James Skeath - Academia.edu (original) (raw)

Papers by James Skeath

ABSTRACTTheDrosophilaventral nerve cord (VNC), the fly equivalent of the spinal cord, is composed... more ABSTRACTTheDrosophilaventral nerve cord (VNC), the fly equivalent of the spinal cord, is composed of thousands of neurons that are born from a set of individually identifiable stem cells. The VNC harbors neuronal circuits required for the execution of vital behaviors, such as flying and walking. Taking advantage of the lineage-based functional organization of the VNC and genetic tools we developed, we investigated the molecular and developmental 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 7B lineage, Unc-4 promotes proper neuronal projections to the leg neuropil, the hub for leg-related neuronal circuits and a specific flight-related take-off behavior. We also uncovered that Unc-4 acts peripherally to promote the development of proprioceptive sense organs and the abilities of flies to execute spec...

In developing embryos, cells receive and interpret positional information as they become or- gani... more In developing embryos, cells receive and interpret positional information as they become or- ganized into discrete patterns and structures. One excel- lent model for understanding the genetic regulatory mechanisms that pattern cellular fields is the regulation and function of the achaete-scute complex (AS-C) in the developing nervous system of the fruit fly, Drosophila melanogaster. Three structurally homologous proneural genes-achaete (ac),

zfh-1 is a member of the zfh family of proteins, which all contain zinc finger and homeodomains. ... more zfh-1 is a member of the zfh family of proteins, which all contain zinc finger and homeodomains. The roles and mechanisms of action of most family members are still unclear. However, we have shown previously that another member of the family, the vertebrate ZEB protein, is a transcriptional repressor that binds E box sequences and inhibits myotube formation in cell

Molecular biology of the cell, 2014

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintain... more 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 wor...

Nature, Jan 11, 1997

Asymmetric cell division is a general process used in many developmental contexts to create two d... more Asymmetric cell division is a general process used in many developmental contexts to create two differently fated cells from a single progenitor cell. Intrinsic mechanisms like the asymmetric transmission of cell-fate determinants during cell division, and extrinsic cell-interaction mechanisms, can mediate asymmetric divisions. During embryonic development of the Drosophila central nervous system, neural stem cells called neuroblasts divide asymmetrically to produce another multipotent neuroblast and a ganglion mother cell (GMC) of more restricted developmental potential. Intrinsic mechanisms promote asymmetric division of neuroblasts: for example, the transcription factor Prospero localizes to the basal cell cortex of mitotic neuroblasts and then segregates exclusively into the GMC, which buds off from the basal side of the neuroblast. In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. Here we report the identi...

Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, an... more 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.

Developmental cell, Jan 8, 2014

Signaling pathways and small RNAs direct diverse cellular events, but few examples are known of d... more 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 tr...

Developmental Biology, 2005

The Drosophila columnar genes are key regulators of neural precursor formation and patterning alo... more 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

The International Journal of Developmental Biology, 2007

The Drosophila embryonic Central Nervous System (CNS) develops from the ventrolateral region of t... more 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.

Proceedings of the National Academy of Sciences, 2007

Mechanisms regulating CNS pattern formation and neural precursor formation are remarkably conserv... more 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.

Mechanisms of Development, 2004

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cell... more 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, b-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. q

Mechanisms of Development, 1999

In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or... more In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or cyclin A, we observe that several ganglion mother cells (GMCs) fail to divide. Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; we have determined that depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1, numb mutant embryos, we observe binary cell fate changes ('B' to 'A') in several GMCs as well. Finally, we have demonstrated that expression of Delta in the mesoderm is sufficient to attain both sibling fates. Our results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, our results indicate that Delta signal can be received from outside the sibling pair.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts whic... more The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.

Immunology and Cell Biology, 1991

This study compares the fine specificities of the primary and secondary tluorcscein (FITC)-specif... more This study compares the fine specificities of the primary and secondary tluorcscein (FITC)-specific immiinoglobulin M (IgM) reperloires in BALB/c mouse serum and monoclonal aniibodies (MoAb) and has found reproducible, immunization-dependent diflerences. FITC and four of its homologucs: iodoacetamido lluoresccin (lAF). dichlorotriazinyl aminoduorcsccin (DTAF). substituted rhodaminc isothiocyanale (XRITC) and telramethyl rhodaniine isothiocvanate (TRITC). each conjugated to bovine serum albumin (BSA). were used to determine reactivity patterns of serum IgM from mice immunized once or twice with FITC-haemocyanin (FITC-Hy). Reactivity patterns were also obtained Tor 20 IgM MoAb. eight ofwhich were produced by lusionsofSI'2/0 myeloma cells with splenocyles from mice immunized once (primary) and 12 from mice immunized twice (secondary) wilh FITC'-Hy. Each Mo.Ab exhibited a unique line speciticily paltern. evidence of extensive heterogeneity in the FlTC-specilic repcrioire. Reactivities of IgM MoAb wilh certain homologues were found to be more characteristic of either Ihe primary or secondary response. Polyclonal serum IgM also showed reproducible immunization-dependent variations in fine specificity. Such a pattern could result from idiotypic suppression of primary antibodies, from the expansion of subsets of IgM memory cells utilizing novel genes and/or from somatic mutation absent in primary IgM antibodies.

Genes & Development, 1996

After invagination of the mesodermal primordium in the gastrulating Drosophila embryo, the intern... more After invagination of the mesodermal primordium in the gastrulating Drosophila embryo, the internalized cells migrate in a dorsolateral direction along the overlying ectoderm. This movement generates a stereotyped arrangement of mesodermal cells that is essential for their correct patterning by later position-specific inductive signals. We now report that proper mesodermal cell migration is dependent on the function of a fibroblast growth factor (FGF) receptor encoded by heartless (htl). In htl mutant embryos, the mesoderm forms normally but fails to undergo its usual dorsolateral migration. As a result, cardiac, visceral, and dorsal somatic muscle fates are not induced by Decapentaplegic (Dpp), a transforming growth factor beta family member that is derived from the dorsal ectoderm. Visceral mesoderm can nevertheless be induced by Dpp in the absence of htl function. Ras1 is an important downstream effector of Htl signaling because an activated form of Ras1 partially rescues the htl mutant phenotype. The evolutionary conservation of htl function is suggested by the strikingly similar mesodermal migration and patterning phenotypes associated with FGF receptor mutations in species as diverse as nematode and mouse. These studies establish that Htl signaling provides a vital connection between initial formation of the embryonic mesoderm in Drosophila and subsequent cell-fate specification within this germ layer.

Genes & Development, 1991

Adult Drosophila possess a large number of sensory organs, including large and small bristles and... more Adult Drosophila possess a large number of sensory organs, including large and small bristles and other types of sensilla, each arising from a single mother cell at particular positions in a reproducible pattern. Genetic studies have shown that sensory organ pattern formation is partly coordinated by a number of structurally similar, potential heterodimer-forming, helix-loop-helix (HLH) regulatory proteins. Here, by localizing regulatory gene expression during the development of normal and mutant imaginal discs, we show that two positive regulators of sensory neurogenesis, the proneural achaete and scute proteins, initially trans-activate each other and are transiently expressed in identical patterns, including clusters of wing ectodermal cells and the individual sensory mother cells that arise from them. Two negative regulators, hairy and extramacrochaete, suppress sensory neurogenesis by selectively repressing achaete and scute gene expression, respectively, but in different spatial domains and at different developmental stages. Surprisingly, we also find that the level of achaete-scute activity influences the level of hairy expression, thereby providing feedback control upon achaete-scute activity and sensory organ formation. Some or all of these interactions may involve specific dimerization reactions between different combinations of HLH proteins.

Gene Expression Patterns, 2005

The achaete-scute (ac/sc) genes are a highly conserved family of transcription factors that play ... more 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.

Developmental Dynamics, 2008

The Drosophila central nervous system is an excellent model system in which to resolve the geneti... more 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.