1
Q
is the eye part of the brain?
A
yes- it is part of the central nervous system
2
Q
what is the most highly conserved part of the nervous system?
A
the brain
3
Q
is the fish eye similar to the human eye? why is the zebrafish better than a mouse for this study
A
ys- it has good visual acuity, colour vision. The mouse isn’t good because the mouse has poor vision - nocturnal animal
4
Q
why are zebrafish a good fish organism ?
A
they are very tough and easy to look after
5
Q
what are the main reasons why zebrafish are used a model system?
A
- vetrebrates- share the body plan
- they have rapid development - day or so development to swimming from egg
- available all year round
6
Q
why are amphibians good for vert model organisms? why are they not?
A
- used to find organiser
- good for misimpression etc- injecting RNA etc
- they are weak at genetic studies- can do it but it is hard bcause they have long life cycles- generation times, tetraploid
7
Q
why are birds good vert model organism? why not?
A
not good for genetic analysis- egg gets in the way- very hard
- good for classical genetics
8
Q
why are mice a good vert model system?
A
they are extremely good for genetics- can do very elegant studies.
- a downfall is that they are bad to study early development because the little mouse is inside the mother
- the mouse is large too so hard to observe development
- optically transparent- can see every single cell in the fish
- excellent genetics- can inject RNA MOs and make transgenics
- good embryology - good stages
- there is a great deal of conservation
9
Q
what are the 4 main processes in zebrafish development?
A
epiboly, involution, convergence and extension movements
10
Q
is the yolk independent from the embryo in zebrafish, why is this significant?
A
the cells do not contain yolk- develop on top of the yolk so the embryo is basically transparent
11
Q
how can you observe the types of cell movements that occur in the embryo during different stages ind development ?
A
- you can actually use a microscope and zoom in to follow different cell divisions just with the eye
- light sheet imaging: allow you to very rapidly image many points in space at almost the same time: the light shines through the sample and illuminates and things are in focus at one time. You can then use a nuclear label (such as DAPI) within an embryo and then follow them over time in the entire embryo. Then you can I’ve each dot an identity and code for different parameters: direction, speed etc.
12
Q
how can you use light sheet microscopy and computer programmes to look at different cell characteristics during development.
A
: the light shines through the sample and illuminates and things are in focus at one time. You can then use a nuclear label (such as DAPI) within an embryo and then follow them over time in the entire embryo. Then you can I’ve each dot an identity and code for different parameters: direction, speed etc. - you can do this for an entire embryo
- you can also do this in a reverse way and home in on nucleus of cells of one tissue type- for example in the ear and then rewinds and look where these cells came from
- you can then compare this to mutants and see how development changes
13
Q
how can you use microscopy to look at the changes in proliferation in the embryo?
A
- use sheet microscopy for the entire embryo
- you can label the nuclei for different phases of the cel cycle- green for cell in proliferative and red for post mitotic- do this by tagging cell cycle proteins with different colours by producing transgenic translational reporter constructs and injected- because these proteins are degraded at the different cycle stages
- you can then see that as development more cell types become post mitotic and the order in which tissues that do this
- you can identify regions where proliferation is ongoing- in the stem cell sight
14
Q
how can mutations to do with the optic chiasm and some brain defects be viewed
A
can be viewed just b under the microscope- can object dyes into the neurons to stain them
15
Q
what type of mutation was found by using a stain of the brain connections?
A
pax2 who causes defects in optic chiasm
16
Q
what stain can be used to view chiasm?
A
Bodipy-ceramide labeling
17
Q
once you have found pax2 involved in commissar formation, how can you find other genes that ma be involved in the same pathways
A
you can do s mutant screen for other mutants with defects for the commissure and find these genes which ay also be involved - can cone them and test if in the same pathway
18
Q
what is the name of a screen that looks for mutations with phenotypes on a background?
A
an enhancer screen
19
Q
what can enhancer screens be used for?
A
finding components of a pathway
20
Q
why are fish good for enhancer screens?
A
cause when you use an enhancer screen you want a homo for the background and a homo for the new mutation - this is 1/4 x 1/4= 1/16 of the offspring - you would have to do this with several breedings with mice etc- bt not for fish- or for dros because the have large clutches
21
Q
on what background did a WNT signalling component become apparent?
A
on a TCF3a on a background
22
Q
how do you carry out genetic mapping?
A
- classical genetic mapping/ positional cloning: the basic idea is that you try to map which chromosome and on which chromosme the mutation is on- it assess how close the mutation is to a DNA marker that you know the position of- ou can use brittle and red eye and if these are close togetherr then you will normally get parental phenotypes and very rarely would you get recombination. But if they were on separate chromosomes then you get recombination . you can then work out the recomb rate and work out how far different markers are from each other
- now ou can sequence the genomes and look for regions of the genome where all the markers are homozygous- no recombination between matrnral and patternal- homozygous mapping
23
Q
what are SSLPs?
A
- (simple sequence length polymorphisms) little stretches of repetitive DNA that can vary quite a lot- can PCR p this bit of sequence and you will see variation
24
Q
what are CA repeats
A
organaisms have different lengths of CA repeats in their DNA
25
how can you use genetic markers for mapping?
- you first sequence sing PCR different areas in the genome of your two parents, then you can look in the offspring how much variation there is between these areas- has there been recombination or not
26
what is homozygosity mapping?
- sequencing the genomes of F2s that all express the mutation
- then looking for areas of the genome with high homozygosity- that is areas where there has been no recombination between maternal and paternal chromosome tat is common across all of the F2s.
- this gives you an idea hat the alley lies in this region then you can find candidate genes within this region and then you can use RNAi or Mo etc depending on the organism- maybe even crispr- to see if this mimics the phenotype
27
once you have a lift of candidate genes, how do you go about decided on your candidate?
- where is our gene expressed, is it where we would think it was
- can test a large number of mutants and see if in all of these this allele is present
- want to phenocopy the mutant- antisense or you could generate a new allele in the same gene and ask if it produces the same phenotype
- sequence the gene and look for mutation - is it a missense- change in protein structure, or is it a stop codon
- if we put back a wild type version of the gene, does it rescue it?
28
how can/ you knock out a gene function
- create an antisense oligonucleotide targeted against the AUG are of a given gene- morphosinos block translation and can block splicing
- you can do a ubiquitous specific knock down by injected into the entire embryo earl in development or a mosaic knock down by precise injection of antisense
29
how can you do a gain of function technique with zebrafish?
you can inject RNA or DNA ubiqitously and in a mosiac pattern
30
what is a high throughput` laborious method of reverse genetics in zebrafish?
you mutinies a male fish and create a collection of thousands of fish carrying different mutations- you then calculate that within this pool of fish you have a mutation for every gene in this pool of fish- have to do 5-1000 fish, then you PCR around your gene of interest for very fish and identify which of the sperm samples has a mutation in your gene of interest and use in vitro fert to generate a line of fish
31
what are the three main reverse genetics tools?
zinc-fingers, TALENs and CRISPr.
32
what is a method of looking at the actions of mutant cells in an organism?
take labelled coloured mutant cells from one and inject into another coloured labeled embryo
33
why would you want o insert a host that was one colour with the cells from a donor of a different coloured organism?
morphology of individual cells, what do mutant cells do when they are surrounded by wild type cells, what do cancer cells do , during embryology which cells of the transplant contribute to what
34
how can fish be used in drugs?
fish is smaller enough to be put into a 96 well plate- can do drug screening on entire embryo- screen for interesting phenotype- drugs that inhibit melanoma.
35
why is doing drug screens on fish good?
* development
* timing of action
* dose control
* epistasis-like experiments with mutants • disrupt all copies of given gene or family
36
ow can you use transgenics in fish?
- you can take the enhancer from a gene that is in specific cell-type- hook this up to a report gene, inject into the fish and it will be incorporated and will be incorporated into the fish
- this can be used to look at the movement of different cell types to an wound site because the little fish is see through and you can clip a fin and watch the cells of different transgenic fish which express labelled different immune cells
37
what can single cel electroporation allow you to do?
expression of DNA in indiividua cells in the intact brain- label one cell with reporter gene construct
38
what types of behaviours can be viewed in fish and how can you measure brain activity?
- hunting, escape, social, tracking , etc
| - use a calcium fluoresce indicator in the brain and look at what neurons are firing
39
how does the eye develop?
- the neural plate folds and simultaneously there is an out pocketing of neural tissue on the two sides which will form the eyes
- the neural ectoderm contacts the surface ectoderm , this forms the covering of the eye: the lens and cornea and the rest of the retina (neural and pigmented) come from the brain tissue.
eventually the connection between the eye and the Brain degenerates into the optic nerve but during development it is called the optic stalk before it differentiates
40
within the neural plate, where can the cells that will form the eyes be found?
?they will be found as a single field of cells in the anterior of the neural plate
41
do the eyes come from two separate fields of cells or one
- just one- in the anterior neural plate- called the eye field
42
what results if ou can't divide the eye field
cyclopia
43
how can you view the development of the eye field into two separate fields?
you can engineer a transgenic embryo expressing a reporter downstream of a romper for a gene just expressed in the eye field
44
what is the process of the eye field splitting called?
envagination
45
after optic vesicle evagination, what happens?
patterning and invagination
46
how can you use fish models to look at eye development ?
you can make fish models and then her look at the pathways involved or screen for drugs that ameliorate or do enhancer suppressor screens etc.
47
what 4 types of eye disorder types can you replicate in fish models?
eye specification
optic vesicle evagination
patterning and invagination of the optic cup
cell cycle exit, neurogenesis, vasculogenesis
48
ow can you find out which areas of the neural plate in the late gastrula contribute to different parts of the embryo?
you can label the cells inject dye and then see where they end up
49
what is a marker of the eye field?
rx3
50
ho can you mark the eye field?
- in situ
| - reporter gene - transcriptional
51
How has the WNT signalling pathway been implicated in eye formation and how was this shown?
it is involved in the development process: mastermind mutation is in axin1 and tcf3a mutant both result in lack of eyes - both the inhibition of activation and activation of pathway can result in dyfunctional eye development- the eyes are being turned into other territories of the neural plate this is because the WNT pathway is involved in patterning the AP axis
52
what did they find when they outcrossed a tcf3a mutant?
- they found that when they cleaned up the background they got their eye back his suggested the mutation was genetically sensitised.
53
when they found that the cf3a was a sensitised mutant what did the suspect might be happening and how did they test this?
they knew that tcf3a was part of a large family of transcription factors and so looked to see whether they made a tcf3a and tcf3b mutant, did this cause a full mutant? yes it did!
54
after finding that a tcf3a and tcf3b double mutants gave a full phenotype, what did they postulate in relation to how a tcf3a mutation alone could exist without giving a phenotype et did when with tcf3b and how did they test this?
they proposed that maybe when tcf3a was mutated, this resulted in a doubling of the tcf3b expression to compensate- but this was found to not be the cause- when they measured the transcript levels perhaps by using an RNA assay- they found that only the tcf3a levels were reduced
55
once they found that the tcf3b wasn't compensating for tcf3a, how did they find that rx3 was in fact reduced too in these mutants ?
they were doing controls and found that rx3 was reduced
| in transcription levels
56
when you knock out or down rx3 in mice, frogs and mouse, what do you get?
no eyes
57
when they looked at tcf3a mutants at eye development, what was seen?
they found that t the early stages there was no rx3 at all so were initially showing a really strong phenotypes but then later (a day) at day 3, there was no phenotype. at a day the eye was smaller than normal but catching up.
58
how did they test how well the ey as at compensating for a damaged eye early on?
they ablated around a 2 thirds of the after 12 hours and at day 4 it was basically regenerated
59
following the studies on Tcf3a, what did they find out about eye development?
it is very robust- one or a few smaller preteritions can be compensated for but when threes more than a few then it can't be compensated
60
how did they test whether the tcf3a function in eye field specification was cel autonomous or induced?
made a genetic osaic- they made a green fluo fly which were mutant for tcf3a and the red cells were normal in the eye field. They showed that the green cells will not turn on rx3 even when they are close to wild type cells tells us the cf3a is required cell autonomoulsy to promote the formation of eye tissue
61
how did they create and view the mosaics for TCF3a and wild type eye fields?
they inserted mutant eye fields cells from a TCF3a mutant which were expressing GFP, into a wild type eye field and then waited for a bit and then in situ to show the expression of rx3 and compared this to the cells in the area that were expressing GFP and they found that these did not overlap at all- TCF3A could not be induced
62
what genes are missing from TCF3a pathway?
they know that it is a repressor so it must be repressing something to stimulate the eye field and rx3
63
how can they try to uncover which genes are compensating for the TCF3a mutant? what did they find when they did this?>
they can carry out a enhancer screen which will be found in 1/16 offspring from the F3s. they found hesx1.
64
ow can you follow the movement of cells in the ee field during development?
create a transgenic zebrafish which expresses GFP downstream of an rx3 promoter
65
what do you find when you follow the fate of rx3 expressing cells of the eye field?
there is very little mixing with other surrounding brain tissue- they responds in a very coherent way within the neural epithelium within the neural plate they behave as an entity and dont mix
66
when they were trying to determine whether the eye field cells mixed with other brain tissue how did they determine this?
they looked to see if the rx3 expressed cells mixed and then looked to see if emx3 (label of telencephalon cells which surround the eye field) were moving to the eye field- no for both
67
at pathway is implicated in keeping cells together?
the eph ephrin pathway
68
how did they determine whether the eph/ephrin pathway was involved in eye field coherence?
they looked at the expression of the eph ephrin genes (eph4) that s expression is disturbed in a rrx3 mutant- it is normally expressed adjacent to the eye below it - but it is dispersed within the eye field in the rx3 mutants
69
once they had found that the eph4 pathway may be involved, how did they test this further?
they used gene reporters to label where the eph's were expressed and their ligands- they found the they were complimentary- the efn ligand was expressed in the eye field and the eph4a was expressed in the telencephalon surrounding
70
how does the efn in the eye field and the Eph in the telencephalon work? how did they prove this?
there were examples of the EFN binding to the EPH and there being a two way signal in which they are repelled from each other
- they had a control- when they mess around the EPH/Ephrin pathway, the specification is the same- it is not a patterning defect
- they then used in in situ and labelled the eye field movements by labelling EFN3B and showed that when you use EFN MO or EPH MO, you get integration of the the eye tissue into the brain and it is generally disrupted
71
how did they show that cell segregation of EPHB4 and EFNB2 cells is an active process?
- they labelled rx3 cells using a gene reporter and then either used labelled EPHB4 cells with RRP or EFNB2 cells with RFP, that when you transplanted either of these and intermingled the cell types into he eye field, initially they can intermingle but over time- the innapropriate cells are either sucked in or pushed out in the late gastrula- can do this in one organism with a double reporter i think
72
how did they identify that there were two different cell populations in the eye field during optic vesicle formation?
- they labelled the membrane with RFP and found that there seemed to be nice polarise mesenchyme around the outside and less so cells in the middle
73
what did they notice about the core cells and how?
they labelled the cells with membrane marker and then tracked them over time - the found that they seemed to disappear
74
how do the core cells of there eye field disappear?
they undergo a mesenchymal to epithelial transition
75
how did they think that the core cells of the eye field/ were becoming epithelial
they thought they were gaining polarity by interacting with the iECM on one side of the cell
76
how did they label the ECM during the onset of morphogenesis in the eye? what did they find?
they labelled laminin- they found that matrix begins to build up and me laid down as the eye field starts undergo morphogenesis of the optic vesicle
77
how did they interrupt the ECM of the eye field during the formation of the optic vesicle in the eye field?
they used an MO against lamin- they see there are patches of apical markers all over the cells in the eye field- even in the basal surface so these cells are orientated in the wrong direction where as normally there is apical protein localised at the apical side of the epithelium of the eye field this tells us the basal lamina is giving ells sh cues required for morphogenesis
. But even without this there is still some morphogenes and there is still polarisation and outgrowth of the eyes
78
how does the eye field undergo morphogenesis and become two different eye fields?
1) As neurulation starts, the eye primordium converges less than other NP domains"
"
"
2) Precocious assembly of the ECM around the eye field promotes elongation and organisation of marginal cells"
"
3) The cells not in contact with the ECM end up in the core of the eye field and become integrated in the forming optic vesicles by intercalation"
"
"
"
4) Apical retraction shortens optic vesicle cells and draws them laterally into the vesicles"
79
how can ou view cell intercalation?
you can label the cell membrane and follow the cells
80
what is coloboma?
e failure of the choroid fissure to close
81
how did they first implicate periocular mesenchyme cells in the closure of the choroid fissure?
they used actin labelled with gap to view the different components of he closure and they observed a group of cells that migrated in and invaded the closing fissure
82
what are POM thought to do maybe?
these mesenchymal cells might secrete priests which get rid of the CEM between the choroid fissure.
83
how did they go about determingin the role of the POMs?
- they tried to kill them y doing a Imxb MO and saw that you dont get a closure
84
what gene did they find to be expressed by the POM cells and how did they look at a knock down?
AP2 they did an MO and found that this gene sensitises it and then tried with AP2a and AP2c MO
85
how did they look at whether AP2 can work as a enhancer mutation of knock down?
Partial abrogation of AP2 activity synergizes with mutations that normally don’t cause coloboma.
86
does the zebrafish visual system maintain populations of neuronal set cells throughout life?
yes
87
where are stem cells found in the eye
cilliary marginal zone
88
how does the eye grow?
in concentric rings ike a tree
89
how can you identify where the stem cells are in the eye?
in situ for stem cell markers
90
how can you label the cycle of proliferation of the eye stem cells
an use a proliferative marker and then marker for cells exiting the cell cycle and look at the areas where these cells are- concentric rings
91
how can you study a mutation that may hinder the stem cells in the eye? what did they do this with and what did they find~
use proliferative markers and markers for cell cycle exit and compare in wild type and mutant. they did this with Flo mutant- the stem cells are still there- prolif marker is reduced and markers for exiting the cell cycle have gone all together- problem with this stage transitioning and see some cell death too
92
when they found that flo mutant eye stem cells were dying, how did they see what affect apoptosis was having? what did they find?
- they inhibited apoptosis by using p53 MO. They see that there perfectly restore proliferation (ccnD1 as a marker) but not can make the transition to differentiation ( use a differentiation marker- cdkn1 marker). You see in wild type the there is prolif then diff. You see in the flo mutant there is prolif then stops and cells die. You see in flo+ p53 apoptosis inhibitor that the cells can't differentiate but can't die to continue expressing prolif marker
93
how did they test whether it was the surrounding differenting cells in the flo mutant that were preventing the stem cells from transitioning from proliferation to differentiation ? what did they conclude? why does this make sense ?
- they used a genetic mosaic: transplanted cells into the eye field on the embryo and see if they contribute to the retina as control
- when they put mutant cells into a wild type environment- many of the cells instead of dying, are integrated perfectly
- this shows that it is not autonomous and that it is induced
- they now think that the differentiated cells are telling the proliferating cells to move towards differentiation and they dont do this in the flo mutant
- if you have many proliferative cells this is not a safe thing to have uncontrolled- it is good to have the differential retina cells telling the proliferaitn cells when to progress to diff
Mechanisms o f Development
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