Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling.

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial-mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal-endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

Transcriptome analysis of the zebrafish atoh7-/- Mutant, lakritz, highlights Atoh7-dependent genetic networks with potential implications for human eye diseases.

Expression of the bHLH transcription protein Atoh7 is a crucial factor conferring competence to retinal progenitor cells for the development of retinal ganglion cells. Several studies have emerged establishing ATOH7 as a retinal disease gene. Remarkably, such studies uncovered ATOH7 variants associated with global eye defects including optic nerve hypoplasia, microphthalmia, retinal vascular disorders, and glaucoma. The complex genetic networks and cellular decisions arising downstream of atoh7 expression, and how their dysregulation cause development of such disease traits remains unknown. To begin to understand such Atoh7-dependent events in vivo, we performed transcriptome analysis of wild-type and atoh7 mutant (lakritz) zebrafish embryos at the onset of retinal ganglion cell differentiation. We investigated in silico interplays of atoh7 and other disease-related genes and pathways. By network reconstruction analysis of differentially expressed genes, we identified gene clusters enriched in retinal development, cell cycle, chromatin remodeling, stress response, and Wnt pathways. By weighted gene coexpression network, we identified coexpression modules affected by the mutation and enriched in retina development genes tightly connected to atoh7. We established the groundwork whereby Atoh7-linked cellular and molecular processes can be investigated in the dynamic multi-tissue environment of the developing normal and diseased vertebrate eye.

Mechanotransduction in cardiovascular morphogenesis and tissue engineering.

Cardiovascular morphogenesis involves cell behavior and cell identity changes that are activated by mechanical forces associated with heart function. Recently, advances in in vivo imaging, methods to alter blood flow, and computational modelling have greatly advanced our understanding of how forces produced by heart contraction and blood flow impact different morphogenetic processes. Meanwhile, traditional genetic approaches have helped to elucidate how endothelial cells respond to forces at the cellular and molecular level. Here we discuss the principles of endothelial mechanosensitity and their interplay with cellular processes during cardiovascular morphogenesis. We then discuss their implications in the field of cardiovascular tissue engineering.

Mechanically activated piezo channels modulate outflow tract valve development through the Yap1 and Klf2-Notch signaling axis.

Mechanical forces are well known for modulating heart valve developmental programs. Yet, it is still unclear how genetic programs and mechanosensation interact during heart valve development. Here, we assessed the mechanosensitive pathways involved during zebrafish outflow tract (OFT) valve development in vivo. Our results show that the hippo effector Yap1, Klf2, and the Notch signaling pathway are all essential for OFT valve morphogenesis in response to mechanical forces, albeit active in different cell layers. Furthermore, we show that Piezo and TRP mechanosensitive channels are important factors modulating these pathways. In addition, live reporters reveal that Piezo controls Klf2 and Notch activity in the endothelium and Yap1 localization in the smooth muscle progenitors to coordinate OFT valve morphogenesis. Together, this work identifies a unique morphogenetic program during OFT valve formation and places Piezo as a central modulator of the cell response to forces in this process.

Hemodynamic Forces Sculpt Developing Heart Valves through a KLF2-WNT9B Paracrine Signaling Axis.

Hemodynamic forces play an essential epigenetic role in heart valve development, but how they do so is not known. Here, we show that the shear-responsive transcription factor KLF2 is required in endocardial cells to regulate the mesenchymal cell responses that remodel cardiac cushions to mature valves. Endocardial Klf2 deficiency results in defective valve formation associated with loss of Wnt9b expression and reduced canonical WNT signaling in neighboring mesenchymal cells, a phenotype reproduced by endocardial-specific loss of Wnt9b. Studies in zebrafish embryos reveal that wnt9b expression is similarly restricted to the endocardial cells overlying the developing heart valves and is dependent upon both hemodynamic shear forces and klf2a expression. These studies identify KLF2-WNT9B signaling as a conserved molecular mechanism by which fluid forces sensed by endothelial cells direct the complex cellular process of heart valve development and suggest that congenital valve defects may arise due to subtle defects in this mechanotransduction pathway.

The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages.

Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP) transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC) containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers.

Asymmetric inheritance of the apical domain and self-renewal of retinal ganglion cell progenitors depend on Anillin function.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.

Collective epithelial migration drives kidney repair after acute injury.

Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney's remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI.