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.

Convertase-dependent regulation of membrane-tethered and secreted ligands tunes dendrite adhesion.

During neural development, cellular adhesion is crucial for interactions among and between neurons and surrounding tissues. This function is mediated by conserved cell adhesion molecules, which are tightly regulated to allow for coordinated neuronal outgrowth. Here, we show that the proprotein convertase KPC-1 (homolog of mammalian furin) regulates the Menorin adhesion complex during development of PVD dendritic arbors in Caenorhabditis elegans. We found a finely regulated antagonistic balance between PVD-expressed KPC-1 and the epidermally expressed putative cell adhesion molecule MNR-1 (Menorin). Genetically, partial loss of mnr-1 suppressed partial loss of kpc-1, and both loss of kpc-1 and transgenic overexpression of mnr-1 resulted in indistinguishable phenotypes in PVD dendrites. This balance regulated cell-surface localization of the DMA-1 leucine-rich transmembrane receptor in PVD neurons. Lastly, kpc-1 mutants showed increased amounts of MNR-1 and decreased amounts of muscle-derived LECT-2 (Chondromodulin II), which is also part of the Menorin adhesion complex. These observations suggest that KPC-1 in PVD neurons directly or indirectly controls the abundance of proteins of the Menorin adhesion complex from adjacent tissues, thereby providing negative feedback from the dendrite to the instructive cues of surrounding tissues.

Heparan sulfates and heparan sulfate proteoglycans in hematopoiesis.

ABSTRACT: From signaling mediators in stem cells to markers of differentiation and lineage commitment to facilitators for the entry of viruses, such as HIV-1, cell surface heparan sulfate (HS) glycans with distinct modification patterns play important roles in hematopoietic biology. In this review, we provide an overview of the importance of HS and the proteoglycans (HSPGs) to which they are attached within the major cellular subtypes of the hematopoietic system. We summarize the roles of HSPGs, HS, and HS modifications within each main hematopoietic cell lineage of both myeloid and lymphoid arms. Lastly, we discuss the biological advances in the detection of HS modifications and their potential to further discriminate cell types within hematopoietic tissue.

Correction: It's All in Your Mind: Determining Germ Cell Fate by Neuronal IRE-1 in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1004747.].

Whole-animal connectomes of both Caenorhabditis elegans sexes.

Knowledge of connectivity in the nervous system is essential to understanding its function. Here we describe connectomes for both adult sexes of the nematode Caenorhabditis elegans, an important model organism for neuroscience research. We present quantitative connectivity matrices that encompass all connections from sensory input to end-organ output across the entire animal, information that is necessary to model behaviour. Serial electron microscopy reconstructions that are based on the analysis of both new and previously published electron micrographs update previous results and include data on the male head. The nervous system differs between sexes at multiple levels. Several sex-shared neurons that function in circuits for sexual behaviour are sexually dimorphic in structure and connectivity. Inputs from sex-specific circuitry to central circuitry reveal points at which sexual and non-sexual pathways converge. In sex-shared central pathways, a substantial number of connections differ in strength between the sexes. Quantitative connectomes that include all connections serve as the basis for understanding how complex, adaptive behavior is generated.

Specific N-glycans regulate an extracellular adhesion complex during somatosensory dendrite patterning.

N-glycans are molecularly diverse sugars borne by over 70% of proteins transiting the secretory pathway and have been implicated in protein folding, stability, and localization. Mutations in genes important for N-glycosylation result in congenital disorders of glycosylation that are often associated with intellectual disability. Here, we show that structurally distinct N-glycans regulate an extracellular protein complex involved in the patterning of somatosensory dendrites in Caenorhabditis elegans. Specifically, aman-2/Golgi alpha-mannosidase II, a conserved key enzyme in the biosynthesis of specific N-glycans, regulates the activity of the Menorin adhesion complex without obviously affecting the protein stability and localization of its components. AMAN-2 functions cell-autonomously to allow for decoration of the neuronal transmembrane receptor DMA-1/LRR-TM with the correct set of high-mannose/hybrid/paucimannose N-glycans. Moreover, distinct types of N-glycans on specific N-glycosylation sites regulate DMA-1/LRR-TM receptor function, which, together with three other extracellular proteins, forms the Menorin adhesion complex. In summary, specific N-glycan structures regulate dendrite patterning by coordinating the activity of an extracellular adhesion complex, suggesting that the molecular diversity of N-glycans can contribute to developmental specificity in the nervous system.

The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning.

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.

HSMotifDiscover: identification of motifs in sequences composed of non-single-letter elements.

SUMMARY: The functional sub-string(s) of a biopolymer sequence defines the specificity of its interaction with other biomolecules and is often referred to as motifs. Computational algorithms and software have been broadly developed for finding such motifs in sequences in which the individual elements are single characters, such as those in DNA and protein sequences. However, there are more complex scenarios where the motifs exist in non-single-letter contexts, e.g. preferred patterns of chemical modifications on proteins, DNAs, RNAs or polysaccharides. To search for those motifs, we describe a new method that converts the modified sequence elements to representative single-letter codes and then uses a modified Gibbs-sampling algorithm to define the position specific scoring matrix representing the motif(s). As a proof of principle, we describe the implementation and application of an R package for discovering heparan sulfate (HS) motifs in glycan sequences, which are important in regulating protein-protein interactions. This software can be valuable for analyzing high-throughput glycoprotein binding data using microarrays with HS oligosaccharides or other biological polymers. AVAILABILITY AND IMPLEMENTATION: HSMotifDiscover is freely available as an open source R package released under an MIT license at https://github.com/bioinfoDZ/HSMotifDiscover and also available in the form of an app at https://hsmotifdiscover.shinyapps.io/HSMotifDiscover_ShinyApp/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Four specific immunoglobulin domains in UNC-52/Perlecan function with NID-1/Nidogen during dendrite morphogenesis in Caenorhabditis elegans.

The extracellular matrix is essential for various aspects of nervous system patterning. For example, sensory dendrites in flies, worms and fish have been shown to rely on coordinated interactions of tissues with extracellular matrix proteins. Here we show that the conserved basement membrane protein UNC-52/Perlecan is required for establishing the correct number of the highly ordered dendritic trees in the somatosensory neuron PVD in Caenorhabditis elegans This function is dependent on four specific immunoglobulin domains, but independent of the known functions of UNC-52 in mediating muscle-skin attachment. Intriguingly, the four conserved immunoglobulin domains in UNC-52 are necessary to correctly localize the basement membrane protein NID-1/Nidogen. Genetic experiments further show that unc-52, nid-1 and genes of the netrin axon guidance signaling cassette share a common pathway to establish the correct number of somatosensory dendrites. Our studies suggest that, in addition to its role in mediating muscle-skin attachment, UNC-52 functions through immunoglobulin domains to establish an ordered lattice of basement membrane proteins, which may control the function of morphogens during dendrite patterning.

Reduced Insulin/Insulin-Like Growth Factor Receptor Signaling Mitigates Defective Dendrite Morphogenesis in Mutants of the ER Stress Sensor IRE-1.

Neurons receive excitatory or sensory inputs through their dendrites, which often branch extensively to form unique neuron-specific structures. How neurons regulate the formation of their particular arbor is only partially understood. In genetic screens using the multidendritic arbor of PVD somatosensory neurons in the nematode Caenorhabditis elegans, we identified a mutation in the ER stress sensor IRE-1/Ire1 (inositol requiring enzyme 1) as crucial for proper PVD dendrite arborization in vivo. We further found that regulation of dendrite growth in cultured rat hippocampal neurons depends on Ire1 function, showing an evolutionarily conserved role for IRE-1/Ire1 in dendrite patterning. PVD neurons of nematodes lacking ire-1 display reduced arbor complexity, whereas mutations in genes encoding other ER stress sensors displayed normal PVD dendrites, specifying IRE-1 as a selective ER stress sensor that is essential for PVD dendrite morphogenesis. Although structure function analyses indicated that IRE-1's nuclease activity is necessary for its role in dendrite morphogenesis, mutations in xbp-1, the best-known target of non-canonical splicing by IRE-1/Ire1, do not exhibit PVD phenotypes. We further determined that secretion and distal localization to dendrites of the DMA-1/leucine rich transmembrane receptor (DMA-1/LRR-TM) is defective in ire-1 but not xbp-1 mutants, suggesting a block in the secretory pathway. Interestingly, reducing Insulin/IGF1 signaling can bypass the secretory block and restore normal targeting of DMA-1, and consequently normal PVD arborization even in the complete absence of functional IRE-1. This bypass of ire-1 requires the DAF-16/FOXO transcription factor. In sum, our work identifies a conserved role for ire-1 in neuronal branching, which is independent of xbp-1, and suggests that arborization defects associated with neuronal pathologies may be overcome by reducing Insulin/IGF signaling and improving ER homeostasis and function.

Intrinsic and extrinsic mechanisms of dendritic morphogenesis.

The complex, branched morphology of dendrites is a cardinal feature of neurons and has been used as a criterion for cell type identification since the beginning of neurobiology. Regulated dendritic outgrowth and branching during development form the basis of receptive fields for neurons and are essential for the wiring of the nervous system. The cellular and molecular mechanisms of dendritic morphogenesis have been an intensely studied area. In this review, we summarize the major experimental systems that have contributed to our understandings of dendritic development as well as the intrinsic and extrinsic mechanisms that instruct the neurons to form cell type-specific dendritic arbors.

Diverse roles for glycosaminoglycans in neural patterning.

The nervous system coordinates the functions of most multicellular organisms and their response to the surrounding environment. Its development involves concerted cellular interactions, including migration, axon guidance, and synapse formation. These processes depend on the molecular constituents and structure of the extracellular matrices (ECM). An essential component of ECMs are proteoglycans, i.e., proteins containing unbranched glycan chains known as glycosaminoglycans (GAGs). A defining characteristic of GAGs is their enormous molecular diversity, created by extensive modifications of the glycans during their biosynthesis. GAGs are widely expressed, and their loss can lead to catastrophic neuronal defects. Despite their importance, we are just beginning to understand the function and mechanisms of GAGs in neuronal development. In this review, we discuss recent evidence suggesting GAGs have specific roles in neuronal patterning and synaptogenesis. We examine the function played by the complex modifications present on GAG glycans and their roles in regulating different aspects of neuronal patterning. Moreover, the review considers the function of proteoglycan core proteins in these processes, stressing their likely role as co-receptors of different signaling pathways in a redundant and context-dependent manner. We conclude by discussing challenges and future directions toward a better understanding of these fascinating molecules during neuronal development. Developmental Dynamics 247:54-74, 2018. © 2017 Wiley Periodicals, Inc.

The proprotein convertase KPC-1/furin controls branching and self-avoidance of sensory dendrites in Caenorhabditis elegans.

Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the 'menorin' pathway to control branching and growth of somatosensory dendrites in PVD.

It's all in your mind: determining germ cell fate by neuronal IRE-1 in C. elegans.

The C. elegans germline is pluripotent and mitotic, similar to self-renewing mammalian tissues. Apoptosis is triggered as part of the normal oogenesis program, and is increased in response to various stresses. Here, we examined the effect of endoplasmic reticulum (ER) stress on apoptosis in the C. elegans germline. We demonstrate that pharmacological or genetic induction of ER stress enhances germline apoptosis. This process is mediated by the ER stress response sensor IRE-1, but is independent of its canonical downstream target XBP-1. We further demonstrate that ire-1-dependent apoptosis in the germline requires both CEP-1/p53 and the same canonical apoptotic genes as DNA damage-induced germline apoptosis. Strikingly, we find that activation of ire-1, specifically in the ASI neurons, but not in germ cells, is sufficient to induce apoptosis in the germline. This implies that ER stress related germline apoptosis can be determined at the organism level, and is a result of active IRE-1 signaling in neurons. Altogether, our findings uncover ire-1 as a novel cell non-autonomous regulator of germ cell apoptosis, linking ER homeostasis in sensory neurons and germ cell fate.

Conservation of anatomically restricted glycosaminoglycan structures in divergent nematode species.

Heparan sulfates (HS) are glycosaminoglycans of the extracellular matrices and characterized by complex modification patterns owing to sulfations, epimerization, and acetylation. Distinct HS modification patterns have been shown to modulate protein-protein interactions during development in general and of the nervous system in particular. This has led to the heparan sulfate code hypothesis, which posits that specifically modified HS epitopes are distributed in a tissue and cell-specific fashion to orchestrate neural circuit formation. Whether an HS code exists in vivo, how specific or how evolutionarily conserved the anatomical distribution of an HS code may be has remained unknown. Here we conduct a systematic comparison of HS modification patterns in the nematode Caenorhabditis elegans using transgenic expression of 33 different HS-specific single chain variable fragment antibodies. We find that some HS modification patterns are widely distributed in the nervous system. In contrast, other HS modification patterns appear highly cell-specific in both non-neuronal and neuronal cells. Some patterns can be as restricted in their localization as to single neurites or synaptic connections between two neurons. This restricted anatomical localization of specific HS patterns can be evolutionarily conserved over a span of 80-100 million years in the divergent nematode species Caenorhabditis briggsae suggesting structural and, possibly functional conservation of glycosaminoglycan structures similar to proteins. These findings suggest a HS code with subcellularly localized, unique glycan identities in the nervous system.

Direct visualization of specifically modified extracellular glycans in living animals.

Modification patterns of heparan sulfate coordinate protein function in metazoans, yet in vivo imaging of such non-genetically encoded structures has been impossible. Here we report a transgenic method in Caenorhabditis elegans that allows direct live imaging of specific heparan sulfate modification patterns. This experimental approach reveals a dynamic and cell-specific heparan sulfate landscape and could in principle be adapted to visualize and analyze any extracellular molecule in vivo.

C. elegans bicd-1, homolog of the Drosophila dynein accessory factor Bicaudal D, regulates the branching of PVD sensory neuron dendrites.

The establishment of cell type-specific dendritic arborization patterns is a key phase in the assembly of neuronal circuitry that facilitates the integration and processing of synaptic and sensory input. Although studies in Drosophila and vertebrate systems have identified a variety of factors that regulate dendrite branch formation, the molecular mechanisms that control this process remain poorly defined. Here, we introduce the use of the Caenorhabditis elegans PVD neurons, a pair of putative nociceptors that elaborate complex dendritic arbors, as a tractable model for conducting high-throughput RNAi screens aimed at identifying key regulators of dendritic branch formation. By carrying out two separate RNAi screens, a small-scale candidate-based screen and a large-scale screen of the ~3000 genes on chromosome IV, we retrieved 11 genes that either promote or suppress the formation of PVD-associated dendrites. We present a detailed functional characterization of one of the genes, bicd-1, which encodes a microtubule-associated protein previously shown to modulate the transport of mRNAs and organelles in a variety of organisms. Specifically, we describe a novel role for bicd-1 in regulating dendrite branch formation and show that bicd-1 is likely to be expressed, and primarily required, in PVD neurons to control dendritic branching. We also present evidence that bicd-1 operates in a conserved pathway with dhc-1 and unc-116, components of the dynein minus-end-directed and kinesin-1 plus-end-directed microtubule-based motor complexes, respectively, and interacts genetically with the repulsive guidance receptor unc-5.

Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Dendrite morphogenesis in Caenorhabditis elegans.

Since the days of Ramón y Cajal, the vast diversity of neuronal and particularly dendrite morphology has been used to catalog neurons into different classes. Dendrite morphology varies greatly and reflects the different functions performed by different types of neurons. Significant progress has been made in our understanding of how dendrites form and the molecular factors and forces that shape these often elaborately sculpted structures. Here, we review work in the nematode Caenorhabditis elegans that has shed light on the developmental mechanisms that mediate dendrite morphogenesis with a focus on studies investigating ciliated sensory neurons and the highly elaborated dendritic trees of somatosensory neurons. These studies, which combine time-lapse imaging, genetics, and biochemistry, reveal an intricate network of factors that function both intrinsically in dendrites and extrinsically from surrounding tissues. Therefore, dendrite morphogenesis is the result of multiple tissue interactions, which ultimately determine the shape of dendritic arbors.

RABR-1, an atypical Rab-related GTPase cell non-autonomously restricts somatosensory dendrite branching.

Neurons are highly polarized cells with dendrites and axons. Dendrites, which receive sensory information or input from other neurons, often display elaborately branched morphologies. While mechanisms that promote dendrite branching have been widely studied, less is known about the mechanisms that restrict branching. Using the nematode Caenorhabditis elegans, we identify rabr-1 (for Rab-related gene 1) as a factor that restricts branching of the elaborately branched dendritic trees of PVD and FLP somatosensory neurons. Animals mutant for rabr-1 show excessively branched dendrites throughout development and into adulthood in areas where the dendrites overlay epidermal tissues. Phylogenetic analyses show that RABR-1 displays similarity to small GTPases of the Rab-type, although based on sequence alone, no clear vertebrate ortholog of RABR-1 can be identified. We find that rabr-1 is expressed and can function in epidermal tissues, suggesting that rabr-1 restricts dendritic branching cell-non-autonomously. Genetic experiments further indicate that for the formation of ectopic branches rabr-1 mutants require the genes of the Menorin pathway, which have been previously shown to mediate dendrite morphogenesis of somatosensory neurons. A translational reporter for RABR-1 reveals a subcellular localization to punctate, perinuclear structures, which correlates with endosomal and autophagosomal markers, but anticorrelates with lysosomal markers suggesting an amphisomal character. Point mutations in rabr-1 analogous to key residues of small GTPases suggest that rabr-1 functions in a GTP-bound form independently of GTPase activity. Taken together, rabr-1 encodes for an atypical small GTPase of the Rab-type that cell-non-autonomously restricts dendritic branching of somatosensory neurons, likely independently of GTPase activity.