Immobilization of C. elegans with different concentrations of an anesthetic for time-lapse imaging of dynamic protein trafficking in neurons.

Here we compare the percentage of anterograde and retrograde trafficking events as well as the average velocity of these events in worms immobilized with microbeads or 0.5-7.5 mM tetramisole. Our results show that the percentage and average velocity of TIR-1 ::GFP moving events in the C. elegans AWC axons are not significantly different between worms immobilized with 7.5 mM tetramisole and other conditions. Our results suggest that 7.5 mM tetramisole, compared to 0.5 mM, 1 mM, and 2 mM tetramisole, does not have a significant effect on the axonal transport of TIR-1 ::GFP along the AWC axons.

CEPsh glia development is not required for general AWC identity or AWC asymmetry.

The Caenorhabditis elegans VAB-3/Pax6 homeodomain protein was previously shown to play a role in both the development of cephalic sheath (CEPsh) glia and asymmetric differentiation of AWC olfactory neuron subtypes AWC ON /AWC OFF . Here we show that vab-3 is not required for the specification of general AWC identity. We also show that some vab-3 mutant alleles with defective CEPsh glia development displayed wild-type AWC asymmetry. These results suggest that vab-3 has separable roles in CEPsh glia development and AWC asymmetry. Together, our results suggest that general AWC identity and AWC asymmetry are not dependent on the development of CEPsh glia.

mNG-tagged mls-2 knock-in alleles in C. elegans.

The Caenorhabditis elegans HMX/NKX MLS-2 transcription factor was previously shown to play sequential roles in AWC general identity and the stochastic AWCON/AWCOFF subtype choice during embryogenesis. Here we analyze the expression pattern of endogenous mls-2 during AWC development using mNeonGreen (mNG) knock-in strains. Similar to transgenic GFP::MLS-2, functional mNG::MLS-2 knock-in displayed nuclear localization in AWC precursor cells but was not observed in AWC during the later embryonic stage. These results suggest that the expression of mls-2 is below the detectable level and/or the stability of MLS-2 protein is very low in AWC cells.

Analysis of unc-62 expression pattern in C. elegans embryonic AWC neurons.

The Caenorhabditis elegans UNC-62 homothorax/Meis/TALE homeodomain protein functions sequentially to regulate general identity of the AWC olfactory neuron pair and the stochastic choice of asymmetric AWC subtypes during embryogenesis. Here we analyze the expression pattern of unc-62 during AWC development using an integrated unc-62::GFP fosmid rescuing transgene. UNC-62::GFP was not detected in AWC neurons in early or late embryos. These results are consistent with previous single-cell RNA sequencing data and also suggest an undetectable level of unc-62 expression and/or low stability of UNC-62 protein in AWC neurons during embryogenesis.

Synergistic roles of homeodomain proteins UNC-62 homothorax and MLS-2 HMX/NKX in the specification of olfactory neurons in Caenorhabditis elegans.

General identity of the Caenorhabditis elegans AWC olfactory neuron pair is specified by the OTX/OTD transcription factor CEH-36 and the HMG-box transcription factor SOX-2, followed by asymmetrical differentiation of the pair into two distinct subtypes, default AWCOFF and induced AWCON, through a stochastic signaling event. The HMX/NKX transcription factor MLS-2 regulates the expression of ceh-36 to specify general AWC identity. However, general AWC identity is lost in only one of the two AWC cells in the majority of mls-2 null mutants displaying defective general AWC identity, suggesting that additional transcription factors have a partially overlapping role with MLS-2 in the specification of general AWC identity. Here, we identify a role of unc-62, encoding a homothorax/Meis/TALE homeodomain protein, in the specification of general AWC identity. As in mls-2 null mutants, unc-62 null mutants showed an incomplete penetrance in loss of general AWC identity. However, unc-62; mls-2 double mutants display a nearly complete penetrance of identity loss in both AWC cells. Thus, unc-62 and mls-2 have a partially overlapping function in the specification of general AWC identity. Furthermore, our genetic results suggest that mls-2 and unc-62 act cell autonomously in promoting the AWCON subtype. Together, our findings reveal the sequential roles of the unc-62 and mls-2 pair in AWC development, specification of general AWC identity in early embryogenesis, and asymmetric differentiation of AWC subtypes in late embryogenesis.

A universal transportin protein drives stochastic choice of olfactory neurons via specific nuclear import of a sox-2-activating factor.

Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olfactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like transcription factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olfactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.

Engulfing cells promote neuronal regeneration and remove neuronal debris through distinct biochemical functions of CED-1.

Two important biological events happen coincidently soon after nerve injury in the peripheral nervous system in C. elegans: removal of axon debris and initiation of axon regeneration. But, it is not known how these two events are co-regulated. Mutants of ced-1, a homolog of Draper and MEGF10, display defects in both events. One model is that those events could be related. But our data suggest that they are actually separable. CED-1 functions in the muscle-type engulfing cells in both events and is enriched in muscle protrusions in close contact with axon debris and regenerating axons. Its two functions occur through distinct biochemical mechanisms; extracellular domain-mediated adhesion for regeneration and extracellular domain binding-induced intracellular domain signaling for debris removal. These studies identify CED-1 in engulfing cells as a receptor in debris removal but as an adhesion molecule in neuronal regeneration, and have important implications for understanding neural circuit repair after injury.

Asymmetric development of the nervous system.

The human nervous system consists of seemingly symmetric left and right halves. However, closer observation of the brain reveals anatomical and functional lateralization. Defects in brain asymmetry correlate with several neurological disorders, yet our understanding of the mechanisms used to establish lateralization in the human central nervous system is extremely limited. Here, we review left-right asymmetries within the nervous system of humans and several model organisms, including rodents, Zebrafish, chickens, Xenopus, Drosophila, and the nematode Caenorhabditis elegans. Comparing and contrasting mechanisms used to develop left-right asymmetry in the nervous system can provide insight into how the human brain is lateralized. Developmental Dynamics 247:124-137, 2018. © 2017 Wiley Periodicals, Inc.

Mechanisms controlling diversification of olfactory sensory neuron classes.

Animals survive in harsh and fluctuating environments using sensory neurons to detect and respond to changes in their surroundings. Olfactory sensory neurons are essential for detecting food, identifying danger, and sensing pheromones. The ability to sense a large repertoire of different types of odors is crucial to distinguish between different situations, and is achieved through neuronal diversity within the olfactory system. Here, we review the developmental mechanisms used to establish diversity of olfactory sensory neurons in various model organisms, including Caenorhabditis elegans, Drosophila, and vertebrate models. Understanding and comparing how different olfactory neurons develop within the nervous system of different animals can provide insight into how the olfactory system is shaped in humans.

ERG-28 controls BK channel trafficking in the ER to regulate synaptic function and alcohol response in C. elegans.

Voltage- and calcium-dependent BK channels regulate calcium-dependent cellular events such as neurotransmitter release by limiting calcium influx. Their plasma membrane abundance is an important factor in determining BK current and thus regulation of calcium-dependent events. In C. elegans, we show that ERG-28, an endoplasmic reticulum (ER) membrane protein, promotes the trafficking of SLO-1 BK channels from the ER to the plasma membrane by shielding them from premature degradation. In the absence of ERG-28, SLO-1 channels undergo aspartic protease DDI-1-dependent degradation, resulting in markedly reduced expression at presynaptic terminals. Loss of erg-28 suppressed phenotypic defects of slo-1 gain-of-function mutants in locomotion, neurotransmitter release, and calcium-mediated asymmetric differentiation of the AWC olfactory neuron pair, and conferred significant ethanol-resistant locomotory behavior, resembling slo-1 loss-of-function mutants, albeit to a lesser extent. Our study thus indicates that the control of BK channel trafficking is a critical regulatory mechanism for synaptic transmission and neural function.

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

Stochastic left-right neuronal asymmetry in Caenorhabditis elegans.

Left-right asymmetry in the nervous system is observed across species. Defects in left-right cerebral asymmetry are linked to several neurological diseases, but the molecular mechanisms underlying brain asymmetry in vertebrates are still not very well understood. The Caenorhabditis elegans left and right amphid wing 'C' (AWC) olfactory neurons communicate through intercellular calcium signalling in a transient embryonic gap junction neural network to specify two asymmetric subtypes, AWCOFF (default) and AWCON (induced), in a stochastic manner. Here, we highlight the molecular mechanisms that establish and maintain stochastic AWC asymmetry. As the components of the AWC asymmetry pathway are highly conserved, insights from the model organism C. elegans may provide a window onto how brain asymmetry develops in humans.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

Postmitotic diversification of olfactory neuron types is mediated by differential activities of the HMG-box transcription factor SOX-2.

Diversification of neuron classes is essential for functions of the olfactory system, but the underlying mechanisms that generate individual olfactory neuron types are only beginning to be understood. Here we describe a role of the highly conserved HMG-box transcription factor SOX-2 in postmitotic specification and alternative differentiation of the Caenorhabditis elegans AWC and AWB olfactory neurons. We show that SOX-2 partners with different transcription factors to diversify postmitotic olfactory cell types. SOX-2 functions cooperatively with the OTX/OTD transcription factor CEH-36 to specify an AWC "ground state," and functions with the LIM homeodomain factor LIM-4 to suppress this ground state and drive an AWB identity instead. Our findings provide novel insights into combinatorial codes that drive terminal differentiation programs in the nervous system and reveal a biological function of the deeply conserved Sox2 protein that goes beyond its well-known role in stem cell biology.

C. elegans SoxB genes are dispensable for embryonic neurogenesis but required for terminal differentiation of specific neuron types.

Neurogenesis involves deeply conserved patterning molecules, such as the proneural basic helix-loop-helix transcription factors. Sox proteins and specifically members of the SoxB and SoxC groups are another class of conserved transcription factors with an important role in neuronal fate commitment and differentiation in various species. In this study, we examine the expression of all five Sox genes of the nematode C. elegans and analyze the effect of null mutant alleles of all members of the SoxB and SoxC groups on nervous system development. Surprisingly, we find that, unlike in other systems, neither of the two C. elegans SoxB genes sox-2 (SoxB1) and sox-3 (SoxB2), nor the sole C. elegans SoxC gene sem-2, is broadly expressed throughout the embryonic or adult nervous system and that all three genes are mostly dispensable for embryonic neurogenesis. Instead, sox-2 is required to maintain the developmental potential of blast cells that are generated in the embryo but divide only postembryonically to give rise to differentiated neuronal cell types. Moreover, sox-2 and sox-3 have selective roles in the terminal differentiation of specific neuronal cell types. Our findings suggest that the common themes of SoxB gene function across phylogeny lie in specifying developmental potential and, later on, in selectively controlling terminal differentiation programs of specific neuron types, but not in broadly controlling neurogenesis.

Asymmetric neural development in the Caenorhabditis elegans olfactory system.

Asymmetries in the nervous system have been observed throughout the animal kingdom. Deviations of brain asymmetries are associated with a variety of neurodevelopmental disorders; however, there has been limited progress in determining how normal asymmetry is established in vertebrates. In the Caenorhabditis elegans chemosensory system, two pairs of morphologically symmetrical neurons exhibit molecular and functional asymmetries. This review focuses on the development of antisymmetry of the pair of amphid wing "C" (AWC) olfactory neurons, from transcriptional regulation of general cell identity, establishment of asymmetry through neural network formation and calcium signaling, to the maintenance of asymmetry throughout the life of the animal. Many of the factors that are involved in AWC development have homologs in vertebrates, which may potentially function in the development of vertebrate brain asymmetry.

Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1.

Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ("antisymmetries") or found stereotypically on one particular side of an animal ("directional asymmetries"). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. We describe here an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs.

microRNA function in left-right neuronal asymmetry: perspectives from C. elegans.

Left-right asymmetry in anatomical structures and functions of the nervous system is present throughout the animal kingdom. For example, language centers are localized in the left side of the human brain, while spatial recognition functions are found in the right hemisphere in the majority of the population. Disruption of asymmetry in the nervous system is correlated with neurological disorders. Although anatomical and functional asymmetries are observed in mammalian nervous systems, it has been a challenge to identify the molecular basis of these asymmetries. C. elegans has emerged as a prime model organism to investigate molecular asymmetries in the nervous system, as it has been shown to display functional asymmetries clearly correlated to asymmetric distribution and regulation of biologically relevant molecules. Small non-coding RNAs have been recently implicated in various aspects of neural development. Here, we review cases in which microRNAs are crucial for establishing left-right asymmetries in the C. elegans nervous system. These studies may provide insight into how molecular and functional asymmetries are established in the human brain.