Publications by authors named "Andrew D Chisholm"

80 Publications

Caenorhabditis elegans Junctophilin has tissue-specific functions and regulates neurotransmission with extended-synaptotagmin.

Genetics 2021 Apr 19. Epub 2021 Apr 19.

Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA.

The junctophilin family of proteins tether together plasma membrane (PM) and endoplasmic reticulum (ER) membranes, and couple PM- and ER-localized calcium channels. Understanding in vivo functions of junctophilins is of great interest for dissecting the physiological roles of ER-PM contact sites. Here, we show that the sole C. elegans junctophilin JPH-1 localizes to discrete membrane contact sites in neurons and muscles and has important tissue-specific functions. jph-1 null mutants display slow growth and development due to weaker contraction of pharyngeal muscles, leading to reduced feeding. In the body wall muscle, JPH-1 co-localizes with the PM-localized EGL-19 voltage-gated calcium channel and ER-localized UNC-68/RyR calcium channel, and is required for animal movement. In neurons, JPH-1 co-localizes with the membrane contact site protein Extended-SYnaptoTagmin 2 (ESYT-2) in soma, and is present near presynaptic release sites. Interestingly, jph-1 and esyt-2 null mutants display mutual suppression in their response to aldicarb, suggesting that JPH-1 and ESYT-2 have antagonistic roles in neuromuscular synaptic transmission. Additionally, we find an unexpected cell non-autonomous effect of jph-1 in axon regrowth after injury. Genetic double mutant analysis suggests that jph-1 functions in overlapping pathways with two PM-localized voltage-gated calcium channels, egl-19 and unc-2, and unc-68/RyR for animal health and development. Finally, we show that jph-1 regulates the colocalization of EGL-19 and UNC-68 and that unc-68/RyR is required for JPH-1 localization to ER-PM puncta. Our data demonstrate important roles for junctophilin in cellular physiology, and also provide insights into how junctophilin functions together with other calcium channels in vivo.
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http://dx.doi.org/10.1093/genetics/iyab063DOI Listing
April 2021

Form and function of the apical extracellular matrix: new insights from , and the vertebrate inner ear.

Fac Rev 2020 22;9:27. Epub 2020 Dec 22.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.

Apical extracellular matrices (aECMs) are the extracellular layers on the apical sides of epithelia. aECMs form the outer layer of the skin in most animals and line the luminal surface of internal tubular epithelia. Compared to the more conserved basal ECMs (basement membranes), aECMs are highly diverse between tissues and between organisms and have been more challenging to understand at mechanistic levels. Studies in several genetic model organisms are revealing new insights into aECM composition, biogenesis, and function and have begun to illuminate common principles and themes of aECM organization. There is emerging evidence that, in addition to mechanical or structural roles, aECMs can participate in reciprocal signaling with associated epithelia and other cell types. Studies are also revealing mechanisms underlying the intricate nanopatterns exhibited by many aECMs. In this review, we highlight recent findings from well-studied model systems, including the external cuticle and ductal aECMs of , and other insects and the internal aECMs of the vertebrate inner ear.
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http://dx.doi.org/10.12703/r/9-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886070PMC
December 2020

A null mutation of .

MicroPubl Biol 2020 Jun 7;2020. Epub 2020 Jun 7.

Section of Neurobiology, University of California San Diego, La Jolla, CA 92093, United States.

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http://dx.doi.org/10.17912/micropub.biology.000263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297599PMC
June 2020

VWA-8 is a mitochondrial protein.

MicroPubl Biol 2020 Jun 3;2020. Epub 2020 Jun 3.

Section of Neurobiology, University of California San Diego, La Jolla, CA 92093, United States.

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http://dx.doi.org/10.17912/micropub.biology.000264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297598PMC
June 2020

Wounding triggers MIRO-1 dependent mitochondrial fragmentation that accelerates epidermal wound closure through oxidative signaling.

Nat Commun 2020 02 26;11(1):1050. Epub 2020 Feb 26.

Center for Stem Cell and Regenerative Medicine and Department of Cardiology of The Second Affiliated Hospital, Zhejiang University School of Medicine, 310058, Hangzhou, China.

Organisms respond to tissue damage through the upregulation of protective responses which restore tissue structure and metabolic function. Mitochondria are key sources of intracellular oxidative metabolic signals that maintain cellular homeostasis. Here we report that tissue and cellular wounding triggers rapid and reversible mitochondrial fragmentation. Elevated mitochondrial fragmentation either in fzo-1 fusion-defective mutants or after acute drug treatment accelerates actin-based wound closure. Wounding triggered mitochondrial fragmentation is independent of the GTPase DRP-1 but acts via the mitochondrial Rho GTPase MIRO-1 and cytosolic Ca. The fragmented mitochondria and accelerated wound closure of fzo-1 mutants are dependent on MIRO-1 function. Genetic and transcriptomic analyzes show that enhanced mitochondrial fragmentation accelerates wound closure via the upregulation of mtROS and Cytochrome P450. Our results reveal how mitochondrial dynamics respond to cellular and tissue injury and promote tissue repair.
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http://dx.doi.org/10.1038/s41467-020-14885-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044169PMC
February 2020

The mRNA Decay Factor CAR-1/LSM14 Regulates Axon Regeneration via Mitochondrial Calcium Dynamics.

Curr Biol 2020 03 23;30(5):865-876.e7. Epub 2020 Jan 23.

Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

mRNA decay factors regulate mRNA turnover by recruiting non-translating mRNAs and targeting them for translational repression and mRNA degradation. How mRNA decay pathways regulate cellular function in vivo with specificity is poorly understood. Here, we show that C. elegans mRNA decay factors, including the translational repressors CAR-1/LSM14 and CGH-1/DDX6, and the decapping enzymes DCAP-1/DCP1, function in neurons to differentially regulate axon development, maintenance, and regrowth following injury. In neuronal cell bodies, CAR-1 fully colocalizes with CGH-1 and partially colocalizes with DCAP-1, suggesting that mRNA decay components form at least two types of cytoplasmic granules. Following axon injury in adult neurons, loss of CAR-1 or CGH-1 results in increased axon regrowth and growth cone formation, whereas loss of DCAP-1 or DCAP-2 results in reduced regrowth. To determine how CAR-1 inhibits regrowth, we analyzed mRNAs bound to pan-neuronally expressed GFP::CAR-1 using a crosslinking and immunoprecipitation-based approach. Among the putative mRNA targets of CAR-1, we characterized the roles of micu-1, a regulator of the mitochondrial calcium uniporter MCU-1, in axon injury. We show that loss of car-1 results increased MICU-1 protein levels, and that enhanced axon regrowth in car-1 mutants is dependent on micu-1 and mcu-1. Moreover, axon injury induces transient calcium influx into axonal mitochondria, dependent on MCU-1. In car-1 loss-of-function mutants and in micu-1 overexpressing animals, the axonal mitochondrial calcium influx is more sustained, which likely underlies enhanced axon regrowth. Our data uncover a novel pathway that controls axon regrowth through axonal mitochondrial calcium uptake.
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http://dx.doi.org/10.1016/j.cub.2019.12.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147385PMC
March 2020

Inhibition of Axon Regeneration by Liquid-like TIAR-2 Granules.

Neuron 2019 10 1;104(2):290-304.e8. Epub 2019 Aug 1.

Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Phase separation into liquid-like compartments is an emerging property of proteins containing prion-like domains (PrLDs), yet the in vivo roles of phase separation remain poorly understood. TIA proteins contain a C-terminal PrLD, and mutations in the PrLD are associated with several diseases. Here, we show that the C. elegans TIAR-2/TIA protein functions cell autonomously to inhibit axon regeneration. TIAR-2 undergoes liquid-liquid phase separation in vitro and forms granules with liquid-like properties in vivo. Axon injury induces a transient increase in TIAR-2 granule number. The PrLD is necessary and sufficient for granule formation and inhibiting regeneration. Tyrosine residues within the PrLD are important for granule formation and inhibition of regeneration. TIAR-2 is also serine phosphorylated in vivo. Non-phosphorylatable TIAR-2 variants do not form granules and are unable to inhibit axon regeneration. Our data demonstrate an in vivo function for phase-separated TIAR-2 and identify features critical for its function in axon regeneration.
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http://dx.doi.org/10.1016/j.neuron.2019.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813885PMC
October 2019

Corrigendum: A Pipeline for Volume Electron Microscopy of the Nervous System.

Front Neural Circuits 2019 20;13:16. Epub 2019 Mar 20.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

[This corrects the article DOI: 10.3389/fncir.2018.00094.].
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http://dx.doi.org/10.3389/fncir.2019.00016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436607PMC
March 2019

A high-content imaging approach to profile embryonic development.

Development 2019 04 11;146(7). Epub 2019 Apr 11.

Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA

The embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.
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http://dx.doi.org/10.1242/dev.174029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467471PMC
April 2019

A Pipeline for Volume Electron Microscopy of the Nervous System.

Front Neural Circuits 2018 21;12:94. Epub 2018 Nov 21.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using . Recent advances in vEM allow mapping new connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the nervous system and beyond.
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http://dx.doi.org/10.3389/fncir.2018.00094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262311PMC
April 2019

Expanded genetic screening in identifies new regulators and an inhibitory role for NAD in axon regeneration.

Elife 2018 11 21;7. Epub 2018 Nov 21.

Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, United States.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.
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http://dx.doi.org/10.7554/eLife.39756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281318PMC
November 2018

DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature neurons.

J Cell Biol 2019 01 5;218(1):125-133. Epub 2018 Nov 5.

Neuroscience Program, Ottawa Hospital Research Institute, Ottawa, Canada

Neuronal morphology and circuitry established during early development must often be maintained over the entirety of animal lifespans. Compared with neuronal development, the mechanisms that maintain mature neuronal structures and architecture are little understood. The conserved disco-interacting protein 2 (DIP2) consists of a DMAP1-binding domain and two adenylate-forming domains (AFDs). We show that the DIP-2 maintains morphology of mature neurons. loss-of-function mutants display a progressive increase in ectopic neurite sprouting and branching during late larval and adult life. In adults, also inhibits initial stages of axon regeneration cell autonomously and acts in parallel to DLK-1 MAP kinase and EFA-6 pathways. The function of DIP-2 in maintenance of neuron morphology and in axon regrowth requires its AFD domains and is independent of its DMAP1-binding domain. Our findings reveal a new conserved regulator of neuronal morphology maintenance and axon regrowth after injury.
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http://dx.doi.org/10.1083/jcb.201804207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314549PMC
January 2019

Genetic Suppression of Basement Membrane Defects in by Gain of Function in Extracellular Matrix and Cell-Matrix Attachment Genes.

Genetics 2018 04 12;208(4):1499-1512. Epub 2018 Feb 12.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093

Basement membranes are extracellular matrices essential for embryonic development in animals. Peroxidasins are extracellular peroxidases implicated in the unique sulfilimine cross-links between type IV basement membrane collagens. Loss of function in the peroxidasin PXN-2 results in fully penetrant embryonic or larval lethality. Using genetic suppressor screening, we find that the requirement for PXN-2 in development can be bypassed by gain of function in multiple genes encoding other basement membrane components, or proteins implicated in cell-matrix attachment. We identify multiple alleles of , encoding the transmembrane protein myotactin, which suppress phenotypes of null mutants and of other basement membrane mutants such as F-spondin/ These suppressor alleles cause missense alterations in two pairs of FNIII repeats in the extracellular domain; they act dominantly and have no detectable phenotypes alone, suggesting they cause gain of function. We also identify suppressor missense mutations affecting basement membrane components type IV collagen (, ) and perlecan (), as well as a mutation affecting spectraplakin (), a component of the epidermal cytoskeleton. These suppressor alleles do not bypass the developmental requirement for core structural proteins of the basement membrane such as laminin or type IV collagen. In conclusion, putative gain-of-function alterations in matrix proteins or in cell-matrix receptors can overcome the requirement for certain basement membrane proteins in embryonic development, revealing previously unknown plasticity in the genetic requirements for the extracellular matrix.
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http://dx.doi.org/10.1534/genetics.118.300731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887144PMC
April 2018

A Neuronal piRNA Pathway Inhibits Axon Regeneration in C. elegans.

Neuron 2018 02 27;97(3):511-519.e6. Epub 2018 Jan 27.

Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

The PIWI-interacting RNA (piRNA) pathway has long been thought to function solely in the germline, but evidence for its functions in somatic cells is emerging. Here we report an unexpected role for the piRNA pathway in Caenorhabditis elegans sensory axon regeneration after injury. Loss of function in a subset of components of the piRNA pathway results in enhanced axon regrowth. Two essential piRNA factors, PRDE-1 and PRG-1/PIWI, inhibit axon regeneration in a gonad-independent and cell-autonomous manner. By smFISH analysis we find that prde-1 transcripts are present in neurons, as well as germ cells. The piRNA pathway inhibits axon regrowth independent of nuclear transcriptional silencing but dependent on the slicer domain of PRG-1/PIWI, suggesting that post-transcriptional gene silencing is involved. Our results reveal the neuronal piRNA pathway as a novel intrinsic repressor of axon regeneration.
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http://dx.doi.org/10.1016/j.neuron.2018.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866297PMC
February 2018

An Antimicrobial Peptide and Its Neuronal Receptor Regulate Dendrite Degeneration in Aging and Infection.

Neuron 2018 01;97(1):125-138.e5

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA; Department of Neurobiology and Duke Institute for Brain Sciences, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

Infections have been identified as possible risk factors for aging-related neurodegenerative diseases, but it remains unclear whether infection-related immune molecules have a causative role in neurodegeneration during aging. Here, we reveal an unexpected role of an epidermally expressed antimicrobial peptide, NLP-29 (neuropeptide-like protein 29), in triggering aging-associated dendrite degeneration in C. elegans. The age-dependent increase of nlp-29 expression is regulated by the epidermal tir-1/SARM-pmk-1/p38 MAPK innate immunity pathway. We further identify an orphan G protein-coupled receptor NPR-12 (neuropeptide receptor 12) acting in neurons as a receptor for NLP-29 and demonstrate that the autophagic machinery is involved cell autonomously downstream of NPR-12 to transduce degeneration signals. Finally, we show that fungal infections cause dendrite degeneration using a similar mechanism as in aging, through NLP-29, NPR-12, and autophagy. Our findings reveal an important causative role of antimicrobial peptides, their neuronal receptors, and the autophagy pathway in aging- and infection-associated dendrite degeneration.
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http://dx.doi.org/10.1016/j.neuron.2017.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757245PMC
January 2018

A toolkit for GFP-mediated tissue-specific protein degradation in .

Development 2017 07 15;144(14):2694-2701. Epub 2017 Jun 15.

Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of proteins.
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http://dx.doi.org/10.1242/dev.150094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536931PMC
July 2017

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in .

Front Neurosci 2017 10;11:263. Epub 2017 May 10.

Section of Neurobiology, Division of Biological Sciences, University of CaliforniaSan Diego, CA, USA.

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode to test regeneration after neuronal injury , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins , and , and the putative oxidoreductase . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of or was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of suppressed the defective regrowth in mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.
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http://dx.doi.org/10.3389/fnins.2017.00263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423972PMC
May 2017

The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans.

Genetics 2016 Nov;204(3):849-882

Department of Pathology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.
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http://dx.doi.org/10.1534/genetics.115.186262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105865PMC
November 2016

Tissue-specific regulation of alternative polyadenylation represses expression of a neuronal ankyrin isoform in epidermal development.

Development 2017 02 13;144(4):698-707. Epub 2017 Jan 13.

Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA

Differential mRNA polyadenylation plays an important role in shaping the neuronal transcriptome. In , several ankyrin isoforms are produced from the locus through alternative polyadenylation. Here, we identify a key role for an intronic polyadenylation site (PAS) in temporal- and tissue-specific regulation of UNC-44/ankyrin isoforms. Removing an intronic PAS results in ectopic expression of the neuronal ankyrin isoform in non-neural tissues. This mis-expression underlies epidermal developmental defects in mutants of the conserved tumor suppressor death-associated protein kinase We have previously reported that the use of this intronic PAS depends on the nuclear polyadenylation factor SYDN-1, which inhibits the RNA polymerase II CTD phosphatase SSUP-72. Consistent with this, loss of blocks ectopic expression of neuronal ankyrin and suppresses epidermal morphology defects of These effects of are mediated by autonomously in the epidermis. We also show that a peptidyl-prolyl isomerase PINN-1 antagonizes SYDN-1 in the spatiotemporal control of neuronal ankyrin isoform. Moreover, the nuclear localization of PINN-1 is altered in mutants. Our data reveal that tissue and stage-specific expression of ankyrin isoforms relies on differential activity of positive and negative regulators of alternative polyadenylation.
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http://dx.doi.org/10.1242/dev.146001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312038PMC
February 2017

DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair.

Elife 2016 09 23;5. Epub 2016 Sep 23.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, San Diego, United States.

Epidermal barrier epithelia form a first line of defense against the environment, protecting animals against infection and repairing physical damage. In death-associated protein kinase (DAPK-1) regulates epidermal morphogenesis, innate immunity and wound repair. Combining genetic suppressor screens and pharmacological tests, we find that DAPK-1 maintains epidermal tissue integrity through regulation of the microtubule (MT) cytoskeleton. epidermal phenotypes are suppressed by treatment with microtubule-destabilizing drugs and mimicked or enhanced by microtubule-stabilizing drugs. Loss of function in , the member of the Patronin/Nezha/CAMSAP family of MT minus-end binding proteins, suppresses epidermal and innate immunity phenotypes. Over-expression of the MT-binding CKK domain of PTRN-1 triggers epidermal and immunity defects resembling those of mutants, and PTRN-1 localization is regulated by DAPK-1. DAPK-1 and PTRN-1 physically interact in co-immunoprecipitation experiments, and DAPK-1 itself undergoes MT-dependent transport. Our results uncover an unexpected interdependence of DAPK-1 and the microtubule cytoskeleton in maintenance of epidermal integrity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053806PMC
http://dx.doi.org/10.7554/eLife.15833DOI Listing
September 2016

Regulation of Microtubule Dynamics in Axon Regeneration: Insights from C. elegans.

F1000Res 2016 27;5. Epub 2016 Apr 27.

Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.

The capacity of an axon to regenerate is regulated by its external environment and by cell-intrinsic factors. Studies in a variety of organisms suggest that alterations in axonal microtubule (MT) dynamics have potent effects on axon regeneration. We review recent findings on the regulation of MT dynamics during axon regeneration, focusing on the nematode Caenorhabditis elegans. In C. elegans the dual leucine zipper kinase (DLK) promotes axon regeneration, whereas the exchange factor for Arf6 (EFA-6) inhibits axon regeneration. Both DLK and EFA-6 respond to injury and control axon regeneration in part via MT dynamics. How the DLK and EFA-6 pathways are related is a topic of active investigation, as is the mechanism by which EFA-6 responds to axonal injury. We evaluate potential candidates, such as the MT affinity-regulating kinase PAR-1/MARK, in regulation of EFA-6 and axonal MT dynamics in regeneration.
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http://dx.doi.org/10.12688/f1000research.8197.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892358PMC
June 2016

CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins.

Elife 2016 06 2;5. Epub 2016 Jun 2.

Section of Neurobiology, University of California, San Diego, Division of Biological Sciences, San Diego, United States.

Axon injury triggers dramatic changes in gene expression. While transcriptional regulation of injury-induced gene expression is widely studied, less is known about the roles of RNA binding proteins (RBPs) in post-transcriptional regulation during axon regeneration. In C. elegans the CELF (CUGBP and Etr-3 Like Factor) family RBP UNC-75 is required for axon regeneration. Using crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq) we identify a set of genes involved in synaptic transmission as mRNA targets of UNC-75. In particular, we show that UNC-75 regulates alternative splicing of two mRNA isoforms of the SNARE Syntaxin/unc-64. In C. elegans mutants lacking unc-75 or its targets, regenerating axons form growth cones, yet are deficient in extension. Extending these findings to mammalian axon regeneration, we show that mouse Celf2 expression is upregulated after peripheral nerve injury and that Celf2 mutant mice are defective in axon regeneration. Further, mRNAs for several Syntaxins show CELF2 dependent regulation. Our data delineate a post-transcriptional regulatory pathway with a conserved role in regenerative axon extension.
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http://dx.doi.org/10.7554/eLife.16072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946901PMC
June 2016

Targeted Mutagenesis of Duplicated Genes in Caenorhabditis elegans Using CRISPR-Cas9.

J Genet Genomics 2016 Feb 25;43(2):103-6. Epub 2016 Jan 25.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Division of Biological Sciences, Section of Neurobiology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jgg.2015.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291165PMC
February 2016

Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG.

Sci Rep 2016 Feb 10;6:21271. Epub 2016 Feb 10.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093.

The genetically encoded photosensitizer miniSOG (mini Singlet Oxygen Generator) can be used to kill cells in C. elegans. miniSOG generates the reactive oxygen species (ROS) singlet oxygen after illumination with blue light. Illumination of neurons expressing miniSOG targeted to the outer mitochondrial membrane (mito-miniSOG) causes neuronal death. To enhance miniSOG's efficiency as an ablation tool in multiple cell types we tested alternative targeting signals. We find that membrane targeted miniSOG allows highly efficient cell killing. When combined with a point mutation that increases miniSOG's ROS generation, membrane targeted miniSOG can ablate neurons in less than one tenth the time of mito-miniSOG. We extend the miniSOG ablation technique to non-neuronal tissues, revealing an essential role for the epidermis in locomotion. These improvements expand the utility and throughput of optogenetic cell ablation in C. elegans.
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http://dx.doi.org/10.1038/srep21271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4748272PMC
February 2016

Pan-neuronal imaging in roaming Caenorhabditis elegans.

Proc Natl Acad Sci U S A 2016 Feb 28;113(8):E1082-8. Epub 2015 Dec 28.

Department of Physics, Harvard University, Cambridge, MA 02138; Center for Brain Science, Harvard University, Cambridge, MA 02138;

We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.
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http://dx.doi.org/10.1073/pnas.1507109113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776525PMC
February 2016

The Caenorhabditis elegans Ephrin EFN-4 Functions Non-cell Autonomously with Heparan Sulfate Proteoglycans to Promote Axon Outgrowth and Branching.

Genetics 2016 Feb 8;202(2):639-60. Epub 2015 Dec 8.

Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, Georgia 30144

The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system.
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http://dx.doi.org/10.1534/genetics.115.185298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788240PMC
February 2016

Axon injury triggers EFA-6 mediated destabilization of axonal microtubules via TACC and doublecortin like kinase.

Elife 2015 Sep 4;4. Epub 2015 Sep 4.

Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, United States.

Axon injury triggers a series of changes in the axonal cytoskeleton that are prerequisites for effective axon regeneration. In Caenorhabditis elegans the signaling protein Exchange Factor for ARF-6 (EFA-6) is a potent intrinsic inhibitor of axon regrowth. Here we show that axon injury triggers rapid EFA-6-dependent inhibition of axonal microtubule (MT) dynamics, concomitant with relocalization of EFA-6. EFA-6 relocalization and axon regrowth inhibition require a conserved 18-aa motif in its otherwise intrinsically disordered N-terminal domain. The EFA-6 N-terminus binds the MT-associated proteins TAC-1/Transforming-Acidic-Coiled-Coil, and ZYG-8/Doublecortin-Like-Kinase, both of which are required for regenerative growth cone formation, and which act downstream of EFA-6. After injury TAC-1 and EFA-6 transiently relocalize to sites marked by the MT minus end binding protein PTRN-1/Patronin. We propose that EFA-6 acts as a bifunctional injury-responsive regulator of axonal MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after injury.
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http://dx.doi.org/10.7554/eLife.08695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596636PMC
September 2015

Epidermal Wound Healing in the Nematode .

Adv Wound Care (New Rochelle) 2015 Apr;4(4):264-271

Section of Cell and Developmental Biology, Section of Neurobiology, Division of Biological Sciences, University of California San Diego , La Jolla, California.

Healing of epidermal wounds is a fundamentally conserved process found in essentially all multicellular organisms. Studies of anatomically simple and genetically tractable model invertebrates can illuminate the roles of key genes and mechanisms in wound healing. The nematode skin is composed of a simple epithelium, the epidermis (also known as hypodermis), and an associated extracellular cuticle. Nematodes likely have a robust capacity for epidermal repair; yet until recently, relatively few studies have directly analyzed wound healing. Here we review epidermal wound responses and repair in the model nematode . Wounding the epidermis triggers a cutaneous innate immune response and wound closure. The innate immune response involves upregulation of a suite of antimicrobial peptides. Wound closure involves a Ca-triggered rearrangement of the actin cytoskeleton. These processes appear to be initiated independently, yet, their coordinated activity allows the animal to survive otherwise fatal skin wounds. Unanswered questions include the nature of the damage-associated molecular patterns sensed by the epidermis, the signaling pathways relaying Ca to the cytoskeleton, and the mechanisms of permeability barrier repair.
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http://dx.doi.org/10.1089/wound.2014.0552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398003PMC
April 2015

Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4).

Genetics 2015 May 24;200(1):237-53. Epub 2015 Mar 24.

Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host-pathogen interactions.
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http://dx.doi.org/10.1534/genetics.114.174110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423366PMC
May 2015

Methods for skin wounding and assays for wound responses in C. elegans.

J Vis Exp 2014 Dec 3(94). Epub 2014 Dec 3.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego;

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca(2+) (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.
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http://dx.doi.org/10.3791/51959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396949PMC
December 2014
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