Retinal Development

Question 1

Which area of the developing CNS gives rise to the retina?

Answer 1

The diencephalon

Question 2

The extension of the CNS which forms the retina begins to form right after…

Answer 2

Neural tube formation

Question 3

The first sign of the developing retina in a human embryo occurs on this day, usually

Answer 3

22

Question 4

Invagination of the lens placode and evagination of the optic vesicle forms this structure

Answer 4

Optic cup

Question 5

The mutation which results in small eyes is called…

Answer 5

Micropthalmia

Question 6

The mutation causing micropthalmia is called…

Answer 6

Ocular retardation: mutant gene is orJ

Question 7

Another name for Chx10 is…

Answer 7

Vsx2

Question 8

The orJ mutation is a mutation affecting this protein…

Answer 8

Chx10

Question 9

Describe the mouse orJ mutation: how does it result in micropthalmia?

Answer 9

There is a tyrosine (Y) at 176, which results in a stop codon, resulting in a truncated Chx10 protein

Question 10

Define the human micropthalmia mutation

Answer 10

R200Q (arginine to glutamine) - same as mouse results in a truncated protein

Question 11

Chx10 is exclusively expressed in this area of the eye…

Answer 11

The retina

Question 12

If Chx10 is only expressed in the retina, why are there mutations to the lens and the cornea in micropthalmia?

Answer 12

The retina secretes different signaling factors: a lack of signals could result in structural defects

Key: secondary, cell non-autonomous effects

Question 13

When in retinal development is Chx10 expressed?

Answer 13

Early, when the retina is mostly comprised of progenitor cells

Question 14

What are the 7 primary cell types of the retina?

Answer 14

Rods, cones, bipolar cells, horizontal cells, amacrine cells, muller glia, ganglion cells

Question 15

Rods are good for detecting…

Answer 15

Low light at night

Question 16

Cones are good for detecting…

Answer 16

Colour, good for daytime vision

Question 17

What are the 3 types of cone?

Answer 17

Each type expresses a different opsin, for R G or B light

Question 18

In what layer of the retina do photoreceptors synapse to bipolar cells?

Answer 18

In the inner nuclear layer

Question 19

Where are the cell bodies of muller glia located?

Answer 19

Inner nuclear layer

Question 20

What is the proposed purpose of muller glia?

Answer 20

Form a scaffolding for other retinal cell types and scavenge ions

Question 21

Which cells are responsible for lateral inhibition?

Answer 21

Horizontal cells

Question 22

Bipolar cells form circuits with these two cell types

Answer 22

Amacrine and Ganglion cells

Question 23

What is the output cell of the retina which projects towards the brain?

Answer 23

Ganglion cells

Question 24

Thinking about the interactions of Chx10 with other proteins, why do people with the micropthalmia mutation have small eyes?

Answer 24

Chx10 normally inhibits Kip1 which is a cdk inhibitor. With kip1 active, cdk and cyclin D1 will be inactive and the cell will not progress to S phase, cells will exit the cell cycle (G0) and will differentiate prematurely into retinal cells

decreased proliferation because progenitor pool is depleted

Question 25

What happens to a Chx10-Kip1 double mutant? Is the retina larger? What does this mean?

Answer 25

The phenotype is actually more similar to wild type, don't get enlarged retina There must be another factor acting redundantly with Kip1 to regulate cell cycle exit

Question 26

What is unusual about the bipolar cells of Chx10 mutants?

Answer 26

They don't have bipolar cells

Question 27

What molecule is required for bipolar cell specification?

Answer 27

Chx10

Question 28

What are 2 roles for Chx10 in retinal development?

Answer 28

1. regulate cell cycle exit 2. specification of bipolar cells

Question 29

Describe this experiment (not the results, just the methodology)

Answer 29

Authors were interested in learning about retinal progenitor divisions and engineered a retrovirus which would stain progenitors, but injected such a low amount such that it would affect only one cell. Then, the authors visualized the divisions of that cell because they were stained with beta-galactosidase

Question 30

What were the key results of this experiment?

Answer 30

Progenitors gave rise to the following cell types: Rods Bipolar cells Amacrine cells Muller glia

Question 31

Why did the authors of this experiment not observe any cones or ganglion cells which arose from progenitors?

Answer 31

There is temporal ordering of retinal cells during development - those cells are born earlier than the cells they observed

Question 32

A single retinal progenitor cell can give rise to (a single/multiple) type(s) of retinal cell

Answer 32

Multiple

Question 33

What is a key factor which regulates retinal progenitor multipotency (ability to give rise to multiple cell types)

Answer 33

Pax6

Question 34

What kind of protein is Pax6?

Answer 34

Homeodomain transcription factor

Question 35

What does it mean to say that retinal progenitors are thought to progress through multiple states of "competence"

Answer 35

At different times depending on the factors present (and the concentration of these factors), retinal progenitors may choose to differentiate into different retinal cell types. These windows are overlapping

Question 36

What cell types are born "early"

Answer 36

Ganglion cells, cones, horizontal cells, amacrine cells

Question 37

What cell types are born "late"

Answer 37

Rods, muller, bipolar cells

Question 38

A loss of function of Pax6 leads to...

Answer 38

Anopthalmia (being born with no eyes)

Question 39

Define conditional gene targeting

Answer 39

Making constructs where genes can be temporarily or conditionally knocked out so that loss of a gene does not adversely affect other aspects of development in a model

Question 40

What are the 2 requirements for cre-lox site specific recombination?

Answer 40

DNA that you want to remove must be flanked by loxP sites ("floxed") Need tissue-specific or cell-specific cre recombinase expression

Question 41

How does cre remove DNA?

Answer 41

Cleaves DNA at the loxP sites so that floxed region of DNA will be excised

Question 42

What are P0 and alpha of the Pax6 gene?

Answer 42

P0 = promoter alpha = enhancer

Question 43

In a transgene construct of P0 and alpha (Pax6 gene) where they are combined upstream of the LacZ marker, where do these regulatory regions direct transcription to?

Answer 43

The distal retina

Question 44

How was the alpha region of Pax6 identified?

Answer 44

Among several species, this intron region was highly conserved: must have important regulatory role

Question 45

In conditional knockouts of Pax6, which retinal cell types are severely downregulated? Which are not affected?

Answer 45

Loss: bipolar, ganglion, muller, PRs Unaffected: Amacrine

Question 46

If all retinal cells express Pax6, and Pax6 is required for differentiation into different retinal cell types, how are these different types specified?

Answer 46

Pax6 works in concert with other downstream TFs at different times to create all the different cell fates

Question 47

What are the 2 categories of downstream TFs that Pax6 (and other related molecules) activate to induce different retinal cell fates?

Answer 47

Basic helix loop helix Homeodomain TFs

Question 48

What does crispr stand for?

Answer 48

Clustered regularly-interspaced short palindromic repeats

Question 49

Where does crispr come from?

Answer 49

Bacterial adaptive immunity system