was not reduced, indicating that R7 cells were mis-specified into pR7, rather than yR7 being specifically eliminated. ss mutant mitotic clones were morphologically wild type; however, Rh3 was always present in mutant R7 cells (marked by the absence of ß-galactosidase (ß-gal) expression), whereas Rh4 was always lost (Wernet, 2006).

To test whether the R7 ss phenotype was cell autonomous, individual mutant R7 cells were generated using the MARCM technique. All mutant R7 cells [marked by the presence of green fluorescent protein (GFP) expression] contained Rh3 and never Rh4, demonstrating that ss is required cell autonomously in R7 to induce Rh4 expression. DRA ommatidia were correctly specified in ss mutant eyes, since Rh3 was expressed normally in both DRA R7 and R8 cells. Therefore, ss is necessary for the establishment of the yR7 subtype without affecting PR fate specification (Wernet, 2006).

The ommatidial subtypes are first specified in R7, which then instruct R8. Therefore, ss mutant eyes should exhibit a rhodopsin phenotype in R8. In wild types, ~30% of R8 cells contain Rh5, and the remaining ~70% contain Rh6. In ss mutant eyes, the large majority (up to 95%) of R8 contained Rh5, with some R8 still containing Rh6. However, most of these remaining yR8 cells were located in the dorsal third of the eye. In this part of the retina, instruction of pale R8 (pR8) by pR7 is less efficient, resulting in ommatidia with odd-coupled (Rh3/Rh6) rhodopsin expression. In ss mutants, the frequency of such ommatidia was significantly increased in the dorsal region. To test whether the R8 opsin phenotype of ss mutants resulted from the inability of some mutant R7 cells to properly instruct R8, rather than from ss being directly required in R8, sevenless; spineless (sev; ss) double-mutant eyes were generated (Wernet, 2006).

These eyes, which lacked R7 cells, always exhibited the sev single-mutant phenotype, with virtually all R8 cells containing Rh6. This indicates that ss is required in R7 for the formation of the yR7 subtype, and consequently for the formation of yR8, without being directly required in R8 PRs (Wernet, 2006).

Whether ss was also sufficient to induce the y ommatidial subtype was tested. Overexpression of ss in all developing PRs using a strong LGMR (long glass multiple reporter)-Gal4 driver and UAS-ss (LGMR.ss flies) resulted in a rough eye phenotype, as well as a dramatic rhodopsin phenotype: Rh4 was activated in all PRs throughout the eye (R1-R6 as well as R7 and R8), as revealed by ectopic expression of an Rh4-GFP reporter in many PRs per ommatidium compared with wild type. To avoid the strong phenotype in the eye, ss was misexpressed using the weaker, variegated GMR driver, sGMR (short GMR)-Gal4³¹ (sGMR.ss flies). This led to strong ectopic induction of Rh4 in many PRs without severely affecting retinal morphology. This ectopic induction of Rh4 was also observed in sev mutants, and was thus independent of R7. Rh3 was still detected in some R7 in sGMR.ss flies, presumably due to the lack of variegated Gal4 expression in these cells, whereas Rh4 was expanded to some outer PRs. However, co-localization of Rh3 and Rh4 was never observed, confirming that gain of Rh4 in R7 cells always leads to the exclusion of Rh3. In contrast, gain of Rh4 in outer PRs did not lead to the exclusion of Rh1; frequent coexpression of Rh1 and Rh4 was observed (Wernet, 2006).

Using an Rh4-lacZ reporter construct in LGMR.ss flies, it was found that ß-gal-positive PR axons projected to both lamina and medulla, confirming the expansion of Rh4 into outer PRs. However, Rh4-expressing outer PRs were not transformed into genuine R7 cells, since they maintained their lamina projections. Notably, DRA inner PRs were the only cells not expressing Rh4, suggesting that the DRA fate, specified by the gene hth, antagonizes ss function. Expression of Rh3 and Rh5 was completely lost (including in the DRA, where no rhodopsin was detected), while Rh6 expression was found in most R8 cells. This resulted in R8 coexpressing Rh4 and Rh6, demonstrating that the 'one sensory receptor per cell' rule can be broken in Drosophila PRs, as has been shown in other insects. Therefore, ectopic induction of the yR7 fate by ss specifically excludes the formation of pR7 cells. As a consequence, R8 cells expressing Rh5 are not induced, with most R8 expressing Rh6. Rh6 was never found in outer PRs, supporting the hypothesis that ss is required only in R7 for the choice between Rh3 and Rh4, and not directly in R8 for the Rh6 choice. In LGMR.ss flies, the specification of outer versus inner PRs (markers spalt and seven up) or of R7 versus R8 (prospero and senseless) was normal. Thus, ss acts by segregating ommatidial subtypes downstream of early PR specification events (Wernet, 2006).

Colour PR cell fate determination seems to be a late event in PR differentiation. To test whether ss can transform the R7 fate at late stages of development, the PanR7-Gal4 driver (which is also expressed in DRA R8 cells) was used. Late mis-expression of ss induced the y fate (Rh4) in all R7 cells, whereas Rh3 was absent. Opsin expression in the DRA was also altered, with Homothorax-positive cells (both R7 and R8) now expressing Rh4. Hence, it is possible to reprogramme the R7 fate at later stages of differentiation, as PanR7-Gal4 becomes activated at the time of rhodopsin expression. Surprisingly, expression of R8 rhodopsins outside the DRA was not affected, since the distribution of Rh5 and Rh6 resembled the wild type. As a result, many ommatidia manifested the very unusual coupling of Rh4 in R7 and Rh5 in R8. Therefore, although ss is able to reprogramme all R7 late in development, R8 cannot revert their fate once they have been instructed to become pR8, and they maintain Rh5. Two antagonistic genes expressed in either of the two R8 subtypes have been identified that act together as a molecular consolidation system responsible for this inertia of R8. To confirm that late expression of ss exclusively in R7 is sufficient to transform R7, ss was mis-expressed in ss^D115.7 mutants using PanR7-Gal4. This was sufficient to induce Rh4 and to repress Rh3. R8 were again not reprogrammed and exhibited the ss mutant phenotype, with many R8 cells expressing Rh5 (Wernet, 2006).

All of the results presented above strongly indicated that ss must be expressed in the y subtype of R7 at some point during pupal development. Since several attempts to generate an anti-Spineless antibody had failed, in situ hybridization was used to detect ss messenger RNA in the retina at mid-pupation. At ~50% pupation, ss mRNA was detected in four neuronal cells per ommatidial cluster, one PR and three bristle cells. The PR was also labelled by anti-Prospero, confirming its identity as R7. Although the expression levels of ss in bristle cells seemed uniformly high, levels of ss expression varied considerably among R7 cells, ranging from very faint to very strong in 60%-80% of R7. A 1.6 kilobase 'eye enhancer' fragment (ss_eye) was also identified within the ss promoter that drives PR-specific expression. After crossing ss eye-Gal4 to UAS-ß-gal::NLS (nuclear localization sequence) reporters, PR-specific ss expression was first detected at mid-pupation-that is, approximately one day before rhodopsins are expressed, and before any visible molecular or morphological distinction between ommatidial subtypes. A single PR per ommatidium, which was identified as R7 through co-staining with Prospero, expressed ss. Thus, the ss eye enhancer recapitulates endogenous ss expression in PRs. ss expression was detected in 60%-80% of R7, correlating well with the distribution of Rh4 in adult retina. Like Rh4-expressing ommatidia, ß-gal-positive ommatidia were more abundant in the dorsal half of the eye, and no ß-gal expression was detected in the DRA (marked by Homothorax), where Rh4 is also never expressed. ss eye-Gal4 expression was detectable for only ~2 h at midpupation (Wernet, 2006).

Although it was not possible to directly co-stain for ss and Rh4 (which starts to be expressed one day later during pupation), it seems that at mid-pupation a short pulse of ss is deployed in a large subset of R7, which will become yR7 (Wernet, 2006).

Whether a short pulse of ectopic ss expression was able to modify the entire retinal mosaic was tested using a heat shock-Gal4 driver (hs-Gal4) to temporally control ss expression (hs.ss flies). A 30-min heat shock at ~50% pupation indeed resulted in an increase of Rh4 expression with a concomitant reduction of Rh3 in adults. The phenotype varied extensively, from only R7 cells expressing Rh4 (~25% of the flies analysed had Rh4 in most R7), to almost every PR expressing Rh4. In contrast, a 30-min pulse of ss in one-day-old adult flies had no effect. Heat shocks during larval or early pupal stages were lethal. Thus, PRs are extremely sensitive to a short pulse of ss during mid-pupation, at the time when endogenous ss is normally expressed. To further study the mechanism of the stochastic choice between p and y ommatidia, the retinal mosaic was examined in different mutant backgrounds. Flies heterozygous for ss^D115.7 had fewer Rh4-expressing R7 cells. Since the ss^D115.7 allele affects only the ss coding sequence, heterozygous flies have two functional promoters, only one of which produces a functional protein, suggesting that the non-productive promoter might sequester limiting factor(s) that regulate(s) the expression levels of ss. If this hypothesis is correct, addition of extra copies of the ss promoter should have a similar effect. Indeed, the addition of two functional copies of the ss eye enhancer (ss eye-Gal4) in an otherwise wild-type background also caused a significant reduction of the yR7 subtype. Therefore, the level of Spineless expression is important for the induction of the yR7 fate, which is less efficient in cells where the amount of Spineless is reduced (Wernet, 2006).

Retinal patterning in Drosophila reveals an original mechanism for how PR mosaics can be generated: stochastic expression of a single transcription factor (Spineless) acts as a binary switch that transforms the seemingly homogeneous compound eye into a mosaic, distinguishing p and y subtypes. However, subtype specification and rhodopsin expression can be separated, since ss expression in yR7 has ceased well before the time of rhodopsin expression. Additional factors are therefore required downstream of ss to ensure expression of adult p- and y-specific markers such as rhodopsins and additional screening pigments. A revised two-step model is proposed for the stochastic specification of p and y ommatidia. First, R7 are stochastically divided into two subtypes by the induction of ss in yR7. ss-positive R7 express Rh4, whereas the remaining R7 choose the pR7 fate and express Rh3 by default. Second, only those R7 cells that did not express ss (pR7) retain the ability to induce the pR8 fate (Rh5), whereas yR8 express Rh6 by default. The 'default states' of R7 (Rh3) and R8 (Rh6) therefore belong to opposite subtypes. Expression of R8 rhodopsin genes is maintained by a bistable regulatory loop containing the genes warts and melted34. Notably, the localized specification of polarization-sensitive DRA ommatidia by hth antagonizes the stochastic choice executed by ss, placing these two genes into a new regulatory relationship during retinal patterning. Therefore, the role of the transcription factor Spineless is to generate the retinal mosaic required for fly colour vision by distinguishing yR7 from pR7 cell fates, and preventing R7 from instructing the underlying R8 cells. Mosaic expression of sensory receptors has been described in detail for the olfactory system of both vertebrates and insects, and random PR mosaics have been described for humans and amphibians, as well as insects. Two transcription factors have been shown to regulate the specification of blue versus red/green cone cell fates in mammals. Upon mutation of either - the human nuclear receptor NR2E3 (also known as PNR) or the rodent thyroid hormone ß2 receptor - the number of blue cones is dramatically increased at the expense of green cones, leading to 'enhanced S-cone syndrome'. It should be noted that this retinal phenotype bears important similarity to the altered ommatidial mosaic in Drosophila ss mutants, where long wavelength-sensitive y ommatidia are lost at the expense of the short wavelength-sensitive p type (Wernet, 2006).

The stochastic cell fate choice occurs at the level of the ss promoter: the very short pulse of ss expression at mid-pupation is not only controlled temporally, but its levels are also critical, and only ~70% of R7 receive enough Spineless to commit to the yR7 fate. Elucidating the mechanism that controls ss expression will shed some light into the fascinating process of stochastic gene expression, and the identification of its downstream targets will provide insights into consolidation and maintenance of cell fates (Wernet, 2006).

The bHLH-PAS protein Spineless is necessary for the diversification of dendrite morphology of Drosophila dendritic arborization neurons

Dendrites exhibit a wide range of morphological diversity, and their arborization patterns are critical determinants of proper neural connectivity. How different neurons acquire their distinct dendritic branching patterns during development is not well understood. This study reports that Spineless (Ss), the Drosophila homolog of the mammalian aryl hydrocarbon (dioxin) receptor (Ahr), regulates dendrite diversity in the dendritic arborization (da) sensory neurons. In loss-of-function ss mutants, class I and II da neurons, which are normally characterized by their simple dendrite morphologies, elaborate more complex arbors, whereas the normally complex class III and IV da neurons develop simpler dendritic arbors. Consequently, different classes of da neurons elaborate dendrites with similar morphologies. In its control of dendritic diversity among da neurons, ss likely acts independently of its known cofactor tango and through a regulatory program distinct from those involving cut and abrupt. These findings suggest that one evolutionarily conserved role for Ahr in neuronal development concerns the diversification of dendrite morphology (Kim, 2006).

The ss protein is present at nearly the same level in all da neurons and acts cell-autonomously to dictate their dendritic complexity, while different da neurons exhibit different sensitivity to the level of Ss, and even the bipolar td neuron can respond to elevated ss activity by increasing dendritic complexity (Kim, 2006).

Previous studies in C. elegans have demonstrated essential roles for invertebrate homologs of Ahr in neuronal cell fate determination. For example, ahr-1 regulates the differentiation program of a subclass of neurons that contact the pseudocoelomic fluid, and both ahr-1 and aha-1 specify GABAergic neuron cell fate in C. elegans. The dramatic changes in the dendrite morphologies of the da neurons, however, are not due to an all-or-nothing change in cell fate because the da neurons in ss mutants displayed normal class-specific expression patterns of the molecular markers Ab and Cut and normal axon projection patterns characteristic of individual da neurons. However, this also does not assume that a partial cell fate change has not occurred. One reflection of the ss function as a transcription factor is the altered expression levels of GFP in the class I Gal4221 reporter, with increased levels of expression in all class IV neurons and essentially no expression in the dorsal class I neuron ddaD and the ventral class I neuron vpda in ss mutants. It will be of interest to further characterize the genetic basis for this Gal4 reporter, to determine whether this regulation constitutes a partial cell fate alteration or transcriptional regulation of genes downstream from ss in the execution of adjustment of dendritic complexity (Kim, 2006).

There is an emerging theme that ss functions to diversify neuronal differentiation by expanding the photopigment repertoire of R7 photoreceptors in the Drosophila eye and by diversifying da neuron dendritic morphologies. Recent studies have demonstrated that the entire retinal mosaic pattern required for color vision in Drosophila is regulated by ss. In the Drosophila retina, two types of ommatidia form the wild-type retinal mosaic: 'pale' and 'yellow.' In ss^D115.7 mutants, the yellow ommatidial subtype is lost and normally yellow R7 cells are misspecified into the pale subtype. As a result, nearly all R7 cells adopt the pale subtype, leading to loss of the retinal mosaic pattern. Thus, the pale R7 subtype represents the R7 'default state' (Kim, 2006 and references therein).

The overall lack of dendritic diversity in the da neurons in ss mutants is suggestive of the hypothesis that ss, an ancient, evolutionarily conserved gene, may act to convert a primordial dendrite pattern (perhaps a default state) to different complexities for different neurons in the peripheral nervous system. The loss-of-function ss phenotype in the da neuron dendrites might reflect such a primordial pattern as the dendrites in the mutant are devoid of specific morphological features that define distinct neuronal subclasses. In support of this notion, dendrites of the different classes of da neurons share similar morphological characteristics and elaborate similar numbers of total branches in ss mutants. The ability of ss to regulate the complexity and diversity of this dendrite pattern, by limiting dendritic branching to shape the simpler arbors of the class I and class II neurons and by promoting class-specific terminal branching to shape the more complex arbors of the class III and class IV neurons, is quite unique. Of the many mutants that affect multiple classes of da neurons, the great majority affect da neurons with simple or complex dendritic arbors the same way; that is, causing them to all become simpler or more complex. The ss phenotype of making simple dendritic arbors more complex and complex arbors simpler is very unusual among the many mutants affecting dendrite complexity. It thus seems likely that the distinct dendritic patterns rely not only on a cohort of gene activities specifying the mechanics of dendrite outgrowth and branching, but also a genetic program that diverts the generic primordial mode of dendritic formation to a diverse range of dendritic patterns (Kim, 2006).

How might spineless exert its functions? Unlike the homeodomain protein Cut, which promotes dendritic complexity in a specific direction, ss functions in an opposing manner in different cell types to regulate dendritic diversity. How might ss function differently in different neuronal cell types? One possibility is that ss is activated by different ligands in different neurons. ss is incapable of binding dioxin and other related compounds, suggesting that other, as yet unidentified ligands are required for its activation. Previous reports have suggested that ss and other invertebrate homologs of Ahr are activated by an endogenous ligand or that no ligand is required at all. Recent studies have shown that Ahr can accumulate in the nucleus upon activation by the second messenger cyclic AMP (cAMP), although it is not yet known whether cAMP signaling can activate ss in Drosophila. Thus, it is conceivable that ss is activated by different upstream factors in different cell types. It will be of interest to test in future studies whether in different neuronal cell types ss is activated by different ligands or upstream second messengers and whether ss acts in concert with regulatory programs for cell fate specification to dictate dendritic complexity (Kim, 2006).

In the canonical Ahr signaling pathway, Ahr requires the appropriate cofactor for its proper function. Members of the bHLH-PAS protein family are able to heterodimerize with other bHLH-PAS proteins. Previous studies have shown that, upon ligand binding, Ahr is translocated to the nucleus, where it heterodimerizes with Arnt to form a transcriptionally active complex. However, tango, the Drosophila homolog of Arnt, is likely not required for the regulation of dendritic morphogenesis, indicating that ss is probably not acting through its canonical signaling pathway to specify dendritic complexity. In Sf9 cells, ss can act independently of tgo to enhance expression of a reporter in the absence of a ligand. Furthermore, Ahr is unable to interact with Arnt upon activation by cAMP. Although Ahr, Arnt, and the Arnt homolog Arnt2 are widely distributed throughout the rat brain, Ahr does not preferentially colocalize with either Arnt or Arnt2. Ahr is also expressed in specific regions of the rat brain where neither Arnt nor Arnt2 is expressed. These studies support the notion that ss can act independently of tgo in certain developmental contexts. Tgo can heterodimerize with other bHLH-PAS proteins in addition to ss. It is conceivable that ss may act with different heterodimerization partners to mediate its different functions in different cell types (Kim, 2006).

EVOLUTIONARY HOMOLOGS

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. Met flies are resistant to the toxic and morphogenetic effects of JH and several JHAs, but not to other classes of insecticide. Biochemical studies reveal a target-site resistance mechanism, that of reduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. This property of reduced JH binding was cytogenetically localized to the Met region on the X chromosome and can account for the resistance. Possible identities for this binding protein include either an accessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein in vertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

The Met+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of Met flies with a Met+ cDNA results in susceptibility to methoprene, indicating that the cDNA encodes a functional Met+ protein. Met shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating Met in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Met shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene has recently been cloned, and DARNT has higher homology to vertebrate ARNT than does Met, suggesting that DARNT, not Met, may function like ARNT in flies. Met homology to these proteins includes the bHLH region that is involved in DNA binding (30-38% identity), the PAS-A region (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Met gene is the same as for other bHLH-PAS genes (Ashok, 1998).

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans are encoded by the genes ahr-1 and aha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation. This requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C. elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by beta-naphthoflavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators (Powell-Coffman, 1998).

Evolution of aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, the PAS homology domain of AHRs from several species of early vertebrates have been sequenced and phylogenetic analyses of these AHR amino acid sequences have been performed in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences have been identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, are found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that are proposed have arisen by gene duplication early in vertebrate evolution. Overall, these analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function (Hahn, 1997).

Molecular logic behind the three-way stochastic choices that expand butterfly colour vision

Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors. This study investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. The Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (Spineless^ON) or an ultraviolet (UV)-sensitive (Spineless^OFF) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes (Perry, 2016).

Sensory neuron fates are distinguished by a transcriptional switch that regulates dendrite branch stabilization

Sensory neurons adopt distinct morphologies and functional modalities to mediate responses to specific stimuli. Transcription factors and their downstream effectors orchestrate this outcome but are incompletely defined. This study shows that different classes of mechanosensory neurons in C. elegans are distinguished by the combined action of the transcription factors LIM-type homeodomain protein MEC-3, bHLH PAS domain protein AHR-1, and Zn finger/homeodomain factor ZAG-1. Low levels of MEC-3 specify the elaborate branching pattern of PVD nociceptors, whereas high MEC-3 is correlated with the simple morphology of AVM and PVM touch neurons. AHR-1 specifies AVM touch neuron fate by elevating MEC-3 while simultaneously blocking expression of nociceptive genes such as the MEC-3 target, the claudin-like membrane protein HPO-30, that promotes the complex dendritic branching pattern of PVD. ZAG-1 exercises a parallel role to prevent PVM from adopting the PVD fate. The conserved dendritic branching function of the Drosophila AHR-1 homolog, Spineless, argues for similar pathways in mammals (Smith, 2013).

Sensory neurons display a wide range of morphological motifs and functional modalities that serve to transduce diverse types of external stimuli into specific physiological responses. Transcription factors define both the identity and number of each type of sensory neuron and thus are critical determinants of organismal behavior. The downstream pathways that distinguish the architectural and functional properties of different sensory neuron classes are largely unknown, however. This study shows that the conserved transcription factors MEC-3, AHR-1 and ZAG-1, function together to define distinct sensory neuron fates in C. elegans and identify downstream targets that are necessary for these roles (Smith, 2013).

The MEC-3 LIM homeodomain protein is expressed in both touch receptor neurons (TRNs) and in PVD but is responsible for distinctly different sets of characteristics displayed by these separate classes of mechanosensory neurons. In PVD neurons, MEC-3 promotes the creation of a highly branched dendritic arbor and nociceptive responses to harsh stimuli, whereas in the TRNs, MEC-3 is necessary for light touch sensitivity and for the adoption of a simple, unbranched morphology. Genetic ablation of mec-3 or its upstream regulator, the POU domain protein UNC-86, disrupts the function and morphological differentiation of both of these types of mechanosensory neurons. How are these different MEC-3-dependent traits produced? The results suggest that low levels of MEC-3 are sufficient to specify the PVD fate, whereas elevated MEC-3 drives TRN differentiation. The existence of this threshold effect is also supported by the finding that overexpression of MEC-3 induces TRN-specific gene expression in the PVD-like FLP neuron. This simple model is not sufficient, however, to explain why PVD nociceptor genes, which are turned on by low levels of MEC-3, are actually repressed in the TRNs as MEC-3 expression is elevated. The current findings now provide a mechanism for this effect. In the light touch AVM neuron, AHR-1 elevates MEC-3 expression while simultaneously blocking downstream MEC-3 targets that drive PVD branching and nociceptor function. It is suggested that ZAG-1 may exercise a similar role in PVM. This mechanism is robust because each of these TRNs is effectively transformed into a functional PVD-like neuron when either ahr-1 or zag-1 is genetically eliminated. Thus, this work has revealed the logic of alternative genetic regulatory pathways in which a single type of transcription factor (e.g., MEC-3) can specify the differentiation of two distinct classes of mechanosensory neurons. A related mechanism accounts in part for the dose-dependent effects of the homeodomain transcription factor Cut on the branching complexity of larval sensory neurons in Drosophila. The transcription factor Knot/Collier is selectively deployed in Type IV da neurons to antagonize expression of Cut targets that produce the dendritic spikes that are characteristic of Type III da neurons. In this case, however, Knot does not regulate Cut expression but functions in a parallel pathway. The finding that the Zinc-finger transcription factor ZAG-1 is required to prevent the PVM touch neuron from adopting a PVD nociceptor fate mirrors the recent observation that genetic ablation of the mammalian ZAG-1 homolog Zfhx1b (Sip1, Zeb2) results in cortical interneurons adopting the fate of striatal GABAerigic cells (McKinsey, 2013). The current results are suggestive of a potentially complex regulatory mechanism in which AHR-1 and ZAG-1 inhibit expression of nociceptor genes (e.g., hpo-30) whereas MEC-3 activates transcription of these targets. Additional upstream regulators of mec-3, UNC- 86, and ALR-1, are also likely involved in this pathway (Smith, 2013).

Although transcription factors are well-established determinants of sensory neuron fate, the downstream pathways that they regulate are largely unknown. As a solution to this problem for MEC-3, a cell-specific profiling strategy was used to detect mec-3-regulated transcripts in the PVD neuron. A combination of RNAi and mutant analysis was used to identify the subset of targets that affect PVD branching morphogenesis. Additional experiments with one of these hits, the claudin-like protein HPO-30, revealed a key role in the generation of PVD branches. It is noted that HPO-30 is expressed in the FLP neuron, where it also mediates the higher order branching morphology shared by FLP and PVD. Time-lapse imaging has revealed that PVD lateral or 2 branches may adopt either of two different modes of outgrowth along the inside surface of the epidermis: (1) fasciculation with existing motor neuron commissures or (2) independent extension as noncommissural or 'pioneer' dendrites. The results show that the principle role of HPO-30 is to stabilize pioneer 2 branches and, thus, that additional unknown factors may drive fasciculation with motor neuron commissures. Because claudins serve as key constituents of junctions between adjacent cells, it seems likely that HPO-30 functions in this case to link growing 2 dendrites with the nematode epidermis. It is noted that an additional membrane component, the LRR protein DMA-1, displays a mutant PVD branching phenotype strongly resembling that of Hpo-30 and therefore could also function in this pathway. The intimate association of topical sensory arbors with the skin and the broad conservation of junctional proteins across species point to the likelihood that homologs of HPO-30/Claudin and similar components could be widely utilized to pattern sensory neuron morphogenesis (Smith, 2013).

ahr-1 encodes a member of the bHLH-PAS family of transcription factors and is the nematode homolog of the aryl hydrocarbon receptor (AHR) protein. In mammals, AHR is activated by the xenobiotic compound dioxin to trigger a wide range of pathological effects. Invertebrate AHR proteins are not activated by dioxin, which suggests that this toxin-binding function represents an evolutionary adaptation unique to vertebrates. An ancestral role for AHR is suggested by AHR mutants in C. elegans and Drosophila that display distinct developmental defects in which a given cell type or tissue adopts an alternative fate. For example, stochastic expression of the Drosophila AHR homolog, Spineless, promotes the adoption of one specific photoreceptor sensory neuron identity at the expense of another (Smith, 2013).

The current results parallel those findings with the demonstration that AHR-1 function is required in C. elegans to distinguish between alternative types of mechanosensory neurons; in ahr-1 mutants, the unbranched light touch neuron, AVM, is transformed into a functional homolog of the highly branched PVD nociceptor. This role for ahr-1 in C. elegans is particularly notable because the AHR-1 homolog, Spineless, also regulates branching complexity in Drosophila. In spineless (Ss) mutants, Class I and II sensory neurons, which normally display simple branching patterns, adopt more complex dendritic arbors. This phenotype resembles the current finding in C. elegans that the simple morphology of the AVM neuron is transformed into the highly branched architecture of the PVD nociceptor in ahr-1 mutants. Ss mutants in Drosophila also show the opposite phenotype of more complex class III and class IV da neurons assuming simpler branching patterns, which could therefore reflect an additional role for spineless in this context of promoting the creation of dendritic branches. On the basis of these results, it is suggested that the striking conservation of the shared role of AHR homologs in regulating sensory neuron fate and branching complexity in nematodes and insects argues that this function is evolutionarily ancient and, thus, that the downstream effectors that have been identified in C. elegans may also pattern the dendritic architecture of vertebrate sensory neurons (Smith, 2013).

Expression of aryl hydrocarbon receptor

The basic helix-loop-helix-PAS (bHLH-PAS) protein ARNT is a dimeric partner of the Ah receptor (AHR) and hypoxia inducible factor 1alpha (HIF1alpha). These dimers mediate biological responses to xenobiotic exposure and low oxygen tension. The recent cloning of ARNT and HIF1 (homologs (ARNT2 and HIF2alpha) indicates that at least six distinct bHLH-PAS heterodimeric combinations can occur in response to a number of environmental stimuli. In an effort to understand the biological relevance of this combinatorial complexity, their relative expression at a number of developmental time points was characterized by parallel in situ hybridization of adjacent tissue sections. In general, there is limited redundancy in the expression of these six transcription factors and each of these bHLH-PAS members displays a unique pattern of developmental expression emerging as early as embryonic day 9.5 (Jain, 1998).

Mutation of aryl hydrocarbon receptor

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis (Schmidt, 1996).

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice die shortly after birth, whereas survivors reached maturity and are fertile. The Ahr-/- mice show decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice are reduced in size by 50 percent and showed bile duct fibrosis. Ahr-/- mice are also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system (Fernandez-Salguero, 1995).

Ah receptor/Arnt heterodimers

The human aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator protein (Arnt - Drosophila homolog: Tango) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of the Ahr/Arnt heterodimeric transcription factor. Specific transcriptional activation mediated by AhR/Arnt heterodimer (which is a functional indicator of receptor expression) was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AhR and Arnt display constitutive transcriptional activity that is not augmented by the addition of AhR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis do respond to AhR agonists and have lower levels of background beta-galactosidase activity. In the yeast system, hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone are effective AhR agonists for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activate transcription in yeast coexpressing AhR and Arnt. Indole-3-carbinol is an exceptionally potent AhR agonist in yeast. This yeast system is useful for the study of AhR/Arnt protein complexes, and may prove to be generally applicable to the investigation of other multiprotein complexes (Miller, 1997).

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatory proteins: the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nuclear translocator (Arnt). Ligation-mediated PCR technique was used to analyze dioxin-induced changes in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in its native chromosomal setting. Dioxin-induced binding of the AhR/Arnt heterodimer to enhancer chromatin is associated with a localized (about 200 bp) alteration in chromatin structure that is manifested by increased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosome by AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure and accessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by a different, more indirect mechanism, because they are induced from a distance and do not reflect a local effect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatin structure at the enhancer and promoter are graded, mirroring the graded induction of Cyp1A1 transcription by dioxin (Okino, 1995).

Function of Ah receptor/Arnt heterodimers: a role for phosphorylation

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a heterodimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/Arnt dimer can be decreased by treatments that cause the down-regulation of protein kinase C and decrease the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription carried out by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid cause two- to threefold increases in TCDD-dependent transcription, at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubles TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increase TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of either okadaic acid or cyclosporin A, both of which substantially stimulate TCDD-dependent transcription. Neither of the inhibitors significantly increase the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increases the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription (Li, 1997).

Interaction of Ahr with HSP90

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding is localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt, compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity, as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than does the corresponding region of Arnt (Fukunaga, 1995).

Expression of a series of Ah receptor (AhR) deletion mutants in an in vitro translation system has been previously used to map several functional domains of the murine AhR. In this report, quantitative immunoprecipitation of 90-kDa heat shock protein (hsp90) from reticulocyte lysate allowed a measurement of expression levels for the AhR and AhR deletion mutants, complexed with hsp90. After translation of a series of deletion mutants it was determined that there are two distinct domains important in forming a stable AhR/hsp90 complex, corresponding to amino acid sequences 1-166 and 289-347 of the AhR. Neither Arnt, nor Per are able to stably interact with hsp90. Thus, the AhR appears to be a unique member of the PAS domain family of proteins that binds a known ligand and stably interacts with hsp90 (Perdew, 1996).

Neural function of ahr-1 in C. elegans

The aryl hydrocarbon receptors (AHR) are bHLH-PAS domain containing transcription factors. In mammals, they mediate responses to environmental toxins such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Such functions of AHRs require a cofactor, the aryl hydrocarbon receptor nuclear translocator (ARNT), and the cytoplasmic chaperonins HSP90 and XAP2. AHR homologs have been identified throughout the animal kingdom. The C. elegans orthologs of AHR and ARNT, ahr-1 and aha-1, regulate GABAergic motor neuron fate specification. Four C. elegans neurons known as RMED, RMEV, RMEL and RMER express the neurotransmitter GABA and control head muscle movements. ahr-1 is expressed in RMEL and RMER neurons. Loss of function in ahr-1 causes RMEL and RMER neurons to adopt a RMED/RMEV-like fate, whereas the ectopic expression of ahr-1 in RMED and RMEV neurons can transform them into RMEL/RMER-like neurons. This function of ahr-1 requires aha-1, but not daf-21/hsp90. These results demonstrate that C. elegans ahr-1 functions as a cell-type specific determinant. This study further supports the notion that the ancestral role of the AHR proteins is in regulating cellular differentiation in animal development (Huang, 2004).

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of dioxins and related compounds. Dioxins have been shown to cause a range of neurological defects, but the role of AHR during normal neuronal development is not known. This study investigated the developmental functions of ahr-1, the Caenorhabditis elegans aryl hydrocarbon receptor homolog. ahr-1:GFP is expressed in a subset of neurons, and animals lacking ahr-1 function have specific defects in neuronal differentiation, as evidenced by changes in gene expression, aberrant cell migration, axon branching, or supernumerary neuronal processes. In ahr-1-deficient animals, the touch receptor neuron AVM and its sister cell, the interneuron SDQR, exhibit cell and axonal migration defects. Dorsal migration of SDQR is mediated by UNC-6/Netrin, SAX-3/Robo, and UNC-129/TGFbeta, and this process requires the functions of both ahr-1 and its transcription factor dimerization partner aha-1. A role for ahr-1 during the differentiation of the neurons that contact the pseudocoelomic fluid has also been document. In ahr-1-deficient animals, these neurons are born but they do not express the cell-type-specific markers gcy-32:GFP and npr-1:GFP at appropriate levels. Additionally, it was shown that ahr-1 expression is regulated by the UNC-86 transcription factor. It is proposed that the AHR-1 transcriptional complex acts in combination with other intrinsic and extracellular factors to direct the differentiation of distinct neuronal subtypes. These data, when considered with the neurotoxic effects of AHR-activating pollutants, support the hypothesis that AHR has an evolutionarily conserved role in neuronal development (Qin, 2004).

C. elegans ahr-1 is orthologous to the mammalian aryl hydrocarbon receptor, and it functions as a transcription factor to regulate the development of certain neurons. This study describes the role of ahr-1 in a specific behavior: the aggregation of C. elegans on lawns of bacterial food. This behavior is modulated by nutritional cues and ambient oxygen levels, and aggregation is inhibited by the npr-1 G protein-coupled neuropeptide receptor gene. Loss-of-function mutations in ahr-1 or its transcription partner aha-1 (ARNT) suppress aggregation behavior in npr-1-deficient animals. This behavioral defect is not irreparable. Aggregation behavior can be restored to ahr-1-deficient animals by heat-shock induction of ahr-1 transcription several hours after ahr-1-expressing neurons have normally differentiated. ahr-1 and aha-1 promote cell-type-specific expression of soluble guanylate cyclase genes that have key roles in aggregation behavior and hyperoxia avoidance. Aggregation behavior can be partially restored to ahr-1 mutant animals by expression of ahr-1 in only 4 neurons, including URXR and URXL. It is concluded that the AHR-1:AHA-1 transcription complex regulates the expression of soluble guanylate cyclase genes and other unidentified genes that are essential for acute regulation of aggregation behavior (Qin, 2006).

Aryl hydrocarbon receptor activation by cAMP vs. dioxin: Divergent signaling pathways

Even before the first vertebrates appeared on earth, the aryl hydrocarbon receptor (AHR) gene was present to carry out one or more critical life functions. The vertebrate AHR then evolved to take on functions of detecting and responding to certain classes of environmental toxicants. These environmental pollutants include polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), polyhalogenated hydrocarbons, dibenzofurans, and the most potent small-molecular-weight toxicant known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). After binding of these ligands, the activated AHR translocates rapidly from the cytosol to the nucleus, where it forms a heterodimer with aryl hydrocarbon nuclear translocator, causing cellular responses that lead to toxicity, carcinogenesis, and teratogenesis. The nuclear form of the activated AHR/aryl hydrocarbon nuclear translocator complex is responsible for alterations in immune, endocrine, reproductive, developmental, cardiovascular, and central nervous system functions whose mechanisms remain poorly understood. Here, it is shown that the second messenger, cAMP (an endogenous mediator of hormones, neurotransmitters, and prostaglandins), activates the AHR, moving the receptor to the nucleus in some ways that are similar to and in other ways fundamentally different from AHR activation by dioxin. It is suggested that this cAMP-mediated activation may reflect the true endogenous function of AHR; disruption of the cAMP-mediated activation by dioxin, binding chronically to the AHR for days, weeks, or months, might be pivotal in the mechanism of dioxin toxicity. Understanding this endogenous activation of the AHR by cAMP may help in developing methods to counteract the toxicity caused by numerous environmental and food-borne toxic chemicals that act via the AHR (Oesch-Bartlomowicz, 2005).

REFERENCES

Search PubMed for articles about Drosophila spineless

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Brown, R. P., McDonnell, C. M., Berenbaum, M. R. and Schuler, M. A. (2005). Regulation of an insect cytochrome P450 monooxygenase gene (CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades. Gene 358: 39-52. Medline abstract: 16099607

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