InteractiveFly: GeneBrief

Forkhead box P: Biological Overview | References

BIOLOGICAL OVERVIEW

FoxP2 is a highly conserved vertebrate transcription factor known for its importance in human speech and language production. Disruption of FoxP2 in several vertebrate models indicates a conserved functional role for this gene in both sound production and motor coordination. Although FoxP2 is known to be strongly expressed in brain regions important for motor coordination, little is known about FoxP2's role in the nervous system. The recent discovery of the well-conserved Drosophila melanogaster homolog, FoxP, provides an opportunity to study the role of this crucial gene in an invertebrate model. It was hypothesized that, like FoxP2, Drosophila FoxP is important for behaviors requiring fine motor coordination. This study used targeted RNA interference to reduce expression of FoxP and assay the effects on a variety of adult behaviors. Male flies with reduced FoxP expression exhibit decreased levels of courtship behavior, altered pulse-song structure, and sex-specific motor impairments in walking and flight. Acute disruption of synaptic activity in FoxP expressing neurons using a temperature-sensitive shibire allele dramatically impaired motor coordination. Utilizing a GFP reporter to visualize FoxP in the fly brain reveals expression in relatively few neurons in distributed clusters within the larval and adult CNS, including distinct labeling of the adult protocerebral bridge - a section of the insect central complex known to be important for motor coordination and thought to be homologous to areas of the vertebrate basal ganglia. The results establish the necessity of this gene in motor coordination in an invertebrate model and suggest a functional homology with vertebrate FoxP2 (Lawton, 2014).

FoxP2 is a vertebrate transcription factor best known for its importance in speech and language production in humans. Its role in human behavior was originally discovered in a multigenerational family whose affected members have a severe speech and language disorder throughout life, and underlying this deficit is a single point mutation in the DNA binding domain of FOXP2 (Lai, 2001). Since this discovery, independent mutations and truncations of FoxP2 have been linked to disorders with specific impairment in production of fluent speech. Across vertebrate models, FoxP2 is remarkably well conserved, both in amino acid sequence and brain expression patterns (Lawton, 2014).

FoxP2 effects on vocal production are not unique to humans. As a parallel to learned human speech, knockdown of FoxP2 in male zebra finch chicks during the critical song learning period significantly alters the structure of their crystallized adult song (Haesler, 2007). This result closely resembles impairments seen in humans, indicating that FoxP2 may play a conserved functional role in vocal production. In mice, a variety of FoxP2 mutations and deletions have demonstrated effects on development and behavior. FoxP2 null mice are developmentally delayed and die within 3 weeks of birth, indicating a crucial role of FoxP2 in early postnatal life (Groszer, 2008; Fujita, 2008; Shu, 2005). In contrast, mice heterozygous for functional FoxP2 were developmentally normal but exhibited a variety of other deficits, such as a reduction in the amplitude of ultrasonic vocalizations, abnormal synaptic plasticity, and deficits in motor skill learning. From this variety of work in vertebrates, it is suggested that FoxP2 plays a role in fine motor control, which may have provided a neural substrate for development of complex vocalizations such as language. Despite these insights into the potentially conserved role of FoxP2 in sound production and fine motor control, the precise function of this gene remains poorly understood (Lawton, 2014).

A gene in the fruit fly Drosophila melanogaster has been identified as a closely related homolog to the vertebrate FoxP subfamily (Santos, 2011). This Drosophila FoxP is highly similar in sequence to the vertebrate FoxP2, and is highly expressed in the nervous system (Lee, 2004). The discovery of this invertebrate homolog in a genetically tractable organism such as the fly provides new possibilities for functional analysis and understanding of the evolutionary importance of the FoxP2 gene. Sound production as a means of social communication is crucial to many species of invertebrates. Like many insects, male fruit flies produce an acoustic signal in the form of a courtship song. In the presence of a virgin female, a male will initiate a sequence of courtship behaviors including a unilateral wing vibration to produce a pulse song with a precise species-specific inter-pulse-interval (IPI). Both the courtship sequence and pulse song are highly stereotyped and easily quantifiable. Based on evidence in vertebrates, it was hypothesized that the Drosophila FoxP gene is also important for courtship song production and fine motor control in the fly. Therefore attempts were made to characterize the behavioral role and expression pattern of this gene in fruit flies in order to better understand the development of fine motor circuits in insects and ultimately identify the potentially conserved developmental and molecular roles of FoxP2 across organisms (Lawton, 2014).

Using RNA interference (RNAi) to knockdown FoxP levels, the behavioral effects of reduced FoxP were studied in the context of courtship, locomotion, and flight behaviors. Deficits were found in all of these behaviors in adults, with males more strongly affected than females in these assays. In parallel, a FoxP antibody and a FoxP-Gal4 line were generated. FoxP-Gal3 combined with two different UAS-GFP lines allowed for visualization of the expression pattern of FoxP in the larval and adult CNS. These molecular tools revealed that FoxP is limited to a relatively small subset of neurons in the brain and ventral ganglion, which appear in several distinct clusters throughout. Particularly strong expression was evident in the protocerebral bridge, part of the central complex, which is thought to be involved in sensory-motor integration, and has been compared to the vertebrate basal ganglia. When the FoxP-Gal4 was used with a conditional temperature sensitive UAS-shibire line to transiently disrupt neurotransmission in FoxP expressing neurons in adults, dramatic effects were observed on motor coordination. The results provide the first functional characterization of FoxP2 in invertebrates and suggest an intriguing homology with this crucial human speech and language gene (Lawton, 2014).

Successful partial knockdown of FoxP using RNAi in flies mimics heterozygous mutations in humans and mice, as well as RNAi used in zebra finch. In this study, no gross developmental abnormalities were apparent in knockdown flies. These flies, as in vertebrates with partial FoxP2 deficiency, show specific motor deficits without any obvious external morphological aberrations. With the stronger 29°C knockdown of Act5c > RNAi pupal lethality was seen in males -- this is consistent with results in homozygous null mutant FoxP2^-/- mice, which have severe developmental defects and die around 3 weeks of age. FoxP may be playing an essential role in neural development and it is possible that FoxP plays distinct roles in various tissues at different time points in development, as this is a common trait of transcription factors. The crucial timing of FoxP action will be an important question to address in future studies (Lawton, 2014).

Given the role of FoxP2 in human speech, zebra finch song learning, and possibly mouse pup cries, it was predicted that FoxP would also play a role in sound production in insects. The current results indicate that this is indeed the case, although given the other motor impairments, the effects on courtship and song are likely related to motor coordination impairments, rather than a specific disruption of the song circuitry. This is supported by previously reported results in mice, where effects on pup cries were one of many deficits, including motor skill learning and synaptic plasticity. Partially reduced FoxP does not completely eliminate song production in the majority of flies, but does change song structure, which may also indicate disrupted fine motor control rather than a more general inability to produce or maintain movement. Strongly convincing support for this idea comes from the dramatic effects on walking using the FoxP > Shi^ts1 flies. These results demonstrate that FoxP expressing neurons are crucial for maintaining proper motor coordination, but without abolishing movement completely. This result is especially notable for being an acute disruption of FoxP neurons, rather than a chronic disruption throughout development using the RNAi knockdown. This reduces the likelihood of compensatory mechanisms and off target effects. The importance of FoxP expression in adulthood was examined using a temperature sensitive Gal80^ts line in combination with UAS-GAL4 to allow RNAi mediated FoxP knockdown only in adults. These adult-only FoxP knockdown animals were then tested both for courtship index and activity level. In both assays no difference was seen in behavior, indicating the likelihood that FoxP is playing a role predominately in development (Lawton, 2014).

Unexpectedly, it was observed that impairment in the majority of motor tasks was more severe in males than females. In both the walking and flight assays, males were strongly affected, with only a slight but significant impairment in Act5c > RNAi females. Elav > RNAi females exhibit no significant difference in the flight assay, and only a slight decrease in walking activity level. This is likely because the neural elav-GAL4 is a weaker driver than Act5c. The possibility cannot be ruled out of a greater sensitivity to the RNAi in males, but it may be that FoxP itself plays different roles in the two sexes. While the majority of vertebrate FoxP2 studies have not addressed sex specific behavioral differences, a few studies have found differences in expression level of FoxP2 in rodents and humans, as well as a behavioral difference in pup calls between the sexes. Although no obvious difference was apparent when visually comparing FoxP expression patterns in male and female Drosophila brains, greater FoxP expression intensity was consistently seen in female brains. This may explain the more severe behavioral effects on males given that they have less FoxP and possibly a greater sensitivity to the RNAi. This would also make sense as to why no sex differences were seen in the adult shibirets experiments. Alternatively, the sex difference could be due to as yet unknown gender specific splice variants, as is the case with the well-studied Drosophila Fruitless transcription factor. It is already known that normal locomotion in flies is sexually dimorphic, which is not unexpected given their many non-overlapping behaviors such as male courtship and fighting and female egg laying. These sex differences raise the intriguing question of whether any gender differences might be observed in future FoxP2 studies in vertebrates (Lawton, 2014).

As in vertebrates, FoxP is strongly expressed in a variety of regions in the brain. Although the majority of cells co-express anti-FoxP and the FoxP-Gal4 driving GFP, a small discrepancy was seen in staining pattern between the two molecular tools used in this study. This may be due to differential stability of FoxP and GFP in these neurons, greater sensitivity of the anti-GFP antibody as compared to the FoxP antibody, or that the promoter that was used in the GAL4 construct does not fully accurately reproduce the FoxP expression pattern. Overall the staining pattern closely matches, which indicates that FoxP cells were targeted specifically, but there is a possibility that the non-overlapping cells are involved in the dramatic behavioral phenotypes observed in the shibire^ts experiments. Given other data indicating coordination impairments when not using the FoxP-Gal4 line, it is suspected that this is not the case, but the discrepancy must be kept in mind in future interpretations. Further studies dissecting the effects of FoxP in different regions of the brain may be able to resolve this concern (Lawton, 2014).

In both larvae and adult a relatively small number of neurons expressing FoxP were seen, but these were distributed throughout a wide area of the CNS. This is similar to the expression pattern seen with in situ hybridization in the honeybee Apis mellifera (Kiya, 2008). In adults some of these neurons are organized in distinct clusters, suggesting that FoxP may have direct behavioral effects though a specific identifiable motor-related network. Expression does not appear to overlap with the well-studied mushroom bodies, known to be involved in learning and memory, which was also consistent with the honeybee staining (Lawton, 2014).

Notably, FoxP appears to be strongly expressed in projections within the distinctive protocerebral bridge (PB) structure, which is part of the central complex (CX). FoxP-CD8::GFP flies show distinct external bilateral clusters that are potentially projecting into the PB. This is similar to recent findings of late-born external projecting neural lineages into the PB. FoxP may be responsible for development of specific subsets of these neurons. A recently constructed wiring diagram of the PB as well as establishment of the developmental origins of CX neurons, may allow for future studies to determine the exact FoxP neurons which contribute to their PB expression. The function of the PB itself has not been extensively studied, although the CX as a whole is thought to be involved in higher locomotion control and has been implicated in courtship song production in both Drosophila and grasshoppers. Some work has specifically examined the role of the PB using mutants with structural defects specific to the bridge -- early studies with the no-bridge mutant indicate deficits in learning, walking speed, and courtship. A more recent study implicated this structure in sensorimotor integration as demonstrated in a gap crossing assay. Two types of bridge mutants showed deficits in correct aiming when attempting gap crossing (Triphan, 2010). Although some of these deficits are consistent with the current results, it is not possible to know at this point if the behavioral deficits seen in FoxP knockdown animals are due to disturbed activity in the PB or in one of the other unidentified FoxP expressing neuronal clusters, but it is an intriguing possibility that the role played by FoxP in motor coordination may be related to its expression in this part of the CX. Importantly, the CX has been compared to the vertebrate basal ganglia and the PB to the striatum, which in vertebrates is known to be important for motor coordination and shows strong FoxP2 expression. This work further adds to the idea of a deep homology in brain structure and function of the insect CX and vertebrate basal ganglia (Lawton, 2014).

This work establishes the behavioral effects of FoxP knockdown in an invertebrate with functional parallels to the vertebrate FoxP2. Further work addressing the timing of FoxP action, which neuron types express FoxP, and the mechanisms of action of this gene in the nervous system, may all contribute to understanding the importance of this gene in Drosophila brain development, as well as provide valuable insight into the evolutionarily conserved functions of the FoxP homologs across invertebrates and vertebrates (Lawton, 2014).

FoxP influences the speed and accuracy of a perceptual decision in Drosophila

Decisions take time if information gradually accumulates to a response threshold, but the neural mechanisms of integration and thresholding are unknown. This study characterized a decision process in Drosophila that bears the behavioral signature of evidence accumulation. As stimulus contrast in trained odor discriminations decreased, reaction times increased and perceptual accuracy declined, in quantitative agreement with a drift-diffusion model. FoxP mutants took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult, low-contrast tasks. RNA interference with FoxP expression in αβ core Kenyon cells, or the overexpression of a potassium conductance in these neurons, recapitulated the FoxP mutant phenotype. A mushroom body subdomain whose development or function require the transcription factor FoxP thus supports the progression of a decision toward commitment (DasGupta, 2014).

Decisions take time if information gradually accumulates to a response threshold, but the neural mechanisms of integration and thresholding are unknown. This study characterized a decision process in Drosophila that bears the behavioral signature of evidence accumulation. As stimulus contrast in trained odor discriminations decreased, reaction times increased and perceptual accuracy declined, in quantitative agreement with a drift-diffusion model. FoxP mutants took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult, low-contrast tasks. RNA interference with FoxP expression in αβ core Kenyon cells, or the overexpression of a potassium conductance in these neurons, recapitulated the FoxP mutant phenotype. A mushroom body subdomain whose development or function require the transcription factor FoxP thus supports the progression of a decision toward commitment (DasGupta, 2014).

Integrator models of perceptual decision-making predict that reaction times will vary with the quality of sensory information: Easy decisions, based on clear evidence, will be fast; difficult decisions, based on uncertain evidence, will be slow. This prediction was tested in fruit flies, using a reaction time version of an olfactory discrimination task. Flies were analyzed individually in narrow chambers, which were perfused with odor-air mixtures whose convergence defined a 7-mm-wide decision zone. With odors present, the flies slowed upon entry into the decision zone, paused near the interface, and exited after committing to a choice. The time between entry and exit was quantitated as the reaction time (DasGupta, 2014).

Flies were trained to avoid a specific concentration of 4-methylcyclohexanol (MCH) and had to distinguish the reinforced concentration from a lower concentration of the same odor. The difficulty of discrimination was titrated by varying the MCH concentration ratio during testing. At a reinforced MCH intensity of ~12 parts per billion, corresponding to 10^-4 volumes of saturated vapor per volume of air, flies achieved accuracies of nearly 100% at large concentration differences (concentration ratio of comparison to reinforced odor of 0.1 to 0.2) but performed randomly when the odor concentrations differed by only 10% (concentration ratio 0.9). Reaction times increased as a function of difficulty, suggesting that the flies compensated for low stimulus contrast in difficult tasks by gathering information for longer. Because overall odor concentrations were highest in the low-contrast tasks that took the longest to complete and because only relative stimulus contrast, not absolute stimulus intensity, affected reaction times, the data favor integration over probability summation: If the probability of odor detection were rate-limiting, reaction time would be expected to correlate inversely with stimulus intensity (DasGupta, 2014). A drift-diffusion model of evidence integration captured the empirical relationship between difficulty and performance. Drift-diffusion models decompose reaction times into decision and residual times. The residual time encompasses sensory and motor latencies, procrastination, and time required to indicate a choice. The decision time constitutes the integration period per se. Its duration depends on the mean rate at which evidence accrues (the drift rate) and the level of the decision criterion (the bound height). Intuitively, the weaker the sensory evidence, the lower will be the drift rate, the longer the response time, and the poorer the decision accuracy. The model that was used formalizes this intuition by scaling the drift rate in proportion to stimulus contrast, which was quantified as |log (odor concentration ratio)|. Estimation of the three free model parameters (drift rate, bound height, and residual time) from reaction time measurements generates a prediction of the corresponding decision accuracies. The modeled chronometric and psychometric functions provided satisfying simultaneous fits to the performance data (DasGupta, 2014).

At all difficulty levels, the reaction time distributions exhibited positive skew, a characteristic of information accumulation to threshold. The drift-diffusion model explains the origin of the asymmetry: Equal differences in drift rate generate unequal differences in reaction time at the intersection with the response bound. In contrast, transit times of nondecision zones located off center had nearly symmetrical distributions that did not vary with task difficulty (DasGupta, 2014).

To examine whether the relationship between task difficulty and reaction time generalized, two tasks were designed other than intensity discrimination. In a masking odor task, shock-reinforced MCH was presented in a ubiquitous background of 3-octanol (OCT). Difficulty was adjusted by varying the level of masking odor while keeping constant the concentration of cue. Flies took longer to respond to cues hidden in a high level of background than to salient cues and did so with lower accuracy. In binary mixture discriminations, the closer the proportions of MCH and OCT, the lower the accuracy and speed of discrimination (DasGupta, 2014).

A pilot analysis of 41 strains carrying candidate mutations implicated the transcription factor FoxP in the decision process. FoxP^5-SZ-3955 mutants learned to distinguish the shock-reinforced concentration of MCH with the same accuracy as wild-type flies but took longer to decide. The defect was subtle in easy discriminations (concentration ratio 0.1 to 0.4) but glaring in difficult tasks (concentration ratio 0.7 to 0.9) (DasGupta, 2014).

Mutating FoxP might alter any one of several processes that affect performance in this assay: the abilities to learn from shock reinforcement, walk to and from the odor interface, detect olfactory cues, and decide. Learning and locomotor deficits could be ruled out by examining the accuracy scores and transit time distributions, respectively; both were identical in FoxP^5-SZ-3955 mutants and wild-type flies. Mutants detected odors with the same sensitivity as wild-type controls: Diluting all odors 1000-fold had similar effects on either genotype. Where mutant and wild-type flies clearly differed, however, was in the dependence of reaction time on stimulus contrast: In mutants, narrowing the odor concentration difference caused disproportionate increases in reaction time. A drift-diffusion model identified two changes that can account for this phenotype: a 38% drop in drift rate and a - perhaps compensatory - increase in the height of the response bound. The reduction in drift rate suggests that FoxP mutants are impaired in the accumulation and/or retention of sensory information in the buildup to a choice (DasGupta, 2014).

The FoxP mutant phenotype was confirmed with two independently generated alleles. Heterozygous carriers of any one of these alleles performed like wild-type controls in easy discriminations (concentration ratio 0.1) but displayed prolonged reaction times in difficult tasks (concentration ratio 0.7). Homozygous or transheterozygous carriers of two mutant alleles exhibited pronounced difficulty-dependent speed and, in some allelic combinations, also accuracy deficits. The association of similar phenotypes with different mutant alleles, and the lack of complementation between alleles, tie the defect in decision formation firmly to the FoxP locus (DasGupta, 2014).

To identify, label, and manipulate sites of FoxP action in the brain, a FoxP promoter fragment was used to direct the expression of GAL4. FoxP-GAL4-driven transgene expression was confined to two subsets of Kenyon cells (KCs), the principal intrinsic neurons of the mushroom bodies: ~80 KCs whose axons extend into the cores of the α and β lobes, and ~100 KCs innervating the γ lobes. Given their positions as third-order olfactory neurons, the FoxP-GAL4-expressing KCs could transmit sensory data to downstream integrators. Alternatively, the FoxP-GAL4-positive KCs themselves could integrate olfactory signals, or the representations of momentary and accumulated sensory evidence might be entwined within the KC population. Because both representations require externally or recurrently evoked electrical activity α (DasGupta, 2014).

Therefore the inwardly rectifying potassium channel Kir2.1 under FoxP-GAL4 control was targeted to αβ core (αβ_c) and γ KCs while tuning expression levels with temperature-sensitive GAL80^ts. Flies expressing low Kir2.1 levels behaved like homozygous FoxP^5-SZ-3955 mutants: Reaction times increased in a difficulty-dependent manner relative to parental controls, but accuracy was maintained. Boosting the expression of Kir2.1 exacerbated this phenotype: Despite a further increase in reaction times, FoxP>Kir2.1 flies now performed near chance level in difficult discriminations, echoing the accuracy defects of severe FoxP alleles(DasGupta, 2014).

To bolster and refine the identification of FoxP-GAL4-positive KCs as sites of FoxP action, the consequences were compared of introducing Kir2.1 with those of reducing FoxP expression, using a panel of GAL4 lines whose expression domains included all or parts of the FoxP-GAL4 pattern: OK107-GAL4 targets all KCs, NP6024-GAL4 and NP7175-GAL4 label αβ_c neurons, and NP1131-GAL4 marks γ KCs. Knockdown of FoxP in αβ_c, but not γ, KCs prolonged reaction times in difficult discriminations, mirroring the differential impact of Kir2.1 on these neuronal populations. Attempts to disrupt FoxP expression with the help of FoxP-GAL4 itself produced marginal effects, probably due to inadequate FoxP^RNAi levels. Consistent with this interpretation, significant decision phenotypes were seen with all three of the GAL4 drivers capable of expressing high levels of FoxP^RNAi in αβ_c KCs (DasGupta, 2014).

The evolution of a decision toward commitment requires the progression of neural activity from a choice-neutral to a choice-specific state. Mutations in FoxP evidently slow this progression, at least in part by interfering with the function of αβ_c neurons. The same neurons have been implicated in value-based decisions, such as choices between odors associated with punishments of differing severity. It remains untested whether value judgments also incur a difficulty-dependent cost of decision time. Nonetheless, the available evidence suggests that ~80 FoxP-GAL4-positive αβ_c KCs form part of a versatile decision circuit that processes sensory information in one context and remembered value in another (DasGupta, 2014).

As a transcription factor, FoxP could act during development to specify synaptic connections and/or throughout life to regulate neuronal function. Vertebrate FoxP homologs have been linked to both types of processes and have been attributed critical roles in cognitive development, vocal communication, and motor control. A potential commonality between these processes and decision-making is their unfolding over time: Neurons representing trains of thought, strings of syllables, chains of motor commands, or accumulating evidence must all step through ordered activity sequences. It is therefore tempting to speculate that understanding the function of an ancestral FoxP gene might reveal fundamentals of temporal processing (DasGupta, 2014).

Drosophila FoxP mutants are sufficient in operant self-learning

Intact function of the Forkhead Box P2 (FOXP2) gene is necessary for normal development of speech and language. This important role has recently been extended, first to other forms of vocal learning in animals and then also to other forms of motor learning. The homology in structure and in function among the FoxP gene members raises the possibility that the ancestral FoxP gene may have evolved as a crucial component of the neural circuitry mediating motor learning. This study reports that genetic manipulations of the single Drosophila orthologue, dFoxP, disrupt operant self-learning, a form of motor learning sharing several conceptually analogous features with language acquisition. Structural alterations of the dFoxP locus uncovered the role of dFoxP in operant self-learning and habit formation, as well as the dispensability of dFoxP for operant world-learning, in which no motor learning occurs. These manipulations also led to subtle alterations in the brain anatomy, including a reduced volume of the optic glomeruli. RNAi-mediated interference with dFoxP expression levels copied the behavioral phenotype of the mutant flies, even in the absence of mRNA degradation. These results provide evidence that motor learning and language acquisition share a common ancestral trait still present in extant invertebrates, manifest in operant self-learning. This 'deep' homology probably traces back to before the split between vertebrate and invertebrate animals (Mendoza, 2014).

This study sought to discover whether manipulations of the Drosophila FoxP gene could affect learning with reference to its own body, 'self-learning', more than other forms of operant learning. In the operant self-learning paradigm that was used, the tethered fly's attempts to turn are measured by a torque meter and divided into two domains, roughly corresponding to 'left' and 'right' turns, respectively. Fixed in space in a featureless environment, the fly alternates between turning directions in a highly variable, random-like fashion. Making punishment by an infrared heat beam contingent on one set of maneuvers (e.g., right turning attempts), leads to a reduction in the variability of the behavior as the fly restricts its turning maneuvers to the unpunished side (i.e., left turning attempts). This reduction outlasts the application of the heat beam after a given training period and the flies restrict their yaw torque to the previously unpunished side, even when the heat is permanently switched off (Mendoza, 2014).

Three insertions were selected within the dFoxP locus. Prior to testing, all three isoforms had been introgressed into the Canton S wild type strain, homogenizing the genetic background of all four lines. Two of the lines (FoxP^f03746 and FoxP^c03619) did not express any detectable level of the novel intron-retention isoform mRNA. These two lines also showed a significant impairment in flight initiation and maintenance, suggesting that this isoform may be involved in flight performance. A recent study supports the notion of a broader function of the different dFoxP isoforms in motor control above and beyond operant self-learning. Coincidentally, this study and the current study also serve to suggest that the dFoxP-dependent phenotype described in another, yet more recent report may potentially also be due to motor issues, in contrast to the assertions of those authors. The operant self-learning phenotype of FoxP³⁹⁵⁵ mutant flies was uncovered in the heterozygous state over a deficiency spanning the dFoxP locus, solidly tying the phenotype to this dFoxP manipulation (Mendoza, 2014).

Line FoxP³⁹⁵⁵ expressed a mutated isoform B mRNA, such that the putative isoform B protein was truncated and the C-terminal amino acid altered, potentially affecting protein function. There did not appear to be any change in the regulation of isoform B expression in this line as the expression levels were similar to the CS control strain on the mRNA level. This structural modification with its specific effect on operant self- but not world-learning is a reminder that many specific behavioral mutants are often due to structural mutations, commonly affecting only a subset of a gene's isoforms. Targeting isoform B with a specific RNAi construct in order to probe for regulatory effects of dFoxP, yielded a phenocopy of the FoxP³⁹⁵⁵ mutant behavioral phenotype, albeit without any detectable knock-down of the mRNA. It cannot be ruled out that the knock-down did take place although at undetectable levels due to the already low levels of isoform B expression. The alternative also remains that hypothetical extra-neuronal expression could have masked any knock-down in the neuronal tissue. Finally, mismatches between the RNAi construct sequence and the target sequence can bias the RNAi process towards posttranscriptional silencing rather than mRNA degradation. Sequencing of the strains used for the RNAi experiments indeed revealed two such polymorphisms leading to mismatches of the target region with the RNAi construct, potentially biasing the RNAi process towards sequestration (Mendoza, 2014).

It is thus plausible that the RNAi method employed here affected the protein, but not the overall mRNA levels of isoform B, explaining the behavioral phenotype. In the review process of this manuscript, an antibody against dFoxP isoform B was described, such that it will now be possible to test this hypothesis. Interestingly, none of the six currently available RNAi lines targeting the dFoxP locus show any detectable knock-down of any of the three isoforms (replicated in two different laboratories, each using a different pan-neuronal driver line), despite one of them also showing a behavioral phenotype in two studies, as well as mRNA knock-down using semi-quantitative PCR. As similar dFoxP mRNA knock-downs have been observed using semi-quantitative PCR which were not confirmed by qPCR, it is possible to cautiously speculate that the knock-down observed by the previous study. may be a false positive as well, with the behavioral phenotypes in both reports potentially explained by polymorphic mismatches in the target region. For these reasons, it can only be tentatively concluded that besides the structural manipulations, also regulatory manipulations of only isoform B expression may affect operant self-learning. The most parsimonious explanation of the common phenotypes after various dFoxP manipulations is that dFoxP is indeed necessary for operant self-learning (Mendoza, 2014).

Volumetric analyses suggest a role of Foxp³⁹⁵⁵ on brain development in Drosophila, analogous to the role FOXP2 plays in vertebrates. Specifically, flies with the mutant FoxP³⁹⁵⁵ allele have smaller optic glomeruli. So far, very little is known about the functional role of optic glomeruli and, thus, of their relevance for operant self-learning. It has been hypothesized that optic glomeruli may form information hubs by virtue of containing the terminals of many projection neurons within the ipsilateral brain hemisphere. Optic glomeruli have been discovered in dipterans and it is not known if other insect orders possess optic glomeruli. Nevertheless, it is interesting to note that a conspicuous cluster of AmFoxP-expressing neurons is located near the optic lobes of the honeybee. Notably, no significant change was observed in the protocerebral bridge, a neuropil in which a recently developed dFoxP-GAL4 driver line was reported to express (Mendoza, 2014).

It deserves to be emphasized, however, that the overall structure of the brains of FoxP³⁹⁵⁵ mutant flies appears completely normal to the human eye. Only numerical analyses are capable of detecting the subtle changes this mutation causes to brain anatomy. More severe alleles of the dFoxP locus have been observed to lead to more severe anatomical defects (Mendoza, 2014).

dFoxP was targeted with genetic manipulations because of the conserved role of the FoxP gene family in vocal learning. These manipulations were each tested in two very similar operant learning paradigms that differ in their conceptual similarity to language acquisition (Mendoza, 2014).

Operant self-learning in Drosophila parallels the operant feedback structure of vocal learning in humans or songbirds in that no other external cues are contingent upon the feedback. This conceptual similarity is now supported by the parallel biological similarity of both vocal and operant self-learning requiring FoxP function. Interestingly, Protein Kinase C (PKC), the only other known molecular component of the self-learning mechanism, has also been implicated in vocal learning in songbirds (Mendoza, 2014).

The technically all but identical operant world-learning task, in contrast, not only differs conceptually from vocal learning - determined by two comparable rates of productive elongation an- but the biological requirements are also different: neither dFoxP nor PKC function is necessary. Recent experiments reporting unaffected Pavlovian conditioning in FoxP³⁹⁵⁵ mutant flies (DasGupta, 2014) confirm the notion that FoxP is specifically required for self-learning. PKC is not required for other forms of world-learning in flies either, but in other model systems, the data are less straightforward, with varying requirements of varying PKC isoforms for various phases of memory induction and/or maintenance having been reported, depending on preparation, type of training, time of testing, brain region and method of PKC manipulation. There is only one experiment where PKC requirement has been compared between self- and world-learning. This experiment in mice supports the view that PKC-activity in the cerebellum is not required for world-learning but for self-learning, analogous to the requirements in flies. Conversely, the components that are required for operant world-learning in flies are the same that are required for many other forms of learning in many other model systems, such as classical (Pavlovian) conditioning, sensitization or some forms of habituation, e.g., the type 1 adenylyl cyclase, encoded by the fly gene rutabaga, which is dispensable for self-learning (Mendoza, 2014).

Thus, it is postulated that the converging evidence from multiple model systems concerning PKC and FoxP provides first insights into a core set of mechanisms that are specifically required for operant self-learning, and not for other forms of (associative) learning, such as world learning (Mendoza, 2014).

This study found that not only self-learning but also habit formation is impaired in FoxP³⁹⁵⁵ mutant flies, indicating that habit formation may be mediated by the same biochemical processes as operant self-learning, as had been hypothesized before. If wild-type flies are trained in world-learning for eight minutes (i.e., with colors) and then tested for their turning preference in the absence of the colors, there is no preference, demonstrating a hierarchical learning system where external cues are preferentially memorized/retrieved (world-learning) over behavioral cues (self-learning). However, if training in the world-learning situation (i.e., with colors) is extended to 16 minutes and then the same preference test for turning is performed (i.e., without the colors), then a preference for turning towards the previously unpunished direction can be observed. Because self-learning is manifest already after eight minutes of training (i.e., without colors), the presence and learning of the colors during training must have inhibited self-learning, apparently via a neuropil in the fly brain termed mushroom-bodies. Extended training can overcome this inhibition and lead to habit formation. These habits lead to reduced generalization in 'habit interference' experiments when an orthogonal behavior is used to control the colors after extended world-learning. This study shows that flies with a mutated dFoxP gene are impaired in habit formation, i.e., extended operant world-learning leads to a reduced preference for the previously unpunished yaw torque domain in a test without colors. Interestingly, the preference for the unpunished turning direction in the mutant flies is statistically significant, suggesting that perhaps flies with the mutated gene product are still able to encode some memory, albeit at greatly reduced efficiency. This finding also indicates that perhaps extended self-learning (i.e., without colors) might also be able to yield significant learning scores in FoxP³⁹⁵⁵ mutant flies. These flies will therefore be tested not only in extended self-learning, but also in habit interference experiments in future work (Mendoza, 2014).

Vocal learning has been characterized as an automatization of behavior, akin to habit formation in non-vocal mammals. Analogous to the conceptual and biological similarity of self-learning across taxa, both vocal learning and habit formation in vertebrates also share biological substrates in cortico-striatal circuits, where FoxP2 is expressed. The finding that the FoxP³⁹⁵⁵ allele is also involved in habit formation in invertebrates not only further supports the homology between PKC/FoxP-mediated operant self-learning in flies and vocal learning in birds and humans, but also prompts the hypothesis that habit formation may require a FoxP gene in vertebrates as well (Mendoza, 2014).

Thus, several bodies of evidence can be drawn from, spanning multiple vertebrate and invertebrate model systems and humans, when it is concluded that the current results strongly support the hypothesis that the FoxP-dependent component of language evolved from an ancestral operant self-learning mechanism. Interestingly, preliminary data from non-vocal vertebrate motor learning further corroborate this hypothesis. Moreover, following this extension of the 'motor learning hypothesis' of FoxP function, one may predict language and/or motor skill deficits in patients with mutations in other FOXP paralogues, anticipating that some of the ancestral function is conserved. Indeed, the symptoms of patients with mutations in the closest paralogue of FOXP2, FOXP1, include language and motor skill impairments. In fact, the Drosophila isoform probably involved in self-learning is not present in FoxP2 genes, but in FoxP1. Because FoxP gene products form homo- as well as heterodimers , it is tempting to speculate that all three brain-expressed FoxP paralogues (1, 2 and 4) may perform similar tasks in a degenerate fashion, with the neuronal circuits controlling language being more susceptible to disruption than other FoxP-expressing circuits (Mendoza, 2014).

Autism spectrum disorders and schizophrenia are being discussed as diametric malfunctions on a continuous scale of 'sense of self'. Given the implication of members of the FoxP gene family in both disorders, it is interesting to note that the operant self-learning mechanism appears to be selectively engaged when the content of the learning task concerns the organism itself and not when it concerns non-self entities (Mendoza, 2014).

In contrast to Chomsky's assertion that it is 'completely meaningless to speak of extrapolating this concept of operant to ordinary verbal behavior', this study was able to show that a transcription factor known for its specific involvement in language is also involved in operant conditioning, specifically operant self-learning. This result is consistent with Skinner's description of language as an operant behavior, while nevertheless espousing Chomsky's view of inborn components in language acquisition. In particular, the results emphasize the speech component in the evolution of language: "In many respects the story of the evolution of language must begin with the evolution of serial motor activity and its nervous control" (Mendoza, 2014).

Identification of FoxP circuits involved in locomotion and object fixation in Drosophila

The FoxP family of transcription factors is necessary for operant self-learning, an evolutionary conserved form of motor learning. The expression pattern, molecular function and mechanisms of action of the Drosophila FoxP orthologue remain to be elucidated. By editing the genomic locus of FoxP with CRISPR/Cas9, this study found that the three different FoxP isoforms are expressed in neurons, but not in glia and that not all neurons express all isoforms. Furthermore, FoxP expression was found in the protocerebral bridge, the fan-shaped body and in motor neurons, but not in the mushroom bodies. Finally, this study discovered that FoxP expression during development, but not adulthood, is required for normal locomotion and landmark fixation in walking flies. While FoxP expression in the protocerebral bridge and motor neurons is involved in locomotion and landmark fixation, the FoxP gene can be excised from dorsal cluster neurons and mushroom-body Kenyon cells without affecting these behaviours (Palazzo, 2020).

The genomic locus of the Drosophila FoxP gene was edited in order to better understand the expression patterns of the FoxP isoforms and their involvement in behaviour. The isoforms differ with respect to their expression in neuronal tissue. For instance, isoform B (FoxP-iB) expression was found in neuropil areas such as the superior medial protocerebrum, the protocerebral bridge, the noduli, the vest, the saddle, the gnathal ganglia and the medulla, while areas such as the antennal lobes, the fan-shaped body, the lobula and a glomerulus of the posterior ventrolateral protocerebrum contain other FoxP isoforms but not isoform B. Previous results that FoxP is expressed in a large variety of neuronal cell types was corroborated. Genomic manipulations created several new alleles of the FoxP gene which had a number of behavioural consequences that mimicked other, previously published alleles. Specifically, it was found that constitutive knock-out of either FoxP-IB alone or of all FoxP isoforms affects several parameters of locomotor behaviour, such as walking speed, the straightness of walking trajectories or landmark fixation. Mutating the FoxP gene only in particular neurons can have different effects. For instance, knocking FoxP out in neurons of the dorsal cluster (where FoxP is expressed) or in MB Kenyon cells (where no FoxP expression was detected) had no effect in Buridan's paradigm, despite these neurons being required for normal locomotion in Buridan's paradigm. By contrast, without FoxP in the protocerebral bridge or motor neurons, flies show similar locomotor impairments as flies with constitutive knock-outs. These impairments appear to be due to developmental action of the FoxP gene during larval development, as no such effects can be found if the gene is knocked out in all cells in the early pupal or adult stages (Palazzo, 2020).

The exact expression pattern of FoxP has been under debate for quite some time now. Initial work combined traditional reporter gene expression with immunohistochemistry. A previous study created a FoxP-Gal4 line where a 1.5 kb fragment of genomic DNA upstream of the FoxP coding region was used to drive Gal4 expression. These the resulting expression pattern was validated with the staining of a commercial polyclonal antibody against FoxP. The same antibody was used in the current work and observed perfect co-expression with the reporter. The previous description of the FoxP expression pattern as a small number of neurons distributed in various areas of the brain, particularly in the protocerebral bridge, matches the current results (Palazzo, 2020).

Subsequent reports on FoxP expression patterns also used putative FoxP promoter fragments to direct the expression of Gal4. One study used a 1.4 kb sequence upstream of the FoxP transcription start site, while another used 1.9 kb. The larger fragment contained the sequences of the two previously used fragments. The latest study reporting on FoxP expression in Drosophila avoided the problematic promoter fragment method and instead tagged FoxP within a genomic segment contained in a fosmid, intended to ensure expression of GFP-tagged FoxP under the control of its own, endogenous regulatory elements. This study was the first to circumvent the potential for artifacts created either by selection of the wrong promoter fragment or by choosing an inappropriate basal promoter with the fragment. However, since they also used insertion of a transgene, their expression pattern, analogous to that of a promoter fragment Gal4 line, may potentially be subject to local effects where the fosmid with the tagged FoxP was inserted (Palazzo, 2020).

In an attempt to eliminate, the last source of error for determining the expression pattern of FoxP in Drosophila, CRISPR/Cas9 with homology-directed repair was used to tag FoxP in situ, avoiding both the potential local insertion effects of the previous approaches and without disrupting the complex regulation that may occur from more distant parts in the genome. For instance, in human cells, there are at least 18 different genomic regions that are in physical contact with the FOXP2 promoter, some of which act as enhancers. The effects of these regions may be disrupted even if the entire genomic FoxP locus were inserted in a different genomic region as in. Interestingly, the first promoter fragment approach and the fosmid approach agree both with the most artefact-avoiding genome editing approach and the immunohistochemistry with an antibody validated by at least three different FoxP-KO approaches. This converging evidence from four different methods used in three different laboratories suggests that FoxP is expressed in about 1800 neurons in the fly nervous system, of which about 500 are located in the ventral nerve cord. Expression in the brain is widespread with both localized clusters and individual neurons across a variety of neuronal cell types. Notably, the four methods also agree that there is no detectable FoxP expression in the adult or larval MBs. By contrast, in honey bees, there is converging evidence of FoxP expression in the MBs (Palazzo, 2020).

This comparison of the current data with the literature prompts the question why two different promoter fragment approaches suggested FoxP expression in the MBs (confirmed by a ribosome-based approach) when there is no FoxP protein detectable there (Palazzo, 2020).

A first observation used the classic hsp70-based pGaTB vector to create a Gal4 line, while two other studies used the more modern Drosophila synthetic core promoter (DSCP)-based pBPGUw vector. The two vectors differ with regard to their effects on gene expression. In addition to carrying two different basal promoters, the modern pBPGUw sports a 3'UTR that is designed to increase the longevity and stability of the mRNA over the pGaTB vector, which can result in twofold higher Gal4 levels (Palazzo, 2020).

This observation is complemented by single-cell transcriptome data. FoxP RNA can be detected in more than 4100 brain cells, likely overcounting the actual FoxP expression more than threefold. For instance, FoxP RNA is detected in over 1000 glial cells where none of the published studies has ever detected any FoxP expression (Palazzo, 2020).

Taking these two observations together, it becomes plausible that there may be transient, low-level FoxP transcription in some MB neurons (and likely thousands of other cells as well), which in wild-type animals rarely leads to any physiologically relevant FoxP protein levels in these cells. Only when gene expression is enhanced by combining some arbitrary promoter fragments with genetically engineered constructs designed to maximize Gal4 yield such as the pBPGUw vector, such transient, low-abundance mRNAs may be amplified to a detectable level (Palazzo, 2020).

These considerations may also help explain why the ribosome-based method of was able to detect FoxP RNA in MB Kenyon cells: the transcript that was detected may have been present and occupied by ribosomes, but ribosomal occupancy does not automatically entail translation. It remains unexplained, however, how a previous study failed to detect all those much more strongly expressing and numerous neurons outside of the MBs. All of the above is consistent with other insect species showing FoxP expression on the protein level in their MBs, as only limited genetic alterations would be needed for such minor changes in gene expression (Palazzo, 2020).

The stochasticity of gene expression is a well-known fact and known to arise from the transcription machinery. Post-transcriptional gene regulation is similarly well-known. It is thus not surprising if it is observed that many cells often express transcripts that rarely, if ever, are translated into proteins. The final arbiter of gene expression must therefore remain the protein level, which is why this study validated the expression analysis with the appropriate antibody. On this decisive level, FoxP has not been detected in the MBs at this point (Palazzo, 2020).

The genome editing approach allowed distinguishing of differences in the expression patterns of different FoxP isoforms. The isoform specifically involved in operant self-learning, FoxP-iB, is only expressed in about 65% of all FoxP-positive neurons. The remainder express either FoxP-iA or FoxP-iIR or both. Neurons expressing only non-iB isoforms are localized in the antennal lobes, the fan-shaped body, the lobula and a glomerulus of the posterior ventrolateral protocerebrum. Combined with all three isoforms differing in their DNA-binding FH box, the different expression patterns for the different isoforms adds to the emerging picture that the different isoforms may serve very different functions (Palazzo, 2020).

Alterations of FoxP family genes universally result in various motor deficits on a broad scale in humans and mice for both learned and innate behaviours. Also in flies, manipulations of the FoxP locus by mutation or RNA interference have revealed that FoxP is involved in flight performance and other, presumably inborn, locomotor behaviours as well as in motor learning tasks (Palazzo, 2020).

The locomotor phenotypes described so far largely concerned the temporal aspects of locomotion, such as initiation, speed or duration of locomotor behaviours. Using Buridan's paradigm, this study reports that manipulations of FoxP can also alter spatial aspects of locomotion, such as landmark fixation or the straightness of trajectories. The results further exemplify the old insight that coarse assaults on gene function such as constitutive knock-outs of entire genes or isoforms very rarely yield useful, specific phenotypes. Rather, it is often the most delicate of manipulations that reveal the involvement of a particular gene in a specific behaviour. This fact is likely most often due to the pleiotropy of genes, often paired with differential dominance which renders coarse neurogenetic approaches useless in most instances, as so many different behaviours are affected that the specific contribution of a gene to a behavioural phenotype becomes impossible to dissect (Palazzo, 2020).

In the case of FoxP, it was already known, for instance, that the different isoforms affect flight performance to differing degrees and that a variety of different FoxP manipulations affected general locomotor activity. This study shows that a complete knock-out of either FoxP-iB or all isoforms affected both spatial and temporal parameters of locomotion, but the insertion mutation FoxP3955 did not alter stripe fixation. Remarkably, despite the ubiquitous and substantial locomotor impairments after nearly any kind of FoxP manipulation be it genomic or via RNAi reported in the published literature failed to detect the locomotor defects of these flies (Palazzo, 2020).

While some of the manipulations used in this study did not affect locomotion significantly (e.g. knock-out in MBs or DCNs), most of them affected both spatial and temporal locomotion parameters, despite these parameters commonly not co-varying. Thus, while one would expect these behaviours to be biologically separable, the manipulations carried out in this study did not succeed in this separation (Palazzo, 2020).

Taken together, the results available to-date reveal FoxP to be a highly pleiotropic gene with phenotypes that span both temporal and spatial domains of locomotion in several behavioural modalities, lifespan, motor learning, social behaviour and habituation. It is straightforward to conclude that only precise, cell-type-specific FoxP manipulations of specific isoforms will be capable of elucidating the function this gene serves in each phenotype. With RNAi generally yielding varying levels of knock-down and, specifically, with currently available FoxP RNAi lines showing only little, if any, detectable knock-down with RT-qPCR, CRISPR/Cas9-mediated genome editing lends itself as the method of choice for this task. Practical considerations when designing multi-target gRNAs for FoxP prompted testing of the CRISPR/Cas9 system as an alternative to RNAi with an isoform-unspecific approach first, keeping the isoform-specific approach for a time when more experience in this technique has been collected. In a first proof-of-principle, CRISPR/Cas9 was used to remove FoxP from MB Kenyon cells, DCNs, motor neurons and the protocerebral bridge (Palazzo, 2020).

MBs have been shown to affect both spatial and temporal aspects of locomotion reported a subtle structural phenotype in a subset of MB Kenyon cells that did not express FoxP. As detailed above, two groups have reported FoxP expression in the MBs and it appears that some transcript can be found in MB Kenyon cells. With a substantial walking defect both in FoxP3955 mutant flies (which primarily affects FoxP-iB expression) and in flies without any FoxP, together with the MBs being critical for normal walking behaviour, the MBs were a straightforward candidate for a cell-type-specific FoxP-KO. However, flies without FoxP in the MBs walk perfectly normally. There are two possible reasons for this lack of an effect of this manipulation: either FoxP protein is not present in MBs or it is not important in MBs for walking. While at this point it is not possible to decide between these two options, the expression data concurring with those from previous studies suggest the former explanation may be the more likely one. Remarkably, a publication that did report FoxP expression in the MBs did not detect the walking deficits in FoxP3955 mutant flies despite testing for such effects. Motor aberrations as those described here and in other FoxP manipulations constitute a potential alternative to the decision-making impairments ascribed to these flies (Palazzo, 2020).

DCNs were recently shown to be involved in the spatial component (landmark fixation) of walking in Buridan's paradigm, but removing FoxP from DCNs showed no effect, despite abundant FoxP expression in DCNs. It is possible that a potential effect in stripe fixation may have been masked by already somewhat low fixation in both control strains. On the other hand, even at such control fixation levels, significant increases in stripe deviation can be obtained. Before this is resolved, one explanation is that FoxP is not required in these neurons for landmark fixation in Buridan's paradigm, while the neurons themselves are required (Palazzo, 2020).

Motor neurons are involved in all aspects of behaviour and have been shown to be important for operant self-learning. With abundant expression of FoxP in motor neurons, these neurons are considered a prime candidate for a clear FoxP-cKO phenotype. Indeed, removing FoxP specifically from motor neurons only, mimicked the effects of removing the gene constitutively from all cells. It is noteworthy that this manipulation alone was sufficient to affect both temporal and spatial parameters, albeit only one of the two driver lines showed clear-cut results. One would not necessarily expect motor neurons to affect purportedly 'higher-order' functions such as landmark fixation. It is possible that the higher tortuosity in the trajectories of the flies where D42 was used to drive the UAS-gRNA construct is largely responsible for the greater angular deviation from the landmarks in these flies and that this tortuosity, in turn is caused by the missing FoxP in motor neurons. Alternatively, D42 is also driving in non-motor neurons where FoxP is responsible for landmark fixation. The driver line C380 showed similar trends, albeit not quite statistically significant at an alpha value of 0.5%, suggesting that potentially the increased meander parameter may be caused by motor neurons lacking FoxP (Palazzo, 2020).

The protocerebral bridge is not only the arguably most conspicuous FoxP-positive neuropil, it has also been reported to be involved in temporal aspects of walking. Moreover, the protocerebral bridge provides input to other components of the central complex involved in angular orientation. Similar to the results in motor neurons, removing FoxP from a small group of brain neurons innervating the protocerebral bridge, phenocopies constitutive FoxP mutants (Palazzo, 2020).

Taken together, the motor neuron and protocerebral bridge results suggest that both sets of neurons serve their locomotor function in sequence. At this point, it is unclear which set of neurons precedes the other in this sequence (Palazzo, 2020).

There is ample evidence that the FoxP family of transcription factors acts during development in a variety of tissues. What is less well known is if adult FoxP expression serves any specific function. A recent study in transgenic mice in operant conditioning and motor learning tasks showed postnatal knock-out of FOXP2 in cerebellar and striatal neurons affected leverpressing and cerebellar knock-out also affected motor-learning. At least for these tasks in mammals, a FoxP family member does serve a postnatal function that is independent of brain development (brain morphology was unaltered in these experiments). Also in birds, evidence has been accumulating that adult FoxP expression serves a song plasticity function. The temporally controlled experiments in this study suggest that at least locomotion in Buridan's paradigm can function normally in the absence of FoxP expression in the adult, as long as FoxP expression remains unaltered during larval development. Future research on the role of FoxP in locomotion and landmark fixation hence needs to focus on the larval development before pupation (Palazzo, 2020).

Alternative splicing and gene duplication in the evolution of the FoxP gene subfamily

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1-P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system. In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. This gene was characterized, and it was shown to generate by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, this phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA. With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, this study has established a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, a scenario is presented for the structural evolution of this gene class, and new previously unsuspected levels of regulation for FoxP1 in the vertebrate system was revealed (Santos, 2011).

Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability

Mutations of the FOXP2 gene impair speech and language development in humans and shRNA-mediated suppression of the avian ortholog FoxP2 disrupts song learning in juvenile zebra finches. How diminished FoxP2 levels affect vocal control and alter the function of neural circuits important to learned vocalizations remains unclear. This study shows that FoxP2 knockdown in the songbird striatum disrupts developmental and social modulation of song variability. Recordings in anesthetized birds show that FoxP2 knockdown interferes with D1R-dependent modulation of activity propagation in a corticostriatal pathway important to song variability, an effect that may be partly attributable to reduced D1R and DARPP-32 protein levels. Furthermore, recordings in singing birds reveal that FoxP2 knockdown prevents social modulation of singing-related activity in this pathway. These findings show that reduced FoxP2 levels interfere with the dopaminergic modulation of vocal variability, which may impede song and speech development by disrupting reinforcement learning mechanisms (Murugan, 2013).

The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice

Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some which have been implicated in a number of neurodevelopmental disorders. This study shows that the sushi repeat-containing domain protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. It was also shown that FoxP2 modulates synapse formation through regulating SRPX2 levels, and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. The results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders (Sia, 2013).

This study has shown that SRPX2 is a sushi domain protein involved in synapse formation. In invertebrates, sushi domain proteins have been shown to cluster AChRs at synapses in C. elegans, and the Drosophila homolog, Hikaru Genki, is localized to the nascent synaptic cleft. In vertebrates, sushi domain proteins are primarily studied as regulators of the classical complement cascade. The current results suggest that sushi domain proteins may also play roles in regulating synaptic development and organization in vertebrates. In addition, as genes of the classical complement cascade have been shown to regulate synapse elimination, it is speculated that SRPX2 may act through modulation of components of the complement cascade (Sia, 2013).

To date, FoxP2 is the only gene that has been shown to be involved in a human monogenic language disorder, although the cellular mechanisms involved remain obscure. Previous studies have suggested that FoxP2 may regulate neurite growth, dendritic morphology and synaptic physiology of basal ganglia neurons. This study shows that FoxP2 can regulate synaptogenesis of excitatory synapses in cortical neurons through SRXP2. While activity-regulated transcription factors have been shown to regulate synaptogenesis, developmental synapse formation can occur in the complete absence of activity, and it is unclear whether such synapse formation is also regulated by activity-independent transcription factors. This study shows that FoxP2 is an activity-independent transcription factor that regulates synaptogenesis through SRPX2. In conclusion, this study suggests that FoxP2 can affect the development of language-related neural circuitry through regulating synaptogenesis, and that SRPX2 may be involved in the pathogenesis of language disorders (Sia, 2013).

The forkhead transcription factors, Foxp1 and Foxp2, identify different subpopulations of projection neurons in the mouse cerebral cortex

Foxp1 and Foxp2, which belong to the forkhead transcription factor family, are expressed in the developing and adult mouse brain, including the striatum, thalamus, and cerebral cortex. Recent reports suggest that FOXP1 and FOXP2 are involved in the development of speech and language in humans. Although both Foxp1 and Foxp2 are expressed in the neural circuits that mediate speech and language, including the corticostriatal circuit, the functions of Foxp1 and Foxp2 in the cerebral cortex remain unclear. To gain insight into the functions of Foxp1 and Foxp2 in the cerebral cortex, Foxp1- and Foxp2-expressing cells were characterized in postnatal and adult mice using immunohistochemistry. In adult mice, Foxp1 was expressed in neurons of layers III-VIa in the neocortex, whereas the expression of Foxp2 was restricted to dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32)(+) neurons of layer VI. In addition, Foxp2 was weakly expressed in the neurons of layer V of the motor cortex and hindlimb and forelimb regions of the primary somatosensory cortex. Both Foxp1 and Foxp2 were expressed in the ionotropic glutamate receptor (GluR) 2/3(+) neurons, and colocalized with none of GluR1, gamma-aminobutyric acid, calbindin, and parvalbumin, indicating that expression of Foxp1 and Foxp2 is restricted to projection neurons. During the postnatal stages, Foxp1 was predominantly expressed in Satb2(+)/Ctip2(-) corticocortical projection neurons of layers III-V and in Tbr1(+) corticothalamic projection neurons of layer VIa. Although Foxp2 was also expressed in Tbr1(+) corticothalamic projection neurons of layer VI, no colocalization of Foxp1 with Foxp2 was observed from postnatal day (P) 0 to P7. These findings suggest that Foxp1 and Foxp2 may be involved in the development of different cortical projection neurons during the early postnatal stages in addition to the establishment and maintenance of different cortical circuits from the late postnatal stage to adulthood (Hisaoka, 2010).

A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. This study introduced these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, medium spiny neurons were found to have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans (Enard, 2009).

Coordinated actions of the Forkhead protein Foxp1 and Hox proteins in the columnar organization of spinal motor neurons

The formation of locomotor circuits depends on the spatially organized generation of motor columns that innervate distinct muscle and autonomic nervous system targets along the body axis. Within each spinal segment, multiple motor neuron classes arise from a common progenitor population; however, the mechanisms underlying their diversification remain poorly understood. This study shows that the Forkhead domain transcription factor Foxp1 plays a critical role in defining the columnar identity of motor neurons at each axial position. Using genetic manipulations, it was demonstrated that Foxp1 establishes the pattern of LIM-HD protein expression and accordingly organizes motor axon projections, their connectivity with peripheral targets, and the establishment of motor pools. These functions of Foxp1 act in accordance with the rostrocaudal pattern provided by Hox proteins along the length of the spinal cord, suggesting a model by which motor neuron diversity is achieved through the coordinated actions of Foxp1 and Hox proteins (Rousso, 2008).

Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1

The precision with which motor neurons innervate target muscles depends on a regulatory network of Hox transcription factors that translates neuronal identity into patterns of connectivity. A single transcription factor, FoxP1, coordinates motor neuron subtype identity and connectivity through its activity as a Hox accessory factor. FoxP1 is expressed in Hox-sensitive motor columns and acts as a dose-dependent determinant of columnar fate. Inactivation of Foxp1 abolishes the output of the motor neuron Hox network, reverting the spinal motor system to an ancestral state. The loss of FoxP1 also changes the pattern of motor neuron connectivity, and in the limb motor axons appear to select their trajectories and muscle targets at random. These findings show that FoxP1 is a crucial determinant of motor neuron diversification and connectivity, and clarify how this Hox regulatory network controls the formation of a topographic neural map (Dasen, 2008).

Search PubMed for articles about Drosophila FoxP

Dasen, J. S. et al. (2008). Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell 134: 304-316. PubMed Citation: 18662545

DasGupta, S., Ferreira, C. H. and Miesenbock, G. (2014). FoxP influences the speed and accuracy of a perceptual decision in Drosophila. Science 344: 901-904. PubMed ID: 24855268

Enard, W., et al. (2009). A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell 137(5): 961-71. PubMed Citation: 19490899

Fujita, E., Tanabe, Y., Shiota, A., Ueda, M., Suwa, K., Momoi, M. Y. and Momoi, T. (2008). Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells. Proc Natl Acad Sci U S A 105: 3117-3122. PubMed ID: 18287060

Groszer, M., et al. (2008). Impaired synaptic plasticity and motor learning in mice with a point mutation implicated in human speech deficits. Curr Biol 18: 354-362. PubMed ID: 18328704

Haesler, S., Rochefort, C., Georgi, B., Licznerski, P., Osten, P., Scharff, C. (2007) Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X. PLoS Biol 5: e321. PubMed ID: 18052609

Hisaoka, T., Nakamura, Y., Senba, E. and Morikawa, Y. (2010). The forkhead transcription factors, Foxp1 and Foxp2, identify different subpopulations of projection neurons in the mouse cerebral cortex. Neuroscience 166(2): 551-63. PubMed Citation: 20040367

Kiya, T., Itoh, Y., Kubo, T. (2008) Expression analysis of the FoxP homologue in the brain of the honeybee, Apis mellifera. Insect Mol Biol 17: 53-60. PubMed ID: 18237284

Lai, C. S., Fisher, S. E., Hurst, J. A., Vargha-Khadem, F., Monaco, A. P. (2001) A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 413: 519-523. PubMed ID: 11586359

Lawton, K. J., Wassmer, T. L., Deitcher, D. L. (2014) Conserved role of Drosophila melanogaster FoxP in motor coordination and courtship song. Behav Brain Res. PubMed ID: 24747661

Lee, H. H., Frasch, M. (2004) Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns. Dev Dyn 229: 357-366. PubMed ID: 14745961

Mendoza, E., Colomb, J., Rybak, J., Pfluger, H. J., Zars, T., Scharff, C. and Brembs, B. (2014). Drosophila FoxP mutants are sufficient in operant self-learning. PLoS One 9: e100648. PubMed ID: 24964149

Murugan, M., Harward, S., Scharff, C. and Mooney, R. (2013). Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability. Neuron 80: 1464-1476. PubMed ID: 24268418

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date revised: 27 July 2014

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