王歐力 助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University Institute of Molecular & Cellular Biology College of Life Science Laboratory course: Model organism C. elegans Week1: 1. Special features of C. elegans 2. Worm maintaining 3. Male anatomy and L4 stage 4. Neurobiology

Short introduction to C. elegans In 1965 Sydney Brenner looked around for a “minimal animal” nearly as simple as E. coli to study genetics and molecular biology He has chosen C. elegans due to its small size (1 mm) and light-thru features allowing visualization and mapping of each cell in the living animal Exactly 959 cells form a working nervous system, muscles, sexual organs and intestine with many features similar to humans More than 10,000 worms can grow on a single petri dish reproducing rapidly (from egg to mature animal in 3.5 days) Nobel Prize 2002 to S. Brenner, H. R. Horvitz and J. Sulston on their work of organ development and apoptosis in C. elegans Nobel Prize 2006 to A. Fire and C. Mellow on their discovery of RNAi in C. elegans Nobel Prize 2008 to, e.g., M. Chalfie for expressing GFP in specific C. elegans cells C. elegans survived the Space Shuttle Columbia disaster in February 2003 Movie crawl.mov

Introduction to Caenorhabditis elegans Caeno = recent, rhabditis = rod, elegans = nice C. elegans is a member of the family Rhabditidae, a large and diverse group of nematodes found in the soil It is 1 mm long, bacteriovorous (eat bacteria) and is transparent (very suitable for GFP expression) In the lab C. elegans is fed by E. coli mutants (OP50) that have a uracil biosynthesis defect to ensure that the bacteria do not outgrow the worm Some rhabditids are pathogenic or parasitic on animals, but C. elegans does not harm humans C. elegans is abundantly found in compost heaps or other nutrient-rich environments; it has been isolated from temperate regions world-wide In the soil, C. elegans associates with woodlice to use it as a transport host cm 1 mm worm sit on top of the woodlice (Crustacea)

A bird’s eye view of C. elegans Movie BIRDSEYEMOVIE.mov

The cuticle contains 4 openings: the mouth, the vulva, the anus and the excretory pore Egg expelled from the vulva Scale bars: 10 µm Basic anatomy

Hermaphrodite Male Movie male maleHalf.mov When hermaphrodite mate with male, 50% of the progeny will be males! (self-fertilization produces only 0.1% males)

General biology of C. elegans Development of all 959 somatic cells has been traced back from their appearance in the embryo until their localization in the adult (“wiring diagram” or cell lineage) All synaptic connections made by the 302 neurons are known Through coordinated muscular contraction the two bulbed-pharynx aspirates the bacteria which are then crushed in the grinder The pharynx is a nearly autonomous organ with its own nervous and muscle system The pumping of the grinder can be observed by DIC microscopy Movie pumping pumping.mov Basic feature of the hermaphrodite:

Special features of male anatomy Only one X-chromosome (XO => Hermaphrodite is XX) Different gross morphology and behaviors from hermaphrodites Slimmer than hermaphrodites (no eggs) and a clear (white) ventral gonad The hermaphrodite gonad is U-shaped while the male gonad is J-shaped U-shaped gonad in hermaphrodites J-shaped gonad in males

Stress induces throwing of males Males occur infrequently by hermaphrodite self-fertilization to a very low extent (0.1%) Ways to increase the number of males:  Using him mutations (him = high incidence of males) => these mutations increase the frequency of of X-non-disjunction => up to 30% males  Male mating: mating hermaphrodites + males increases number of males up to 50%  Heat-shock: exposure of hermaphrodites to 30°C for several hours increases  Exposure to ethanol increases the number of males Oliver’s brother (to the right) and his friend

How to cultivate C. elegans? 5.5 cm petri dish with agar

E. coli on top of the agar serves as food

Bacterial lawn (smear) Eggs Larvae Adults Movie wormsonplate.mov

4 larval stages (“juveniles”) “L4 mark”: White triangle- type spot at vulva

C. elegans neurobiology The C. elegans nervous system is simple and well described It consists of 302 neurons interconnected by chemical and electrical (gap junctions) synapses The position and the characteristic of each neuron does not vary from animal to animal which makes it a very good model organism for studying neuronal processes Even though the neuronal system is simple, it regulates a wide variety of behaviors: different mechanosensory responses chemosensory responses thermotaxis complex responses to food (habituation and learning) locomotion egg-laying and defecation complex male mating behavior

Neurons are paired head vulva tail head vulva tail Basic features of the nervous system

Neurons are paired Basic features of the nervous system P = posterior A = anterior L = lateral M = microtubule cell N = interneuron R/L = right/left

Dorsal nerve cord Ventral nerve cord Nerve ring (not shown) Basic features of the nervous system sublaterals

Commissures Basic features of the nervous system Some ventrally located motor neurons make commissures to the dorsal nerve cord Interestingly, most commissures are located on the right side of the worm

Head neurons Head sensory neurites are dendrites Nerve ring The nerve ring contains mostly sensory neurons and almost all interneurons It is the brain of the worm

Features of the nervous system Neurons can be categorized in: interneurons, sensory neurons and motorneurons interneuron sensory neuron motorneuron

Motor neurons Innervate body-wall, pharyngeal, egg-laying and defecation muscles Cell bodies of motore neurons are located in the ventral cord: some of them send processes to the dorsal cord via commissures Most of the commissures travel along the right side of the animal Many right sided commissures Less left sided commissures

Motor circuit hierarchy Major elements of the neuronal circuits in mechanosensory, chemosensory and thermosensory behaviors Sensory neurons Head interneurons Command interneurons Pool of B- or A-type motor neurons