The nematode even-skipped homolog vab-7 regulates gonad and vulva position in Pristionchus pacificus

In free-living nematodes, developmental processes like the formation of the vulva, can be studied at a cellular level. Cell lineage and ablation studies have been carried out in various nematode species and multiple changes in vulval patterning have been identified. In Pristionchus pacificus, vulva formation differs from Caenorhabditis elegans with respect to several autonomous and conditional aspects of cell fate specification. To understand the molecular basis of these evolutionary changes, we have performed a genetic analysis of vulva formation in P. pacificus. Here, we describe two mutants where the vulva is shifted posteriorly, affecting which precursor cells will form vulval tissue in P. pacificus. Mutant animals show a concomitant posterior displacement of the gonadal anchor cell, indicating that the gonad and the vulva are affected in a similar way. We show that mutations in the even-skipped homolog of nematodes, vab-7, cause these posterior displacements. In addition, cell ablation studies in the vab-7 mutant indicate that the altered position of the gonad not only changes the cell fate pattern but also the developmental competence of vulval precursor cells. Investigation of Cel-vab-7 mutant animals showed a similar but weaker vulva defective phenotype to the one described for Ppa-vab-7.     

Characterization of the C. elegans erlin homologue

Background

Ovarian cancer is one of the most lethal malignancies in women, as it is frequently detected at an advanced stage, and cancers often become refractory to chemotherapy. Evidence suggests that dysregulation of pro-apoptotic genes plays a key role in the onset of chemoresistance. The secreted Frizzled-Related Protein (sFRP) family is pro-apoptotic and also a negative modulator of the Wnt signalling cascade. Studies have demonstrated that the re-expression of sFRPs, in particular sFRP4, is associated with a better prognosis, and that experimentally induced expression results in cell death.

Results

In vitro experimental models determined that sFRP4 was differentially expressed in chemosensitive (A2780) and chemoresistant (A2780 ADR and A2780 Cis) ovarian cell lines, with chemosensitive cells expressing significantly higher levels of sFRP4. Transfection of the chemoresistant cell lines with sFRP4 significantly increased their sensitivity to chemotherapy. Conversely, silencing of sFRP4 expression in the chemosensitive cell line resulted in a corresponding increase in chemoresistance. Comparison of sFRP4 expression in tumour biopsies revealed a positive trend between sFRP4 expression and tumour grade, with mucinous cyst adenocarcinomas exhibiting significantly decreased sFRP4 levels compared to mucinous borderline tumours.

Conclusions

This study indicates a role for sFRP4 as a predictive marker of chemosensitivity in ovarian cancer and suggests that this pathway may be worth exploiting for novel therapies.     

The C. elegans homologue of the spastic paraplegia protein, spastin, disassembles microtubules

Mutations in human spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia. Sequence analysis revealed that spastin contains the AAA (ATPases associated with diverse cellular activities) domain in the C-terminal region. Recently, it was reported that spastin interacts dynamically with microtubules and displays microtubule-severing activity. A plausible Caenorhabditis elegans homologue of spastin (SPAS-1) has been identified by homology search and phylogenetic analyses. To understand the function of the spastin homologue, we characterized the spas-1 deletion mutant and analyzed spas-1 expression regulation in C. elegans. SPAS-1 was localized with cytoskeletons at the perinuclear region. We found that microtubules were intensely stained at the centrosomal region in the deletion mutant. Furthermore, overexpression of SPAS-1 caused disassembly of microtubule network in cultured cells, while ATPase-deficient SPAS-1 did not. These results indicate that C. elegans SPAS-1 plays an important role in microtubule dynamics. We also found that two kinds of products were generated from spas-1 by alternative splicing in a developmental stage-dependent manner.     

UNC-119 suppresses axon branching in C. elegans

The architecture of the differentiated nervous system is stable but the molecular mechanisms that are required for stabilization are unknown. We characterized the gene unc-119 in the nematode Caenorhabditis elegans and demonstrate that it is required to stabilize the differentiated structure of the nervous system. In unc-119 mutants, motor neuron commissures are excessively branched in adults. However, live imaging demonstrated that growth cone behavior during extension was fairly normal with the exception that the overall rate of migration was reduced. Later, after development was complete, secondary growth cones sprouted from existing motor neuron axons and cell bodies. These new growth cones extended supernumerary branches to the dorsal nerve cord at the same time the previously formed axons retracted. These defects could be suppressed by expressing the UNC-119 protein after embryonic development; thus demonstrating that UNC-119 is required for the maintenance of the nervous system architecture. Finally, UNC-119 is located in neuron cell bodies and axons and acts cell-autonomously to inhibit axon branching.     

Syndecan regulates cell migration and axon guidance in C. elegans

During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. As there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. Here, we show that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, our experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans.     

The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2 mediated axon guidance

The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding. In the SDQL neuron, LAD-2 mediates dorsal axon guidance via the secreted MAB-20/Sema2 and PLX-2 plexin receptor, the functions of which have largely been characterized in epidermal morphogenesis. We use targeted misexpression experiments to provide in vivo evidence that MAB-20/Sema2 acts as a repellent to SDQL. Coimmunoprecipitation assays reveal that MAB-20 weakly interacts with PLX-2; this interaction is increased in the presence of LAD-2, which can interact independently with MAB-20 and PLX-2. These results suggest that LAD-2 functions as a MAB-20 coreceptor to secure MAB-20 coupling to PLX-2. In vertebrates, L1 binds neuropilin1, the obligate receptor to the secreted Sema3A. However, invertebrates lack neuropilins. LAD-2 may thus function in the semaphorin complex by combining the roles of neuropilins and L1CAMs.     

Sensory activity affects sensory axon development in C. elegans

The simple nervous system of the nematode C. elegans consists of 302 neurons with highly reproducible morphologies, suggesting a hard-wired program of axon guidance. Surprisingly, we show here that sensory activity shapes sensory axon morphology in C. elegans. A class of mutants with deformed sensory cilia at their dendrite endings have extra axon branches, suggesting that sensory deprivation disrupts axon outgrowth. Mutations that alter calcium channels or membrane potential cause similar defects. Cell-specific perturbations of sensory activity can cause cell-autonomous changes in axon morphology. Although the sensory axons initially reach their targets in the embryo, the mutations that alter sensory activity cause extra axon growth late in development. Thus, perturbations of activity affect the maintenance of sensory axon morphology after an initial pattern of innervation is established. This system provides a genetically tractable model for identifying molecular mechanisms linking neuronal activity to nervous system structure.     

The FGD homologue EXC-5 regulates apical trafficking in C. elegans tubules

Maintenance of the shape of biological tubules is critical for development and physiology of metazoan organisms. Loss of function of the Caenorhabditis elegans FGD protein EXC-5 allows large fluid-filled cysts to form in the lumen of the single-cell excretory canal tubules, while overexpression of exc-5 causes defects at the tubule's basolateral surface. We have examined the effects of altering expression levels of exc-5 on the distribution of fluorescently-marked subcellular organelles. In exc-5 mutants, early endosomes build up in the cell, especially in areas close to cysts, while recycling endosomes are depleted. Endosome morphology changes prior to cyst formation. Conversely, when exc-5 is overexpressed, recycling endosomes are enriched. Since FGD proteins activate the small GTPases CDC42 and Rac, these results support the hypothesis that EXC-5 acts through small GTPases to move material from apical early endosomes to recycling endosomes, and that loss of such movement is likely the cause of tubule deformation both in nematodes and in tissues affected by FGD dysfunction such as Charcot–Marie–Tooth Syndrome type 4H.     

Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

The molecular and cellular mechanisms that allow adult-stage neurons to regenerate following damage are poorly understood. Recently, axons of motoneurons and mechanosensory neurons in adult C. elegans were found to regrow after being snipped by femtosecond laser ablation. Here, we explore the molecular determinants of adult-stage axon regeneration using the AVM mechanosensory neurons. The first step in AVM axon development is a pioneer axonal projection from the cell body to the ventral nerve cord. We show that regeneration of the AVM axon to the ventral nerve cord lacks the deterministic precision of initial axon development, requiring competition and pruning of unwanted axon branches. Nevertheless, axons of injured AVM neurons regrow to the ventral nerve cord with over 60% reliability in adult animals. In addition, in contrast to initial development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but independent of UNC-129/TGF-beta repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes heavily dependent on UNC-34/Ena and CED-10/Rac actin regulators. Thus, C. elegans may be used as a genetic system to characterize novel cellular and molecular mechanisms underlying adult-stage nervous system regeneration.     

The C. elegans LIM homeobox gene lin-11 specifies multiple cell fates during vulval development

LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lin-11 have previously been shown to alter the cell division pattern of a subset of the 2 degrees lineage vulval cells. We demonstrate multiple functions of lin-11 during vulval development. We examined the fate of vulval cells in lin-11 mutant animals using five cellular markers and found that lin-11 is necessary for the patterning of both 1 degrees and 2 degrees lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, we show that lin-11 is initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2 degrees lineage cells. Using a conditional RNAi approach, we show that lin-11 regulates vulval morphogenesis. Finally, we show that LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning.     

UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans.

The unc-6 gene is required for the guidance of pioneer axons and migrating cells along the body wall in C. elegans. In mutants, dorsal and ventral migrations are disrupted, but longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by two independent transposon insertions. Based on genomic and cDNA sequencing, the gene encodes a novel laminin-related protein, UNC-6 (591 amino acids). The N-terminus is homologous to the N-termini (i.e., domains V1, V-1, V-2, and V-3) of laminin subunits, while the C-terminus is a unique domain. We propose that UNC-6 is a component of an extracellular matrix cue that guides dorsoventral migrations on the epidermis.     

Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position

Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon outgrowth. We show here that the C. elegans fibroblast growth factor receptor (FGFR), EGL-15, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-15 tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate outgrowth of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-15(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon outgrowth but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-15(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position.     

Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C. elegans

Secreted proteins of the Wnt family affect axon guidance, asymmetric cell division, and cell fate. We show here that C. elegans Wnts acting through Frizzled receptors can shape axon and dendrite trajectories by reversing the anterior-posterior polarity of neurons. In lin-44/Wnt and lin-17/Frizzled mutants, the polarity of the PLM mechanosensory neuron is reversed along the body axis: the long PLM process, PLM growth cone, and synapses are posterior to its cell body instead of anterior. Similarly, the polarity of the ALM mechanosensory neuron is reversed in cwn-1 egl-20 Wnt double mutants, suggesting that different Wnt signals regulate neuronal polarity at different anterior-posterior positions. LIN-17 protein is asymmetrically localized to the posterior process of PLM in a lin-44-dependent manner, indicating that Wnt signaling redistributes LIN-17 in PLM. In this context, Wnts appear to function not as instructive growth cone attractants or repellents, but as organizers of neuronal polarity.     

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.