L8 : Introduction to Zebrafish Model System Flashcards
Why were zebrafish initially investigated?
Primarily understood embyronic development through invertebrates including drosophila and C. elegans
Zebrafish used as vertebrate species parallel to drosophila
What are properties of the zebrafish model system?
- Small (30-40mm) : efficient use of space
- Tolerant
- Large egg clutches (normally ~200)
- Non-seasonal : eggs spawned daily, all year round
- Fast growing : from single cell to free swimming within week
- Almost transparent : can track individual cells
- External fertilisation : no opaque womb, easy access
- Easy to create transgenics
- Knock in/outs possible (mostly CRISPR/Cas9)
Note: ZFEs permeable to small molecules in water, allowing testing of drugs on vertebrate development
What is the midblastula transition?
Point in development at which waves of division become asynchronous
Occurs when zygotic transcription takes over from maternal transcription
What is a major driver behind studying development?
Cancer = temporally ectopic development
Cancer cells behave similarly to cells undergoing development
What are some problems with zebrafish as model organisms?
- Small embryos : some manipulations are difficult
- Slow maturing (~3 mo) : slow compared to flies/worms
- Sex determination not X/Y : often problematic sex ratios
- Not amniotes/mammals : often findings need further validation, no womb/amniotic fluid
- Duplicated genome : redundancy between chronologues
- Limited behavioural repertoire : more than flies/worms, less than rodents
What are the main techniques used in zebrafish studies?
- Microinjection of DNA, RNA, morpholinos etc.
- Cell transplantation
- Time lapse microscopy
- Individual cell tracking
- In situ hybridisation
- Immunohistochemisty
- Forward/reverse genetic screens
- Transgenics
What are 2 examples of how transgenetics is used in zebrafish?
GFP in subset of brain cells
Can be used dynamically, examining DCamp and indicating calcium influx in brain
What is gastrulation?
Formation of germ layers from undifferentiated embryonic blastula
Gastrula (3-layers) composed of: endoderm, ectoderm, mesoderm
By the end, differentiation into distinct cell lineages has begun
What is the process of gastrulation in zebrafish?
Starts at around 50% epiboly (% yolk covered) and after ~5 h at 28*c
- Cells ingressing at node form mesoderm, from which endoderm and mesoderm will form
- Enveloping layer cells and remaining superficial epiblast cells form ectoderm
What are the 5 stages of gastrulation?
- Invagination
- Involution
- Ingression
- Delamination
- Epiboly
