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Notch signaling is involved in the development of almost all organ systems and is required post‐developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1‐4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss‐ or gain‐of‐function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine‐tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy. [ABSTRACT FROM AUTHOR]

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well. [ABSTRACT FROM AUTHOR]

Johnston's organ, the Drosophila auditory organ, is anatomically very different from the mammalian organ of Corti. However, recent evidence indicates significant cellular and molecular similarities exist between vertebrate and invertebrate hearing, suggesting that Drosophila may be a useful platform to determine the function of the many mammalian deafness genes whose underlying biological mechanisms are poorly characterized. Our goal was a comprehensive screen of all known orthologues of mammalian deafness genes in the fruit fly to better understand conservation of hearing mechanisms between the insect and the fly and ultimately gain insight into human hereditary deafness. We used bioinformatic comparisons to screen previously reported human and mouse deafness genes and found that 156 of them have orthologues in Drosophila melanogaster. We used fluorescent imaging of T2A-GAL4 gene trap and GFP or YFP fluorescent protein trap lines for 54 of the Drosophila genes and found 38 to be expressed in different cell types in Johnston's organ. We phenotypically characterized the function of strong loss-of-function mutants in three genes expressed in Johnston's organ (Cad99C, Msp-300, and Koi) using a courtship assay and electrophysiological recordings of sound-evoked potentials. Cad99C and Koi were found to have significant courtship defects. However, when we tested these genes for electrophysiological defects in hearing response, we did not see a significant difference suggesting the courtship defects were not caused by hearing deficiencies. Furthermore, we used a UAS/RNAi approach to test the function of seven genes and found two additional genes, CG5921 and Myo10a, that gave a statistically significant delay in courtship but not in sound-evoked potentials. Our results suggest that many mammalian deafness genes have Drosophila homologues expressed in the Johnston's organ, but that their requirement for hearing may not necessarily be the same as in mammals. [ABSTRACT FROM AUTHOR]

EZH1, a polycomb repressive complex-2 component, is involved in a myriad of cellular processes. EZH1 represses transcription of downstream target genes through histone 3 lysine27 (H3K27) trimethylation (H3K27me3). Genetic variants in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. Here we report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo missense variant in EZH1 through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and an analogous somatic or germline mutation in EZH2 has been reported in patients with B-cell lymphoma or Weaver syndrome, respectively. Human EZH1/2 are homologous to fly Enhancer of zeste (E(z)), an essential gene in Drosophila, and the affected residue (p.A678 in humans, p.A691 in flies) is conserved. To further study this variant, we obtained null alleles and generated transgenic flies expressing wildtype [E(z)WT] and the variant [E(z)A691G]. When expressed ubiquitously the variant rescues null-lethality similar to the wildtype. Overexpression of E(z)WT induces homeotic patterning defects but notably the E(z)A691G variant leads to dramatically stronger morphological phenotypes. We also note a dramatic loss of H3K27me2 and a corresponding increase in H3K27me3 in flies expressing E(z)A691G, suggesting this acts as a gain-of-function allele. In conclusion, here we present a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila. [ABSTRACT FROM AUTHOR]

Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function. [ABSTRACT FROM AUTHOR]

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD. Author summary: Alzheimer's disease (AD) is the most common neurodegenerative disease affecting the aging population. Although many genetic factors have been implicated in its pathogenesis, in vivo functions of many of these genes have not been well defined. In this study, we investigated the function of TM2D3, a conserved gene that has been implicated in late-onset AD through an exome-wide association study, and two closely related genes, TM2D1 and TM2D2, using the fruit fly Drosophila melanogaster. In addition to exhibiting a previously reported maternal-effect neuurodevelopmental phenotype caused by Notch signaling defects during embryogenesis, fly TM2D3 mutants are short lived and display age-dependent motor and electrophysiological defects, providing the first link between this gene and age-dependent neurological phenotypes. Furthermore, TM2D1 and TM2D2 knockout flies phenotypically mimic the loss of TM2D3. Triple knockout of all three TM2D genes resemble the single knockouts, suggesting that these genes likely function together. Together with functional data that implicates TM2D3 in a biological process that is liked to the γ-secretase, a protease that is involved in AD in addition to being required for proper Notch signaling, we propose that all three TM2D family genes may be involved in AD pathogenesis, which warrants further investigation through human genetics studies. [ABSTRACT FROM AUTHOR]

Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000-13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support. [ABSTRACT FROM AUTHOR]

The fruit fly, Drosophila melanogaster, has been used to understand fundamental principles of genetics and biology for over a century. Drosophila is now also considered an essential tool to study mechanisms underlying numerous human genetic diseases. In this review, we will discuss how flies can be used to deepen our knowledge of infectious disease mechanisms in vivo. Flies make effective and applicable models for studying host-pathogen interactions thanks to their highly conserved innate immune systems and cellular processes commonly hijacked by pathogens. Drosophila researchers also possess the most powerful, rapid, and versatile tools for genetic manipulation in multicellular organisms. This allows for robust experiments in which specific pathogenic proteins can be expressed either one at a time or in conjunction with each other to dissect the molecular functions of each virulent factor in a cell-type-specific manner. Well documented phenotypes allow large genetic and pharmacological screens to be performed with relative ease using huge collections of mutant and transgenic strains that are publicly available. These factors combine to make Drosophila a powerful tool for dissecting out host-pathogen interactions as well as a tool to better understand how we can treat infectious diseases that pose risks to public health, including COVID-19, caused by SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Since its discovery in Drosophila, the Notch signaling pathway has been studied in numerous developmental contexts in diverse multicellular organisms. The role of Notch signaling in nervous system development has been extensively investigated by numerous scientists, partially because many of the core Notch signaling components were initially identified through their dramatic 'neurogenic' phenotype of developing fruit fly embryos. Components of the Notch signaling pathway continue to be expressed in mature neurons and glia cells, which is suggestive of a role in the post-developmental nervous system. The Notch pathway has been, so far, implicated in learning and memory, social behavior, addiction, and other complex behaviors using genetic model organisms including Drosophila and mice. Additionally, Notch signaling has been shown to play a modulatory role in several neurodegenerative disease model animals and in mediating neural toxicity of several environmental factors. In this paper, we summarize the knowledge pertaining to the post-developmental roles of Notch signaling in the nervous system with a focus on discoveries made using the fruit fly as a model system as well as relevant studies in C elegans, mouse, rat, and cellular models. Since components of this pathway have been implicated in the pathogenesis of numerous psychiatric and neurodegenerative disorders in human, understanding the role of Notch signaling in the mature brain using model organisms will likely provide novel insights into the mechanisms underlying these diseases. [ABSTRACT FROM AUTHOR]

We performed an exome-wide association analysis in 1393 late-onset Alzheimer’s disease (LOAD) cases and 8141 controls from the CHARGE consortium. We found that a rare variant (P155L) in TM2D3 was enriched in Icelanders (~0.5% versus <0.05% in other European populations). In 433 LOAD cases and 3903 controls from the Icelandic AGES sub-study, P155L was associated with increased risk and earlier onset of LOAD [odds ratio (95% CI) = 7.5 (3.5–15.9), p = 6.6x10-9]. Mutation in the Drosophila TM2D3 homolog, almondex, causes a phenotype similar to loss of Notch/Presenilin signaling. Human TM2D3 is capable of rescuing these phenotypes, but this activity is abolished by P155L, establishing it as a functionally damaging allele. Our results establish a rare TM2D3 variant in association with LOAD susceptibility, and together with prior work suggests possible links to the β-amyloid cascade. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I (CI) is an essential component in energy production through oxidative phosphorylation. Most CI subunits are encoded by nuclear genes, translated in the cytoplasm, and imported into mitochondria. Upon entry, they are embedded into the mitochondrial inner membrane. How these membrane-associated proteins cope with the hydrophilic cytoplasmic environment before import is unknown. In a forward genetic screen to identify genes that cause neurodegeneration, we identified sicily, the Drosophila melanogaster homologue of human C80RF38, the loss of which causes Leigh syndrome. We show that in the cytoplasm, Sicily preprotein interacts with cytosolic Hsp90 to chaperone the CI subunit, ND42, before mitochondrial import. Loss of Sicily leads to loss of CI proteins and preproteins in both mitochondria and cytoplasm, respectively, and causes a CI deficiency and neurodegeneration. Our data indicate that cytosolic chaper-ones are required for the subcellular transport of ND42. [ABSTRACT FROM AUTHOR]

Transport of newly synthesized Rhodopsin upon light stimulation in adult Drosophila photoreceptors is mediated by a Crag/Rab11-dependent vesicular trafficking process. [ABSTRACT FROM AUTHOR]