A Network of HSPG Core Proteins and HS Modifying Enzymes Regulates Netrin-Dependent Guidance of D-Type Motor Neurons in Caenorhabditis elegans

Abstract

Heparan sulfate proteoglycans (HSPGs) are proteins with long covalently attached sugar side chains of the heparan sulfate (HS) type. Depending on the cellular context HS chains carry multiple structural modifications such as sulfate residues or epimerized sugars allowing them to bind to a wide range of molecules. HSPGs have been found to play extremely diverse roles in animal development and were shown to interact with certain axon guidance molecules. In this study we describe the role of the Caenorhabditis elegans HSPG core proteins Syndecan (SDN-1) and Glypican (LON-2) and the HS modifying enzymes in the dorsal guidance of D-type motor axons, a process controlled mainly by the conserved axon guidance molecule UNC-6/Netrin. Our genetic analysis established the specific HS code relevant for this axon guidance event. Using two sensitized genetic backgrounds, we isolated novel components influencing D-type motor axon guidance with a link to HSPGs, as well as new alleles of several previously characterized axon guidance genes. Interestingly, the dorsal axon guidance defects induced by mutations in zfp-1 or lin-35 depended on the transgene oxIs12 used to visualize the D-type motor neurons. oxIs12 is a large multi-copy transgene that enlarges the X chromosome by approximately 20%. In a search for genes with a comparable phenotype we found that a mutation in the known dosage compensation gene dpy-21 showed similar axon guidance defects as zfp-1 or lin-35 mutants. Thus, derepression of genes on X, where many genes relevant for HS dependent axon guidance are located, might also influence axon guidance of D-type motor neurons.

Neuronal Migration Is Regulated by Endogenous RNAi and Chromatin-Binding Factor ZFP-1/AF10 in Caenorhabditis elegans

“…With the discovery that ZFP-1 negatively regulates pdk-1, a component of the IIS pathway, via direct repression of pdk-1 transcription (Mansisidor et al 2011), we were prompted to investigate the connection between ZFP-1 and IIS during HSN migration. We found that the zfp-1 (ok554) loss-of-function mutant (Cui et al 2006;Avgousti et al 2013;Cecere et al 2013) and the zfp-1(op481) mutant (Gysi et al 2013)-both of which lead to the generation of mRNAs that contain premature stop codons after the PHD fingers-display HSN undermigration defects (Figure 1, A-C). We used a tryptophan hydroxylase GFP reporter (tph-1:: gfp) that marks the HSNs in adult animals for scoring the migration phenotype.…”

“…Moreover, the op481 allele was shown to exhibit an identical axon guidance phenotype as the ok554 allele of zfp-1 (Gysi et al 2013). Surprisingly, however, the axon guidance phenotype in the zfp-1(ok554); sdn-1(zh20) double mutant was dependent upon the presence of the integrated transgene used to visualize the axon guidance defects (Gysi et al 2013).…”

“…Moreover, the op481 allele was shown to exhibit an identical axon guidance phenotype as the ok554 allele of zfp-1 (Gysi et al 2013). Surprisingly, however, the axon guidance phenotype in the zfp-1(ok554); sdn-1(zh20) double mutant was dependent upon the presence of the integrated transgene used to visualize the axon guidance defects (Gysi et al 2013). Therefore, to exclude the possibility that the tph-1::gfp transgene was enhancing or causing the zfp-1 mutant undermigration phenotype, we stained wild-type worms and zfp-1 mutant worms with an anti-serotonin antibody (Garriga et al 1993).…”

“…The zfp-1(op481) allele was originally isolated in a mutagenesis screen performed to identify genetic enhancers of a null mutation in the heparan sulfate proteoglycan core protein SDN-1, which is involved in D-type motor axon guidance (Gysi et al 2013). Moreover, the op481 allele was shown to exhibit an identical axon guidance phenotype as the ok554 allele of zfp-1 (Gysi et al 2013).…”

Hypergravity hinders axonal development of motor neurons in Caenorhabditis elegans

“…Interestingly, we did not detect any DD/VD axonal 287 defects at either 1G or 100G in this strain (data not shown). However, a previous report 288 cautioned that the oxIs12 transgenic strain affected the expression of X-linked genes involved in 289 axon guidance such as unc-6/netrin and lon-2/glypican genes, altering axon guidance phenotypes 290 in different genetic backgrounds (Gysi et al 2013). In addition, previous studies using electron 291 microscopy have shown that the DD motor neurons can develop in slightly different locations in 292 individual animals (White et al 1976), and the number of axons innervating the dorsal nerve 293 cord can also vary in individual animals (Hedgecock et al 1990).…”