James Meir et al. (2003) International Worm Meeting "Characterization of mutants regulating synaptic remodeling of DD motor neurons"

James Meir, Nikki Alvarez, Yishi Jin

[International Worm Meeting, 2003]

Neurons communicate with their targets through specific synaptic connections. Such connections are usually stably maintained. In some examples, neurons have been observed to re-select their synaptic targets in response to developmental needs. The mechanisms underlying such neuronal plasticity are not known. We have focused on the GABAergic DD motor neurons to study the process of re-specification of synaptic partners. DD neurons are born in embryo, and form synapses on ventral body muscles in L1 larvae. During the L1 to L2 transition, DD neurons remove their ventral synapses and form synaptic connection exclusively to dorsal body muscles (1). Using a synaptobrevin::GFP fusion marker driven by a DD specific promoter (a generous gift of Chris Li) (2), we are able to follow the event of re-specification of DD synaptic choice and to search for mutants disrupting the synaptic pattern of DD neurons at different stages. We isolated five mutants, ju297, ju298, ju334, ju483, and ju484, from such a screen, all of which have synapse-like puncta along both dorsal and ventral processes of DD neurons in older larvae and adults. Complementation tests and genetic mapping place them into four loci. ju297 maps between unc-23 and dpy-11 on chromosome V. ju298 and ju483 map between let-502 and dpy-5 on chromosome I. ju334 maps near acr-2 on chromosome X. ju484 maps between dpy-13 and unc-24 on chromosome IV, and is covered by mDf9 and mDf4. The phenotype of ju484 homozygous mutant and ju484/ Df are indistinguishable, suggesting that it may be a null mutation. Double mutants between any two of these mutants resemble single mutant suggesting that genes defined by these mutants may function in the same pathway. We have attempted transgenic rescue experiments. However, these mutants display low germ-line transmission of transgenes as well as unstable rescuing activity. We are currently using high density single nucleotide polymorphisms in CB4856 and RC301 strains to map each gene. 1.White, J. G. et al., (1978) Nature 271: 764-766. 2.Nonet, M. L., (1999) J. Neurosci. Methods, 89(1): 33-40.

James Meir et al. (2002) West Coast Worm Meeting "Characterization of mutants regulating synaptic remodeling of DD motor neurons"

Nikki Alvarez, Yishi Jin, James Meir

[West Coast Worm Meeting, 2002]

Information flow of the neural network depends on maintaining the polarity of individual neurons as well as on precise selection of synaptic partners. Certain neurons, however, re-specify their synaptic targets in response to developmental or environmental cues. The development of DD motor neuron circuitry in C. elegansaccounts for a striking example of neural synaptic remodeling (1). DD neurons are born in embryo, and initially form synapses on ventral muscles in L1 stage. During the L1 to L2 transition, DD neurons remove their ventral synapses and re-establish synaptic connection to dorsal muscles without changing their cell morphology. Therefore, DD motor neurons re-specify their synaptic partners by way of reversing their neural polarity. Using a synaptobrevin::gfp fusion marker driven by a DD specific promoter (a generous gift of Chris Li) (2), we are able to follow DD synaptic remodeling event and to search for mutants disrupting the remodeling process. We isolated two mutants, ju298and ju334, both of which have synapse-like puncta along both dorsal and ventral processes of DD neurons in older larvae and adults. Phenotypic analysis of these two mutants suggests that the establishment of DD neuron polarity may require two independent steps separated by the synaptic remodeling event. In L1 larvae of ju298 homozygote, the DD neurons show partial defect in their synaptic pattern, suggesting that ju298 may be required for the polarity establishment in both pre- and post-DD synaptic remodeling. ju298 maps to the center of chromosome I. In ju334 mutant, although the distribution of the synapse-like puncta in adult DD neurons is similar to that in ju298, the polarity of DD neurons in L1 larvae and the onset of DD remodeling appear to be normal. ju334 may be specifically required for DD neuron polarity establishment in the post- but not pre-DD synaptic remodeling. We mapped ju334between -2.66 and -2.95 on chromosome X, and are currently performing transgenic rescuing experiments.

Lianna Wong et al. (2003) International Worm Meeting "FMI-1, the C. elegans homolog of Flamingo, is important for axon guidance and synapse formation"

Yishi Jin, Jim Rader, Julie Ahringer, Alexander Goncharov, Nikki Alvarez, Mei ZHEN, Lianna Wong, Gian Garriga

[International Worm Meeting, 2003]

To understand the processes of axon guidance and synapse formation, we performed two independent genetic screens. One was for mutants defective in axon guidance, focusing on the HSN axons. The HSNs are a bilaterally symmetric pair of serotonergic motor neurons that innervate the vulval muscles and stimulate egg laying. The mature HSN axon morphology can be visualized using gmIs1, a GFP reporter that illuminates the HSN cell bodies and axons. In wild-type animals, each HSN axon extends ventrally to the ventral nerve cord (VNC), turns anteriorly and extends along the VNC to the nerve ring. At the vulva, the HSN axons branch and elaborate varicosities. Two of the mutants isolated, gm188 and gm204, have abnormal HSN axons that circle around the vulva, elaborate unusually long branches, and cross between the left and right VNC tracts multiple times.The second screen was for mutants defective in synapse formation, using the synaptic vesicle marker Punc-25-SNB-1::GFP. In wild-type animals, this marker labels the presynaptic terminals of VD and DD motor neurons, and GFP appears as discrete puncta spaced evenly along the ventral and dorsal nerve cords. Two mutants isolated, ju43 and ju58, frequently lose SNB-1::GFP puncta in the presynaptic regions of VD1 and VD2 neurons, while the puncta from the remaining VDs exhibit subtle abnormalities in morphology and spacing. Some mutant animals also lack SNB-1::GFP puncta in presynaptic regions of DD neurons. Some of these neurons show axon outgrowth defects which could account for the loss of synapses. Specific synaptic defects are also observed in preliminary ultrastructural analysis.These mutations map to the same region of chromosome V and fail to complement. We mapped gm188 to a region containing 30 ORFs. Unable to achieve rescue of the mutant phenotype by cosmid injection, we phenocopied the HSN axon phenotype by RNAi to F15B9.7. We have identified molecular lesions in F15B9.7 for three of the four alleles.F15B9.7 encodes the C. elegans homolog of Flamingo, hereby designated FMI-1, a large, seven-pass transmembrane cadherin involved in the planar polarity pathway and dendritic morphogenesis in Drosophila. We are currently making GFP fusions and antibodies to study expression and localization of FMI-1 in C. elegans.

Delich, Cassandra et al. (2020) MicroPubl Biol

Hebbar, Shilpa, Delich, Cassandra, Zinovyeva, Anna, Dillon, Annabelle, Haskell, Dustin, Winans, Noah

[MicroPubl Biol, 2020]

MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally repress gene expression (Gebert and MacRae, 2018). While many miRNA genes and their families have been analyzed for function (Miska et al. 2007, Alvarez-Saavedra and Horvitz 2010), there are microRNA genes for which loss of function alleles have not yet been generated. There are no available alleles for the C. elegans mir-49 gene. Using CRISPR-Cas9 genome editing, we generated two deletion alleles, zen99 and zen102, that disrupt the C. elegans mir-49 gene (Fig 1A). mir-49(zen99) and mir-49(zen102) delete 56 base pairs and 58 base pairs from the mir-49 locus, respectively (Fig 1B and Fig 1C). Each deletion nearly completely removes both strands generated by the mir-49 locus, mir-49-3p and mir-49-5p. Both mir-49 alleles are homozygous viable and appear to be superficially wild type. Careful phenotypic analysis will be important to characterize the effects of the two mir-49 deletions.

Yang, Bing et al. MicroPubl Biol

Yang, Bing, McJunkin, Katherine

[MicroPubl Biol, 2020]

The mir-35-42 family of microRNAs (miRNAs) acts redundantly to ensure embryonic viability in C. elegans (Alvarez-Saavedra and Horvitz 2010). We are interested in defining the essential targets that must be repressed by the mir-35-42 family. Our previous work suggested that NHL (ring finger b-box coiled coil) domain containing 2 (nhl-2) may be one such target because genome editing attempts to delete the mir-35-42 seed binding region in the nhl-2 3UTR were unsuccessful (McJunkin and Ambros 2017). The same CRISPR reagents were successful at creating such a deletion in a background containing an NHL-2 CDS deletion (nhl-2(ok818)) (McJunkin and Ambros 2017). Together, we took these results to mean that derepression of nhl-2 induced lethality or sterility, preventing our isolation of the deletion lines in the wild type context. More recently, CRISPR genome editing reagents and protocols have become many-fold more efficient, most notably by injection of recombinant Cas9 RNPs pre-loaded with synthetic guide RNAs (gRNAs) (Paix et al. 2014). Using injection of Cas9/gRNA RNPs, we have succeeded in deleting and mutating the mir-35-42 seed binding region in the nhl-2 3UTR in a wild type background (see alleles in Figure 1A). Because such alleles were previously difficult to generate, we quantified their fecundity and embryonic viability (which are two aspects of physiology affected by mir-35 family mutations) (Alvarez-Saavedra and Horvitz 2010; McJunkin and Ambros 2014) to see if they were impaired, but we found these animals to be wild type (Figure 1B). Therefore, our original interpretation that the difference in CRISPR editing between wild type and nhl-2(ok818) backgrounds was due to negative selection of miRNA binding site mutations in the wild type background was incorrect. One possible explanation for the observed difference in editing may be alterations in chromatin structure induced by the 1.5kb nhl-2(ok818) deletion. Indeed, nucleosome position and dynamics have been shown to alter efficiency of Cas9 cleavage (Chen et al. 2016; Horlbeck et al. 2016; Isaac et al. 2016; Hinz et al. 2016; Daer et al. 2017; Yarrington et al. 2018; Kim and Kim 2018). Thus, differences in genome editing efficiencies between genetic backgrounds should be interpreted with caution.

Eric A Miska et al. (2005) International Worm Meeting "Functional analysis of the microRNA genes of C. elegans"

Victor Ambros, Nelson Lau, David Bartel, Ezequiel Alvarez-Saavedra, Andrew Hellman, Eric A Miska, Bob Horvitz, Allison Abbott

[International Worm Meeting, 2005]

The heterochronic genes lin-4 and let-7 encode small (21-22 nt) non-protein coding regulatory RNAs. Strains carrying a mutation in either of these genes are heterochronic, displaying retarded development with some cell lineages having an altered temporal pattern of cell division and differentiation. lin-4 and let-7 normally inhibit translation of target genes that when mutated lead to a phenotype opposite that of lin-4 and let-7 mutants: precocious development and early expression of certain paths of cell division and differentiation. More recently, molecular and bioinformatic approaches have identified many genes encoding small RNAs in C. elegans, Drosophila and mammals. All of these genes encode 21-25 nt RNAs derived from longer transcripts that contain partially double-stranded RNAs. These small RNAs, termed microRNAs (miRNAs), define a large, new class. To understand the biology of the C. elegans microRNA genes, we decided to combine the generation of loss-of-function mutants with GFP expression studies and target prediction using bioinformatics. We have generated deletion strains corresponding to 92 microRNAs, covering the majority of known microRNA genes. We will present an overview of the classes of mutant phenotypes we have observed. (for information about the mir-35 and the let-7 families of microRNAs, see also abstracts by Alvarez-Saavedra et al. and Abbott et al.). One focus will be the issue of redundancy within families of microRNA genes. This study represents the first comprehensive analysis of microRNA function.

W. Robert Shaw et al. (2007) International Worm Meeting "Role of the mir-51 Family of microRNAs in Caenorhabditis elegans Development."

W. Robert Shaw, Eric A. Miska

[International Worm Meeting, 2007]

MicroRNAs (miRNAs) are a class of small regulatory RNA that inhibit target gene expression post-transcriptionally. Several hundreds of miRNAs have been discovered in diverse organisms but their biological roles are largely unknown. Caenorhabditis elegans </I> mutants with deletions in most miRNA genes have been generated by a deletion screen (1) as a resource for the study of miRNA function. The mir-51 family of miRNAs is a highly conserved family of six miRNAs that share a ''seed'' region (seven continuous base pairs at their 5'' end at bases 2 to 8). Animals lacking individual members of the mir-51 family of miRNAs show no obvious abnormal phenotypes, likely because these six miRNAs act redundantly. Mutant strains in which multiple mir-51 family members have been deleted show gradually more severe defects in growth rate and development as further family members are deleted, culminating in embryonic or early larval lethality. This series correlates with the relative expression levels of the miRNAs (1). Mutant phenotypes can be rescued by short genomic regions encoding mir-51 family miRNAs. Bioinformatic miRNA target prediction has provided lists of candidate miRNA target mRNAs. These targets are being tested using a combination of RNAi, fluorescent protein reporter constructs and antibodies. In addition, we are carrying out enhancer and suppressor screens to identify direct targets of the mir-51 miRNA family. (1) Miska, E. A., Alvarez-Saavedra, E. A., Abbott, A. L., Lau, N. P., Hellman, A. B., McGonagle, S., Bartel, D. P., Ambros, V. R., Horvitz, H. R. - unpublished results.

Eric A Miska et al. (2003) International Worm Meeting "Mutant phenotypes of the microRNA genes of C. elegans"

Bob Horvitz, Victor Ambros, Allison Abbott, David P Bartel, Eric A Miska, Ezequiel Alvarez-Saavedra, Nelson Lau

[International Worm Meeting, 2003]

Recently, molecular and bioinformatic approaches have identified many genes encoding 21-25 nt RNAs in C. elegans, Drosophila and mammals. All of these RNAs are derived from longer transcripts that form hairpin loops. These small RNAs, termed microRNAs (miRNAs, mirs), define a large, new class of genes. To date the only genetically characterized microRNA genes in any organism are the C. elegans genes lin-4 and let-7. Both are heterochronic genes, which regulate the timing of development. To learn more about the biological roles of microRNAs, we have generated 34 deletion strains corresponding to loss-of function mutations in 40 microRNAs. We have developed an efficient robotics system for the isolation of deletion mutants and are continuing to generate additional microRNA deletion strains. We have examined the 34 deletion strains for general appearance, movement, viability, fertility, egg laying, dauer formation, pumping and defecation. We have also assayed response to body touch, to drugs such as aldicarb, levamisole and ivermectin, and to osmotic barriers. In addition, we are studying genetic interactions of the microRNA genes to uncover synthetic phenotypes. So far we have focused on genes involved in microRNA/RNAi-processing, heterochrony, cell-death, dauer-formation, vulval development and the microRNA genes themselves. We will present the initial analysis of several mutant phenotypes. We will demonstrate how the microRNAs mir-48 and mir-84 interact with genes of the heterochronic pathway. In addition, we will show that microRNAs are required for embryonic and germline development. Finally, we will present data indicating redundancy between members of several families of microRNAs. Additional information about both the technology and our phenotypic characterization will be presented by Alvarez-Saavedra et al. (see abstract).

Allison L Abbott et al. (2002) East Coast Worm Meeting "IDENTIFICATION AND FUNCTIONAL ANALYSIS OF microRNA GENES IN C. elegans AND D. melanogaster."

Daniel Hopkins, Lorenzo Sempere, Rosalind C Lee, Allison L Abbott, Ann Lavanway, Nicholas S Sokol, Peter T Williams, David Jewell, Victor Ambros

[East Coast Worm Meeting, 2002]

lin-4 and let-7 are the founding members of a new family of regulatory RNAs, the microRNAs, many dozens of which have been identified in worms, flies and humans 1-3 . A total of 60 C. elegans microRNA genes have been published previously2,3 . In order to develop a comprehensive catalog of C. elegans miRNAs, we are continuing to identify new miRNA sequences from C. elegans cDNA libraries and from genomic sequence conservation between C. elegans and C. briggsae. So far we have identified 65 distinct new microRNA genes, bringing the total to at least 125 in worms. We confirmed the expression of all of these new microRNA genes using Northern blot analysis and are characterizing their patterns of expression during development. 47 of the new miRNA sequences occur in single copy in the C. elegans genome, while the remaining 18 sequences are found in multiple loci. Some microRNA genes are clustered, suggesting coordinate regulation by a shared promoter. Using RNA folding programs, we find that approximately 70% of these new miRNAs are predicted to be processed from stem-loop hairpin structures, like lin-4 and let-7. However, a significant portion of these miRNAs are not predicted to fold into a hairpin precursor. Interestingly, both hairpin and non-hairpin miRNAs exhibit Dicer-dependent production of the ~22nt mature miRNA. To aid in analyzing the whole family of microRNA genes, we have developed web interfaces to customized high-throughput Blast and mfold servers, and microViewer, a Java program tailored for microRNA annotation. In order to obtain mutants in microRNA genes, we are generating deletion mutations (for details, see abstract by Alvarez-Saavedra et al. ) and analyzing candidate genetic loci that map in close proximity to cloned microRNA genes. Lastly, expression profiles of microRNAs conserved between flies and worms are being compared. For example, mir-1, 2 and 87 are expressed throughout development in both flies and worms whereas mir-34 is enriched at later stages. These experiments should help elucidate the biological function of members of this new class of regulatory molecules.

Huarcaya Najarro, Elvis et al. (2009) International Worm Meeting "The flamingo protocadherin regulates cell migration, axon guidance and synapse formation in both cell-autonomous and non-autonomous fashion."

Goncharov, Alexandr, Huarcaya Najarro, Elvis, Ackley, Brian, Jin, Yishi

[International Worm Meeting, 2009]

The flamingo/starry night protocadherin has been demonstrated to function in the planar cell polarity pathway and separately in axon target recognition. We find that mutants for the C. elegans homolog of flaming, fmi-1/cdh-6, demonstrate multiple defects in the patterning of the nervous system. Mutations in fmi-1 cause abnormal distribution of synaptic vesicle clusters in the GABAergic neurons, misrouting of the DD and VD neurons and positioning defects in the DD cells. Additionally, electron microscopy analysis showed that mutations in fmi-1 cause a failure in synaptic target recognition. In order to determine where fmi-1 functions, we carried out expression analysis of fmi-1 by using the putative fmi-1 promoter to drive expression of GFP and co-injecting the type D-type motor neuron marker Punc-25::mCherry or cholinergic neuron marker Pacr-2::mCherry. These studies suggest that fmi-1 is expressed in the DD GABAergic, and most, if not all of the cholinergic motorneurons, as well as other cells. However, we find no evidence that the postembryonically derived VD neurons express fmi-1. Since the axon pathfinding defects in D-type motor neurons are primarily observed in VD neurons, this observation raises the possibility that fmi-1 functions in a non-cell autonomous manner. Further, knockdown of fmi-1 using dsRNA expressed specifically in the cholinergic neurons caused synaptic defects in the VD, but not DD neurons. This indicates that fmi-1 likely mediates signaling between the cholinergic and GABAergic neurons to establish the requisite patterning of these neurons. For at least the VDs, fmi-1 appears to act non-autonomously. Results from the Hutter lab and Garriga lab indicate fmi-1 acts cell autonomously in patterning of interneurons and the migration of the HSN respectively. We''d like to thank M. Zhen for the isolation of two fmi-1 alleles and N. Alvarez for some preliminary characterization of the axon guidance defects.