Abstract
The purpose of this thesis work was to hilight the importance of hemodynamic forces in normal heart development. These forces, caused by the contractions of the cardiac tube since the earliest steps of embryogenesis, will act on the primitive vasculature and generate a mechanical stress constituting a differenciation signal for the endothelial cells. The transduction of this signal, mediated by the non-selective mechanosensitive ion channel Piezo, will induce a change in the genetic expression profile within the endothelium and surrounding tissues, and therefore contribute to the development of specialised cardiac structures. By using several in situ hybridisation technics coupled with pharmacological treatments and antisense morpholino knock-down injections, we could reveal in zebrafish that the cardiac expression of aggrecan a, a gene coding a proteoglycane allowing for tissue compression resistance, depends on hemodynamic forces. The expression of this gene, restraint to the outflow tract (OFT) of the heart, is found to be localised in cells surrounding the endothelium. CRISPR/Cas9 mutagenesis knock-out and morpholino injection induced knock-down of aggrecan a provoke the apparition of a phenotype characterised by a heart edema and developmental and cardiac chambers arrangement defects. Continuing with knock-down experiments on zebrafish, we demonstrated that the loss of expression of aggrecan a results in OFT underdevelopment, and that this hypoplasia strongly affects cardiac performance. These experimental results are concordant with the transcriptomics analysis on human valve tissue, showing a dramatic loss of expression of the AGGRECAN gene in the aortic valves of patients suffering from type 0 bicuspidy. This strongly suggests the association of this gene’s expression abnormalities with the development of this type of congenital heart defects in Humans. In a similar manner, we performed chromogenic and fluorescent in situ hybridisation experiments in order to reveal the cardiac expression profile of the spp1 gene. This gene codes an extracellular matrix protein involved in mineralisation and cell migration, and is found to be expressed at the level of the atrioventricular and aortic valve formation sites. Antisense morpholino injections induced the loss of expression of tnnt2, a gene that is essential for cardiac contraction, and allowed us to confirm that the expression of spp1 is hemodynamically dependent. The knock-down of piezo1b gave similar results, thus confirming its mechanotransducing role. The knock-down of spp1 by morpholino injection revealed dysmorphic aortic valve cusps development at 6 days when observed in vivo on a biphotonic microscopy setup. In parallel, we created a spp1 knock-out line using CRISPR/Cas9 mutagenesis, then confirmed the insertion of a premature STOP codon in the coding sequence of spp1 by sequencing. However, we were not able to detect any phenotype in these fish, suggesting the emergence of a compensation phenomenon that degrades nonsense mRNA (Nonsense-Mediated mRNA Decay). In order to cope with this issue, we strated new knock-out experiments, using a « RNA-less » spp1 promoter sequence excision strategy. Finally, we identified human mutations in the SPP1 orthologous gene in patients suffering from congenital aortopathies, including a rare 4 nucleotides deletion that a priori induces the loss of the C-terminal part of the protein, involved in the binding of CD44. These results show the interest of the zebrafish as a model for mutations validation and determination of their impact on congenital heart defects in Humans.