Final Report Summary - ACMO (Systematic dissection of molecular machines and neural circuits coordinating C. elegans aggregation behaviour)
The human brain contains 100 billion nerve cells. Understanding how such a complex structure functions in health and disease is daunting. Fortunately, many molecules in our brain are conserved in simpler animals, such as the worm Caenorhabditis elegans. Studying this experimentally tractable animal can quickly and cost-effectively provide insights into how our brains work.
In C. elegans behaviors can be linked to small numbers of identified neurons connected by few, anatomically defined, chemical and electrical connections. A particularly intricate C. elegans behavior, aggregation, is governed by multiple sensory cues, including ambient O2, noxious stimuli, food stimuli, and pheromones.
We sought to deconstruct aggregation in molecular and circuitry terms.
We isolated >800 aggregation-defective mutants following chemical mutagenesis, and sequenced their genomes using the Illumina platform. We created a bioinformatics pipeline that enabled us, without preliminary experimental work, to predict the lesion that caused the aggregation phenotype in many of these strains. We confirmed these predictions by transgenic rescue, or by creating de novo knockouts of the predicted gene. For this purpose, we adapted CRISPR/Cas9 technology for C. elegans.
To predict groups of genes that work together we clustered mutants by their phenotypic similarity. We assembled hardware and created software that enabled rapid, high dimension analysis of behavioral phenotypes for the hundreds of mutants we studied. By high dimension I mean simultaneous analysis of multiple sensory responses and multiple behavioral features.
One group of genes identified interleukin-17 (IL-17), its two co-receptors, and a signalling pathway acting downstream of the co-receptors, including C. elegans orthologs of human ACT1, IRAK and IκBζ. Il-17 is a major pro-inflammatory cytokine associated with many auto-immune diseases; it has been studied almost exclusively in the immune system. Very little is known about its functions in the nervous system. We showed that IL-17 act like a neuromodulator, changing circuit gain, most likely by altering intrinsic cell excitability.
Other groups highlighted molecules required to localize soluble guanylate cyclases and cGMP channels to the dendritic ending, and molecules required for GPCR biogenesis. Multiple genes we identified had no previously known function in any animal. We are currently using biochemistry to define better the functions of these genes.
As part of our characterization of mutants we imaged, for the first time in any animal, physiological cGMP signalling in a neuron in vivo, and simultaneously measured Ca2+ responses. By visualizing cGMP dynamics in different mutants, we provided mechanistic insights into how graded tonic responses are encoded. We also showed that cGMP and Ca2+ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, but varies substantially across individuals, despite animals being genetically identical and similarly reared. We are studying this mysterious inter-individual variation further.
In parallel with our genetics, we developed methods to image neural activity in freely moving animals using genetically encoded sensors. Using these methods we identified a tonically signalling circuit that switches the global behavioral and physiological state of C. elegans in response to an environmental cue, resetting the salience or valence of sensory cues, metabolism, ageing, and immune responses. This provides us with a genetically tractable model to study how such global states are assembled in neural circuits, and how they are modified by experience, directions we are pursuing further.
In C. elegans behaviors can be linked to small numbers of identified neurons connected by few, anatomically defined, chemical and electrical connections. A particularly intricate C. elegans behavior, aggregation, is governed by multiple sensory cues, including ambient O2, noxious stimuli, food stimuli, and pheromones.
We sought to deconstruct aggregation in molecular and circuitry terms.
We isolated >800 aggregation-defective mutants following chemical mutagenesis, and sequenced their genomes using the Illumina platform. We created a bioinformatics pipeline that enabled us, without preliminary experimental work, to predict the lesion that caused the aggregation phenotype in many of these strains. We confirmed these predictions by transgenic rescue, or by creating de novo knockouts of the predicted gene. For this purpose, we adapted CRISPR/Cas9 technology for C. elegans.
To predict groups of genes that work together we clustered mutants by their phenotypic similarity. We assembled hardware and created software that enabled rapid, high dimension analysis of behavioral phenotypes for the hundreds of mutants we studied. By high dimension I mean simultaneous analysis of multiple sensory responses and multiple behavioral features.
One group of genes identified interleukin-17 (IL-17), its two co-receptors, and a signalling pathway acting downstream of the co-receptors, including C. elegans orthologs of human ACT1, IRAK and IκBζ. Il-17 is a major pro-inflammatory cytokine associated with many auto-immune diseases; it has been studied almost exclusively in the immune system. Very little is known about its functions in the nervous system. We showed that IL-17 act like a neuromodulator, changing circuit gain, most likely by altering intrinsic cell excitability.
Other groups highlighted molecules required to localize soluble guanylate cyclases and cGMP channels to the dendritic ending, and molecules required for GPCR biogenesis. Multiple genes we identified had no previously known function in any animal. We are currently using biochemistry to define better the functions of these genes.
As part of our characterization of mutants we imaged, for the first time in any animal, physiological cGMP signalling in a neuron in vivo, and simultaneously measured Ca2+ responses. By visualizing cGMP dynamics in different mutants, we provided mechanistic insights into how graded tonic responses are encoded. We also showed that cGMP and Ca2+ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, but varies substantially across individuals, despite animals being genetically identical and similarly reared. We are studying this mysterious inter-individual variation further.
In parallel with our genetics, we developed methods to image neural activity in freely moving animals using genetically encoded sensors. Using these methods we identified a tonically signalling circuit that switches the global behavioral and physiological state of C. elegans in response to an environmental cue, resetting the salience or valence of sensory cues, metabolism, ageing, and immune responses. This provides us with a genetically tractable model to study how such global states are assembled in neural circuits, and how they are modified by experience, directions we are pursuing further.