Multiple essential roles for primary cilia in heart development

Abstract

BackgroundThe primary cilium is a microtubule-based, plasma membrane-ensheathed protrusion projecting from the basal bodies of almost all cell types in the mammalian body. In the past several years a plethora of papers has indicated a crucial role for primary cilia in the development of a wide variety of organs. We have investigated heart development in cobblestone, a hypomorphic allele of the gene encoding the intraflagellar transport protein Ift88, and uncovered a number of the most common congenital heart defects seen in newborn humans.MethodsWe generated serial sections of mutant cobblestone and wild type embryos in the region encompassing the heart and the cardiac outflow tract. The sections were further processed to generate three-dimensional reconstructions of these structures, and immunofluorescence confocal microscopy, transmission electron microscopy, and in situ hybridization were used to examine signal transduction pathways in the relevant areas. Whole mount in situ hybridization was also employed for certain developmental markers.ResultsIn addition to an enlarged pericardium and failure of both ventricular and atrial septum formation, the cobblestone mutants displayed manifold defects in outflow tract formation, including persistent truncus arteriosus, an overriding aorta, and abnormal transformation of the aortic arches. To discern the basis of these anomalies we examined both the maintenance of primary cilia as well as endogenous and migratory embryonic cell populations that contribute to the outflow tract and atrioventricular septa. The colonization of the embryonic heart by cardiac neural crest occurred normally in the cobblestone mutant, as did the expression of Sonic hedgehog. However, with the loss of primary cilia in the mutant hearts, there was a loss of both downstream Sonic hedgehog signaling and of Islet 1 expression in the second heart field, a derivative of the pharyngeal mesoderm. In addition, defects were recorded in development of atrial laterality and ventricular myocardiogenesis. Finally, we observed a reduction in expression of Bmp4 in the outflow tract, and complete loss of expression of both Bmp2 and Bmp4 in the atrioventricular endocardial cushions. Loss of BMP2/4 signaling may result in the observed proliferative defect in the endocardial cushions, which give rise to both the atrioventricular septa as well as to the septation of the outflow tract.ConclusionsTaken together, our results potentially identify a novel link between Sonic hedgehog signaling at the primary cilium and BMP-dependent effects upon cardiogenesis. Our data further point to a potential linkage of atrioventricular septal defects, the most common congenital heart defects, to genes of the transport machinery or basal body of the cilia.

Cilia and coordination of signaling networks during heart development

“…Furthermore, endocardial cells derived from Ift88/Tg737 −/− mice show disturbances in EndoMT. 136,142,151 Taken together, this Figure 5. examples of signaling networks involved in development of specific anatomical structures of the heart.…”

“…Components in this pathway localize to primary cilia in embryonic hearts as well as in cardiomyocytes differentiating from stem cells in vitro (Fig. 4), 21,22,142 and defective ciliary assembly is associated with defective cardiomyogenesis 21 as well as Hh-related heart defects that might be independent of nodal cilia, including ventricular and endocardial cushion-derived defects. 122,142,154 Further, 60% of patients with mutations in genes encoding the EVC proteins, EVC1/EVC2, which interact with Smo at the primary cilium to transduce Hh signaling [155][156][157][158][159] , display CHD, including AVSD and ASD.…”

“…Consequently, defects in ciliary assembly or turnover of Hh components in the cilium lead to a plethora of developmental disorders and diseases, including CHD. 21,22,136,142 A number of observations have specifically linked cardiac primary cilia to the regulation of Hh signaling. Components in this pathway localize to primary cilia in embryonic hearts as well as in cardiomyocytes differentiating from stem cells in vitro (Fig.…”

“…21,136 Mutations in other genes involved in ciliogenesis also causes CHD. Ift88/Tg737 −/− mouse embryos with stunted cilia develop severe CHD including atrial septal defects (ASD), VSD, atrioventricular septal defect (AVSD), and OFT septal defects 21,136,142 ; defects that are all associated with malformed endocardial cushions (Fig. 3).…”

“…140 Interestingly, mouse endothelial cells that form stunted primary cilia (e.g derived from the Tg737 orpk mutant) or are mutated in Pkd1 show impaired response to shear stress, 141 and mice with mutations in Kif3a, Lrd, Pkd2, 136 and Ift88/Tg737 21,142 have defects in chamber maturation. 21,136,142 A variety of different vesicle trafficking pathways regulate the assembly, maintenance, and sensory function of cilia. 143,144 Often, the proximal part of the cilium is placed within an invagination of the plasma membrane known as the ciliary pocket, which functions as the ciliary platform for exocytosis of Golgi-derived vesicles and for clathrin-dependent endocytosis (CDE).…”