Zebrafish - LR asymmetry

Question 1

Give an example of left right defects in humans. And a physiological behaviour involving left right asymmetry.

Answer 1

• Unilateral spatial neglect
  
  • right hemisphere damage results in patients not being able to process information coming from the left side of space
• Dolphins use their left eye to observe familiar objects and their right for unfamiliar objects

Question 2

Describe the dorsal diencephalon conduction system in zebrafish.

Answer 2

• The epithalamus is composed of the pineal complex and the habenular nuclei
• The pineal complex:
  
  • The parapineal innervates eclusively the left side of the epithalamus
  • The parapineal delaminates from the midline and migrates leftwards
    
    • This was found by fate map analysis with fluorescent dyes
• The habenulae nuclei:
  
  • Asymmetric neuroanatomy and molecular signature
    
    • DAPI and neuropil staining by immunohistochemical staining
    • WISH showd genetic markers to be expressed asymmetrically. E.g. lov
    • Afferent projections from mitral cells to the rHb and parapineal projections to the lHb
    • Staining for dyes DiI and DiD shows efferent projections from LR habenular asymmetries translate to IPN DV innervation asymmetries

Question 3

Which signalling pathways govern parapineal migration?

Answer 3

• WISH showed asymmetrically expressed Nodal pathway genes in the epithalamus
  
  • Learning from mutants
    
    • ​Laterality of epithalmic asymmetries is randomised in LZoep and ntl mutants
• Fgf pathway genes are also expressed in the epithalamus
  
  • Fgf receptors expressed in the pineal and parapineal anlage
  • Fgf8 expression is asymmetrical to the left
  • Fgf8 expression becomes asymmetric in nodal signalling mutants
  • Learning from mutants
    
    • ​The parapineal is present but remains in the midling in fgf8 mutants
    • Can rescue parapineal migration by implanting an Fgf soaked bead
    • Therefore, Fgf signalling is required for parapineal migration
      *

Question 4

What are the functional asymmetries of the habenulae nuclei and how have they been studied?

Answer 4

• Using GCaMP and two-photon microscope to visualise neuronal activity in living embryos it was found that the functional and anatomical asymmetries are linked.
• Light and odour responses are LR lateralised in WT brains

Question 5

What signalling pathways govern the devopment of an asymmetric habenula? Notch signalling (1)

Answer 5

• Classic birthdate analysis with BrdU pulse-chase experiment
  
  • ​Measure the dilution of BrdU as cells divide. If the cell differentiates instead of proliferates the BrdU remains.
  • Found that left neurons differentiated earlier than right neurons. There was two waves of differentiation.
• The Notch pathway genes are expressed in the epithalamus
  
  • Constiuitive activtion of Notch at 32hpf blocks left-type fates and favours right-type
  • Learning from mutants
    
    • ​​Consituitive inactivation of Notch signalling in mindbomb mutants favours early symmetic neurogenesis and thus left-type fates
  • Thus Notch maintains cell proliferation and prevents differentiation
  • It is unknown what is regulating Notch signalling on the left and right
  • Thus Notch signalling may explain kinectics of cell prolifertation BUT does not govern cell fate

Question 6

Described in basic terms the Notch signalling pathway.

Answer 6

• When considering binary cell fate decisions, especially neuronal progenitors proliferating of differentiating, think Wnt signalling!
• Notch signalling works by cell-cell contact

1. Two ligands, delta and notch are present in both cells but to various factors one cell will have slightly more delta ligand than the other.
2. The delta ligand binds to the notch ligand on the continuous cell. This triggers a cascade of events.
3. The notch receptor is partial cleaved
4. The notch intracellular domain (NCID) gets translocated into the nucleus
5. It then activates the expression of downstream target genes.
6. There is a negative feedback loop to maintain system

Question 7

Describe in basic terms the Wnt signalling pathway.

Answer 7

• Wnt is morphogen that orks over a long distance

1. A ligand (Wnt) binds to its receptor frizzled that causes a cascade of events and inactivates beta catenin repressor complex
2. This leads to an accumulation of beta catenin
3. Once a critical level of beta catenin is reached it is translocated into the nucleus
4. In the nucleus it binds to transcription factors (LEF/TCF) and acts as a cofactor for target genes

• Beta catenin is constiuitively produced in the cell however, its levels are kept low by a complex that binds to it and marks it for degradation. Axin is protein in the beta cantenin complex.

Question 8

What signalling pathways govern habenula asymmetric fates. (2) Wnt signalling.

Answer 8

• The Wnt signalling pathway
  
  • Learning from mutants
    
    • ​Mbl is a mutant defective Axin and therefore has constitutive activation of beta cantenin regardless of Wnt or not. The mutant phenotype is a double right habenula
    • Tcf7l2 is a mutant with a defect in the TCF transcription factor that beta cantenin bind to and therefore has constituive inactivation of beta cantenin regardless of the presence of Wnt. Antiobody staining showed that Tcf7l2 is expressed in the neurons when they undergo differentiation. The mutant phenotype is a double left habenula
    • Crossed both mutants to generate a double mutant and carried out epistasis assays to see if the proteins were in the same pathway. A double left habenula phenotype was found. Therefore, TCF must act downstream of axin
    • This was confirmed using a heat shock promoter linked to a dominant negative version of the tcf7l2. This also lead to a double left phenotye.
  • Using drugs that inhibit the Wnt signalling pathway confirmed its involvement
    
    • This method also allowed for temporal control and thus found out when the Wnt signalling pathway is important in cell fate decisions of the habenula
  • Transplanted mutant Tcf7l2 cells into WT environment.
    
    • ​These mutant cells act autonomously and express markers of left-sided neurons
• *

Question 9

What is the relation between the parapineal migration and the habenular asymmetic development?

Answer 9

• Parapineal and habeunlar asymmetries are concordant.
• Ablating the parapineal the habenular shows a reduction left-type fates and increase in right-type.
• Pitx2MO = enlarged parapineal and increased left-type fates
• Pitx2MO AND pp partial ablation = normal habenular phenotypes
• Transplanted PP to the right = left type fates n the right side of the epithalamus
• Future questions:
  
  • Does the parapineal secreate an inducing factor that determines left-type fate? Or does asymmetric innervations determine left-type fate?
  • What is the connection between Notch and Wnt? Is it the parapineal? (At the moment we have no molecular evidence just embryological)

Question 10

Summarise the our knowledge of the events that occur to develop an asymmetric epithalamus.

Answer 10

• First, Nodal signalling must be expressed asymmetrically along with Fgf to initiate parapineal migration to the left.
• Second, Notch signalling is important to confer neurogenic potential between left and right sides. In parrallel or complementary fashion, Wnt signalling is working in a cell autonomous fashion to allocate cell fates.