Introduction

The Zhen Lab

Links

Research Overview

The research focus of the Zhen lab is how neurons develop, connect and regulate the development and function of animals. We mainly use the C. elegans nervous system as a model. C. elegans is an excellent model for live imaging and molecular genetic studies; its compact and fully sequenced genome, as well as the fast life cycle, allows the application of powerful forward and reverse genetic tools; the synaptic connectivity of its simple nervous system has been deduced through EM reconstructions; the transparency of the animal, and the small number of neurons and synapses allow the high resolution imaging at single neuron and single synapse resolution in intact animals; cell biology and electrophysiology tools, such as calcium imaging and intracellular recording, have recently become available in C. elegans, expanding the horizon of functional analyses of nervous system development and function.

We have developed a number of green and red fluorescent protein markers that allow direct visualization of different neuronal structures in live C. elegans. We are utilizing these markers to uncover signalling pathways that determine the differentiation, maturation and stabilization of neuronal polarity, ciliary development and synapses. A standard approach we are using is to chemically mutagenize genes in animals, and to screen for mutants that have abnormal neuronal development such as synaptic structures (as revealed by fluorescent markers). Genetic mapping and molecular cloning of these genes will thus elucidate essential molecular mechanisms for synaptic development. Through cutting-edge biochemistry, electronmicroscopy, live imaging and electrophysiology analyses, we further dissect the mechanisms through which these proteins regulate neuronal development and function.

Using these tools, we have identified several novel yet evolutionarily conserved genes and their genetic pathways that regulate various aspects of neuronal development and function.

In collaboration with other groups, we have also extended our investigation on the role of cilia, in particular, intraflagella transport in development and human disorder called ciliopathy. We have also begun a systematic approach to identify neural-specific alterative splicing events, and investigate the mechanisms that regulate neural-specific splicings.

Our current research projects aim at addressing the following questions:

1. How is neuronal polarity regulated?
Neurons are polarized cells that develop two types of morphologically and functionally differentiated processes, called axons and dendrites, respectively. In vivo mechanisms through which axonal and dendritic development are regulated are poorly understood. We identified a Ser/Thr kinase, SAD-1, that plays an essential role to establish polarity in C. elegans neurons. SAD-1-family kinases are recently shown to regulate polarity of vertebrate neurons also. We further show that SAD-1 regulates neuronal polarity through its interaction with neurabin (NAB-1), a scaffold protein, to restrict the delivery of axonal components to axonal processes. We are currently identifying upstream regulators of SAD-1 kinase activity, as well as downstream effectors (substrates) of SAD-1.
(Publications: Crump et al., 2001, Neuron; Hung et al., 2007, Development; Kim et al., 2008, Neural Development; Kim et al., in preparation)

2. Mechanisms that regulate synapse development
Neurons communicate through specialized subcellular structures called synapses. The development of synaptic structures is regulated by both intrinsic and extrinsic singals/mechanisms. We identified a novel, neural-specific E3 ubiquitin ligase complex that is required for proper synapse development. Two novel proteins, RPM-1 and FSN-1, together with Cullin and SKP, form a novel SCF-like complex at the presynaptic termini. In the absence of these key components of this complex, some synapses fail to develop properly, while others show further expansion. We have identified, and are currently examining the function of components of two distinct signalling pathways through which this SCF-like complex regulate synapse development.
(Publications: Zhen et al., Neuron 2000; Liao et al., Nature 2004; Patel et al., Nature Neuroscience 2007; Gao et al., Cell death and differentiation 2008; Hung, Liao et al., in preparation)

3. Molecular mechanisms that regulate active zone differentiation and synaptic activity
Through the genetic screen for mutations with defective vesicle marker expression, we identified SYD-2, a scaffold protein that localizes specifically to the active zone, and required for active zone development. With SYD-2, we further developed a fluorescent marker that allows us to visualize active zones in live animals. Using the markers, we showed that in GABAergic neuromuscular junctions, synapses develop in size, incorporating more active zone components throughout development. We further identified several mutants with defective active zone marker expression but normal vesicle markers. We have cloned three novel genes that regulate active zone development, and are currently determining their mechanisms.
In this screen we discovered the function of a new family of cation ion channel called NCA. Through calcium imaging and electrophysiology, we showed that the level of NCA channel activity correlates with neuronal excitability. We showed that this NCA channel complex, composed of NCA, along with two accessory subunits UNC-79 and UNC-80, localized along axons, near but excluded from synapses; these NCA channels propagate neuronal excitability from cell body to synapses. We are further examining cellular mechanisms through which these channels regulate neuronal activity.
(Publications: Zhen and Jin, Nature 1999; Yeh et al., J. of Neuroscience 2005; Yeh et al., PLos Biology 2008; Liewald et al., Nature Method 2008; Yeh et al., Journal of Neuroscience 2009 in press; Kawano, Po et al., in preparation; Bouhours et al. in preparation)

4. Molecular mechanisms that regulate endocytosis
At synapses, the released synaptic vesicles are replenished mainly through a process called endocytosis. This is an important process to ensure the recycling of vesicle for sustained neuronal activity. We identified a mutant called syd-9 that has severely diffused distrubution of vesicle proteins. We showed by phenotypic and genetic analyses that SYD-9 is a novel regulator, likely through splicing, for endocytosis.
(Publications: Wang et al., PNAS 2006; Wang et al., Traffic 2008)

5. How does cilium regulate development
Bardet-Biedl Syndrome (BBS) is a complex and pleiotropic human disorder. Progressive blindness, digit anomalies, obesity, cognitive impairment and kidney failure are key diagnostic hallmarks. The first insight into the pleiotropic nature of BBS resulted from animal model studies, which revealed most BBS proteins as part of an intracellular transport system required for the maintenance and function of cilia, an organelle projecting from the surface of virtually all eukaryotic cell types. BBS is thus proposed to be a 'ciliopathy', where abnormal ciliary functions in different tissues lead to diverse clinical features. Caenorhabditis elegans is the animal model that pioneered the discovery of the ciliopathy nature of BBS.
In collaboration with Dr. Elise Heon’s lab, we are utilizing the C. elegans model to investigate the heterogeneity nature of BBS syndrome. In particular, we have isolated, cloned and are currently characterizing a novel genetic modifier for BBS-related phenotypes in C. elegans.
(Publication: Li et al., PLos Genetics 2008; Mok et al., in preparation)

6. Alternative splicing – how does it regulate neuronal development
Alternative splicing (AS) is the process where several messenger RNA (mRNA) transcripts arise from a single precursor-mRNA (pre-mRNA) through differential splice site usage. In humans more than two thirds of known genes are alternatively spliced and evidence has shown that AS generates increased transcriptional and proteomic diversity, particularly in neurons. Little is known about how regulated alternative splicing contributes to development and function in living organisms. Most of the key proteins involved in regulating alternative splicing, including tissue-specific splicing factors, are conserved between C. elegans and mammals. This conservation of the splicing machinery, coupled with the ability to use forward and reverse genetic strategies, makes C. elegans an excellent model organism to discover and characterize AS regulation in the nervous system of live animals.
In collaboration with Dr. Blencow’s lab, we are using a combination of microarray and reporter-based assays to identify novel cases of spatiotemporally regulated splicing events in C. elegans neurons. Following their identification, the functional consequences of these splicing events on animal behaviour and nervous system morphology will be analyzed through genetic, in vivo imaging, and biochemical approaches. (Publication: Calarco et al., in preparation)

C. elegans

FSN-1::GFP

Fluorescent markers are developed to illuminate neuronal structures

Synapse mutants are identified under high magnification