Planarian collagens and their roles during regeneration

Abstract

The behaviour of cells is regulated by the microenvironment they reside in, which consists of various signalling molecules, supporting cells and the extracellular matrix. The ECM acts as both a structural element and regulator of biochemical signals to influence cell behavior. There is an increasing significance of the role of the ECM during repair and regeneration, with the hopes of developing suitable scaffolds for regenerative medicine. Understanding how the ECM guides regeneration would enable us to better address the complications associated with wound repair and regeneration in humans. Planarians are free-living flatworms that possess incredible regenerative ability due to a large population of heterogeneous pluripotent stem cells residing throughout the entire animal that are recruited during regeneration. While there is a growing understanding of the molecular signals that regulate planarian regeneration, the role of the ECM in planarian regeneration and regulation of stem cells is still unclear. Previous proteomic analysis of the regenerating blastema of the planarian Dugesia japonica showed that DjCol-2, DjCol-4 and DjCol-6 are dynamically upregulated, suggesting that collagens plays a role in the regenerative process. Further analysis of an EST library of planarian transcripts revealed a total of nine fibrillar-related collagen chains (DjCol-1 to -9), which possess conserved sequence characteristics with mammalian fibrillar collagens, with the exception of interrupted triple helixes typically not present in vertebrate fibrillar collagens. Overall conservation of gene and protein structure suggests similar functions would exist between different species. Using the planarian as a regenerative model could enable us to potentially identify novel functions of fibrillar collagens that could have similar roles in mammalians systems. Dj-collagen is exclusively expressed by the subepidermal and dorso-ventral muscle cell population, which are located surrounding the stem cell population in the mesenchymal space. During regeneration, Dj-collagen expression is upregulated in the blastema and is likely activated during the middle stages of muscle differentiation. Immunostaining of DjCol-2 shows it is localised around the body-wall and dorso-ventral muscle fibers, and is secreted concomitantly with the elongation of muscle fibers during regeneration, suggesting a potential role in providing structural support and guiding muscle fiber regeneration. Interestingly, functional analysis reveals a potential role of Dj-Collagen in guiding brain regeneration. This is demonstrated through combinatorial Dj-collagen knockdown which leads to slower regeneration rate of the eyes and evidence of abnormal brain patterning. Knockdown of MyoD, a muscle differentiation transcription factor, also leads to similar defects. These findings suggest that muscle fiber organisation together with the presence of collagen potentially influences the guidance of axons as an upstream regulator of neural regeneration, which in turn regulates regeneration of other tissues, indicating the importance of structural organisation in regulating the restoration of functional tissues. Fibrillar collagens are traditionally known for their structural properties and our study suggests an additional role of collagens in providing instructional cues to direct the regenerative process. The mechanisms behind collagen-directed guidance of planarian neural regeneration is still unclear and this study opens the door to further investigation into the relationship between Dj-Collagen, muscle formation and neural regeneration.