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. Overall design: 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.

自噬-溶酶体降解（autophagy-lysosomal degradation）是一类进化保守的生物学过程，对细胞稳态、细胞分化及应激存活具有关键意义，尤其与心血管系统的病理生理学紧密关联。实验与临床观察均证实，自噬-溶酶体降解可调控正常心脏形态发生，特别是心脏瓣膜的发育进程。然而，目前仍未明确是哪些细胞上调了自噬-溶酶体降解，以及该过程具体依赖哪些细胞生物学事件。
本研究构建了新型斑马鱼（zebrafish）转基因模型，可在体内（in vivo）可视化自噬体（autophagosome）与溶酶体（lysosome），并追踪二者在仔鱼心脏中的时序分布与细胞定位。借此，我们明确了自噬体与溶酶体囊泡形成的动力学特征，并观察到房室瓣（atrioventricular valves）与球室瓣（bulboventricular valves）发育阶段中溶酶体囊泡的显著聚集。随后，我们以spns1突变体（spns1 mutant）作为溶酶体功能受损（lysosomal impairment）的斑马鱼模型，探究了溶酶体降解在心血管发育中的功能作用。研究发现，spns1突变体表现出形态与功能均异常的心脏发育表型，包括心内膜组织（endocardial organization）紊乱、心脏瓣膜形成（cardiac valve formation）受阻以及高发生率的血液逆流（retrograde blood flow）现象。单核转录组分析（single-nuclear transcriptome analysis）显示，突变仔鱼心脏中存在心内膜特异性的溶酶体相关基因表达差异，以及Notch1信号通路（notch1 signaling）的异常改变。进一步实验证实，心内膜特异性过表达spns1与Notch1可挽救瓣膜形成与功能缺陷，以及整体心脏形态发生异常。综上，本研究完善了斑马鱼心脏发育过程中自噬与溶酶体相关事件的描述，并揭示了溶酶体加工过程作为Notch1信号通路上游事件，在心脏瓣膜形成中发挥的细胞自主功能（cell-autonomous role）。
**实验设计概述**：于受精后3天（3 dpf）通过手动解剖获取野生型同窝仔鱼（sibling）与spns突变体斑马鱼的仔鱼心脏。每个实验组（野生型同窝组、突变体组）设置2个生物学重复，每个重复包含4组混合样本，每组混合样本由50个心脏组织制备。使用Chromium细胞核分离试剂盒（Chromium Nuclei Isolation Kit）制备浓度为3800~4000个细胞核/μL的单细胞核悬液。依照4Chromium Next GEM Single Cell Multiome ATAC + Gene Expression试剂盒的标准流程，完成转座反应（Transposition）、GEM生成与条形码标记（GEM generation & barcoding）、反转录（reverse transcription），以及基因表达与ATAC测序文库（gene expression and ATAC libraries）的构建。将cDNA文库（cDNA libraries）混合后，采用Illumina NovaSeq 6000测序平台（Illumina NovaSeq 6000），搭载Illumina NovaSeq 6000 S1试剂盒v1.5（100循环，Illumina, 20028319），以300 pM的上样浓度（loading concentration）进行不对称双端双索引测序（asymmetric paired-end and dual indexed）。