Dataset/Excel tables of (i) Table S3: List of proteins found in adult LO nuclei, (ii) Table S4: List of proteins found in WT and Δlux OMVs.

To provide a window into the embryonic preparation of the animal’s immune system for initial colonization of host tissues by symbiotic bacteria, we used the Euprymna scolopes/Vibrio fischeri (squid-vibrio) light-organ model association. With high-throughput chromosome conformation capture (Hi-C), open-chromatin region and transcriptomic profiling, we characterized key regulatory landscapes around light organ genes. The tissues that will eventually harbor the symbionts had strong expression of antimicrobial proteins (AMPs) immediately before hatching. A similar pattern was found in mouse embryos, suggesting evolutionary conservation of a critical event that prepares the host for initial interaction with the microbial world. Using confocal microscopy, we then focused on a light-organ-specific AMP, a galaxin (EsGal), to characterize the transcript and protein biogeographic patterns during embryogenesis and postembryogenesis. We observed coincident EsGal increases with embryonic crypt development, suggesting co-evolution of these nascent symbiotic features. Just before hatching, abundant EsGal was transported into the nucleus of the crypt epithelia, a behavior not uncommon in AMPs. We then localized EsGal postembryonically under three conditions, animals uncolonized, or colonized by WT V. fischeri or by a dark mutant, which is defective in symbiosis-induced phenotypes. Whereas uncolonized and WT colonized tissues retained nuclear localization of EsGal, the protein was in low concentration in dark-mutant nuclei. NanoSIMS experiments and TEM showed that dark mutants are defective in normal import of outer-membrane-vesicle cargo, which is exported by WT V. fischeri and taken up by host epithelia. This study provides evidence that late-stage embryonic and postembryonic activities of AMPs play an important role in normal establishment of animal symbioses.