InteractiveFly: GeneBrief

bric à brac 1 : Biological Overview | References

Gene name - bric à brac 1

Synonyms -

Cytological map position - 61F1

Function - transcription factor

Keywords - leg, ovary, regulation of species specific sexually dimorphic pigmentation

Symbol - bab1

FlyBase ID: FBgn0004870

Genetic map position -

Classification - BTB/POZ domain

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Dembeck, L. M., Huang, W., Carbone, M. A. and Mackay, T. F. (2015). Genetic basis of natural variation in body pigmentation in Drosophila melanogaster. Fly (Austin): [Epub ahead of print]. PubMed ID: 26554300
Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. A genome wide association (GWA) analysis of variation in abdominal pigmentation was performed using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). The large effects of regulatory variants were confirmed in bab1, t and e. These analyses were, however, imperfect proxies for the effects of segregating variants. This study describes the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. The effects on pigmentation of 28 genes implicated by the DGRP GWA study were replicated, including bab1, t and e and seven genes previously validated by RNAi and/or mutant analyses. Many additional loci were identified. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.

Li, Q., Barish, S., Okuwa, S., Maciejewski, A., Brandt, A. T., Reinhold, D., Jones, C. D. and Volkan, P. C. (2016). A functionally conserved gene regulatory network module governing olfactory neuron diversity. PLoS Genet 12: e1005780. PubMed ID: 26765103
Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1-4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, it was reported that a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. This study shows that Rn, along with BarH1/H2 (Bar), Bric-a-brac/ (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of these data, a molecular map is proposed that defines each unique SOP fate. These results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, this model illustrates how conserved developmental strategies are used to generate neuronal diversity.

Camino, E. M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M. and Williams, T. M. (2015). The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans. PLoS Genet 11: e1005136. PubMed ID: 25835988
The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. This study investigated how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. The data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-a-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, it was discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

The gene bric à brac (bab) is required for pattern formation along the proximal-distal axis of the leg and antenna of Drosophila (Godt, 1993). The French name bric à brac refers to the disorganized structure of the bab mutant ovaries. In bab mutant legs, the bristle pattern of the three central tarsal segments is transformed towards the pattern of the most proximal tarsal segment. bab function is dosage dependent and is required in a graded manner for the specification of tarsal segments. The graded requirement for bab correlates with its graded expression pattern, suggesting that the concentration of Bab protein specifies segment identity in the tarsus (Godt, 1993). In addition, bab mutant legs and antennae have segmentation defects. In addition, ovarian terminal filament formation depends on the Bab protein, which is expressed in the nuclei of terminal filament cells and is cell autonomously required (Godt, 1995). Disruption of terminal filament formation, together with defects of basal and interfollicular stalk development, leads to disruption of ovariole formation and female sterility in bab mutants. bab encodes a nuclear protein that contains a highly conserved BTB/POZ domain. The BTB/POZ protein-protein interaction domain is involved in homomeric and heteromeric associations with other BTB/POZ domains. Bab physically interacts with Bip2, a novel Drosophila TATA-box protein associated factor also termed (TAFII), also named dTAFII155. This interaction provides a direct link between BTB/POZ transcription factors and the basal transcriptional machinery (Pointud, 2001).

bab is required and expressed in a distinct proximal-distal domain of the limbs; the central region of the tarsus of the leg and the basal cylinder of the antenna. The domain of bab activity in limbs is apparently identical to the domain defined by the phenotype and expression pattern of the zinc finger transcription factor rotund (once mistakenly thought to be a RAS GTPase activator). In addition, this leg domain is characterized by the gene deadpan, which is expressed in a distal circumferential stripe in each of the segments TS1 to TS4. bab and rotund appear to act rather late in limb development, in contrast to genes that control the whole proximal-distal axis and appear to be required from embryogenesis onward, such as Distal-less and wingless. The subdivision of the tarsal primordium is a late event in the pattern formation of the leg and is also an evolutionarily recent step. Primitive insects only have one tarsal segment and the number of tarsal segments differs widely among more advanced insects. Taken together, this indicates that the bab/rotund domain is a distinct field for pattern formation during leg and antenna development (Godt, 1993).

Comparison of the bristle pattern in the tarsal segments of wild-type and bab mutant flies suggests that loss-of-function bab mutations cause a homeotic transformation of the three central tarsal segments (TS2-TS4) towards the basitarsus (TS1). This indicates a serial homology of segments in the tarsus. A similar conclusion was reached by Curt Stern in 1954, based on the finding of an extra sex comb on TS2 in extra sex comb-aristapedia double mutants. It may be assumed, therefore, that TS1-TS4 have the same basic pattern information that is modulated by bab activity in TS2-TS4 in order to give them a specification different from that of TS1. The data suggest a role for bab as a homeotic gene that is required along the proximal-distal axis of the legs to direct the developmental fate of TS2, TS3, and TS4 (Godt, 1993 and references therein).

The haploinsufficient transformation effect of bab alleles indicates that the specification of the tarsal segments depends on bab dosage. Also, the phenotypic series of bab alleles can be interpreted as a consequence of sequentially reduced levels of bab expression. The comparison of different bab alleles indicates a graded requirement for bab activity along the proximal-distal axis that becomes apparent in the higher sensitivity of TS2 compared to TS3 and TS4 transformation towards TS1. Given the dosage dependence, a simple explanation for this observation would be a graded distribution of the bab gene product along the proximal-distal axis. This explanation is corroborated by the different levels of bab expression that are observed in TS1- TS4. TS1 contains the lowest level of bab product and is considered the ground state. TS2, which is most sensitive to homeotic transformation to TS1, has a lower level of bab expression than TS3 and TS4. It is proposed, therefore, that the sensitivity to homeotic transformation towards TS1 is correlated with the concentration of the Bab protein in the different tarsal segments. It is unclear if this correlation holds for TS4, which contains equal or slightly lower levels of bab expression than TS3. In TS4, whose small size seems to allow the production of only one SCB, the usage of this morphological marker might not be sensitive enough to always detect a transformation to TS1 (Godt, 1993).

Is bab a morphogen? There are two possible explanations for how bab may act in a concentration-dependent manner for the specification of tarsal segments. bab may promote a binary decision between a TS1 fate and a non-TS1 fate, or bab may act as a morphogen and provide different positional values for the specification of TS2, TS3 and TS4. The latter possibility is supported by the graded distribution of the bab product. In addition, the analysis of metathoracic tarsal segments of weak and intermediate bab mutants shows a change in the bristle pattern of TS3 that can be interpreted as a transformation of TS3 towards TS2 rather than TS1. It is suggested, therefore, that bab may provide, depending on its concentration, different positional values for the specification of TS2 and TS3. It will be possible to assess the ability of the Bab protein to act as a morphogen by studies of bab overexpression and by examination of segment-specific molecular markers in bab mutants (Godt, 1993).

bab is the first gene for which a graded expression along the proximal-distal axis is described. The Bab protein distribution in the tarsal primordium reflects the distribution of the bab transcript, which indicates that the pattern of bab expression is regulated at the transcriptional level. The polar coordinate model proposes the formation of distal limb structures under the control of a circular coordinate which integrates anterior-posterior and dorsal-ventral positional values. For example, mutations in wg (a gene that encodes a secreted and diffusible protein and is believed to define circumferential positional values) have a drastic effect on the proximal-distal axis. However, it is not clear how genes like wg could induce a gradient of a nuclear protein along the proximal-distal axis. Whether Dll, which likely acts upstream of bab and is proposed to provide positional information along the proximal-distal axis, directly regulates bab expression is not known, but considering that the tarsus is a distinct Dll expression domain in the third larval instar, Dll might be involved in defining the domain and/or the pattern of bab expression (Godt, 1993).

The analysis of bab mutations indicates that bab has, in addition to its requirement for segment specification, a second function in limb development. bab mutations cause segmentation defects in the central region of the tarsus and in the homologous part of the antenna, the basal cylinder. Because strongly hypomorphic bab mutations cause more severe segmentation defects as hemizygotes than as homozygotes, it is assumed that amorphic bab mutations might lead to even stronger defects in segmentation (Pointud, 2001).

At the onset of metamorphosis, the bab product is distributed in a wave-like pattern in the tarsal primordium. Each tarsal fold shows a bell-shaped expression pattern of bab with the maximum level of expression at the ridge and the minimum in the furrow. Considering the segmentation defects in the tarsus of bab mutants, it is proposed that the wave-like pattern may be involved in the segmentation process of the tarsus. A number of theoretical models have utilized chemical wave patterns to explain how segmentation could occur. In these models, the waves compose a prepattern that reflect the structures that will develop, and the segment boundaries are proposed to be specified by the troughs or peaks of the wave pattern. Analysis of the molecular mechanisms of segmentation in the Drosophila embryo has shown that wave-like prepatterns are not part of the segmentation process. The finding that a wave-like pattern of bab exists in the developing tarsus suggests that segmentation in the tarsus occurs by a mechanism that differs from segmentation in the embryo (Godt, 1993).

In this context, it has to be asked whether the wave-like bab expression pattern can be considered a prepattern for segmentation of the tarsus. The wave-like pattern of the bab product at the onset of metamorphosis develops from a rather uniform distribution in the mid third larval instar. The wave-like pattern appears to form in parallel to the development of the tarsal folds. This, together with the observation that the segments of the tarsus seem to be already defined by the mid third larval instar, suggests that the wave-like distribution of the bab product is not likely to be a classical prepattern. In contrast to the embryo, however, pattern formation in the tarsal primordium occurs in a proliferating epithelium where a stable molecular prepattern is not expected. It has been suggested that new positional values in the growing imaginal disc are generated by intercalation between values that already exist. The development of the wave-like bab expression pattern may reflect this process. Early folding defects in the tarsal region of bab mutant imaginal discs indicate that bab is involved in the morphogenetic folding process. It is therefore proposed that in the growing field of the tarsal primordium, the morphogenetic process of segmentation and the development of the bab gene expression pattern may occur in a mutually dependent manner (Godt, 1993).

Genetic control and evolution of sexually dimorphic characters in Drosophila

Sexually dimorphic abdominal pigmentation and segment morphology evolved recently in the melanogaster species group of the Drosophila. These traits are controlled by the bric-à-brac gene, which integrates regulatory inputs from the homeotic and sex-determination pathways. bab expression is modulated segment- and sex-specifically in sexually dimorphic species, but is uniform in sexually monomorphic species. It is suggested that bab has an ancestral homeotic function, and that regulatory changes at the bab locus played a key role in the evolution of sexual dimorphism. Pigmentation patterns specified by bab affect mating preferences, suggesting that sexual selection has contributed to the evolution of bab regulation (Kopp, 2000).

A key challenge in evolutionary biology is to identify genetic events responsible for morphological change, and to understand how changes at the molecular level affect development and translate into phenotypic diversity. To achieve this, two distinct approaches have been pursued in recent years: (1) comparative studies have revealed strong correlations between the expression patterns of individual regulatory genes during development and differences in morphology; (2) direct genetic analysis has been used to estimate the number and identity of genetic loci that contribute to morphological variation within and between species. Despite their respective successes, the two approaches remain far apart because of their different scales of analysis. Comparative studies have concentrated mainly on slowly evolving traits among high-level taxa, but genetic analyses are only possible among closely related species that produce viable and fertile hybrids (Kopp, 2000).

An approach to bridging this gap between evolutionary genetics and comparative embryology is to analyze and compare the development of rapidly evolving morphological traits. In many animals, secondary sexual characteristics evolve rapidly, making them good candidates for analysis. One such character in Drosophila is the pigmentation of adult abdominal segments. In D. melanogaster, abdominal pigmentation is sexually dimorphic. Segments 1 to 6 in females and 1 to 4 in males carry only a posterior stripe of dark pigment. However, segments 5 and 6 (A5 and A6) in males are completely pigmented, giving the species its name. This pattern is of recent evolutionary origin; in most Drosophila species, male-specific pigmentation is absent, so that females and males are pigmented identically. To understand how this new pattern originated and evolved, the regulatory circuit that controls its development has been characterized, and its operation has been compared in sexually dimorphic and monomorphic species (Kopp, 2000).

The development of sexually dimorphic external characteristics is controlled by the doublesex (dsx) gene. Alternative splicing of the dsx transcript produces a male-specific product in males (dsxM), and a female-specific product in females (dsxF). Loss of dsx function in females results in the development of male-like pigmentation, which can be suppressed by heat-shock dsxF transgenes. Male-specific pigmentation is therefore expressed by default, and must be actively repressed by dsxF (Kopp, 2000).

Thus, the development of sexually dimorphic pigmentation requires integration of homeotic and sex determination gene inputs. In investigating how this integration is achieved, a newly evolved genetic circuit has been discovered that appears to be responsible for the origin of male-specific pigmentation (Kopp, 2000).

A gene near the left tip of the third chromosome contributes to the variation in female abdominal pigmentation. In investigating this genetic region, it was found that loss of one copy of the bab locus results in the development of male-specific pigmentation in females, but has no effect on the male abdomen. Ectopic pigmentation in heterozygous bab females is suppressed by reducing the dosage of Abd-B, but is not eliminated by loss of omb. This suggests that bab+ represses the development of male-specific pigmentation in females by opposing the function of Abd-B. The bab locus contains two closely related genes, bab1 and bab2, which encode putative transcription factors with multiple roles in development. Ectopic pigmentation in females increases in the order bab1/+ < bab1/bab1 bab1bab2/+ bab1bab2/bab1, indicating that both genes are involved in repressing male pigmentation. For simplicity, the entire locus has been treated as one gene, bab, unless noted otherwise (Kopp, 2000).

The expression pattern of bab at the pupal stage when the adult epidermis develops reflects its sex- and segment-specific function. In females, bab expression is strongest in segments A2 and A3, and progressively weaker in A4, A5 and A6. In males, bab expression is considerably weaker than in females in all segments. Most strikingly, it is completely absent from A5 and A6. This pattern of bab repression correlates with the presence of sex-specific pigmentation in males, and its absence in females (Kopp, 2000).

To test whether bab+ is sufficient to repress pigmentation, the bab genes were ectopically expressed in the pupal abdomen. Low-level expression of bab+ results in the loss of male-specific pigmentation, but has no other effects on external morphology, indicating that differential regulation of bab plays a central role in establishing sexual dimorphism. bab+ can also repress non-sex-specific pigment stripes when expressed at a higher level. This suggests that bab+ acts as a general repressor of pigmentation, but that its effects are overridden by omb in the posterior part of each segment. Consistent with this, complete loss of both bab genes results in ectopic pigmentation of A2 to A7 in both sexes. This phenotype is not caused by expansion of Abd-B expression, which appears normal in these mutants. In bab homozygotes, the intensity of pigmentation is higher in the more posterior segments than in those more anterior. This suggests that pigmentation does not develop by default in the absence of bab, but is actively promoted by Abd-B and abd-A (Kopp, 2000).

The sexually dimorphic repression of bab in the posterior abdomen suggests that bab integrates the homeotic and sex determination regulatory inputs. To test this, bab expression was examined in Abd-B and dsx mutant backgrounds. Ectopic expression of Abd-B in A3 and A4 eliminates bab expression from these segments in males, and downregulates it in females. Conversely, bab is derepressed in A5-A7 in the mutants that lack Abd-B function in these segments. Together, these results indicate that bab expression in A5 and A6 is normally repressed by Abd-B. The slight downregulation of bab in A4 suggests that it is also weakly repressed by abd-A (Kopp, 2000).

In dsx-intersexes, bab is expressed in a male-like pattern, suggesting that dsxF upregulates bab transcription in females. Abd-B and abd-A expression is identical in males, females and dsx -intersexes, indicating that bab is regulated independently by homeotic and sex-determination inputs. dsxDominant intersexes, which express both male- and female-specific dsx products, also show male-like expression of bab, indicating that dsxM can interfere with dsxF function. The two dsx isoforms encode transcription factors that bind the same DNA sequence, but have opposite effects on gene expression. dsx-intersexes differ from males in having a small unpigmented region at the anterior-lateral margin of A5, suggesting that dsxM may have a slight negative influence on bab expression (Kopp, 2000).

These results suggest that bab+ regulates sexually dimorphic pigmentation by integrating regulatory inputs from the homeotic genes and the sex determination pathway. In this regulatory circuit, bab+ acts as a general repressor of pigmentation, and Abd-B and abd-A promote pigmentation in both sexes. In addition, Abd-B, and to a lesser extent abd-A, repress bab transcription. In males, this results in the absence of bab from A5 and A6, allowing Abd-B and abd-A to promote pigmentation in these segments. However, in females, dsxF prevents bab transcription from being completely repressed by the homeotic genes. As a result, bab is present in A5 and A6 in females, where it blocks the ability of Abd-B and abd-A to promote pigmentation. In A2-A4, abd-A alone is not sufficient either to repress bab or to overcome its inhibitory effect on pigmentation; thus, only the omb-dependent striped pigmentation is generated. Because Abd-B, abd-A and dsx encode transcription factors, they may regulate bab expression directly (Kopp, 2000).

The central role of bab as an integrator of homeotic and sex-determination gene inputs suggests that changes in bab regulation may have been responsible for the evolution of sexually dimorphic pigmentation. In the subgenus Sophophora, male-specific pigmentation is present only in the melanogaster species group. Within this group, sexual dimorphism is seen in all species of the melanogaster subgroup and the closely related oriental subgroups, whereas the ananassae and montium subgroups contain both sexually dimorphic and sexually monomorphic species (Kopp, 2000).

In species with male-specific pigmentation of A5 and A6, bab expression is absent or strongly downregulated in these segments in males, but not in females. Moreover, in the sexually monomorphic species outside the melanogaster species group, bab expression is identical in both sexes and in all segments from A2 to A7. This correlation suggests that changes in the regulation of bab by Abd-B and dsx played an important role in the origin of sexually dimorphic pigmentation (Kopp, 2000).

bab+ regulates segment shape and bristle and trichome patterns in a manner reciprocal to Abd-B. Loss of bab+ function in females enhances posterior characteristics in A6, A7 and A8. No phenotype is seen in males, consistent with the absence of bab expression in posterior segments. Conversely, ectopic expression of bab transforms A6 and A7 to a more anterior identity in both males and females. These observations suggest that bab+ acts as an antagonist of Abd-B homeotic function, and that posterior abdominal characters are determined by the balance between Abd-B and bab activities (Kopp, 2000).

This model predicts that evolutionary changes in bab regulation should result in morphological transformation of Abd-B-expressing segments. Indeed, the entire suite of characteristics that distinguishes A5 and A6 from the more anterior segments in D. melanogaster is of recent evolutionary origin. In D. willistoni, bab is expressed strongly in A5 and A6 in males, whereas Abd-B is expressed in the same pattern as in D. melanogaster. As predicted, A5 and A6 are almost identical to the more anterior, non-Abd-B-expressing segments in the males of this species. In contrast, the melanogaster species group shows great diversity of bristle and trichome patterns in posterior abdominal segments. The two main lineages within this group show different patterns of evolution. In the clade composed of the melanogaster and oriental subgroups, male-specific pigmentation and bristle and trichome patterns have evolved in a concerted fashion. However, in the ananassae + montium lineage, these characteristics vary independently of each other, and sexually dimorphic bristle and trichome patterns are sometimes observed in species that do not show visible modulation of bab expression. This suggests that evolutionary changes have occurred not only in bab regulation, but also in the target genes of bab and in other genes regulated by Abd-B and dsx . Suppression of A7 development in males has occurred earlier in evolution than visible modulation of bab expression, despite the ability of bab to override this suppression (Kopp, 2000).

The rapid evolution of sexually dimorphic pigmentation and segment morphology may have been driven by sexual selection. Whether male-specific pigmentation confers a competitive advantage in D. melanogaster males was tested. Surprisingly, UAS-bab2 males, which lack male-specific pigmentation but are otherwise normal, enjoy the same mating success as wild-type males. Thus, although male pigmentation may have been important in the past, it appears to have little or no effect on female mating preferences in extant D. melanogaster (Kopp, 2000).

However, D. melanogaster males discriminate strongly against heterozygous bab females, which have ectopic male-specific pigmentation but are otherwise normal, compared with females with lightly pigmented A5 and A6. Importantly, white mutant males, which are visually impaired, mate equally with bab/+ and lightly pigmented females, suggesting that discrimination against bab heterozygous females is due to their pigmentation. These results suggest that female pigmentation is important in determining their attractiveness to males, and that the absence of male-specific pigmentation in females may be maintained by sexual selection (Kopp, 2000).

These findings indicate that changes in bab regulation have played an important part in the evolution of abdominal segment morphology. The presence of bab expression in all Drosophila species examined suggests that its roles in antagonizing the homeotic function of Abd-B and repressing pigmentation are ancestral. However, in the ancestral condition, bab expression was independent of Abd-B and dsx, resulting in sexually monomorphic pigmentation and segment morphology. In the melanogaster species group, bab evolved to be under the control of Abd-B and dsx. This eliminated bab from Abd-B-expressing segments in the male and resulted in a major transformation of male segment morphology. Subsequent diversification of pigmentation, bristle and trichome patterns was probably driven both by the fine-tuning of bab regulation and by changes in the downstream targets of bab and Abd-B (Kopp, 2000).

Two features of this genetic circuit make it highly plastic and evolvable: (1) the adult phenotype is sensitive to quantitative changes in bab expression; (2) the level of bab expression is determined by the balance between Abd-B and dsxF inputs. If bab is regulated directly by Abd-B and dsx, then the evolution of sexually dimorphic pigmentation and segment morphology may ultimately be traced to the acquisition and modification of binding sites for the Abd-B and Dsx proteins in the cis-regulatory region of bab. Thus, even a subtle molecular change could be expressed phenotypically and become subject to selection (Kopp, 2000).

This evolutionary model is further supported by the presence of intraspecific genetic variation in sexually dimorphic pigmentation in many extant species. In at least one case, there is strong evidence that allelic differences at the bab locus contribute to this variation. Females found in natural populations of D. melanogaster vary widely in the extent of A6 pigmentation, ranging from near-zero to 100%. The locus with the largest effect on this variation has been mapped to the exact position of bab. These observations suggest that sexually dimorphic pigmentation evolved through fixation of intraspecific genetic variants at the bab locus (Kopp, 2000).

Fixation of new bab alleles was probably driven initially by 'runaway' sexual selection. In this case, a slight female preference for a weakly pronounced male character would initiate a positive feedback loop that would rapidly increase both the expression of the male character and the female preference for it. This self-reinforcing mechanism can drive rapid character divergence and create new species through sexual isolation. Male-specific pigmentation could evolve by this mechanism, with increasingly discriminating females selecting for increasingly dark males. However, once fixed, sexual characteristics can lose their significance as they are overtaken by newly evolving signals and as females become habituated and 'resistant' to old characters. This may explain the finding that male pigmentation has no effect on mating success in extant D. melanogaster (Kopp, 2000).

Whereas the runaway model explains the evolution of male sexual characters, it does not account for the absence of these characters in females, that is, sexual dimorphism. However, sexual dimorphism can be produced effectively by counter-selection against male-specific traits in females. Consistent with this, D. melanogaster males discriminate against females that have male-like pigmentation. In most Drosophila species, including D. melanogaster, males seek out females at feeding sites and attempt to court as many as possible. Courting other males is not only disadvantageous in competition for females, but may also carry a direct cost. Thus, males are probably selected for an ability to avoid courting other males, and pigmentation may be used to identify females at a distance (Kopp, 2000).

The evolution of bab regulation offers a tractable model of how selection creates new morphological characters through changes in DNA sequence. Analysis of the cis-regulatory elements of bab in sexually dimorphic and monomorphic species will help to clarify the molecular basis of morphological divergence between these taxa (Kopp, 2000).



The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, the evolution of the genetic switch controlling dimorphic bab expression has been identified and traced. The HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSXF activate bab expression whereas in males DSXM directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits (Williams, 2008).

bab expression in the abdominal epidermis is regulated by two separate CREs, one of which directs gene expression in the anterior abdomen of both sexes, and a second, dimorphic element that regulates female-specific gene expression in segments A5-A7. The dimorphic element, when bound by ABD-B and sex-specific isoforms of the DSX protein, acts as a genetic switch that allows pigmentation in males and represses pigmentation in females. Changes in the activities of both CREs have evolved in the course of the origin of the trait from a monomorphic ancestor. Furthermore, dimorphic CRE function evolved by multiple fine-scale changes within the CRE. These results bear on understanding of how sexually dimorphic traits develop, how new sex- and segment-restricted traits arise, and how CRE functions evolve (Williams, 2008).

Sex-restricted traits are the product of differences in gene expression between sexes, therefore, understanding how such traits develop requires the identification of those genes with sex-limited expression and elucidation of the genetic and molecular mechanisms governing their regulation. This study showed that dimorphic bab expression is regulated by a discrete CRE whose activity is combinatorally regulated by the direct inputs of both region- (ABD-B) and sex-specific (DSX) transcription factors. In females, ABD-B acts in concert with the DSXF isoform through binding sites in the dimorphic element to activate bab expression in the posterior segments. Whereas in males, ABD-B activity is overridden by the repressive activity of the DSXM isoform which binds to the same sites as DSXF and hence, permits the formation of the male-specific posterior pigmentation (Williams, 2008).

The genetic pathways that regulate sex-determination and sexual differentiation differ greatly across the animal kingdom, so this mode of male-specific trait regulation in Drosophila may not apply in detail to other animals. However, the integration of region- and sex-specific regulatory inputs must be a requirement for the production of dimorphic traits. It is suggested that the integration of such combinatorial inputs by cis-regulatory elements, as demonstrated for bab, is a general feature of genetic switches within the pathways regulating the production of dimorphic traits (Williams, 2008).

The origins of sexually dimorphic traits have long been of central interest in evolutionary biology. One of the key questions that Darwin grappled with, as have many others subsequently, was whether dimorphic traits are limited to one sex at their origin, or whether these traits first appear in both sexes and then become restricted to one sex. This question has been particularly important and challenging in terms of genetics and evolutionary theory, as it has not been resolved previously how the effects of mutations could be restricted to one sex (Williams, 2008).

In the simplest genetic scenarios of sexual dimorphism, male-limited traits are the products of the male-limited expression of specific genes. The main evolutionary question then, as it has been phrased in classical genetic terms, is whether male-limited gene expression evolves via: (1) 'alleles' that are expressed only in males; or (2) alleles expressed in both sexes which are then suppressed in females or promoted in males. The elucidation of the regulation and evolution of male-specific pigmentation provides a unique opportunity to reconstruct the genetic path of the evolution of a dimorphic trait (Williams, 2008).

Although posterior male-specific pigmentation is a relatively simple, two-dimensional morphological trait, it is clear that it did not originate via just one of the alternative genetic paths above. Rather, the evolution of this trait has involved three paths: the evolution of male-limited gene expression, of female-limited gene expression, and of non-sex-restricted gene expression. Specifically, this study shows that in the course of the evolution from a monomorphically pigmented ancestor, the activity of the female-specific bab dimorphic CRE expanded into segments A6 and A5 and that the activity of the monomorphic bab anterior CRE retreated from segments A6 and A5 of both sexes. These two combined changes produced the sex-specific repression of bab expression in male segments A5 and A6. In addition, in previous work it was shown that the yellow pigmentation gene gained high-level expression in segments A5 and A6 via the acquisition of ABD-B binding sites in a specific yellow gene CRE, whose activity was male-limited due to repression by Bab (which is apparently indirect) (Williams, 2008).

It is important to underscore that none of the genes in this newly-evolved regulatory circuit are globally restricted in their expression to one sex. Rather, the sex-specific features of their expression are controlled by modular CREs that are physically separate from those controlling gene expression in other developing body regions. The properties of these CREs resolve the question of how the effects of mutations can be restricted to one sex. Namely, mutations in a CRE that is under the direct (the female-specific bab dimorphic element) or indirect (the male-specific yellow CRE) control of an effector of sex determination will have sex-limited effects on gene expression. The findings here are a further demonstration of the general principle of how the modular CREs of pleiotropic genes enable the modification of gene expression in and morphology of one body part independent of other body parts, or in this case, the same body part in the opposite sex (Williams, 2008).

It is also notable that none of the CREs analyzed are new to the dimorphically pigmented melanogaster species group. It is clear, then, that the ancestral dimorphic CRE was active in segment A7 and modified to govern sexually dimorphic pigmentation in segments A6 and A5. Thus, in this example, one path is seen to evolving a new dimorphic trait is via the co-option of genetic components that regulate other pre-existing dimorphic traits (Williams, 2008).

One of the major questions concerning the evolution of gene expression is how new gene expression patterns arise. The two most obvious mechanisms would appear to be the gain of new regulatory elements or the gain of new transcription factor-CRE linkages. While the deep ancestry of the dimorphic element ruled out the former, it was expected that the novel sex- and segment-specific regulation of this CRE by DSX and ABD-B in the D. mel. lineage would require the gain of binding sites for these two transcription factors. However, it was found that the both DSX binding sites and most ABD-B sites were present in D. wil. and other monomorphic species and therefore were present in the last common ancestor of both monomorphic and dimorphic species. Thus, the expansion of the dimorphic CRE activity was not due to the wholesale gain of new DSX and ABD-B binding sites (Williams, 2008).

Rather, it was discovered that the expanded, high level activity of the D. mel. dimorphic CRE in segments A6 and A5, relative to the A7-restricted activity of the D. wil. element, was due to an amalgam of changes involving the number, polarity, and topology of transcription factor binding sites. The evolution of dimorphic CRE activity demonstrates how changes beyond the simple gain or loss of binding sites shape CRE evolution. Similarly, changes in the topology and helical phasing of transcription factor binding sites have shaped the evolution of a genetic switch controlling galactose utilization in yeast (Hittinger, 2007). These studies strongly support the view that the relationship between function and sequence variation in CREs is complex. A vast body of work on eukaryotic and prokaryotic transcriptional regulation has shown that binding site polarity and spacing influences the output of regulatory elements. Therefore, it is suggested that one important, but generally unappreciated, class of functionally relevant mutations in CRE and trait evolution involves sequences outside of transcription factor binding sites. CREs thus present a very large target area for potential functionally relevant mutations that quantitatively modulate gene expression and trait development (Williams, 2008).

Finally, these observations concerning the mechanisms underlying the expansion of dimorphic CRE activity help to shed light on another general aspect of the evolution of animal body plans -- the evolution of segmental traits. A large number of studies have demonstrated that some of the major differences among arthropod and vertebrate body plans have involved evolutionary shifts in the spatial boundaries of gene expression along the main body axis. However, the path by which such gene expression patterns are shifted has not been elucidated in any molecular detail. It is submitted here that the expansion of the activity of the dimorphic element from the A7 segment into A6 and A5 is a model of this process. The remodeling of the dimorphic CRE in the course of evolution illustrates that one way such shifts can be accomplished is through numerous small, quantitative incremental changes in the activity of Hox-regulated CREs (Williams, 2008).

Recurrent modification of a conserved cis-regulatory element underlies fruit fly pigmentation diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In fruit flies, the Bric-a-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (Abd-B) and sex-determination (Dsx) transcription factors. This study finds that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, this study demonstrates that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeds with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, these findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function are accompanied by deep conservation of key regulatory linkages (Rogers, 2013).

In the D. melanogaster pigmentation network, the bab genes function as an Input-Output node through the dimorphic element's integration of patterning inputs that include body plan (ABD-B) and sex determination (DSX) pathway inputs. These inputs are converted into a female-specific pattern of expression that culminates in the repression of the differentiation genes yellow and tan in females. In principle, changes in the expression or activity of a patterning gene, differentiation gene, or the Input-Output gene (bab) could alter pigmentation phenotypes. In application though, it is logical that bab expression and dimorphic element encodings were modified as those alterations minimize negative pleiotropic effects while being sufficient to alter the female pigmentation phenotype. For example, ectopic yellow expression fails to create additional melanic pigmentation, and changes in either DSX or ABD-B expression result in ectopic abdominal pigmentation in addition to several other trait phenotypes. Thus, sufficiency for pigmentation is counterbalanced by the negative pleiotropic affects for these genes. In contrast, increased Bab expression in the A5 and A6 segments is sufficient to suppress pigmentation, and ectopic abdomen pigmentation develops in bab heterozygous and homozygous null mutant females (Rogers, 2013).

Bab though is not dedicated to pigmentation. In the pupa, Bab expression includes the leg tarsal segments, abdomen epidermis, sensory organ precursor cells, oenocytes, and dorsal abdominal muscles, and each of these expression patterns are governed by a modular CRE (s). Thus, Bab itself is highly pleiotropic, however it's CREs are far less pleiotropic. For this reason, mutations altering female pigmentation would maximize sufficiency and minimize pleiotropy if they occurred in the dimorphic element, an expectation borne out in this study. Pigmentation of the A5 and A6 segments, though, is only one of many traits influenced by the regulatory activity of the dimorphic element. This CRE drives Bab expression in the female A7 and A8 segments, regulating numerous female-specific traits, including the size, shape, trichome density, and bristle morphologies of the resident dorsal tergites and ventral sternites. As expression in these more posterior segments require the ABD-B and DSX regulatory linkages, these regulatory linkages remain highly pleiotropic. For this reason, it seems logical that evolution would disfavor mutations that have deleterious consequences to these linkages and favor mutations that alter other CRE properties. This scenario reflects how dimorphic element function was modified in both the intraspecific and interspecific comparisons presented presented in this study as well as the long term conservation of the ABD-B and DSX linkages previously described (Rogers, 2013).

The current findings provide a unique contrast with previous investigations of the relationship between CRE conservation and CRE evolution. Although Drosophila non-coding DNA, including CRE sequences, evolves slower than synonymous sites, several well studied CREs were found to undergo substantial sequence evolution without matching regulatory activity evolution. During Drosophila embryonic development, the pair-rule gene even-skipped (eve) is expressed in seven stripes along the anteroposterior axis, with the second stripe of eve expression being specified by the stripe 2 element (S2E) CRE. In D. melanogaster, the S2E possesses binding sites for four transcription factors that collectively specify the eve expression output. The orthologous S2E from the species D. pseudoobscura differs in sequence for numerous binding sites, the overall content of binding sites, and spacing between conserved binding sites, yet the orthologous S2Es function equivalently in vivo. Hence, the S2E is an exemplar as to how selection acting at the level of the character (eve stripe expression) can accommodate a surprising amount of CRE evolution. Similarly, CRE sequence evolution without corresponding functional evolution was found between Drosophila species for the sparkling (spa) CRE that directs cone cell expression for the dPax2 gene. The content and spatial proximity of binding sites for neurogenic ectoderm enhancers (NEEs) evolved in order to conserve expression pattern outputs in response to changing regulatory inputs. These case studies, demonstrate how CRE sequence conservation is not a prerequisite for CRE functional conservation (Rogers, 2013).

In contrast, this study found little divergence in the content and sequence of known binding sites for the D. melanogaster dimorphic element alleles and orthologous sequences. At the sequence level, these CRE alleles and orthologs respectively posses identities of ~98% and ~80%. Indeed, the vast majority of binding sites in the dimorphic element have been conserved for over 30 million years, showing conservation to D. willistoni. At the functional level, these CREs exhibited striking differences in their regulatory activities. Thus, in contrast to S2E, spa, and the NEEs, the dimorphic element demonstrates how CREs can derive dramatic changes in function that drive phenotypic divergence, with little-to-no alteration to the characterized pre-existing regulatory linkages (Rogers, 2013).

While the regulatory activity of the Light and Dark dimorphic elements alleles correlated with female A5 and A6 pigmentation, some outcomes suggest that these variant sequences are affected by other features within or perhaps outside of the bab locus. For instance, the Light 2 and Dark 2 alleles exhibit the highest and lowest regulatory activities respectively. Surprisingly, the Light 1 and Dark 1 alleles and their intermediate regulatory activities are associated with the more extreme Light and Dark female pigmentation phenotypes. At the expression level, Bab1 and Bab2 showed similar patterns in females from the Light 1 (prominent expression in segments A5 and A6) and Dark 1 (reduced expression is A5 and A6) strains. In the Dark 2 strain, Bab1 but not Bab2 expression was reduced in females. Several possible explanations might explain the uncoupled expression of the Bab paralogs in Dark 2. For example, it is possible that a separate, as of yet unidentified CRE controls Bab2 expression. However, a screen of the entire ~160 kb locus failed to identify such a CRE. A second possibility is that a mutation(s) in the Dark 2 allele has paralog-specific regulatory effects, perhaps by modifying an interaction with the promoter for bab1 but not that of bab2 (Rogers, 2013).

Another possible explanation would involve the existence of CREs that coordinate communication between bab1 and bab2. In such a scenario, the Dark 2 allele could contain mutations that alter interaction with coordinating elements to result in paralog-specific expression patterns in the female A5 and A6 segments. This possibility is consistent with observations of bab locus evolution in another population where females differ in A6 segment pigmentation. For this population, fine-scale genetic mapping found that three disparate non-coding regions of the bab locus collaborate to compose a major effect QTL. One of these regions spans the dimorphic element, though no mutations reside with this CRE's core element. The other two regions include an intergenic sequence between bab1 and bab2 and a large sequence that includes the bab2 promoter. In the future, it will be important to understand what roles these other regions serve, and how they may interact with polymorphisms in the dimorphic element to produce paralog-specific effects on gene expression (Rogers, 2013).

With the centrality of CREs and their evolution to the diversification of phenotypic traits, a major obstacle to reaching this goal is understanding the processes by which CRE regulatory logics were modified to contemporary forms. Often studies of CRE evolution involve comparisons of two divergent derived regulatory states, where one sequence assumes the role of a surrogate for the ancestral function. This approach has been successful in making inferences about the ancestral states for regulatory linkages and identifying gains and losses of other key derived transcription factor binding sites. However, it is important to acknowledge a key limitation of this comparative approach; a CRE derived from an outgroup species that serves as a surrogate for the ancestor has also evolved along a unique lineage since divergence (Rogers, 2013).

Studies into the evolution of divergent protein activities encountered a similar problem when comparing extant proteins forms. For several cases, key amino acid residues necessary for a derived function were identified. When substituted into the surrogate ancestral protein, these changes were insufficient to impart the derived function and thereby indicating that the paths of evolution were more intricate. As a solution, the reconstruction of ancestral protein sequences, combined with functional testing of inferred ancestral proteins has allowed a more realistic simulation of evolutionary events. As a result, inferences about the paths of protein evolution were made that likely would not have been found from comparisons of extant proteins (Rogers, 2013).

A more ideal research program to study CRE evolution would include reconstruction of ancestral CREs as a starting point to trace the paths of evolutionarily relevant mutations. Few studies have used CRE reconstruction. For one study, a novel optic lobe expression pattern for the D. santomea Nep-1 gene occurred via the modification of a CRE that drove an eye field pattern of expression for an ancestor that existed ~0.5 million years ago. Importantly, by reconstructing and evaluating the ancestral CRE, the wrong conclusion - that this optic lobe activity evolved de novo – was avoided and the correct conclusion was found - a latent optic lobe CRE activity was augmented into a robust derived state. In the current study, had the Concestor element not been reconstructed, the Dark 1 and Dark 2 dimorphic element sequences would have been considered hypomorphic CRE alleles compared to the robust wild type-like activity of the Light 1 and Light 2 alleles. The Light alleles possessed activities more similar to a previously characterized dimorphic element allele and consistent with the narrative of D. melanogaster being a sexually dimorphic species where females lack posterior abdominal pigmentation. Reconstruction of the dimorphic element revealed a more complex reality, where neither alleles were good surrogates for the ancestral state. Using ancestral sequences as a starting point, this study found that the evolutionary paths for these alleles to be short in number of steps (one to two mutations) and in time frame (in the last ~60,000 years). Thus, demonstrating how simple and rapid an existing CRE regulatory logic can evolve (Rogers, 2013).

The cases of Nep1 optic lobe CRE and the bab dimorphic element evolution demonstrate the utility for reconstructing ancestral CRE states; though it must be pointed out that these cases involved comparisons of very closely-related species/populations. As a result of these short time frames for divergence, the extant CRE forms differ at fewer than two percent of the nucleotide sites. This made possible ancestral sequence reconstruction by the principle of parsimony. However, not all compelling instances of functional CRE evolution occur over similarly short time frames. Therefore, studies will need to reconstruct CREs that existed further in the past and for which the method of parsimony will need to be replaced by methods of maximum likelihood-based inference coupled with the testing of multiple alternate reconstructions (Rogers, 2013).

Transcriptional Regulation

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 a 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).

Distal-less protein can be detected in a central domain in leg discs throughout most of larval development; in mature discs this domain corresponds to the distal-most regions of the leg: the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. Dll3 is the strongest hypomorph in which all of the tarsus is deleted and the tibia and femur are reduced in size. The expression of two genes required for the patterning of the tarsus, al and bric a brac (bab) was examined in Dll3 leg discs. In wild-type discs, al is expressed in the center of the disc and bab in the rest of the presumptive tarsus. In Dll3 leg discs no al or bab expression can be detected in the center of the discs (Campbell, 1998).

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).

Targets of Activity

The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. This study investigated how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. The data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-a-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, this study discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages (Camino, 2015).

This study has traced the evolutionary history of two CREs required for a novel trait, and show that they have recently evolved similar expression patterns through remarkably different architectures in a common trans-regulatory landscape. The data indicates that the tergite-wide activities of the yBE and t_MSE did not exist in the monomorphic ancestor for Sophophora, but evolved in the lineage leading to the common ancestor of the melanogaster species group. The results support a scenario where the subsequent expansion and contraction of male pigmentation pattern was driven primarily by alteration of the trans-regulators, whereas repeated losses involved both cis- and trans-evolution with respect to these CREs. Though the t_MSE and yBE drive coordinated patterns of gene expression, striking differences were found in their upstream regulators and direct regulatory linkages. These results bear on the understanding of how new gene regulatory networks form, diversify, and how coordinated regulatory activities can arise through the independent evolution of unique regulatory codes (Camino, 2015).

Hox transcription factors play a prominent role in generating the differences in serially homologous animal body parts, and the origin of novelties. The diversification of homologous parts can be driven by changes in the spatial domains of Hox protein expression, as has been shown for crustacean appendage morphology, snake limblessness, and for the water strider appendage ground plan. Changes in the downstream Hox targets are evident in cases such as the hindwings of insects, and for fruit fly tergite pigmentation. The origin of novel structures can also be traced to the co-option of Hox proteins, as exemplified by cases such as the Photuris firefly lantern and the sex combs residing on the forelegs of certain Drosophila species. For many of these evolved traits, the molecular mechanisms by which Hox expression patterns and target genes evolve remain unknown (Camino, 2015).

While mechanistic studies on the evolution of Hox-regulated CREs remain limited, several target gene CREs have been thoroughly characterized and serve as exemplars of Hox-regulation during development. Hox proteins can interact with CRE binding sites as monomers or through cooperative interactions with Hox-cofactors. The activity of these bound complexes can be further modulated through interactions with collaborating transcription factors. However, to date, few direct Hox target linkages have been traced to their evolutionary beginnings. Expression of yellow in the male A5 and A6 segments required the gain of two binding sites for Abd-B, but it remains uncertain whether these binding events require cooperative interactions with Hox cofactors and which transcription factors are acting as collaborators (Camino, 2015).

The t_MSE presented an opportunity to study how a second Hox-responsive CRE evolved in parallel to the activity at yellow. This study shows that Abd-A and Abd-B respectively are necessary and sufficient for t_MSE regulatory activity. However, the ablation of the resident Hox sites had little effect on this CRE's activity in the A5 and A6 segments, though mutations to nearby CRE sequences resulted in dramatically reduced activity. This result strongly implies that both Abd-A and Abd-B indirectly activate the t_MSE through a downstream factor or factors. While it can't be entirely ruled out that these factors are operating directly through other non-canonical Hox sites, the gel shift assays did not provide convincing evidence that such sites exist. While the Hox sites were not necessary for activation in the A5 and A6 segments, their ablation resulted in a drastic gain of regulatory activity in the A4 and A3 segments, a setting in which Abd-A is the only Hox protein present. This indicates that Abd-A is a direct repressor of t_MSE function in these anterior abdomen segments. The observed dichotomy in Abd-A function can be explained by at least two-not necessarily mutually exclusive-scenarios. First, in the A5 and A6 segments Abd-B may not act as a direct activator of the t_MSE but its occupancy of Hox sites might preclude the direct repressive effects of Abd-A. Secondly, Abd-A may interact cooperatively or collaboratively with other transcription factors in the more anterior segments to impart repression. The results with Hth support this second scenario (Camino, 2015).

The Hox co-factors Hth and Exd were prime candidates to mediate the context-dependent modulation of Abd-A activity. First, RNAi suppression of hth and exd expression each resulted in ectopic pigmentation (Rogers, 2014) and t_MSE activity in the male A4 and A3 segments. Furthermore, inspection of the t_MSE sequence revealed sites characteristic of Hth (AGACAG) and Exd (GATCAT) binding that reside in close proximity to Hox sites. This site content and arrangement is strikingly similar to that found in an abdominal-repressive module for the CRE controlling thoracic Distalless expression. Along a similar vein, this study shows that the ablation of the Hth-like site led to an anterior expansion in t_MSE activity similar to that induced by the Hox site mutations. This outcome supports the interpretation that the more recent origin of the t_MSE involved the formation of novel regulatory linkages with Hox proteins and Hox cofactors (Camino, 2015).

Morphological traits result from the activities of gene regulatory networks, in which each network is governed by a trans-regulatory tier of transcription factors and cell signaling components that ultimately regulate the expression of a set of differentiation genes. For animals, the trans-regulatory genes are remarkably conserved. It is plausible that the origin of new morphologies occurs through the formulation of new gene regulatory networks, while diversification and losses in traits would likely occur through the modification and dismantling of extant networks. The empirical evaluation of such trends of network evolution necessitates the study of trait evolution at the level of networks, CREs, and their encoded binding sites for multiple animal lineages, traits, and evolutionary time frames. The Drosophila pigmentation system is particularly well poised to make pioneering contributions to this growing body of knowledge (Camino, 2015).

The most recent common ancestor of monomorphic and dimorphic Sophophora lineages was inferred to have possessed monomorphic tergite pigmentation, in the context of an otherwise invariant morphological landscape, in which segment number and form has remained conserved at the genus level. Hence, the origin of this novel pigmentation trait may be expected to have co-opted spatial and sex-specific patterning mechanisms that shape the conserved abdomen features. Comparative analysis of orthologous yellow and tan non-coding sequences indicate that these co-option events involved the origination of novel CRE activities that connected a trans-regulatory tier of Hox, Hox-cofactors, and the Bab proteins to these key differentiation genes that encoded pigmentation enzymes (Camino, 2015).

The patterns of regulatory activity for the orthologous tan and yellow sequences support some additional inferences about the early events in this dimorphic trait's origin. While the t_MSE abdominal activity was strikingly lower in D. pseudoobscura and D. willistoni, the D. pseudoobscura yellow body element was active (albeit with expanded activity). These outcomes support at least two evolutionary scenarios. One scenario is a sequence of events where the origination of the t_MSE and y_BE in the lineage of D. pseudoobscura was followed by a secondary loss of the t_MSE. This scenario is supported by a previous observation of dimorphic Bab expression in the D. pseudoobscura abdomen, backing the notion that this species' broad pattern of monomorphic abdominal pigmentation evolved from a dimorphic ancestral state. For the other scenario, the body element-like regulatory activity of D. pseudoobscura could be due to this CRE's origin preceding that of the t_MSE. Distinguishing between these two scenarios will require a more rigorous comparison of the pigmentation phenotypes and networks within the melanogaster and obscura species groups. The outcomes would provide a more nuanced understanding of the early evolutionary history for the derived sexually dimorphic pigmentation network (Camino, 2015).

Tergite pigmentation evolution in the Sophophora subgenus has been relatively well-studied, and the accumulated results frame an extended perspective of trait evolution within a common network. Trans-evolution at the bric-à-brac (bab) locus has been found to be a major driver for the diversification of female tergite pigmentation. This study, in addition to previous studies, indicates that trans-evolution at as of yet unidentified loci may have played prominent roles in the diversification of male-limited tergite pigmentation. Regarding the repeated losses in male pigmentation, the current results are consistent with a scenario where both trans- and cis-evolution occurred, though the targets of cis-evolution have alternated between tan and yellow. While cis-evolution has been identified for a case of monomorphic gain (ebony) in tergite pigmentation, and for a case of monomorphic loss (ebony and tan), the full wealth of case studies portend to a more prominent role for evolutionary changes in the trans-regulatory tier of the pigmentation gene network. However, it is important to note that many of these case studies only assessed the activities of transgenes in D. melanogaster. While similarities in CRE activity might be indicative that expression divergence occurred through trans-evolution, it does not rule out the possibility that cis-changes occurred at other regions in the pigmentation enzyme gene loci, or that expression divergence results from combined cis- and trans-changes. In the future, it will be important to validate or reject the prominent role for trans-regulatory evolution by the reciprocal tests of CREs in species with the contrasting patterns of pigmentation. Two studies where CREs were tested in species with contrasting pigmentation phenotypes, showed that trans-regulatory evolution was a major driver for diversification of fruit fly wing spot patterns by modifying Distalless and wingless expression (Arnoult, 2013; Werner, 2010). Thus it appears the notion of a “conserved trans-landscape” requires more scrutiny (Camino, 2015).

In this study, and elsewhere, experiments indicate that pigmentation losses are associated with and perhaps result from both changes in the trans-regulatory tier and in the cis-regulatory regions of the yellow and tan genes. Interestingly, some instances of trans-regulatory modifications that cause loss of gene expression appear to leave perfectly good CREs intact. The current data provides a second instance in which loss of expression occurred without the loss of the encoded CRE. The yBE was found to be conserved in D. santomea, which diverged from D. yakuba ~400,000 years ago. The activity for this CRE has also remained for D. ananassae since its divergence from a pigmented ancestor. In contrast, D. kikkawai has lost pigmentation while still expressing tan in the abdomen through a perfectly active t_MSE. These results suggest that these CREs were maintained within the population for long periods of time, perhaps indicating additional functions that promote the preservation of these CREs' ancestral potential. Furthermore, the observed heterogeneity of changes in cis and trans to yellow and tan were at first surprising. However, study of the binding site architecture at the yBE and t_MSE provided key clues as to why their evolution may often be uncoupled (Camino, 2015).

The coordinated expression of genes is a ubiquitous theme in developmental biology. Gene expression is finely regulated during development through the activities of CREs that are individually encoded as evolved combinations of transcription factor binding sites (regulatory logic). A compelling question is whether such synchronized expression results from the independent evolution of CREs with similar logics. This question was previously pursued for CREs of regulatory genes coordinately expressed in the developing fruit fly neurogenic ectoderm. In this case, the coordinately activated CREs are encoded by a common regulatory logic, or a so called 'cis-regulatory module equivalence class'. However, the neurogenic ectoderm CREs are deeply conserved, and arose in the distant past (over 230 million years ago) (Camino, 2015).

The recently evolved male-specific expression patterns for tan and yellow present a case in which the evolutionary formation of coordinated regulation can be observed over shorter time-scales. Though both the t_MSE and yBE0.6 drive reporter expression in the dorsal A5 and A6 segment epidermis of males during late pupal development, this study found their regulatory logic to be surprisingly dissimilar. Whereas the yBE0.6 is directly activated by Abd-B, the results indicate that the t_MSE is indirectly activated by Abd-B and Abd-A, and is directly repressed in more anterior body segments by Abd-A and seemingly Hth. Thus, this study provides an example that illustrates how coordinated expression evolved through the evolution of very different binding site architectures and logic (Camino, 2015).

The disparity of regulatory logic governing the yBE0.6 and t_MSE sheds light on the evolutionary tendencies of gene regulatory networks. The incipient stages of the dimorphic pigmentation network's origin involved the derivation of CREs that generate similar patterns through distinct combinations of binding sites. This evolutionary history establishes a 'branched' network in which several of the possible trans-regulatory alterations are incapable of generating coordinated shifts in the expression patterns for co-expressed genes. Hence, an emerging theme from the work in this system is that the differences in regulatory logic of yBE and t_MSE may necessitate changes in one CRE or the other, but is unable to be altered through a common trans regulator that influences both CRE's patterning. Future studies are needed to substantiate the occurrence and identity of the trans changes altering this network's structure. As other recently derived morphological traits are resolved to the level of binding sites within their networks, it will be instructive to see whether similar branched networks and paths of cis and trans evolution permeate their origin and diversification. The net results may reveal general principles of gene regulatory network evolution (Camino, 2015).

Protein Interactions

The BTB/POZ domain is an evolutionarily conserved protein-protein interaction domain present in the N-terminal region of numerous transcription factors involved in development, chromatin remodeling, and human cancers. This domain is involved in homomeric and heteromeric associations with other BTB/POZ domains. The Drosophila BTB/POZ proteins Bric à brac 1 (Bab1) and Bric à brac 2 (Bab2) are developmentally regulated transcription factors which are involved in pattern formation along the proximo-distal axis of the leg and antenna, in the morphogenesis of the adult ovaries, and in the control of sexually dimorphic characters. Partners of the Bab1 protein have been identified by using the two-hybrid system. The characterization of one of these proteins, called BIP2 for BAB interacting protein 2, is presented. BIP2 is a novel Drosophila TATA-box protein associated factor (TAFII), also named dTAFII155. The BTB/POZ domains of BAB1 and BAB2 are sufficient to mediate a direct interaction with BIP2/dTAFII155. This provides a direct link between these BTB/POZ transcription factors and the basal transcriptional machinery (Pointud, 2001).

Many of the BTB/POZ transcription factors have been shown to be transcriptional repressors. Some of them, but not all, mediate their repressive activity, via their BTB/POZ domain, by recruiting histone deacetylase complexes to promoters of their respective target genes, thereby inducing a repressive chromatin state. Other BTB/POZ proteins function as transcriptional activators. The BTB/POZ domains of BAB1 and BAB2 are sufficient to interact with a potential transcriptional co-activator, the TAFII factor BIP2/ dTAFII155. As BIP2/dTAFII155 is a component of TFIID (Gangloff, 2001), this interaction provides a direct link between the BTB/POZ transcription factors and the basal transcriptional machinery and suggests a new mechanism for the transcriptional regulation by BTB/POZ proteins. Interestingly, interactions between BTB/POZ domains and TAF proteins may mediate either a transcriptional activation or repression. Classical transcriptional activators act through a direct interaction between their activation domain and the co-activators of the basal transcriptional machinery. A number of TAFs have been identified as direct transcriptional targets of activators in vitro (Pointud, 2001).

One interpretation of the interaction between a given BTB/POZ domain and a TAFII is that this BTB/POZ protein is an activator that directly interacts with TFIID or other TBP-free TAFII-containing complexes to activate the transcription of its target genes. An alternative possibility is that the interaction between a BTB/POZ domain and a TAFII mediates repression or anti-activation by interfering with the basal transcriptional machinery. It has been shown that the co-repressor N-CoR can directly interact with TFIIB, hTAFII32, and hTAFII70 to mediate signals from repressors to the basal machinery. BIP2/dTAFII155 and its homolog hTAFII140 contain a PHD-finger at their respective C-termini. While the function of the PHD-finger is unknown, it has been suggested that it is involved in protein-protein interactions. This domain is present in proteins associated with a repressor activity, like histone deacetylases, and in interactions with heterochromatin. The BIP2/dTAFII155 and hTAFII140 proteins could mediate, through their PHD fingers, interactions between BTB/POZ proteins, and repressor activities. BTB/ POZ domains could mediate repression of transcription by recruiting histone deacetylase complexes to generate a close conformation of the chromatin, and/or by direct contacts with members of the basal transcriptional machinery, locking them into a nonfunctional complex or conformation (Pointud, 2001).

Very few non-BTB/POZ proteins have been identified as partners of the BTB/POZ domains of Drosophila proteins. The interaction of the Tramtrack BTB/POZ domain with the corepressor dCtBP (C-terminal binding protein) and the BTB/POZ domain of GAGA with the heterologous proteins N-CoR and SMRT in in vitro experiments have been briefly reported. Since the BTB/POZ domains of the Drosophila nuclear proteins are strongly conserved in evolution, it is probable that other Drosophila BTB/POZ proteins interact directly, via their BTB/POZ domain, with one of the central components of the preinitiation complex, like BIP2/dTAFII155, to regulate the transcription of their target genes. Since a given TAF can interact with more than one activator and a single activator can also contact multiple TAFs, it is suggested that BIP2/dTAFII155 could interact with other Drosophila BTB/POZ proteins and/or that different BTB/POZ domains can contact different TAFs. The BTB/POZ transcriptional repressor HIC-1 and its avian homolog gammaFBP-B do not seem to mediate their repressive effect by recruiting histone deacetylase complexes, since they fail to interact with the co-repressors SMRT, N-CoR, and mSIN3A and as their transcriptional repression is unaffected by Histone Deacetylase (HDAC) inhibitors. The repressive function of HIC-1 and gammaFBP-B could be mediated by a direct interaction between their BTB/POZ domains and components of the basal transcriptional machinery such as the vertebrate homolog of BIP2/dTAFII155 (Pointud, 2001).

Although the BTB/POZ domain of BAB1 is sufficient for the interaction with BIP2/dTAFII155, in vitro experiments show that the regions of BAB1 adjacent to the BTB/POZ domain facilitate this interaction. This observation could reflect different folding properties of the different BAB1 fusion proteins or could imply a direct involvement of the BTB/POZ adjacent regions to stabilize the interaction with BIP2/dTAFII155. This could indicate that the regions adjacent to the BTB/POZ domain could give another level of specificity to the BTB/POZ proteins for an interaction with a given protein like a TAF (Pointud, 2001).

The crystal structure of the BTB/POZ domain of promyelocytic leukaemia zinc finger (PLZF) revealed a tightly intertwined dimer with a surface-exposed groove. This pocket is composed of some of the most conserved residues of the BTB/POZ sequence and has a high charge density. The minimal domain of the BIP2/dTAFII155 protein required for the interaction with the BTB/POZ domains of the BAB proteins corresponds to a highly charged region comprising multiple lysine, glutamic acid and aspartic acid residues (aa 859-1091). This charged region of BIP2/dTAFII155 is well conserved in its homologs hTAFII140 and mTAFII140 (Gangloff, 2001). The charged-exposed groove of the BTB/POZ dimer may bind this charged region of BIP2/ dTAFII155 or hTAFII140 through electrostatic interactions (Pointud, 2001).

The BAB proteins may regulate the expression of several genes at different steps of Drosophila development. The BAB2 protein binds to several sites on polytene chromosomes. During leg morphogenesis, bab is required for segmentation and for the specification of segment identity in the tarsus. bab is also required for adult ovary morphogenesis. Kopp (2000) has shown that the BAB proteins are involved in the genetic control of the sexually dimorphic abdominal pigmentation and the abdominal A5-A7 segment morphology in Drosophila melanogaster. Since BIP2/dTAFII155 is expressed in all the tissues that express and require BAB, it is possible that BIP2/dTAFII155 mediates all the BAB functions. The BAB proteins could be transcriptional activators as well as transcriptional repressors, depending on the promoter considered and it will be of great interest to identify BAB target genes and cis-regulatory sequences recognized by the BAB proteins. However, the BAB proteins, unlike most of the other BTB/POZ proteins, do not contain a zinc finger DNA binding domain but a new type of DNA binding domain that is currently being characterizing. Clearly, the identification of bab downstream genes is crucial to understand whether the interaction between BIP2/dTAFII155 and the BTB/POZ domain of the BAB proteins is critical for BTB/ POZ-mediated transcriptional activation or repression (Pointud, 2001).


Larval and Pupal

The babP enhancer trap line expresses ß-galactosidase (ß-gal) in the leg and antenna imaginal discs. Flies of the line babA128, which have a similar ß-gal expression pattern but are phenotypic wild type as homozygotes, were chosen for a detailed analysis of the ß-gal expression pattern to avoid any effects that might be caused by haploinsufficiency associated with the babP mutation. Imaginal discs from the early third instar larva until the mid prepupa [6 hours post puparium formation (PP)] were studied. The distribution of the bab transcript was also examined by tissue in situ hybridization to imaginal discs from mid and late third larval instar using bab-specific DNA probes. In addition, a polyclonal antibody (anti-BAB r2), which is directed to a protein that corresponds to part of the defined open reading frame, was used to study the bab expression pattern in imaginal discs in the third larval instar and throughout the prepupal stage. The patterns of expression of ß-gal in the enhancer trap lines and of the bab transcript and the BAB protein were found to be equivalent in leg and antenna imaginal discs (Godt, 1993).

From mid third larval instar onward the bab product is present in a concentric domain around the center of the imaginal discs, a region that corresponds to parts of the tarsal primordium of the legs, and the subdistal structures of the antenna. No staining is detected in the center of the imaginal discs, which is the primordium of TS5 of the leg and the arista of the antenna (Godt, 1993).

At mid third larval instar, ß-gal is weakly expressed in the furrow between the two central folds of the leg imaginal disc. The staining level becomes stronger during late third larval instar, when the domain where bab is expressed in the leg imaginal disc gives rise to three additional folds, the primordia of TS2 to TS4. In addition to the region of TS2 to TS4, bab product is also found in the distal margin of TS1. In anti-Bab r2 stainings and RNA in situ hybridizations, no signal was detected prior to this stage. During evagination in the prepupal stage, when the tarsal segments have expanded, bab is expressed in the region from distal TS1 through TS4. The exact boundaries of the expression domain are difficult to localize because the staining drops strongly towards the edges of the domain. Differences in the distribution of the bab product in the imaginal discs for different leg pairs were not observed. The oldest antibody-stained discs that were examined were from 6 hours old prepupae, when the evagination is completed. To correlate the bab expression domain to the future segments is more difficult in the antenna than in the leg imaginal disc, however, strong ß-gal activity is detectable in the two segments of the basal cylinder in the adult antenna of babA128 flies, suggesting that the two rings of strong anti-ß-gal staining in the antenna disc correspond to the primordia of these structures. The analysis of the distribution of the bab transcript and protein in leg and antenna imaginal discs shows that bab expression is restricted to those regions of the antenna and leg discs that are affected by bab mutations (Godt, 1993).

In the bab expression domain that extends from distal TS1 through TS4, the bab product is found in all cells but the amount differs from cell to cell. At mid third instar, the bab expression domain comprises less than 20 cell diameters along the proximal-distal axis and shows a higher level of staining distally than proximally. During the late third larval instar, when the tarsal primordium grows by cell division and becomes folded, bab expression becomes stronger and more differentiated. The staining level is highest in TS4 and TS3, lower in TS2 and lowest in the distal margin of TS1. Instead of a simple distal-proximal gradient, however, cells within each of the folds show different staining intensity. At puparium formation when the tarsal folds are established and the evagination process starts, the bab expression pattern appears to be fully developed and remains stable throughout the early prepupal stage. In the expanding tarsal primordium, the bab expression domain comprises about 40 cell diameters along the proximal-distal axis. The complex distribution of the bab product has two characteristics. (1) A wave-like pattern is observed in the expression domain. Each tarsal fold in the bab domain shows a bell-shaped distribution of staining intensity. The cells at the ridges of the segmental folds show a much higher staining level than the cells in the furrows. (2) There is a graded distribution of the bab product throughout the expression domain. The highest levels of the bab product are found at the ridge of TS3. TS2 shows lower expression levels than TS3, and the ridge of TS1 has the lowest levels. The staining intensity in TS4 was never found to be higher than in TS3 but either equal or lower (Godt, 1993).

In order to facilitate a cell-by-cell analysis of the complex bab expression pattern, sections of anti-ß-gal-stained prepupal leg discs of bab A128 flies were examined with an image processor which translated the staining intensity of each cell into a color value. The graded distribution of staining intensity was reproduced in all leg discs examined by this method (Godt, 1993).


The first bab allele, babP, was isolated from an enhancer trap screen as a female-semisterile mutation caused by disorganized mutant ovaries. Twenty-four additional bab alleles that have a stronger female-sterile phenotype were isolated by remobilization of the P-element insert in the bab P line. Examination of these alleles showed that they also cause defects in the leg and the antenna of the fly. An additional mutation, the EMS-induced babE1 mutation was identified as a bab allele on the basis of its mutant phenotype and because it fails to complement other bab alleles. A deficiency, Df(3L)babPG (61D3-E1; 61F5-8), was isolated that uncovers the bab locus. Two more P-element insertions, P[lacZ,ry+]A30 and P[IArB]A128.1F3 (the latter designated as babA128) map to the same chromosomal position and express the lacZ reporter gene in a pattern similar to babP but do not cause a mutant phenotype in homozygous flies. The isolated bab mutations were classified as having either a strong, intermediate or weak mutant leg phenotype according to the severity of the leg defects. The homozygous mutant phenotype of strong bab alleles is slightly weaker than the transheterozygous phenotype of these alleles over Df(3L)babPG suggesting that these bab alleles are strongly hypomorphic (Godt, 1993).

bab mutations cause homeotic transformation of the bristle pattern of tarsal segments. Homozygotes and transheterozygotes of strong bab mutations show a change of the bristle pattern of tarsal segments (TS) TS2, TS3 and TS4 towards the most proximal tarsal segment, TS1. TS1, also called the basitarsus, can be distinguished from the other tarsal segments by specific bristle markers. The most prominent marker of TS1 is the sex comb on the prothoracic (front) legs of males. The sex comb is a longitudinal row of about eleven blunt, black sex comb bristles (SCB), which is located in the distal region of TS1. In addition to the normal sex comb on TS1, males homozygous for a strong bab mutation exhibit ectopic sex combs on TS2, TS3 and occasionally TS4 of the prothoracic legs. The ectopic sex combs were defined as such based on the color, shape, orientation and position of the bristles. The ectopic sex comb on TS2 usually contains 5-6 SCB, half as many as are present in the normal sex comb on TS1. The ectopic sex comb on TS3 is even smaller and usually contains 1-2 SCB; the one on TS4 not more than one SCB (Godt, 1993).

The appearance of ectopic sex combs in distal tarsal segments indicates a homeotic transformation of the bristle pattern of TS2, TS3 and TS4 towards TS1. This conclusion is supported by the observation that the homeotic transformation is not limited to the SCB but includes other characteristics of TS1, and that the transformation is neither sex nor leg specific. In wild-type flies, the ventral bristles of the prothoracic TS1 are arranged in tightly packed transverse rows. In contrast, the distal tarsal segments of the prothoracic legs have separated longitudinal columns of single bristles. In strong bab mutant flies, both male and female, the normal bristles on the ventral side of TS2 and TS3 are replaced by a larger number of bristles that are arranged in such transverse rows. The analysis of the bristle pattern of TS4 was difficult because of its small size and because it is fused to TS5 in strong bab mutants; however, the bristle pattern of TS4 is clearly affected by bab mutations as indicated by the occasional appearance of a SCB. In contrast, no alterations in the bristle pattern could be detected in the size-reduced TS5, which suggests that the identity of TS5 is not affected. Similar to the prothoracic legs, in metathoracic legs (third leg pair) the bristle pattern of TS1 is repeated in the distal tarsal segments TS2-TS4. The bristle pattern in the mesothoracic tarsal segments is indistinguishable and therefore not accessible to an analysis (Godt, 1993).

The size of the basitarsus in wild-type and bab mutant legs is much larger than that of the other tarsal segments. Although the bristle pattern is changed in TS2-TS4 of bab mutant flies, the size of these segments is not enlarged to the size of TS1, suggesting that the specification of the tarsal segments has changed upon a segment primordia of normal size. The analysis of the phenotype of strong loss-of-function bab alleles demonstrates that the bristle pattern of the distal tarsal segments TS2, TS3 and TS4 is transformed towards the bristle pattern of the basitarsus, indicating that the bab gene is required for the specification of the three central tarsal segments (Godt, 1993).

In addition to its recessive phenotype, strong bab mutations cause a dominant mutant phenotype. Heterozygous males have an ectopic sex comb on TS2, indicating a homeotic transformation of TS2 towards TS1. The same dominant effect is also detected in hemizygous flies that carry a wild-type chromosome over the deficiency Df(3L)babPG or over the terminal deletion of the translocation T(3;Y)A114 (61A1; 61F). This indicates that the dominant phenotype is caused by a reduction of the gene dosage of bab, and defines bab as a haploinsufficient gene. In contrast to the recessive phenotype, only the TS2 bristle pattern has changed, and on TS2 itself the ectopic sex comb is the only clear indication of a homeotic transformation. This shows that the dominant phenotype of strong bab mutations is similar to but weaker than the recessive phenotype of strong bab alleles. A comparison between the recessive and dominant phenotype of strong bab mutations indicates that TS2 is more sensitive to transformation towards TS1 than either TS3 or TS4. This effect can be interpreted as a graded requirement for bab along the proximal-distal axis of the tarsus (Godt, 1993).

The graded requirement for bab activity is supported by the analysis of weaker bab alleles. Flies homozygous for babPR11 or babPR30 have small ectopic sex combs on TS2. Therefore, the recessive phenotype of these bab alleles is comparable to the dominant phenotype of strong bab alleles. A stronger transformation of distal tarsal segments was observed with the alleles babE1 and babPR23 (Godt, 1993).

On the prothoracic legs of flies homozygous for these alleles, TS2 exhibits a sex comb with usually 4 SCB as well as transverse bristle rows. On TS3, however, neither a sex comb nor transverse bristle rows were observed. In the metathoracic legs of wild-type flies, TS1 contains transverse bristle rows with more than 7 bristles per row; TS2 contains somewhat irregular transverse rows containing 3 and 4 bristles, whereas TS3 and TS4 lack transverse row bristles. The larger number of 4 and 5 bristles in the transverse rows on TS2 of babE1 and babPR23 metathoracic legs suggests a transformation of TS2 towards TS1 as described previously for the prothoracic leg. The additional appearance of transverse rows on TS3 (2 and 3 bristles per row) indicates that TS3 also has taken on the identity of a more proximal tarsal segment. This observation might suggest that TS3 of metathoracic legs is more sensitive than TS3 of the prothoracic legs to transformation towards TS1. Alternatively, it might suggest that TS3 is transformed towards TS2, a transformation that would not be identifiable on the prothoracic legs because of a lack of distinguishing markers between TS2 and TS3. The analysis of bab mutants of different phenotypic strength indicates a gradual transformation of the bristle pattern of the tarsal segments (Godt, 1993).

Strong bab alleles produce a complete fusion of tarsal segments TS5 and TS4 in all six legs as homozygotes. The segmental joint is missing between TS5 and TS4, and the fused double segment is shorter and thicker than that of TS4 and TS5 together in wild-type legs. In flies carrying a strong bab allele over Df(3L)babPG, TS4 and TS3, and TS3 and TS2 are partially fused as well. In weaker bab mutants, TS5 and TS4 are only partially fused and this occurs with incomplete penetrance (Godt, 1993).

The analysis of different bab alleles shows that the extent of tarsal fusion and homeotic transformation are related. These two phenotypic traits, however, overlap spatially only in the strong bab mutant phenotype. In weaker bab mutants, the transformation is only seen in the proximal region of the tarsus and the segmental fusion only in the distal region. This separation suggests that these defects are not dependent on one another (Godt, 1993).

In addition to the segmentation defects that are associated with loss of tarsal joints, bab mutant flies occasionally produce legs with a kink in TS3 or legs where structures of the tarsus that lie distal to the position where the kinks occur are completely missing. The defects might be caused by the cell death, which can be detected in the imaginal primordium of TS3. They are observed more frequently in the metathoracic legs than in other legs and usually only in one leg of a fly. They appear rarely (<1%) in the case of the described strong bab mutations, which are derived from the babP mutation, but occur frequently in flies of the genotype babE1/ Df(3R)babPG (Godt, 1993).

Defects in the legs of bab mutants are restricted to a specific subdistal domain of the tarsus. Structures proximal to this domain as well as the most distal structure of the leg, the claw organ, are not affected. In addition to the leg, bab mutations affect the morphology of the antenna. The antenna of Drosophila is a structure homologous to the leg and similarly subdivided into different segments. The basal cylinder of the antenna, which consists of two small segments, has been shown to be homologous to TS2-TS4. Strong bab mutations cause defects in the basal cylinder of the antenna, suggesting that bab is required in a homologous region of the leg and antenna. Defects at the arista, the distalmost structure of the antenna, and at structures lying proximal to the basal cylinder were not observed. The two segments of the basal cylinder are fused to a variable degree to each other and to the arista in strong bab mutants. This segmentation defect is accompanied by loss of the segmental joints. Based on available morphological markers, no homeotic transformation of the basal cylinder was detected (Godt, 1993).

The penetrance and strength of the dominant leg phenotype is temperature sensitive. Flies heterozygous for a strong bab allele develop legs with a stronger homeotic defect at 30°C than at lower temperatures. The temperature sensitivity is not allele specific and can be seen in hemizygous Df(3L)babPG/+ flies as well. The strong allele babPRDS was used to perform temperature-shift experiments for which the penetrance of ectopic sex combs was chosen as the phenotypic parameter. Along with its penetrance, the size of an ectopic sex comb changes gradually. The temperature-sensitive period of the dominant homeotic bab phenotype centers on the prepupal stage and defines a critical period for tarsal segment specification. Because the temperature sensitivity is not an allele-specific effect, it cannot be certain whether it is caused by the Bab protein. However, bab is expressed in the leg imaginal disc at the corresponding stage, which suggests that the phenocritical period is the likely time of bab requirement (Godt, 1993).

The adult ovary of Drosophila is composed of approximately 20 parallel repetitive structures called ovarioles. At the anterior tip of each ovariole is a stack of 8-9 disc-shaped cells, called the terminal filament. Ovariole morphogenesis starts with the formation of the terminal filaments. Using two enhancer trap markers for terminal filament cells, it has been shown that terminal filaments form in a progressive manner from medial to lateral across the ovary and that the number of terminal filament cells in a developing stack increases gradually. This process occurs during the second half of the third larval instar. One of these enhancer trap mutations, which is in the bric à brac gene, demonstrates that this gene is necessary for terminal filament formation and that a terminal filament cell cluster is required for ovariole morphogenesis to take place (Sahut-Bernola, 1995).

The Drosophila ovary consists of repeated units, the ovarioles, where oogenesis takes place. The repetitive structure of the ovary develops de novo from a mesenchymal cell mass, a process that is initiated by the formation of a two-dimensional array of cell stacks, called terminal filaments, during the third larval instar. The morphogenetic process leading to the formation of terminal filaments has been studied and it has been found that this involves recruitment, intercalation and sorting of terminal filament cells. Two other types of cell stacks that participate in ovary morphogenesis, the basal stalks and interfollicular stalks, also form by cell rearrangement utilizing a convergence and extension mechanism. Terminal filament formation depends on the Bric à brac protein, which is expressed in the nuclei of terminal filament cells and is cell autonomously required. Disruption of terminal filament formation, together with defects of basal and interfollicular stalk development, leads to disruption of ovariole formation and female sterility in bric à brac mutants (Godt, 1995).

During the third larval instar distinct mesodermal cell populations become apparent in the ovary. By puparium formation the different cell populations are arranged in layers along the anterior-posterior axis (King, 1970). There are three cell populations in the anterior region of the ovary. The anterior most cells, referred to as cap cells, participate in the formation of the peritoneal sheath which envelops the whole ovary. The apical cells migrate between the terminal filaments and form the epithelial sheaths that divide the ovary into ovarioles. Terminal filaments are already differentiated cell stacks. The central region is occupied by the germ cells intermingled with somatic cells that are believed to give rise to the precursors of the follicle cells and the interfollicular stalk cells in the germarium. The posterior region consists of at least two distinct cell populations: the basal stalk primordium and the basal cells that will form the calyx of the oviduct. During the larval and prepupal stages the fat body is attached to the lateral side of an ovary (Godt, 1995).

Three types of cell stacks are found in a pupal ovary, and each forms at a different stage of ovary development. The terminal filaments (TFs), stacks of 8-9 cells, form during the third larval instar. Short TFs have already formed at mid third larval instar. The basal stalks (BSs), stacks of approximately 30 cells, are made during the early pupal phase; and the interfollicular stalks (IFSs), each of which comprises 6-8 cells, are generated from the mid pupal stage onward (Godt, 1995).

bab mutant females are sterile and have ovaries of an aberrant morphology. In comparison to the cone-shaped wild-type adult ovary, the bab mutant ovary is very small and abnormally shaped. Ovaries that are homozygous for strong hypomorphic alleles, which do not express the Bab protein at a detectable level, have a severely reduced ovariole number or lack ovarioles altogether. A dominant haploinsufficient effect of bab mutations, previously described for legs, has not been observed for ovaries. The bab mutant adult ovary phenotype is complex. This study analyzes the requirement of bab on the development of TFs, BSs and IFSs (Godt, 1995).

Ovary defects in bab mutants are first detected in the third larval instar. Wild-type ovaries take on an oval shape when the TFs develop. In contrast, bab mutant ovaries maintain a nearly round shape, and analysis shows that TFs fail to form. Enhancer traps that express lacZ in TFs were used to study the bab mutant ovary phenotype. These included the strong allele babPRDS, as well as AD47, XA42 and B1-93F, which map to other loci and which were used in the background of the ß-gal negative babPR24 and babPR72 mutations. The analysis shows that TF cells are affected in a strong hypomorphic bab mutant to a variable degree. (1) The number of cells that express the four TF markers is severely reduced in the TF region as compared to wild-type. (2) Most of these cells have aberrant morphogenetic properties. They have a rounded cell shape and form loose aggregates that do not differentiate into stacks. (3) A very few TF cells have a flattened cell shape and form short and irregular stacks of 2-5 cells. This probably reflects some residual bab activity in the examined bab mutants; however, study of bab null alleles will be necessary to exclude the possibility that TF formation is to a minor degree independent of bab function. (4) babPRDS hemizygous ovaries have cells in the posterior region that express lacZ, which is not seen in babPRDS heterozygous ovaries. These cells show a lower staining level than the anterior cells, have a rounded shape and show signs of degeneration. Because the posterior cells also express a TF marker unrelated to bab the simplest assumption is that these are TF cells that are misplaced due to cellular defects which would also explain the strongly reduced number of TF cells in the anterior region. Alternatively, TF-specific genes might be ectopically expressed in another cell population in bab mutant ovaries and the reduced number of TF cells might be due to cell death or an altered cell fate (Godt, 1995).

To study whether bab is autonomously required in TF cells, a mosaic analysis was conducted using the strong allele babPRDS which also serves as a TF cell marker. babPRDS homozygous cells stain detectably stronger in an anti-ß-gal assay than heterozygous cells and can therefore be easily identified. Clones were induced at early or mid second or early third larval instar, and 11, 58, and 15 mosaic ovaries were recovered, respectively. Unstained cells, which lost the marker and are wild type, and light-colored heterozygous cells have a flattened shape and form normal TFs in the mosaic ovaries. Most of the darker stained bab mutant cells have a rounded cell nucleus, are scattered or form loose aggregates in the TF region, but do not form normal stacks. Occasionally, dark stained cells were found integrated into a TF. This finding is not surprising because in non-mosaic bab mutant ovaries a few TF cells form rudimentary stacks. Interestingly, the alignment of TF cells in a mosaic stack is disturbed at the point where a mutant cell is located, which can lead to a kink in the TF. Furthermore, the mosaic ovaries contain in addition to the mutant cells in the TF region lacZ expressing cells ectopically in a more posterior region of the ovary similar to non-mosaic bab mutant ovaries. These observations show that bab mutant TF cells display autonomously aberrant morphogenetic features (Godt, 1995).

Disruption of TF formation is the earliest defect observed in bab mutant ovaries. Subsequently, defects develop involving other somatic cell populations. Staining with anti-Fasciclin III reveals defects in the BS primordium from puparium formation onwards. In wild type, Fasciclin III is expressed in the BS primordium and the apical cell population at puparium formation. In strong bab mutant ovaries, no cells or only a small number of cells are stained in the BS region and the stained apical population is strongly reduced in size, which causes the remaining TF cells to be located more anteriorly than in wild type. Increased cell death is observed in both regions. In wild-type adult ovarioles, the follicles are separated from each other by interfollicular stalks. The remaining ovarioles in bab mutant adult ovaries contain follicles of different developmental stages, which are partially or completely fused and of irregular shape. To identify IFS cells in the disorganized ovaries of bab mutants, the enhancer trap B1-93F was used as a genetic marker. Stained cells are found between adjacent follicles of bab mutant ovarioles. These cells however, are not aligned in a single row but are organized as an irregular band. The larger number of ß-gal-positive cells per cluster, as compared to a wild-type IFS, seems not to rely on an altered cell fate decision between IFS and polar follicle cells, which have been shown to derive from common precursors. Even though the IFS cells do exist in the rudimentary ovarioles of bab mutant ovaries, they are not able to arrange into a stack. This phenotypic trait is similar to the failure to form TFs in the larval ovary (Godt, 1995).

bab plays a central role in ovary morphogenesis and is the first gene described to control this process. bab mutant females are sterile and have small and disorganized ovaries. The absence of ovarioles in bab mutant ovaries results from developmental defects in morphogenesis during the third larval instar and early pupal stages. The primary defect appears to be the failure to form TFs, which correlates with the specific expression of the bab protein in the nuclei of TF cells, beginning with their appearance during the third larval instar. Phenotypic analysis shows that bab is required for TF formation. bab mutant TF cells have an abnormal shape, are not able to form normal stacks, and appear to be partially located in ectopic positions in the ovary, indicating that they are affected in their morphogenetic properties. This phenotype is cell autonomously expressed. In addition to the TFs, other somatic cell populations in the ovary are also affected at the onset of metamorphosis. The BS primordium and the apical cell population are strongly reduced in size. Because the bab protein is only detected in TF cells and appears to be cell autonomously required, one possibility is that the absence of TFs is responsible for these additional phenotypic traits. Alternatively, bab may be directly involved in the proper development of other somatic cell types in the ovary, including the IFS cells. The babP enhancer trap expresses ß-gal not only in TFs but also at lower levels in BSs, in forming IFSs, as well as in some other somatic cells of the adult ovary. In addition, the recently identified BTB-II transcript that is related to bab, based on the same chromosomal map position, sequence homology and an overlapping expression pattern, is expressed in TFs, the apical cells and the BS primordium of the prepupal ovary, and bab mutations may affect both transcripts (Godt, 1995).

A characteristic defect of oogenesis in bab mutants is fused follicles. This is at least partially attributable to a failure in IFS formation, although the IFS cells, identified with a genetic cell marker, are present at their proper location. IFS cells, like TF cells, are not able to arrange into stacks. This is different from the phenotype of mutations in the neurogenic genes, where the fusion of follicles results from the absence of IFS cells due to an altered cell fate. Therefore, if bab acts directly in IFS development it is expected to function downstream of the neurogenic genes in this process (Godt, 1995).

bric à brac locus acts as a homeotic and morphogenetic regulator in the development of ovaries, appendages and the abdomen. It consists of two structurally and functionally related genes, bab1 and bab2, each of which encodes a single nuclear protein. Bab1 and Bab2 have two conserved domains in common, a BTB/POZ domain and a Psq domain, a motif that characterizes a subfamily of BTB/POZ domain proteins in Drosophila. The tissue distribution of Bab1 and Bab2 overlaps, with Bab1 being expressed in a subpattern of Bab2. Analysis of a series of mutations indicates that the two bab genes have synergistic, distinct and redundant functions during imaginal development. Interestingly, several reproduction-related traits that are sexually dimorphic or show diversity among Drosophila species are highly sensitive to changes in the bab gene dose, suggesting that alterations in bab activity may contribute to evolutionary modification of sex-related morphology (Couderc, 2002).

During embryogenesis, bab2 is zygotically expressed in a complex pattern, whereas bab1 is not expressed at a detectable level. bab seems to have no essential function during embryonic development since even mutants that lack both bab genes are not embryonic lethal. During post-embryonic stages, bab2 is expressed in a broader range of tissues than bab1 and generally shows a higher level of expression. In larval and prepupal ovaries, bab1 transcript and protein are only detected in cells that form the terminal filaments. The expression of bab2 is more complex. At early to mid third larval instar, prominent bab2 expression is seen in the developing terminal filaments and in a population of cells termed 'swarm cells'. Swarm cells migrate from anterior to posterior past the cluster of germ cells during third larval instar. They produce the basal stalks, a pupal-specific tissue, and may also contribute to tissues of the adult ovary. The highest level of bab2 expression in the swarm cells is seen during their migration. bab2 is also expressed in the apical cells of the larval ovary. After terminal filaments have formed, apical cells migrate between the terminal filaments posteriorly and form the outer sheaths of the egg tubes. The level of expression in these cells increases during the third larval instar and is highest at the time the cells begin their posterior migration. bab2 expression is also seen in the interstitial cells that intermingle with the germ cells. bab mutant ovaries not only display defects in terminal filament formation but also in other cell populations of the ovary, such as the apical cells and the basal stalk primordium. If the development of these cell populations depends on the presence of terminal filaments, then the observed defects in these cell populations could be a secondary effect of bab mutations. Alternatively, bab may be directly required for the development of these cell populations since the apical cells and swarm cells express bab2 (Couderc, 2002).

bab1 and bab2 transcripts are expressed in a similar pattern in the tarsal primordium of leg imaginal discs. Similar to the protein distribution of Bab1, Bab2 protein is expressed in a graded manner in the tarsal primordium, with the concentration of Bab2 highest in tarsal segments TS3 and TS4, lower in TS2 and even lower in TS1. However, the differences in the level of expression between the tarsal segments are not as pronounced as with Bab1. Both Bab proteins are enriched in the ridges compared to the furrows of the tarsal folds. In contrast to Bab1, Bab2 expression is not restricted to TS1-4 but is also found in the proximal region of TS5, in the peripodial membrane, and in the periphery of the leg imaginal disc that gives rise to thorax structures. No morphological defects have been observed in derivatives of leg imaginal discs outside the tarsus (Couderc, 2002).

Both bab genes are expressed in the female and male genital discs. The genital discs give rise to the internal and external structures of the genitalia, the A8 and A9 tergites and the anal plates. The strongest expression of Bab proteins in the female genital disc is found in the primordium of the vaginal plates and the A8 tergite, structures that are affected in bab mutants. In the male genital disc, bab expression is mainly seen in a region of the male genital primordium. In addition, in the central nervous system (CNS), Bab1 and Bab2 are distributed in a similar pattern. bab-expressing cells are found in the central brain hemispheres and the thoracic ganglia of late 3rd instar larvae and prepupae. In bab mutants, no gross morphological defects were observed in histological sections of the prepupal CNS, and it remains uncertain whether bab has a function in the brain (Couderc, 2002).

In summary, the expression pattern of bab1 during imaginal development can be described as a subpattern of the bab2 expression pattern. In tissues that require bab function for development, bab1 and bab2 are usually co-expressed (Couderc, 2002).

To determine the roles of bab1 and bab2 in mediating bab function, a molecular and phenotypic analysis of mutations in the bab locus was conducted. The P-element insertion of babP maps close to the 5' end of the first intron of bab1. babP does not affect the transcription of bab2 but results in the loss of the bab1 5.4 kb transcript and the appearance of an abundant 2.6 kb transcript. This shorter bab1 transcript is detected in babP heterozygotes and homozygotes, and is detected only with probes located upstream of the insertion. Characterization of the 3' end of this 2.6 kb mRNA by 3' RACE-PCR showed that this transcript is a hybrid of the first bab1 exon and a region of the P[ry+, lacZ] construct. The 5' splice site of the bab1 transcript that normally functions to splice out the large first intron of bab1 and the 3' splice site of the l(3)S12 gene, contained in the P[ry+, lacZ] construct just upstream of the rosy gene, are spliced together. This demonstrates that the P[ry+, lacZ] insertion causes aberrant splicing and transcription termination of the bab1 transcript. A similar event has been reported for a PlacZ allele of the psq gene. The 2.6 kb truncated bab1 transcript is more abundant than the 5.4 kb RNA suggesting that it might be more stable than the wild-type transcript. Translation of this transcript would produce a protein that contains the BTB domain of Bab1 but not the BabCD. A Bab1-specific antibody directed to a domain of the Bab1 protein that is encoded by the truncated transcript, however, did not detect any protein in babP homozygous flies. These results suggest that the babP P-element insertion severely disrupts or abolishes the function of the bab1 gene. Nevertheless, babP homozygous flies display ovary defects of only intermediate strength and reveal no leg defects. This indicates that a loss of bab1 does not produce a bab null mutant phenotype and suggests that a second gene is involved in bab function (Couderc, 2002).

This hypothesis is corroborated by the analysis of another P-element insertion, babA128 that maps 57 bp from the 5' end of the bab1 transcript. Only one phenotypic trait of bab mutants, an abdominal pigmentation defect in females, is associated with this insertion. Because homozygous babA128 flies have no ovary or leg defects, it was surprising to find that Bab1 protein is not detectable in babA128 mutant tissues. A strong reduction in the amount of bab1 transcript, as seen by in situ hybridization and RNA blot analysis, indicates that the babA128 insertion interferes with the transcription of bab1, reducing Bab1 to undetectable levels. By contrast, flies that are heterozygous for strong bab mutations, such as babPR72, or deletions of the bab locus, such as Df(3L)Fpa1, have reduced but clearly detectable levels of Bab1, and nevertheless show leg defects in addition to defects in abdominal pigmentation. Taken together, analysis of the mutations babP and babA128 strongly suggests that bab1 is not the only gene involved in bab function (Couderc, 2002).

That bab2 is involved in bab function was confirmed by a protein analysis of bab mutants. The wild-type Bab2 protein is detected in immunoblots as a band of approximately 145 kDa using different anti-Bab2 antibodies that recognize either the N- or C-terminal region of Bab2. An analysis of bab mutants revealed that the alleles babE1, babE4 and babE5 affect the Bab2 protein. In a homozygous babE1 mutant, Bab2 protein is reduced to barely detectable levels. babE4 and babE5 mutants produce truncated Bab2 proteins. By contrast, Bab1 expression appears normal in these three mutants, shown by tissue immunostaining, since the anti-Bab1 antibody does not produce a signal in immunoblots. No change in the size of Bab2 and the expression level of either Bab1 or Bab2 was detected in babE3 and babE6 mutants. babE1, babE4, and babE5 mutants display developmental defects in ovaries, legs and the abdomen, demonstrating that the bab2 gene plays an essential role in development and that it is functionally related to bab1 (Couderc, 2002).

The strongest bab mutations previously published have both a strong ovary and leg phenotype. Further analysis of two of these mutations, babPRDS and babPR72, revealed that they affect the expression of both bab1 and bab2. They each lack detectable amounts of Bab1 and have reduced levels of Bab2. The mutant phenotypes caused by babPRDS or babPR72 are slightly enhanced in trans to a large deletion (Df(3L)babPG), indicating that these mutations are not null for the bab locus (Couderc, 2002).

Additional bab alleles were isolated and studied to find one that completely lacks bab activity. Two deletions, Df(3L)Fpa1 and Df(3L)Fpa2, that were isolated based on a dominant female pigmentation defect, extend into the bab locus from opposite sides, each having a breakpoint in the bab locus. Df(3L)Fpa2 deletes bab2 completely, and deletes the 5' region of bab1, including the BTB domain. Df(3L)Fpa1 deletes bab1, and has a breakpoint in the second intron of bab2, deleting everything downstream of the BTB domain. In Df(3L)Fpa1/Df(3L)Fpa2 transheterozygotes and in flies homozygous for the mutation babAR07, neither Bab1 nor Bab2 are detected. The phenotype of these genotypes is stronger than that of previously described mutations. Since these flies lack both bab1 and bab2 function, this phenotype corresponds to the null phenotype of the bab locus (Couderc, 2002).

To further analyze the function of the two bab genes, the phenotypic series of bab mutations and the bab null mutant phenotype were studied. bab null mutants are semi-viable. They develop into pharate adults but often have difficulties in eclosing from the pupal case, which may be a result of their leg defects. bab null mutants display defects in ovaries, tarsal segments, antennae, abdominal segments and female genital disc derivatives (Couderc, 2002).

Based on the phenotypic series of bab alleles of varying strength, four phenotypic classes of bab mutant adult ovaries have been defined. (1) Females with a weak bab mutant ovary phenotype are fertile but have ovaries that are somewhat smaller than wild-type ovaries, are slightly irregular, and rounded at the anterior end, owing to defects in terminal filament formation. The ovarioles contain normal-looking follicles and mature oocytes. (2) Female flies with an intermediate bab mutant ovary phenotype are semi-sterile to sterile. The ovaries have a very irregular shape and are substantially smaller than wild-type ovaries. They contain a reduced number of ovarioles that are abnormally oriented with the germaria often not located at the anterior end but inside the ovary. (3) Females with a strong bab mutant ovary phenotype do not lay eggs. The ovaries are very small and contain only one to two ovarioles of very abnormal structure and orientation. Only very few and defective follicles are found in these ovarioles. (4) In bab null mutants, the ovaries are even smaller and no developing follicles have been observed (Couderc, 2002).

The bab mutant leg phenotype that involves all three leg pairs in both females and males has two characteristics: (1) a fusion of tarsal segments, characterized by a shortening of tarsal segments and a loss of tarsal joints; and (2) a transformation of the bristle pattern of distal tarsal segments toward the bristle pattern of the first tarsal segment. Most sensitive to a fusion are tarsal segments TS5 and TS4. The stronger the bab mutation, the further proximal the fusion extends. In bab null mutants, TS5 to TS2 are frequently fused into a single segment. Sensitivity to a transformation of the bristle pattern of tarsal segments decreases from proximal to distal, involving only TS2 in weak bab mutants and TS2-4 in strong bab mutants. This can best be seen using the prominent sex comb bristles of the prothoracic legs of males as a marker, and the transverse bristle rows of the pro- and meta-thoracic legs of both sexes. In a bab null mutant, the bristle pattern of TS2-4 is transformed; however, the sex combs are often eliminated, owing to the shortening and fusing of the tarsal segments. A thickening of the distal tarsal segments seen in bab mutant legs is an additional indication that the distal tarsal segments are transformed towards the identity of the first tarsal segment, which in wild type is much thicker than the distal tarsal segments (Couderc, 2002).

Wild-type females have eight tergites formed by abdominal segments A1-A8, whereas wild-type males have seven tergites corresponding to A1-A6 + A9. In females, the tergites of A1-A6 each show a darkly pigmented posterior and lightly pigmented anterior band. The two tergite plates of A7 are variably pigmented and A8 has a light coloration. The tergites of A1-A4 in males are similarly pigmented as in females, whereas the tergites of A5-A6 + A9 are darkly pigmented throughout. bab null mutants display a change in the pigmentation pattern of both sexes. A thorough dark pigmentation is found in A3-A8/A9 and is seen with low penetrance also in A2. Ectopic dark pigmentation in A2 and A3 is usually patchy and restricted to the anterior margin. The phenotypic series of bab mutants shows that the sensitivity towards a change in pigmentation decreases from posterior to anterior, with A6 being more sensitive than A5, and A5 more sensitive than more anterior segments. In weak bab mutants, a change in pigmentation is therefore seen only in females. In summary, this indicates that loss of bab function leads to a transformation of the pigmentation from a female to a male-like pattern as well as from an anterior to a posterior-like pattern. The bab locus regulates the pattern and amount of pigmentation in all abdominal segments (except for A1), and suppresses dark pigmentation in the anterior region of abdominal segments in both sexes with the exception of A5 + A6 in males (Couderc, 2002).

Except for a change in the pigmentation pattern, the morphology of A2-A5 appears to be normal in both sexes of bab null mutants. However, the posterior segments A6-A8 show additional morphological abnormalities, most of which are restricted to females. The trichome pattern of A6 is affected in both sexes. In bab null mutants, trichomes are not restricted to the anterior and lateral margin of the A6 tergite as in wild type, but are found at a low density throughout the tergite, similar to the normal trichome pattern of A5. This suggests a posterior-to-anterior transformation of the trichome pattern. Furthermore, the A6 tergite of bab mutant females is broader (anteroposterior) than the more anterior tergites in contrast to wild type, which together with the heavy pigmentation gives this tergite a male-like appearance (Couderc, 2002).

In contrast to A1-A6, in which the two primordia of each tergite fuse into a single plate, the A7 tergite consists of two loosely connected triangular plates in wild-type females that show small slightly twisted bristles and often two to three larger bristles. In bab null mutants, the A7 plates are fused into a continuous plate, are considerably broader (anteroposterior) than in wild type, and display an increased number of large bristles. These morphological changes suggest a transformation of A7 towards a more anterior segment fate. Furthermore, instead of the small pale bristles, which are characteristic of an A8 tergite in wild-type females, larger pigmented and slightly twisted bristles are found in a bab mutant A8 tergite. Such bristles are similar to those normally found in A7 of females or in A9 of males, again suggesting homeotic transformations. In addition, the two rows of thorn bristles seen on the vaginal plates of wild-type females are replaced in bab mutants by a different type of bristle which is longer and twisted (Couderc, 2002).

bab mutants also display defects in the sternites of the abdominal segments. Shape, pigmentation, and bristle pattern of the A6 and A7 sternites in females are different from wild type and show similarities to the A6 sternite in males. Both sternites are more strongly pigmented, and the number of bristles is considerably decreased compared with wild type. Taken together, the alterations in the shape and the bristle and trichome patterns of posterior segments indicate that loss of bab function causes posterior-to-anterior transformations of some abdominal features (opposite to the change of the pigmentation pattern), and also transformations from a female to a male-like morphology (Couderc, 2002).

Ubiquitous overexpression of a UAS-bab2 transgene under control of Hsp70-Gal4 causes reduced viability, a general reduction in the pigmentation of the cuticle and bristles, and defective macrochaetae when flies are raised at a constant temperature of 25°C. In the abdomen of either sex, the posterior dark pigmentation of A6 is reduced or missing, and little of the dark pigmentation is left in A5 and A4. Tergites anterior to A4 are less affected than the posterior tergites. In both bab loss- and gain-of-function experiments, pigmentation in posterior segments is more strongly affected than in anterior segments, indicating a graded requirement for bab along the anteroposterior axis. Similar phenotypic effects were observed in bab1 overexpression experiments. Together, loss- and gain-of-function studies show that the bab locus is a suppressor of dark cuticle pigmentation in the fly (Couderc, 2002).

When flies carrying UAS-bab2 under the control of Hsp70-Gal4 were shifted to 32°C during the late 3rd instar/pupal stages, they showed a 'split-tergite'-phenotype in addition to the loss of pigmentation. Here, the tergite primordia of all abdominal segments do not fuse, a trait normally only found in A7. The split-tergite phenotype is also seen when UAS-bab2 expression is driven by a bab1-Gal4 transgene, and is therefore likely not an artifact of the heat shock but a consequence of bab2 overexpression. These data suggest that bab plays a role in tergite morphogenesis and is required to prevent a fusion of the A7 tergite primordia (Couderc, 2002).

To analyze the relative functions of bab1 and bab2, and to look for possible interactions, the phenotypic effects of bab mutations were compared in ovaries and legs, and their complementation behavior was studied. This study involved mutations that affect bab1 (babP and babA128), bab2 (babE1, babE4, and babE5), bab1 and bab2 (babPRDS and babPR72), or mutations null for bab1 and bab2 (babAR07 and deletions of the bab locus), and some molecularly uncharacterized bab alleles (Couderc, 2002).

All five EMS alleles (babE series of alleles) were isolated based on dominant leg defects. babE1 is the strongest EMS allele, causing a strong recessive phenotype in ovaries and legs; babE4 and babE5 produce an intermediate, and babE6 and babE3 a weak recessive phenotype in those organs. The phenotype seen in flies transheterozygous for any two of the EMS alleles is intermediate in strength to the phenotypes displayed by the homozygotes, indicating that the phenotypic effect of the EMS alleles is additive. The EMS alleles in trans to the strongest allele babE1 produce a mutant phenotypic series that is comparable with, but less severe than, each EMS allele in trans to a deletion of the bab locus. Since babE3 and babE6 do not complement babE1 but complement babP they should represent bab2 mutations like the other EMS alleles. Therefore, all bab alleles isolated on the basis of a dominant mutant leg phenotype are mutations in bab2 (Couderc, 2002).

All bab alleles that were isolated as excision derivatives of babP (the babPR alleles) show non-complementation in trans to each other or in trans to a deletion of the bab locus, and display a normal phenotypic series. The babPR alleles that cause a strong mutant phenotype, such as babPR72 and babPRDS, not only reduce the expression level of bab1, but also of bab2. Mutations in bab that affect bab1 but have no detectable effect on the expression of bab2, such as babP and babA128, have a considerably weaker mutant phenotype. No bab mutation has been identified that affects only bab1 and causes strong mutant defects in ovaries and/or legs. Therefore, it is proposed that bab2 plays a predominant role in exerting bab function in ovarian and particularly in leg development (Couderc, 2002).

Effects of bab1 and bab2 mutations on abdominal pigmentation were compared. Mutations in either bab gene cause dominant and more pronounced recessive pigmentation defects. Females homozygous for the strong bab2 allele babE1 display a uniformly dark pigmentation of tergites in A5 and A6, and females homozygous for the intermediate allele babE5 show uniformly dark pigmentation in A6 and partial ectopic pigmentation in A5. By contrast, bab1 mutant females that are homozygous for babP or babA128 show ectopic dark pigmentation in the tergites of A6 and A7. These observations suggest that there is an overlapping and differential requirement for bab1 and bab2 in abdominal segments (Couderc, 2002).

To gain a better understanding of the relationship of the two bab genes, mutations in bab1 and bab2 were tested for complementation. Partial non-complementation is observed between bab1 and bab2 alleles in ovaries and legs. (1) Flies carrying a bab1 and a bab2 mutation in trans display a mutant ovary phenotype, although it is weaker than the one observed in flies homozygous for either the bab1 or the bab2 mutation. This may be caused by an interaction of these alleles with the wild-type copy of bab1 and/or bab2, since flies heterozygous for a deletion of the bab locus do not have ovary defects. (2) Although bab1 mutations produce neither dominant nor recessive leg defects in a background that is wild-type for bab2, flies carrying a bab1 mutation in trans to a bab2 mutation show leg defects that are stronger than the dominant leg defects caused by the bab2 mutation in trans to a wild-type chromosome. This suggests that bab1 is functionally active although not essential in leg development. These complementation data point to functional dependency between mutations in bab1 and bab2, the nature of which remains to be explored (Couderc, 2002).

In conclusion, the two bab genes seem partially redundant since the strongest developmental defects in ovaries, legs and the abdomen associated with the bab locus have been observed only in mutants that are null for bab1 and bab2, and that both bab genes are required for normal bab function. The two bab genes are not functionally equivalent, however. (1) There is an overlapping but also differential requirement for bab1 and bab2 in the pigmentation of different abdominal segments, with A7 being more dependent on bab1 and A5 on bab2 activity. (2) Ovarian defects are seen with mutations affecting either bab1 or bab2, but loss-of-function of bab2 causes a more severe phenotype. Since the function of the bab locus is strongly dose/concentration dependent, the predominance of bab2 in regulating ovarian development may be a result of the higher expression level of bab2. Furthermore, the differences in the ovarian expression patterns may have functional significance. Cis-regulatory differences, however, cannot sufficiently explain the differential requirement of bab1 and bab2 in leg development. Although both genes are similarly expressed in leg imaginal discs, a bab1 knockout does not cause a mutant leg phenotype, whereas even weak bab2 mutants display dominant leg defects. This indicates that only bab2 plays an essential role in leg development and suggests a qualitative divergence in the function of Bab1 and Bab2 proteins. Taken together, it is proposed that bab1 and bab2 have not only developed differences in transcriptional regulation but also differences in protein function that could be responsible for changes in the interaction with other transcription factors and/or DNA-binding sites (Couderc, 2002).

bab acts as a homeotic regulator, since bab mutations cause homeotic transformations in the legs and the abdomen. Here, it has been shown that the homeotic transformations in the abdomen of bab mutants are complex. bab loss-of-function mutants display a combination of anterior-to-posterior transformations (pigmentation), posterior-to-anterior transformations (bristles, trichomes and segment shape and size), and female to male transformations (pigmentation, bristles and segment shape and size). bab seems to be mainly required in the posterior segments A5-A8. This domain that is mostly controlled by the Hox gene Abdominal-B (Abd-B), whose loss-of-function causes posterior to anterior transformations of segment identity. It has been demonstrated that bab expression is repressed by Abd-B, either directly or indirectly, in posterior abdominal segments at the late pupal stage. Since bab acts as a suppressor of pigmentation, the repression of bab expression by Abd-B function leads to the complete pigmentation of the A5 and A6 tergites in wild-type males. In females, the repression of bab by Abd-B is counteracted by the female specific doublesex (dsxF) gene product. It is unlikely, however, that Abd-B is a general repressor of bab activity, since bab mutants show not only anterior-to-posterior but also posterior-to-anterior transformations in the abdomen. This indicates that the regulation of bab activity is complex. Abd-B in conjunction with co-regulators might repress or activate bab function, dependent on the cell type and on the developmental time at which specific morphological features are specified. It is proposed that the differential, fine-tuned spatial and temporal regulation of bab expression plays a crucial role in providing morphological diversity between the abdominal segments along the anteroposterior axis and between the sexes. Similar to the abdomen, bab plays a role in the generation of morphological diversity between distal segments in the leg. bab is part of a network of transcription factors that divide the proximodistal axis into successively smaller domains, leading to the formation and specification of the different leg segments (Couderc, 2002).

bab also plays a role as a morphogenetic regulator of development. Previous studies have indicated that bab controls cell rearrangements during terminal filament formation in the ovary. bab is also required for the proper folding of leg imaginal discs, which may be important for tarsus segmentation. Furthermore, bab negatively regulates the fusion of the tergite primordia in the abdomen, a process that is also controlled by the Hox genes. This suggests that the Bab transcription factors control the morphogenetic behavior of cells in different developmental processes. It will be a future challenge to determine whether bab directly regulates expression of proteins that mediate cell shape changes and cell movements (Couderc, 2002).

Flies of the Drosophila family show substantial intraspecific and interspecific variation in sex-related traits, including sex combs and abdominal pigmentation, as well as male genital structures and number of ovarioles. Variation in these traits can affect mate choice and fertility, and thus reproductive success. Furthermore, there is evidence that divergence of phenotypic traits related to reproduction in combination with ecologically adaptive divergence in sexual selection can lead to reproductive isolation and speciation. Interestingly, bab controls the morphology of several traits that are involved in reproduction and that show rapid evolutionary divergence. bab regulates the formation of the reproductive organ in females, since bab is required for terminal filament formation and consequently for the development of ovarioles in the ovary. bab mutations of increasing strength cause a decrease in the number of ovarioles, raising the possibility that bab might be involved in determining ovariole number in Dm. Moreover, bab controls several secondary sexual traits. bab activity suppresses sex combs on tarsal segments distal to TS1. bab may also be involved in determining the number of sex comb bristles in TS1, since overexpression of Bab2 in TS1 causes a reduction in the number of sex comb bristles compared with wild type. Furthermore, bab regulates sexually dimorphic bristle and trichome patterns and the pigmentation of posterior abdominal segments. A comparison of abdominal pigmentation and bab expression pattern between the two sexes of different members of the Drosophila species group demonstrates a striking correlation between phenotypic differences and bab expression patterns, suggesting a causal relationship. Since bab loss- and gain-of-function mutations have pleiotropic effects on the development of reproduction-related characteristics, evolutionary alterations in bab function could lead to a diversification of multiple sex traits (Couderc, 2002).

The bab locus appears to have two important properties that make it suitable to cause variation in the development of morphological traits. (1) Because the bab locus represents a tandem duplication, redundancy between bab1 and bab2 may have facilitated fast molecular modifications, resulting in the observed alterations of the expression level and pattern of bab1 and bab2 and their functional diversification. One potential consequence, for example, would be that abdominal pigmentation could change independent of the leg pattern through mutations in bab1, since this gene is no longer essential for leg development. (2) bab function is highly dose dependent. bab is haploinsufficient, and bab mutations cause dominant homeotic transformations of adult characteristics that do not interfere with viability in laboratory cultures. The expression profile of bab in imaginal discs and the abdomen is graded, and differences in bab concentration determine morphology of legs and abdomina. Since concentration matters, small variations in the expression level or shape of the bab gradient could lead to morphological diversification. Taken together, the data suggest bab is an important regulator of reproduction-related characteristics in Dm, and therefore may play an active role in the variation, divergence and speciation in the genus Drosophila (Couderc, 2002).

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation

Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. This study greatly expands this network through an RNAi-screen of 558 transcription factors. Twenty-eight genes were identified, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. Many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. Pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution (Rogers, 2014).


Search PubMed for articles about Drosophila bric à brac 1

Camino, E. M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M. and Williams, T. M. (2015). The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans. PLoS Genet 11: e1005136. PubMed ID: 25835988

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Biological Overview

date revised: 21 November 2016

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