Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy

Area of Science:

• Developmental Biology
• Genetics
• Molecular Biology

Background:

• Forward genetic screens using N-ethyl-N-nitrosourea (ENU) mutagenesis are powerful for gene discovery.
• Identifying specific mutations after ENU mutagenesis can be challenging.

Purpose of the Study:

• To identify a gene responsible for heterotaxy and congenital heart defects using ENU mutagenesis and next-generation sequencing.
• To elucidate the function of the identified gene, Megf8, in embryonic left-right patterning.

Main Methods:

• ENU mutagenesis in mice to induce mutations.
• Positional cloning and massively parallel DNA sequencing to identify the causative mutation.
• Morpholino knockdown in zebrafish embryos.
• Confocal microscopy and videomicroscopy in mouse mutants.

Main Results:

• A novel ENU-induced mutation causing heterotaxy and heart defects was mapped to a 2.2-Mb interval on mouse chromosome 7.
• Massively parallel sequencing identified a mutation in the gene Megf8 (C193R).
• Megf8 knockdown in zebrafish caused heterotaxy; mouse mutants exhibited defects in Nodal signaling propagation without affecting nodal cilia motility.
• Megf8 protein was found to translocate to the nucleus and colocalize with chromatin remodeling proteins Gfi1b and Baf60C.

Conclusions:

• Megf8 is an essential regulator of embryonic left-right patterning.
• The study demonstrates the utility of massively parallel sequencing for identifying ENU-induced mutations.
• Megf8's nuclear localization and interaction with chromatin remodelers suggest a novel mechanism in developmental gene regulation.