Tanimoto, R. | bioRxiv Science

bioRxiv2026

A versatile fluorescent probe for imaging fibrillar collagen during avian morphogenesis

Fibrillar collagen architecture provides a fundamental structural framework for vertebrate tissues. Understanding how collagen matrices are organized and remodeled requires high-resolution imaging approaches capable of resolving their three-dimensional architecture and developmental dynamics in vivo. We previously established that the fluorescent probe DAF FM DA enables sensitive visualization of collagen structures in zebrafish, amphibian, and mouse tissues. However, whether this approach is applicable to avian embryos - an important model system in morphogenesis research - has remained unclear. Here we demonstrate that DAF FM DA specifically and robustly labels collagen matrices in quail embryonic tissues. Using this method, lattice like stromal networks in the cornea and spongy matrices in the limb cartilage primordium were clearly visualized throughout deep tissue regions. This imaging strategy provides a practical and versatile tool for investigating collagen matrix development in vertebrate morphogenesis using avian embryos.

developmental biology↗

bioRxiv2026

A collagen orientation switch reshapes fin architecture

The orientation and distribution of fibrillar collagen are critical determinants of the shape and mechanical properties of bones and organs.1-3 However, how they are spatially organized within tissues is still poorly understood,4-7 as visualizing these collagen architectures remains challenging. Actinotrichia (AT), the spear-shaped fibrillar collagen structures located at the distal tips of fish fins, are easily observable due to their large size and distinctive morphology8-14 and have recently emerged as a model system for studying collagen fiber organization.15-19 In this study, we generated knockout lines for the fish-specific extracellular matrix (ECM) genes actinodin1 and actinodin2 (and1/2), which are lost in tetrapods.12 Loss of these genes dramatically altered the orientation of collagen fibers, thereby inducing changes in fin morphology. In the wild-type fins, AT are orderly arranged beneath the epidermis, forming layers parallel to the fin surface, and their individual fibers radiate distally toward the fin tip. In contrast, double knockout (dKO) of and1/2 results in overall fin reduction accompanied by increased thickness. Examination of the collagen structure distribution revealed the presence of aberrant collagen fibers oriented perpendicular to the fin epidermis. Moreover, the vertically oriented fibers contributed to thickening of the mesenchymal region in which they were distributed. The number of abnormal fibers increased with the severity of and1/2 deficiency, suggesting that collagen fibers in fins inherently tend to align perpendicular to the epidermis when these genes are absent. Furthermore, in tetrapods lacking the and gene family--specifically amphibians, the tetrapod group most closely related to fish20--examination of the developing limb, the organ homologous to paired fins in fish,21 revealed collagen fibers oriented perpendicular to the epidermis. The distribution pattern also resembled that observed in the fin buds of and1/2 dKO fish. Together, these findings highlight collagen patterning alterations as a previously unrecognized factor contributing to the evolutionary divergence between thinned fins and thickened limbs. Moreover, the identification of mutants that dramatically alter collagen fiber orientation is unprecedented, suggesting that analysis of Actinodin (And) function unveil the mechanisms underlying collagen matrix formation.22-28

developmental biology↗