The pericardium forms as a distinct structure during heart formation

The heart integrates diverse cell lineages into a functional unit, including the pericardium, a mesothelial sac that supports heart movement, homeostasis, and immune responses. However, despite its critical roles, the developmental origins of the pericardium remain uncertain due to disparate models. Here, using live imaging, lineage tracking, and single-cell transcriptomics in zebrafish, we find the pericardium forms within the lateral plate mesoderm from dedicated anterior mesothelial progenitors and distinct from the classic heart field. Imaging of transgenic reporters in zebrafish documents lateral plate mesoderm cells that emerge lateral of the classic heart field and among a continuous mesothelial progenitor field. Single-cell transcriptomics and trajectories of hand2-expressing lateral plate mesoderm reveal distinct populations of mesothelial and cardiac precursors, including pericardial precursors that are distinct from the cardiomyocyte lineage. The mesothelial gene expression signature is conserved in mammals and carries over to postnatal development. Light sheet-based live-imaging and machine learning-supported cell tracking documents that during heart tube formation, pericardial precursors that reside at the anterior edge of the heart field migrate anteriorly and medially before fusing, enclosing the embryonic heart to form a single pericardial cavity. Pericardium formation proceeds even upon genetic disruption of heart tube formation, uncoupling the two structures. Canonical Wnt/β-catenin signaling modulates pericardial cell number, resulting in a stretched pericardial epithelium with reduced cell number upon canonical Wnt inhibition. We connect the pathological expression of secreted Wnt antagonists of the SFRP family found in pediatric dilated cardiomyopathy to increased pericardial stiffness: sFRP1 in the presence of increased catecholamines causes cardiomyocyte stiffness in neonatal rats as measured by atomic force microscopy. Altogether, our data integrate pericardium formation as an independent process into heart morphogenesis and connect disrupted pericardial tissue properties such as pericardial stiffness to pediatric cardiomyopathies. We sought to probe whether pericardial progenitors are a transcriptionally distinct cell population in the emerging LPM and what gene expression signature defines them. We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture. We asked if this differential transcriptome signature between the myocardium and pericardium is also detectable in mammalian systems and remains in more mature hearts. We compiled cluster-defining LPM gene lists for myocardium versus mesothelium/pericardium as informed from our zebrafish scRNA-seq and prior work. We then compared the expression of these select gene lists to bulk RNA-sequencing of isolated pericardia and left ventricle myocardia from neonatal 3-week-old rats (Rattus norvegicus).

心脏将多种细胞谱系整合为功能单元，包括心包（pericardium）——一种支持心脏运动、内稳态与免疫应答的间皮囊。尽管心包发挥着关键作用，但其发育起源因存在多种迥异的研究模型仍未明确。本研究以斑马鱼（zebrafish）为模型，借助活体成像、谱系示踪与单细胞转录组学（single-cell transcriptomics）技术，发现心包起源于侧板中胚层（lateral plate mesoderm）中特化的前体间皮祖细胞，且其来源与经典心野相互独立。通过对斑马鱼转基因报告株的成像分析，我们观测到侧板中胚层细胞出现在经典心野的外侧，且处于连续的间皮祖细胞区域内。对表达hand2的侧板中胚层进行单细胞转录组学分析及细胞轨迹推演，揭示了间皮与心脏前体细胞的不同亚群，其中心包祖细胞与心肌细胞谱系相互独立。间皮基因表达特征在哺乳动物中保守存在，并延续至出生后发育阶段。基于光片活体成像（light sheet-based live-imaging）与机器学习（machine learning）辅助的细胞追踪实验表明，在心管形成过程中，位于心野前缘的心包祖细胞会向前方及内侧迁移，随后融合并包裹胚胎心脏，形成单个心包腔。即便通过遗传手段干扰心管形成，心包的发育仍可正常进行，说明二者的发育过程相互解耦。经典Wnt/β-连环蛋白（Wnt/β-catenin）信号通路可调控心包细胞数量：抑制经典Wnt信号通路会导致心包上皮细胞数量减少，且上皮组织呈现拉伸状态。我们将小儿扩张型心肌病（pediatric dilated cardiomyopathy）中观测到的SFRP家族（SFRP family）分泌型Wnt拮抗剂异常表达与心包硬度升高关联起来：在儿茶酚胺水平升高的条件下，SFRP1可通过原子力显微镜（atomic force microscopy）检测到新生大鼠心肌细胞硬度升高。综上，本研究数据将心包形成作为独立过程整合至心脏形态发生中，并将心包硬度等受损的心包组织特性与小儿心肌病关联起来。我们旨在探究心包祖细胞是否为新兴侧板中胚层中转录特征独特的细胞群，以及何种基因表达特征可定义这群细胞。我们针对受精后10小时（尾芽期，原肠胚形成末期）的斑马鱼侧板中胚层细胞开展了基于10x基因组学（10xGenomics）的单细胞RNA测序（single-cell RNA-sequencing，scRNA-seq）分析，此时侧板中胚层已凝聚为双侧条纹状结构。我们进一步探究了心肌与心包之间的差异转录组特征是否在哺乳动物系统中同样存在，并在更成熟的心脏中得以保留。我们基于斑马鱼单细胞转录组数据及前期研究结果，整理了心肌与间皮/心包的集群特异性基因列表。随后，我们将这些筛选出的基因列表的表达情况，与取自3周龄新生褐家鼠（Rattus norvegicus）的分离心包与左心室心肌的批量RNA测序（bulk RNA-sequencing）结果进行了比对。