A nonneural miRNA cluster mediates hearing via repression of two neural targets.

We show here that mir-279/996 are absolutely essential for development and function of Johnston's organ (JO), the primary proprioceptive and auditory organ in Drosophila Their deletion results in highly aberrant cell fate determination, including loss of scolopale cells and ectopic neurons, and mutants are electrophysiologically deaf. In vivo activity sensors and mosaic analyses indicate that these seed-related miRNAs function autonomously to suppress neural fate in nonneuronal cells. Finally, genetic interactions pinpoint two neural targets (elav and insensible) that underlie miRNA mutant JO phenotypes. This work uncovers how critical post-transcriptional regulation of specific miRNA targets governs cell specification and function of the auditory system.

Lay Definitions of Intelligence, Knowledge, and Memory: Inter- and Independence of Constructs.

The present study examined how lay participants define the following concepts used widely in psychology: being intelligent, knowing, and remembering. In the scientific community, knowledge overlaps with the contents of semantic memory, crystallized intelligence reflects the accumulation of knowledge, knowledge and event memory interact, and fluid intelligence and working memory correlate. Naturally, the lay public has implicit theories of these constructs. These theories mainly distinguish between intelligent and unintelligent behaviors and tend to include characteristics outside psychometric studies of intelligence, such as emotional intelligence. Here, we asked lay participants from the online platform Prolific to explain "what does being intelligent mean to you?" as well as "knowing" and "remembering" to understand their degree of alignment with theoretical conceptualizations in the research community. Qualitative coding of participant definitions showed that intelligence and knowledge are closely related, but asymmetrically-when defining what it means to be intelligent, participants reference knowledge, but intelligence is not considered in explaining knowing. Although participants note that intelligence is multi-faceted and related to problem-solving, there is an emphasis (in terms of frequency of mentions) on the crystallized side of intelligence (i.e., knowledge). A deeper understanding of lay participants' mental models of these constructs (i.e., their metacognitions) is essential for bridging gaps between experts and the general public.

Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity.

Proper function of cell signaling pathways is dependent upon regulated membrane trafficking events that lead to the endocytosis, recycling, and degradation of cell surface receptors. The endosomal complexes required for transport (ESCRT) genes play a critical role in the sorting of ubiquitinated cell surface proteins. CHMP2BIntron5 , a truncated form of a human ESCRT-III protein, was discovered in a Danish family afflicted by a hereditary form of frontotemporal dementia (FTD). Although the mechanism by which the CHMP2B mutation in this family causes FTD is unknown, the resulting protein has been shown to disrupt normal endosomal-lysosomal pathway function and leads to aberrant regulation of signaling pathways. Here we have misexpressed CHMP2BIntron5 in the developing Drosophila external sensory (ES) organ lineage and demonstrate that it is capable of altering cell fates. Each of the cell fate transformations seen is compatible with an increase in Notch signaling. Furthermore, this interpretation is supported by evidence that expression of CHMP2BIntron5 in the notum environment is capable of raising the levels of Notch signaling. As such, these results add to a growing body of evidence that CHMP2BIntron5 can act rapidly to disrupt normal cellular function via the misregulation of critical cell surface receptor function.

A Functional Analysis of the Drosophila Gene hindsight: Evidence for Positive Regulation of EGFR Signaling.

We have investigated the relationship between the function of the gene hindsight (hnt), which is the Drosophila homolog of Ras Responsive Element Binding protein-1 (RREB-1), and the EGFR signaling pathway. We report that hnt mutant embryos are defective in EGFR signaling dependent processes, namely chordotonal organ recruitment and oenocyte specification. We also show the temperature sensitive hypomorphic allele hntpebbled is enhanced by the hypomorphic MAPK allele rolled (rl1 ). We find that hnt overexpression results in ectopic DPax2 expression within the embryonic peripheral nervous system, and we show that this effect is EGFR-dependent. Finally, we show that the canonical U-shaped embryonic lethal phenotype of hnt, which is associated with premature degeneration of the extraembyonic amnioserosa and a failure in germ band retraction, is rescued by expression of several components of the EGFR signaling pathway (sSpi, Ras85DV12 , pntP1 ) as well as the caspase inhibitor p35 Based on this collection of corroborating evidence, we suggest that an overarching function of hnt involves the positive regulation of EGFR signaling.

Expression of mutant CHMP2B linked to neurodegeneration in humans disrupts circadian rhythms in Drosophila.

Mutations in CHMP2B, an ESCRT-III (endosomal sorting complexes required for transport) component, are associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Neurodegenerative disorders including FTD are also associated with a disruption in circadian rhythms, but the mechanism underlying this defect is not well understood. Here, we ectopically expressed the human CHMP2B variant associated with FTD (CHMP2BIntron5) in flies using the GMR-GAL4 driver (GMR>CHMP2BIntron5) and analyzed their circadian rhythms at behavioral, cellular, and biochemical level. In GMR>CHMP2BIntron5 flies, we observed disrupted eclosion rhythms, shortened free-running circadian locomotor period, and reduced levels of timeless (tim) mRNA-a circadian pacemaker gene. We also observed that the GMR-GAL4 driver, primarily known for its expression in the retina, drives expression in a subset of tim expressing neurons in the optic lobe of the brain. The patterning of these GMR- and tim-positive neurons in the optic lobe, which appears distinct from the putative clusters of circadian pacemaker neurons in the fly brain, was disrupted in GMR>CHMP2BIntron5 flies. These results demonstrate that CHMP2BIntron5 can disrupt the normal function of the circadian clock in Drosophila.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye.

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control.

miRNA suppression of a Notch repressor directs non-neuronal fate in Drosophila mechanosensory organs.

Although there is abundant evidence that individual microRNA (miRNA) loci repress large cohorts of targets, large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. Here, we identify a rare case of developmental cell specification that is highly dependent on miRNA control of an individual target. We observe that binary cell fate choice in the Drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. The mir-279/996 defect phenocopies Notch loss of function during the sheath-neuron cell fate decision, suggesting the miRNAs facilitate Notch signaling. Consistent with this, mir-279/996 knockouts are strongly enhanced by Notch heterozygosity, and activated nuclear Notch is impaired in the miRNA mutant. Although Hairless (H) is the canonical nuclear Notch pathway inhibitor, and H heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-Notch signaling, H/+ does not rescue mir-279/996 mutants. Instead, we identify Insensible (Insb), another neural nuclear Notch pathway inhibitor, as a critical direct miR-279/996 target. Insb is posttranscriptionally restricted to neurons by these miRNAs, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. Thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving miRNA-mediated suppression of a Notch repressor to assign non-neuronal cell fate.

Insensitive is a corepressor for Suppressor of Hairless and regulates Notch signalling during neural development.

The Notch intracellular domain functions as a co-activator for the DNA-binding protein Suppressor of Hairless (Su(H)) to mediate myriad cell fate decisions. Notch pathway activity is balanced by transcriptional repression, mediated by Su(H) in concert with its Drosophila corepressor Hairless. We demonstrate that the Drosophila neural BEN-solo protein Insensitive (Insv) is a nuclear factor that inhibits Notch signalling during multiple peripheral nervous system cell fate decisions. Endogenous Insv was particularly critical when repressor activity of Su(H) was compromised. Reciprocally, ectopic Insv generated several Notch loss-of-function phenotypes, repressed most Notch targets in the E(spl)-C, and opposed Notch-mediated activation of an E(spl)m3-luc reporter. A direct role for Insv in transcriptional repression was indicated by binding of Insv to Su(H), and by strong chromatin immunoprecipitation of endogenous Insv to most E(spl)-C loci. Strikingly, ectopic Insv fully rescued sensory organ precursors in Hairless null clones, indicating that Insv can antagonize Notch independently of Hairless. These data shed first light on the in vivo function for a BEN-solo protein as an Su(H) corepressor in the Notch pathway regulating neural development.

Discrete regulatory regions control early and late expression of D-Pax2 during external sensory organ development.

The transcription factor D-Pax2 is required for the correct differentiation of several cell types in Drosophila sensory systems. While the regulation of its expression in the developing eye has been well studied, little is known about the mechanisms by which the dynamic pattern of D-Pax2 expression in the external sensory organs is achieved. Here we demonstrate that early activation of D-Pax2 in the sensory organ lineage and its maintenance in the trichogen and thecogen cells are governed by separate enhancers. Furthermore, the initial activation is controlled in part by proneural proteins whereas the later maintenance expression is regulated by a positive feedback loop.

The transcription factor D-Pax2 regulates crystallin production during eye development in Drosophila melanogaster.

The generation of a functioning Drosophila eye requires the coordinated differentiation of multiple cell types and the morphogenesis of eye-specific structures. Here we show that D-Pax2 plays a significant role in lens development through regulation of the Crystallin gene and because Crystallin is also expressed in D-Pax2(+) cells in the external sensory organs. Loss of D-Pax2 function leads to loss of Crystallin expression in both eyes and bristles. A 2.3 kilobase (kb) upstream region of the Crystallin gene can drive GFP expression in the eye and is dependent on D-Pax2. In addition, D-Pax2 binds to an evolutionarily conserved site in this region that, by itself, is sufficient to drive GFP expression in the eye. However, mutation of this site does not greatly affect the regulatory region's function. The data indicate that D-Pax2 acts to promote lens development by controlling the production of the major protein component of the lens. Whether this control is direct or indirect remains unresolved.

Frequent unanticipated alleles of lethal giant larvae in Drosophila second chromosome stocks.

Forty years ago, a high frequency of lethal giant larvae (lgl) alleles in wild populations of Drosophila melanogaster was reported. This locus has been intensively studied for its roles in epithelial polarity, asymmetric neural divisions, and restriction of tissue proliferation. Here, we identify a high frequency of lgl alleles in the Bloomington second chromosome deficiency kit and the University of California at Los Angeles Bruinfly FRT40A-lethal P collection. These unrecognized aberrations confound the use of these workhorse collections for phenotypic screening or genetic mapping. In addition, we determined that independent alleles of insensitive, reported to affect asymmetric cell divisions during sensory organ development, carry lgl deletions that are responsible for the observed phenotypes. Taken together, these results encourage the routine testing of second chromosome stocks for second-site alleles of lgl.