Lecture 1: Neural Development and Cell Biology

Question 1

How does a cell differentiate?

Answer 1

Differential gene expression causes a cell to differentiate. It terms when and where a gene will be expressed. Controlled by different mechanisms.

Question 2

What are the mechanisms behind cell differentiation?

Answer 2

Cell proliferation, cell specialization, cell interaction, and cell movement

Question 3

What is cell proliferation?

Answer 3

Cells multiplying due cell division

Question 4

What is cell interaction?

Answer 4

The way that cells talk to each other. If a cell is placed in the same scenario, it should react the same.

Question 5

What is cell specialization?

Answer 5

The identity of the cell and where they become competent in their cell type.

Question 6

What is cell movement?

Answer 6

How cells are able to morph and reorganize

Question 7

What is accomplished through the development process?

Answer 7

Pattern formation of cells, cells become specialized, the embryo morphs and changes its’ shape, and cells differentiate.

Question 8

How do cells become different?

Answer 8

Through different cascading mechanisms that are informed by the DNA found in the nucleus of the cell (i.e. SHH, Wnt).

Epigenetic control determines how, when and where the gene will be expressed.

Question 9

How do one cell become two different cells?

Answer 9

Autonomous Mechanism: asymmetrical cell division where daughter cells receive different contents from parent cell. Sister cells are different.

Conditional Mechanism: symmetrical cell division where one daughter cell becomes different because of an external stimuli after initial cell division from parent.

Question 10

How does the nervous system begin to form?

Answer 10

Through gastrulation the cells are rearranged into 3 cell (germ) layers: ectoderm, endoderm, and mesoderm. These cells can induce, respond, and signal to each other.

Question 11

What does the frog gastrulation model show us?

Answer 11

Demonstrates how cells divide, gastrulation, and where neural cells are situated on the blastula.

Used to identify the development of the different nervous system parts.

Egg -> cell division -> blastula -> gastrulation begins

Question 12

How was the location of the nervous system discovered?

Answer 12

The Organizer Experiment revealed that early neural cells where found at the dorsal lip of the blastopore region.

Question 13

What is the organizer experiment?

Answer 13

During gastrula phase, the piece above the blastopore (dorsal lip) was taken from a donor embryo and placed in a host embryo. A second nervous system was created because implanted cells induced cells in the host embryo.

Question 14

How would you probe a nervous system, pre gastrula?

Answer 14

You can’t. Mesodermal cells are needed. Isolate the animal cap and place in dish and you’ll only get epidermal tissue.

Question 15

What role does mesodermal tissue play in nervous tissue creation?

Answer 15

When you wait until gastrula, you get nerve tissue suggesting you need an inductive signal from the mesoderm to ectoderm.

Question 16

How would you test to make sure mesodermal cells are necessary for nerve tissue?

Answer 16

1. Extract mesodermal cells from pre gastrula so it never interacts with ectoderm. Observe if you get nerve tissue.
2. Take mesodermal tissue at different stages and add to extracted animal cap to see if you get nervous system.

Question 17

How did they identify which genes were neural tissue inducing?

Answer 17

Li and UV Light:
Embryo in UV Light -> no NS, ventralized embryo
Embryo in Li -> hyper dorsalized nervous system (big)

Best of both worlds: Extract PolyA mRNA that were expected to give rise to the protein required for NS. Took the mRNA and converted them into cDNA. PolyA mRNA was injected in UV embryo and correct NS defect. They located the noggin protein through in situ hybridization.

Question 18

How would you use in situ hybridization to locate a protein or signaling molecule?

Answer 18

Create a RNA probe that is complementary to the DNA sequence you are trying to locate. Inject the cDNA and see if it binds to the DNA or RNA of organism. The probe, if connected with target RNA, will react with color or fluorescent light. (NOGGIN).

Question 19

Where do you find noggin?

Answer 19

The signaling molecule is expressed in the organizer region, above the dorsal lip of the blastopore.

Question 20

What is cell signaling?

Answer 20

How a cell receives, interpret, and transmit signals with the environment. It involves reception, transduction, and response.

Receiving cell detects that there is a signal from the receptor to inside of the cell. Happens on the plasma membrane

Cell signaling cascade begins.

Transcription factors are involved, work to to change the expression.

Question 21

What is the TGF beta/Smad signaling pathway?

Answer 21

An example of cell signaling. TGF beta is a signaling molecule that binds to proteins on the cell membrane. TGF beta causes the dimers to interact and phosphorylate each other. Smad is downstream unphosophorylated. Smad is activated by phosphorylation and is able to move into the nucleus to begin transcription. This pathway gives rise to epidermal tissue.

Question 22

Explain BMP signaling.

Answer 22

BMP (Bone Morphogenic Protein) is present in ectodermal tissue that become epidermal cells. Through BMP signaling, chordin, noggin, and follistatin inhibit BMP, preventing the cascading pattern to occur.

When ectodermal cells are disassociated, you get neural tissue. When close together with BMP, you get epidermis. When disassociated and add back BMP, you get epidermis.

Question 23

What is induction?

Answer 23

When one group of cells sends signals to another group of cells in order to change the embryo.

Question 24

What is competence?

Answer 24

If a tissue can respond to another cell’s inducing signal.

Question 25

What does having competence require?

Answer 25

It needs transcription factors, receptor signaling molecule, and depends on the responding molecule.

Question 26

What is the role of transcription factors?

Answer 26

The regulate the transcription of the gene, contains information about how the gene will be expressed.

Question 27

What is the Wnt pathway responsible for?

Answer 27

The rostrocaudal formation. Wnt is secreted as a gradient and induces neural cells along the rostrocaudal axis and mid brain.

TF: Otx2 responds lower Wnt activity (Wnt inhibitors) and Gbx2 responds to increased levels of Wnt activity. Engrailed TF is expressed in the midbrain-hindbrain region.

Question 28

What is Fibroblast Growth Factor responsible for?

Answer 28

FGF gives rise to different cells, depends on which direction it is being secreted. (Dopaminergic and serotonergic).

Question 29

What are Hox genes?

Answer 29

(Homeobox genes) regulate cellular identity along the A/P axis. Where the gene is expressed correlates to where it is found on the body.

Question 30

How is cell fate determines along the dorsal/ventral axis?

Answer 30

Sonic the Hedgehog which is found in the notochord and floor plate.

Question 31

Explain SHH signaling.

Answer 31

SHH leads to the expression or activation of a gene. Once SHH molecule binds to protein, cascade begins creating a gradient of SHH along the D/V axis. In situ hybridization is used to locate it in the notochord (development of spinal cord region).

Question 32

How are gene expression borders sharpened?

Answer 32

Through mutual repression along neural tube. Two TF are antagonistic towards each other, slowing down the transcription of the other.

Question 33

Why is DNA regulation important?

Answer 33

So that cells can act and perform different from each other which is necessary for development. Allows cell to react (change) to their environment.

Question 34

What is pronuclear injection?

Answer 34

Introduce a gene from from another organism/animal to another. (Genetically modified organism)

Question 35

Why use pronuclear injection?

Answer 35

Look at gene function (gain in function). Observe how the gene is expressed, level of expression and how it is passed down.

Question 36

What is DNA Homologous Recombination?

Answer 36

Gene editing tool to examine the necessity of a gene. Facilitates gene knock out.

Question 37

What is Non Homologous End Joining ?

Answer 37

DNA repair where proteins trim off a few nucleotides (AT,CG) and fuses the broken ends back together. Not perfect.

(Think CRSPR/Cas9 tool) - takes advantage of this function because it can totally knock out a gene, suspending its function.

Question 38

Describe CRISPR/Cas9 and what it’s used for.

Answer 38

KNOCK OUT: A guide DNA (sgRNA) is created to hybridize to the target gene in the DNA. sgRNA is attached to Cas9, an enzyme protein (endonuclease), that then is taken to the target gene and Cas9 snips the target gene. Once cut, the cell will try to repair the blunt ends (NHEJ) . Because of its inconsistency, the gene is often turned off.

Helps to understand what happens in the organism when the target gene has been turned off or changed.

Question 39

How would you create neomycin resistance?

Answer 39

You could use DNA Homologous Recombination. Create a gene that is resistant to neomycin (NEO) and inject it into cells. If the gene is incorporated into the genome, you can place those cells in neomycin and observe which cells will die. Cells that remain express neomycin resistance and have successfully incorporated the gene.

Question 40

How would you knock in a gene?

Answer 40

Using CRISPR/CAS9. First, knock out the gene and then to put back its function, you could create the complementary RNA, and create a cassette to interject in the repair process to incorporate the gene.

Question 41

Cre/LoxP System

Answer 41

Helps to determine when and where a gene will be expressed. Cre recognizes loxP. Used to study later aspects.

Tamoxifen can induce Cre.

Question 42

Flourscent tags

Answer 42

They can be knocked in using Cas9 or pronuclear injection, naturally occurring sequences. If you wanted a specific protein, use the cre lox system.