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[
International Worm Meeting,
2019]
The pharyngeal nervous system is composed of only 20 neurons belonging to 14 different types, which form a self-contained circuit that is almost independent from the somatic nervous system. This simplicity makes it possible to analyze it in a comprehensive way. Moreover, all pharyngeal neurons directly connect to end organs and can be considered polymodal with sensory-motor characteristics (see abstract by S.J. Cook and S.W.Emmons), a feature that is reminiscent of primitive nervous systems. Thus, understanding how pharyngeal neurons are specified during development might shed light on fundamental aspects of neuronal development.
ceh-34, a homeodomain transcription factor of the Six family, is continuously expressed in all pharyngeal neurons and no other neurons outside of the pharynx. Remarkably, we have found that in
ceh-34 mutants, pharyngeal neurons are generated, but fail to express a wide array of terminal identity genes, including neurotransmitter pathway genes, indicating that
ceh-34 acts as a pharyngeal neuron master regulator ("terminal selector"). Moreover, a conditional AID-based allele demonstrates that
ceh-34 is continuously required during the life of the worm to maintain pharyngeal neuron identity. We hypothesize that
ceh-34 acts together with other transcription factors to form a combinatorial code that gives each pharyngeal neuron its unique identity. We have found several other homeodomain transcription factors expressed in subsets of pharyngeal neurons (see abstract by M. Reilly and O.Hobert) and we are doing a mutant analysis to test whether they also play a role in the pharyngeal nervous system specification. Moreover, we are performing forward genetic screens to find additional factors in an unbiased way. So far we have identified four mutants that appear to affect I2 neuron identity. We are in the process of pinpointing the causal molecular lesions and further characterizing these new mutants. We hope our efforts will lead to a comprehensive understanding of the regulatory code that dictates pharyngeal neuron development.
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Bonnington, R.C., Emmons, S.W., Kim, B., Hall, D.H., Sammut, M., Cook, S.J., Barrios, A., Molina-Garcia, L., Poole, R.J., Khambhaita, K.
[
International Worm Meeting,
2017]
We have recently characterised differentiated glial cells that divide to produce neurons, the mystery cells of the male (MCMs). Here we present a previously undescribed direct glia-to-neuron cell fate switch, revising the total number of male neurons to 387 and glia to 90. Studies of vertebrate neural development have revealed that differentiated glia can act as neural precursors, however the cellular and molecular mechanisms have not been fully determined. To identify the genetic factors that regulate these glia-to-neuron cell fate switches, we have generated a collection of no mystery cell (nom) mutants and assessed their role in both glia-to-neuron cell fate switches. In early L4, the male amphid socket (AMso) glial cells divide asymmetrically. We observe increased nuclear localization of the Wnt-signalling effector POP-1/TCF, in the anterior daughter, the self-renewed AMso than in the posterior MCMs. The proneural factor
hlh-14/Ascl1 is then transiently expressed in the MCMs, prior to neuronal differentiation. From a GFP-based forward genetic screen of 4000 genomes we have isolated nine nom mutants in which the MCMs fail to be specified. We have identified mutants that affect AMso division, MCM neuronal differentiation and MCM neuronal subtype specification. Two AMso division mutants,
nom-5 and
nom-8 were mapped-by-sequence to the
cdk-4 locus. CDK-4 is required for G1-S progressions in postembryonic blast cells but surprisingly, our alleles only appear to affect the AMso division. Moreover, no loss of glial fate or acquisition of neuronal characteristics was observed in the undivided AMso, suggesting that MCM specification requires DNA replication or the asymmetric segregation of neural factors. Following Sulston's preliminary observations, we find that the male phasmid socket one (PHso1) glial cells become bona fide neurons during the L4 stage and name them phasmid neuron D (PHD). Importantly, this transition is direct, not requiring cell division. We establish their developmental lineage, identity as cholinergic neurons, connectivity and provide insight into their function during mating. (see poster by R.C. Bonnington). PHD specification is unaffected in
cdk-4 mutants and the majority of nom mutants tested. This suggests that largely distinct genetic mechanisms regulate the two events. The male provides two glia-to-neuron cell fate switches that occur by seemingly independent cellular and genetic mechanisms. We are continuing to investigate the regulatory strategies that confer neurogenic potential to differentiated glia.
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[
International Worm Meeting,
2021]
Neuronal plasticity and circuit stability are fundamental properties of brain development and function. Activity-dependent changes to neuronal connectivity often occur within a defined time window, also known as a developmental plasticity window. How neuronal activity contributes to such precise timing of neural circuit rewiring is a central question in neuroscience. Ultrastructural connectomic studies that began nearly 50 years ago revealed that during the first larval stage, the C. elegans locomotor circuit undergoes dramatic synaptic rewiring known as 'DD synapse remodeling' as postembryonic motor neurons are born to establish the mature motor circuit (White et al., 1978). Live imaging studies subsequently showed that presynaptic terminals in DD motor neurons are progressively removed from the ventral side and new synapses are formed at dorsal locations from mid to late L1 stages (Hallam and Jin, 1998). The precise timing of DD synapse remodeling has been shown to depend on several transcriptional programs and can be modulated by neuronal activity. However, it remains unclear which form of neuronal activity affects the time window of this developmental plasticity, and how neuronal activity is molecularly coupled to transcription regulation. To address this, we are using fluorescently tagged reporters for in vivo detection of the key transcription factors, such as LIN-14 and UNC-30. To precisely determine L1 developmental stages, we use P cell nuclear migration and divisions using Nomarski optics (Sulston and Horvitz, 1977). We have also generated a nuclear calcium sensor to measure activity in DD neurons during synapse remodeling. We will present our detailed findings on the changes in nuclear calcium dynamics before, during and after DD synapse remodeling. References: Hallam, S.J., and Y. Jin. 1998. Nature. 395(6697):78-82. Sulston, J.E., and H.R. Horvitz. 1977. Dev. Biol. 56:110-156. White, J.G., D.G. Albertson, and M.A. Anness. 1978. Nature. 271:764-766.
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[
European Worm Meeting,
2006]
Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (
brc-1 and
brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require
brc-1,
brd-1,
ubc5(
let-70),
mre-11 and
atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.
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[
International Worm Meeting,
2013]
C. elegans P-granules are essential for both the development and maintenance of the germline tissue. Disruption of P-granule formation interferes with proper germ cell proliferation and differentiation, resulting in sterility (1, 2). P-granule assembly requires structural scaffold proteins PGL-1 and PGL-3 (3, 4). These nematode-specific paralogs are sufficient to form granules in cells and multimerize through self-association (5, 6). We aim to understand the structural organization of the P-granule scaffold to better understand how the organelle regulates mRNA trafficking and turnover. We are able to express and purify recombinant PGL-1 and PGL-3. Full-length recombinant protein self-assembles into large soluble aggregates. Protease digestion analyses identify a single domain that dimerizes in solution. We are currently trying to obtain a high-resolution crystal structure of the protease-protected fragment, as well as determine the role of the N- and C- terminal regions in scaffold oligomerization. Several different types of RNA granules are required in eukaryotes for cell homeostasis, differentiation, and response to stress. The fundamental mechanisms involved in P-granule organization will undoubtedly shed light on other granules involved in RNA regulation.
References:
1. Updike, D., Strome, S. (2010) J Androl 31: 53-60.
2. Voronina, E., Paix, A., Seydoux, G. (2012) Development 139: 3732-3740.
3. Kawasaki, I., et al. (1998) Cell 94: 635-645.
4. Kawasaki, I., et al. (2004) Genetics 167: 645-661.
5. Updike, D.L., Hachey, S.J., Kreher, J., Strome, S. (2011) J Cell Biol 192: 939-948.
6. Hanazawa, M., Yonetani, M., Sugimoto, A. (2011) J Cell Biol 192: 929-937..
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Berger, D.R., Wang, J., Cook, S.J., Holmyard, D., Lichtman, J.W., Neubauer, M., Laskova, V., Zhen, M., Emmons, S.W., Mulcahy, B., Samuel, A.D.T., Kersen, D., Chisholm, A.D., Schalek, R.L., Koh, W.X., Qian, J., Hall, D.H., Mitchell, J.K., Chang, M., Witvliet, D.
[
International Worm Meeting,
2017]
When animals are born, neuronal networks are already in place to integrate sensory cues and effect appropriate behavioral or homeostatic responses. During the course of postnatal development, synapses and circuits undergo refinement and remodeling, updating or adapting sensorimotor behaviors. Rules for developmental remodeling are poorly characterized, because a circuit-level analyses at multiple time points, in multiple animals are missing. We use an isogenic C. elegans N2 population to examine how the neural circuit changes during development. At birth, C. elegans has 218 neurons (excluding CAN); 82 new neurons are incorporated into the nervous system before the end of larval development. These include sensory, motor, and interneurons that are located throughout the body, suggesting a system-wide modification of the juvenile circuit during post-embryonic development. Using serial-section electron microscopy, we mapped the synaptic connectivity in the head and tail ganglia for five animals, from hatching to late larval development. These datasets allow us to separate the connectome into core connections, synapses that are present throughout the lifetime, transient connections, synapses that are present only during early or late development, and variable connections, synapses that are not conserved between animals. We propose that the core connections may drive hard-wired behaviors, and developmentally regulated connections exert stage-specific roles, while variable connections may confer individual variability and/or be stochastic in nature. Our current data analyses lead to two notions: First, developmentally regulated connections are enriched for synapses between sensory neurons, and with neuromodulatory neurons, while connections between interneurons are remarkably stable. This implies that for C. elegans, core circuits for decision-making are already established at birth, but their modulation and multi-sensory integration are refined or shaped during development. Second, variable connections, comprising about half of the connection edges in the published adult dataset, are prominent. Stochastic or experience dependent variability in connections may contribute to variability of behaviors set by hard-wired core connections.
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Neubauer, M., Witvliet, D., Mitchell, J.K., Maeng, S., Meirovitch, Y., Chisholm, A.D., Wang, M.D., Shavit, N., Wang, Z., Ho, C.Y., Samuel, A.D.T., Lichtman, J.W., Koh, W.X., Schalek, R.L., Rehaluk, C., Mulcahy, B., Holmyard, D., Berger, D.R., Zhen, M., Cook, S.J.
[
International Worm Meeting,
2019]
Nervous system development is widely thought to be optimized for precise wiring and efficient networks. Maintaining plasticity, however, is necessary for animals to adapt, learn, and evolve. This is especially evident during postembryonic development when newly born neurons are integrated into functioning neurocircuits. Rules for circuit plasticity and variability during and after development are poorly because circuit-level analyses across multiple animals and developmental time points are missing. Using electron microscopy, we mapped the connectome of the central nervous system for eight isogenic C. elegans animals spanning from birth to adulthood. These datasets reveal a highly dynamic circuit architecture, with a five-fold increase in synapse number across postembryonic development. Synapses are built and removed for multiple reasons: (1) to strengthen a remarkably stable core circuitry for decision-making, (2) to fine-tune multi-sensory integration and motor responses by building new unique cell-cell connections, (3) to streamline circuit computations, and (4) to fill connectivity gaps as a result of evolutionary pressures to hatch before initial wiring is complete. This remodeling is driven independently of the contact area between neurons and instead is dependent on neuron class and centrality. Remodeling is overrepresented between connections from sensory neurons to motor and interneurons, while connections between interneurons are remarkably stable. Well-connected, central neurons (typically interneurons), are more heavily regulated than other neurons, but surprisingly this regulation does not originate from other central neurons. Thus, the driving force for synaptic growth is not uniform, and may depend on neuron identity, suggesting an underpinning by genetic or functional identity. Together, our findings show that even in one of the most deterministic animals, nervous system wiring retains a high degree of plasticity. The connectome should be separated into core connections, that are present throughout life, developmentally changing connections, that depend on the stage of development, and variable connections, that are not conserved among individuals. Core connections are the strongest class of connections, but they make up less than half of all connections. The prevalence of wiring plasticity implicates relevance to nervous system adaptation and variability. Our data will be available on nemanode.org before the end of the summer.
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[
International Worm Meeting,
2021]
The nature of the relationship between neural circuits and the resulting animal behavior is a key question in neurobiology. It was previously established that the specific synaptic and cellular properties of neural networks can be widely disparate, yet maintain similar function (Prinz et al., 2004). It is therefore clear that some features of a network's structure are important to retain certain functional features. The sensitivity of such networks to changes in the topology have not been characterized. To explore the contribution of topology to the network's performance, we focused here on the circuit for nociceptive behaviors in C. elegans. The neurons of the circuit are shared between the two sexes, but their connectivity is different (Cook et al., 2019). The behaviors that result from these circuits are sexually dimorphic as well. The distinct network topologies and behavioral outputs make this circuit a good example for exploring the relationship between structure and function in neural networks. We simulated the response of the nociceptive circuits to external stimuli, in males and in hermaphrodites, using a wide range of realistic values for the circuit's biophysical parameters (synaptic strengths, conductivity, membrane time constants, etc.). We then searched for the parameters' space in which the activity of the motor output neurons in the simulation would match the worms' behaviors in experimental observations. We found an overlap between the sexes in terms of the synaptic and cellular parameters that allow for the correct behavior of the network. Moreover, our results suggest that the connectivity alone might be sufficient to explain the behavioral differences between the sexes. Notably, more stringent requirements of the models' performance suggests that the connections in this network cannot be all excitatory, as has been commonly assumed, or that additional inhibitory neurons must play an important role in shaping the circuit's response to tail stimulation. Future analysis will further explore the relations between the network's topology and the joint activity patterns of the neurons as measured by calcium imaging.
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Nguyen, Kenneth, Bloniarz, Adam E., Brittin, Christopher A., Cook, Steven J., Hall, David H., Emmons, Scott W., Xu, Meng, Jarrell, Travis A., Wang, Yi
[
International Worm Meeting,
2013]
The innate behavioral repertoires of the two sexes of a species are guided by differing reproductive priorities. C. elegans male copulation is controlled by a neural network in the tail in which a majority of the neurons and muscles are specific to the male. But known differences in olfactory preferences and exploratory tendencies emanate from behaviors controlled by circuits in the head, where the complement of neurons is nearly identical in the two sexes. We determined connectivity in the anterior nervous system of the adult male from a 1,500 section-long thin section EM series extending from near the tip of the nose, through the nerve ring, and part way into the retrovesicular ganglion. This region contains the bulk of the synapses, excluding ventral cord nmj's. To make a comparison to the hermaphrodite, we re-reconstructed legacy Cambridge micrographs using our software, which allows us to score synaptic weights (see abstract by Cook et al). While our analysis is at an early stage, we can already see the essential result: in the adjacency matrices that display the connectivity, it is difficult to spot differences that appear greater than would be expected given the inherent variability of neuronal wiring. Known circuits in navigation and other responses are conserved. Thus behavioral differences likely emerge from differing circuit properties rather than differing connectivity. There are two possible exceptions: AIM synapses onto AIB and RIA synapses onto RIB in the male only. One set of male-specific synapses expected involves the male-specific head CEM sensory neurons, and the tail EF interneurons, which receive extensive input from the copulatory circuits in the tail and extend processes through the ventral nerve cord into the nerve ring. Both of these neuron classes have as their strongest targets the AVB command interneurons for forward locomotion. This suggests one of their functions may be to inhibit forward locomotion when a hermaphrodite is sensed or during copulation. They make additional connections to be further explored.
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[
International Worm Meeting,
2021]
Sexually reproducing animals display sexually dimorphic behaviors, geared towards reproductive success. Are there differences in the way the two sexes interpret and respond to the same aversive input? To address this question we analyzed the worm's avoidance responses to hazardous conditions. C. elegans generates an escape response to aversive stimuli by integrating sensory information from the polymodal nociceptive ASH head neurons and tail neurons, and conveying it to the main reversal interneuron AVA. The recent full mapping of the male connectome (Cook et al. 2019) suggests that the sex-shared neurons in the avoidance circuit are dimorphically connected, e.g. ASH to AVA connection is predicted to exist only in hermaphrodites. We measured the response of both sexes to the aversive stimuli SDS and glycerol using a behavioral tail-drop assay. We found that the two sexes exhibit dose-dependent sexually dimorphic responses to the aversive stimuli - across multiple nociceptive modalities, hermaphrodites exhibited a lower pain threshold than males. The behavioral differences and the suggested anatomical maps prompted us to functionally deconstruct the avoidance circuit. To examine potential sexual dimorphism at the sensory level, we compared ASH receptor expression levels (OCR-2, OSM-9, OSM10, QUI-1, ODR-3, GPA-3), ASH glutamatergic secretion by imaging the pHluorin sensor, and neuronal activation by calcium imaging in both sexes. We found that the ASH sensory neuron is non-dimorphic for all these parameters and responds similarly in the two sexes. Furthermore, we activated ASH optogenetically, thus bypassing the sensory input level, and found that hermaphrodites responded with a reversal at a lower LED intensity compared to males, in agreement with the tail-drop assay. Lastly, imaging of the downstream AVA interneuron revealed a stronger and longer response to the stimulus in hermaphrodites compared to males, further pointing to the connectivity and interneuron levels as the key sources for dimorphism in the circuit. Together, our results suggest that dimorphic responses to noxious cues arise due to neuronal circuit dimorphism downstream of sensory processing. We hypothesize that differences in circuit connectivity, rather than sensory perception per se, allow for sex-specific behavioral adaptation.