Muscle functions as a connective tissue and source of extracellular matrix in planarians

Abstract

Regeneration and tissue turnover require new cell production and positional information. Planarians are flatworms capable of regenerating all body parts using a population of stem cells called neoblasts. The positional information required for tissue patterning is primarily harbored by muscle cells, which also control body contraction. Here we produce an in silico planarian matrisome and use recent whole-animal single-cell-transcriptome data to determine that muscle is a major source of extracellular matrix (ECM). No other ECM-secreting, fibroblast-like cell type was detected. Instead, muscle cells express core ECM components, including all 19 collagen-encoding genes. Inhibition of muscle-expressed hemicentin-1 ( hmcn-1 ), which encodes a highly conserved ECM glycoprotein, results in ectopic peripheral localization of cells, including neoblasts, outside of the muscle layer. ECM secretion and hmcn-1- dependent maintenance of tissue separation indicate that muscle functions as a planarian connective tissue, raising the possibility of broad roles for connective tissue in adult positional information.

Planarian EGF repeat-containing genes megf6 and hemicentin are required to restrict the stem cell compartment

“…We noted that two genes, both containing multiple EGF-repeat domains, produced identical, unusual morphological phenotypes upon knockdown: wrinkling of the epidermis as well as unpigmented spots on the dorsal side ( Fig 1A). These two genes were identified as megf6 and hemicentin; these findings confirmed recent observations that knockdown of hemicentin in planarians results in wrinkled epidermis [26]. The wrinkling phenotype was 100% penetrant after 9 feeds of RNAi (0/150 animals in control RNAi, 150/150 in megf6 RNAi, 150/150 in hemicentin RNAi), whereas the appearance of unpigmented spots was only about 50-60% penetrant (0/150 animals in control RNAi, 86/150 in megf6 RNAi, 72/150 in hemicentin RNAi).…”

“…This organization appeared disrupted following knockdown of megf6 or hemicentin, with prog-2 + and agat-3 + cells intermingled rather than ordered by cell type, indicating that localization of these cell types in the parenchyma was altered ( Fig 2B). We also noted that ectopic prog-2 + and agat-3 + cells were sometimes located near ectopic neoblasts as previously observed, indicating that entire ectopic epithelial lineages may be produced apical to the muscle layer ( Fig 2C) [26].…”

“…This finding was corroborated by previously-published single-cell RNA-sequencing data (scRNAseq), which detected megf6 expression in pharynx cells, but also detected lowlevel expression in epidermal and intestinal tissue (S1 Fig) [30]. By contrast, hemicentin expression was almost entirely in muscle cells (S1 Fig) [26]. Interestingly, within the pharynx, hemicentin was co-expressed with the muscle cell marker collagen whereas megf6 expression was not, indicating that these two genes were expressed by different cell populations in the same location ( Fig 1B and 1C, S1 Fig).…”

“…We found that cells appeared in ectopic locations regardless of the proximity of their normal location to the basement membrane. As previously observed following knockdown of hemicentin [26], piwi-1 + neoblasts were detected in clusters near the dorsal epidermis of megf6 or hemicentin knockdown animals, whereas in control animals neoblasts were restricted to the parenchyma ( Fig 2A). Cells stained with prog-2 or agat-3, marking early and late progenitors of the epidermal lineage, respectively, were found adjacent to the epidermis independent of piwi-1 + neoblasts, indicating that these cells were not merely the progeny of ectopic stem cells but also migrated ectopically ( Fig 2B).…”

“…Unlike most of the ectopic cell types, ectopic piwi-1 + cells in megf6 or hemicentin RNAi animals formed clusters. One possible explanation for this was that individual ectopic neoblasts proliferated and formed colonies outside of the normal neoblast compartment, a hypothesis supported by the previous observation of ectopic dividing cells in hemicentin knockdown animals [26]. To test whether ectopic piwi-1 + cells were proliferative, we co-stained planarians with piwi-1 and the G2/M-phase marker phosphorylated-histone H3 on serine 10 (H3P).…”