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

Abstract

Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENUinduced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8 C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.cardiogenesis ͉ left-right ͉ nodal

Large-scale identification of chemically induced mutations in Drosophila melanogaster

“…Thus far, several proof-of-principle studies, each applying different approaches to successfully map a chemically induced mutation using WGS, have been documented in the literature (Sarin et al 2008Blumenstiel et al 2009;Zhang et al 2009;Earley and Jones 2011;Fairfield et al 2011;Andrews et al 2012;Bull et al 2013). In general, a subset of SNVs is first removed based on assay-specific criteria upon which some form of mapping is performed to reduce the number of candidate mutations.…”

Revealing ETC-1922159 Affected Unknown 3<em>^rd</em> order WNT10B-X-X Combinations, in Silico

“…Tsukamoto et al 150 observe overexpression of CLN1 in colorectal cancer which encodes PPT for removal of fatty acids from fattyacylated cysteineresidues in proteins 148 . Consistent with these, ETC-1922159 treatment of colorectal cancer cells lead to the the down regulation of PPT1 and the pipeline indicates this with a low rank of 91.Cortactin-binding protein 2 is encoded by CTTNBP2 and has been found to be up regulated in APC driven tumorigenesis Gaspar et al 151 154 . MEGF8 has been found to be significantly associated with colorectal cancer cases in 155 .…”

“…Cortactin-binding protein 2 is encoded by CTTNBP2 and has been found to be up regulated in APC driven tumorigenesis Gaspar et al 151 154 . MEGF8 has been found to be significantly associated with colorectal cancer cases in 155 .…”

“…MEGF8 encoded Multiple Epidermal Growth Factor-like Domains 8, is a single pass membrane protein that facilitates cell communication and developmental regulation 154 . MEGF8 has been found to be significantly associated with colorectal cancer cases in 155 .…”

<em>Ex vivo</em> Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

“…The simple protocol developed for imaging the mouse airway epithelia provides an efficient method to interrogate airway cilia motility in mutant and knockout mouse models and require only basic skills in mouse tracheal tissue dissection and ex vivo imaging of airway cilia motility with high resolution videomicroscopy. This protocol was established and refined during a large-scale mouse mutagenesis screen to allow rapid evaluation of motile cilia function (cilia beat frequency, cilia beat shape, cilia generated flow) in mutants with congenital heart disease associated with heterotaxy [2][3][4][5] .…”