Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ZEBRA-VALVE (“Quantitative analysis of the molecular and cellular behaviors associated with cardiac valve morphogenesis in pathological and regenerative contexts”)

Teaser

Heart valve (HV) morphogenesis relies on a coordinated interplay between transcription factors and mechanical forces generated by the blood flow. HVs ensure optimal blood circulation and avoid reverse blood. Industrialised countries have a prevalence of HV disease estimated at...

Summary

Heart valve (HV) morphogenesis relies on a coordinated interplay between transcription factors and mechanical forces generated by the blood flow. HVs ensure optimal blood circulation and avoid reverse blood. Industrialised countries have a prevalence of HV disease estimated at 2.5%, moreover HV replacement is one of the most common cardiac interventions. Valve diseases have their origin during embryogenesis, either as signs of abnormal developmental processes or the aberrant re-expression of foetal gene programs normally quiescent in adulthood.
The zebrafish is a good model organism to study heart development, despite having one atrium and one ventricle instead of two, it shares the same three cardiac tissue layers (endocardium, myocardium and epicardium) with the human heart. The atrium collects the deoxygenated blood and sends it to the ventricle through the atrioventricular valve (AVV). This valve is structurally similar to mammalian valves, suggesting conservation of the cellular and molecular events involved in its formation. The zebrafish AVV model has proven to be powerful in the study of the effect of mechanical forces on HV development. While mammals have a very limited regenerative capacity, zebrafish is one of the most widely used models for regeneration. However, it is unclear if HV have the ability to regenerate. Most importantly, the regenerative potential of valvulogenic endocardial cells (EdC) has never been explored, despite being crucial to the development of novel therapies. This project aimed to investigate the spatial and temporal nature of EdC behaviours necessary for HV morphogenesis in normal, pathological and regenerative contexts.
In the developing heart, the heartbeat and the blood flow signal to the EdCs through mechanosensitive proteins which in turn modulate the genetic program controlling valvulogenesis. However, the precise dynamics of the cellular events involved are difficult to describe owing to the location deep within the cardiac cavity and the constant motion of the beating heart. Dr. Vermot’s laboratory has recently demonstrated that oscillatory flow is essential for early valve morphogenesis and that the EdCs are able to discriminate between small changes in oscillatory flow, which leads to different cells responses. The flow-responsive transcription factor krüppel-like factor 2a (Klf2a), for example, is important during valvulogenesis. Removing klf2a expression is known to result in malformed valves. However, how mechanical forces influence key cellular processes underlying AVV morphogenesis and the full genetic network activated by oscillatory flow in EdCs is poorly understood. It is essential to determine how mechanical forces control pathway activation and morphogenesis in vivo because the mechanical stimuli experienced by EdCs are too complex to be faithfully reproduced in vitro.

Work performed

To address our first aim “The identification of genetic markers of valves progenitors”, we optimized a high throughput extraction technique for zebrafish hearts at 48 hours post fertilization (hpf), and developed a protocol for endothelial cells (EdCs) mRNA extraction. We performed RNA-seq in EdCs isolated from klf2a; klf2b double mutant and control embryonic hearts to study the genes controlled by the flow-responsive transcription factors Klf2a and 2b. The differential expression analysis of these data allowed us to identify genes involved in valve morphogenesis downstream a mechanical stimuli. We have validated some of these genes using molecular techniques.
The speed in which the zebrafish heart beats causes motion blur and loss of spatial resolution during traditional confocal imaging. To reach a comprehensive description of EdCs organization during AVV morphogenesis, we developed novel strategies for live imaging to perform high-resolution time-lapse imaging and photoconversion approaches. Processing the microscopy data to gain real biological insight represents a computational challenge, to overcome this, we established a pipeline of data alignment and analysis in collaboration with Dr Liebling’s lab. These new approaches allowed us to study AVV morphogenesis at an unprecedented temporal and spatial resolution during normal development. In addition to the characterization of normal valvulogenesis, we analysed klf2a; klf2b double mutant valve morphology phenotypes (Figure 1).
Primary cilia are immotile microtubule-based organelles that are known to act as mechanical sensors. Recently, mutations in cilia-related proteins have been linked to Congenital Heart Diseases (CHDs). As part of the characterization of the role of mechanical forces during AVV development, we explored a possible role of cilia-related proteins during valve morphogenesis. Surprisingly, we did not find a valve phenotype in these cilia mutants but a strong proepicardial phenotype. The proepicardium is an extra-cardiac transient cluster of heterogeneous cells that give rise to the epicardium. During development this cardiac cell layer plays an essential role in myocardial maturation, coronary vessel formation and contribute to the formation of the cardiac valves in the mouse. Given that the epicardium has a crucial role during heart regeneration we further explored the cilia mutant phenotype.
To better understand the function of cilia in the developing heart, we not only took advantage of our previously developed protocols, but also established collaborations with several other laboratories in order to explore if this function is conserved in mouse and human cell lines. The joint work in this project allowed us to uncover a novel role for the Intraflagellar transport complex B proteins regulating the Hippo pathway effector Yap1 during cardiogenesis.
The results of this work have been presented at the Weinstein Cardiovascular Conference 2018, one of most prestigious cardiovascular meeting in the world, as well as in the Tri-Regio Developmental and Stem Cell Biology Meeting 2018.

Final results

For technical limitations, most of the previous valvulogenesis studies have been performed using fixed samples or stopped hearts raising the concerns that these can lead to misinterpretations pertaining to valve morphology. Similar problems are faced by other cardiovascular developmental studies. The live imaging protocols and pipeline of data alignment and analysis that we developed will be valuable not only for the study of valve morphogenesis, but for cardiovascular research in general. Likewise, the protocols developed for high throughput extraction technique for zebrafish hearts and endothelial cells (EdCs) mRNA extraction can be used for other cardiovascular research topics.
The new genes involved in valve morphogenesis identified throughout this project have improved our understanding of the fine regulation active during valvulogenesis. These genes have the potential to contribute to improvement of generating prosthetic valves, which is a big challenge in tissue engineering research.
During this project we have created a strong international network, establishing collaborations with Dr Meilhac (Paris, France), Dr Delaval (Montpellier, France), Dr Mercader (Bern, Switzerland) and Dr Lecaudey (Frankfurt, Germany) laboratories among others. Through this European network of colleagues, I was able to learn new techniques and develop new hypothesis about valve development and signalling thereby actively participating and improving the European science.

Website & more info

More info: https://twitter.com/Zebra_Valve.