Methods in Development

Question 1

How did we study development in the past, anatomically and experimentally?

Answer 1

1. Anatomical
   a. Descriptive embryology
   b. Normal morphology and histology
   c. Fate mapping
2. Experimental
   a. Grafting
   b. Treatment with substances that affect normal embryogenesis

Question 2

What are the benefits of using light microscopy?

Answer 2

Allows us to analyse specimens with good detail and resolution

Question 3

What can we do with fluorescent tags?

Answer 3

• We can label cellular/subcellular structures with fluorescent tags
• We can follow developmental events over time

Question 4

What is fate mapping?

Answer 4

• Following cells to see where they end up (their fate)
• Defines patterns of cell migration
• Defines origins of cells in formed structures

Question 5

Give some examples of markers/labels to follow cell fate

Give some genetic markers

Answer 5

o Chemical markers
o Vital dyes (Nile blue sulphate - surface)
o Radiolabel (nucleotides)
o Carbocyanine dyes (DiI, DiO)
o Fluorescent dextrans (fluorescein, rhodamine)
o Enzymes (Horseradish peroxidase)

• Genetic markers - integration of GFP and its variants, beta-galactosidase into:
o Retroviruses
o Chimeras
o Transgenics

Question 6

What is prospective cell mapping?

Answer 6

When we label cells in the early embryo and see how they develop

Question 7

How do we graft a small piece of quail embryo into a chick embryo?

How can we identify the tissue we grafted?

Answer 7

• This graft of cells will integrate into the tissue and will give rise to structures in a normal way
• Quail and chick cells are not identical, and there are some proteins that are expressed in quail cells and not chick cells. We can use this to our advantage by using antibodies that bind to quail cells over chick cells to find the quail cells in our chick embryos
• This allows us to determine the cell fate
• Another way of detecting quail cells is by looking at the organisation of there DNA as they usually have condensed DNA that is not present in chick cells.

Question 8

How does the notochord induce formation of neurones in the neural tube?

What happens when we graft the notochord lateral to the neural tube?

Define competence

Answer 8

• There is an important group of cells forming a structure called the notochord for neural tube patterning
• The neural tube is a tissue that runs along the dorsal midline of the vertebrae embryo and underneath the neural tube lays a structure that runs along the posterior axis called the notochord
• The notochord is a source of signals that has inductive properties that is important to establish the fate of the neurones that are derived from ventral portion of the developing neural tube
• This was shown in experiments where a piece of notochord was grafted to the lateral side of the embryo which was able to induce the formation of ventral fated neurones out of the lateral portion of the neural tube
• Competence is the ability to respond to an inductive effect, these experiments show us that the whole neural tube is competent to respond to the signals from the notochord that form under the inductive process
• Highlights that different tissues respond differently to inductive signals

Question 9

Define competence

Describe competence in relation to the neural tube

Answer 9

Competence: ability to respond to an inductive signal

2. The neural tube is competent to respond to the inductive signal released by the notochord by generating ventrally fated neurones
3. Other parts of the embryo may respond differently to the same inductive signal by generating different types of cells – different potential

Question 10

Do cells become more restricted in there potential during development?

What does restriction depend on?

Answer 10

Yes,

Restriction in potential often depends on inductive interactions from neighbouring cells

Question 11

Explore induction, competence and potential in an experiment

What are explants and what do they show us?

What happens when 2 explants are put together?

Answer 11

• This is a blastula xenopus embryo
• The vegetal cells will form the endoderm and the animal cap cells will give rise to the ectoderm and mesoderm cells
• By taking grafts from this blastula and growing them as explants we can see what region gives rise to which cell type.
• Scientists also looked at what happens when two explants are put together, they took vegetal cells and placed them next to the animal cap cells
• They found that a portion of cells from the animal cap where giving rise to mesodermal derivatives
• The vegetal tissue is the source of a mesoderm-inductive signal
• Normally it would only give rise to ectoderm derivatives

They went even further and combined different portions of the vegetal part of the embryo with the animal cap

They found dorsal and ventral have different inductive properties and give rise to different derivatives

Question 12

What is activin?

Answer 12

• Activin (morphogen) is the mesoderm inducing signal and it works in a concentration-dependent way

Question 13

What does high and low concentrations of activins give form?

Answer 13

high concentrations would give rise to dorsal mesodermal derivatives while low concentrations would induce ventral mesodermal derivatives

Question 14

What does activin do in the developing embryo?

Answer 14

Activin expressed in vegetal cells is inducing mesodermal cell fates from the animal cap cells

Activin generates a dorsal and ventral gradient so higher levels of activin are received by cells in the dorsal region and and low levels are reicieved by ventral region

Question 15

How can we identify genes that control development?

Answer 15

• Genetic analysis
o Mutagenesis screens (forward genetics) – Random mutagenesis and selection by phenotypic analysis
o Reverse genetics - Mutation of specific DNA sequences and analysing the phenotype
o Transgenics
• Molecular methods
o Methods to identify genes with restricted expression patterns in the embryo and genetic interactions

Question 16

How can gene lines be analysed?

Answer 16

o complementation analysis
o genetic mapping
o positional cloning

Question 17

How do we identify phenotypes?

Answer 17

• Morphological defects
• Changes in other genes’ expression
• Changes in the expression of a transgene highlighting a particular tissue/cell type

Question 18

How is forward genetics different to reverse?

Answer 18

• Reverse genetics allows us to generate specific mutations in particular regions of the genome using molecular techniques

forward is random

Question 19

How is a reverse genetic mutation made?

What are the 3 main tools used?

Answer 19

• A cut is made in the specific region of the genome
• This will activate repair machinery in the cell which is not very efficient as it causes mistakes to be made
• This results in a mutation being inserted in the region that has been targeted by the editing tools
• There are three main tools:
o ZFNs
o TALENs
o CRISPRs

Question 20

What are ZFNs and TALENS?

Answer 20

• ZFNs: zinc finger nucleases

* TALENs: transcription activator-like effector nucleases

Question 21

How do ZFNs and TALENS work?

Answer 21

• We design these proteins that recognise specific DNA sequences and are attached to an endonuclease domain
• they design the proteins in pairs so they can recognise sequences on both sides of the sequence
• The proteins will recognise the targets and the endonucleases will work as a pair of scissors and cut
• The repair machinery will then follow

Question 22

What does the CRISPR-Cas9 tool do?

Answer 22

Induces mutations

Question 23

How does CRISPR-Cas9 work?

Answer 23

1. The target specific sequence is an oligonucleotide we have developed that will recognise the specific sequence we want to target. It is bound to another RNA (tracr) which allows the RNA to assemble to Cas9 (this has endonuclease activity).
2. This complex will target regions in the genome that is complementary to our guide RNA.
3. Repair mechanism imprecisely form mutations in the genome
4. We then analyse the phenotypic outcome to see if the technique has worked.

Question 24

How can we tell if our mutations have worked/ done anything?

Answer 24

• Morphological defects
• Changes in other genes’ expression
• Changes in the expression of a transgene highlighting a particular tissue/cell type

Question 25

How can we determine the exact expression domain within an embryo?

Answer 25

• Determining the exact expression domains within the embryo o in situ hybridisation to detect mRNA o Immunohistochemistry to detect protein

Question 26

How does In situ hybridisation and immunostaining work?

Answer 26

1. The gene is first transcribed to generate Mrna 2. It will then be translated to form a protein 3. In situ hybridisation detects the distribution of the mrna or proteins which allows us to determine which tissues are expressing a particular gene. In situ hybridisation – we design a probe (an RNA molecule) which is complementary to a fragment of the mrna (that we want to identify and detect). We generate this probe using modified nucleotides (which are attatched to a molecule called dioxychain). We can then use anitbodies that can detect dioxychains. So we put the probe into our tissue and bind to the mrna and we then incubate the tissue with an anitbody that recognises dioxychain. The anitbody is usually conjugated with an enzyme called alkaline phosphotase (forms a coloured precipitate). Immunohistochemistry – this detects the presence of a particular protein instead of mrna. We use antibodies that have been designed to recognise and bind to the protein that we want to detect. We first incubate our tissue with this primary antibody that will bind to the protein wherever it is accumulating. We then use a secondary antibody that is direct to the first antibody that is either attached to AP or fluroscent tags to form a coloured precipitate AP = alkaline phosphotase

Question 27

What is subtractive hybridization? What does this allow us to do?

Answer 27

Subtractive hybridization is a technique used to isolate a DNA segment that is missing from one particular sample of DNA. * Identify differences in gene expression between regions of the embryo * Identify differences in gene expression between normal and mutant embryos

Question 28

How is subtractive hybridization performed?

Answer 28

* Isolate the mRNA from the two samples * We then retrotranscribe this to make cDNA and modify one sample eg tagging using biotin * Hybridise the two samples of DNA * Remove the genes common to both samples (in this example, using streptavidin) * Isolate remaining genes = then identify them

Question 29

How does microarray work?

Answer 29

1. We isolate the mrna 2. Retrotranscribe the mrna into cdna and also label with a fluorescent tags (red and green) 3. We then combine the targets, we hybridise to a microarray (a slide full of wells, in each there are DNA fragments that represent one particular gene) 4. In each well is a particular gene 5. One colour indicates one gene and the other indicates the other 6. If the represented in the well is represented in our probe then our probs will bind to it and flurores

Question 30

How does RNAseq work?

Answer 30

1. We start with two different tissues/ embryos 2. We isolate the rna from the tissues 3. Generate cdna fragements and then fragment it with linkers added to every single fragment of cdna 4. We then sequence the fragments 5. We can then assemble the reads we get (exome) (transcriptome) 6. We can then analyse and compare the levels of expression of each expression from each conditions and compare levels of expression of every gene in our sample

Question 31

What is an activator cascade?

Answer 31

Working out gene order by genetic epistasis: • This allows us to work out the order of genes working in the same pathway • We have a sequence of molecules that regulate each other to produce a segment of an organism • If we have mutant with a lack of B we can see what we need to add to rescue the formation of the segment • This is called an activator cascade

Question 32

Genetic epistasis in repressive pathways: comparing opposite phenotypes between single and double mutants

Answer 32

On image

Question 33

What is a mis-expression study? Give an example

Answer 33

* Express a gene in a region of the embryo (or at a time) where it is normally not expressed * Allows us to study the effect on development and/or expression of other genes Cell transplantation: • We will transplant a group of cells expressing a particular gene in a region of an embryo where the gene is not expressed • We normally inject mrna with a green fluorescent protein attached or something that allows us to label the cells • We then transplant the cells into a host embryo, which might repress the formation of a structure such as the eye which tells us that the absence of the gene is needed for eye formation

Question 34

How can we cause mis expression of proteins with TFs?

Answer 34

Take a repressor TF from one gene and inject it into another gene and observe changes

Question 35

How can we alter molecular pathways at a signalling level

Answer 35

Dominant negative Constitutively active