Spinster homolog 1 (spns1)-dependent endocardial autophagy-lysosomal pathway drives valve morphogenesis through the control of Notch1 signaling.

Autophagy-lysosomal degradation is an evolutionarily conserved process key to cellular homeostasis, differentiation, and stress survival, which is particularly important to the pathophysiology of the cardiovascular system. What is more, both experimental and clinical observations indicate that autophagy-lysosomal degradation affects correct cardiac morphogenesis, and in particular valve development. However, it is still unclear which cells upregulate autophagy-lysosomal degradation and for which specific cellular processes it is required. Here, we introduce novel zebrafish transgenic models to visualize autophagosomes and lysosomes in vivo and to follow their temporal and cellular localization in the larval heart. This allowed us to determine the kinetics of autophagosome and lysosome vesicle formation and to observe significant accumulation of lysosomal vesicles during the development of the atrioventricular and bulboventricular valves. We then addressed the functional role of lysosomal degradation in cardiovascular development using a spns1 mutant as a zebrafish model of lysosomal impairment. We found that spns1 mutants displayed morphologically and functionally abnormal heart development, including abnormal endocardial organization, impaired cardiac valve formation and high incidence of retrograde blood flow. Single-nuclear transcriptome analysis revealed endocardial-specific differences in the expression of lysosome-related genes and alterations of notch1 signaling in the mutant larval heart. Further, endocardial-specific overexpression of spns1 and notch1 rescued features of valve formation and function as well as overall cardiac morphogenesis. Altogether, our study provides an improved description of the autophagy and lysosomal events that take place during zebrafish heart development and reveals a cell-autonomous role of lysosomal processing during cardiac valve formation upstream of notch1 signaling. Sibling and spns mutant zebrafish larval hearts were obtained at 3 dpf by manual dissection. Two replicates, each consisting of four pools(50 hearts), were obtained for each experimental group (sibling, mutant). Single nuclei suspensions containing 3800-4000 nuclei/ µL were prepared using the Chromium Nuclei Isolation Kit. The Transposition, GEM generation & barcoding, reverse transcription, and preparation of the gene expression and ATAC libraries was performed according to the 4Chromium Next GEM Single Cell Multiome ATAC + Gene Expression. The cDNA libraries were pooled and sequenced with a loading concentration of 300 pM, asymmetric paired-end and dual indexed, using an Illumina NovaSeq 6000 S1 Reagent Kit v1.5 100 cycles (Illumina, 20028319) on an Illumina NovaSeq 6000.