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.

Fig. 1.

Recovery of Megf8 Mutation (A). A 15 BAC contig was obtained spanning the 2.2-Mb critical region containing the mutation in chromosome 7 (25, 837,390–28,028,384 b, NCBI m37 assembly). Sequencing showed a T to C substitution, causing cysteine (C) to arginine (R) amino acid replacement in Megf8, and another C to A substitution in a noncoding intergenic region. (B) Megf8 encodes a protein containing EGF, EGF-calcium, EGF-like, EGF-laminin, kelch, plexin, and CUB domains, with the C193R substitution situated in the second conserved EGF domain. The mutated cysteine is highlighted in the comparative sequence alignment.

Fig. 2.

Morpholino knockdown of Megf8 causes situs discordance of heart tube and foregut. Zebrafish embryos injected with control or Megf8 morpholinos were examined for heart and gut situs defects using RNA in situ hybridization analysis with Nkx2.5 (A–D) and Foxa3 (E–H) probes to visualize the heart tube and gut, respectively. In the majority of zebrafish embryos injected with control morpholino, the heart tube exhibited normal rightward looping delineating a S-shaped heart tube (A), whereas the foregut showed normal leftward looping (E). Megf8 morpholino injection resulted in randomization of the direction of looping for both the heart tube (B–D) and foregut (F–H). Megf8 morphants show normal (B and F), reduced or no looping (C and G), or reversed looping (D and H). (I) The distribution of heart and foregut looping pattern observed in the control and Megf8 morphants is summarized in the bar graph. Note that 74% of Megf8 morphants show heterotaxy, as indicated by discordance in their heart and gut situs. (A–D) Embryos shown in ventral view with anterior at the top. (E–H) Embryos shown in ventral view with anterior at the top. (Scale bar: 100 μm.)

Fig. 3.

In situ hybridization analysis show altered expression of left determinant genes in Megf8^m/m embryos. (A and B) Nodal is expressed at the node (arrowhead) and in the left LPM (arrow) in 4 somite stage wild-type embryo (A). In Megf8^m/m embryos, Nodal expression was preserved at the node (arrowhead), but absent in the LPM. (C and D) In wild-type embryos, Lefty1 and 2 are normally expressed in the floor plate (arrowhead) and left LPM (arrow), respectively (C), but, in Megf8^m/m mutants, expression of both Lefty 1 and 2 are lost (D). (E and F) Cryptic expression in the node and LPM show no change in Megf8^m/m mutants (F) vs. wild-type (E) embryos. (G–I). Wildtype embryos show Pitx2 expression in the left LPM (arrow in G), but this is either lost (H) or bilateral (arrowheads in I) in Megf8^m/m mutant embryos. Pitx2 expression in the head mesenchyme is unchanged. (J and K). Gdf1 expression in the node and LPM is not affected in Megf8^m/m (K) vs. wild-type (J) embryos. (A, B, E, and F) Dorsal view. (C, D, and G–J) Ventral view. WT, wild-type; left (L)–right (R) orientation indicated by double arrowheads. (Scale bar: 100 μm.)

Fig. 4.

Distribution of Megf8 in embryos and cultured cells. (A–C). Immunostaining with Megf8 antibody of an embryo at E8 show ubiquitous Megf8 expression. Regions 1 and 2 in A are enlarged in B and C. Punctate staining is observed in the nuclei (arrowheads in B and C), and there is also diffuse cytoplasmic staining. (D–F). Confocal imaging of MEFs (D and E) or NIH 3T3 (F) cells immnostainined with a Megf8 antibody show Megf8 distributed as punctate spots in the nuclei, largely in regions with low level DAPI staining. Coimmunostaining with Gfi1b antibody showed extensive colocalization of Megf8 with Gfi1b (D and E). (G–J). Confocal imaging of MEFs immunostained with antibodies to Megf8 (red), Gfi1b (green), and Baf60C (blue) showed some regions in the nucleus where all 3 proteins are colocalized in punctate spots (see arrowhead).