Biology subjects

Lundquist, E. A.

Publications and source records attributed to Lundquist, E. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Domain-specific mutations in unc-6/Netrin differentially affect dorsal-ventral axon pathfinding in Caenorhabditis elegans

UNC-6/Netrin is a conserved regulator of dorsal-ventral axon and cell migrations. UNC-6 is composed of a Laminin N-terminal domain (LN), three epidermal growth factor repeats (EGF), and a Netrin C terminal domain (NC). Here, we identified missense mutations in distinct UNC-6 domains and assessed their roles in dorsal VD/DD motor axon guidance and ventral AVM axon guidance. A missense mutation in a conserved residue of the LN domain (G289D) resulted in dorsal and ventral axon guidance defects similar to unc-6 null. A distinct missense mutation in the LN domain (S120F) was hypomorphic and strongly perturbed ventral AVM axon guidance with minimal effects on dorsal VD/DD axon guidance, showing that S120F is predominantly required for ventral guidance. Missense mutations altering conserved cysteine residues involved in di-sulfide bonding in the EGF domains were analyzed. EGF1(C321G) caused both ventral and dorsal axon guidance defects albeit weaker than unc-6 null, indicating that EGF1 is required for both. EGF2(C347Y) strongly affected dorsal VD/DD axon guidance similar to unc-6 null, with weaker perturbation of ventral AVM axon guidance. Previous results revealed that EGF3(C410Y) specifically disrupted dorsal axon guidance, a result that we confirmed. Our studies using missense mutations in the endogenous unc-6 locus complement previous structure-function studies using transgenic expression, and identify domains specifically required for ventral AVM guidance (S120Y in the LN domain) and dorsal VD/DD axon guidance (C410Y in EGF3). The crystal structure of UNC-6 indicates conserved N-linked glycosylation at N114 and N128. Mutation of these sites in UNC-6 had no effect on dorsal ventral axon guidance, showing that they do not play a major role. However, the N114 and N128 mutations interacted genetically with unc-40 and unc-5 mutations, indicating that these glycosylation sites indeed have a role in UNC-6 signaling. Our results will inform studies on how these distinct UNC-6 domains interact with guidance receptors (e.g. UNC-40/DCC and UNC-5) and other extracellular molecules to mediate dorsal-ventral axon guidance.

developmental biology↗

bioRxiv2026

EFN-4/Ephrin converges with SAX-3/Robo, UNC-6/Netrin, and Heparan Sulfate Proteoglycan signaling to control MAB-5/Hox-dependent posterior Q neuroblast migration in Caenorhabditis elegans

Hox genes have been broadly implicated in nervous system development, but the molecular and genetic mechanisms that act downstream of Hox factors remain to be identified. The MAB-5 antennapedia-like Hox transcription factor is both necessary and sufficient to cause posterior migration of the Q neuroblast descendants in Caenorhabditis elegans. In response to MAB-5, the left-side QL descendants QL.a and QL.ap undergo a three-stage migration process, with each stage characterized by a posterior lamellipodial protrusion followed by cell body migration. The QL.ap cell differentiates into the PQR neuron posterior to the anus. Previous studies showed that the MAB-5-regulated gene efn-4/Ephrin was required for the third and final stage of QL.ap migration, with efn-4 mutation resulting in placement of PQR immediately anterior to the anus. This subtle and previously-undescribed phenotype opens the possibility that other known neuronal development genes could be involved. In this work, we screened known signaling mutants for third-stage PQR migration defects. We found that mutations in SAX-3/Robo signaling, UNC-6/Netrin signaling, and heparan sulfate proteoglycans (HSPGs) all displayed third-stage PQR migration defects. The effects in single mutants were weak compared to efn-4, and double mutant analysis revealed lack of genetic synergy, consistent with all of these molecules converging on a common pathway. This genetic analysis is consistent with physical interaction studies in vitro from another group that suggest that these molecules form connected communities of interacting extracellular domains, raising the possibility that they are all components of a large extracellular signaling complex required for posterior QL.ap migration. In this model, we envision that MAB-5/Hox drives EFN-4/Ephrin expression in QL.ap, which then seeds the formation of an extracellular signaling complex containing SAX-3/Robo signaling, UNC-6/Netrin signaling, and HSPGs that drives posterior lamellipodial formation and posterior migration.

developmental biology↗

bioRxiv2025

Single-cell transcriptomic profiling of C. elegans Q neuroblast lineage during migration and differentiation

Proper migration and differentiation of neuroblasts into neurons are essential for the development of a healthy nervous system. In this context, the asymmetrical migration of Caenorhabditis elegans Q neuroblasts provides a powerful model for studying the genetic aspects of neuronal migration in vivo at single-cell resolution. We isolated Q lineage cells at various stages of development using FACS and employed single-cell RNA sequencing to investigate the molecular mechanisms underlying the migration and differentiation of these neuroblasts. We created a robust transcriptomic differentiation map of the Q neuroblast lineage and used established markers to identify each cell in the lineage. Our results revealed novel genes not previously described on these cells and linked the expression of known genes to specific stages of Q lineage progression. Furthermore, functional enrichment and imaging provided evidence that the parent Q cells are initially specified with an epithelial-like identity and undergo epithelial-mesenchymal transition during the early stages of migration. We also identified novel Wnt-related mechanisms, including left-right asymmetric expression of cwn-1 and cwn-2, and the involvement of the Wnt/{beta}-catenin asymmetry pathway in the Q lineage. Our work offers a high-resolution view of neuroblast development, showcasing the power of single-cell transcriptomics to reveal stage-specific regulatory programs. Author SummaryCell migration and differentiation are critical steps for nervous system formation. Mapping changes in single-cell behavior during this process can greatly improve our understanding of neuronal development. However, the complexity of the human brain presents significant challenges to analyzing these processes in individual cells. Here, we used the Q neuroblast lineage from the nematode worm Caenorhabditis elegans as a model system to investigate the molecular dynamics of neurodevelopment using single-cell RNA sequencing. By analyzing over 6,000 individual cells from the Q lineage, we generated a detailed map showing how each cell in this lineage changes over time. This approach allowed us to identify new genes that had not previously been associated with this system and to identify the expression patterns of known genes at different stages of lineage development. By looking at gene functions at different stages, we also discovered that the parent cells in the lineage initially exhibit an epithelial-like identity before transitioning to neuroblasts and later becoming neurons. Overall, our study gives a clear picture of how gene expression changes as this neuroblast lineage develops, enhancing our understanding of how different genetic programs can influence neural development.

developmental biology↗

bioRxiv2025

VAB-8/KIF26, LIN-17/Frizzled, and EFN-4/Ephrin, control distinct stages of posterior neuroblast migration downstream of the MAB-5/Hox transcription factor in Caenorhabditis elegans

Hox transcription factors are involved in neuronal and neural crest development and differentiation, including migration, but the genetic programs employed by Hox genes to regulate terminal differentiation remain to be defined. In C. elegans, the Antennapedia-like Hox factor MAB-5 is both necessary and sufficient to induce posterior migration of the Q lineage neuroblasts and neurons downstream of canonical Wnt signaling. Q lineage fluorescence-activated cell sorting and RNA seq in mab-5 loss-of-function and gain-of-function backgrounds revealed genes with expression in the Q lineage dependent upon MAB-5. Here, the roles of three mab-5 target genes in QL lineage posterior migration are delineated, vab-8/KIF26, lin-17/Fz, and efn-4/Ephrin. First, live, time-lapse imaging of QL.a and QL.ap posterior migration revealed that this migration occurs in three distinct stages: QL.a migration posterior to QL.p; after QL.a division, posterior migration of QL.ap to a region immediately anterior to the anus; and final migration of QL.ap posterior to the anus to the final position where it differentiates into the PQR neuron. vab-8 affected each of the three stages, lin-17 affected stages two and three, and efn-4 was required for the third stage of posterior QL.ap migration. Thus, different MAB-5 target genes control distinct stages of posterior migration. mab-20/Semaphorin, a known interaction partner with efn-4, also affected only the third stage similar to efn-4. Suppression of mab-5 gof posterior migration confirmed that these genes act downstream of mab-5 in posterior migration. Possibly, VAB-8/KIF26 trafficks distinct molecules to the plasma membrane that mediate distinct stages of migration, including LIN-17/Fz and EFN-4. Surpisingly, failure of stages two and three led to the premature extension of a posterior dendritic protrusion, which normally forms after QL.ap had migrated to its final position and PQR differentiation begins. This suggests a link between migration and differentiation, where differentiation is delayed while migration proceeds. In sum, this works delineates a transcriptional program downstream of mab-5/Hox that controls posterior neuroblast migration, in response to Wnt signaling.

developmental biology↗

bioRxiv2024

Short- and long-range roles of UNC-6/Netrin in dorsal-ventral axon guidance in vivo in Caenorhabditis elegans

Recent studies in vertebrates and Caenorhabditis elegans have reshaped models of how the axon guidance cue UNC-6/Netrin functions in dorsal-ventral axon guidance, which was traditionally thought to form a ventral-to-dorsal concentration gradient that was actively sensed by growing axons. In the vertebrate spinal cord, floorplate Netrin1 was shown to be largely dispensable for ventral commissural growth. Rather, short range interactions with Netrin1 on the ventricular zone radial glial stem cells was shown to guide ventral commissural axon growth. In C. elegans, analysis of dorsally-migrating growth cones during outgrowth has shown that growth cone polarity of filopodial extension is separable from the extent of growth cone protrusion. Growth cones are first polarized by UNC-6/Netrin, and subsequent regulation of protrusion by UNC-6/Netrin is based on this earlier-established polarity (the Polarity/Protrusion model). In both cases, short-range or even haptotactic mechanisms are invoked: in vertebrate spinal cord, interactions of growth cones with radial glia expressing Netrin-1; and in C. elegans, a potential close-range interaction that polarizes the growth cone. To explore potential short-range and long-range functions of UNC-6/Netrin, a potentially membrane-anchored transmembrane UNC-6 (UNC-6(TM)) was generated by genome editing. unc-6(tm) was hypomorphic for dorsal VD/DD axon pathfinding, indicating that it retained some unc-6 function. Polarity of VD growth cone filopodial protrusion was initially established in unc-6(tm), but was lost as the growth cones migrated away from the unc-6(tm) source in the ventral nerve cord. In contrast, ventral guidance of the AVM and PVM axons was equally severe in unc-6(tm) and unc-6(null). Together, these results suggest that unc-6(tm) retains short-range functions but lacks long-range functions. Finally, ectopic unc-6(+) expression from non-ventral sources could rescue dorsal and ventral guidance defects in unc-6(tm) and unc-6(null). Thus, a ventral directional source of UNC-6 was not required for dorsal-ventral axon guidance, and UNC-6 can act as a permissive, not instructive, cue for dorsal-ventral axon guidance. Possibly, UNC-6 is a permissive signal that activates cell-intrinsic polarity; or UNC-6 acts with another signal that is required in a directional manner. In either case, the role of UNC-6 is to polarize the pro-protrusive activity of UNC-40/DCC in the direction of outgrowth.

developmental biology↗

bioRxiv2023

MAB-5/Hox regulates the Q neuroblast transcriptome, including cwn-1/Wnt, to mediate posterior migration in Caenorhabditis elegans

Neurogenesis involves the precisely-coordinated action of genetic programs controlling large-scale neuronal fate specification down to terminal events of neuronal differentiation. The Q neuroblasts in C. elegans, QL on the left and QR on the right, divide, differentiate, and migrate in a similar pattern to produce three neurons each. However, QL on the left migrates posteriorly, and QR on the right migrates anteriorly. The MAB-5/Hox transcription factor is necessary and sufficient for posterior Q lineage migration, and is normally expressed only in the QL lineage. To define genes controlled by MAB-5 in the Q cells, fluorescence-activated cell sorting was utilized to isolate populations of Q cells at a time in early L1 larvae when MAB-5 first becomes active. Sorted Q cells from wild-type, mab-5 loss-of-function (lof), and mab-5 gain-of-function (gof) mutants were subject to RNA-seq and differential expression analysis. Genes enriched in Q cells included those involved in cell division, DNA replication, and DNA repair, consist with the neuroblast stem cell identity of the Q cells at this stage. Genes affected by mab-5 included those involved in neurogenesis, neural development, and interaction with the extracellular matrix. cwn-1, which encodes a Wnt signaling molecule, showed a paired response to mab-5 in the Q cells: cwn-1 expression was reduced in mab-5(lof) and increased in mab-5(gof), suggesting that MAB-5 is required for cwn-1 expression in Q cells. MAB-5 is required to prevent anterior migration of the Q lineage while it transcriptionally reprograms the Q lineage for posterior migration. Functional genetic analysis revealed that CWN-1 is required downstream of MAB-5 to inhibit anterior migration of the QL lineage, likely in parallel to EGL-20/Wnt in a non-canonical Wnt pathway. In sum, work here describes a Q cell transcriptome, and a set of genes regulated by MAB-5 in the QL lineage. One of these genes, cwn-1, acts downstream of mab-5 in QL migration, indicating that this gene set includes other genes utilized by MAB-5 to facilitate posterior neuroblast migration.

developmental biology↗

bioRxiv2023

SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans

In the Polarity/Protusion model of growth cone migration away from the guidance cue UNC-6/Netrin, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge of the growth cone. UNC-5 also inhibits growth cone protrusion ventrally based upon this polarity. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

developmental biology↗

bioRxiv2023

A short isoform of the UNC-6/Netrin receptor UNC-5 is required for growth cone polarity and robust growth cone protrusion in Caenorhabditis elegans.

UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance. Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B cause loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion. In sum, these results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion.

developmental biology↗

bioRxiv2022

TOM-1/Tomosyn acts with the UNC-6/Netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans

In the polarity/protrusion model of growth cone repulsion from UNC-6/Netrin, UNC-6 first polarizes the VD growth cone via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. Through the UNC-40/DCC receptor, UNC-6 stimulates protrusion dorsally, and through UNC-5 inhibits protrusion ventrally and laterally, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule + end entry into the growth cone. To explore the role of vesicle fusion in growth cone protrusion, we analyzed tom-1/tomosyn mutants. Tomosyn normally occludes formation of the SNARE complex by interacting with and inhibiting Syntaxin and thus preventing vesicle fusion. VD growth cones of tom-1 null mutants were similar to wild-type. However, tom-1 null mutants suppressed the effects of constitutively-activated MYR::UNC-5, which alone causes small growth cones with little protrusion. This suggests that TOM-1 is normally required for the inhibitory effects of MYR::UNC-5 on growth cone protrusion. tom-1 encodes long and short isoforms, and results here indicate that tom-1S is required downstream of UNC-5 to inhibit protrusion, whereas the tom-1L has a pro-protrusive role. unc-64/Syntaxin mutants displayed reduced growth cone protrusion, suggesting that TOM-1 inhibits growth cone protrusion by inhibiting UNC-64/Syntaxin, similar to its role in neurotransmission. TOM-1L, TOM-1S, and UNC-64/Syntaxin were all required for VD growth cone polarity of protrusion, indicating that regulated vesicle fusion is required for the establishment and/or maintenance of VD growth cone polarity. These studies show that, in addition to effects on actin and microtubules, UNC-5 might inhibit VD growth cone protrusion by inhibiting growth cone vesicle fusion and thus the ability of growth cones to add plasma membrane necessary for protrusive growth.

developmental biology↗

bioRxiv2021

The PH/MyTH4/FERMmolecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of C. elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally-expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild-type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

neuroscience↗

bioRxiv2021

Caenorhabditis elegans ETR-1/CELF has broad effects on the muscle cell transcriptome, including genes that regulate translation and neuroblast migration

Migration of neuroblasts and neurons from their birthplace is central to the formation of neural circuits and networks. ETR-1 is the Caenorhabditis elegans homolog of the CELF1 (CUGBP, ELAV-like family 1) RNA-processing factor involved in neuromuscular disorders. etr-1 regulates body wall muscle differentiation. Our previous work showed that etr-1 in muscle has a non-autonomous role in neuronal migration, suggesting that ETR-1 is involved in the production of a signal emanating from body wall muscle that controls neuroblast migration and that interacts with Wnt signaling. etr-1 is extensively alternatively-spliced, and we identified the viable etr-1(lq61) mutant, caused by a stop codon in alternatively-spliced exon 8 and only affecting etr-1 isoforms containing exon 8. We took advantage of viable etr-1(lq61) to identify potential RNA targets of ETR-1 in body wall muscle using a combination of fluorescence activated cell sorting (FACS) of body wall muscles from wild-type and etr-1(lq61) and subsequent RNA-seq. This analysis revealed genes whose splicing and transcript levels were controlled by ETR-1 exon 8 isoforms, and represented a broad spectrum of genes involved in muscle differentiation, myofilament lattice structure, and physiology. Genes with transcripts underrepresented in etr-1(lq61) included those involved in ribosome function and translation, similar to potential CELF1 targets identified in chick cardiomyocytes. This suggests that at least some targets of ETR-1 might be conserved in vertebrates, and that ETR-1 might generally stimulate translation in muscles. As proof-of-principle, a functional analysis of a subset of ETR-1 targets revealed genes involved in AQR and PQR neuronal migration. One such gene, lev-11/tropomyosin, requires ETR-1 for alternative splicing, and another, unc-52/perlecan, requires ETR-1 for the production of long isoforms containing 3 exons. In sum, these studies identified gene targets of ETR-1/CELF1 in muscles, which included genes involved in muscle development and physiology, and genes with novel roles in neuronal migration.

developmental biology↗

bioRxiv2021

The Collagens DPY-17 and SQT-3 Direct Anterior-Posterior Migration of the Q Neuroblasts in C. elegans.

Cell adhesion molecules and their extracellular ligands control morphogenetic events such as directed cell migration. The migration of neuroblasts and neural crest cells establishes the structure of the central and peripheral nervous systems. In C. elegans, the bilateral Q neuroblasts and their descendants undergo long-range migrations with left/right asymmetry. QR and descendants on the right migrate anteriorly, and QL and descendants on the left migrate posteriorly, despite identical patterns of cell division, cell death, and neuronal generation. The initial direction of protrusion of the Q cells relies on the left/right asymmetric function of the transmembrane receptors UNC-40/DCC and PTP-3/LAR in the Q cells. Here we show that Q cell left/right asymmetry of migration is independent of the GPA-16/G pathway that regulates other left/right asymmetries including nervous system L/R asymmetry. No extracellular cue has been identified that guides initial Q anterior versus posterior migration. We show that the Collagens DPY-17 and SQT-3 control initial Q direction of protrusion. Genetic interactions with UNC-40/DCC and PTP-3/LAR suggest that DPY-17 and SQT-3 drive posterior migration and might act with both receptors or in a parallel pathway. Analysis of mutants in other Collagens and extracellular matrix components indicated that general perturbation of Collagens and the ECM did not result in directional defects, and that the effect of DPY-17 and SQT-3 on Q direction is specific. DPY-17 and SQT-3 are components of the cuticle, but a role in the basement membrane cannot be excluded. Possibly, DPY-17 and SQT-3 are part of a pattern in the cuticle and/or basement membrane that is oriented to the anterior-posterior axis of the animal and that is deciphered by the Q cells in a left-right asymmetric fashion. Alternatively, DPY-17 and SQT-3 might be involved in the production or stabilization of a guidance cue that directs Q migrations. In any case, these results describe a novel role for the DPY-17 and SQT-3 Collagens in directing posterior Q neuroblast migration. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

developmental biology↗

bioRxiv2020

The predicted RNA-binding protein ETR-1/CELF1 acts in muscles to regulate neuroblast migration in Caenorhabditis elegans

Neuroblast migration is a critical aspect of nervous system development (e.g., neural crest migration). In an unbiased forward genetic screen, we identified a novel player in neuroblast migration, the ETR-1/CELF1 RNA binding protein. CELF1 RNA binding proteins are involved in multiple aspects of RNA processing including alternative splicing, stability, and translation. We find that a specific mutation in alternatively-spliced exon 8 results in migration defects of the AQR and PQR neurons, and not the embryonic lethality and body wall muscle defects of complete knockdown of the locus. Surprisingly, ETR-1 was required in body wall muscle cells for AQR/PQR migration (i.e. it acts cell non-autonomously). Genetic interactions indicate that ETR-1 acts with Wnt signaling, either in the Wnt pathway or in a parallel pathway. Possibly, ETR-1 is involved in the production of a Wnt signal or a parallel signal by the body wall muscles that controls AQR and PQR neuronal migration. In humans, CELF1 is involved in a number of neuromuscular disorders. If the role of ETR-1/CELF1 is conserved, these disorders might also involve cell or neuronal migration. Finally, we describe a technique of amplicon sequencing to detect rare, cell-specific genome edits by CRISPR/Cas9 in vivo (CRISPR-seq) as an alternative to the T7E1 assay.

developmental biology↗

bioRxiv2019

RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in C. elegans

UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions suggest that RHO-1 and RHGF-1 act with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.\n\nAuthor SummaryNeural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion.

neuroscience↗

bioRxiv2018

Novel exc Genes Involved in Formation of the Tubular Excretory Canals of C. elegans

Regulation of luminal diameter is critical to the function of small single-celled tubes, of which the seamless tubular excretory canals of C. elegans provide a tractable genetic model. Mutations in several sets of genes exhibit the Exc phenotype, in which canal luminal growth is visibly altered. Here, a focused reverse genomic screen of genes highly expressed in the canals found 24 genes that significantly affect luminal outgrowth or diameter. These genes encode novel proteins as well as highly conserved proteins involved in processes including gene expression, cytoskeletal regulation, vesicular movement, and transmembrane transport. In addition, two genes act as suppressors on a pathway of conserved genes whose products mediate vesicle movement from early to recycling endosomes. The results provide new tools for understanding the integration of cytoplasmic structure and physiology in forming and maintaining the narrow diameter of single-cell tubules.

developmental biology↗

bioRxiv2018

UNC-6/Netrin and its Receptors UNC-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion

Many axon guidance ligands and their receptors have been identified, but it is still unclear how these ligand-receptor interactions regulate events in the growth cone, such as protrusion and cytoskeletal arrangement, during directed outgrowth in vivo. In this work, we dissect the multiple and complex effects of UNC-6/Netrin on the growth cone. Previous studies showed that in C. elegans, the UNC-6/Netrin receptor UNC-5 regulates growth cone polarity, as evidenced by loss of asymmetric dorsal F-actin localization and protrusion in unc-5 mutants. UNC-5 and another UNC-6/Netrin receptor UNC-40/DCC also regulate the extent of protrusion, with UNC-40/DCC driving protrusion and UNC-5 inhibiting protrusion. In this work we analyze the roles of UNC-6/Netrin, UNC-40/DCC, and UNC-5 in coordinating growth cone F-actin localization, microtubule organization, and protrusion that results in directed outgrowth away from UNC-6/Netrin. We find that a previously-described pathway involving the UNC-73/Trio Rac GEF and UNC-33/CRMP that acts downstream of UNC-5, regulates growth cone dorsal asymmetric F-actin accumulation and protrusion. unc-5 and unc-33 mutants also display excess EBP-2::GFP puncta, suggesting that MT + end accumulation is important in growth cone polarity and/or protrusion. unc-73 Rac GEF mutants did not display excess EBP-2::GFP puncta despite larger and more protrusive growth cones, indicating a MT-independent mechanism to polarize the growth cone and to inhibit protrusion, possibly via actin. Finally, we show that UNC-6/Netrin and UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of F-actin asymmetry and MT + end entry. Our data suggest a model in which UNC-6/Netrin polarizes the growth cone via UNC-5, and then regulates a balance of pro- and anti-protrusive forces driven by UNC-40 and UNC-5, respectively, that result in directed protrusion and outgrowth.

neuroscience↗

bioRxiv2018

Intermediate filaments EXC-2 and IFA-4 Maintain Luminal Structure of the Tubular Excretory Canals in Caenorhabditis elegans

The excretory canals of Caenorhabditis elegans are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts that lack a complete terminal web at the apical surface, and accumulate filamentous material in the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes intermediate filament protein IFC-2. EXC-2/IFC-2 protein, fluorescently tagged via CRISPR/Cas9, is located at the apical surface of the canals independently of other intermediate filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either protein results in narrow but shortened canals. These results are consistent with a model whereby three intermediate filaments in the canals, EXC-2, IFA-4, and IFB-1, restrain swelling of narrow tubules in concert with actin filaments that guide the extension and direction of tubule outgrowth, while allowing the tube to bend as the animal moves.\n\nArticle SummaryThe C. elegans excretory canals form a useful model for understanding formation of narrow tubes. exc-2 mutants start to form normal canals that then swell into fluid-filled cysts. We show that exc-2 encodes a large intermediate filament (IF) protein previously not thought to be located in the canals. EXC-2 is located at the apical (luminal) membrane, binds to another IF protein, and appears to be one of three IF proteins that form a flexible meshwork to maintain the thin canal diameter. This work provides a genetically useful model for understanding the interactions of IF proteins with other cytoskeletal elements to regulate tube size and growth.

developmental biology↗

bioRxiv2018

The Atypical Rho GTPase CHW-1 Works With SAX-3/Robo to Mediate Axon Guidance in Caenorhabditis elegans

During development, neuronal cells extend an axon towards their target destination in response to a cue to form a properly functioning nervous system. Rho proteins, Ras-related small GTPases that regulate cytoskeletal organization and dynamics, cell adhesion, and motility, are known to regulate axon guidance. Despite extensive knowledge about canonical Rho proteins (RhoA/Rac1/Cdc42), little is known about the Caenorhabditis elegans (C. elegans) atypical Cdc42-like family members CHW-1 and CRP-1 in regards to axon pathfinding and neuronal migration. chw-1(Chp/Wrch) encodes a protein that resembles human Chp (Wrch-2/RhoV) and Wrch-1 (RhoU), and crp-1 encodes for a protein that resembles TC10 and TCL. Here, we show that chw-1 works redundantly with crp-1 and cdc-42 in axon guidance. Furthermore, proper levels of chw-1 expression and activity are required for proper axon guidance. When examining CHW-1 GTPase mutants, we found that the native CHW-1 protein is likely partially activated, and mutations at a conserved residue (position 12 using Ras numbering, position 18 in CHW-1) alter axon guidance and neural migration. Additionally, we showed that chw-1 genetically interacts with the guidance receptor sax-3 in PDE neurons. Finally, in VD/DD motor neurons, chw-1 works downstream of sax-3 to control axon guidance. In summary, this is the first study implicating the atypical Rho GTPases chw-1 and crp-1 in axon guidance. Furthermore, this is the first evidence of genetic interaction between chw-1 and the guidance receptor sax-3. These data suggest that chw-1 is likely acting downstream and/or in parallel to sax-3 in axon guidance.

developmental biology↗