C. Elegans and its adaptations

c. elegans

• invertebrate

• roundworm - not an earthworm

• belongs to the phylum nematoda and the taxonomic group ecydosozoa

• have non segmented bodies

• found nearly everywhere: fruit, water, soil, animals, plants

• most abundant animal on earth

discovery

• N2 bristol strain is the wildtype strain

• Hawaiian strains are social worms and cluster together on the plate

• horwitz and sulston mapped how cells mature into tissues and organs

culture

• free living - can be grown on an agar plate

• feed on E.coli OP50

• maintained at 20 degrees

• NGM plates (nematode growth media)

• can be frozen up to -80 for stock miantenace

• strains can be acquired in post

sexual forms

HEMAPHORODITE

• XX

• can produce sperm and oocytes which it can fertilise and lay

• all genetically identical - useful progeny

MALE

• cant produce their own eggs

• useful for gene knockouts

• has rays which contain sensory neurons

• uses the fan to locate the position of the vulva so it can fertilise

anatomy

• pharynx: muscular structure innervated by 20 neurons which is important for sucking up bacteria

• reproductive tissue like conveyer belt which is geared up to propogate and produce progeny (300-350)

• cylindrical body shape

• dark cuticle around the outside which is selectively pereable

  • keeps things out

  • hydrostatic pressure

• below the cuticle is hyperdermis and body wall muscle cells

• within the muscular structure is the gut and the intestines which are bathed in fluid

  • this fluid is the closest thing to blood and is used for long distance signalling

muscle types

• phalangeal muscle - main one for feeding

• body wall muscle

• head and neck muscle

• vulval muscle: main one for reproduction

• intestinal muscle

• anal muscle

nervous system

• 302 neurons in hemaphorodites

• nerve rings and nerve chords

  • lots of interneurons in the nerve ring

  • ventral and dorsal nerve chords which are interneurons

• can be broken down into somatic (282) and pharangeal neurons (20) with one neuron linking the two

• have motor neurons, sensory and interneurons

• know the connectome: anatomical connection for all the neurons

• labelled by dorsal, ventral left or right

sensory neurons

• 12 amphid neurons in the head which are bilaterally paired —> detect external cues

• ciliarry endings which extend to the nose and are externally exposed

neurotransmitters

similar nervous system to us with a few differences

• 2 that we dont have: tyramine and octopamine (acts as NAdr)

• conservation across nt synthesis ( packed in vesicles and taken up)

• conserved enzymes which break down nts

• neuropeptide that modulates synapses

genetics

• first multicellular organism to have genome sequenced

• 100MB genome

• 5 pairs of chromosomes

• we can do genetic screening and gene knockouts

• GENETIC SCREENING: expose worm to mutant and allow it to lay eggs —> look for phenotype that is different and examine genome to see what has changed

• REVERSE GENETIC SCREEN: have a gene of interest —> mutate the gene and insert it into worm to see how it impacts the worm

• MICROINJECTION: transform worms to express certain proteins

pharynx and feeding

• predominatly hemaphorodites used

• two roles of the pharynx: peristalsis and pumping

• pharynx found in transparent region (can be used to measure activity)

• darker region is the intestine

• draws bacteria into the pharynx to the intestine

• pharynx is a muscular structure controlled by the NS

MECHANISM FOR FEEDING

• sequential relaxation and contraction is required to suck in bacteria through bucal opening

  • when the muscle is contracted the lumen opens

• spits out the fluid the bacteria is in to condense it down

  • the muscle is relaxed and the lumen shrinks to spit out fluid

• peristalsis moves pellet through the corpus (made up of the procorpus and the metacorpus) , through the isthmus which connects the corpus to the grinder

• ground down by grinder in terminal bulb

• contraction and relaxation of muscle controlled by gap junctions

albertson and thompson

• characterised structures of teh NS

• 20 neurons NS

  • characterised by motor neurons, interneurons and dual function neurons

  • sometimes neurons ahve to do more than one job

muscle anatomy

• surround the lumen of the pharynx

• 8 diff muscle cell types

• PM - pharangeal muscle

• triradial symmetry - around 3 axes of symmetry

  • radially orientated muscles in corpus and terminal bulb needed for opening and closing of lumen by relaxing and contracting

  • longitudinal muscles move bacteria aling the lumen of the isthus

• linked by electrical synapse —> NOTE: some muscles controlled by chemical and electrical synapse, but all motor neurons controlled by chemical synapse)

• sieve structures which allow the worm to keep the bacteria and remove the liquid

• terminal bulb is a rigid structure which is the extension of external cuticle which opens and closes in the grinder (contraction of the muscle causes opening of the lumen)

laser ablation

laser ablation allows you to remove any neuron in the nervous system and observe what happens:

M4 neurone

• coordinates peristalsis

• is we laser ablate the pharynx becomes stuffed, unable to move bacteria into the gut

• doesnt develop into adulthood

• M3 also has a role in rate of pumping

MC

• laser ablation leads to a slower feeding rate

• coordinates feeding rate

• is a cholinergic neuron

genetic ablation

• can do gene knockouts and observe what happens

• release of neurotransmitters requires primary docking and release of vesicle —> unc13 protein is important for this

UNC13

• sits within the vesicle docking complex

• gene knockout impacts behaviour of the worm

• disrupted release of vesicles which impacts NS and rate of pumping

• unc13 mutant has slower rate of feeding

• can still pump with disrupted NS

pharangeal NS

• required for modulating the activity of the pharynx

• as its transparent we can use GFP reporter to tell us which cells express which genes

• Eat 4 is a glutamate transporter which loads up vesicles with glutamate

• allows us to assign nts identity to neurons

• donw via engineered plasmids which can be injected into worms reproductive system

• can start to map out NS with their nts

  • either serotonin, glutamate, cholinergic

• link between extrapharangeal NS and pharangeal NS (can still pump if its disrupted)

• has the ability to respond to neurohormonal signalling (signals that act on tissue from outside the tissue and can do long distance signalling)

quantifying NS activity

• can make counts of activty of the grinder - gives a readout for how fast the worm is feeding

• measured in pumps/min (1 grinder movement i open - close - open)

• done on age synchronised worms)

• amphids are exposed to external environment - amphids feed into sensory neurone (ADF) which releases serotonin to MC (motor neurone important for determining feeding rate)

• MC is a cholinergic neuron (receives serotonin through ser 7 5HT receptor which is a Gas GPCR)

• MC neurons cause increase feeding arte when signalled to with serotonin

mechanism

• mutant without tph1 mutant cant biosynthesise 5HT —> the worm no longer pumps at high rate in food prescence

• nicotinic receptor eat 2 responds to the Ach, when Eat 2 is ablated feeding behaviour is disrupted

• this pathway is important for high rate of feeding on food

off food

• turns down feeding activity

• M3 motor neuron sends an inhibitory signal to supress the pharangeal pumping

• signals through choinergic glutamate gated ion channel

• can remove EAT 4 from M3 (vesicle loader) and causes high rate of pumping

• Eat 4 is important for loading glutamate which is responsible for supressing pumping

egg laying physiology

• HSN neuron connects pharynx to vulva and coordinates egg laying

• needs to adapt where it lays progeny into environment where it will best survive

anatomy of C, elegans reproductive system

• 2 U shaped gonad arms (branches of the reproductive system that converge onto the vulva and uterus) - one in posterior and one in anterior

• 2 so that if one is damaged it can still lay eggs

• DTC continually produces mitotic cells which undergo meiosis

• like a production line, cells produced, cells mature, cells fertilised, move into the uterus (oocytes —> embryos (when fertilised) —> eggs)

• can produce around 300 progeny on its own

• uterine and vulval muscles, 2 types of each

  • uterine muscles like an elastic band round the uterus and helps squeexe out eggs

  • vulval muscle cells involved in opening and closing of vulva

HSN neuron

• HSNL and HSNR act as a bilateral pair

• control egg laying physiology

• laser ablation of HSN neurons impact egg laying behaviour

• male worm doesnt have HSN neuron - are deleted as they develop

• HSN neurons express arrestin which can be tagged with GFP to determine where neurons go

• HSN express tph1 (tryptophan hydroxylase) which causes tryptophan to be converted to 5HT and these neurons also secrete 5HT

laser ablation of HSN

• if the left is removed it can still lay eggs, same if right removed

• only if both are ablated is egg laying affected

egg laying

• connected circuit between neurons and muscles

• HSN speaks to VC neurone but also some muscle cells (via chemical synapse)

• also electrical synpased between muscles to allow co-ordinated movement

• each of the groups of muscle cells have different roles

• eggs lay in certain patterns (bursts)

• bursts are intersperced with inactive phases

response to sensory cues

• can change the rate of egg laying in reponse to: salt conc. impact of food

• in absence of bacteria: retain eggs

• straved of food: eggs retained but develop into larvae which will eat the adult worm from the inside out (bag of worms)

pharmacological experiment

• serotonin released into synapse and induces signalling, then reuptaken

• use drugs such as impipramine whih inhibits reuptake —> increased electrical activity of postsynaptic cells

• when exposed to serotonin increased egg laying rate, same as when imipramine is used ( this is because serotonin is used at the synpase and imipramine blocks reuptake)

inactive and active states

• genetic knockout of serotonin signalling and laser ablation have the same effect

• active phase is the same but much longer inactive period

• hypothesised that serotonin is required for the switch between active and inactive states

• burst in active phase is driven by Ach

neuropeptides

• class of signalling molecules

• neuromodulators which turn up/down signalling at synapses

• can cause slower neuromodulatory signalling

• signal through GPCRs

• can conduct neurohormonal signalling too

• 4-20AAs which are derived from larger precursors

• 3 families of neuropeptides:

  • insulin related peptides

  • non insulin related and non FMRFamide

  • FMRFamide

genetic screening

• form mutants and search for mutant phenotype

• can then map the mutated phenotype

• in mutants where egg laying was defective mutated genes were egl3 and egl21

• these code for enzymes which cleave the precursor

• mutants which dont produce flp neuropeptide still lay eggs and egg laying is still clustered into active and inactive phases but inactive phase is lomger

• flp and serotonin are both important for egg laying

• as serotonin concentration increases so does rate of egg laying but this is not observed with flp1 —> flp1 needed to enhance egg laying by serotonin

• flp1 expressed in some of the amphineurons that detect food so may be involved in food dependentt responses

• egg laying rate decreases with removal of food

• flp1 mutants have low egg laying rate on food and dont adapt to off food therefore flp1 important in adaptive behaviour

• AIA and AIY neurons in nose express flp1 which signals to HSN which controls vulval muscle cells to adapt physiology

role of EGL4

• identified from a screen

• mutants have lower rate of egg laying and dont adapt when theres no food around

• also have reduced egg laying rate in response to serotonin

• Egl4 required for the mediating induction of active state by serotonin

• Egl4 is a serine threonine kinase also called protein kinase G

• role in neural plasticity and regulation of gene transcription

• Egl4 translocated to the nucleus and adapts response of sensory neuron to odours

• has 2 domains: one binds cGMP, the other acts as a kinase

• expressed in neurons required to detect food cues (heavily expressed in sensory and interneurons)

development of C. elegans

• eggs remain in adult for 150 mins

• 3 days from egg to adult at 20 degrees

• period of cell division, followed by period of elongation and metamorphosis

• muscles activated so worm starts to move around within the egg (quickening phase)

• then bursts out of egg shell (shell provides a barrier for the worm as it develops)

• after the worm hatches it progresses L1-L4 where theres 4 larval stages

• development can take diff amounts of time depending on temp

• outer cuticle is shed (acts to protect worm)

• dauer is an alternative state that the worm can enter depending on the environment

• L4 stage is characterised by the appearance of a vulva

L1 arrest

• if theres a lack of food L1 remains as L1

• can survive up to 2-3 weeks with no food

• post embryonic development not initiated

• if food is present development continues

late L4 development

• if the worm is starved at late L4 they will still molt into adults ut enter an adult reproductive dispause (stop producing embryos)

• prolonged starvation can lead to shrinkage of the germ line, progranmmed cell death —> reduction of germ cells

• distal tip cells shut down to reduce no of germ line cells passing through

• if exposed to food, ability of reproductive tissues to develop and lay eggs restored

dauer

• if its L1 and encounters factors below will malt and become dauer

  • crowding

  • starvation

  • high temp

• worms can survive many months and can exit when conditions improve —> doesnt ipact on lifespan/dvelopment out of dauer state

• dauer state is good at survival

• incubated with SDS detergent which cuases denaturation of proteins (worms incubated in this really good at survival and can resist harsh chemicals)

• dauer state considered state of quiescence

  • quiescnec defined as: dormant state in which metabolism is slowed down in response to stress

  • dormant state ended when period of stress is relieved and worms can return to their normal activity

• also seen in lots of other types of nematode

environmental cues

• food, temp, pheramones - sensed by sensory neurons and integrated to produce behaviour)

• the maount of pheramones a worm detects gives a readout of how many worms are in surrounding environment

• once a worm has developed into a normal L2 it will keep developing into L4

• dauer adjusts the metabolism and morphology

c. elegans in the wild

• have a boom and bust lifesyle

• find food, lays eggs, increased population which results in less food

• ferociuos feeders, not conservative and sop when food is low eneters the dauer state which initiates behaviours to where it will hunt and attempt to find food

• L2 dauer finds new food rich environment which is beneficial for survival and proliferation

• the new environment must have:

  • low population

  • bacteria rich

morphological/metabolic change

• thinner

• arrests germ line development

• shrinkage of muscle, intestine, hyperdermal cells

• encapsulation of worms nose from cuticle

• also closes off the other end of the gut

• thicker cuticle

  • more protective

  • prevents fluid leaving

  • helps the worm locomote

• pharynx constricted and doesnt pup to conserve energy

• faster

physiology of quiescent stage

NORMAL STATE

• bacteria taken in and nutrients distributed to muscle

• uses TCA cycle to generate ATP

• ATP used for growth, development, movement

DAUER

• metabolism slowed down

• increased lipid stoage, conversion of glycogen which is distributed to muscle cells

• no anaerobic metabolism

• reliant on energy stores

signalling pathways

• DAF = dauer forming genes

• genes involves in pheramone production, cillium structure

• diff genes form cilia for detection of sensory cues

• DATD drives the formation into dauers

• DATC are constituatively expressed

DAF 2

• expressed ubiquitously throughout

• this is good as allows for coordination of tissues

• DAF2 gene encodes an insulin receptor which recieves insulin from a sensory neurone —> allows to signal to other cells

• DAF2 is a tyrosine kinase receptor

• number of components in insulin pathway preserved in mammalian signalling pathway

• insulin (when food is around) activates DAF2 —> activates kinase signalling cascade which activates foxo TF

• foxo cant translocate into nucleus which drives reproduction

  • so when insulin is low foxo can move to nucleus and inhibit hormone biosynthesis

  • when insulin is high foxo activates and cant enter nucleus so hormones synthesised as usual