Zebrafish - Eye development

Question 1

What are the stages of eye development?

Answer 1

1. Eye field specification
   
   1. Eye specification genes are turned on in the anterior neural plate
2. Optic vesicle evagination
   
   1. All of the cells forming the eye constitue a single field of cells. If this fails to seperate then two eye will not be formed
3. Optic Cup formation: Patterning and Invagination

Question 2

Why is it important to study eye development? And why is the zebrafish a good model?

Answer 2

• To understand congenital eye diseases seen in humans
• anopthalmia
  
  • absence of an eye
• microphthalmia
  
  • small eye
• coloboma
  
  • As the eye is forming the sphere fails to fuse
• Retinal degenerations
  
  • Glaucoma
  • Macular degeneration
• Cancers
  
  • Retinblastoma
• The zebrafish is a good model because there are mutant lines that have the same phenotypes seen in the human diseases

Question 3

What did the mutants masterblind and hdl show?

Answer 3

Both these mutants had no eyes - anopthalmia. Was found to be due to enhanced Wnt signalling.

• mbl: had a mutation in the axin gene
• hdl: had a mutation in the tcf3a gene
• Both genes are reuired in the Wnt signalling pathway

Question 4

What did outcrossing the tcf3a mutant show?

Answer 4

The phenotype was lost after just three outcrosses. The mutation on its own isn’t enough to cause the phenotype.

Question 5

Describe the story of the tcf3a mutant. Eye field specification.

Answer 5

• Annalysied transcript levels of Tcf genes and eye genes in Tcf3a-/-
  
  • Analysed with qPCR (quantative PCR, aka. real-time PCR)
  • One of the control eye genes, Rx3, was found to be much lower than it should have been.
  • rx3 expression and eye field is reduced
• Rx3 is a transcription factor required for normal eye development. Very conserved. Needed in mammals also.
• rx3 expression recovers to normal at 3dpf
• Optic vesicle ablation (embryological technique) of WT embryos (2/3) also had a complete recovery of eye by 4dpf. This highlighted how robust eye development is.
• Tcf3a function in eye field specification was found to be self-autonomous
  
  • found this by analysing 2 genetic mosaics. 1) tcf3a-/- cells to WT embryos and 2) WT cells to tcf3a-/- embryos.
  • There was no expression of rx3 where there was tcf3a-/- cells
• Current research. tcf3a promotes eye field specification
  
  • tcf3a –> eye field/rx3
  • but tcf3a is repressor. So tcf3a –| X –| eye field/rx3
• Finally, wanted to find out what genes compensate when tcf3a is absent.
• Did a mutangenesis screen on the tcf3a mutant background (enhancer screen)
  
  • enhanced phenotype meant a double phenotype (1/16 mendelian ratio). Could identify other genes important in eye formation
  • hesx1 was the first identified eyeless enhancer of tcf3a

Question 6

Describe the story of the rex3 mutants and eye field evagination.

Answer 6

• Created a transgenic animal that had GFP linked to a rex3-specifc reporter
  
  • ​Showed that segregation of domains is maintaned throughout morphogenesis. I.e the eye field is a coherent structure during morphogeneis
  • Wanted to know how this was being regulated
• In rx3 mutants there was failed eye morphogenesis
• Genes encoding Eph/ephrin are misregulated in rx3 mutants
• Eph/ephrin pathway was known to be important in eye development. Repulsion between Eph in the telencephalon and ephrin in the eye field maintains seperation between cell populations.
• In Rx3 mutants there is no affect to neural plate patterning (i.e eye field specification). Therefore, it must be at the level of evagination.
• A graft of cells expressing the Eph receptor placed in the eye field showed the movement of these cells out of the eye field and into the telecenapholon.
• There are two populations of cells in the eye field. Core cells and marginal cells.
• Core cells transform into marginal cells as eye development progresses. They integrate into the optic vesicle (neuroepithelium) by gradual intercalation.
• (The extracellular matrix is a source of signals instructing polarity. Epithelial cells have a basal and apical polarity)
• As soon as the eye field begins to evaginate there is a very strong laying down of extracellular matrix
• Is the precocious laying down of ECM important for morphogeneis?
• By genetically depleteing laminin1 (proteins of the ECM) it was found that cell shape and coordination of polarity was disrupted.
• The basal lamina is giving the cells the cues to properly polarise during morphogenesis.

Question 7

Describe the story of optic cuff fusion. (Reverse genetic techniques)

Answer 7

• In coloboma (most common form of blindness in children) the eye the optic cuff fail to fuse
• The closing of the choroid fissue is invaded by highly motile periocular mesenchymal cells
  
  • what is their function?
  • One theory: they secrete proteases that dissolve the ECM that needs to be removed for choroid fusion.
• AMOs against genes (AP2) required for POM cells disrupts their function and a severe phenotype occurs (the retina is completely open)
  
  • However this is not conclusive evidence for what is happening at the point of fusion. POM cells may be required in development before fusion.
• Brachio-oculo-facial syndrome is linked to mutations in tfap2.
  
  • One of the phenotypes is coloboma
  • There is great variance in phenotypes why?
  • Partial abrogation of AP2 activity synergizes with mutations that normally do not cause coloboma. This could explain variation
• Remaining questions
  
  • How does POM influence retina so that choroid fussion occurs?
  • what cell shape changes in retina and POM drives closure?

Question 8

What is a stem cell? And why are they required?

Answer 8

1. A cell that has self-renewal potential and has multipotency or pluripotency, i.e has the ability to generate a variety of cell types.
2. They are needed to maintain tissues with a high-turnover rate and to regenerate tissues upon injury

Question 9

Why are the zebrafish a good model to study neuronal stem cell biology?

Answer 9

The zebrafish visual system maintains populations of neuonal stem cells throughout life. The cillary marginal zone (CMZ) is the stem cell niche.

Question 10

Describe the story of the forward genetic screen that uncovered stem cell mutants.

Answer 10

• A forward genetic screen was performed to identify to animals with eye growth defects.
• flo mutant had diminshed eye growth
• flo encodes a protein required for cell cycle progression and is expressed in highly proliferative tissues
• flo CMZ cells fail to perform transition from proliferation to differentiation.
• Cells at the interface between the differentiated retina tissue and the CMZ undergo apoptosis in flo mutants
• Dying cells are unable to differentiate. What happens if apoptosis is inhibited?
  
  • inhibited apoptosis by p53AMOs
  • proliferation occurs but no differentiation
• In flo mutants the cells die before they can transition from proliferation to differentiation
• In absence of apoptosis (p53AMO), flo mutant retinal cells can cycle but still do not exit cell cycle (no differentiation)
  
  • does their defective cell cycling behaviour make them intrinsically incapable of the proliferative program and differentiating? Or are their non-cell autonomous signals
• They observed how small groups of cells interact with the surrounding environment by transplanting modified cells into a host embryo to generate a mosaic fish.
  
  • ​graft can be mutant and WT - carry put experiment in both directions
  • ​flo cells in WT retina differentiate normally. Suggesting that local signals in WT CMZ resuce the mutant cells to differentiate normally
  • Environmentally driven differentiation: a mechanism contributing to self-regulating ize control? - ensures that any cells with potentially oncogenic mutations that escape apoptosis are driven to differentiation
• Unanswered questions:
  
  • Which extrinsic signals drives transition from proliferation to differentiation?