****** The image of graphs is not part of the question You found a new zebrafish
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You found a new zebrafish mutant that suffers from microphthalmia (small eyes). You want to determine what is wrong with these eyes. Your two hypotheses are A) excessive cell death has lead to the microphthalmia phenotype or B) reduced cell proliferation during development has lead to the microphthalmia phenotype. 1) What experiment (technique) would you use to test each hypothesis? 2) How would you interpret the results (If I saw "X" I would conclude "Y")?Explanation / Answer
Microphthalmia is a developmental abnormality of eyes that arises before birth. Eyes are abnormally small and they have malformation in this disorder.
The affected individuals may have unilateral microphthalmia (single eye appears small) or bilateral microphthalmia (both the eyes are affected). Most extreme condition leads to anophthalmia (absence of eyes).
Zebra fish are usually used as experimental animal models to study the genetic causes of this abnormality.
As microphthalmia is a genetic disorder that occur due to chromosomal abnormality, the genetic screening has to be done at different stages of embryogenesis.
Microphthalmia results from mutations in genes during embryogenesis. Hence you have to carry out experiments at several stages of embryogenesis and find out the reason for this disorder.
Hypothesis A: To study that excessive cell death lead to microphthalmia phenotype:
Excessive exposure to the chemicals or drugs during embryogenesis causes cell death. The excessive cell death may some times lead to congenital malformations.
Teratogen induced apoptosis study is a technique to study this hypothesis.
For example, you may induce p53 dependent apoptosis by using teratogens (an agent that causes malformation in embryo development). p53 is a gene that regulates cell death or apoptosis.
Expose the early embryo to teratogenic stress. Study the extent of p53 expression at the early stage of embryo by RNA isolation and blotting techniques In-vitro.
Carryout teratogenic evaluation studies with zebra fish animal models using the techniques like gene allelotyping, immunostaing , TUNEL assay. TUNEL assay helps in detecting apoptotic cells that undergo extensive DNA degradation during apoptosis.
You can coclude that microphthalmia phenotype is a result of excessive cell death if apoptosis induced by the teratogen treatment.
X- Apoptosis induced by teratogenic treatment; Y- Excessive cell death lead to microphthalmia phenotype.
Hypothesis B: Reduced cell proliferation during embryogenesis lead to microphthalmia phenotype:
There are reports proving that reduced cell proliferation during embryogenesis may not result in microphthalmia in zebra fish.
You may expose the early embryo to chemicals like ethanol and study its effect on microphthalmia phenotype.
Analyse the cells by TUNEL assay by dying the cells.
X- Cell death not observed (If the number of TUNEL positive cells is not high); but still resulted in microphthalmia phenotype; Y- Reduced cell proliferation during embryogenesis does not lead to microphthalmia phenotype
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