Knobel KM et al. (1998) West Coast Worm Meeting "VD GROWTH CONES ARE DYNAMIC MOTILE STRUCTURES IN VIVO"

Jorgensen EM, Knobel KM, Bastiani MJ

[West Coast Worm Meeting, 1998]

We want to understand how growth cones behave in the complex environment of a living animal. How fast do they migrate? What behaviors do they exhibit during migration? How do these behaviors relate to the underlying substratum? We have begun by characterizing the growth cones belonging to the 13 post-embryonic VD motor neurons. An unc-47::GFP reporter construct is expressed in the cytoplasm of these neurons shortly after their cell bodies are born. First we determined that the anterior VD growth cones begin migrating at 15.5 hours after hatching. The various VD growth cones take from 1 to 2.5 hours to complete migration from the ventral to the dorsal nerve cord. Thus, the rate of outgrowth is somewhat dependent upon where the cell body of the growth cone is located. What behaviors do VD growth cones exhibit during axonogenesis? When we looked at individual worms during VD outgrowth, a number of different growth cone shapes were obvious. Some growth cones were round, others were anvil-shaped. To determine the temporal relationship between these shapes we immobilized L1's just prior to the onset of outgrowth and made time-lapse movies of migrating growth cones using laser-scanning confocal microscopy (Thomas et al., 1996). From these movies we were able to assign a temporal sequence to the growth cone shapes we observed. VD growth cones become visible when they turn away from the ventral nerve cord and begin migrating dorsally along the hypodermal ridge. These rapidly migrating growth cones are round. Round growth cones extend and retract lamellipodia in all directions along the hypodermis. Midway between the ventral and dorsal nerve cords VD growth cones briefly stall and spread out, forming anvil-shaped structures. Why do VD growth cones change from rounded to anvil-shaped structures in this location? The lateral nerve cord runs along the hypodermis midway between the dorsal and ventral nerve cords. Using ceh-23::GFP to mark the lateral CAN process, we determined that VD growth cones spread out along the lateral process upon contact. After briefly stalling VD growth cones extend a lamellipodia beyond the lateral process toward the dorsal nerve cord. They quickly resume their rapidly migrating rounded shape once they have moved beyond the lateral nerve cord. A second anvil is formed when the growth cone has migrated about 3/4 of the way toward the dorsal nerve cord. In this region the growth cone is forced to migrate between the dorsal body wall muscle and the hypodermis. We used Nomarski imaging to confirm that VD growth cones spread out along the dorsal body wall muscle. These anvil-shaped growth cones behave differently than those observed at the midway point. Instead of projecting a lamellipodia toward the dorsal cord, we see thin finger-like extensions projecting dorsally from the leading edge of the anvil. One of these fingers extends farthest: just as it reaches the dorsal cord all other secondary fingers retract and the body of the anvil growth cone collapses. We are currently trying to determine why VD growth cones send fingers, not lamellipodia, dorsally. Next, a new growth cone emerges at the tip of the finger. This newly formed growth cone migrates to the dorsal cord. In summary, VD growth cones migrating in vivo are dynamic structures. They assume a highly motile rounded shape when migrating rapidly across the open hypodermis. When they contact new substrates VD growth cones stall, spread out and form anvils. The anvil structure remains until lamellipodia or finger-like extensions project dorsally and the growth cone resumes its rapidly migrating form. Thomas, C., DeVries, P., Hardin, J., and White, J. (1996). Four-Dimensional Imaging: Computer Visualization of 3D Movements in Living Specimens. Science 273, 603-607

B Walthall et al. (1999) International C. elegans Meeting "Convergent Genetic Programs Create the Synaptic Pattern that Distinguishes the VD and DD Motor Neurons from One Another"

B Walthall, M Zhou

[International C. elegans Meeting, 1999]

How do genes orchestrate the many different features necessary to assemble and maintain functional neural circuits? One particularly important challenge is the establishment of diverse but specific synaptic patterns among neurons and their targets. The VD and DD motor neurons (mns) together form a cross-inhibitory network that maintains the proper phase relationship between the waves of dorsal and ventral muscular contraction that create the sinuous patterns of locomotion. The VD mns release the inhibitory neurotransmitter GABA onto ventral muscle as a result of dorsal cholinergic input from excitatory mns. Conversely, the DD mns inhibit dorsal muscle in response to input from ventral excitatory mns. The transcription factor Unc-30 is expressed shortly after the birth of both the VD and DD mns and is necessary for proper structural and functional features (McIntire, 1993; Jin, 1994, 1999). More than 15 genes are known to be regulated either directly or indirectly by Unc-30 and mutations in these genes produce identical defects in both mn classes. Reduction of inhibition on either the dorsal or ventral side (either surgically or genetically) produces asymmetric patterns of movement. Thus the analysis of asymmetric unc mutants could identify genes that effect either the synaptic specificity of the DD or the VD mns. unc-55 mutants exhibit a strong ventral asymmetric pattern of locomotion. In these mutants the VD mns adopt the DD mn synaptic pattern. Unc-55 is a transcription factor that is expressed in the postembryonic VD mns and their sisters, the AS mns, shortly after their birth (10 hrs after hatching). We report here experiments examining interactions among unc-30 and unc-55 . To test whether Unc-30 regulates the expression of unc-55 in the VD mns we placed an unc-55:gfp reporter in an unc-30(e191) mutant background. We observed gfp expression in VD and AS mns suggesting that the two genes are regulated independently of one another. unc-55:gfp expression is characterized by rapid onset and dissipation in the ventral cord mns, suggesting temporal regulation. However, unc-55:gfp expression in an unc-55(jd8) mutant background was brighter and lasted longer than usual, indicating that Unc-55 negatively regulates its own expression.

Sarah Anthony et al. (2008) C.elegans Neuronal Development Meeting "Identification of Synaptic Remodeling Genes by Expression Profiling of unc-55 GABAergic Neurons"

David Miller, Bill Walthall, Kathie Watkins, Sarah Anthony, Joseph Watson

[C.elegans Neuronal Development Meeting, 2008]

Neurons adopt polarized morphologies to create separate axonal and dendritic compartments. The ability to reorganize these compartments in response to injury or developmental cues is an evolutionarily conserved feature of neurons. One striking example of this phenomenon occurs in the C. elegans GABAergic nervous system. Dorsal D (DD) motor neurons initially adopt a ventral polarity to innervate ventral body wall muscle. At the end of the first larval stage, DD motor neurons are remodeled to synapse with dorsal muscles. This polarity switch is coincident with the birth of VD motor neurons, which innervate ventral muscle. In unc-55 mutants, VD motor neurons mimic the dorsal polarity of DD motor neurons. UNC-55, a member of the COUP family of nuclear hormone receptors, is expressed in VD motor neurons, where it appears to function as a transcriptional repressor. Thus, we propose that UNC-55 functions as a transcriptional switch to repress a synaptic remodeling program in VD motor neurons. In this model, UNC-55 targets are likely to fulfill crucial roles in the synaptic remodeling program. With the goal of identifying these genes, we have employed the mRNA tagging method to obtain larval gene expression profiles of wild-type and unc-55 GABAergic motor neurons. Comparison of these profiles has uncovered 118 enriched transcripts and 11 depleted transcripts in unc-55 DD and VD motor neurons. We predict that the enriched transcripts represent pro-remodeling genes normally repressed by UNC-55 in VD motor neurons. Likely UNC-55 targets uncovered in our study include novel, uncharacterized genes, as well as genes with known roles in synaptogenesis, cytoskeleton rearrangement, and cell-cell communication. Currently, we are confirming regulation of these genes by UNC-55 and testing for function in the DD synaptic remodeling program. Our study is expected to reveal key downstream effectors of neuronal polarity.

Sarah C. Anthony et al. (2007) International Worm Meeting "Microarray profiling of C. elegans GABAergic motor neurons to reveal synaptic remodeling genes."

Sarah C. Anthony, Bill Walthall, Stephen E. Von Stetina, Joseph D. Watson, David M. Miller III

[International Worm Meeting, 2007]

Neurons adopt polarized morphologies to set the direction of information flow in the nervous system. Neurons in diverse species are universally capable of altering axonal versus dendritic compartments in response to injury or through developmentally regulated programs. One striking example of this phenomenon is the remodeling of GABAergic motor neurons in C. elegans. Dorsal D or DD motor neurons initially innervate ventral muscle but switch polarity after hatching to synapse with dorsal muscle. This polarity switch occurs as ventral D (VD) motor neurons arise in L1 larvae to innervate ventral muscles. In unc-55 mutants, VD motor neurons adopt the dorsal polarity of DD motor neurons thereby resulting in simultaneous innervation (inhibition) of dorsal muscles by both DD and VD motor neurons. UNC-55, a member of the COUP family of nuclear hormone receptors, is expressed specifically in VD motor neurons. Thus, UNC-55 appears to function as a transcriptional switch that prevents VD motor neurons from adopting the dorsal polarity of DD motor neurons (Zhou and Walthall, 1998). Our goal is to identify UNC-55 regulated genes that govern D motor neuron synaptic remodeling. For this purpose, we are using the mRNA tagging method to generate microarray profiles of GABAergic neurons. We used the D motor neuron-specific promoter, C04G2.1, to drive expression of epitope-tagged poly-A-Binding Protein (3X FLAG-PAB-1) in DD and VD motor neurons. An mRNA tagging profile of wildtype L2 larvae using this strain detects established GABAergic and neuronal transcripts including unc-55. With this reference profile of normal D-type motor neurons, we can now identify dynamically regulated genes in unc-55 gain-of-function and loss-of-function mutants. This strategy is expected to reveal UNC-55 target genes with key roles in motor neuron synaptic remodeling.

Alcantara, A. et al. (2017) International Worm Meeting "The Effects of Altered Gravity on Aging in Motor Neurons."

Lee, J.I., Alcantara, A., Kalichamy, S.

[International Worm Meeting, 2017]

As the world's population increases, humans will begin to populate space. With today's technological developments, migration of humanity in space might be possible in the near future. Still, human bodies are not conditioned for a long-term altered gravity environment, and the full effects on developmental and physiological process is not known. We wanted to know whether long-term changes in gravity could affect aging and lifespan, which is relatively unexplored. Previously, we showed that hypergravity can affect development of the DD/VD motor neurons in C. elegans. Using a similar experimental paradigm, we exposed C. elegans to 100G hypergravity and observed the DD/VD motor neurons using a (p)unc-25::GFP reporter strain. DD/VD motor neurons normally show progressive defects during the aging process including axonal beading and formation of extraneous neurites similar to aging in other neurons. Upon hypergravity exposure, adults showed a 12% increase of DD/VD axon aging phenotypes even after only 4 days. Genetic experiments with aging mutants are ongoing currently. To understand the effects of microgravity on aging and lifespan, we can simulate microgravity in the laboratory using a clinostat. Currently, we are determining a suitable clinostat condition by varying its speed and measuring the expression of E. coli and C. elegans known microgravity marker genes. After obtaining simulated microgravity conditions, we will conduct experiments to study the microgravity effects on aging in DD/VD motor neurons and even C. elegans lifespan with the eventual hope of confirming any results in space aboard the International Space Station.

Campbell, Richard et al. (2015) International Worm Meeting "UNC-62 alternative transcripts differentially regulate UNC-55 expression and neural differentiation of GABAergic VD motor neurons."

Walthall, Walter W., Campbell, Richard

[International Worm Meeting, 2015]

The animal's sinuous locomotion is a direct result of a cross activational/inhibitory network of motor neurons in the ventral nerve cord (VNC). This is network is composed of cholinergic (excitatory) inputs and GABAergic (inhibitory) inputs that originate from distinct classes and subclasses of motor neurons. The excitatory motor neurons are comprised of the A (for backward locomotion) and B(for forward locomotion). The inhibitory motor neurons are comprised of the D class motor neurons which are predicted to be required for both backward and forward locomotion. Both cholinergic and GABAergic classes can be further subdivided by innervation type and developmental origin. The GABAergic DD subclass is embryonic and innervates dorsal muscle in adult animals while the VD subclass is postembryonic and innervates ventral muscle. Both the DD and VD motor neurons express the transcription factor, UNC-30 which is necessary and sufficient for post mitotic differentiation GABAergic D class motor neurons. The VD motor neurons express a nuclear hormone receptor, UNC-55 which prevents the VD motor neurons from innervating the dorsal muscle and assuming a DD like fate through the suppression of various UNC-30 transcriptional targets. Interestingly, UNC-55 is not transcriptionally regulated by UNC-30. Thus in the DD and VD motor neurons which share very similar developmental mechanisms, how is the expression of UNC-55 regulated in the VNC?To address this question, the transcriptional activation of unc-55 and the differentiation of the VD motor neurons was tested utilizing bioinformatics, mutant analyses of candidate regulators, promoter dissection, exon specific disruption utilizing CRISPR/Cas9 and GFP constructs. Several putative transcription factor binding sites (cis sites) corresponding to the MEIS domain transcription factor, UNC-62 were found on the unc-55 promoter. Different mutant alleles of UNC-62 were found to repress, activate or have no effect on punc-55::gfp expression. Interestingly, each mutant allele of UNC-62 disrupts alternatively spliced or transcribed exons. Dissection of the unc-55 promoter identified multiple regions and putative cis sites that correlate with the UNC-62 mutant expression patterns. These data provide evidence that different isoforms of UNC-62 are required for activation and repression of unc-55 transcription and subsequently VD motor neuron differentiation. Isoform specific transcriptional regulation of differentiating neurons is a relatively unexplored area of research and represents an interesting mechanism for differentiation in neuronal subtypes.

Kim, BanSeok et al. (2021) International Worm Meeting "Spaceflight effects on muscle size in C. elegans"

Alcantara, Alfredo Jr., Higashitani, Atsushi, Moon, Je-Hyun, Kim, BanSeok, Lee, JinIl

[International Worm Meeting, 2021]

Many countries are planning on space colonization, researching space habitation and even privatizing space travel. However, if we travel to space in the future, our bodies will be exposed to space environment during long-term space flight. Previous studies have revealed that space microgravity induces muscle atrophy. To understand how microgravity affects muscle and motor neuron that traverses body wall muscle, we focused on the GABAergic motor neurons in C. elegans, the DD/VD neurons. We sent C. elegans to the ISS in space to find any changes in DD/VD neurons and muscles. The worms who were born in microgravity environment were frozen after six days from birth and parental worms who experienced both 1G and microgravity condition were frozen together, and we received these samples. While we were analyzing the neurons as well as muscle size and shape by immunostaining, we were faced with a difficulty seeing GFP of DD/VD neurons because of their weak signal. Thus, we could check only muscle size of C. elegans using phalloidin staining method. Preliminary results show that space microgravity-exposed worms aboard the ISS may show differences in size compared to ground-control samples. We are currently analyzing muscle size in other space samples, as well as DD/VD motor neuron development. Keywords : space flight, microgravity, muscle size

Mulcahy, Ben et al. (2017) International Worm Meeting "The Dynamics of Synaptic Remodeling: insights from the C. elegans Motor Circuit."

Koh, WanXian, Fetter, Rick, Holmyard, Douglas, Chang, Maggie, Schroff, Hari, Lichtman, Jeff, Schalek, Richard, Christensen, Ryan, Witvliet, Daniel, Chisholm, Andrew, Bermant, Peter, Zhen, Mei, Mitchell, James, Samuel, Aravi, Qu, Yixin, Mulcahy, Ben

[International Worm Meeting, 2017]

During development, neural circuits undergo constant changes: they incorporate new neurons, and maintain, prune or remodel existing synapses. The small and rapidly developing nervous system of C. elegans allows us to address the developmental dynamics of synapses and neurons in the context of an intact neural ensemble. The vast majority of C. elegans postembryonic neurons are motor neurons. Previous studies suggest that at least one group of motor neurons, DD, reverse their neurite polarity entirely: they innervate ventral body wall muscles in the first larval stage, whereas they innervate dorsal muscles in adults. Their role of innervating ventral muscle is taken over by a group of post-embryonically born motor neurons, VD. Precisely how and when such processes occur, without disrupting motility as the motor circuit transitions into the adult form, is poorly described. We used serial section electron microscopy to reconstruct DD and VD motor neurons across development to define the beginning and ending of remodeling. We identified a series of elegantly coordinated synaptogenesis and remodeling events where synaptogenesis events follow different sequential orders for embryonic (DD) and post-embryonic (VD) neurons. Developing VD neurons have some interaction with DD neurons that may help to guide neurite extension and synaptogenesis. These events implicate an elegant mechanism that allows a gradual and seamless transition of the motor circuit to take place without disrupting its functional output.

Julie McCleery et al. (2005) International Worm Meeting "Regulation of GABAergic motor neuron fate specification by the sumoylation pathway and chromatin modifying factors"

Julie McCleery, Chris Li, Yishi Jin, James Meir

[International Worm Meeting, 2005]

The C. elegans ventral cord GABAergic DD and VD neurons have identical cell morphology but differ in several aspects. They are born from separate lineages at different stages of development, they form synapses to opposite body wall muscles and the juvenile DDs undergo developmental synaptic remodeling during L1 to L2 larval development (1). Thus far, only two genes have been shown to be differentially expressed in these two groups of neurons. The unc-55/coupTF nuclear hormone receptor is expressed in the VDs (2) and the flp-13 gene is expressed in the DDs (3). We have identified a consensus COUPII DNA binding site in the flp-13 promoter. In unc-55 mutants, flp-13 promoter driven transgenes are ectopically expressed in VD neurons suggesting that unc-55 specifies VD neuron fate by repressing DD neuron-specific genes including flp-13. To further understand the mechanism that distinguishes DD and VD neurons, we performed a genetic screen utilizing a Pflp-13-SNB-1::GFP marker. We isolated a group of mutants that show ectopic expression of the Pflp-13-SNB-1::GFP marker in VD neurons. These genes encode the small ubiquitin-like-modifier (SUMO) E1 and E2 enzymes, aos-1 and ubc-9, the ATRX homolog SWI/SNF chromatin remodeler xnp-1, and the MYST histone acetyltransferase mys-1. Sumoylation has been shown to play a role in transcriptional repression by modification of nuclear receptor transcription factors and chromatin modifiers. Our double mutant analysis is consistent with this possibility in that it indicates that the chromatin modifiers and sumoylation pathway components may function in the same pathway, and that this pathway may modulate the VD neuron specification program controlled by unc-55. P-xnp-1-xnp-1-GFP is altered in sumo E1 and E2 mutants indicating XNP-1 may be modified by sumoylation. We are currently performing biochemical experiments to determine if XNP-1, MYS-1 or UNC-55 may interact with the sumo E2 enzyme UBC-9.1.White J Nature 271, 764-766 (1978)2.Zhou MH, Walthall WW The Journal of Neuroscience 18(24), 10438-10444 (1998)3.Kyuhyung K, Li C The Journal of Comparative Neurology 475, 540-550 (2004)

Li L et al. (1995) International C. elegans Meeting "MOSAIC ANALYSIS OF unc-55: A GENE INVOLVED IN SYNAPTIC SPECIFICITY"

Walthall WW, Li L

[International C. elegans Meeting, 1995]

The DD and VD motoneurons (mns) form reciprocal synaptic patterns that result in the formation of a cross inhibitory circuit that is essential for normal movement. Despite different lineal origins the two classes of mns share a number of features related to their structure and function. Mutations in unc-55 alter the synaptic patterns of the VD mns so that it becomes identical to that of the DD mns. Here we performed a mosaic analysis and determined that unc-55 (+) must be expressed in the VD but not the DD mns. In this study, a che-3 mutant was used as a marker because it blocks uptake of FITC by chemosensory neurons, which are lineally related to the D mns. We constructed a duplication strain, unc-55(e1170); che-3(nr2288); hDp69. unc mosaics (n=5) were observed that had lost the duplication in the four phasmid neurons, indicating that the duplication loss had occurred before the AB.p division. In contrast, wild-type mosaic animals (n=17) had lost the duplication in a subset of phasmid neurons, indicating that the duplication loss had occurred after the division of AB.p. These results suggest that a primary target of unc-55 is the descendants of AB.p. Both the DD and VD mn are derived from this lineage. Further analysis showed that 9 of 17 wild-type mosaics had lost the duplication at or after the birth of AB.plp or AB.prp, the lineage from which the DD mns are derived. Thus loss of the duplication in the DD mns had no affect on their synaptic pattern. Therefore, unc-55 must be expressed in the VD mns and is necessary for their synaptic pattern.