O-GlcNAcylation Dampens Dpp/BMP Signaling to Ensure Proper Drosophila Embryonic Development.

BMP (bone morphogenetic protein) signaling activity is precisely controlled by both pathway agonists and antagonists. Here, we identify a previously unrecognized BMP signaling antagonist. We demonstrate that the Drosophila BMP type I receptor Sax (Saxophone) functions as a Dpp (Decapentaplegic) receptor in Drosophila embryos, but that its activity is normally inhibited by the O-linked glycosyltransferase Sxc (Super sex combs). In wild-type embryos, Sax activity is inhibited, and the BMP type I receptor Tkv (Thickveins) is the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal is transduced by both Tkv and Sax, and elevated Dpp signaling results in embryonic lethality. We also demonstrate that Sxc O-glycosylates Sax and observe elevated Dpp signaling in response to maternal restriction of dietary sugar. These findings link fertility to nutritive environment and point to Sax signaling as the nutrient-sensitive branch of BMP signaling.

Etiology and treatment of adrenoleukodystrophy: new insights from Drosophila.

Adrenoleukodystrophy (ALD) is a fatal progressive neurodegenerative disorder affecting brain white matter. The most common form of ALD is X-linked (X-ALD) and results from mutation of the ABCD1-encoded very-long-chain fatty acid (VLCFA) transporter. X-ALD is clinically heterogeneous, with the cerebral form being the most severe. Diagnosed in boys usually between the ages of 4 and 8 years, cerebral X-ALD symptoms progress rapidly (in as little as 2 years) through declines in cognition, learning and behavior, to paralysis and ultimately to a vegetative state and death. Currently, there are no good treatments for X-ALD. Here, we exploit the Drosophila bubblegum (bgm) double bubble (dbb) model of neurometabolic disease to expand diagnostic power and therapeutic potential for ALD. We show that loss of the Drosophila long-/very-long-chain acyl-CoA synthetase genes bgm and/or dbb is indistinguishable from loss of the Drosophila ABC transporter gene ABCD Shared loss-of-function phenotypes for synthetase and transporter mutants point to a lipid metabolic pathway association with ALD-like neurodegenerative disease in Drosophila; a pathway association that has yet to be established in humans. We also show that manipulation of environment increases the severity of neurodegeneration in bgm and dbb mutant flies, adding even further to a suite of new candidate ALD disease-causing genes and pathways in humans. Finally, we show that it is a lack of lipid metabolic pathway product and not (as commonly thought) an accumulation of pathway precursor that is causative of neurometabolic disease: addition of medium-chain fatty acids to the diet of bgm or dbb mutant flies prevents the onset of neurodegeneration. Taken together, our data provide new foundations both for diagnosing ALD and for designing effective, mechanism-based treatment protocols.This article has an associated First Person interview with the first author of the paper.

Lef1-dependent hypothalamic neurogenesis inhibits anxiety.

While innate behaviors are conserved throughout the animal kingdom, it is unknown whether common signaling pathways regulate the development of neuronal populations mediating these behaviors in diverse organisms. Here, we demonstrate that the Wnt/ß-catenin effector Lef1 is required for the differentiation of anxiolytic hypothalamic neurons in zebrafish and mice, although the identity of Lef1-dependent genes and neurons differ between these 2 species. We further show that zebrafish and Drosophila have common Lef1-dependent gene expression in their respective neuroendocrine organs, consistent with a conserved pathway that has diverged in the mouse. Finally, orthologs of Lef1-dependent genes from both zebrafish and mouse show highly correlated hypothalamic expression in marmosets and humans, suggesting co-regulation of 2 parallel anxiolytic pathways in primates. These findings demonstrate that during evolution, a transcription factor can act through multiple mechanisms to generate a common behavioral output, and that Lef1 regulates circuit development that is fundamentally important for mediating anxiety in a wide variety of animal species.

Developing sustainable research careers for KL2 scholars: The importance of an inclusive environment and mentorship.

INTRODUCTION: The National Clinical and Translational Science Award (CTSA) Consortium 2.0 has developed common metrics as a collaborative project for all participating sites. Metrics address several important aspects and functions of the consortium, including workforce development. The first workforce development metrics to be proposed for all CTSA hubs include the proportion of CTSA-supported trainees and scholars with sustainable careers in translational research and the diversity and inclusiveness of programs. METHODS AND RESULTS: The University of Utah Center for Clinical and Translational Science (CCTS), a CTSA hub, has been actively engaged in mentoring translational scientists for the last decade. We have developed programs, processes, and institutional policies that support translational scientists, which have resulted in 100% of our KL2 scholars remaining engaged in translational science and in increasing the inclusion of individuals under-represented in medicine in our research enterprise. In this paper, we share details of our program and what we believe are evidence-based best practices for developing sustainable translational research careers for all aspiring junior faculty members. CONCLUSIONS: The University of Utah Center for Clinical and Translational Science has been integral in catalyzing interactions across the campus to reverse the negative trends seen nationally in sustaining clinician scientists. Our programs and processes can serve as a model for other institutions seeking to develop translational scientists.

Modeling congenital disease and inborn errors of development in Drosophila melanogaster.

Fly models that faithfully recapitulate various aspects of human disease and human health-related biology are being used for research into disease diagnosis and prevention. Established and new genetic strategies in Drosophila have yielded numerous substantial successes in modeling congenital disorders or inborn errors of human development, as well as neurodegenerative disease and cancer. Moreover, although our ability to generate sequence datasets continues to outpace our ability to analyze these datasets, the development of high-throughput analysis platforms in Drosophila has provided access through the bottleneck in the identification of disease gene candidates. In this Review, we describe both the traditional and newer methods that are facilitating the incorporation of Drosophila into the human disease discovery process, with a focus on the models that have enhanced our understanding of human developmental disorders and congenital disease. Enviable features of the Drosophila experimental system, which make it particularly useful in facilitating the much anticipated move from genotype to phenotype (understanding and predicting phenotypes directly from the primary DNA sequence), include its genetic tractability, the low cost for high-throughput discovery, and a genome and underlying biology that are highly evolutionarily conserved. In embracing the fly in the human disease-gene discovery process, we can expect to speed up and reduce the cost of this process, allowing experimental scales that are not feasible and/or would be too costly in higher eukaryotes.

Neurodegeneration in a Drosophila model of adrenoleukodystrophy: the roles of the Bubblegum and Double bubble acyl-CoA synthetases.

Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum(bgm) and double bubble(dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivois causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6(encoding a very-long-chain ACS), a human homolog of bgm and dbb.

The leukodystrophies.

Leukodystrophies are a group of genetically determined disorders that affect development or maintenance of central nervous system myelin. Leukodystrophies have a reported incidence of 1 in 7500 live births, but fewer than half of patients receive a specific diagnosis. In this review, the authors discuss types of leukodystrophies: their prevalence, clinical presentation, symptoms, and diagnosis, as well as current and future treatments. Diagnosis is based on a combination of history, exam, radiological, and laboratory findings, including genetic testing. Leukodystrophies can present at any age from infancy to adulthood, with variability in disease progression and clinical presentation, ranging from developmental delay to seizures to spasticity. Although there are few cures, there are significant opportunities for care and improvements in patient well-being. Their high prevalence, combined with rapid advances in imaging, genetics, and potential treatments, makes an understanding of the leukodystrophies necessary for care providers in genetics and neurology.

Mummy, A UDP-N-acetylglucosamine pyrophosphorylase, modulates DPP signaling in the embryonic epidermis of Drosophila.

The evolutionarily conserved JNK/AP-1 (Jun N-terminal kinase/activator protein 1) and BMP (Bone Morphogenetic Protein) signaling cascades are deployed hierarchically to regulate dorsal closure in the fruit fly Drosophila melanogaster. In this developmental context, the JNK/AP-1 signaling cascade transcriptionally activates BMP signaling in leading edge epidermal cells. Here we show that the mummy (mmy) gene product, which is required for dorsal closure, functions as a BMP signaling antagonist. Genetic and biochemical tests of Mmy's role as a BMP-antagonist indicate that its function is independent of AP-1, the transcriptional trigger of BMP signal transduction in leading edge cells. pMAD (phosphorylated Mothers Against Dpp) activity data show the mmy gene product to be a new type of epidermal BMP regulator - one which transforms a BMP ligand from a long- to a short-range signal. mmy codes for the single UDP-N-acetylglucosamine pyrophosphorylase in Drosophila, and its requirement for attenuating epidermal BMP signaling during dorsal closure points to a new role for glycosylation in defining a highly restricted BMP activity field in the fly. These findings add a new dimension to our understanding of mechanisms modulating the BMP signaling gradient.

Programmed ribosomal frameshifting in the expression of the regulator of intestinal stem cell proliferation, adenomatous polyposis coli (APC).

A programmed ribosomal frameshift (PRF) in the decoding of APC (adenomatous polyposis coli) mRNA has been identified and characterized in Caenorhabditis worms, Drosophila and mosquitoes. The frameshift product lacks the C-terminal approximately one-third of the product of standard decoding and instead has a short sequence encoded by the -1 frame which is just 13 residues in C. elegans, but is 125 in D. melanogaster. The frameshift site is A_AA.A_AA.C in Caenorhabditids, fruit flies and the mosquitoes studied while a variant A_AA.A_AA.A is found in some other nematodes. The predicted secondary RNA structure of the downstream stimulators varies considerably in the species studied. In the twelve sequenced Drosophila genomes, it is a long stem with a four-way junction in its loop. In the five sequenced Caenorhabditis species, it is a short RNA pseudoknot with an additional stem in loop 1. The efficiency of frameshifting varies significantly, depending on the particular stimulator within the frameshift cassette, when tested with reporter constructs in rabbit reticulocyte lysates. Phylogenetic analysis of the distribution of APC programmed ribosomal frameshifting cassettes suggests it has an ancient origin and raises questions about a possibility of synthesis of alternative protein products during expression of APC in other organisms such as humans. The origin of APC as a PRF candidate emerged from a prior study of evolutionary signatures derived from comparative analysis of the 12 fly genomes. Three other proposed PRF candidates (Xbp1, CG32736, CG14047) with switches in conservation of reading frames are likely explained by mechanisms other than PRF.

Raw mediates antagonism of AP-1 activity in Drosophila.

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. Here we show that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. Our studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, we provide the first evidence that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, our mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor.

Head involution in Drosophila: genetic and morphogenetic connections to dorsal closure.

Dorsal closure and head involution are complex morphogenetic processes that occur nearly simultaneously, midway through Drosophila embryonic development. While dorsal closure has been studied extensively in terms of both its morphology and genetics, head involution has not been described comprehensively. A thorough review of the literature nonetheless reveals considerable information regarding the genetic components of head involution. In several instances, authors have made explicit references to head involution in regard to mutant phenotypes; in others, we have made this connection. Here we collect, catalogue, and discuss published head involution studies. In considering and integrating the data, an enhanced appreciation of the molecular mechanisms underlying head involution and its molecular kinship with dorsal closure has emerged.

scylla and charybde, homologues of the human apoptotic gene RTP801, are required for head involution in Drosophila.

We employed robotic methods and the whole-genome sequence of Drosophila melanogaster to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, we identified a pair of genes, scylla (scyl) and charybde (chrb), that code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, our studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, our studies reveal roles for the scyl and chrb gene products as links between patterning and cell death.

Amnioserosa is required for dorsal closure in Drosophila.

Dorsal closure in the fruit fly Drosophila melanogaster is a complex morphogenetic process, driven by sequential signaling cascades and involving multiple forces, which contribute to cell movements and rearrangements as well as to changes in cell shape. During closure, lateral epidermal cells elongate along the dorsoventral axis and subsequently spread dorsally to cover the embryonic dorsal surface. Amnioserosal cells, which are the original occupants of the most dorsal position in the developing embryo, constrict during closure; thus, the increase in epidermal surface area is accommodated by a reduction in the amnioserosal surface area. Several of the epidermal requirements for closure have been established in functional assays. In contrast, amnioserosal requirements for closure have remained elusive, in part because laser ablation and clonal approaches are limited to only subsets of amnioserosal cells. Here, we report our use of the UAS-GAL4 system to target expression of the cell autonomous toxin Ricin-A to all cells of the amnioserosa. We show that ablation of the amnioserosa leads to clear defects in dorsal closure and, thus, directly demonstrate a role for the amnioserosa in dorsal closure. We also show that DJNK (Drosophila Jun N-terminal kinase) signaling, an epidermal trigger of closure, is unaffected by amnioserosal ablation. These data, together with our demonstration that amnioserosal ablated and Dpp signaling mutant embryos exhibit shared loss-of-function phenotypes, point to a requirement for the amnioserosa in dorsal closure that is downstream of Dpp, perhaps as part of a paracrine response to this signaling cascade.

Profiling patterned transcripts in Drosophila embryos.

Here we describe a high-throughput screen to isolate transcripts with spatially restricted patterns of expression in early embryos. Our approach utilizes robotic automation for rapid analysis of sequence-selected cDNAs in a whole-mount in situ hybridization assay. We determined the spatial distribution of a random collection of 778 different genes from an embryonic cDNA library and show that a significant fraction of these exhibit patterned profiles of expression. In addition, gene ontology studies revealed groups of gene products exhibiting shared expression patterns, providing new insights into the largely overlooked effector molecules that function in development. As described in this paper, automated hybridization to whole-mount embryos in situ proved to be straightforward and provided us with a very powerful method for the global survey of gene expression in early embryos. From the perspective of biological significance, our finding that many spatially restricted transcripts correspond to loci encoding novel transcripts that have not been previously identified in nearly saturating genetic screens for maternal effect and zygotic lethals is particularly notable.

B56-associated protein phosphatase 2A is required for survival and protects from apoptosis in Drosophila melanogaster.

Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. B56:PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase DRICE: B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.