Methods in Development

Question 1

State how development was anatomically described in the past.

Answer 1

• Descriptive embryology
• Normal morphology and histology
• Fate mapping.

(All processes used today.)

Question 2

State how development was experimentally studied in the past.

Answer 2

• Grafting

- Treatment with substances that affect normal embryogenesis

Question 3

List how descriptive anatomy is produced today

Answer 3

֍ Light and electron microscopy- can see formation and organization of tissues, even at sub-cellular resolution with e-
֍ Histological sections or whole specimens
֍ We can label cellular/subcellular structures with fluorescent tags
֍ We can follow developmental events over time e.g. using transgenics e.g. growing blood vessels in zebrafish embryo
֍ Zebrafish antibody labelled for tubulin and SV2 expressed in central nervous system, antibodies attached to fluorescent tags so we can now analyze distribution of these proteins with a lot of detail in the developing tissue.
֍ Can label individual cells e.g. 1 mechanism has been labelled in a cell, revealing huge complexity of the dendritic projections in these type of cells

Question 4

What is grafting and what are the 2 different types?

Answer 4

Moving a piece of a tissue from one embryo to another.

• Orthotopic - same place
• Heterotopic - different place

Question 5

What experiment led to the discovery of induction?

Answer 5

Spemann and Mangold 1923. The discovery of the Spemann-Mangold organizer introduced the concept of induction in embryonic development. Now integral to the field of developmental biology, induction is the process by which the identity of certain cells influences the developmental fate of surrounding cells.

Question 6

Define: Fate

Answer 6

What will normally happen to a cell during development.

Question 7

What does fate mapping and lineage analysis help us to do?

Answer 7

• Defines patterns of cell migration

* Defines origins of cells in formed structures

Question 8

What are the chemical ways we can do fate mapping?

Answer 8

• Chemical markers
– Vital dyes (Nile blue sulphate - surface)
– Radiolabel (nucleotides) – not used very much anymore
– Carbocyanine dyes (DiI, DiO)
– Fluorescent dextrans (fluorescein, rhodamine) – very much used
– Enzymes (Horseradish peroxidase)

Question 9

What are the genetic ways we can do fate mapping?

Answer 9

• Genetic markers (GFP and its variants, beta-galactosidase)
– Retroviruses
– Chimeras
– Transgenics
– Integrate fragments of DNA encoding for fluorescent proteins into the genome of the embryos where we want to do fate mapping. Normally the fragments integrate and follow the regulatory instructions of the genome around them, leading to them being expressed in certain tissues/ cells as the embryo develops  can follow structures over time.

Question 10

How can we do photoconversion in fate mapping?

Answer 10

Transgenic zebrafish embryos express Kaede, fluorescent protein which come from coral. Normally glows green, upon illumination with UV light the protein will change confirmation and start glowing red. Great advantage for fate mapping. E.g. can illuminate whole zebrafish with kaede, then UV light a small portion of cells so they go red and follow those red cells over time.

Question 11

What is retrospective fate mapping?

Answer 11

start at the end point of some development, label all cells in the embryo with green fluorescent protein. Run movie backwards to follow the labelled cells to see where they come from. Could use to follow the derivatives, see where any structure in the mature embryo come from.

Question 12

What is chimeric fate mapping?

Answer 12

Grafting a piece of tissue from one species onto another e.g. quail onto chick.
Can use differing antibodies to differentiate
Can use DNA organisation to differentiate as quail DNA is more condensed around nucleioli

Question 13

What is a variation of traditional chimeric fate mapping?

Answer 13

Variation of this technique: dye labelling and grafting. Use 2 embryos from the same species, one with dye labelling. Graft dye-labelled embryo tissue into unlabelled embryo and follow growth.

Question 14

What can grafting tissues help us do?

Answer 14

Tells us things like were the cells already committed/ specified to a cell fate or were they still plastic enough to integrate into another part of the embryo and do whatever the new embryo was doing in new region.

Question 15

What is competence?

Answer 15

the ability of a tissue to respond to an inductive signal

Question 16

What is potential?

Answer 16

The ability to respond to an inductive signal. Different potential: parts of the embryo may respond differently to the same inductive signal

Question 17

What affect’s a cell’s potential?

Answer 17

• Cells often become progressively more restricted in their potential during development – eventually will unipotent
• Restriction in potential often depends on inductive interactions from neighbouring cells

Question 18

What’s a morphogen?

Answer 18

A morphogen is a substance whose non-uniform distribution governs the pattern of tissue development, establishing positions of the various specialized cell types within a tissue.

Question 19

Give an example of morphogenesis during development?

Answer 19

Activin is expressed in vegetal cells is inducing mesodermal fates in the immediately adjacent animal cap cells. However, activin isn’t expressed equally in all vegetal cells, it generates a dorsal- ventral gradient in the vegetal pole of the cells so higher levels of activin are received by the cells adjacent to the vegetal pole in the dorsal region, and lower levels by the cells in the opposite pole of the embryo.

Dorsal portion: induced muscle and notochord tissues.
Ventral portion: induced blood derivatives

Question 20

How can we decide if there’s been a change in phenotypic outcome?

Answer 20

• Morphological defects e.g. defects in central nervous system
• Changes in other genes’ expression: look in F3+
• Changes in the expression of a transgene highlighting a particular tissue/cell type – transgenics

Question 21

What genetic methods can we use to identify genes controlling development?

Answer 21

– Mutagenesis screens (forward genetic screens) – Random mutagenesis and selection by phenotypic analysis – select genes that may affect particular organs/ developmental processes but we generate the mutants by random mutagenesis, see phenotype then go back and see what caused it
– Reverse genetics - Mutation of specific DNA sequences and analysing the phenotype
– Transgenics
- both by CRISPR, ZFN, TALENS

Question 22

What molecular methods can we use to identify genes controlling development?

Answer 22

– Methods to identify genes with restricted expression patterns in the embryo and genetic interactions

Question 23

What are ZFNs and TALENS?

Answer 23

• ZFNs: zinc finger nucleases
• TALENs: transcription activator-like effector nucleases
• Proteins that recognise specific DNA sequences, attached to an endonuclease domain
• Are a pair, read target from both sides.
• Once target recognised, will act like a pair of scissors and will make a cut in DNA, and the cell machinery will do the rest by improperly repairing the defect, inserting some sort of mutation.

Question 24

Describe the process of CRISPR?

Answer 24

1. Design a guide RNA (oligonucleotide)

2.