Question

You have recently identified a maternal effect mutation in Xenopus that results in a complete lack of endoderm. You map the mutation and find two genes with point mutations that change the amino acid sequence in the protein, but don’t know which mutation is causing the phenotype. Both genes have unknown functions. Design an experiment or series of experiments that would allow you to 1) discern which gene is the cause of the mutation, 2) determine if it most likely functioned as a cell autonomous determinant or morphogen, and 3) if this gene was sufficient promote endodermal fates

Answer 1

1. To determine the causative mutation, we can obtain mutants for each gene from the stock center and analyze their phenotypes. Or else, we can complement the double mutant with a WT copy of each gene. Whichever gene complements the mutant phenotype, that gene itself is responsible for the mutant phenotype.

2. To determine if the given protein is acting autonomous or non-cell autonomous manner, we can perform several experiments.
i. Prepare transcription reporter fusion and translation reporter fusion transgenics and observe their expression to see whether the protein is found in regions where the promoter is not active. This reveals information about the migration of the protein
ii. We have to cross-link the protein with a heavy tag such as 3X-GFP and restrict its migration/expression to a particular cell type and observe its ability to complement the mutant phenotype

3. Over express the given gene in other types of embryonic cells and observe the phenotype whether they are exhibiting the endodermal fate.
If so, it is sufficient to determine the endodermal fate.