Skin-derived cues control arborization of sensory dendrites in Caenorhabditis elegans.

Sensory dendrites depend on cues from their environment to pattern their growth and direct them toward their correct target tissues. Yet, little is known about dendrite-substrate interactions during dendrite morphogenesis. Here, we describe MNR-1/menorin, which is part of the conserved Fam151 family of proteins and is expressed in the skin to control the elaboration of "menorah"-like dendrites of mechanosensory neurons in Caenorhabditis elegans. We provide biochemical and genetic evidence that MNR-1 acts as a contact-dependent or short-range cue in concert with the neural cell adhesion molecule SAX-7/L1CAM in the skin and through the neuronal leucine-rich repeat transmembrane receptor DMA-1 on sensory dendrites. Our data describe an unknown pathway that provides spatial information from the skin substrate to pattern sensory dendrite development nonautonomously.

Correction: The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

[This corrects the article DOI: 10.1371/journal.ppat.1009350.].

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.

The Caenorhabditis elegans RIG-I Homolog DRH-1 Mediates the Intracellular Pathogen Response upon Viral Infection.

Mammalian retinoic acid-inducible gene I (RIG-I)-like receptors detect viral double-stranded RNA (dsRNA) and 5'-triphosphorylated RNA to activate the transcription of interferon genes and promote antiviral defense. The Caenorhabditis elegans RIG-I-like receptor DRH-1 promotes defense through antiviral RNA interference (RNAi), but less is known about its role in regulating transcription. Here, we describe a role for DRH-1 in directing a transcriptional response in C. elegans called the intracellular pathogen response (IPR), which is associated with increased pathogen resistance. The IPR includes a set of genes induced by diverse stimuli, including intracellular infection and proteotoxic stress. Previous work suggested that the proteotoxic stress caused by intracellular infections might be the common trigger of the IPR, but here, we demonstrate that different stimuli act through distinct pathways. Specifically, we demonstrate that DRH-1/RIG-I is required for inducing the IPR in response to Orsay virus infection but not in response to other triggers like microsporidian infection or proteotoxic stress. Furthermore, DRH-1 appears to be acting independently of its known role in RNAi. Interestingly, expression of the replication-competent Orsay virus RNA1 segment alone is sufficient to induce most of the IPR genes in a manner dependent on RNA-dependent RNA polymerase activity and on DRH-1. Altogether, these results suggest that DRH-1 is a pattern recognition receptor that detects viral replication products to activate the IPR stress/immune program in C. elegansIMPORTANCEC. elegans lacks homologs of most mammalian pattern recognition receptors, and how nematodes detect pathogens is poorly understood. We show that the C. elegans RIG-I homolog DRH-1 mediates the induction of the intracellular pathogen response (IPR), a novel transcriptional defense program, in response to infection by the natural C. elegans viral pathogen Orsay virus. DRH-1 appears to act as a pattern recognition receptor to induce the IPR transcriptional defense program by sensing the products of viral RNA-dependent RNA polymerase activity. Interestingly, this signaling role of DRH-1 is separable from its previously known role in antiviral RNAi. In addition, we show that there are multiple host pathways for inducing the IPR, shedding light on the regulation of this novel transcriptional immune response.

The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival.

A sperm that fertilizes an egg has successfully survived multiple checkpoints within the female reproductive tract, termed pre-fertilization events. The leukocytic response is a pre-fertilization event in which sperm trigger an immune response that promotes homing of circulating leukocytes to the uterine lumen to destroy most sperm. Various glycoconjugates decorate the sperm surface, including sialic acids, which are abundant at the sperm surface where they cap most glycan chains and regulate sperm migration through cervical mucus, formation of the sperm oviductal reservoir, and sperm capacitation. However, the role of sperm-associated sialic acids in the leukocytic reaction remains unknown. The cognate endogenous binding partners of sialic acids, sialic acid-binding immunoglobulin-like lectins (Siglecs) play a pivotal role in regulating many immune responses. Here we investigated whether sperm-associated sialic acids inhibit activation of neutrophils, one of the major immune cells involved in the leukocytic reaction. We used in vitro interactions between sperm and neutrophils as well as binding assays between sperm and recombinant Siglec-Fc chimeric proteins to measure interactions. Moreover, we examined whether Siglecs are expressed on human and mouse endometria, which have a role in initiating the leukocytic reaction. Surprisingly less sialylated, capacitated, sperm did not increase neutrophil activation in vitro However, we observed expression of several Siglecs on the endometrium and that these receptors interact with sialylated sperm. Our results indicate that sperm sialic acids may interact with endometrial Siglecs and that these interactions facilitate sperm survival in the face of female immunity.

Sugar-coated sperm: Unraveling the functions of the mammalian sperm glycocalyx.

Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol-anchored glycoproteins and glycolipids) and glycan-rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm-associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions.

Insights from C. elegans into Microsporidia Biology and Host-Pathogen Relationships.

Microsporidia are poorly understood, ubiquitous eukaryotic parasites that are completely dependent on their hosts for replication. With the discovery of microsporidia species naturally infecting the genetically tractable transparent nematode C. elegans, this host has been used to explore multiple areas of microsporidia biology. Here we review results about microsporidia infections in C. elegans, which began with the discovery of the intestinal-infecting species Nematocida parisii. Recent findings include new species identification in the Nematocida genus, with more intestinal-infecting species, and also a species with broader tissue tropism, the epidermal and muscle-infecting species Nematocida displodere. This species has a longer polar tube infection apparatus, which may enable its wider tissue range. After invasion, multiple Nematocida species appear to fuse host cells, which likely promotes their dissemination within host organs. Localized proteomics identified Nematocida proteins that have direct contact with the C. elegans intestinal cytosol and nucleus, and many of these host-exposed proteins belong to expanded, species-specific gene families. On the host side, forward genetic screens have identified regulators of the Intracellular Pathogen Response (IPR), which is a transcriptional response induced by both microsporidia and the Orsay virus, which is also a natural, obligate intracellular pathogen of the C. elegans intestine. The IPR constitutes a novel immune/stress response that promotes resistance against microsporidia, virus, and heat shock. Overall, the Nematocida/C. elegans system has provided insights about strategies for microsporidia pathogenesis, as well as innate defense pathways against these parasites.

Identification of a unique Alu-based polymorphism and its use in human population studies.

Alu elements represent a family of short interspersed DNA elements (SINEs) found in primate genomes. These are members of a group of transposable elements that integrate into the genome by the process of retrotransposition. Recent integrations of Alu elements within the human genome have generated presence/absence variants useful as DNA markers in human population studies as well as in forensic and paternity analyses. Besides the ease of use, this type of marker is unique because the absence of the Alu represents the ancestral form. We have identified an Alu-based polymorphism that consists of four alleles in which we can predict the evolutionary order. Additionally, we have developed a simple PCR plus restriction endonuclease assay to readily distinguish the four alleles. We have thus far analyzed DNA from a small set of samples comprising ten different ethnic groups. The three populations of African descent exhibited a relatively low frequency of the absence allele in contrast to the other populations, as well as being the only populations in which all four alleles were identified. One presence allele was not found in both European Caucasian and South American populations that were sampled, whereas a different presence allele was not observed among the sampled Asian populations. Additionally, the four-allele system identified variations among populations not observed by simply scoring as presence/absence variants. Therefore, extending beyond the two-allele dimorphic Alu system further elucidates population variations. These features afford this marker as a unique tool in the study of both global and regional analyses of human populations.

Coordination of Heparan Sulfate Proteoglycans with Wnt Signaling To Control Cellular Migrations and Positioning in Caenorhabditis elegans.

Heparan sulfates (HS) are linear polysaccharides with complex modification patterns, which are covalently bound via conserved attachment sites to core proteins to form heparan sulfate proteoglycans (HSPGs). HSPGs regulate many aspects of the development and function of the nervous system, including cell migration, morphology, and network connectivity. HSPGs function as cofactors for multiple signaling pathways, including the Wnt-signaling molecules and their Frizzled receptors. To investigate the functional interactions among the HSPG and Wnt networks, we conducted genetic analyses of each, and also between these networks using five cellular migrations in the nematode Caenorhabditis elegans We find that HSPG core proteins act genetically in a combinatorial fashion dependent on the cellular contexts. Double mutant analyses reveal distinct redundancies among HSPGs for different migration events, and different cellular migrations require distinct heparan sulfate modification patterns. Our studies reveal that the transmembrane HSPG SDN-1/Syndecan functions within the migrating cell to promote cellular migrations, while the GPI-linked LON-2/Glypican functions cell nonautonomously to establish the final cellular position. Genetic analyses with the Wnt-signaling system show that (1) a given HSPG can act with different Wnts and Frizzled receptors, and that (2) a given Wnt/Frizzled pair acts with different HSPGs in a context-dependent manner. Lastly, we find that distinct HSPG and Wnt/Frizzled combinations serve separate functions to promote cellular migration and establish position of specific neurons. Our studies suggest that HSPGs use structurally diverse glycans in coordination with Wnt-signaling pathways to control multiple cellular behaviors, including cellular and axonal migrations and, cellular positioning.

Recent integrations of mammalian Hmg retropseudogenes.

We propose that select retropseudogenes of the high mobility group nonhistone chromosomal protein genes have recently integrated into mammalian genomes on the basis of the high sequence identity of the copies to the cDNA sequences derived from the original genes. These include the Hmg1 gene family in mice and the Hmgn2 family in humans. We investigated orthologous loci of several strains and species of Mus for presence or absence of apparently young Hmg1 retropseudogenes. Three of four analysed elements were specific to Mus musculus, two of which were not fixed, indicative of recent evolutionary origins. Additionally, we datamined a presumptive subfamily (Hmgz) of mouse Hmg1, but only identified one true element in the GenBank database, which is not consistent with a separate subfamily status. Two of four analysed Hmgn2 retropseudogenes were specific for the human genome, whereas a third was identified in human, chimpanzee and gorilla genomes, and a fourth additionally found in orangutan but absent in African green monkey. Flanking target-site duplications were consistent with LINE integration sites supporting LINE machinery for their mechanism of amplification. The human Hmgn2 retropseudogenes were full length, whereas the mouse Hmg1 elements were either full length or 3'-truncated at specific positions, most plausibly the result of use of alternative polyadenylation sites. The nature of their recent amplification success in relation to other retropseudogenes is unclear, although availability of a large number of transcripts during gametogenesis may be a reason. It is apparent that retropseudogenes continue to shape mammalian genomes, and may provide insight into the process of retrotransposition, as well as offer potential use as phylogenetic markers.

Complex cooperative functions of heparan sulfate proteoglycans shape nervous system development in Caenorhabditis elegans.

The development of the nervous system is a complex process requiring the integration of numerous molecular cues to form functional circuits. Many cues are regulated by heparan sulfates, a class of linear glycosaminoglycan polysaccharides. These sugars contain distinct modification patterns that regulate protein-protein interactions. Misexpressing the homolog of KAL-1/anosmin-1, a neural cell adhesion molecule mutant in Kallmann syndrome, in Caenorhabditis elegans causes a highly penetrant, heparan sulfate-dependent axonal branching phenotype in AIY interneurons. In an extended forward genetic screen for modifiers of this phenotype, we identified alleles in new as well as previously identified genes involved in HS biosynthesis and modification, namely the xylosyltransferase sqv-6, the HS-6-O-sulfotransferase hst-6, and the HS-3-O-sulfotransferase hst-3.2. Cell-specific rescue experiments showed that different HS biosynthetic and modification enzymes can be provided cell-nonautonomously by different tissues to allow kal-1-dependent branching of AIY. In addition, we show that heparan sulfate proteoglycan core proteins that carry the heparan sulfate chains act genetically in a highly redundant fashion to mediate kal-1-dependent branching in AIY neurons. Specifically, lon-2/glypican and unc-52/perlecan act in parallel genetic pathways and display synergistic interactions with sdn-1/syndecan to mediate kal-1 function. Because all of these heparan sulfate core proteins have been shown to act in different tissues, these studies indicate that KAL-1/anosmin-1 requires heparan sulfate with distinct modification patterns of different cellular origin for function. Our results support a model in which a three-dimensional scaffold of heparan sulfate mediates KAL-1/anosmin-1 and intercellular communication through complex and cooperative interactions. In addition, the genes we have identified could contribute to the etiology of Kallmann syndrome in humans.

Distinct 3-O-sulfated heparan sulfate modification patterns are required for kal-1-dependent neurite branching in a context-dependent manner in Caenorhabditis elegans.

Heparan sulfate (HS) is an unbranched glycosaminoglycan exhibiting substantial molecular diversity due to multiple, nonuniformly introduced modifications, including sulfations, epimerization, and acetylation. HS modifications serve specific and instructive roles in neuronal development, leading to the hypothesis of a HS code that regulates nervous system patterning. Although the in vivo roles of many of the HS modifications have been investigated, very little is known about the function of HS 3-O-sulfation in vivo. By examining patterning of the Caenorhabditis elegans nervous system in loss of function mutants of the two 3-O-sulfotransferases, hst-3.1 and hst-3.2, we found HS 3-O-sulfation to be largely dispensable for overall neural development. However, generation of stereotypical neurite branches in hermaphroditic-specific neurons required hst-3.1, hst-3.2, as well as an extracellular cell adhesion molecule encoded by kal-1, the homolog of Kallmann Syndrome associated gene 1/anosmin-1. In contrast, kal-1-dependent neurite branching in AIY neurons required catalytic activity of hst-3.2 but not hst-3.1. The context-dependent requirement for hst-3.2 and hst-3.1 indicates that both enzymes generate distinct types of HS modification patterns in different cell types, which regulate kal-1 to promote neurite branching. We conclude that HS 3-O-sulfation does not play a general role in establishing the HS code in C. elegans but rather plays a specialized role in a context-dependent manner to establish defined aspects of neuronal circuits.