Three-dimensional imaging reveals distinct muscle-vessel-nerve relationships during dorsal and ventral muscle splitting in the chicken forelimb

During vertebrate limb development, muscles, blood vessels, and nerves undergo coordinated morphogenesis. Limb muscles form through progressive splitting of the dorsal and ventral muscle masses, but the three-dimensional relationships between muscle splitting and surrounding tissues remain incompletely understood. Although blood vessels have been implicated in muscle splitting, whether this association is shared among different muscle masses is unclear. Here, we established a three-dimensional imaging approach for chicken embryonic forelimbs by combining fluorescent vascular labeling, whole-mount immunostaining, tissue clearing, and confocal microscopy. We compared three hydrophilic clearing methods, ScaleS, CUBIC, and SeeDB2, for compatibility with fluorescent ink, LCA-lectin, and PKH26 vascular labeling. SeeDB2 preserved all three vascular signals. We then developed a modified SeeDB2 protocol incorporating blood decolorization and used it to examine muscle, vessel, and nerve relationships from E5.0 to E6.0. In the ventral muscle mass, multiple vascular extensions were observed around the splitting region at E5.5. In contrast, no prominent vascular extension was detected around the splitting region of the dorsal muscle mass despite ongoing splitting at the same stage. Nerve projections also showed distinct spatial patterns between the dorsal and ventral muscle masses. These findings indicate that vascular extension is not a universal feature of limb muscle splitting and suggest that individual splitting events occur within distinct local tissue environments. Our three-dimensional approach provides new morphological insight into the coordinated development of muscle, vasculature, and peripheral nerves in the embryonic limb.