In a study published in Cell, a team led by Prof. ZHOU Bin from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, along with Prof. Ralf H. Adams from the Max Planck Institute for Molecular Biomedicine, Germany, reported the absence of lymphatic vessels in homeostasis and regenerating bone and their abnormal invasion under pathological conditions by using dual-recombinase-mediated genetic strategies.

The lymphatic system, composed of capillary lymphatics, collecting vessels, and ducts lined by lymphatic endothelial cells (LECs), performs fluid homeostasis and immune surveillance. Although lymphatics are abundant in organs such as skin, heart, lung, and intestine, whether they exist in bone has been debated for decades.

Previous studies rely on single markers to identify LECs in bone, but these markers lack cell lineage specificity: lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) labels immune cells, podoplanin (PDPN) labels mesenchymal cells, and prospero-related homeobox 1 (PROX1) labels neurons, cardiomyocytes, hepatocytes, and skeletal myocytes.

In this study, the researchers found that single-recombinase-mediated lineage tracing tools suffer from non-specific labeling, which greatly interferes with lineage tracing results. Re-analysis of single-cell RNA sequencing data confirmed that most PROX1⁺ cells lack Cdh5 (vascular endothelial marker) expression, indicating contamination from non-endothelial lineages.

The researchers developed a dual-recombinase (Cre-loxP and Dre-rox) system, LEC-iCre (Cdh5-Dre;Prox1-RSR-CreER), which restricts labeling to cells co-expressing Cdh5 and PROX1, greatly improving labeling accuracy and specificity.

Using this system, under homeostasis, LECs were exclusively localized to periosteal fibrous layer and surrounding connective tissues, and were absent from trabecular bone, cortical bone, and bone marrow. Following irradiation injury, LECs remained restricted to periosteum and did not invade bone at various stages after transplantation. After fracture injury, although LECs expanded significantly in periosteum and callus, they never invaded bone tissue. This finding demonstrates that during post-injury bone regeneration, lymphatic vessels do not enter bone to participate in repair.

In GLA/GSD mouse models carrying the Pik3ca^H1047R activating mutation, the researchers captured the entire process of lymphatic invasion. Using the dual-recombinase system for LEC-specific expression of the mutant gene, they observed that periosteal LECs proliferated, sequentially breached the periosteum, eroded cortical bone, and ultimately invaded the bone marrow. This finding demonstrates that intraosseous lymphatics in GLA/GSD originate from active invasion of extraosseous lymphatics.

This study resolves the issue regarding the existence of lymphatic vessels in bone, and reveals a pathological mechanism for GLA and GSD. The newly developed lineage tracing strategies provide new technical methods for studying lymphatic vessel function, related regulatory mechanisms, and lymphatic-associated diseases.