Abstract
The regeneration of the sponge Hymeniacidon heliophila was investigated using the in vitro microexplant model. This model differs from cell dissociation, surface ablation, and fragment culture models because it preserves the choanosome attached to the substrate, maintaining the apical-basal axis while entirely lacking the ectosome and the water flow polarity (ostia-osculum). The complete ectosome regenerative development was assessed using stereoscopy and scanning electron microscopy until complete adult formation. The observed stages of ectosome regeneration from the preserved choanosome align with existing data on sponge regeneration. Epithelization begins ubiquitously across the microexplant surface and expands to fully encase the microexplant. Two key processes were noticed: (1) the formation of an extracellular layer at the surface and (2) the appearance of discrete patches of pinacoderm over the extracellular matrix, probably by mesenchymal-to-epithelial transition. In more advanced regeneration stages, ectosomal spicules align transversely to the pinacoderm, with their tips protruding outward. These spicules provide structural support for the developing subdermal space while the choanosome concurrently becomes functional. Additionally, outer cells were identified in both adult microexplants and naturally growing individuals. Further efforts are needed to distinguish whether these outer cells are epibionts or autologous. As final considerations, the data were discussed from an evolutionary perspective that regeneration in sponges is an inherent feature linked to the emergence of multicellularity in animals. This process represents a primordial mechanism for stabilizing metazoan multicellular organization.