Publications by authors named "James B Skeath"

36 Publications

A genetic screen for regulators of muscle morphogenesis in Drosophila.

G3 (Bethesda) 2021 May 16. Epub 2021 May 16.

Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA.

The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendon cells. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including back seat driver, kon-tiki, thisbe, and tumbleweed, arguing our screen had the depth and precision to uncover myogenic genes. We also identified new alleles of spalt-major, barren, and patched that presumably disrupt independent muscle morphogenesis pathways. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be mapped and characterized. Our screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that regulate skeletal muscle topology.
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http://dx.doi.org/10.1093/g3journal/jkab172DOI Listing
May 2021

Unc-4 acts to promote neuronal identity and development of the take-off circuit in the CNS.

Elife 2020 03 27;9. Epub 2020 Mar 27.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

The ventral nerve cord (VNC) is composed of thousands of neurons born from a set of individually identifiable stem cells. The VNC harbors neuronal circuits required to execute key behaviors, such as flying and walking. Leveraging the lineage-based functional organization of the VNC, we investigated the developmental and molecular basis of behavior by focusing on lineage-specific functions of the homeodomain transcription factor, Unc-4. We found that Unc-4 functions in lineage 11A to promote cholinergic neurotransmitter identity and suppress the GABA fate. In lineage 7B, Unc-4 promotes proper neuronal projections to the leg neuropil and a specific flight-related take-off behavior. We also uncovered that Unc-4 acts peripherally to promote proprioceptive sensory organ development and the execution of specific leg-related behaviors. Through time-dependent conditional knock-out of Unc-4, we found that its function is required during development, but not in the adult, to regulate the above events.
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http://dx.doi.org/10.7554/eLife.55007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156266PMC
March 2020

GABA-A receptor and mitochondrial TSPO signaling act in parallel to regulate melanocyte stem cell quiescence in larval zebrafish.

Pigment Cell Melanoma Res 2020 05 11;33(3):416-425. Epub 2019 Nov 11.

Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA.

Tissue regeneration and homeostasis often require recruitment of undifferentiated precursors (adult stem cells; ASCs). While many ASCs continuously proliferate throughout the lifetime of an organism, others are recruited from a quiescent state to replenish their target tissue. A long-standing question in stem cell biology concerns how long-lived, non-dividing ASCs regulate the transition between quiescence and proliferation. We study the melanocyte stem cell (MSC) to investigate the molecular pathways that regulate ASC quiescence. Our prior work indicated that GABA-A receptor activation promotes MSC quiescence in larval zebrafish. Here, through pharmacological and genetic approaches we show that GABA-A acts through calcium signaling to maintain MSC quiescence. Unexpectedly, we identified translocator protein (TSPO), a mitochondrial membrane-associated protein that regulates mitochondrial function and metabolic homeostasis, as a parallel regulator of MSC quiescence. We found that both TSPO-specific ligands and induction of gluconeogenesis likely act in the same pathway to promote MSC activation and melanocyte production in larval zebrafish. In contrast, TSPO and gluconeogenesis appear to act in parallel to GABA-A receptor signaling to regulate MSC quiescence and vertebrate pigment patterning.
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http://dx.doi.org/10.1111/pcmr.12836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176537PMC
May 2020

Maintenance of Melanocyte Stem Cell Quiescence by GABA-A Signaling in Larval Zebrafish.

Genetics 2019 10 23;213(2):555-566. Epub 2019 Aug 23.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

In larval zebrafish, melanocyte stem cells (MSCs) are quiescent, but can be recruited to regenerate the larval pigment pattern following melanocyte ablation. Through pharmacological experiments, we found that inhibition of γ-aminobutyric acid (GABA)-A receptor function, specifically the GABA-A ρ subtype, induces excessive melanocyte production in larval zebrafish. Conversely, pharmacological activation of GABA-A inhibited melanocyte regeneration. We used clustered regularly interspaced short palindromic repeats/Cas9 to generate two mutant alleles of , a subtype of GABA-A receptors. Both alleles exhibited robust melanocyte overproduction, while conditional overexpression of inhibited larval melanocyte regeneration. Our data suggest that signaling is necessary to maintain MSC quiescence and sufficient to reduce, but not eliminate, melanocyte regeneration in larval zebrafish.
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http://dx.doi.org/10.1534/genetics.119.302416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781893PMC
October 2019

The extracellular metalloprotease AdamTS-A anchors neural lineages in place within and preserves the architecture of the central nervous system.

Development 2017 09 31;144(17):3102-3113. Epub 2017 Jul 31.

Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.

The extracellular matrix (ECM) regulates cell migration and sculpts organ shape. AdamTS proteins are extracellular metalloproteases known to modify ECM proteins and promote cell migration, but demonstrated roles for AdamTS proteins in regulating CNS structure and ensuring cell lineages remain fixed in place have not been uncovered. Using forward genetic approaches in , we find that reduction of function induces both the mass exodus of neural lineages out of the CNS and drastic perturbations to CNS structure. Expressed and active in surface glia, acts in parallel to and in opposition to / and - to keep CNS lineages rooted in place and to preserve the structural integrity of the CNS. / and - are known to promote tissue stiffness and oppose the function of , which reduces tissue stiffness. Our work supports a model in which AdamTS-A anchors cells in place and preserves CNS architecture by reducing tissue stiffness.
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http://dx.doi.org/10.1242/dev.145854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611953PMC
September 2017

Rapid generation of hypomorphic mutations.

Nat Commun 2017 01 20;8:14112. Epub 2017 Jan 20.

Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
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http://dx.doi.org/10.1038/ncomms14112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263891PMC
January 2017

Collaborative Control of Cell Cycle Progression by the RNA Exonuclease Dis3 and Ras Is Conserved Across Species.

Genetics 2016 06 30;203(2):749-62. Epub 2016 Mar 30.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3'-5' exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, we find that absence of dis3 activity inhibits cell division. We uncover a conserved CDK1 phosphorylation site that when phosphorylated inhibits Dis3's exonuclease, but not endonuclease, activity. Leveraging this information, we show that Dis3's exonuclease function is required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, we find that modest reduction of dis3 function enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, we find that dis3 and ras genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms.
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http://dx.doi.org/10.1534/genetics.116.187930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896191PMC
June 2016

Deletion of Rb1 induces both hyperproliferation and cell death in murine germinal center B cells.

Exp Hematol 2016 Mar 1;44(3):161-5.e4. Epub 2015 Dec 1.

Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO. Electronic address:

The retinoblastoma gene (RB1) has been implicated as a tumor suppressor in multiple myeloma (MM), yet its role remains unclear because in the majority of cases with 13q14 deletions, un-mutated RB1 remains expressed from the retained allele. To explore the role of Rb1 in MM, we examined the functional consequences of single- and double-copy Rb1 loss in germinal center B cells, the cells of origin of MM. We generated mice without Rb1 function in germinal center B cells by crossing Rb1(Flox/Flox) with C-γ-1-Cre (Cγ1) mice expressing the Cre recombinase in class-switched B cells in a p107(-/-) background to prevent p107 from compensating for Rb1 loss (Cγ1-Rb1(F/F)-p107(-/-)). All mice developed normally, but B cells with two copies of Rb1 deleted (Cγ1-Rb1(F/F)-p107(-/-)) exhibited increased proliferation and cell death compared with Cγ1-Rb1(+/+)-p107(-/-) controls ex vivo. In vivo, Cγ1-Rb1(F/F)-p107(-/-) mice had a lower percentage of splenic B220+ cells and reduced numbers of bone marrow antigen-specific secreting cells compared with control mice. Our data indicate that Rb1 loss induces both cell proliferation and death in germinal center B cells. Because no B-cell malignancies developed after 1 year of observation, our data also suggest that Rb1 loss is not sufficient to transform post-germinal center B cells and that additional, specific mutations are likely required to cooperate with Rb1 loss to induce malignant transformation.
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http://dx.doi.org/10.1016/j.exphem.2015.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789175PMC
March 2016

A requirement for ERK-dependent Dicer phosphorylation in coordinating oocyte-to-embryo transition in C. elegans.

Dev Cell 2014 Dec;31(5):614-28

Department of Genetics, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Graduate School of Biomedical Sciences, Houston, TX 77030, USA; Center for Genetics and Genomics, UT MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

Signaling pathways and small RNAs direct diverse cellular events, but few examples are known of defined signaling pathways directly regulating small RNA biogenesis. We show that ERK phosphorylates Dicer on two conserved residues in its RNase IIIb and double-stranded RNA (dsRNA)-binding domains and that phosphorylation of these residues is necessary and sufficient to trigger Dicer's nuclear translocation in worms, mice, and human cells. Phosphorylation of Dicer on either site inhibits Dicer function in the female germline and dampens small RNA repertoire. Our data demonstrate that ERK phosphorylates and inhibits Dicer during meiosis I for oogenesis to proceed normally in Caenorhabditis elegans and that this inhibition is released before fertilization for embryogenesis to proceed normally. The conserved Dicer residues, their phosphorylation by ERK, and the consequences of the resulting modifications implicate an ERK-Dicer nexus as a fundamental component of the oocyte-to-embryo transition and an underlying mechanism coupling extracellular cues to small RNA production.
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http://dx.doi.org/10.1016/j.devcel.2014.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261158PMC
December 2014

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II.

Mol Biol Cell 2014 Oct 30;25(19):2956-69. Epub 2014 Jul 30.

ICCE Institute, Washington University School of Medicine, St. Louis, MO 63110 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 BRIGHT Institute, Washington University School of Medicine, St. Louis, MO 63110

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin-containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin-containing vesicular trafficking during AJ remodeling in live epithelia.
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http://dx.doi.org/10.1091/mbc.E14-04-0894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4230585PMC
October 2014

Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system.

Development 2014 Mar;141(5):1011-21

Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA.

Most neurons of the adult Drosophila ventral nerve cord arise from a burst of neurogenesis during the third larval instar stage. Most of this growth occurs in thoracic neuromeres, which contain 25 individually identifiable postembryonic neuronal lineages. Initially, each lineage consists of two hemilineages--'A' (Notch(On)) and 'B' (Notch(Off))--that exhibit distinct axonal trajectories or fates. No reliable method presently exists to identify these lineages or hemilineages unambiguously other than labor-intensive lineage-tracing methods. By combining mosaic analysis with a repressible cell marker (MARCM) analysis with gene expression studies, we constructed a gene expression map that enables the rapid, unambiguous identification of 23 of the 25 postembryonic lineages based on the expression of 15 transcription factors. Pilot genetic studies reveal that these transcription factors regulate the specification and differentiation of postembryonic neurons: for example, Nkx6 is necessary and sufficient to direct axonal pathway selection in lineage 3. The gene expression map thus provides a descriptive foundation for the genetic and molecular dissection of adult-specific neurogenesis and identifies many transcription factors that are likely to regulate the development and differentiation of discrete subsets of postembryonic neurons.
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http://dx.doi.org/10.1242/dev.102178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929408PMC
March 2014

Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of nitric oxide synthase and Fd59a/Fox-D.

Dev Biol 2014 Apr 7;388(1):117-33. Epub 2014 Feb 7.

Department of Genetics, Washington University School of Medicine, St. Louis 4566, Scott Avenue, St. Louis, MO 63110, USA. Electronic address:

Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, and Tail-up (Islet) to guide the stereotyped differentiation, connectivity, and function of a subset of neurons in Drosophila. The role of Hb9 in directing neuronal differentiation is well documented, but the lineage of Hb9(+) neurons is only partly characterized, its regulation is poorly understood, and most of the downstream genes through which it acts remain at large. Here, we complete the lineage tracing of all embryonic Hb9(+) neurons (to eight neuronal lineages) and provide evidence that hb9, lim3, and tail-up are coordinately regulated by a common set of upstream factors. Through the parallel use of micro-array gene expression profiling and the Dam-ID method, we searched for Hb9-regulated genes, uncovering transcription factors as the most over-represented class of genes regulated by Hb9 (and Nkx6) in the CNS. By a nearly ten-to-one ratio, Hb9 represses rather than activates transcription factors, highlighting transcriptional repression of other transcription factors as a core mechanism by which Hb9 governs neuronal determination. From the small set of genes activated by Hb9, we characterized the expression and function of two - fd59a/foxd, which encodes a transcription factor, and Nitric oxide synthase. Under standard lab conditions, both genes are dispensable for Drosophila development, but Nos appears to inhibit hyper-active behavior and fd59a appears to act in octopaminergic neurons to control egg-laying behavior. Together our data clarify the mechanisms through which Hb9 governs neuronal specification and differentiation and provide an initial characterization of the expression and function of Nos and fd59a in the Drosophila CNS.
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http://dx.doi.org/10.1016/j.ydbio.2014.01.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003567PMC
April 2014

Loss of the spectraplakin short stop activates the DLK injury response pathway in Drosophila.

J Neurosci 2013 Nov;33(45):17863-73

Department of Developmental Biology, Hope Center for Neurological Disorders, and Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. Here we describe a new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, we identify a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. We demonstrate that both phenotypes are the result of overactivation of the DLK signaling pathway. We further show that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons.
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http://dx.doi.org/10.1523/JNEUROSCI.2196-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818558PMC
November 2013

Expression and function of scalloped during Drosophila development.

Dev Dyn 2013 Jul 3;242(7):874-85. Epub 2013 Jun 3.

Department of Biology, Dickinson College, Carlisle, Pennsylvania 17013, USA.

Background: The scalloped (sd) and vestigial (vg) genes function together in Drosophila wing development. Little is known about sd protein (SD) expression during development, or whether sd and vg interact in other developing tissues. To begin to address these questions, we generated an anti-SD antibody.

Results: During embryogenesis, SD is expressed in both central and peripheral nervous systems, and the musculature. SD is also expressed in developing flight appendages. Despite SD expression herein, the peripheral nervous system, musculature, and dorsal limb primordia appeared generally normal in the absence of sd function. SD is also expressed in subsets of ventral nerve cord cells, including neuroblast 1-2 descendants and ventral unpaired median motor neurons (mVUMs). While sd function is not required to specify these neurons, it is necessary for the correct innervation of somatic muscles by the mVUMs. We also show that SD and vg protein (VG) are co-expressed in overlapping and distinctive subsets of cells in embryonic and larval tissues.

Conclusions: We describe the full breadth of SD expression during Drosophila embryogenesis, and identify a requirement for sd function in a subset of motor neurons. This work provides the necessary foundation for functional studies regarding the roles of sd during Drosophila development.
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http://dx.doi.org/10.1002/dvdy.23942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887039PMC
July 2013

Molecular organization of Drosophila neuroendocrine cells by Dimmed.

Curr Biol 2011 Sep 1;21(18):1515-24. Epub 2011 Sep 1.

Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Background: In Drosophila, the basic-helix-loop-helix protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, DIMM confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state.

Results: We first identified 134 transcripts upregulated by DIMM in embryos and then evaluated them systematically using diverse assays (including embryo in situ hybridization, in vivo chromatin immunoprecipitation, and cell-based transactivation assays). We conclude that of eleven strong candidates, six are strongly and directly controlled by DIMM in vivo. The six targets include several large dense-core vesicle (LDCV) proteins, but also proteins in non-LDCV compartments such as the RNA-associated protein Maelstrom. In addition, a functional in vivo assay, combining transgenic RNA interference with MS-based peptidomics, revealed that three DIMM targets are especially critical for its action. These include two well-established LDCV proteins, the amidation enzyme PHM and the ascorbate-regenerating electron transporter cytochrome b(561-1). The third key DIMM target, CAT-4 (CG13248), has not previously been associated with peptide neurosecretion-it encodes a putative cationic amino acid transporter, closely related to the Slimfast arginine transporter. Finally, we compared transcripts upregulated by DIMM with those normally enriched in DIMM neurons of the adult brain and found an intersection of 18 DIMM-regulated genes, which included all six direct DIMM targets.

Conclusions: The results provide a rigorous molecular framework with which to describe the fundamental regulatory organization of diverse neuroendocrine cells.
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http://dx.doi.org/10.1016/j.cub.2011.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184372PMC
September 2011

Ajuba LIM proteins are negative regulators of the Hippo signaling pathway.

Curr Biol 2010 Apr 18;20(7):657-62. Epub 2010 Mar 18.

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.

The mammalian Ajuba LIM proteins (Ajuba, LIMD1, and WTIP) are adaptor proteins that exhibit the potential to communicate cell adhesive events with nuclear responses to remodel epithelia. Determining their role in vivo, however, has been challenging due to overlapping tissue expression and functional redundancy. Thus, we turned to Drosophila, where a single gene, CG11063 or djub, exists. Drosophila lacking the djub gene or depleted of dJub by RNA interference identify djub as an essential gene for development and a novel regulator of epithelial organ size as a component of the conserved Hippo (Hpo) pathway, which has been implicated in both tissue size control and cancer development. djub-deficient tissues were small and had decreased cell numbers as a result of increased apoptosis and decreased proliferation, due to downregulation of DIAP1 and cyclin E. This phenocopies tissues deficient for Yorkie (Yki), the downstream target of the Hippo pathway. djub genetically interacts with the Hippo pathway, and epistasis suggests that djub lies downstream of hpo. In mammalian and Drosophila cells, Ajuba LIM proteins/dJub interact with LATS/Warts (Wts) and WW45/Sav to inhibit phosphorylation of YAP/Yki. This work describes a novel role for the Ajuba LIM proteins as negative regulators of the Hippo signaling pathway.
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http://dx.doi.org/10.1016/j.cub.2010.02.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855397PMC
April 2010

Sanpodo: a context-dependent activator and inhibitor of Notch signaling during asymmetric divisions.

Development 2009 Dec 11;136(24):4089-98. Epub 2009 Nov 11.

Program in Developmental Biology, Washington University School of Medicine, St Louis, MO 63110, USA.

Asymmetric cell divisions generate sibling cells of distinct fates ('A', 'B') and constitute a fundamental mechanism that creates cell-type diversity in multicellular organisms. Antagonistic interactions between the Notch pathway and the intrinsic cell-fate determinant Numb appear to regulate asymmetric divisions in flies and vertebrates. During these divisions, productive Notch signaling requires sanpodo, which encodes a novel transmembrane protein. Here, we demonstrate that Drosophila sanpodo plays a dual role to regulate Notch signaling during asymmetric divisions - amplifying Notch signaling in the absence of Numb in the 'A' daughter cell and inhibiting Notch signaling in the presence of Numb in the 'B' daughter cell. In so doing, sanpodo ensures the asymmetry in Notch signaling levels necessary for the acquisition of distinct fates by the two daughter cells. These findings answer long-standing questions about the restricted ability of Numb and Sanpodo to inhibit and to promote, respectively, Notch signaling during asymmetric divisions.
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http://dx.doi.org/10.1242/dev.040386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781049PMC
December 2009

dbx mediates neuronal specification and differentiation through cross-repressive, lineage-specific interactions with eve and hb9.

Development 2009 Oct 26;136(19):3257-66. Epub 2009 Aug 26.

Program in Developmental Biology, Washington University School of Medicine, St Louis, MO 63110, USA.

Individual neurons adopt and maintain defined morphological and physiological phenotypes as a result of the expression of specific combinations of transcription factors. In particular, homeodomain-containing transcription factors play key roles in determining neuronal subtype identity in flies and vertebrates. dbx belongs to the highly divergent H2.0 family of homeobox genes. In vertebrates, Dbx1 and Dbx2 promote the development of a subset of interneurons, some of which help mediate left-right coordination of locomotor activity. Here, we identify and show that the single Drosophila ortholog of Dbx1/2 contributes to the development of specific subsets of interneurons via cross-repressive, lineage-specific interactions with the motoneuron-promoting factors eve and hb9 (exex). dbx is expressed primarily in interneurons of the embryonic, larval and adult central nervous system, and these interneurons tend to extend short axons and be GABAergic. Interestingly, many Dbx(+) interneurons share a sibling relationship with Eve(+) or Hb9(+) motoneurons. The non-overlapping expression of dbx and eve, or dbx and hb9, within pairs of sibling neurons is initially established as a result of Notch/Numb-mediated asymmetric divisions. Cross-repressive interactions between dbx and eve, and dbx and hb9, then help maintain the distinct expression profiles of these genes in their respective pairs of sibling neurons. Strict maintenance of the mutually exclusive expression of dbx relative to that of eve and hb9 in sibling neurons is crucial for proper neuronal specification, as misexpression of dbx in motoneurons dramatically hinders motor axon outgrowth.
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http://dx.doi.org/10.1242/dev.037242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739143PMC
October 2009

Vestigial expression in the Drosophila embryonic central nervous system.

Dev Dyn 2008 Sep;237(9):2483-9

Department of Biology, Dickinson College, Carlisle, Pennsylvania 17013, USA.

The Drosophila central nervous system is an excellent model system in which to resolve the genetic and molecular control of neuronal differentiation. Here we show that the wing selector vestigial is expressed in discrete sets of neurons. We track the axonal trajectories of VESTIGIAL-expressing cells in the ventral nerve cord and show that these cells descend from neuroblasts 1-2, 5-1, and 5-6. In addition, along the midline, VESTIGIAL is expressed in ventral unpaired median motorneurons and cells that may descend from the median neuroblast. These studies form the requisite descriptive foundation for functional studies addressing the role of vestigial during interneuron differentiation.
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http://dx.doi.org/10.1002/dvdy.21664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631210PMC
September 2008

Linking pattern formation to cell-type specification: Dichaete and Ind directly repress achaete gene expression in the Drosophila CNS.

Proc Natl Acad Sci U S A 2007 Mar 26;104(10):3847-52. Epub 2007 Feb 26.

Program in Molecular Cell Biology and Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, USA.

Mechanisms regulating CNS pattern formation and neural precursor formation are remarkably conserved between Drosophila and vertebrates. However, to date, few direct connections have been made between genes that pattern the early CNS and those that trigger neural precursor formation. Here, we use Drosophila to link directly the function of two evolutionarily conserved regulators of CNS pattern along the dorsoventral axis, the homeodomain protein Ind and the Sox-domain protein Dichaete, to the spatial regulation of the proneural gene achaete (ac) in the embryonic CNS. We identify a minimal achaete regulatory region that recapitulates half of the wild-type ac expression pattern in the CNS and find multiple putative Dichaete-, Ind-, and Vnd-binding sites within this region. Consensus Dichaete sites are often found adjacent to those for Vnd and Ind, suggesting that Dichaete associates with Ind or Vnd on target promoters. Consistent with this finding, we observe that Dichaete can physically interact with Ind and Vnd. Finally, we demonstrate the in vivo requirement of adjacent Dichaete and Ind sites in the repression of ac gene expression in the CNS. Our data identify a direct link between the molecules that pattern the CNS and those that specify distinct cell-types.
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http://dx.doi.org/10.1073/pnas.0611700104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820672PMC
March 2007

Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila Central Nervous System.

Int J Dev Biol 2007 ;51(2):107-15

Program in Molecular Cell Biology, Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA.

The Drosophila embryonic Central Nervous System (CNS) develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of our knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, we combined loss of function with ectopic activation of Egfr activity. We observe that ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. Through this work, we identified the genetic criteria required for the development of the medial and intermediate column cell fates. We also show that ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. Finally, we demonstrate that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells.
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http://dx.doi.org/10.1387/ijdb.062188gzDOI Listing
July 2007

The identification and expression of achaete-scute genes in the branchiopod crustacean Triops longicaudatus.

Gene Expr Patterns 2005 Jun 9;5(5):695-700. Epub 2005 Apr 9.

Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA.

The achaete-scute (ac/sc) genes are a highly conserved family of transcription factors that play important roles in the development of neural cells in both vertebrates and invertebrates. As such, the study of arthropod ac/sc gene expression during neurogenesis has become a model system for investigating the evolution of neural patterning. To date, ac/sc gene expression has been investigated in insects, chelicerates, and myriapods. Here we present the identification of two ac/sc genes from the branchiopod crustacean Triops longicaudatus. Triops longicaudatus achaete-scute homologs1 and 2 (Tl-ASH1 and Tl-ASH2) exhibit dynamic and distinct expression profiles during Triops neurogenesis. Tl-ASH1 expression initiates in nearly all cells of the neurogenic region and subsequently in clusters of cells evenly spaced along the length of the developing limbs. In contrast, Tl-ASH2 initiates expression after Tl-ASH1. In the CNS, only a subset of Tl-ASH1 cells appears to express Tl-ASH2. Similarly, in the PNS individual Tl-ASH2 positive cells appear to arise from the clusters of Tl-ASH1 expressing cells. Shortly after activating Tl-ASH2 expression, these cells enlarge and divide. The expression dynamics of ac/sc genes in Triops parallel those observed in insects and contrasts with those found in chelicerates and myriapods.
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http://dx.doi.org/10.1016/j.modgep.2005.02.005DOI Listing
June 2005

The Tribolium columnar genes reveal conservation and plasticity in neural precursor patterning along the embryonic dorsal-ventral axis.

Dev Biol 2005 Mar;279(2):491-500

Department of Genetics, Washington University School of Medicine, Campus Box 8232, 4566 Scott Avenue, St. Louis, MO 63110, USA.

The Drosophila columnar genes are key regulators of neural precursor formation and patterning along the dorsal-ventral axis of the developing CNS and include ventral nerve cord defective (vnd), intermediate nerve cord defective (ind), muscle segment homeodomain (msh), and Epidermal growth factor receptor (Egfr). To investigate the evolution of neural pattern formation, we identified and determined the expression patterns of Tribolium vnd, ind, and msh, and found that they are expressed in the medial, intermediate, and lateral columns of the developing CNS, respectively, in patterns similar, but not identical, to their Drosophila orthologs. The pattern of Egfr activity suggests that the genetic regulatory mechanisms that initiate Tc-vnd expression are similar in Drosophila and Tribolium, whereas those that initiate Tc-ind have diverged. RNAi analyses of gene function show that Tc-vnd and Tc-ind promote the formation of medial and intermediate column neural precursors and that vnd-mediated repression of ind establishes the boundary between the medial and intermediate columns. These data suggest that columnar gene expression and function underlie neural pattern formation in Drosophila, Tribolium, and potentially all insects, but that subtle spatiotemporal differences in expression of these genes may produce species-specific morphological differences.
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http://dx.doi.org/10.1016/j.ydbio.2004.12.031DOI Listing
March 2005

Epsin potentiates Notch pathway activity in Drosophila and C. elegans.

Development 2004 Dec;131(23):5807-15

Department of Genetics, Washington University in St Louis, School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA.

Endocytosis and trafficking within the endocytosis pathway are known to modulate the activity of different signaling pathways. Epsins promote endocytosis and are postulated to target specific proteins for regulated endocytosis. Here, we present a functional link between the Notch pathway and epsins. We identify the Drosophila ortholog of epsin, liquid facets (lqf), as an inhibitor of cardioblast development in a genetic screen for mutants that affect heart development. We find that lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple Notch-dependent events in Drosophila. We extended the link between the Notch pathway and epsin function to C. elegans, where the C. elegans lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. Our results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis.
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http://dx.doi.org/10.1242/dev.01459DOI Listing
December 2004

Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development.

Mech Dev 2004 Dec;121(12):1495-507

Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue Box 8232, St Louis, MO 63110, USA.

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. Here, we describe the genetic identification and molecular characterisation of the Drosophila Cullin-3 homologue. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. We also find that Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg, but not eye, imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development.
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http://dx.doi.org/10.1016/j.mod.2004.07.007DOI Listing
December 2004

Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis.

Development 2004 Nov 29;131(21):5233-42. Epub 2004 Sep 29.

Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA.

The regulatory networks acting in individual neurons to control their stereotyped differentiation, connectivity, and function are not well understood. Here, we demonstrate that homeodomain protein Nkx6 is a key member of the genetic network of transcription factors that specifies neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9 to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6 acts in parallel with hb9 to regulate motoneuron fate, we find that Nkx6 plays a distinct role to promote axonogenesis, as axon growth of Nkx6-positive motoneurons is severely compromised in Nkx6 mutant embryos. Furthermore, Nkx6 is necessary for the expression of the neural adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work demonstrates that Nkx6 acts in a specific neuronal population to link neuronal subtype identity to neuronal morphology and connectivity.
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http://dx.doi.org/10.1242/dev.01394DOI Listing
November 2004

The Drosophila RCC1 homolog, Bj1, regulates nucleocytoplasmic transport and neural differentiation during Drosophila development.

Dev Biol 2004 Jun;270(1):106-21

Program in Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA.

The Bj1 gene encodes the Drosophila homolog of RCC1, the guanine-nucleotide exchange factor for RanGTPase. Here, we provide the first phenotypic characterization of a RCC1 homolog in a developmental model system. We identified Bj1 (dRCC1) in a genetic screen to identify mutations that alter central nervous system development. We find that zygotic dRCC1 mutant embryos exhibit specific defects in the development and differentiation of lateral CNS neurons although cell division and the cell cycle appear grossly normal. dRCC1 mutant nerve cords contain abnormally large cells with compartmentalized nuclei and exhibit increased transcription in the lateral CNS. As RCC1 is an important component of the nucleocytoplasmic transport machinery, we find that dRCC1 function is required for nuclear import of nuclear localization signal sequence (NLS)-carrying cargo molecules. Finally, we show that dRCC1 is required for cell proliferation and/or survival during germline, eye and wing development and that dRCC1 appears to facilitate apoptosis.
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http://dx.doi.org/10.1016/j.ydbio.2004.02.011DOI Listing
June 2004

Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila.

Dev Cell 2003 Aug;5(2):231-43

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.

Cellular diversity is a fundamental characteristic of complex organisms, and the Drosophila CNS has proved an informative paradigm for understanding the mechanisms that create cellular diversity. One such mechanism is the asymmetric localization of Numb to ensure that sibling cells respond differently to the extrinsic Notch signal and, thus, adopt distinct fates (A and B). Here we focus on the only genes known to function specifically to regulate Notch-dependent asymmetric divisions: sanpodo and numb. We demonstrate that sanpodo, which specifies the Notch-dependent fate (A), encodes a four-pass transmembrane protein that localizes to the cell membrane in the A cell and physically interacts with the Notch receptor. We also show that Numb, which inhibits Notch signaling to specify the default fate (B), physically associates with Sanpodo and inhibits Sanpodo membrane localization in the B cell. Our findings suggest a model in which Numb inhibits Notch signaling through the regulation of Sanpodo membrane localization.
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http://dx.doi.org/10.1016/s1534-5807(03)00226-0DOI Listing
August 2003

The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification.

Development 2003 Sep;130(18):4373-81

Program in Neuroscience, Washington University School of Medicine, St. Louis, MO 63110, USA.

The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.
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http://dx.doi.org/10.1242/dev.00646DOI Listing
September 2003