Pan neuronal Differentiation

Question 1

Two ways to differentiate cells

Answer 1

1.iPSC-yamanaka factors
2.Direct differentiation

Question 2

Advantages of Direct differentiation

Answer 2

Less time than iPSCs
No intermediate pluripotent/multipotent cell state
Fast & more efficient turnaround time to acquire reprogrammed cell
Reduced tumorigenesis risk
Epigenetic marks are maintained
Can do it directly in vivo and not need to take it out of body and reintegrate in - particularly useless for injuries

Question 3

Disadvantage of Direct differentiation

Answer 3

Low reprogramming efficiency
Poor cell viability during and post-reprogramming
Incomplete neuronal maturation
Issues with general neuronal differentiation/ maturation
Phenotypic instability
Limited neuron types can be generated -Issues with differentiating specific neuron types/ sub-types

Question 4

Why use the optic lobe to study transcriptome?

Answer 4

well-characterized neuronal diversity, extensive genetic toolbox, and detailed connectome.
Drosophila offers a rich genetic toolbox, including Gal4 drivers that can target specific cell types, making it easier to manipulate and study individual neuronal types.

Question 5

+ of using DR as model

Answer 5

1.Half the organisms nerves are involved in the visual system so lots of cells to work with (visual processing occurs in the optic lobes that comprise more than 60% of the brain’s neurons0
2.Drosophila, like vertebres, have an organised retinotopically into repeated modular circuits that process sensory input from the entire visual field
3.Can use many genetic tools
4.Evolutionarily conserved
5.200 cell types= genetic diversity

Question 6

• of using DR as model

Answer 6

-Evolutionary distance from humans
-Lack of complex organs, and environmental sensitivity

Question 7

How is the pan-neuronal program engaged to trigger neuronal differentiation?

Answer 7

Initiate new program
suppress old program

Question 8

which two cell types in the optic lobe begin differentiation

Answer 8

photoreceptor cells and glia cross talk

Question 9

How do Photoreceptors and glia communicate?

Answer 9

differentiate it from the top down using ERK signalling

Question 10

Which signalling pathways are in charge of the differentition of optic lobe

Answer 10

ERK signalling triggered by glia and SHH

Question 11

Which TF from ERK pathway is involed in pan-neruonal differentiation

Answer 11

Pointed P1 (PntP1)

Question 12

Earliest Drosophilia markers of neuronal differentiation

Answer 12

Elav

Question 13

When in development does ERK signalling turn on and how do the know?

Answer 13

early developemnt because of early expression of elav showing up in RNAseq

Question 14

DAM ID

Answer 14

Methylate called DAM used (DAM does not exist in Eukaryotes)

Wherever PntP1 binds to the genome, DAM can find and create methylation sites at sequence GATC. (methylated the A).
Now you chop up the protein and sequence them.

Enzyme used to chop DNA into smaller fragments where there aren’t these sites and sequence them to see where PNpt was binding directly onto the DNA sequence. DAMID gives you a broader profile than CHIP- over the full timeframe of 16 hours compared to chip which would only give you a snapshot of it.
From this they identified 16 genes that were targets of PntP- half are transcription factors

Question 15

What is onecut?

Answer 15

TF found in the photoreceptors of the eye

Question 16

Overexpression of onecut

Answer 16

earlier expression of elav

Question 17

why isn’t onecut expressing elav early on in development?

Answer 17

It gets supppressed by a microRNA to activate at the right time when ERK siganlling acts on it