Spatial transcriptomics reveals novel genes during the remodelling of the embryonic human arterial valves

Abstract

Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development. Non-Technical SummaryCongenital heart defects, particularly those affecting the valves and septa of the heart, are very common. Despite this, few gene variants have been confirmed as disease-causing in human congenital heart (including valve) disease patients. Here we utilise spatial transcriptomics technology, which allows the identification of genes expressed in tissue slices, on embryonic human heart valves and identify a gene dataset that is human arterial valve-specific. We confirm the localisation of key novel genes to the arterial valves and highlight the relevance of the dataset by showing that mice mutant for RBP1, a novel gene identified as being highly differentially expressed in our valve dataset, have previously unidentified arterial valve defects. Using commonly used bioinformatic databases we show that filtering patient genomic data using these terms would likely exclude valve genes and thus may not identify the causative genes. Thus, we confirm that spatial transcriptomics technology can be used to study gene expression in tiny structures such as the developing heart valves and provide a new human embryonic valve dataset that can be used in future genomic studies of patients with congenital valve defects.