title: 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 ENU-induced 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.
authors.affiliation: Laboratory of Developmental Biology, National Heart Lung and Blood Institute, Bethesda, MD 20892-1583, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.forename: Deanne
authors.initials: D
authors.lastname: Alpert
authors.forename: Richard
authors.initials: R
authors.lastname: Francis
authors.forename: Bishwanath
authors.initials: B
authors.lastname: Chatterjee
authors.forename: Qing
authors.initials: Q
authors.lastname: Yu
authors.forename: Terry
authors.initials: T
authors.lastname: Tansey
authors.forename: Steven L
authors.initials: SL
authors.lastname: Sabol
authors.forename: Cheng
authors.initials: C
authors.lastname: Cui
authors.forename: Yongli
authors.initials: Y
authors.lastname: Bai
authors.forename: Maxim
authors.initials: M
authors.lastname: Koriabine
authors.forename: Yuko
authors.initials: Y
authors.lastname: Yoshinaga
authors.forename: Jan-Fang
authors.initials: JF
authors.lastname: Cheng
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.forename: Joel
authors.initials: J
authors.lastname: Martin
authors.forename: Wendy
authors.initials: W
authors.lastname: Schackwitz
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Gunn
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Kramer
authors.forename: Pieter J
authors.initials: PJ
authors.lastname: De Jong
authors.forename: Len A
authors.initials: LA
authors.lastname: Pennacchio
authors.forename: Cecilia W
authors.initials: CW
authors.lastname: Lo
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019521:Body Patterning; D002478:Cells, Cultured; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D005038:Ethylnitrosourea / Q000494:pharmacology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D055457:Nodal Protein / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C539500:Megf8 protein, zebrafish; D008565:Membrane Proteins; D055457:Nodal Protein; D029961:Zebrafish Proteins; D005038:Ethylnitrosourea