Adaptive Physiology of Caenorhabditis Elegans Flashcards
1
Q
Environmental cues in C. elegans
A
Food and population.
2
Q
C. elegans
A
A free-living invertebrate nematode in the ecdysozoa group, they are roundworms with non-segmented bodies found in rotting fruit.
3
Q
A free-living invertebrate nematode in the ecdysozoa group, they are roundworms with non-segmented bodies found in rotting fruit.
A
Sydney Brenner.
4
Q
First studied C. elegans strain
A
N2 Bristol strain.
5
Q
How are C. elegans cultured?
A
On agar plated with NGM maintained at 20C with the food source of OP50 bacteria, large mixed populations with passage between plates possible and can be frozen to -80C for stock maintenance.
6
Q
C. elegans sexual forms
A
Male (XO) and hermaphrodites (XX).
7
Q
Hermaphrodite C. elegans
A
Predominant sexual form, can reproduce asexually with ovaries, vulva, anus and sperm in spermatheca.
8
Q
Male C. elegans
A
Have testis, seminal vesicle, sperm, vas deferens, cloaca, rays, spicule and fan.
9
Q
General anatomy of C. elegans
A
1mm in length, key organs include pharynx, reproductive system, intestine and defecation and they have a cylindrical body shape with several muscle groups that run the length of the worm.
10
Q
Pharyngeal muscle
A
Located in the head and neck for food.
11
Q
Body wall muscle
A
Runs length of body, controlling locomotion and motility.
12
Q
Head and neck muscle
A
Direct the worm.
13
Q
Vulval muscle
A
Involved in egg laying.
14
Q
What muscular groups are present?
A
Pharyngeal, head and neck, body wall, vulval, anal and intestinal.
15
Q
Key features of C. elegans nervous system
A
302 neurons, nerve ring (head with high density of cell bodies) and nerve cords (ventral + dorsal), has a somatic and pharyngeal nervous system.
16
Q
C. elegans neurons
A
Motoneurones, interneurones and sensory neurons that are typically polymodal.
17
Q
Somatic nervous system
A
282 neurons.
18
Q
Pharyngeal nervous system
A
20 neurons.
19
Q
Amphids
A
Sensory neurons in the head that have ciliated endings exposed to environment.
20
Q
Phasmids
A
Posterior sensory neurons.
21
Q
Function of interneurons
A
Integration of inputs.
22
Q
Sensory neurons
A
Amphidial neurons detect external cues from environment with 12 neurons bilaterally paired.
23
Q
Amphidial neurons detect external cues from environment with 12 neurons bilaterally paired.
A
Can directly synapse onto neural cells.
24
Q
What neurotransmitters do C. elegans have?
A
Cholinergic (90-98), GABA (26), Glutamine (72+), Serotonin (11), Tyramine (4), Octopamine (2) and Dopamine (8).
25
C. elegans genome size
100MB, has 5 pairs of chromosomes.
26
Forward genetic screen
Isolation of mutants that show differences in phenotype for the process of interest and then find the associated genes.
27
Backward genetic screen
Mutate a sequence in a known gene and then observe difference in phenotype.
28
Microinjection
Transform worm using DNA engineered in a tube.
29
CRISPR-Cas9
Edit genomic DNA of worms.
30
C. elegans nomenclature
Gene in lower case, allele in brackets and protein in full caps.
31
Technologies for study of worm physiology
Visual markers (gene expression), manual observation of behaviour and laser ablation of neurons.
32
C. elegans muscle
8 muscle cell types, the cells surround a hollow tube bacteria are moved through.
33
Potential muscle orientations
Radial and longitudinal.
34
What neuron is vital for pharynx pumping?
M4 motor neuron, ablation of all other neurons allows pumping.
35
M4 motor neuron function
Performs isthmus peristalsis.
36
What are MC and M4 vital for?
Pharyngeal feeding.
37
What is M3 used for?
Rate of pumping.
38
3 regions of the C. elegans pharynx
Corpus (pro and meta), isthmus and terminal bulb (contains grinder).
39
What do C. elegans nose and mouth lead to?
Pharyngeal lumen.
40
Bacteria movement in C. elegans
Opening of the lumen pulls bacteria suspended in liquid in, through the isthmus into the grinder.
41
What does C. elegans coordinate?
Pumping and peristalsis.
42
How does the C. elegans suck in bacteria?
Contracting muscle cells that surround lumen opening it in the corpus and anterior isthmus and a co-ordinated opening of terminal bulb region.
43
What happens after the bacteria has been sucked in?
It traps bacteria in the lumen and expels liquid with peristalsis then occurring with contraction and relaxing moving bolus down to the grinder.
44
Albertson and Thomson
Used a microscopy approach to construct a map of the C. elegans, discovered that it has motor neurons, interneurons and polymodal neurons with 20 pharyngeal neurons, some include cell pairs.
45
What coordinates pharynx muscle cells?
Pharyngeal nervous system.
46
Longitudinal muscle function
Move bacteria through pharynx.
47
Radial muscle function
Crush/concentrate bacteria.
48
Unc-13
Protein required for vesicle release at presynaptic terminal, when it is knocked out there is reduced pharyngeal activity and body thrashing.
49
Normal pharyngeal pump per minute
211 (+/-4) vs unc13 knockout of 69 (+/-5).
50
Visual Reporters
Assign neurotransmitter identity to certain neurons.
51
eat-4
Loads glutamate into vesicles.
52
Visual report of eat-4
Take regulatory sequence of eat-4 and fuse it to a reporter gene, then microinject it into worms and anywhere the reporter gene is found shows where eat-4 is expressed.
53
What neurotransmitter is eat-4 linked with?
Glutamate (M3).
54
Corpus motor neurons
M1, MC (both cholinergic) and M3 (glutamatergic).
55
Isthmus motor neurons
M2, M4 (both cholinergic) and NSM (serotonergic).
56
Terminal bulb motor neurons
M5 (cholinergic).
57
RIP neuron
Connects pharynx nervous system to somatic nervous system.
58
How is pharyngeal activity quantified?
Pumps/minute with 1 grinder movement (closed-open-closed) is 1 pump with pump rate can be counted in individual worms in a group or moved to a separate plate.
59
Buccal cavity
Contains cilia that are structurally part of sensory neurons.
60
ADF Action
Sensory neuron that can detect cues from bacteria and surrounding environment, releasing serotonin that is picked up by SER7 receptor on MC motor neuron, increasing feeding rate and cholinergic transmission to pharyngeal muscles.
61
SER-7
A serotonin receptor on MC motor neurons.
62
What activates pharyngeal pumping at the cellular level?
A 5-HT/Ach driven circuit, when 5-HT binds SER-7 Gs akoha signalling is activated which releases Ach.
63
What are tph-1 mutants?
Cannot convert tryptophan into 5-HT so cannot activate receptor signalling.
64
What is seen in tph-1 mutants?
Less pharyngeal pumping
65
EAT-2 receptor
Found on pharyngeal muscle and are activated by Ach causing activation of contraction/relax cycle.
66
Pumping rate in EAT-2 mutants
1/5 normal.
67
What happens when food source is removed?
Rapid and dramatic reduction in pumping, rapidly reversed when food is re-introduced.
68
Glutamate in an off-food setting
Modulates timing of pharyngeal relaxation in corpus and promotes rapid relaxation of pharyngeal muscle following contraction.
69
AVR-15
Glutamate gated ion channel that allows glutamate to enter muscle cell, pm4.
70
What does eat-4 encode?
Vesicle glutamate transporter.
71
What happens to eat-4 mutants?
They have a faster recovery and lower initial drop in pumping rate, due to the inhibitory signal being weaker.
72
Hermaphroditic reproductive system
Produces mature gametes, a necessary environment for fertilisation and egg laying and it can produce 300 embryos by fertilising oocytes with self sperm over its life.
73
3 parts of C. elegans reproductive system
Germ line (mitotic cells), gonadal sheath and uterus/egg laying apparatus.
74
C. elegans oocyte
Mature in proximal gonad and then pass into the spermatheca where they are fertilised and move into uterus.
75
Nervous control of C. elegans reproductive system
Control uterine and vulval muscle cells allowing for control of egg laying from vulva, HSN neurons are vital for coordination.
76
Laser ablation of HSN neurons
Causes egg laying defects, if a single HSN is ablated there is little effects suggesting a certain level of autonomous activation.
77
HSN neurons
Exclusive to hermaphrodites, linked to function of neurons in egg laying.
78
Neurophysiology of egg laying
Egg laying is a combined circuit between neurons and muscles with coordinated signalling between them leading to changes in muscle activity.
79
VM1
Opens vulva.
80
VM2
Opens uterus.
81
UM1/UM2
Squeezes uterus.
82
How do C. elegans lay their eggs?
In bursts.
83
What can modulate egg laying?
Mechanical stimulation, salt concentrations and food.
84
How can food be seen to modulate egg laying?
In absence of bacteria, hermaphrodites retain eggs making them appear bloated and once they are placed in the presence of bacteria they will lay eggs.
85
Imipramine
Blocks serotonin reuptake, maintaining activity.
86
Effect of serotonin on egg laying
In the absence of food, applied serotonin increased the number of eggs laid showing it is vital for controlling it.
87
What does serotonin drive in egg laying?
The transition from inactive to the active state.
88
First neuropeptides discovered in mammals
Vasopressin and oxytocin.
89
How many neuropeptide genes does C. elegans have?
113 genes, encoding 250 peptides.
90
3 families of C. elegans neuropeptides
Insulin-like peptides, FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides and Non-insulin and non-FMRFamide-related neuropeptides.
91
Neuropeptide origin
Derived from larger precursor molecules, typically cut down to 4-20 AA sequences.
92
Neuropeptide function
Neuromodulatory function at the synapse, can function as hormones, signal via GPCR and they coordinate locomotion, ethanol response and social behaviour.
93
Amino acid transmission
Very fast with fast vesicle recycling and nanometers of distance travelled to ionotropic receptors.
94
Neuropeptide transmission
Slow vesicle recycling with local diffusion over micrometers to reach GPCR.
95
What was found in forward genetic screen for egg laying mutants?
145 egg laying defects (egl) such as egl21 (carboxypeptidase) and egl3 (proprotein convertase)- both neuropeptides, showing the role they play.
96
flp-1
FMRF-amide like peptide was first neuropeptide extensively studied, it was identified based on analysis of genome sequencing data
97
What happens in flp-1 deletions?
Mutants still lay eggs that are clustered into active and inactive phases, but inactive phases are much longer.
98
flp-1 function
Modulates egg laying and is responsible for enhancing the egg-laying response to serotonin.
99
AIY and AIA
Interneurons responsible for integration.
100
flp-1 mutants
Low egg laying rate on food with the mutants not able to adapt their egg laying rate when off food.
101
Issue with flp-1 implication in egg laying control
It is not expressed in the circuitry controlling egg laying.
102
egl4 mutants
Egg laying phenotype resembling HSN ablated worms so have increased inactive phases and they also have a reduced egg laying response to serotonin.
103
egl4 function
It encodes a cGMP-dependent protein kinase that is Ser/Thr specific kinases which a role in neuronal plasticity and regulation of gene transcription.
104
EGL-4
Translocates to nucleus to adapt response of sensory neurons to odour.
105
Where is egl4 expressed?
Sensory neurons and interneurons downstream of sensory neurons.
106
How long is C. elegans embryonic development?
800 minutes.
107
Larval stages
4 stages, L1-L4.
108
How many days from egg to adult?
3 days at 20C.
109
What is shed at each larval molt?
Outer cuticle.
110
What differentiates stages?
Vulva and size.
111
L1 arrest
If there is little food for hatchlings, they do not initiate post-embryonic development and enter L1 arrest as a starvation response, can survive for 2-3 weeks with no food.
112
Why is post-embryonic development not triggered in L1 arrest?
Not enough energy resources or nutrients available.
113
What stops L1 arrest?
Eating which triggers cell division to occur and kickstarting post-embryonic development.
114
Late L4 development
If starved they will still become adults, but will enter a reproductive diapause and stop embryo production with prolonged starvation shrinking germline with programmed cell death reducing germ cell nuclei but still retains germ cell totipotency.
115
Dauer
Alternative L2 stage when conditions are unfavourable and can exit to L4 when conditions improve where they molt and shed dauer cuticle allowing them to feed and develop into adults.
116
Effect of dauer stage
Time spent has no impact on pattern or sequence of cell division in post-dauer development with no effect on adult lifespan.
117
Cassada and Russel
Found dauer is very resistant to harsh environmental conditions.
118
What can dauer be considered a type of?
Quiescence.
119
What is quiescence?
A dormant state where metabolism and activity are slowed in response to environmental stress.
120
Environmental triggers for dauer formation
Low food, high temperature and high population.
121
What happens once worm has passed L2?
It cannot go back and form a dauer, but can alter development at L4.
122
C. elegans lifecycle in the wild
Boom and bust with colonisation of a food-rich environment causing boom and as the food source decreases, dauer stage is entered as they try to find new sources of food.
123
What do dauers allow for?
C. elegans to propagate and populate a wider area.
124
Dauer anatomy
Thicker and more resilient cuticle that fills the mouth blocking alimentary tract access stopping the worm feeding, along with pharynx constriction so no pumping occurs, preserving energy.
125
What do embryo and L1 larva do in food presence?
Use the glyoxylate cycle to generate carbs from lipid sources.
126
What do L2 and later larva do in food presence?
Shift to aerobic respiration using the Kreb's cycle.
127
L3 larva in food presence
Eat food, distribute it to respiration and TCA cycle that is used for growth, development and locomotion.
128
Dauer metabolism
Slowed down with no aerobic shift, increased lipid stores and conversion to glycogen which is distributed to muscles for energy to perform locomotion.
129
daf genes
Have different functions but involved in convergent pathways essential for survival.
130
How were daf genes identified?
Forward and reverse genetic approaches
131
Daf-d
Responsible for cilia structure and daf3, daf5, daf12 and daf16.
132
daf-2
C. elegans insulin receptor.
133
Sensory neurons that express daf-2
Act as environmental sensors modulating global dauer stage program.
134
DAF-2 activation
Dimer signal molecule binds to RTK, phosphorylating the receptor's Tyr residues, increasing kinase activity and activating it providing a docking site for signalling molecules to bind pTyr allowing signalling into cell.
135
What signalling molecules associated with DAF-2?
PTEN, PDK-1 and ACT-1/2.
136
C. elegans reproductive state induction
Insulin-like peptides activate DAF-2 causing a kinase cascade producing PIP3 and activating SGK and AKT1+2 which phosphorylate DAF-16 so it is retained in cytoplasm so hormone biosynthesis is retained reproductive programs are kept on.
137
C. elegans dauer induction
Low insulin-like peptide levels so no PIP3 produced or SGK and AKT1+2 activated, so no DAF-16 phosphorylation allowing it to enter nucleus and inhibit hormone biosynthesis, activating dauer programs.
138
What activates EAT-2?
Acetylcholine.
139
What laser ablation can increase inactive period?
Ablation of HSN.
