Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

The C. elegans ALA neuron : its transcriptome and role in inducing sleep

A long-standing yet to be accomplished task in understanding behavior is to dissect the function of each gene involved in the development and function of a neuron. The C. elegans ALA neuron was chosen in this study for its known function in sleep, an ancient but less understood animal behavior. Single-cell transcriptome profiling identified 8,133 protein-coding genes in the ALA neuron, of which 57 are neuropeptide-coding genes. The most enriched genes are also neuropeptides. In combination with gain-of-function and loss-of-function assays, here I showed that the ALA-enriched FMRFamide neuropeptides, FLP-7, FLP-13, and FLP-24, are sufficient and necessary for inducing C. elegans sleep. These neuropeptides act as neuromodulators through GPCRs, NPR-7, and NPR-22. Further investigation in zebrafish indicates that FMRFamide neuropeptides are sleep-promoting molecules in animals. To correlate the behavioral outputs with genomic context, I constructed a gene regulatory network of the relevant genes controlling C. elegans sleep behavior through EGFR signaling in the ALA neuron. First, I identified an ALA cell-specific motif to conduct a genome-wide search for possible ALA-expressed genes. I then filtered out non ALA-expressed genes by comparing the motif-search genes with ALA transcriptomes from single-cell profiling. In corroborating with ChIP-seq data from modENCODE, I sorted out direct interaction of ALA-expressed transcription factors and differentiation genes in the EGFR sleep regulation pathway. This approach provides a network reference for the molecular regulation of C. elegans sleep behavior, and serves as an entry point for the understanding of functional genomics in animal behaviors.