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Mechanisms o f Development > Left Right Asymmetry- Zebrafish > Flashcards

Left Right Asymmetry- Zebrafish Flashcards

1
Q

list the asymmetric arrangements of the organs

A
  • the apex of the heart points towards the left
  • the lungs differ with respect to the lobation: two lobes on the right side
  • the stomach and the spleen are positioned on the left, whereas the liver and appendix are found on the right
  • the small intestine and colon coil asymmetrically
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2
Q

why is organ asymmetry good?

A
  • it allows good compaction of the organs
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3
Q

how are ciliated body involved in left right asymmetry?

A
  • they are present in the mouse, xenopus and zebrafish and they beat components to one side and this stimulates the asymmetric expression of genes- a morphogen is thought to be moved across
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4
Q

what is the most important pathway in left right?

A

nodal signalling pathway

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5
Q

which direction is Nodal taken to?

A

left

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6
Q

which direction do the cilia normally beat

A

they send components over to the left side

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7
Q

what is an example of when in nature left right asymmetry is important?

A
  • snakes have evoled to attack the snail from one side so then those snails that has shells that curled in the other direction were selected for
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8
Q

what are the neuroanatomical differences between the brain regions?

A

the left cortex is thicker, they two hemispheres are tilted slightly towards the left.

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9
Q

what is the difference in laterality found in the brain?

A

different areas of the brain respond to different stimuli:

  • language processing is found on the left hemispheres
  • handedness
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10
Q

how did people find functional asymmetries in the brain?

A
  • classical neuroscience: have patients that have lesions in different areas of the brain and test them for different capabilities
  • Broca’s area: people can’t comprehend or produce speak with lesions in left hand side
  • split brain patients: hemispheres have been disconnected by ablation of the corpus callosum and there is no communication between the two sides of the brain
  • hemisphere neglect- lesions in right side of the brain- dont acknowledge the left side of anything
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11
Q

what diseases are associated with altered asymmetry in the brain?

A
  • schizphrenia: reduced palnar asymmetry
  • semantic dementia: atrophy worse on the left side
  • asymmetric progression of alzeihmers
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12
Q

is brain lateralisation widespread in animals?

A
  • recall of long term memories in the fly is asymmetrical
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13
Q

how can you test is insects have asymmetrical CNS?

A
  1. brain of dros that posses a unilateral body that normally only is present singularly in fly brains- there are some mutants that havee bilateral bodies- the ones that have bilateral bodies dont perhaps as well
  2. if you present an odourant to the right or left antennae- their ability to store a long term memory of a stimulus will be recalled better if presented to the right
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14
Q

what is the basic premise to understanding CNS left asymmetries?

A
  • first you have to identify an asymmetrical structure in the CNS that you can follow
  • then you ask questions such as which genes are involved- mutants that disrupt the mutants and the processes these are involved
  • then see when you disrupt the asymmetry what are the behaviour results
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15
Q

what is the epithalamus comprised of?

A

the epithalamus: pineal complex + habenula nuclei

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16
Q

from where in the brain does the epithalamus receive input from?

A
  • olfactory bulb
  • eyes
  • ears
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17
Q

what is in the pineal complex?

A

pineal galnd and the parapineal

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18
Q

what is the most asymmetric structure in the fish brain?

A

parapineal; - innervates exclusively the left side of the epithalamus

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19
Q

do humans have the parapineal gland?

A
  • no
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20
Q

how can the asymmetry of the parapineal be viewed?

A

by using a transgenic line which labels the parapineal

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21
Q

how does the parapineal become asymmetrical

A
  • it migrates from the epithalamus during developme-t the asymmetry develops
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22
Q

what was the first question people asked when seeking to understand the parapineal gland migration? how did they look at this

A

they asked whether the precursor cells come directly from the right or left side. they tested this by doing a fate map with fluo dye- they labelled either the left or the right of the pineal complex and found both left and right contributed to the parapineal

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23
Q

how did they begin to look at the signalling pathways that given parapineal migration? what did they find

A
  • they new that the nodal signalling pathway had been heavily implicated in LR development so they began to look at whether there components of this signalling pathway were expressed at the tissue of interest during the migration
  • they found that pitx2 was only expressed in the left and so was cyclopse (nodal)
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24
Q

how did they begin to use mutants to look at the process of parapineal asymmetrical migration? what did they find

A
  • examine mutants for genes of interest- they looked at nodal signalling mutants and found that the parapineal still migrates but the direction becomes randomised- 50/50 left or right- so the NODAL mutants that were used were LZoep (co-factor of nodal signals) and ntl mutants (target gene)
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25
Q

which nodal mutants were used to look at the role of NODal signalling in parapineal migration?

A

LZoep (co-factor of nodal signals) and ntl mutants (target gene)

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26
Q

so once they knew that NODAL signalling wasn’t involved in the migration itself, how did they go about finding the pathways that stimulate the migration of the parapineal migration? what did they find?

A
  • they already knew that FGF signalling is involved in collective cell migration
  • so they looked whether fgf genes were expressed at the right time in the right place
  • they found that Fgf signals in the neuropeithelium that will give rise to the habenula. Fgf receptors expressed in the pineal and parapineal anlage.
  • Fgf 8 expression slightly asymmetric: stronger in the left side. Fgf8 expression becomes symmetric in nodal signaling mutants.
27
Q

what did they observe in fgf8 mutants?

A
  • when they label the parapineal with trangesnics (to demonstrate there was not a problem with fate or specification) and the surrounded cells as a contract with red- they see that in fgf8 mutants the parapineal does not migrate and that this phenotype can be rescued by using fgf soaked beads
28
Q

how did FGF involved in migration? how did they show that parapineal cells demonstrate such traits. what is the overall idea of FGF nodal signalling in the migration of the parapineal?

A

they are involved in migration and protrusion of leading cells-

  • Tg(dusp6:d2GFP): a transgenic line for visualising Fgf signaling Strong ac?va?on of Fgf signaling is coincident with protrusive ac?vity of leading cells
  • so if there is asymmetrical FGF in nodal mutants then NODAL signalling must regulate left side expressing which stimulates left cell protrusion and migration
29
Q

what is the asymmetry in the hobnail nuclei?

A

the left side is enlarged compared to the right and if you use a marker for for neuropil which demonstrates how neuronal networks are being formed- there is aver clear asymmetry between the left and right

30
Q

what genetic markers are expressed more on the left of the habenula? what about for the other side?

A

dHbl…. dHbM

31
Q

how can the habenula be divided into subdomains? what is its neuroanatomy?

A
  • left type markers are expressed in a more broad domain in the left and in a small domain in the right and right side markers to the opposite
32
Q

what were the two types of asymmetry that were found in the habenula and what were they specifically?

A
  • the neuroanatomical differences: expressed of dHbL vs dHbM more on the left and right respectively
  • functional asymmetries : right side in odour processing and left in light sensing
33
Q

how did they measure the difference in inputs in the habenula

A
  • it is supposed to receive inputs and put out targets to the midbrain.
  • they used a marker that reported the expression of the mitral cells form the olfactory bulb and found that these innervated the right side
  • the parapineal is a photosensor organ that only innervates the left side- transgenics
34
Q

how did they measure which sides of the habenula projected preferentially to certain areas of the brain?

A
  • they used dyes which travel down neurons (Dil or DID) which have different colours and they found when they injected them into the left or right that the left preferentially innervared in part of the interpednculular nucleus and the same for the right
35
Q

what two dyes can be used otlabel projections from the habenula?

A

Dil or DiD

36
Q

how can you see differences in termination of the two sides of the herbal

A

you can label the type of projections that neurons have, you can see that they have different type of termination on the left than on the right (look this up)

37
Q

how can you look at the difference in properties of Hb neurons relative to their left/ right position?

A
  • you can use a calcium sensor transgenic line - GCamp5- you can see see that there is functional preference in the habenula
  • you can test which stimuli elicit difference responses to the fish - light or odour and found that most of the cells respond to light are on the left and odours on the right
  • if you look at the stimulation response in the IPN- you see the areas correspond to the areas which are innervated by the relative left or right side
38
Q

what are the two main ways in which it is thought that the asymmetries arise in the hebnula sides?

A
  • differences in proliferation

- difference in signals

39
Q

what is a classical BrdU analysis? what was found when using this to look at habenula development in asymmetry

A
  • uses BrdU which gets diluted as the cells divide- if cells undergo lots of division then you can’t see brdu anymore. But if BRDU is added to the cell in its final division, it will continue to exhibit BrdU.
  • you can apply BrdU at different time points- wash it way and then let the embryos grow and then you can find out where cells were that were undergoing their final division at the given timepjoint of application
  • he gave the cell cycle marker at different time points and marked when the habenula ended up showing up- at different time points he counted how many BrdU expressing cells there were at each point in th left and the right
  • e found that at the earlier time point- most BrdU was exhibited on the left of the brain and only after this did the cells in the right side differentiatie
40
Q

what is the notch pathway?

A

pathway that requires cell to cell contact- notch ligand and delta is present in both cells- but eventually one cell will have more delta than in the other which will bind to notch activation more in the other cell which then inhibits the expression of delta.

41
Q

what pathway is implicated in cell boundary fates?

A
  • notch pathway
42
Q

what pathway is implicated in cell group migration?

A

FGF

43
Q

why did they think that Notch may be playing a role in habenula formation?

A
  • it is involved in cell bonder fates- maybe it is involved in the different boundaries seen in the asymmetry of the habenula
44
Q

how did they test if notch was involved in habenula development?

A
  • they saw that you have the expression of the notch receptor on both sides of the epithalamus but there is slightly more expression of notch ligand in the right than the left- which indicates that there is more notch signalling on the right
45
Q

how does the role of notch signalling generally give insight and explain why the right side differentiates late ?

A
  • notch is normally involved in maintaining stemness in cells - cells on the right maintain their stemness whereas those on the left dont
46
Q

how did they investigate whether notch signalling influenced the differentiation timing of the two sides of the habenula? (2) what did they find?

A
  • they induce CA of notch in both sides and you lose left fates and only get right fates
  • they there is a mutant that has completely low Notch signalling in both sides- you get two left fates- mind bomb mutant
47
Q

what pathway do they think is responsible for the different left right habenula fates due to the different sides being exposed to it at different times ?

A

WNT pathway

48
Q

what was the first piece of evidence showing that WNT was involved in habenula development? (2 mutants)

A
  • an axon mutant was found which meant that in the mutant the wnt pathway was constantly on. this resulted in a double right habenula
  • if there is mutants in the tcf3a then the WNT is always turned off and then this is the case then there is a double left
  • when you combine both mutants- to see if they are both working together in the same pathway- if you dont have TCF in both then you get double left- downstream - acting in the WNT pathway
49
Q

how can you use heat inducible mutants to study the role of WNT?

A

they did heat shock treatment and over express a dnTCF3a mutant- inactivates the pathway- if they turned off the pathway at a certain time- because WNT is involved in lots of different pathways- you also get a double left habenula

50
Q

how can you use drugs to look at the role of WNT?

A

if you use a drug to inactivate the pathway in a temporal way- you see a double left- using a drug is good because you can narrow down a time point in which the pathway is working

51
Q

why is using a drug good?

A

using a drug is good because you can narrow down a time point in which the pathway is working

52
Q

by using drug WNT inhibitors, when did scientists find that the WNT signal was needed to induce right fate?

A

34-36 hours post fertilisation

53
Q

how can you show that TCF is being activativated in the habenula? what did they find?

A

they used an antibody staining for tcf - they see it becomes activated in the habenula neurons when the neurons start beiing born and you see that more neurons are produced in the left first and then the right catches up

54
Q

what did they see when they investigated whether not having tcf3 would affect the kinetics of proliferation in the habenula? why would they want to do this?

A
  • they counted pku+ cells by using a reporter or immune labelling (this is expressed in habenula cells) and found that wild type and mutants look the same at 32 hpf but if you count have many acquire left vs right fate- you see that there is the same number generally for left and right habenula cells. this shows that the differences seen are not due to a decrease in cell number but due to direct dysruption in left right pattenring
55
Q

which organisms can you make mosaic embryos of every easily?

A

zebrafish and xenopus

56
Q

how did they show that the inability of the right cell cells to express a right side fate in tcf3a mutants was because of an inability to respond to environmental signals?

A
  • they took tcf3a mutants cells from the habenula and injected them into the right habenula to see if they could take cues from the wild type environment. They showed that cells that were transplanted to the right side were unable to respond even though they were in wild type environment
57
Q

how can you measure the difference in WNT signalling in the wild type between the left and right habenula?

A

you can do insitu against TCF3 and find that it is degraded in the left but not in the right

58
Q

how did they start to look at what is modulating the difference in WNT expression between the left and right?

A
  • the first obsveration came from looking at the development of parapineal avuncular neurons: you see that the parapinea and habenular asymmetries are condcordant so maybe the parapineal secretes factors which inhibit wnt?
  • they first tested this by using a laser to kill the parapineal and then do in situ for left vs right then you see that there is a loss of left fates and an increase in right fates
59
Q

how can a pitx2 mutant be used to demonstrate the role of the parapineal in habunlar asymmetries

A

in embryos that was defected forpitx2 genes- there have a large parapineal gland and an increase left fates and reduced right fates and if you restore the size of the parapineal then you can rescue the phenotype seen

60
Q

what happens when you transplant parapineal cells into the right side?

A

induce left cell fates in the right side of the thalamus

61
Q

how can you find novel genes involved in habenula development?

A

screen for reversed laterality or asymmetry

62
Q

is FGF expression asymmetrical?

A

yeah after 24 hours

63
Q

at what time in development does the parapineal gland migrate?

A

between 24 and 48 hours

Mechanisms o f Development (15 decks)

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