Targeted Mutation Reveals Essential Functions of the Homeodomain Transcription FactorShox2in Sinoatrial and Pacemaking Development

Abstract

Background-Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results-To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2 Ϫ/Ϫ embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions-From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

Podoplanin deficient mice show a rhoa‐related hypoplasia of the sinus venosus myocardium including the sinoatrial node

“…In podoplanin mutants we found a hypoplastic sinus venosus myocardium. Another interesting gene involved in the development of the sinus venosus myocardium is Shox2 (Blaschke et al, 2007). Shox2 mutants showed severe hypoplasia of the sinus venosus myocardium comparable to our observations in the podoplanin null mice.…”

“…Lineage tracing experiments studying Isl1 (Cai et al, 2003), Fgf 10 (Kelly, 2005), and Tbx5 (Bruneau et al, 1999;Liberatore et al, 2000), 18 (Christoffels et al, 2006), and Shox2 (Blaschke et al, 2007) have demonstrated the contribution of the second heart field to the formation of the atrial myocardium which has a distinct molecular composition compared with the heart tube derived from the primary heart field (Cai et al, 2003;Kelly, 2005;Anderson et al, 2006).…”

“…Several genes and proteins, considered as early markers of the second heart field at the outflow and inflow tract, have been reported, such as MesP1 (Saga et al, 1999), fibroblast growth factor (Fgf) 8 and 10 (Kelly et al, 2001), BMP-2 and Nkx2.5 (Waldo et al, 2001;Christoffels et al, 2006;Gittenberger-de Groot et al, 2007), Isl1 (Cai et al, 2003), inhibitor of differentiation Id2 (Martinsen et al, 2004), GATA factors targeting Mef2c (Dodou et al, 2004;Verzi et al, 2005), Tbx1, Tbx3, and Tbx18 Christoffels et al, 2006) and Shox2 (Blaschke et al, 2007). Recently, we have added podoplanin to this list as a novel gene in cardiac development.…”

Expression of Id2 in the second heart field and cardiac defects in Id2 knock‐out mice

“…Id2 knockout embryos demonstrated hypoplasia of the atrial myocardium and larger interatrial communications than observed in wildtype animals, a phenotype that was also observed in other models with impaired myocardial contributions from the posterior heart field, including the mice lacking podoplanin, Pdgfr-a, and Shox2 (Blaschke et al, 2007;Douglas et al, 2009;Bax et al, 2010). However, in contrast to observations in podoplanin and Pdgfra knockout embryos, atrioventricular septal defects were not observed in Id2 knockouts.…”

“…We have referred to this contribution as being derived from the posterior heart field (PHF) , as opposed to the anterior heart field recognized at the arterial pole. Myocardium derived from the PHF is characterized by the expression of several genes, including Podoplanin Mahtab et al, 2009), its downstream factor RhoA (VicenteSteijn et al, 2010), HCN4 (Garcia-Frigola et al, 2003), Shox2 (Blaschke et al, 2007), Tbx5 (Puskaric et al, 2010), and Tbx18 (Christoffels et al, 2006). PHF-derived sinus venosus myocardium is furthermore characterized by the lack of expression of the transcription factor Nkx2.5 (Christoffels et al, 2006;Gittenberger-de Groot et al, 2007).…”