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.

The Wnt inhibitor Dkk1 is required for maintaining the normal cardiac differentiation program in Xenopus laevis.

Wnt proteins can activate different intracellular signaling pathways. These pathways need to be tightly regulated for proper cardiogenesis. The canonical Wnt/ß-catenin inhibitor Dkk1 has been shown to be sufficient to trigger cardiogenesis in gain-of-function experiments performed in multiple model systems. Loss-of-function studies however did not reveal any fundamental function for Dkk1 during cardiogenesis. Using Xenopus laevis as a model we here show for the first time that Dkk1 is required for proper differentiation of cardiomyocytes, whereas specification of cardiomyocytes remains unaffected in absence of Dkk1. This effect is at least in part mediated through regulation of non-canonical Wnt signaling via Wnt11. In line with these observations we also found that Isl1, a critical regulator for specification of the common cardiac progenitor cell (CPC) population, acts upstream of Dkk1.

The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis.

Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/ß-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, ß-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation.

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/ß-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/ß-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.

Comparative expression study of sipa family members during early Xenopus laevis development.

The signal-induced proliferation-associated (SIPA) protein family belongs to the RapGAP protein superfamily. Previous studies mainly focused on the expression and function of SIPA genes in vertebrate neuronal tissue. Only limited data about the embryonic expression pattern of the genes are currently available. Our study provides the first expression analysis of sipa1, sipa1l1, sipa1l2, and sipa1l3 during early development of the vertebrate organism Xenopus laevis. In silico, analysis revealed that all genes are highly conserved across species. Semi-quantitative RT-PCR experiments demonstrated that the RNA of all genes was maternally supplied. By whole mount in situ hybridization approaches, we showed that sipa1 is mainly expressed in various sensory organs, the respiratory and blood system, heart, neural tube, and eye. In contrast, sipa1l1 showed a broad expression during development in particular within the brain, somites, eye, and heart. Sipa1l2 was detected in the branchial arches, glomerulus, and the developing eye. In contrast, sipa1l3 revealed a tissue specific expression within the olfactory and otic vesicles, the cranial placodes and ganglia, neural tube, pronephros, retina, and lens. In summary, all sipa gene family members are expressed throughout the whole developing Xenopus organism and might play an important role during vertebrate early embryogenesis.

A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes.

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2ß. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.

Two similar near-infrared (IR) absorbing benzannulated aza-BODIPY dyes as near-IR sensitizers for ternary solar cells.

Ternary composite inverted organic solar cells based on poly(3-hexylthiophen-2,5-diyl) (P3HT) and phenyl-C61-butyric acid methyl ester (PCBM) blended with two different near-infrared absorbing benzannulated aza-BODIPY dyes, difluoro-bora-bis-(1-phenyl-indoyl)-azamethine (1) or difluoro-bora-bis-(1-(5-methylthiophen)-indoyl)-azamethine (2), were constructed and characterized. The amount of these two aza-BODIPY dyes, within the P3HT and PCBM matrix, was systematically varied, and the characteristics of the respective devices were recorded. Although the addition of both aza-BODIPY dyes enhanced the absorption of the blends, only the addition of 1 improved the overall power conversion efficiency (PCE) in the near-infrared (IR) region. The present work paves the way for the integration of near-infrared absorbing aza-BODIPY derivatives as sensitizers in ternary composite solar cells.