Dissecting the molecular mechanisms underlying synpase development and neuronal functions in Caenorhabditiselegans.

摘要

The development and function of the nervous system is under delicate regulation of diverse tissue-derived signals in multi-cellular organisms. In Dr. Nonet's lab, I am using the model organism Caenorhabditis elegans to ask two basic questions: (1) How do different tissues in an organism coordinate to regulate neural functions and behaviors? (2) What controls the development of synapse, the basic unit of the nervous system? These questions divide my dissertation into three parts, with the first two parts related to the first question and the third part to the second question.;In the first part of this dissertation, I present work that demonstrates the role of the C. elegans intestine as an endocrine organ in regulating the rhythmic defecation behavior (Chapter 2). The C. elegans defecation behavior consists of three well-coordinated muscle contractions that enable the nematode to expel intestinal contents out to the environment. Genetic and cell biology analyses showed that the early and late muscle contractions involve activities in the intestine and GABAergic neurons (AVL and DVB), respectively, while it remains unclear how the intestinal event is coordinated with later activation of GABAergic neurons. Using molecular genetics and cell biology approaches, we demonstrate that the exocytic protein AEX-4 and proprotein convertase AEX-5 function in the worm intestine to control the defecation motor program. When expressed in the intestine, AEX-5 is secreted into the pseudocoelom, and this secretion is blocked by AEX-4 disruption. Moreover, we show that the G-protein coupled receptor (GPCR) AEX-2 functions in GABAergic neurons to regulate defecation behavior, and it is genetically downstream of intestinal AEX-4 and AEX-5 signals. We also demonstrate that the stimulatory Galpha pathway relays the AEX-2 signal in GABAergic neurons. Together, our results provide evidence that the C. elegans intestine is able to modulate neuronal function by secretory signals.;In the second part of this dissertation, I present work that demonstrates the role of the C. elegans intestine in modulating the cholinergic neurotransmission (Chapter 3). C. elegans utilizes acetyl choline as a neurotransmitter at its neuromuscular junctions (NMJs) to control muscle contractions and locomotion related behaviors. Using molecular genetics, pharmacological, and physiological approaches, we show that the proprotein convertase AEX-5 is required in the intestine to maintain normal cholinergic transmission in the nematode. In addition, we find that the GPCR AEX-2 functions in the GABAergic neurons to maintain cholinergic transmission level, and the stimulatory Galpha pathway is genetically downstream of AEX-2. Interestingly, we find that although both the defecation motor program and the cholinergic transmission modulation involve intestinal signals and neuronal G-protein pathways, they depend on different downstream molecules: while the defecation requires GABA to activate the enteric muscle contraction in the last step of the defecation, the modulation of cholinergic transmission depends on neuropeptide processing enzymes EGL-3 and EGL-21. As GABAergic neurons do not directly synapse on cholinergic neurons in C. elegans, we speculate that the peptide signals act in a paracrine manner on cholinergic neurons. This suggests the C. elegans intestine could function as an endocrine organ to modulate multiple aspects of neuronal functions.;In the last part of this dissertation, I focus on the early neural development of C. elegans and I present the preliminary work on the focal adhesion complex molecule ZYX-1 for its role in mechanosensory synapse development (Chapter 4). We cloned the zyx-1 allele from the genetic screen that looked for worms defective in PLM synaptic patch formation. Using time course imaging analysis of fluorescence labeled PLM neurons, we show that zyx-1 mutants are able to form synapses during early development, while they fail to maintain the synapse to adulthood. In addition, we demonstrate that ZYX-1 acts cell-autonomously in mechanosensory neurons to regulate PLM synapse maintenance. We are currently working to dissect the molecular mechanisms that underlie ZYX-1's function in synapse maintenance. I expect the study will shed light on our understanding of the molecular mechanisms underlying neural development.