Methods In Development

Question 1

What is fate mapping and why is it important?

Answer 1

fate mapping = process of tracing the developmental outcome of specific cells or regions in an embryo

• helps us understand how cells contribute to tissues/organs during development
• insights into cell fate and lineage tracing

Question 2

How are genetic markers used in fate mapping?

Answer 2

genetic markers (e.g. GFP and beta-galactosidase (lacZ))
- label cells in transgenic organisms
- enables continuous tracking of cells over time
- retroviruses and chimeras (e.g., chick-quail) can also trace cell lineages and developmental paths in embryos

Question 3

common chemical markers used in fate mapping?

Answer 3

vital dyes - label surface cells for tracking tissue development
radiolabelling - labels DNA; study cell division & migration
enzymes - histological analysis
carbocyanine dyes - tracks membrane-bound cells (neuronal studies)
fluorescent dextrans - live imaging of cells

Question 4

What is photoconversion and how does it aid in fate mapping?

Answer 4

photoconversion = involves a fluorescent protein that changes colour when exposed to specific light wavelengths
- allows precise tracking of labelled cells over time
- can study cell lineage and migration

Question 5

How do chimeras help in fate mapping studies?

Answer 5

chimeras = organisms made from cells of two embryos, often from different species (e.g., chick-quail chimeras)
- can trace how cells from one organism contribute to development in another
- insights into cell migration and developmental interactions

Question 6

What are transgenics? How do transgenics contribute to fate mapping?

Answer 6

transgenics = organisms genetically modified to stably express foreign DNA (e.g. GFP reporter genes) - allows long-term, inheritable labelling of specific cells/tissues
- visual tracking of cell migration, development contributions, differentiation
- high-resolution live imaging

Question 7

What is grafting and how is it used in fate mapping?

Answer 7

grafting = transplanting a piece of tissue from one embryo to another
- orthotopic (same location) or heterotopic (different location)
- helps study how tissues behave when placed in different environments & tracks their fate in development

Question 8

What are the classical approaches in descriptive embryology?

Answer 8

• light microscopy (gross morphology)
• electron microscopy (ultrastructural details
• histology (tissue organization)
• fluorescent labelling (live tracking of cells)

provide insights into normal embryonic development and tissue differentiation

Question 9

pros of fate mapping?

Answer 9

• continuous tracking of individual cells or tissue regions over time (e.g., GFP, photoconversion)
• versatile across species and stages of development
• allows specific lineage tracing with genetic markers

Question 10

cons of fate mapping?

Answer 10

1. limited resolution (e.g., dye labelling) = not always high-resolution enough to track single cells in complex tissues
2. short-term vs. long-term tracking = some techniques (vital dyes) are useful only for short-term studies; others (transgenics) better for long-term studies but are more technically challenging
3. potential off-target effects with genetic manipulation

Question 11

What is forward genetics and how is it used to study development?

Answer 11

forward genetics = creating random mutations (by chemicals or radiation) and screening embryos for phenotypic changes

• mutants with abnormal development are isolated = mutated gene identified
• helps discover unknown genes involved in development

Question 12

What is reverse genetics and how does it help study developmental genes?

Answer 12

reverse genetics = known gene mutated/deleted deliberately; resulting phenotype analysed
- directly tests a gene’s specific role in development

Question 13

What are mutagenesis screens and what do they reveal?

Answer 13

mutagenesis screens (forward genetics) = induce random mutations and screening embryos for developmental defects
- reveal genes that are essential for normal embryogenesis

Question 14

how are ZFNs and TALENs used for targeted genome editing in reverse genetics?

Answer 14

consist of a DNA binding domain & cutting domain
- DNA binding domains bind to specific DNA sequences
- cutting domains cut DNA
- cells tries to repair DNA itself but makes mistakes - insertions/mutations

Question 15

How does CRISPR-Cas9 work in gene editing?

Answer 15

• guide RNA leads Cas9 enzyme to a specific DNA sequence - makes a cut
• cell repairs the break via NHEJ (non-homologous end joining; error-prone, knockouts) OR HDR (homology-directed repair; precise edits if a template is given)
• type of repair allows targeted gene disruption/modification

Question 16

how do forward and reverse genetics fundamentally differ?

Answer 16

forward genetics - start with phenotype, find the gene

reverse genetics - start with known gene, create mutation/deletion, observe phenotype

Question 17

pros and cons of CRISPR-Cas9?

Answer 17

pros:
- fast, cheap, efficient
- targets many genes at once

cons:
- off-target effects with unwanted cuts
- not all cells are edited; mosaicism
- HDR/ homology-directed repair is less efficient despite more precise edits

Question 18

What is subtractive hybridisation and what is it used for?

Answer 18

subtractive hybridisation = compares two mRNA samples (e.g. normal vs mutant) to remove common genes and isolate genes differentially expressed

helps identify genes specific to a tissue/condition

Question 19

how does subtractive hybridisation work?

Answer 19

1. isolate mRNA from two samples.
2. make cDNA and tag one sample (e.g., biotin).
3. hybridise together — shared sequences bind.
4. remove common sequences (e.g., via streptavidin).
5. remaining unbound cDNA = differentially expressed genes.

Question 20

How can we study if genes function in the same molecular pathway?

Answer 20

• analysing gene expression patterns
• performing misexpression studies (gain/loss of function)
• conducting genetic epistasis tests

helps infer the order and interaction of genes

Question 21

what is genetic epistasis? how is it used? how is genetic epistasis interpreted in repressive pathways?

Answer 21

genetic epistasis = studies the relationship between two genes by creating single and double mutants and analysing phenotypes

helps determine gene order in regulatory pathways

repressive pathways - if two genes repress each other, double mutants often show the phenotype of the gene acting last - comparing opposite phenotypes in single vs double mutants clarifies regulatory order

Question 22

What are misexpression studies? How do misexpression studies help understand gene function? (gain and loss of function)

Answer 22

misexpression = altering expression of a gene (level/ time/ location) - observe its effect on development & other genes’ expression

gain of function: force expression of a gene in a new place/time - reveals sufficiency (gene is enough on its own to cause a specific developmental process)

loss of function: knockout/ knockdown a gene - shows necessity for a process

Question 23

How can misexpression be achieved experimentally?

Answer 23

retroviruses
injected mRNA
transgenic animals carrying chimeric genes

Question 24

dominant-negative vs dominant-positive construct/ mutant receptor for studying gene expression?

Answer 24

dominant-negative = removes the intracellular signalling domain of a receptor - receptor still binds the ligand but blocks signal transduction; sequestering the ligand and causing a loss of function

dominant-positive = designed to signal even without a ligand; constant pathway activation leading to gain of function phenotypes