This is suggested by a recent study conducted by SATOH A. and his team. The researchers focused on collagen synthesis in axolotls, amphibians with transparent skin and significant regenerative capabilities. They used various imaging techniques on axolotls of different sizes (from 5 to 12 cm) to track the formation of the dermis as they grew, including collagen-specific fluorescent probes (DAR and DAF). These two probes differ in their structure and affinity: DAR is a small peptide molecule capable of recognizing partially unfolded α-helices of collagen, making it an excellent probe for marking procollagen, the immature form still in the process of assembly. Conversely, DAF is an affinity-activated dye that preferentially targets mature fibers, already organized into a stable triple helix. By first injecting a red marker (DAR) and then a second green marker (DAF) a few days later, the researchers were able to distinguish old collagen fibers from new ones, while identifying their degree of maturity. This technique is called pulse-chase and allows us to see where and when collagen is produced.
Surprisingly, researchers observed that the collagen in the skin of young 5 cm axolotls was already well organized into lattice-structured fibers, even in the absence of fibroblasts. By analyzing the expression of the gene Col1a1, which codes for type I collagen, they detected a strong signal in the keratinocytes of the basal layer of the epidermis, the layer in direct contact with the dermis. The presence of procollagen, an immature form of collagen, was also detected in this location using immunofluorescence. Finally, electron microscopy confirmed that these keratinocytes indeed contained procollagen ready to be secreted through the basal membrane. As the axolotl grew, the researchers observed through histological stains, notably Masson's trichrome, a popular method for highlighting collagen fibers, that the dermis transforms and becomes more complex. From a simple initial sheet, it becomes a structure with three well-organized layers:
Stratum baladachinum (SB): a layer located just beneath the epidermis, not very dense and synthesized by keratinocytes.
Spongiosum Stratum (SS): This is an intermediate layer where cells originating from the mesoderm, particularly fibroblasts, begin to appear.
Stratum compactum (SC): A deep, dense, and regular layer, with a highly structured network of orthogonal fibers.
It was only from 8 cm that scientists detected the arrival of mesenchymal cells, namely fibroblasts. These cells penetrate the existing collagen matrix through certain matrix metalloproteinases (MMPs), enzymes capable of locally digesting collagen to allow for cell invasion. Once settled in the dermis, the fibroblasts deploy filopodia, extensions that insert themselves between existing collagen fibers. Thanks to these structures, they modify, thicken, and reorganize the initial network formed by the keratinocytes. The use of electron microscopy allowed for tracking the evolution of collagen fibers during the growth of axolotls. At 5 cm, the fibers are thin and isolated, while at 12 cm, they are significantly thicker and intertwined, especially in the stratum compactum. This change reflects the work of the fibroblasts on the matrix initially produced by the keratinocytes. The figure below summarizes the structural evolution undergone by the collagen.