Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure.

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.

Cardiomyocyte heterogeneity during zebrafish development and regeneration.

Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.

Focal adhesions are essential to drive zebrafish heart valve morphogenesis.

Elucidating the morphogenetic events that shape vertebrate heart valves, complex structures that prevent retrograde blood flow, is critical to understanding valvular development and aberrations. Here, we used the zebrafish atrioventricular (AV) valve to investigate these events in real time and at single-cell resolution. We report the initial events of collective migration of AV endocardial cells (ECs) into the extracellular matrix (ECM), and their subsequent rearrangements to form the leaflets. We functionally characterize integrin-based focal adhesions (FAs), critical mediators of cell-ECM interactions, during valve morphogenesis. Using transgenes to block FA signaling specifically in AV ECs as well as loss-of-function approaches, we show that FA signaling mediated by Integrin α5ß1 and Talin1 promotes AV EC migration and overall shaping of the valve leaflets. Altogether, our investigation reveals the critical processes driving cardiac valve morphogenesis in vivo and establishes the zebrafish AV valve as a vertebrate model to study FA-regulated tissue morphogenesis.

The flow responsive transcription factor Klf2 is required for myocardial wall integrity by modulating Fgf signaling.

Complex interplay between cardiac tissues is crucial for their integrity. The flow responsive transcription factor KLF2, which is expressed in the endocardium, is vital for cardiovascular development but its exact role remains to be defined. To this end, we mutated both klf2 paralogues in zebrafish, and while single mutants exhibit no obvious phenotype, double mutants display a novel phenotype of cardiomyocyte extrusion towards the abluminal side. This extrusion requires cardiac contractility and correlates with the mislocalization of N-cadherin from the lateral to the apical side of cardiomyocytes. Transgenic rescue data show that klf2 expression in endothelium, but not myocardium, prevents this cardiomyocyte extrusion phenotype. Transcriptome analysis of klf2 mutant hearts reveals that Fgf signaling is affected, and accordingly, we find that inhibition of Fgf signaling in wild-type animals can lead to abluminal cardiomyocyte extrusion. These studies provide new insights into how Klf2 regulates cardiovascular development and specifically myocardial wall integrity.

Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration.

Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.