Kulkarni, S.S. et al. (2017) International Worm Meeting "UNC-16 inhibits the function of regeneration promoting isoform of DLK-1."

Sheoran, S., Kulkarni, S.S., Matsumoto, K., Sabharwal, V., Hisamoto, N., Koushika, S.P.

[International Worm Meeting, 2017]

Neurons in the adult nervous system have a limited ability to regenerate after injury. The extent of neuronal regeneration after injury depends on the intrinsic growth potential of neurons and their extracellular environment, both influenced by several genes. We show that UNC-16 plays an inhibitory role during the early stages of neuronal regeneration after axotomy. UNC-16, a C. elegans JIP3 (JNK Interacting Protein 3) homologue, is a scaffolding protein for MAP kinases and binds to the Kinesin-1 and Dynein motors. JIPs are a family of classical scaffolding molecules and they are known to be able to switch their roles from growth promoting to inhibitory based on their levels and times of activation or deactivation. We also show UNC-16's inhibitory role is independent of JNK-1, Kinesin-1 and Dynein but is dependent on Dual Leucine Zipper Kinase-1 (DLK-1). DLK-1 is an essential MAPKKK for neuronal regeneration and has been reported to interact with JIP3. DLK-1 has two isomers, long and short, of these, DLK-1 long promotes regeneration while DLK-1 short inhibits regeneration. We show that UNC-16 inhibits the regeneration promoting activity of DLK-1 long but does not influence the activity of DLK-1 short. We find that UNC-16 promotes the DLK-1 long punctate localization in a concentration dependent manner limiting DLK-1 long availability at the cut site. We also show that UNC-16 levels are responsible for negatively regulating microtubule dynamics at the cut site immediately after axotomy which we think leads to the observed faster rate of neuronal regrowth in unc-16 animals. We suggest a model where UNC-16 may hold DLK-1 long in a complex and restrict the availability of active DLK-1 long and thereby inhibit regeneration. The dual inhibitory control by both UNC-16 and DLK-1 short can calibrate the intrinsic growth promoting function of DLK-1 long in vivo. We thus show that JIP3 could play its inhibitory role to allow tight temporal and spatial control of DLK-1 function.

Kulkarni SS et al. (2021) Genetics "UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons."

Sheoran S, Koushika SP, Hisamoto N, Sabharwal V, Kulkarni SS, Matsumoto K, Basu A, Ghosh-Roy A

[Genetics, 2021]

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditiselegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16's inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.

Wahl G et al. (1998) Parasitology "Onchocerca ochengi: assessment of the Simulium vectors in north Cameroon."

Wahl G, Schmitz A, Ekale D

[Parasitology, 1998]

In the savanna areas of tropical Africa, cattle are frequently infected with the filaria Onchocerca ochengi. This parasite is closely related to Onchocerca volvulus, the causative agent of human onchocerciasis (river blindness), and is capable of developing in the same vector, Simulium damnosum s.l. In North Cameroon, where both O. ochengi and O. volvulus are endemic, we carried out a field study (reported in this and 2 following papers) to examine to which extent the transmission of the 2 parasite species overlap and what influence this has on the epidemiology of human onchocerciasis. In this paper we report our experiments to determine which of the S. damnosum species in North Cameroon act as vectors of O. ochengi, how efficiently they do so and whether other Simulium species play a vector role. To this end, infected cattle were exposed near 5 rivers in different geographical areas. Among 14 Simulium species identified as aquatic and/or adult stages at these rivers, only 6 (S. squamosum, S. damnosum s.s., S. sirbanum, S. bovis, S. wellmanni and S. hargreavesi) were found to bite cattle in important numbers in at least 1 of the sites. The 3 species of the S. damnosum complex were all capable of ingesting microfilariae (mf) of O. ochengi and developing a proportion of them to infective larvae (L3). Whereas S. squamosum and S. damnosum s.s., the prevailing vectors in the Guinea and Sudan savanna respectively, showed a high vector competence (17% of ingested mf developed to L3), S. sirbanum, which was much rarer in both areas, appeared to have a much lower susceptibility (2%). Other boophilic Simulium species were only seen in certain sites and seasons, being either incapable of ingesting important numbers of O. ochengi mf from body regions where these mf were abundant (S. bovis, S. hargreavesi); not able to support the development of ingested mf to L3 (S. wellmanni), or bit cattle preferentially in the ears, where O. ochengi mf do not occur (S. hargreavesi). We conclude that in North Cameroon members of the S. damnosum complex are the only important vectors of O. ochengi, with S. squamosum and S. damnosum s.s. being the main vectors.

Prost A Ann Parasitol Hum Comp "[The different types of human onchocerciasis in west Africa (author's transl)]."

Prost A

[Ann Parasitol Hum Comp]

This study reports the clinical, ophtalmological and parasitological examination for onchocerciasis in 4 882 people, living in 23 villages located in 8 different areas of west Africa. The disease is hyperendemic in the whole sample and no major differences occur when comparing the indices of Onchocerca volvulus infection. However, all the complications (eye, skin and lymph node lesions) are less common in the four forest clusters than in to the four savanna clusters. The disease seems to be relatively well tolerated in areas where transmission of Onchocerca volvulus is carried out by Simulium species other than S. damnosum s.s. and S. sirbanum. The striking epidemiological differences of onchocerciasis is probably associated with several vector-parasite complexes, which may indicate the existence of different strains of O. volvulus.

Gauri Kulkarni et al. (2007) International Worm Meeting "Characterization of clec-1, a gene encoding a putative secreted C-type lectin-like protein from C. elegans."

William Wadsworth, Gauri Kulkarni

[International Worm Meeting, 2007]

Proteins with C-type lectin-like domains (CTLDs) are known to contribute in extracellular matrix organization, cell-cell interactions, pathogen recognition and immune response via binding to sugars, lipids or direct protein-protein interactions in vertebrates. There is a large family of CTLD-containing proteins in C. elegans, however their biological significance is largely unknown. One of the members of this family, clec-38, has been found to be involved in UNC-6/Netrin mediated axon guidance (see abstract by kulkarni et; al, and Li et; al, this meeting). Here we show analysis of clec-1, a gene encoding a putative secreted C-type-lectin like protein. RNAi and deletion allele analysis showed that animals deficient for CLEC-1 cause dumpy body phenotype with cell migration defects. Further, expression analysis using transcriptional gfp-fusion construct, suggested that clec-1 is strongly expressed in hypodermal cells, muscles and glial-like sheath and socket cells. Further analysis is being carried out to study axonal and cell migration defects.

Nicole F Liachko et al. (2005) International Worm Meeting "Characterization of putative DAF-16 target genes"

Siu Sylvia Lee, Nicole F Liachko

[International Worm Meeting, 2005]

The forkhead transcription factor DAF-16 is a key mediator of longevity, metabolism, and development. An informatic search using the known DAF-16 DNA recognition sequence to predict putative DAF-16 target genes has been performed (1). To further characterize the predicted DAF-16 target genes, we are optimizing a chromatin immunoprecipitation (ChIP) procedure using larvae and adult worms to demonstrate the presence of DAF-16 at the promoter of the target genes. In addition, we are using a promoter GFP fusion strategy to examine the expression of a subset of the DAF-16 target genes in worms. This approach allows study of when and where the target genes are expressed, as well as study of the dynamic regulation of the expression of the target genes in reponse to environmental stresses. Preliminary results show cell-type and developmental stage specific expression of the genes of interest. (1) S.S. Lee, S. Kennedy, A.C. Tolonen, G. Ruvkin, Science 300, 644 (2003).

Haichang Li et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Genetic Screen for Modifiers of unc-6 Axon Guidance Function"

William Wadsworth, Haichang Li, Chaunte Cannon, Zhennan Xu, Yan Xu

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The UNC-6/netrin molecule comprises multiple domains that are associated with guidance and axon branching activities. The VI and V domains are similar to the N-termini of the laminin subunits, whereas the UNC-6 C domain is found in proteins that include complement components C3, C4, and C5. The unc-6(rh46) allele encodes substitution A157P within domain VI and renders the UNC-6 guidance activity temperature-sensitive. To further examine the activities and interactions of UNC-6, a genetic screen was undertaken to isolate mutations that suppress the unc-6(rh46) loss-of-guidance phenotype. We screened screened ~ 150,000 mutagenized genomes and recovered 25 mutations. We have identified four intragenic suppressors and all are substitutions of A214 or P216, suggesting that this domain VI site is structurally important for UNC-6/netrin guidance activities. We will also present the cloning and characterization of two extragenic mutations, ur299 and ur320. These mutations can restore the dorsal DA/DB axon migrations in the unc-6(rh46) background and they have other traits that suggest they affect UNC-6 guidance functions. Another gene identified from this screen is clec-38, which is described in another abstract at this meeting (Kulkarni and Wadsworth).

Haichang Li et al. (2005) International Worm Meeting "A Genetic Screen for Suppressors of unc-6 (rh46)"

Yan Xu, William G Wadsworth, Chaunte Cannon, Haichang Li, Gauri Kulkarni

[International Worm Meeting, 2005]

During the development of nervous system, neurons and axons have to navigate long distances in order to reach their final synaptic targets. The unc-6 gene encodes a member of the netrin family of extracellular guidance molecules that guides circumferential cell and axon migration in C.elegans. UNC-6 is a laminin-related protein and comprises multiple domains. Based on the analysis of selective unc-6 loss-of-function alleles and transgenic animals expressing UNC-6 derivatives with domain deletions, we showed that the different direction- and tissue-specific guidance activities of UNC-6 are mediated by distinct domains. However, still little is known about the molecular mechanism that govern different responses individual migrating axons have to these guidance cues. To better understand the mechanisms involved in Netrin/UNC-6 mediated guidance. We have undertaken a genetic suppressor screen in hopes of identifying mutations that can compensate for the defects introduced by the unc-6(rh46) allele. The unc-6(rh46) allele introduces an alanine to proline substitution near the middle of domain VI, which causes a temperature-sensitive loss of guidance activity. In a pilot screen, we have screened over 150,000 mutagenized haploid genomes and 25 possible candidates have been isolated. These mutations are being further characterized for their ability to revert movement and dorsal axon migrations. Some of them are being cloned, for example see the abstract of the ur280 allele by Kulkarni and Wadsworth.

Messina, Kari et al. (2011) International Worm Meeting "Novel Protein SPE-7 is Required for Meiotic Chromosome Segregation and Fibrous Body Assembly."

Shakes, Diane C., Presler, Marc, Messina, Kari

[International Worm Meeting, 2011]

The development of functional spermatozoon from uncommitted germ cells requires the progression of two distinct yet presumably interacting cellular programs: the meiotic and cell differentiation programs of spermatogenesis. One spermatogenesis-defective factor required for the normal progression of both of these programs is SPE-7. spe-7 mutant spermatocytes progress through the early phases of meiosis, detach from the rachis, and set up a metaphase I spindle. However meiotic chromosome segregation is aberrant and the spermatocytes never undergo cytokinesis. Ultimately, the spe-7 spermatocytes arrest with cortically localized actin and microtubules that reassemble into a network-like pattern. Surprisingly, we found that in developing wildtype spermatocytes, SPE-7 localizes to fibrous bodies, the paracrystaline storage form of the major sperm protein, MSP. During the meiotic divisions, the localization of SPE-7 is somewhat dynamic but it ultimately segregates with the fibrous bodies into the budding spermatids before disappearing altogether. This localization pattern is apparently functional as a reexamination of spe-7 spermatocytes revealed that they express MSP but are defective for fibrous body assembly. spe-7 encodes a novel protein with close homologs limited to other Caenorhabditis species. Both germline microarrays and western blots indicate that SPE-7 is expressed specifically during spermatogenesis, and it is one of the many genes regulated by the sperm-specific transcription factor, SPE-44 (see abstract by Kulkarni et al.). We are currently pursuing additional genetic and biochemical studies designed to understand the dynamic localization of SPE-7 as well as its interactions with other known spe genes.

Haichang Li et al. (2007) International Worm Meeting "RPM-1, a putative ubiquitin ligase, regulates SAX-3 and UNC-5 receptor-mediate axon guidance in C. elegans."

Gauri Kulkarni, Haichang Li, William Wadsworth

[International Worm Meeting, 2007]

The development of the nervous system is a complex process involving the interactions of numerous molecules. Axons can navigate long distances in order to reach their final synaptic targets by interacting with many different extracellular guidance cues. UNC-6/netrin is an extracellular matrix guidance cue that directs circumferential axon and cell migrations in C. elegans. Receptor complexes, comprising UNC-5, UNC-40, and SAX-3, modulate growth cone responses to UNC-6. Mechanisms that regulate the formation of these receptor complexes are unknown. We present evidence to show that RPM-1, a putative RING finger/E3 ubiquitin ligase, negatively regulates UNC-5 and SAX-3 activity. Genetic analysis on the unique axon migration pattern of three different neurons, which respond differently to UNC-6 and SLT-1, suggested that RPM-1 negatively regulates UNC-5 and SAX-3. To further test this model, we examined the expression of UNC-5::GFP and SAX-3::GFP in rpm-1 mutant, suggests that UNC-5 and SAX-3 expression is upregulated by loss of function of rpm-1. We have also identified a gene encoding a predicted transmembrane protein with C-type lectin-like domains (see abstract by Kulkarni el al., this meeting). Analyses of clec-38 mutations suggest that the gene regulates UNC-40 receptor signaling. Axon guidance defects caused by loss of rpm-1 function are suppressed by the loss of clec-38 function. Together these results suggest a model that RPM-1 and CLEC-38 negatively regulate a different set of receptors and help control a balance between the axon outgrowth-promoting and -inhibiting activities mediated by the receptors in response to multiple extracellular cues.