C.elegans Flashcards
(93 cards)
1
Q
how many neurons does a c.elegan have ?
A
302
2
Q
how many glial do c.elegans have?
A
56
3
Q
what is good about the development in worms for study?
A
every cell division in development has been documented and the development of every worm is invariant- makes comparing mutants a lot easier
4
Q
why is a worm good even though it doesn’t have many neurons for nervous system study?
A
- it has 118 morphological classes of neurons
5
Q
is the full connectome of the worm known?
A
yes
6
Q
is every single neuron in the worm characterised?
A
yes- they all have names
7
Q
how many neurons in worms exist in bilateral pairs?
A
around 1/3
8
Q
why is knowing the connectome not that helpful?
A
dont know positive or negative interaction for all etc
9
Q
what are the main embryology techniques in the worm?
A
you can do blastomere ablations and manipulations, can do 4D lineaging
10
Q
what are the main transgenics techniques you can use in the worm?
A
transcriptional and translational reporters of gene expression and protein localisation, over expression/ rescue
11
Q
what are the main genetic approaches that are used in the worms?
A
forward and reverse genetic screens, mutant mapping, genetic dissection of regulatory pathways
12
Q
what are the molecular biology techniques that can be used in the worm?
A
RT-PCR, qPCR, FISH, NGS
13
Q
what is laser ablations?
A
you can kill specific cells in an embryo
14
Q
why would you want to do laser ablations? give an example
A
- laser ablations, you know that Abp cells give rise to certain cells but it is surrounded by Aba and p2- if you ablate these surrounding cells, do you get the same fate or is the cell fate not cell autonomous
15
Q
why would you want to remove a cell from a blastomere?
A
to ask about cell intrinsic properties- when alone do its daughter cells change
16
Q
after isolating a blastomere, what can you do and why would you do this?
A
isolate ABa and put it next to p2 and see if its fate is turned into a Abp- like fate, this would suggest that p2 induces the Abp fate and can change ABa fate
17
Q
how can you use 4d lineaging with GFP reporters?
A
you can label certain cells and then follow how the move
- you can follow expression patterns of genes- follow the lineage and identify in exactly which cells of the lineage this gene is expressed
18
Q
how can you use 4d lineaging with mutants?
A
can follow the lineage through an uncover differences in cell lineages of mutants- are cell lost, do they become something else (similar lineage to another cell)?
19
Q
how can you label all the neutrons in a worm?
A
you can put GFP downstream of a pan neuronal reporter
20
Q
how do you inject DNA into a worm to make a transgenic line?
A
you microinject the DNA contrast into the distal arm of the gonad
21
Q
why would you use a trancriptional reporter? what are the downsides?
A
- monitor geen expression
- identify cis-regulatiory analysis (identifyy enhancer regions)
- not all mRNA is translated
- doesn’t show you localisation within the cell
22
Q
why would you use a translational reporter?
A
introduce a GFP in frame with your gene
- monitor your gene expression
- monitor protein localisation
- cis-regulatory analysis
- cover-expression or mutant rescue
23
Q
how can you use translational reporters to understand where a gene is important?
A
you can produce a trasngeic worm that is a mutant for a gene but has a translational reporter for the gene only expressed in a specific tissue due to the promoter that it is downstream of- this allows you to see when the mutant is rescued dependent on whether the green is required
24
Q
how can you control when a gene is expressed in a worm?
A
you can use a translational reporter in a mutant that is downstream of a heat shock promoter
25
when would you want to use a heat shock promoter?
if you restore the expression in the embryo and you get a rescue and not if you restore late instead, then you know the gene is required n development- can do vice versa
26
how can you make a transgenic construct in c.elegans?
Inject into gonad - form an “array” (mini-chromosome) that is inherited in a mosaic fashion - can be generated in a week
• For mendelian segregation gamma/uv-irradiate to integrate into the genome- generates double strand breaks to let the DNA pop into the DNA
• Can also bombard (gold particles) directly into genome
27
what is a fosmid?
5-100 megabases- large piece of DNA- can contain multiple genes- and recombine GFP via HR into the gene of interest and this will allow you to label a gene when you inject this - they generate more complete genetic patterns of reporter
28
why would you want to make a bi-cistronic fosmid?
so that you can transcriptionally report the expression of a protein in a cell but can also allow the protein to localise to its normal site- you make the GFP be attached to a NLS so that will stay in the nucleus and then after the stop codon of your gene that you are labelling you then put an intercistronic region called SL2- this means that when the gene is transcribed, the protein will do its normal thing and the reporter will go into the nucleus
29
in order to have one construct transcribed but two different proteins, how can you do this?
you can place an intercistronic region between the two proteins- the first will have a stop codon - this has been shown as SL2 but an IRES can be used too
30
how can you use transgenics to do promoter analsys to identify cis-regulatory regions- maybe to see which promoter region drives expression in a specific tissue?
you take the upstream region of the gene- hook it up to GFP and make sure that you get expression in the tissue of interest, then you can induce deletions in the promoter - delete a large bit and see you lose expression, then you know that a TF binding site is in there- then you get smaller and smaller each time until you identify which region is important - then you can find the exact region and the sequence- then you can use this and look whether its a motif that is specific for a certain TF
31
why are transgenic worms good for promoter and cis-regulatory element finding?
-transgenic worms are soda quick and easy to make that you can find an element after making many mutations in the region, very quickly
32
how do you carry out a classic F2 forward screen in worms?
- use EMS- introduces mutations every 1000 approx.
- so either the eggs or the spam in a hermaphrodite will carry the mutation
- single a herm and it will self fert and produce a het F1 which you can then self fert after being plated
- this will produce 25% homo mutants
33
how can you use epistasis analysis to order a pathway in worms?
- you mutants that seem to be involved in a similar pathway- for example growth. You can use double mutants which have opposite phenotypes and the double mutant phenotype will normally tell you the downstream component. If you dont have to opposite mutants you can do a gain of function with a loss of function
34
what types of mutations can you get generally?
- null- no signalling
- loss of function- less signalling
- gain of function- increased signalling
- over expression- increased signalling due to double expression
35
how can you use SNP-mapping to clone mutated genes?
- use single nucleotide polymorphism
- you performa mapping cross to a polymorphic strain and isolate mutant recombinants
- you analyse the recombination rates of SNP-markers to find out whether the the gene is by looking at which markers (with known positions) do not recombine in the mutants
- you select for homo mutant phenotype in F2- where you're gene is you will never have had recombination- in this region you will never get the SNPs from th polymorphic strain - you use cloud map and you find where all the reads are the same - no grey SNPS in this region in any of the F2s
36
how can you do a reverse genetic screen?
use RNAi
37
what is determinate development?
intrinsic, cell-autonomous, coupled to lineal descent of a cell, no regulation following cell loss
38
what is inductive development
indeterminate development, non-cell autonomous, no obvious correlation between lineage and cell fate, regulation following cell loss,
39
what worm technique and data is perfect for looking and inducitve vs autonomous cell fate?
- cell ablation and the invariant lineage
40
how can you test whether the cell lineages that result from ABp are inductive or autonomous?
- you can ablate the other cells in the embryo- if it does what it normally does then it isn't inductive but if not then it will be cell- non -autonomous
- you can isolate them rather tan ablate
41
how can you use mutants to look at lineages?
you can look for mutants that have an altered lineage- you get a daughter cell acting like another daughter cell rather than dying- this suggests there is something intrinsic but need to prove that
42
what is an example of a mutant which suggested there was intrinsic cell identity in worms?
- unc-86
43
what is the principle behind determinate asymmetric segregation?
there is an asymmetric component that is refined one side of the cell- resulted in asymmetric daughter cells
44
how can you determine intrinsic vs inductive signalling using forward genetics?
you can design a screen using the bag of worms technique- the lin-2 mutants- this allows a bag of worms to be formed in the F2- if you had an event which gives you Embryonic lethality, you will just get a bag of eggs- this makes it easy to identify the lethal eggs all in one place. They used this to identify embryos that have symmetrical first cell division (normally one bigger than the other). They found Par genes using this method
45
once they had identified mutants which produced symmetrical division of the first cell cleavage- how did they proceed? what did this show?
- they then mapped and cloned the gene then they stained for it
- they found that par-2 was found on the right and par-6 on the left- show that it is determinant
46
are the par genes conserved?
yes- drosophila- polarity
47
how were cell lineage mutants used to look at inductive vs determinate development?
- they found a lit-1 mutant which is a maternal affect lethal mutant that results anterior to posterior transformations in daughter cell fate: EMS divides to give E and MS but in the lit-1 mutant, E adopts the MS fate- shows that this is likely an intrinsic cell feature
- it was a temperature sensiive allele so they could control when this mutation was exposed- you can control when the mutant is expressed- find it works in 6 binary switches that regulate posterior cell fate in a lineage dependent manner
48
how were experiments on pop-1 carried out and why was this significant?
in pop-1 mutants, E seems to acquire an MS fate (from the EMS cell) this results in extra gut at the expense of the pharynx
- they did antibodies for pop-1 and found that pop-1 protein is asymmetrically segregated during multiple division. this suggests that pop-1 and lit-1 may act together as a global binary switch at each cell division in a determinate manner
- they are both components of the WNT signalling pathway
49
how does the pop-1 work in the cell?
- asymmetric segregation works at every single division after the first 2 divisions- there is a pop-1 binary code- in the decent of these cells you can give the cell a 1 or a 0 depending on whether it inherited pop-1- this gives the cell a history which can influence their history
- in the posterior pop-1 acts together with sys-1 to regulate gene expression and in the anterior pop-1 acts as a repressor of REP- so you can see how different cell fates can arise from this
50
what determines Hox gene expression?
position along the segmental body axis
51
what is mab-5 and where is it expressed?
expressed in the posterior of the worm
52
when you look at the lineage of the ma-5 expressing cells, what do you see and how can you start to investigate this?
when you trace back the lineage you see that mab-5 expressing cells come from many different areas of the worm- so is this to do white lineage (intrinsic) or is it to do with a signalling inductive event in the posterior
53
how did they determine whether the expression of mab-5 in the posterior was an inductive event of autonomous?
- they found a particular cell called M that migrates from the anterior to the posterior
- they blocked migration to the posterior ( to maybe block inductive signal) via suing a cytoskeletal drug
- you then do in situ and you see it is still expressing mab-5
- this suggests that there is not a signal in the posterior- autonomous
- they took a V6 cell which expresses mab-5 and had a mutants where you get lots of copies of V6 in lots of different regions- you have some that are placed in an anterior ectopic position express mab-5 even if in the anterior
54
how can you do inductive vs autonomous experiments on the processes behind ABa cell fate? (3) what did this show?
- you can isolate ABa and put in contact with p2 and see if it takes a Abp like fate which is normally the cell that is in contact
- you can use a glass pipette and cause a swapping of the cells- you see that ABa develops like ABp
- this shows that there is an inductive signal
- use blastomere ablations do remove the ABa cells and the p2 cells from around the ABp and see if it behaves like a ABa cell and you do
55
how was glp-1 found? why was this significant?
it is a mutant in which left-sided blastomeres revert to their right-sides lineal equivalents and vice versa- the same thing happens in MS ablations- this suggests that the MS is a signalling source that establishes the fates between the left and right side- find that these are notch signalling pathway components
- were able to show by using ablation and mutants that MS is releasing a notch ligand and MS switches the fate of the cells of the left sided blastomeres - glp-1 is the notch receptor
- this shows us that before this signalling- there are the same- the inductive signal is required
56
how are the four notch pathways though to work in the c.elegans?
- notch 1 is release from p2 to induce the ABp fate
- the notch signals that distinguish cells during divisions- integrate the differences in pop-1 history and then notch changes the fate in a binary fashion to change depending on the left or the right of these daughter cells and then most cells are derived by the integration of these binary signals
57
how can you look at asymmetry in worms?
can look at the expression patterns in the bilateral pairs and how these changes come about
58
why are worms good to look at asymmetry in?
they have bilateral asymmetrical pairs of nerves
59
how many of the neurons in the worm are bilateral pairs?
198/302
60
what neural pair did they look at?
ASE neurons
61
how did they go about looking to see how the ASE type neuron is specifically from ?
- you find a gene that is expressed in the ASE neurons- ASE markers by using SAGE analysis and comparing the transcripts of the ASE to another bilateral gene pair and finding specific genes
- he did promoter analysis of the ASE specific genes- and chop it down until he found the tiny element that still expresses in the ASE
- he found that each one of his genes that is expressed in the ASE there is a common motif in all of these genes
- called this the ASE motif
- this suggests that there is a TF that regulates all of the genes
62
once you know that a single TF controls the expression of all the genes in the ASE neurons, how do you go about identifying this TF?
- you find the area of the genes expressed in the ASE (that are not pan-neuronal genes) and look to see where the regulatory elements in common may be.
- you can do this my computer sequence analysis but perhaps too larger piece of DNA
- so can do the enhancer element deletion/reporter technique to refine the area that is required for ASE binding
- you can then look for mutants which lose the expression of ASE neurons GFP
- you can then see if all of the genes express this- they do!
- they identified Che-1, they tested the preference binding site of this zinc finger TF and found it matched
63
what is the concept of general neuronal specification in the neurons?
there is one TF which is the 'terminal selector' that then auto regulates itself and induces the expression of all of the genes that regulate the specification of that neuron
64
how can you go about finding how neurons becomes asymmetric in worms?
- took an adult animal and did RNAI against che-1 late- took adult animal and did RNAi against che-1 - see that you lose asymmetry- lose both fates
- this show that che-1 is not just required for symmetrically expressed gene but also the asymmetrically expressed genes
65
what is the difference between the ASE L/R neurons?
the ASEL is involved in sodium detection and the and ASER in chloride
66
what were the two models of ASELR asymmetrical patterning?
1. Che-1 is required to turn on some of the left right symmetric genes and then later it isn't involved and you have specific activator so repressors which maintain the asymmetry
2. in parallel: che-1 turns on a bunch or genes and then we can either co activate these genes just in the left or repress those genes just in the right
67
how can you find the regulatory regions of the left right expressed genes? what did this tell us?
- you use the enhancer investigation technique via making deletions int he promoter of ASE L or R genes - for example they knew that gcy-1 was expressed in right genes so they made mutations in its cis-regulatory region -some of these deletions result in the expression of ASER genes in the ASEL neurons- this show that there is a repressor which prevents the expression of right genes in the left.
68
once you know that Che-1 is required to switch all of the genes on and that activators and repressor are involved in controlling the left right specific expression, how do you go about finding which these Tfs are? how could you do this with another method?
- do a screen looking for mutants that change the asymmetry
- see whether there are two left phenotypes or two right phenotypes
- found 24 different genes
- can do this with RNAi
69
what did they eventually find out about how ASE L R fates are determined?
che-1 turns on all of the genes and then certain factors control a feedback loop which determines either the left or right fate
70
how did they order the newly found genes implicated in the ASELR development?
they used epistasis analysis and created a double mutant: use opposite mutants with known opposite mutant (both lsy-22 and lsy-5 display 2-ASEL phenotype) but know that lsy-6 has double ASER- find they are downstream
- then do the same with die-1 and find that die-1 is epistatic
71
what does it mean " what biases the bi-stable feedback loop?"
this means- what results in one cell expressing ASEL and one expressing ASER pathway?
72
do the ASELR neurons come from asymmetric lineages? how doe know?
yes- you can lineage trace them
73
wha are the two model for how the bias feedback loop is formed?
- at a late stage there isa signal which induces the asymmetry in the ASE cells
- there is something much more dependent on the lineage
74
how do you distiguish between the two feedback loop theories using cell fate changes (genetic)?
- can generate ectopic ASEl/ASER lineages (cell ablation or genetic)
- remove all possible signalling cells (ablation)
- they used genetic tricks involving the notch signalling pathway to generate different lineages- when you inhibit the third notch 3 signal, you get ectopic ASER on the left hand side - if it is inductive the cell will express a left fate but if not then it will be indcutive- to do with lineage
- you can also inhibit the first notch signal and you get two ectopic left lineages- one of the right and one on the left in th posterior- to they still all express left markers?
- find it is all determined by the lineage
75
how do you distiguish between the two feedback loop theories using cell fate changes (ablation?
you can ablate all cells but the ABa or ABP and you see that you still get a left ASEL from the ABa and a right from the ABp
76
what told them the ASEL vs R signal was made very early?
the notch 1 signal perturbaion caused 3 left fates causes three ABa to form and another mutant that causes two ABp to form causes 3 ASER
- there is an early signal which raises the cell
- something regulates a very brief signal of lsy-6 expression which regulates the chromatin structure and opens up the lay-6 chromatin in the left- but not in the right
77
how was the MCM cells first noticed and why were they interesting?
they did a nerve stain for PDF-1 and noticed this cell projecting to the brain, they noticed that this supposed neuron shouldn't have been there as there aren't supposed to be any neurons in that region
78
what is the MCM ?
mystery cell of the male
79
what was the first question they asked to find out what the MCM was?
- they asked if the MCM was a neuron- they used a pan neuronal marker
- they found that they exist in a pair and they become present at the late L4 stage of development in the male only- not in hermaphrodite
- they then took an entire set of different neuronal proteins and stained the to see if still these were present in the MCM - synapse secretion molecules
- they then used Electron microscope to look at the neuron and take seriel sections to look for neuron traits- can see presynaptic densities and neuron peptide release: dense core vesicles
they then used the ECM to trace where these neurons were connecting to
80
how did they fin the connections to and from the MCM? what did they find from this?
- they used EM- they found that they were connected to male specific cells- reidve input from male tail sensory neurons- they are responsible for the male being able to detect hermaprhodites - they also saw it was connected to cells related to learning behaviours linked to finding a mate- exploratory behaviour: if you put a male on food and there are no females then the male will leave the food and go look for females but if you put it on food and there is mate then it won't leave- another behaviour - starve worms on a plate and move them to to a plate with high salt then he worms will crawl towards the salt. but you can condition them to learn that salt is associated with no food and can repel them. males ca undergo sexual conditioning: starvation and salt- it trumps the learning of salt of starvtion and males worms will be stretched to salt because there are mates there
81
what are the three learning behaviours of the worm
if you put a male on food and there are no females then the male will leave the food and go look for females but if you put it on food and there is mate then it won't leave- another behaviour - starve worms on a plate and move them to to a plate with high salt then he worms will crawl towards the salt. but you can condition them to learn that salt is associated with no food and can repel them. males ca undergo sexual conditioning: starvation and salt- it trumps the learning of salt of starvtion and males worms will be stretched to salt because there are mates there
82
how do they show that MCMs are required for sexual conditioning?
they ablate the MCMs then they can't do the sexual conditioning- they can sense the hermaphrodites and salt but they dont respond
83
why does it make sense that MCMs dont appear until L4- sexual maturation?
because worms won't care about finding hermaphrodites until they are sexually mature
84
how did they go about finding where the MCMs come from in terms of lineage what did they find
they used a cell division marker to look at the cell division during sexual matuirty- they see a lone pair of cells dividing in the head of the male and not in the head of the hermaphoridte - they show by labelling the non fluorescent and the fluoresce maker slide that these are the the AMso cells - one of the many glial cells
85
what is the AMso?
glial cell-
86
how did they observe the cell division int he AMso cell? what did they see?
they labelled the cell in green and the histones in red and could see the chromosome repllicating and the cell division. - see two cells- one as the MCM - see it starting to express rab-4 after a while- one of the cells from the cell division-
87
what was interesting about the AMso cell in terms of gender?
it only undergoes this division to make the MCM in males- dont see the histones increase in concentration in the hermaphrodites
88
how did they go about looking why the AMso only gives rise to the MCM in males and not hermaphrodite? what did they find?
- they asked whether it was down to genetics- they used a trick in which they converted the AMso cell to a female cell in males - drive a feminisation factor intuit cell- find that it does not divide and if you do the opposite in the male- you see that if you masculanise AMso cell in the herm then you see that it divides.
89
why was finding that the AMso could turn into a neuron interesting?
that a glial cell could transdifferentiate into a neuron
90
how did they test whether the glial cell was fully differentiation? what did they find?
its a socket cell (the AMso) which has a characterise morphology- makes a hole in the skin to allow access of the sensory neurones to the outside world - does it look like this in a male? yes it retains the morphology even when it divides.
- then look at glial specific markers and see at the L3 stage it is expressing glial markers - it does when AMso and early on the MCM expressed some of these makrkers - this suggested that it may be a differentiation
91
what applications are there of glial ells turning into neurons?
Waddington’s epigenetic landscape model. a | In normal development,
• Studying human development (organoids) th
a pluripotent cell (which is represented by a marble at the top of the hill) ‘rolls’ down a landscape that segregates into different groves on the slope. Depending on in which
ty in sw
• Stem cell therapies and cell replacement therapies
groove the marble falls, the cell acquires a distinct tissue-specific fate. b | A differentiated cell, which is reprogrammed to pluripotency, is symbolized by a marble rolling from the
(e.g in vitro neuron production)
bottom of the hill back to the top. From there, it can be redifferentiated into another de
somatic cell type. c | During direct conversion, a tissue-specific cell directly converts into • In vivo stimulation of stem cells differentiation and
m in im ca an
a related tissue-specific cell (symbolized by a marble ‘jumping’ over a low hill) or into a
transdifferentiation
cell type of another germ layer (depicted
92
what types of animals have neural crest cells?
vertebrates
93
how do you insert a fosmid into a genome of the worm?
you inject into the gonad and then expose to gamma irradiation
