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. Megf8C193R 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.
| Original language | English |
|---|---|
| Pages (from-to) | 3219-3224 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 106 |
| Issue number | 9 |
| DOIs | |
| State | Published - Mar 3 2009 |
| Externally published | Yes |
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Cite this
Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy. / Zhang, Zhen; Alpert, Deanne; Francis, Richard; Chatterjee, Bishwanath; Yu, Qing; Tansey, Terry; Sabol, Steven L.; Cui, Cheng; Bai, Yongli; Koriabine, Maxim; Yoshinaga, Yuko; Cheng, Jan Fang; Chen, Feng; Martin, Joel; Schackwitz, Wendy; Gunn, Teresa M.; Kramer, Ken; De Jong, Pieter J.; Pennacchio, Len A.; Lo, Cecilia W.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 9, 03.03.2009, p. 3219-3224.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy
AU - Zhang, Zhen
AU - Alpert, Deanne
AU - Francis, Richard
AU - Chatterjee, Bishwanath
AU - Yu, Qing
AU - Tansey, Terry
AU - Sabol, Steven L.
AU - Cui, Cheng
AU - Bai, Yongli
AU - Koriabine, Maxim
AU - Yoshinaga, Yuko
AU - Cheng, Jan Fang
AU - Chen, Feng
AU - Martin, Joel
AU - Schackwitz, Wendy
AU - Gunn, Teresa M.
AU - Kramer, Ken
AU - De Jong, Pieter J.
AU - Pennacchio, Len A.
AU - Lo, Cecilia W.
PY - 2009/3/3
Y1 - 2009/3/3
N2 - 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. Megf8C193R 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.
AB - 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. Megf8C193R 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.
UR - http://www.scopus.com/inward/record.url?scp=62549105821&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=62549105821&partnerID=8YFLogxK
U2 - 10.1073/pnas.0813400106
DO - 10.1073/pnas.0813400106
M3 - Article
C2 - 19218456
AN - SCOPUS:62549105821
VL - 106
SP - 3219
EP - 3224
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 9
ER -