Muscarinic receptors promote pacemaker fate at the expense of secondary conduction system tissue in zebrafish.

Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.

Centrobin controls primary ciliogenesis in vertebrates.

The BRCA2 interactor, centrobin, is a centrosomal protein that has been implicated in centriole duplication and microtubule stability. We used genome editing to ablate CNTROB in hTERT-RPE1 cells and observed an increased frequency of monocentriolar and acentriolar cells. Using a novel monoclonal antibody, we found that centrobin primarily localizes to daughter centrioles but also associates with mother centrioles upon serum starvation. Strikingly, centrobin loss abrogated primary ciliation upon serum starvation. Ultrastructural analysis of centrobin nulls revealed defective axonemal extension after mother centriole docking. Ciliogenesis required a C-terminal portion of centrobin that interacts with CP110 and tubulin. We also depleted centrobin in zebrafish embryos to explore its roles in an entire organism. Centrobin-depleted embryos showed microcephaly, with curved and shorter bodies, along with marked defects in laterality control, morphological features that indicate ciliary dysfunction. Our data identify new roles for centrobin as a positive regulator of vertebrate ciliogenesis.

Assessing Smoothened-mediated Hedgehog signaling in zebrafish.

Smoothened belongs to the class of atypical G protein-coupled receptors and serves as the transducing molecule in Hedgehog (Hh) signaling. Hh proteins comprise a family of secreted, cholesterol-modified ligands, which act both as morphogens and as signaling molecules. Binding of Hh proteins to their direct receptor, the transmembrane protein Patched-1, relieves Smoothened from tonal inhibition by Patched-1 and causes the translocation of Smoothened into the cilium. Here, the Hh signaling cascade is initiated and results in transcriptional activation of Hh target genes such as gli1 or patched-1. This induces a plethora of physiological outcomes including normal embryonic development, but also cancer, which is the reason why scientists aim to develop strategies to manipulate as well as monitor Smoothened-mediated Hh signaling. The zebrafish has emerged as a valuable tool for the assessment of Smoothened-mediated Hh signaling. In this chapter we thus describe how Smoothened-mediated Hh signaling can be monitored and also quantified using zebrafish embryos.