30We generated new genetic tools to efficiently tag genes in 31 Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions 32 to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 33 gene trapping in vivo based on CRE recombinase to avoid embryo 34 injections. DH significantly increases integration efficiency compared to 35 previous strategies and faithfully reports the expression pattern of genes 36 and proteins. The second technique targets genes lacking coding introns 37 using a two-step cassette exchange. First, we replace the endogenous 38 gene with an excisable compact dominant marker using CRISPR making a 39 null allele. Second, the insertion is replaced with a protein::tag cassette.40 2 This sequential manipulation allows the generation of numerous tagged 41 alleles or insertion of other DNA fragments that facilitates multiple 42 downstream applications. Both techniques allow precise gene manipulation 43 and facilitate detection of gene expression, protein localization and 44 assessment of protein function, as well as numerous other applications. 45 46 47 113 circular plasmid or can be circularized in vivo from an initial insertion locus 114 in the genome through Cre/loxP or Flp/FRT mediated recombination (Diao 115 et al., 2015; Nagarkar-Jaiswal et al., 2015b). Importantly, RMCE cassettes 116 can replace a SIC in either orientation with equal probability due to inverted 117 symmetric attP sequences. Therefore 50% of the insertions are inserted in 118 the opposite orientation of transcription and will not be included in the 119 4 mature mRNA. Hence, only half of all successful exchange events will 120 result in protein or gene trap lines. 121 Here, we show that by combining GFP protein traps and T2A-GAL4 122 gene traps in a single RMCE construct, named Double Header (DH), we 123 significantly increased the number of productive RMCE events for 124 MiMIC/CRIMIC containing genes to generate protein or gene trap alleles. 125 Importantly, we expand the ability to target SICs into genes regardless of 126 the presence of introns to allow access to virtually any gene in the fly 127 genome based on CRISPR/Cas9 mediated HDR. This provides a means to 128 create robust null alleles with simple screening, and to convert the SIC 129 insertion using any DNA, creating scarless modifications to facilitate 130 numerous downstream applications. 131 132 Results 133 134 Double Header (DH) improves the tagging rate of MiMIC containing 135 genes 136 137 SICs in coding introns can be converted into GFP protein traps or 138 T2A-GAL4 gene traps through RMCE. However, because RMCE of SICs in 139 MiMICs and CRIMICs can occur in either orientation, only one out of two 140 events produces a tag that is incorporated in the gene product. Moreover, 141 796 We thank the Bloomington Drosophila Stock Center and the Developmental 797 Studies Hybridoma Bank for antibodies. We thank Zelun Wang for technical 798 help, and Megan Campbell and Shinya Yamamoto for reading the 799 manuscript and providing helpful su...

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Notch signaling research dates back to more than one hundred years, beginning with the identification of the Notch mutant in the fruit fly Drosophila melanogaster. Since then, research on Notch and related genes in flies has laid the foundation of what we now know as the Notch signaling pathway. In the 1990s, basic biological and biochemical studies of Notch signaling components in mammalian systems, as well as identification of rare mutations in Notch signaling pathway genes in human patients with rare Mendelian diseases or cancer, increased the significance of this pathway in human biology and medicine. In the 21st century, Drosophila and other genetic model organisms continue to play a leading role in understanding basic Notch biology. Furthermore, these model organisms can be used in a translational manner to study underlying mechanisms of Notch-related human diseases and to investigate the function of novel disease associated genes and variants. In this chapter, we first briefly review the major contributions of Drosophila to Notch signaling research, discussing the similarities and differences between the fly and human pathways. Next, we introduce several biological contexts in Drosophila in which Notch signaling has been extensively characterized. Finally, we discuss a number of genetic diseases caused by mutations in genes in the Notch signaling pathway in humans and we expand on how Drosophila can be used to study rare genetic variants associated with these and novel disorders. By combining modern genomics and state-of-the art technologies, Drosophila research is continuing to reveal exciting biology that sheds light onto mechanisms of disease.

A consecutive series of 78 patients underwent surgery for 106 aneurysms between 1972 and 1978; this group included 14 patients with subarachnoid hemorrhage who had multiple, unruptured, incidental aneurysms (20 additional aneurysms). These aneurysms were operated on with no mortality. In 15 other patients, who underwent angiography for various nonhemorrhagic disorders, a total of 18 asymptomatic aneurysms were found. These were also operated on with no mortality. Three of the 29 patients had postoperative hemiplegia, which persists in only one. The author recommends that all aneurysms should be considered for operation when diagnosed, if the patient's clinical condition is stable.

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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.

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell–cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine‐tune the pathway in various developmental and post‐developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double‐pass transmembrane protein, was identified in the 1970s as a maternal‐effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling‐dependent single‐minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid‐stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh‐like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular‐trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.

A retrospective analysis of 1171 consecutive percutaneous retrograde brachial and carotid cerebral angiograms was performed on 635 patients, 50.7% of whom were in the sixth decade or older. Symptoms and signs of cerebrovascular disease were the most frequently investigated and diagnosed, accounting for 46.7% of all the angiograms. Despite this relatively high-risk population, we have found direct percutaneous cerebral angiography to have a very low risk. The pros and cons of direct percutaneous versus transfemoral cerebral angiography are discussed. The literature of the previous 10 years is reviewed, and the complication rate of these two techniques is compared.