王歐力 助理教授Oliver I. Wagner, PhDAssistant Professor

National Tsing Hua University

Institute of Molecular & Cellular Biology

College of Life Science

Laboratory course: Model organism C. elegans

Week 2:

1.Sensory neurons2.Behavior3.Worm mating (crossing)

Features of the nervous systemNeurons can be categorized in: interneurons, sensory neurons and motorneurons

interneuron

sensory neuron

motorneuron

can be made visiblewith a special dye: DiI/DiO

Labial process bundles

Amphid process bundles

Ring ganglia

Head neuronsThe nerve ring contains mostly sensory neurons and almost all interneurons

UNC-104:: GFP

Front head Middle head

Posterior head

UNC-104:: GFP

• Amphids: are a pair of laterally located sensilla in the head which are open to outside at the base of the lips• These chemosensory organs can be stained with FITC or DiI • Some mutants fail to be stained => dyf = dye filling (mutant)• Each amphid is made up of 12 sensory neurons (ADF, ADL, ASH…) with ciliated dendrites as well as one sheath and one socket cell• The axon of the amphid is located in the nerve ring

Identifying amphids

• Oocyte nuclei are produced by meiosis at the distal end of the gonad and grow in a syncytium • Just before fertilization the single nuclei are enclosed by a separate plasma membrane• Produced sperm is stored in the spermatheca• After fertilization the egg-shell is added: => self-fertilization produces up to 300 eggs

Reproduction• 99% of adult C. elegans are self-fertilizing hermaphrodites• This feature enables scientists to easily generate homozygous mutant stocks• Hermaphrodites are protandrous: the gonads produces germ cells which first differentiate as sperm (about 250 sperm cells) and then produces eggs• In C. elegans hermaphrodites reproducibility is determined by the sperm supply• C. elegans produce males to about 0.05% which are fully functional but produce sperm only; the sperm is transferred to the hermaphrodites during mating => this feature enables scientists to transfer mutant alleles (to wildtypes or other mutants)

spermatheca

General biology of C. elegans• Fertilization takes place by squeezing mature oocytes through the spermatheca• The eggs are laid through the vulva at a 40-cell stage

• Adult hermaphrodites have about 10 mature eggs inside; the older eggs are laid as fast as new eggs are generated

• Males cannot produce eggs but they can cross-fertilize hermaphrodites makingthem very suitable in genetics to create genetic combinations (male-fertilizing)• The male sperm outcompetes the hermaphrodites sperm during cross-fertilizing• XO combination in male is a spontaneous loss of X chromosome: XX => XO

A hermaphrodite produces up to 1300 eggs

during its lifetime

The C. elegans lifecycle• The 4 larval stages (“juveniles”) are common features of nematodes• The lifecycle takes about 2.5 days at 25°C, 3.5 days at 20°C and 5.5 days at 15°C• C. elegans also has an alternative L3 stage known as dauer (“enduring”) stage• The dauer stage is a metabolic diapause to survive extreme conditions (mainly lackof food); in the wild, the dauer stage might serve to geographically disperse

• The entry into the dauer stage isdetermined by worm-crowding, hightemperature and lack of food• As a dauer, C. elegans can survive for up to 3 month highly extending its lifespan • On exposure to improved conditions (availability of food…) the L3 dauer exits and resumes development• Parasitic nematodes use the dauer to infect hosts

Dauer larvae usually appear dark,thin, rigid and motionless

Recovered dauer larvae retain theirtransparent appearance and begin feeding with increasing motion

During dauer-formation the mouth closes• There is no aging at dauer state! Due to the dauer stage worms can live 10 times longer than their normal lifespan!• Due to the mouth closure the worms are restricted from eating

Imagine a human that is supposed to become 90 years old might become 900 years old

Dauer wormL3 worm

• During post-embryonic development the number of somatic cell nuclei increases to 959 • Total life span under suitable living conditions: 2 weeks (300 eggs every 4 days) = 1300 eggs during a lifetime• Wildtype-worms: Bristol (most commonlyused strain) has been isolated from mushroom compost in Bristol (England) =N2 strain• Other strains isolated from soil and moist environment are C. briggsae and C. remanei

about 1.3 mm in length and 80 µm in diameter

Behavior, learning and memory

Mechanosensing: Avoidance reflex circuitsMechanosensory neurons can sense different strength of touch:

eyelash touch

“Mec response”:• touching the anterior side of the body results in a backward movement• touching the posterior side results in a forward movement

Gentle touch (eyelash) mechanosensory neurons

Harsh touch mechanosensory neurons

Nose touch and osmolarity sensors

Texture sensing neurons

Mechanosensory neurons

Chemotaxis Thermotaxis

Traces of a worm which was allowed to freely move within one hour on a plate with an attractant (B = biotin)(right circle = buffer)

• Traces of a worm in a radial thermal gradient• Chooses an optimal temperature (similar to its cultivation temperature)• Circles in isotherms at that temp.• Can detect thermal gradients < 0.1°C

Simple behavior

• Tapping at the side of the petri dish stimulates backing response • Backing movement decreases if stimulus is continuously applied (every 10 seconds for total 30 minutes)

• If stimulus is applied less often (every 60 seconds) decrease of backing response is less abrupt (indicating habituation and not worm fatigue)• Also habituation can be rapidly abolished with an electroshock stimulus (dishabituation)• Further, worms recover from short-interval habituation faster compared to long-interval habituation

Habituation occurrence can be stored for more than

1 hour in the nervous system

Habituation (non-associative learning)

Classical conditioning (associative learning)

• Non-associative learning (habituation): an animal alters its behavior to a single stimulus• Associative learning: an animal learns to use a previously neutral stimulus to predict a second more significant stimulus• Example: after food deprivation, one ion is associated with food and the second ion is associated with the absence of food => a conditioned animal will move to the ion associated with food (even if no food is present)• lrn-1 and lrn-2 (lrn = learn) are genes involved in learning

Na+ = food Cl- = no food (short incubation)

5 ho

urs

depr

ivat

ion

worms go to Na+

Male mating behavior

Male mating behavior

WAmating.mov