Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy.
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.
Zhang
Zhen
Z
Laboratory of Developmental Biology, National Heart Lung and Blood Institute, Bethesda, MD 20892-1583, USA.
Alpert
Deanne
D
Francis
Richard
R
Chatterjee
Bishwanath
B
Yu
Qing
Q
Tansey
Terry
T
Sabol
Steven L
SL
Cui
Cheng
C
Bai
Yongli
Y
Koriabine
Maxim
M
Yoshinaga
Yuko
Y
Cheng
Jan-Fang
JF
Chen
Feng
F
Martin
Joel
J
Schackwitz
Wendy
W
Gunn
Teresa M
TM
Kramer
Kenneth L
KL
De Jong
Pieter J
PJ
Pennacchio
Len A
LA
Lo
Cecilia W
CW
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*
C539500:Megf8 protein, zebrafish; D008565:Membrane Proteins; D055457:Nodal Protein; D029961:Zebrafish Proteins; D005038:Ethylnitrosourea