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1.
Genetics ; 220(4)2022 04 04.
Article in English | MEDLINE | ID: mdl-35143653

ABSTRACT

During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3, a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3 might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5, a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background. We show that ccd-5 acts in a pathway with cdk-5, aex-3, and unc-5. Navigation defects of follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Axon Guidance/genetics , Axons/metabolism , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Netrins/metabolism , Neurons/metabolism , Receptors, Cell Surface/metabolism
2.
Dev Biol ; 410(1): 56-69, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26703426

ABSTRACT

All 302 neurons in the C. elegans hermaphrodite arise through asymmetric division of neuroblasts. During embryogenesis, the C. elegans ham-1 gene is required for several asymmetric neuroblast divisions in lineages that generate both neural and apoptotic cells. By antibody staining, endogenous HAM-1 is found exclusively at the cell cortex in many cells during embryogenesis and is asymmetrically localized in dividing cells. Here we show that in transgenic embryos expressing a functional GFP::HAM-1 fusion protein, GFP expression is also detected in the nucleus, in addition to the cell cortex. Consistent with the nuclear localization is the presence of a putative DNA binding winged-helix domain within the N-terminus of HAM-1. Through a deletion analysis we determined that the C-terminus of the protein is required for nuclear localization and we identified two nuclear localization sequences (NLSs). A subcellular fractionation experiment from wild type embryos, followed by Western blotting, revealed that endogenous HAM-1 is primarily found in the nucleus. Our analysis also showed that the N-terminus is necessary for cortical localization. While ham-1 function is essential for asymmetric division in the lineage that generates the PLM mechanosensory neuron, we showed that cortical localization may not required. Thus, our results suggest that there is a nuclear function for HAM-1 in regulating asymmetric neuroblast division and that the requirement for cortical localization may be lineage dependent.


Subject(s)
Asymmetric Cell Division , Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Cell Nucleus/physiology , Nerve Tissue Proteins/physiology , Neural Stem Cells/physiology , Neurons/physiology , Amino Acid Sequence , Animals , Caenorhabditis elegans/cytology , Cell Lineage , Molecular Sequence Data , Neurons/cytology
3.
ACS Chem Biol ; 9(10): 2412-20, 2014 Oct 17.
Article in English | MEDLINE | ID: mdl-25101481

ABSTRACT

Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes.


Subject(s)
Aptamers, Nucleotide/metabolism , Caenorhabditis elegans/genetics , Fluorescent Dyes/chemistry , Microscopy, Fluorescence , RNA, Bacterial/chemistry , RNA, Untranslated/chemistry , RNA/isolation & purification , RNA/metabolism , Animals , Aptamers, Nucleotide/chemistry , Benzothiazoles/chemistry , Biotin/metabolism , Caenorhabditis elegans/metabolism , Gonads/metabolism , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Mangifera/chemistry , Quinolines/chemistry , RNA/chemistry , RNA, Bacterial/metabolism , RNA, Untranslated/metabolism , Spinacia oleracea/chemistry
4.
Health Promot Pract ; 15(1): 63-71, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23182862

ABSTRACT

There is gap between the enactment and implementation of local school wellness policies. Building the capacity of promotores to engage parents in strengthening local school wellness policy implementation is an innovative strategy. This evaluation study examines the effects of 6 hours of promotor advocacy training to improve local school wellness policy implementation. Consistent with psychological empowerment theory, the training and the related toolkit were designed to increase promotores' knowledge and self-efficacy to engage parents in advocating for improved local school wellness policy implementation. Pre-post training questionnaires (n = 74), five posttraining participant focus groups, and four staff member focus groups explored changes in promotor and participating organization capacity. Findings show increased participant self-efficacy, knowledge, and attitudes to advocate for improved local school wellness policy implementation. Participating organizations reported intention to continue supporting promotor local school wellness policy advocacy. Findings illuminate strategies to strengthen promotor capacity to engage parents in local school wellness policy advocacy.


Subject(s)
Community Health Workers/education , Community Health Workers/organization & administration , Health Promotion/organization & administration , Inservice Training/organization & administration , School Health Services/organization & administration , Adult , Aged , Diet , Exercise , Female , Health Knowledge, Attitudes, Practice , Humans , Male , Middle Aged , Minority Groups , Nutrition Policy , Parents , Professional Role , Self Efficacy
5.
Dev Biol ; 328(2): 245-56, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19389360

ABSTRACT

In Caenorhabditis elegans, Wnt signaling regulates many asymmetric cell divisions. During embryogenesis, the C. elegans Dishevelled (Dsh) homolog, DSH-2, regulates asymmetric neuroblast division of the ABpl/rpppa blast cell. Dsh is a key intracellular component of both beta-catenin dependent and beta-catenin independent Wnt pathways. In C. elegans, most of the well-characterized asymmetric cell divisions regulated by Wnts are dependent on beta-catenin. In the ABpl/rpppa neuroblast division, however, we determined that DSH-2 regulates cell polarity through a beta-catenin independent Wnt pathway. We also established that the C. elegans Wnt homolog, cwn-1, functions to regulate asymmetric division of the ABpl/rpppa blast cell. Our results indicated that cwn-1 does not act alone in this process, and it functions with another redundant ligand that appears not to be a Wnt. Finally, we show widespread requirements for DSH-2 during embryogenesis in the generation of many other neurons. In particular, DSH-2 function is necessary for the correct production of the embryonic ventral cord motor neurons. This study demonstrates a role for DSH-2 and Wnt signaling in neuronal specification during C. elegans embryogenesis.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Cell Cycle Proteins/physiology , Motor Neurons/physiology , Wnt Proteins/physiology , beta Catenin/physiology , Animals , Animals, Genetically Modified , Body Patterning/physiology , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/genetics , Cell Division , Cell Lineage , Cell Polarity/physiology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Dishevelled Proteins , High Mobility Group Proteins/genetics , High Mobility Group Proteins/physiology , Motor Neurons/cytology , Neurogenesis/physiology , Protein Structure, Tertiary , Signal Transduction , Transcription Factors/genetics , Transcription Factors/physiology , Wnt Proteins/genetics
6.
Dev Biol ; 328(2): 234-44, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19298786

ABSTRACT

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a beta-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/beta-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/beta-catenin, WRM-1/beta-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/beta-catenin asymmetry pathway and regulating anterior-posterior fate decisions required for proper morphogenesis.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Cell Cycle Proteins/physiology , Cytoskeletal Proteins/physiology , Transcription Factors/physiology , Wnt Proteins/physiology , beta Catenin/physiology , Animals , Animals, Genetically Modified , Body Patterning/physiology , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/genetics , Cell Polarity/physiology , DNA-Binding Proteins/physiology , Dishevelled Proteins , Embryo, Nonmammalian/physiology , High Mobility Group Proteins/physiology , Mutation , Protein Structure, Tertiary , Signal Transduction/physiology
7.
Dev Biol ; 284(1): 246-59, 2005 Aug 01.
Article in English | MEDLINE | ID: mdl-15990090

ABSTRACT

Asymmetric cell divisions produce all 302 neurons of the C. elegans hermaphrodite. Here, we describe a role for a C. elegans Dishevelled homolog, DSH-2, in an asymmetric neuroblast division. In dsh-2 mutants, neurons normally descended from the anterior neuroblast daughter of the ABpl/rpppa blast cell were frequently duplicated, while non-neuronal cells produced by the posterior daughter cell were often missing. These observations indicate that in the absence of dsh-2 function, the posterior daughter cell was transformed into a second anterior-like cell. Loss of mom-5, a C. elegans frizzled homolog, produced a similar phenotype. We also show that the DSH-2 protein localized to the cell cortex in most cells of the embryo. In the absence of MOM-5/Fz, DSH-2 was localized to the cytoplasm, suggesting that MOM-5 regulates asymmetric cell division by controlling the localization of DSH-2. Although all neurons in C. elegans are produced by an invariant pattern of cell divisions, our results indicate that cell signaling may contribute to asymmetric neuroblast division during embryogenesis.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Division/physiology , Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Nervous System/embryology , Neurons/cytology , Signal Transduction/physiology , Animals , Animals, Genetically Modified , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/genetics , DNA Primers , Dishevelled Proteins , Microscopy, Fluorescence , Neurons/physiology , RNA Interference
8.
Dev Biol ; 284(2): 301-10, 2005 Aug 15.
Article in English | MEDLINE | ID: mdl-15979607

ABSTRACT

Asymmetric cell division occurs when a mother cell divides to generate two distinct daughter cells, a process that promotes the generation of cellular diversity in metazoans. During Caenorhabditis elegans development, the asymmetric divisions of neural progenitors generate neurons, neural support cells and apoptotic cells. C. elegans HAM-1 is an asymmetrically distributed cortical protein that regulates several of these asymmetric neuroblast divisions. Here, we show that HAM-1 is a novel protein and define residues important for HAM-1 function and distribution to the cell cortex. Our phenotypic analysis of ham-1 mutant embryos suggests that HAM-1 controls only neuroblast divisions that produce apoptotic cells. Moreover, ham-1 mutant embryos contain many unusually large cell-death corpses. An investigation of this corpse phenotype revealed that it results from a reversal of neuroblast polarity. A misplacement of the neuroblast cleavage plane generates daughter cells of abnormal size, with the apoptotic daughters larger than normal. Thus, HAM-1 regulates the position of the cleavage plane, apoptosis and mitotic potential in C. elegans asymmetric cell divisions.


Subject(s)
Apoptosis , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Helminth Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Alleles , Amino Acid Sequence , Amino Acid Substitution , Animals , Arginine/metabolism , Aspartic Acid/metabolism , Base Sequence , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/isolation & purification , Cell Division , Cell Size , Consensus Sequence , Conserved Sequence , Green Fluorescent Proteins/metabolism , Helminth Proteins/chemistry , Helminth Proteins/genetics , Helminth Proteins/isolation & purification , Immunohistochemistry , Models, Biological , Molecular Sequence Data , Mutation, Missense , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Neurons/cytology , Sequence Deletion , Sequence Homology, Amino Acid , Transgenes
9.
Dev Cell ; 7(6): 831-41, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15572126

ABSTRACT

How cells integrate the input of multiple polarizing signals during division is poorly understood. We demonstrate that two distinct Caenorhabditis elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/beta-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. We also find that CKI (KIN-19) plays a role not only in the Wnt/beta-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, we establish a model for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event.


Subject(s)
Proto-Oncogene Proteins/metabolism , Signal Transduction , Spindle Apparatus/metabolism , Alleles , Animals , Blastomeres/metabolism , Caenorhabditis elegans , Caenorhabditis elegans Proteins/metabolism , Cell Division , Centrosome/ultrastructure , Cytoskeletal Proteins/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Mutation , RNA Interference , Trans-Activators/metabolism , Tubulin/metabolism , Wnt Proteins , beta Catenin
10.
Genetics ; 167(3): 1165-76, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15280232

ABSTRACT

We report here that WASP and Ena/VASP family proteins play overlapping roles in C. elegans morphogenesis and neuronal cell migration. Specifically, these studies demonstrate that UNC-34/Ena plays a role in morphogenesis that is revealed only in the absence of WSP-1 function and that WSP-1 has a role in neuronal cell migration that is revealed only in the absence of UNC-34/Ena activity. To identify additional genes that act in parallel to unc-34/ena during morphogenesis, we performed a screen for synthetic lethals in an unc-34 null mutant background utilizing an RNAi feeding approach. To our knowledge, this is the first reported RNAi-based screen for genetic interactors. As a result of this screen, we identified a second C. elegans WASP family protein, wve-1, that is most homologous to SCAR/WAVE proteins. Animals with impaired wve-1 function display defects in gastrulation, fail to undergo proper morphogenesis, and exhibit defects in neuronal cell migrations and axon outgrowth. Reducing wve-1 levels in either unc-34/ena or wsp-1 mutant backgrounds also leads to a significant enhancement of the gastrulation and morphogenesis defects. Thus, unc-34/ena, wsp-1, and wve-1 play overlapping roles during embryogenesis and unc-34/ena and wsp-1 play overlapping roles in neuronal cell migration. These observations show that WASP and Ena/VASP proteins can compensate for each other in vivo and provide the first demonstration of a role for Ena/VASP proteins in gastrulation and morphogenesis. In addition, our results provide the first example of an in vivo role for WASP family proteins in neuronal cell migrations and cytokinesis in metazoans.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Cell Movement/genetics , DNA-Binding Proteins/genetics , Morphogenesis/genetics , Animals , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Cell Division/genetics , DNA Primers , Multigene Family/genetics , Mutation/genetics , Neurons/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction
11.
Biotechniques ; 34(1): 74-8, 80, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12545543

ABSTRACT

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.


Subject(s)
Cloning, Molecular/methods , Luminescent Proteins/genetics , Plasmids/genetics , Polymerase Chain Reaction/methods , Yeasts/genetics , Animals , Base Sequence , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/biosynthesis , Caenorhabditis elegans Proteins/genetics , Cells, Cultured , DNA Primers , Gene Targeting/methods , Green Fluorescent Proteins , Luminescent Proteins/biosynthesis , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Engineering/methods , Quality Control , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombination, Genetic , Sequence Homology , Yeasts/classification
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