Structural and molecular characterization of the chick embryonic cerebrospinal fluid reveals a luminal extracellular matrix network containing SCO-spondin

The development of tubular organs relies on the presence of a transient luminal extracellular matrix within their internal cavities, which regulates both tubular size and morphogenesis. In the neural tube, the brain cavities are filled with the embryonic cerebrospinal fluid (eCSF), a protein-rich fluid known for its trophic and mechanical roles in neuroepithelial development. However, the molecular organization of the eCSF and the mechanisms coordinating its morphogenetic signaling remain poorly understood. Among the proteins detected in the eCSF, SCO-spondin stands out as a giant glycoprotein containing multiple matricellular domains that mediate interactions with different molecules, as well as domains associated with self-assembly. In this study, we investigated whether the eCSF exhibits a defined structural organization and sought to characterize its molecular components and associated interactions, with special emphasis on SCO-spondin. We examined the structural and molecular composition of eCSF in chick embryos using histochemistry, immunohistochemistry, light-sheet microscopy, and scanning electron microscopy. Co-immunoprecipitation followed by tandem mass spectrometry was used to identify molecular interactors of SCO-spondin. Our findings reveal that the eCSF is not a homogeneous fluid but rather an interlaced, sponge-like fibrillar mesh functioning as a luminal extracellular matrix intimately associated with the neuroepithelium. This network comprises SCO-spondin, fibronectin, proteoglycans, and lipophilic aggregates, forming a porous scaffold that may contribute to the retention of fluid and signaling molecules. Histochemical and immunohistochemical analyses of the isolated matrix indicate that it is acellular and glycosylated, with SCO-spondin and fibronectin among its main components. Proteomic analysis identified 74 SCO-spondin interactors, including morphogen carriers such as retinol-binding protein, transthyretin, riboflavin-binding protein, as well as lipoproteins, extracellular vesicles, and structural proteins. These findings suggest that, at early developmental stages, SCO-spondin is associated with macromolecular complexes involved in molecular transport and extracellular organization within the brain cavities. Our findings redefine eCSF as a structured supramolecular network rather than a simple fluid. This network may contribute to the spatial organization of molecular components within the ventricular cavities and their interaction with the neuroepithelium. We propose that the eCSF represents a structured and dynamic luminal extracellular matrix that may play an important role in brain morphogenesis, with SCO-spondin as a key organizing component.