miR-9a minimizes the phenotypic impact of genomic diversity by buffering a transcription factor.

Gene expression has to withstand stochastic, environmental, and genomic perturbations. For example, in the latter case, 0.5%-1% of the human genome is typically variable between any two unrelated individuals. Such diversity might create problematic variability in the activity of gene regulatory networks and, ultimately, in cell behaviors. Using multigenerational selection experiments, we find that for the Drosophila proneural network, the effect of genomic diversity is dampened by miR-9a-mediated regulation of senseless expression. Reducing miR-9a regulation of the Senseless transcription factor frees the genomic landscape to exert greater phenotypic influence. Whole-genome sequencing identified genomic loci that potentially exert such effects. A larger set of sequence variants, including variants within proneural network genes, exhibits these characteristics when miR-9a concentration is reduced. These findings reveal that microRNA-target interactions may be a key mechanism by which the impact of genomic diversity on cell behavior is dampened.

A GoldenBraid cloning system for synthetic biology in social amoebae.

GoldenBraid is a rapid, modular, and robust cloning system used to assemble and combine genetic elements. Dictyostelium amoebae represent an intriguing synthetic biological chassis with tractable applications in development, chemotaxis, bacteria-host interactions, and allorecognition. We present GoldenBraid as a synthetic biological framework for Dictyostelium, including a library of 250 DNA parts and assemblies and a proof-of-concept strain that illustrates cAMP-chemotaxis with four fluorescent reporters coded by one plasmid.

A novel statistical method for interpreting the pathogenicity of rare variants.

PURPOSE: To achieve the ultimate goal of personalized treatment of patients, accurate molecular diagnosis and precise interpretation of the impact of genetic variants on gene function is essential. With sequencing cost becoming increasingly affordable, the accurate distinguishing of benign from pathogenic variants becomes the major bottleneck. Although large normal population sequence databases have become a key resource in filtering benign variants, they are not effective at filtering extremely rare variants. METHODS: To address this challenge, we developed a novel statistical test by combining sequencing data from a patient cohort with a normal control population database. By comparing the expected and observed allele frequency in the patient cohort, variants that are likely benign can be identified. RESULTS: The performance of this new method is evaluated on both simulated and real data sets coupled with experimental validation. As a result, we demonstrate this new test is well powered to identify benign variants, and is particularly effective for variants with low frequency in the normal population. CONCLUSION: Overall, as a general test that can be applied to any type of variants in the context of all Mendelian diseases, our work provides a general framework for filtering benign variants with very low population allele frequency.

Large-scale identification of chemically induced mutations in Drosophila melanogaster.

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of -3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (-70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes.

Gene-specific cell labeling using MiMIC transposons.

Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family.

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster.

BACKGROUND: SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB. RESULTS: SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. CONCLUSIONS: Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV.

Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster.

The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters.

A mouse model of acrodermatitis enteropathica: loss of intestine zinc transporter ZIP4 (Slc39a4) disrupts the stem cell niche and intestine integrity.

Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes.

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

Multiplexed Transgenic Selection and Counterselection Strategies to Expedite Genetic Manipulation Workflows Using Drosophila melanogaster.

We recently described a set of four selectable and two counterselectable markers that provide resistance and sensitivity, respectively, against their corresponding drugs using the model organism Drosophila melanogaster. The four selectable markers provide animals with resistance against G418 sulfate, puromycin HCl, blasticidin S, or hygromycin B, whereas the two counterselection markers make animals sensitive to ganciclovir/acyclovir or 5-fluorocytosine. Unlike classical phenotypic markers, whether visual or fluorescent, which require extensive screening of progeny of a genetic cross for desired genotypes, resistance and sensitivity markers eliminate this laborious procedure by directly selecting for, or counterselecting against, the desired genotypes. We demonstrated the usefulness of these markers with three applications: 1) generating dual transgenic animals for binary overexpression (e.g., GAL4/UAS) analysis in a single step through the process of co-injection, followed by co-selection resulting in co-transgenesis; 2) obtaining balancer chromosomes that are both selectable and counterselectable to manipulate crossing schemes for, or against, the presence of the modified balancer chromosome; and 3) making both selectable and fluorescently tagged P[acman] BAC transgenic animals for gene expression and proteomic analysis. Here, we describe detailed procedures for how to use these drug-based selection and counterselection markers in the fruit fly D. melanogaster when making dual transgenic animals for binary overexpression as an example. Dual transgenesis integrates site-specifically into two sites in the genome in a single step, namely both components of the binary GAL4/UAS overexpression system, via a G418 sulfate-selectable GAL4 transactivator plasmid and a blasticidin S-selectable UAS responder plasmid. The process involves co-injecting the two plasmids, followed by co-selection using G418 sulfate and blasticidin S, resulting in co-transgenesis of the two plasmids in the fly genome. We demonstrate the functionality of the procedure based on the expression pattern obtained after dual transgenesis of the two plasmids. We provide protocols on how to prepare drugged fly food vials, determine the effective drug concentration for markers used during transgenic selection and counterselection strategies, and prepare and confirm plasmid DNA for microinjection, followed by the microinjection procedure itself and setting up crossing schemes to isolate desired progeny through selection and/or counterselection. These protocols can be easily adapted to any combination of the six selectable and counterselectable markers we described or any new marker that is resistant or sensitive to a novel drug. Protocols on how to build plasmids by synthetic-assembly DNA cloning or modify plasmids by serial recombineering to perform a plethora of selection, counterselection, or any other genetic strategies are presented in two accompanying Current Protocols articles. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing drugged fly food vials for transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 2: Determining the effective drug concentration for resistance and sensitivity markers used during transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 3: Preparing and confirming plasmid DNA for microinjection to perform transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 4: Microinjecting plasmid DNA into fly embryos to perform transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 5: Crossing schemes to isolate desired progeny through transgenic selection and counterselection strategies using D. melanogaster.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

Synthetic Assembly DNA Cloning to Build Plasmids for Multiplexed Transgenic Selection, Counterselection or Any Other Genetic Strategies Using Drosophila melanogaster.

We recently described a drug-based selectable and counterselectable genetic platform for the animal model system Drosophila melanogaster, consisting of four resistance and two sensitivity markers that allow direct selection for, or counterselection against, a desired genotype. This platform eliminates the need to identify modified progeny by traditional laborious screening using the dominant eye and body color markers, white+ and yellow+ , respectively. The four resistance markers permit selection of animals using G418 sulfate, puromycin HCl, blasticidin S, or hygromycin B, while the two sensitivity markers allow counterselection of animals against ganciclovir or acyclovir and 5-fluorocytosine. The six markers can be used alone or in combination to perform co-selection, combination selection, and counterselection, as well as co-counterselection. To make this novel selection and counterselection genetics platform easily accessible to and rapidly implementable by the scientific community, we used a synthetic assembly DNA cloning platform, GoldenBraid 2.0 (GB2.0). GB2.0 relies on two Type IIs restriction enzymes that are alternatingly used during successive cloning steps to make increasingly complex genetic constructs. Here we describe, as an example, how to perform synthetic assembly DNA cloning using GB2.0 to build such complex plasmids via the assembly of both components of the binary LexA/LexA-Op overexpression system, a G418 sulfate-selectable LexA transactivator plasmid, and a blasticidin S-selectable LexA-Op responder plasmid. We demonstrate the functionality of these plasmids by including the expression pattern obtained after co-injection, followed by co-selection using G418 sulfate and blasticidin S, resulting in co-transgenesis of both plasmids. Protocols are provided on how to obtain, adapt, and clone DNA parts for synthetic assembly cloning after de novo DNA synthesis or PCR amplification of desired DNA parts and how to assemble those DNA parts into multipartite transcription units, followed by how to further assemble multiple transcription units into genetic constructs of increasing complexity to perform multiplexed transgenic selection and counterselection, or any other genetic strategies using Drosophila melanogaster. The protocols we present can be easily adapted to incorporate any of the six selectable and counterselectable markers, or any other, markers, to generate plasmids of unmatched complexity for various genetic applications. A protocol on how to generate transgenic animals using these synthetically assembled plasmids is described in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Obtaining and cloning a de novo-synthesized DNA part for synthetic assembly DNA cloning Basic Protocol 2: Obtaining and cloning a DNA part amplified by PCR from existing DNA resources for synthetic assembly DNA cloning Alternate Protocol: Obtaining, adapting, and cloning a DNA part amplified by PCR from existing DNA resources for synthetic assembly DNA cloning Basic Protocol 3: Synthetic assembly DNA cloning of individual DNA parts into a multipartite transcription unit Basic Protocol 4: Synthetic assembly DNA cloning of multiple transcription units into genetic constructs of increasing complexity.

Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster.

We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using UC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development.

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids.

Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.

Multiplex Hextuple Luciferase Assaying.

We recently expanded the commonly used dual luciferase assaying method toward multiplex hextuple luciferase assaying, allowing monitoring the activity of five experimental pathways against one control at the same time. In doing so, while our expanded assay utilizes a total of six orthogonal luciferases instead of two, this assay, conveniently, still utilizes the well-established reagents and principles of the widely used dual luciferase assay. Three quenchable D-luciferin-consuming luciferases are measured after addition of D-Luciferin substrate, followed by quenching of their bioluminescence (BL) and the measurement of three coelenterazine (CTZ)-consuming luciferases after addition of CTZ substrate, all in the same vessel. Here, we provide detailed protocols on how to perform such multiplex hextuple luciferase assaying to monitor cellular signal processing upstream of five transcription factors and their corresponding transcription factor-binding motifs, using a constitutive promoter as normalization control. The first protocol is provided on how to perform cell culture in preparation toward genetic or pharmaceutical perturbations, as well as transfecting a multiplex hextuple luciferase reporter vector encoding all luciferase reporter units needed for multiplex hextuple luciferase assaying. The second protocol details on how to execute multiplex hextuple luciferase assaying using a microplate reader appropriately equipped to detect the different BLs emitted by all six luciferases. Finally, the third protocol provides details on analyzing, plotting, and interpreting the data obtained by the microplate reader.

Human pancreatic microenvironment promotes ß-cell differentiation via non-canonical WNT5A/JNK and BMP signaling.

In vitro derivation of pancreatic ß-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent ß-cells are still hindered by incomplete understanding of the microenvironment's role in ß-cell development and maturation. Here, we analyze the human mesenchymal and endothelial primary cells from weeks 9-20 fetal pancreas and identify a time point-specific microenvironment that permits ß-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human ß-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during ß-cell maturation. Interestingly, we also identify the endothelial-derived Endocan as a SST+ cell promoting factor. Overall, our study shows that the pancreatic microenvironment-derived factors can mimic in vivo conditions in an in vitro system to generate bona fide ß-cells for translational applications.

Determining effective drug concentrations for selection and counterselection genetics in Drosophila melanogaster.

We recently integrated into fly genetics a set of four selection and two counterselection markers and their corresponding drugs that can be used individually or in combination. These markers eliminate the need to visually screen progeny. Before using these markers in new genetic backgrounds, effective selection/counterselection concentrations should be established for each marker/drug combination. This protocol describes how to set up, perform, and analyze a drug titration curve to determine the effective selection/counterselection drug concentrations for their corresponding markers. For complete details on the use and execution of this protocol, please refer to Matinyan et al., 2021.

Multiplexed drug-based selection and counterselection genetic manipulations in Drosophila.

The power of Drosophila melanogaster as a model system relies on tractable germline genetic manipulations. Despite Drosophila's expansive genetics toolbox, such manipulations are still accomplished one change at a time and depend predominantly on phenotypic screening. We describe a drug-based genetic platform consisting of four selection and two counterselection markers, eliminating the need to screen for modified progeny. These markers work reliably individually or in combination to produce specific genetic outcomes. We demonstrate three example applications of multiplexed drug-based genetics by generating (1) transgenic animals, expressing both components of binary overexpression systems in a single transgenesis step; (2) dual selectable and counterselectable balancer chromosomes; and (3) selectable, fluorescently tagged P[acman] bacterial artificial chromosome (BAC) strains. We perform immunoprecipitation followed by proteomic analysis on one tagged BAC line, demonstrating our platform's applicability to biological discovery. Lastly, we provide a plasmid library resource to facilitate custom transgene design and technology transfer to other model systems.