Early B cell factor 3 (EBF3) is a member of the highly evolutionarily conserved Collier/Olf/EBF (COE) family of transcription factors. Prior studies on invertebrate and vertebrate animals have shown that EBF3 homologs are essential for survival and that loss-of-function mutations are associated with a range of nervous system developmental defects, including perturbation of neuronal development and migration. Interestingly, aristaless-related homeobox (ARX), a homeobox-containing transcription factor critical for the regulation of nervous system development, transcriptionally represses EBF3 expression. However, human neurodevelopmental disorders related to EBF3 have not been reported. Here, we describe three individuals who are affected by global developmental delay, intellectual disability, and expressive speech disorder and carry de novo variants in EBF3. Associated features seen in these individuals include congenital hypotonia, structural CNS malformations, ataxia, and genitourinary abnormalities. The de novo variants affect a single conserved residue in a zinc finger motif crucial for DNA binding and are deleterious in a fly model. Our findings indicate that mutations in EBF3 cause a genetic neurodevelopmental syndrome and suggest that loss of EBF3 function might mediate a subset of neurologic phenotypes shared by ARX-related disorders, including intellectual disability, abnormal genitalia, and structural CNS malformations.

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

c Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virusblocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system. T he common intracellular bacterium Wolbachia pipientis (1) is maternally inherited through the eggs of its insect hosts. It is able to successfully invade host populations through a range of reproductive manipulations that either directly or indirectly favor its transmission between insect generations (2-8). In its natural host, it has recently been shown that the presence of Wolbachia can block the replication of RNA viruses (9-13). This effect is the basis for the recent development of Wolbachia as a biocontrol approach to block dengue virus (DENV) transmission by the mosquito Aedes aegypti (14,15).The main vector of DENV, the mosquito Aedes aegypti, is not naturally infected with Wolbachia. However, different strains of Wolbachia have recently been artificially introduced from Drosophila melanogaster (Wolbachia strains wMel and wMelPop) or Aedes albopictus (Wolbachia strain wAlbB) into A. aegypti and are stably maintained in laboratory and wild mosquito populations (8,(16)(17)(18). The expectation is that the negative impact that Wolbachia has on DENV replication in the insect will reduce virus transmission to humans and subsequent disease (18)(19)(20).The mechanism(s) that underlies the ability of Wolbachia to affect the replication of DENV appears complex. Using the heterologous association of Wolbachia-infected A. aegypti, transcriptomic and biochemical studies have demonstrated that Wolbachia induces the production of reactive oxygen species (ROS); primes the innate immune system of the mosquito, especially the Toll signaling pathway; and induces the production of various antimicrobial effectors (19)(20)(21)(22)(23). In addition, the use of RNA interference (RNAi) depletion to partially knock down defensin and cecropin genes in Wolbachia-infected A. aegypti lowered resistance to DENV and suggested a role for the innate immune system in mediating virus resistance (22). In contrast, in their natural host D. melanogaster, the same Wolbachia strains do not induce overexpression of immune genes, including the Toll pathway and cecropin-and defensin-encoding genes, yet RNA virus interference, including DENV interference, occurs (23,24). These results demonstrated that induction of the Toll pathway by Wolbachia is not the exclusive mechanism mediating resistance. However, gene expression studies are not sufficient to make a link between a phenotype and a genetic pathway. Since previous studies confirmed the ability of DENV to replicate, and of Wolbachia to block its replication in Drosophila (23), we took advantage of preexisting and wel...

Body size often shows adaptive clines in many ectotherms across altitude and latitude, but little is known about the genetic basis of these adaptive clines. Here we identify a polymorphism in the Dca (Drosophila cold acclimation) gene in Drosophila melanogaster that influences wing size, affects wing:thorax allometry and also controls a substantial proportion of the clinal wing-size variation. A polymorphism in the promoter region of Dca had two common alleles showing strong reciprocal clinal variation in frequency with latitude along the east coast of Australia. The Dca-237 allele increased towards the tropics where wing size is smaller. A within-population association study highlighted that an increase in the frequency of this allele decreased wing size but did not influence thorax size. A manipulated increase in the level of expression of Dca achieved through UAS-GAL4 was associated with a decrease in wing size but had no effect on thorax size. This was consistent with higher Dca expression levels in family lines with higher frequency of the Dca-237 allele. Genetic variation in the promoter region of the Dca gene appears to influence adaptive size variation in the eastern Australian cline of Drosophila melanogaster and accounts for more than 10% of the genetic variation in size within and between populations.

Activation of the Drosophila receptor tyrosine kinase Torso (Tor) only at the termini of the embryo is achieved by the localized expression of the maternal gene Torso-like (Tsl). Tor has a second function in the prothoracic gland as the receptor for prothoracicotropic hormone (PTTH) that initiates metamorphosis. Consistent with the function of Tor in this tissue, Tsl also localizes to the prothoracic gland and influences developmental timing. Despite these commonalities, in our studies of Tsl we unexpectedly found that tsl and tor have opposing effects on body size; tsl null mutants are smaller than normal, rather than larger as would be expected if the PTTH/Tor pathway was disrupted. We further found that whereas both genes regulate developmental timing, tsl does so independently of tor. Although tsl null mutants exhibit a similar length delay in time to pupariation to tor mutants, in tsl: tor double mutants this delay is strikingly enhanced. Thus, loss of tsl is additive rather than epistatic to loss of tor. We also find that phenotypes generated by ectopic PTTH expression are independent of tsl. Finally, we show that a modified form of tsl that can rescue developmental timing cannot rescue terminal patterning, indicating that Tsl can function via distinct mechanisms in different contexts. We conclude that Tsl is not just a specialized cue for Torso signaling but also acts independently of PTTH/Tor in the control of body size and the timing of developmental progression. These data highlight surprisingly diverse developmental functions for this sole Drosophila member of the perforin-like superfamily.MACPF | growth rate | ecdysis | heterochrony