Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila.

FOXP proteins form a subfamily of evolutionarily conserved transcription factors involved in the development and functioning of several tissues, including the central nervous system. In humans, mutations in FOXP1 and FOXP2 have been implicated in cognitive deficits including intellectual disability and speech disorders. Drosophila exhibits a single ortholog, called FoxP, but due to a lack of characterized mutants, our understanding of the gene remains poor. Here we show that the dimerization property required for mammalian FOXP function is conserved in Drosophila. In flies, FoxP is enriched in the adult brain, showing strong expression in ~1000 neurons of cholinergic, glutamatergic and GABAergic nature. We generate Drosophila loss-of-function mutants and UAS-FoxP transgenic lines for ectopic expression, and use them to characterize FoxP function in the nervous system. At the cellular level, we demonstrate that Drosophila FoxP is required in larvae for synaptic morphogenesis at axonal terminals of the neuromuscular junction and for dendrite development of dorsal multidendritic sensory neurons. In the developing brain, we find that FoxP plays important roles in α-lobe mushroom body formation. Finally, at a behavioral level, we show that Drosophila FoxP is important for locomotion, habituation learning and social space behavior of adult flies. Our work shows that Drosophila FoxP is important for regulating several neurodevelopmental processes and behaviors that are related to human disease or vertebrate disease model phenotypes. This suggests a high degree of functional conservation with vertebrate FOXP orthologues and established flies as a model system for understanding FOXP related pathologies.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology.

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy.

A consanguineous family from Pakistan was ascertained to have a novel deafness-dystonia syndrome with motor regression, ichthyosis-like features and signs of sensory neuropathy. By applying a combined strategy of linkage analysis and whole-exome sequencing in the presented family, a homozygous nonsense mutation, c.4G>T (p.Glu2*), in FITM2 was identified. FITM2 and its paralog FITM1 constitute an evolutionary conserved protein family involved in partitioning of triglycerides into cellular lipid droplets. Despite the role of FITM2 in neutral lipid storage and metabolism, no indications for lipodystrophy were observed in the affected individuals. In order to obtain independent evidence for the involvement of FITM2 in the human pathology, downregulation of the single Fitm ortholog, CG10671, in Drosophila melanogaster was pursued using RNA interference. Characteristics of the syndrome, including progressive locomotor impairment, hearing loss and disturbed sensory functions, were recapitulated in Drosophila, which supports the causative nature of the FITM2 mutation. Mutation-based genetic counseling can now be provided to the family and insight is obtained into the potential impact of genetic variation in FITM2.

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry.

The morphology of synapses is of central interest in neuroscience because of the intimate relation with synaptic efficacy. Two decades of gene manipulation studies in different animal models have revealed a repertoire of molecules that contribute to synapse development. However, since such studies often assessed only one, or at best a few, morphological features at a given synapse, it remained unaddressed how different structural aspects relate to one another. Furthermore, such focused and sometimes only qualitative approaches likely left many of the more subtle players unnoticed. Here, we present the image analysis algorithm 'Drosophila_NMJ_Morphometrics', available as a Fiji-compatible macro, for quantitative, accurate and objective synapse morphometry of the Drosophila larval neuromuscular junction (NMJ), a well-established glutamatergic model synapse. We developed this methodology for semi-automated multiparametric analyses of NMJ terminals immunolabeled for the commonly used markers Dlg1 and Brp and showed that it also works for Hrp, Csp and Syt. We demonstrate that gender, genetic background and identity of abdominal body segment consistently and significantly contribute to variability in our data, suggesting that controlling for these parameters is important to minimize variability in quantitative analyses. Correlation and principal component analyses (PCA) were performed to investigate which morphometric parameters are inter-dependent and which ones are regulated rather independently. Based on nine acquired parameters, we identified five morphometric groups: NMJ size, geometry, muscle size, number of NMJ islands and number of active zones. Based on our finding that the parameters of the first two principal components hardly correlated with each other, we suggest that different molecular processes underlie these two morphometric groups. Our study sets the stage for systems morphometry approaches at the well-studied Drosophila NMJ.