The Ciliary Protein Ftm Is Required for Ventricular Wall and Septal Development

Citation: Gerhardt C, Lier JM, Kuschel S, Ru ther U ( The Ciliary Protein Ftm Is Required for Ventricular Wall and Septal Development Christoph Gerhardt 0 Johanna M. Lier 0 Stefanie Kuschel 0 Ulrich Ru ther 0 Robert Dettman, Northwestern University, United States of America 0 Institute for Animal Developmental and Molecular Biology, Heinrich Heine University , Du sseldorf , Germany Ventricular septal defects (VSDs) are the most common congenital heart defects in humans. Despite several studies of the molecular mechanisms involved in ventricular septum (VS) development, very little is known about VS-forming signaling. We observed perimembranous and muscular VSDs in Fantom (Ftm)-negative mice. Since Ftm is a ciliary protein, we investigated presence and function of cilia in murine hearts. Primary cilia could be detected at distinct positions in atria and ventricles at embryonic days (E) 10.5-12.5. The loss of Ftm leads to shortened cilia and a reduced proliferation in distinct atrial and ventricular ciliary regions at E11.5. Consequently, wall thickness is diminished in these areas. We suggest that ventricular proliferation is regulated by cilia-mediated Sonic hedgehog (Shh) and platelet-derived growth factor receptor a (Pdgfra) signaling. Accordingly, we propose that primary cilia govern the cardiac proliferation which is essential for proper atrial and ventricular wall development and hence for the fully outgrowth of the VS. Thus, our study suggests ciliopathy as a cause of VSDs. - One of 100 newborns suffers from a congenital heart defect [1]. Among these human congenital cardiac diseases ventricular septal defects (VSDs) are the most common [2,3] and occur in approximately 1 of 1000 births [4]. The most prevalent VSD subtype is the perimembranous VSD [5,6] which is characterized by the loss of the membranous part of the ventricular septum (VS) and a defect in the development of a second part of the VS - the muscular septum. Interestingly, the membranous VS does not start to grow before the muscular VS generation has been finished [7] indicating that membranous VS development is probably initiated by an interaction of the inlet muscular VS and the atrioventricular endocardial cushion cells (ECCs) [8,9]. The membranous VS arises solely from the ECCs and not from the muscular VS [4]. Although the molecular background of muscular VS development is only poorly understood [10,11], two different hypothesis have been debated for its formation and outgrowth. The first theory describes VS generation as an active process of cell growth in the apical region of the muscular septum [12,13], while the second ascribes muscular septal length gain to a passive process based on the increase of the ventricular cavities. According to this hypothesis, the formation of the muscular septum is carried out by proliferation of cells at distinct regions in the left and right ventricle [1417], so that it consists of cardiomyocytes with both left-ventricular and right-ventricular identities [11,18,19]. Ftm (alias Rpgrip1l)-negative mice display abnormal heart development particularly a VSD and suffer from a dysfunction of primary cilia [20] indicating a potential relation between heart formation and ciliary action. The Ftm protein is localised at the base of cilia [20] and appears to be present at every cilium. The fact that mutations of FTM in humans were already found in ciliopathies like Meckel-Gruber syndrome, Joubert syndrome and nephronophthisis [21,22] accentuates the importance of this gene in human development. Primary cilia are hairlike, 115 mm long protrusions on most vertebrate cells. They function as the cells antenna receiving and mediating signals from the environment. These signals, in turn, control important cellular processes like proliferation, apoptosis, migration, differentiation and cell cycle regulation [23]. Consequently, defective primary cilia provoke severe human diseases [24]. Several signaling pathways are thought to be associated with cilia, including Sonic hedgehog (Shh), plateletderived growth factor receptor a (Pdgfra) as well as canonical and non-canonical Wnt signaling [2535]. While the connection between cilia and Wnt signaling has been frequently discussed and remains the subject of fierce debate [3436], it is well-known that Shh and Pdgfra signaling can be mediated by cilia [25 27,29,30,35]. Shh is a member of the Hedgehog (Hh) family of evolutionary conserved signaling molecules and binds to its receptor Patched (Ptc) which in vertebrates is localized in the ciliary membrane and regulates the activity of Smoothened (Smo), a seven-transmembrane receptor. Recruited to the cilium active Smo invokes Glioblastoma (Gli) transcription factors. In vertebrates three Gli isoforms exist Gli1, 2 and 3. They regulate the expression of Shh target genes like for example Ptc1 and thereby cell differentiation, proliferation, survival and growth [37,38]. Gli1 functions as a constitutive activator [39,40], whereas Gli2 and Gli3 have a Cterminal transcriptional activator domain and a N-terminal transcriptional repressor domain [41]. Full-length Gli3 (Gli3190) protein can be transformed into a transcriptional activator (Gli3-A) most likely by modifications [42,43]. Importantly, the fulllength protein can be proteolytically processed into a transcriptional repressor (Gli3-R, also known as Gli3-83) [44]. The ratio of activator and repressor forms controls cellular processes dependend on Shh signaling. Signaling by Pdgfra relates also to cilia [29]. Pdgfra is localized to cilia and becomes dimerized and phosphorylated after being bound by its ligand Pdgf-AA which also functions as a dimer. Activated Pdgf receptors regulate essential cell processes like proliferation, anti-apoptosis, migration, differentiation, actin reorganization and cell growth [4547]. Stimulation of Pdgfra drives the activation of signal transduction through the Mek1/2-Erk1/2 and Akt/PKB pathways mediated by primary cilia, whereas Pdgfra signaling gets blocked in the absence of cilia [29]. We used Ftm-deficient mice to investigate whether cardiac cilia are functionally involved in heart development, especially in VS formation. Furthermore, we analysed which signals are mediated by these cilia. We were able to identify components of Shh and Pdgfra signaling pathways in or at ventricular cilia giving evidence that these signals are cilia-mediated in embryonic murine hearts. According to ciliary dysfunction caused by Ftm deficiency [21,48,49], Shh and Pdfgra signaling are downregulated in Ftmnegative ventricles. We propose these signaling defects as the cause of reduced ventricular cell proliferation that in turn results in diminished ventricular wall thickness and VSDs. Materials and Methods Ethics Statement and Animal Husbandry All mice (Mus musculus) used in this study were on the C3H background and kept under standard housing conditions with a 12/12 hours dark-light cycle and with food and wa (...truncated)