Axial muscle-fibre orientations in larval zebrafish.

Most teleost fish propel themselves with lateral body waves powered by their axial muscles. These muscles also power suction feeding through rapid expansion of the mouth cavity. They consist of muscle segments (myomeres), separated by connective tissue sheets (myosepts). In adult teleosts, the fast axial muscle fibres follow pseudo-helical trajectories, which are thought to distribute strain (relative fibre length change) approximately evenly across transverse sections during swimming, thereby optimizing power generation. To achieve strain equalization, a significant angle to the longitudinal axis on the frontal plane (azimuth) is necessary near the medial plane, increasing strain. Additionally, a deviation from longitudinal orientation on the sagittal plane (elevation) is required laterally to decrease strain. Despite several detailed morphological studies, our understanding of muscle-fibre orientations in the entire axial musculature of fish remains incomplete. Furthermore, most research has been done in post-larval stages, leaving a knowledge gap regarding the changing axial muscle architecture during larval development. Larval fish exhibit different body size, body shape and swimming kinematics compared to adults. They experience relatively high viscous forces, requiring higher tail-beat amplitudes to overcome increased drag. Additionally, larval fish swim with higher tail-beat frequencies. Histological studies have shown that in larval fish, muscle fibres in the anal region transition from an almost longitudinal orientation to a pseudo-helical pattern by 3 dpf (days post-fertilization). However, these studies were limited to a few sections of the body and were prone to shrinkage and tissue damage. Here, we introduce a novel methodology for quantifying muscle-fibre orientations along the entire axial muscles. We selected 4 dpf larval zebrafish for our analyses, a stage where larvae are actively swimming but not yet free-feeding. High-resolution confocal 3D scans were obtained from four genetically modified zebrafish expressing green fluorescent protein in fast muscle fibres. Fluorescence variation allowed segmentation of individual muscle fibres, which were then converted to fish-bound coordinates by correcting for the fish's position and orientation in the scan, and normalized to pool results across individuals. We show that at 4 dpf, muscle-fibre trajectories exhibit a helical pattern tapering towards the tail. Average fibre angles decrease from anterior to posterior, with azimuth varying over the dorsoventral axis and elevation varying over the mediolateral axis. Notably, only the anteriormost 20% of the body displayed higher azimuth angles near the medial plane. Angles between neighbouring fibres were substantial, particularly at the rim of the epaxial and hypaxial muscles. The revealed muscle-fibre architecture at this age presumably contributes to the swimming performance of these larvae, but that swimming performance is probably not the only driving factor for the fibre pattern. Our methodology offers a promising avenue for exploring muscle-fibre orientations across ontogenetic series and provides a foundation for in-depth functional studies on the role of muscle architecture in facilitating swimming performance of larval fish.