Conserved requirement for EGF-CFC genes in vertebrate left-right axis formation

Abstract

Specification of the left-right (L-R) axis in the vertebrate embryo requires transfer of positional informationfrom the node to the periphery, resulting in asymmetric gene expression in the lateral plate mesoderm. We show that this activation of L-R lateral asymmetry requires the evolutionarily conserved activity of members of the EGF-CFC family of extracellular factors. Targeted disruption of murine Cryptic results in L-R laterality defects including randomization of abdominal situs, hyposplenia, and pulmonary right isomerism, as well as randomized embryo turning and cardiac looping. Similarly, zebrafish one-eyed pinhead (oep) mutants that have been rescued partially by mRNA injection display heterotaxia, including randomization of heart looping and pancreas location. In both Cryptic and oep mutant embryos, L-R asymmetric expression of Nodal/cyclops, Lefty2/antivin, and Pitx2 does not occur in the lateral plate mesoderm, while in Cryptic mutants Lefty1 expression is absent from the prospective floor plate. Notably, L-R asymmetric expression of Nodal at the lateral edges of the node is still observed in Cryptic mutants, indicating that L-R specification has occurred in the node but not the lateral plate. Combined with the previous finding that oep is required for nodal signaling in zebrafish, we propose that a signaling pathway mediated by Nodal and EGF-CFC activities is essential for transfer of L-R positional information from the node.

Laterality defects are influenced by timing of treatments and animal model

“…Because several studies have suggested that absent gene expression correlates with the incidence of isomerisms (Yan et al, 1999; Meno et al, 1998; Ibanes and Izpisua Belmonte, 2009), and because both absent gene expression and isomerisms are common in the mouse model (Figure 4 and data not shown), I also addressed whether there was a direct relationship between the incidence of absent nodal expression and the frequency of isomerisms. When all animals with these two measures were included in this regression analysis, there was no statistically significant relationship observed (R 2 =0.004, p=0.750).…”

“…A considerable number of studies have reported complete absence of nodal, lefty, or pitx2 expression (Field et al, 2011; Oki et al, 2010; Sakano et al, 2010; Gaio et al, 1999; Takeuchi et al, 2007; Zhang et al, 2001; Yan et al, 1999; Tsukui et al, 1999; Collignon et al, 1996; Kramer-Zucker et al, 2005; Krebs et al, 2003; Pennekamp et al, 2002), with some reporting 100% absent expression for all three genes. What does complete absence of gene expression mean?…”

Cardiac patterning and morphogenesis in zebrafish

“…Although significant attention has been paid to zebrafish mutations that randomize L-R asymmetry (e.g. Chen et al, 1997;Yan et al, 1999;Bisgrove et al, 2000;Chin et al, 2000), little is known regarding the molecular mechanisms that translate L-R patterning into directed cardiac morphogenesis. Future studies of mutations that interfere with cardiac looping without disrupting initial L-R patterning will be particularly informative in this regard.…”

Nodal signalling and asymmetry of the nervous system

“…Rotation of the heart cone during jogging might be directing the later torsion of the heart tube leading to D-loop in normal conditions. However, this seems unnecessary as mutants with no jog retain their ability to loop [62,63,[68][69][70]. Indeed, the heart tube in the absence of Nodal signalling shows D-loop in about 70% of embryos and this is maintained even after the heart tube is cultured ex vivo [67].…”