Clec16a-Mediated Mitophagy Modulates Zebrafish Definitive Hematopoiesis

Hematopoietic stem and progenitor cells (HSPCs) generate all blood cell lineages throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely regulated by a variety of signaling pathways. Previous studies have highlighted the essential roles of pattern-recognition receptors such as Toll-like receptors, RIG-I-like receptors, and NOD-like receptors in EHT. However, whether members of the C-type Lectin Receptors (CLRs) family participate in vertebrate embryonic hematopoiesis remains unclear.

To explore the potential pathways involved in hematopoietic ontogeny and differentiation during this stage, we screened the dynamic expression of CLR family genes and other inflammation-related genes in published scRNA-seq data from vertebrate embryos, including zebrafish, mouse, and human, during HSPC development. To verify the expression of CLR family genes in vitro, we used the HSC-like cell differentiation system of mouse and human embryonic stem cells (ESCs). These results suggest that Clec16a is highly expressed during embryonic HSPC development both in vivo and in vitro and may play an important role in the emergence of HSPCs in vertebrates.

In order to validate this hypothesis, we utilized the well-established zebrafish model for studying embryonic hematopoiesis to conduct in vivo functional experiments. First, we synthesized probes from the full-length mRNA of clec16a and utilized them to observe the in-situ expression of clec16a in zebrafish at different developmental stages. The whole-mount in situ hybridization (WISH) experiment demonstrated that clec16a was expressed from the 1-cell stage and, importantly, showed specific expression in the AGM region at the 24-28hpf, which coincides with the onset and generation of HSPCs. Then, morpholinos were utilized to knockdown clec16a in transgenic embryos Tg(runx1/kdrl), Tg(cmyb/kdrl), and Tg(CD41). Then mutants generated by CRISPR-Cas9 were used to perform WISH and qPCR to detect specific markers such as runx1, cmyb (HSPCs), rag1 (lymphocytes), gata1a (erythrocytes), and l-plastin (myelocytes). The results showed that clec16a was required for HSPC emergence, expansion, and differentiation.

Mechanistically, we sorted EGFP⁺ cells in Tg (fli1a:EGFP) zebrafish embryos at 26 hpf to perform RNA-Seq, which was the stage where EHT and these EGFP⁺ cells contained HEs and HSPCs. After bioinformatics analysis of sequencing data and considering published reports, we found that mitophagy in HEs and HSPCs was inhibited in clec16a-deficient zebrafish embryos. Transmission electron microscopy showed that clec16a knockdown led to a reduction in mitochondria in HEs, with disrupted outer membranes, damaged cristae structures, and enlarged intermembrane spaces. Furthermore, immunofluorescence and flow cytometry revealed elevated levels of mitochondrial reactive oxygen species (ROS), and treatment with mitochondrial antioxidants could reduce ROS levels while restoring hematopoiesis. These findings suggest that clec16a regulates EHT and HSPC generation and differentiation through mitophagy.

Furthermore, combined with transcriptomic sequencing and proteomics, we discovering that the key mitophagy gene prkn might play a crucial role. Rescue experiments showed that overexpression of prkn could ameliorate the phenotypes caused by inhibited mitophagy, reduce ROS levels, and restore the hematopoietic defects in clec16a-deficient embryos. Therefore, we propose the existence of a clec16a-prkn-ROS regulatory axis that is essential for the development and differentiation of HSPCs during zebrafish embryogenesis.

In summary, our work is the first to identify the role of the CLRs family gene clec16a in zebrafish embryonic hematopoiesis. We elucidate the mechanism by which clec16a-mediated mitophagy regulates embryonic hematopoiesis through ROS, highlighting the critical role of the mitophagy protein prkn in this process. This study not only provides new theoretical insights into the complex biological process of HSPC emergence, offers a novel perspective on the regulatory network between the innate immune system and embryonic hematopoiesis, but also proposes new approaches for the in vitro cultivation and expansion of HSCs.