THE EVOLUTION OF CELL-CELL ADHESION AND THE ASSEMBLY OF EPITHELIAL TISSUES WERE INTIMATELY COUPLED TO THE EVOLUTION OF COMPLEX MULTICELLULARITY. EPITHELIA HAVE ESSENTIAL ROLES FROM ACTING AS AN ENVIRONMENTAL BARRIER AND PARTITIONING ORGANISMS INTO DISCRETE COMPARTMENTS TO COORDINATING MORPHOGENESIS DURING DEVELOPMENT. WE PROPOSE TO EXPLORE THE EARLY EVOLUTION OF EPITHELIA - STRUCTURES THAT WE PREDICT WOULD BE ADVANTAGEOUS IN COMPLEX LIFE FORMS ANYWHERE TO UNDERSTAND INNOVATIONS IN THE ANIMAL STEM-LINEAGE THAT CONTRIBUTED TO THE EVOLUTION OF ANIMAL BODY PLANS. DEFINING FEATURES OF EPITHELIA ARE THE ADHESION MODULES THAT TETHER CELLS TO EACH OTHER ENABLE FOLDING AND BENDING OF SHEETS AND TUBES OF CELLS DEFINE APICAL/BASAL POLARITY AND SPECIFY GENE EXPRESSION. OUR FOCUS IS ON THE CADHERIN/CATENIN MODULE (CCM) WHICH FORMS THE MOLECULAR FOUNDATION OF ADHERENS JUNCTIONS IN ALL STUDIED BILATERIAN TISSUES AND IS HYPOTHESIZED TO HAVE EVOLVED IN CONCERT WITH ANIMAL MULTICELLULARITY. FORMATION OF THESE JUNCTIONS APPEARS TO BE THE EARLIEST EVENT IN EPITHELIAL MORPHOGENESIS AND IT SERVES AS A SPATIAL LANDMARK FOR FORMATION OF OTHER TYPES OF CELL-CELL JUNCTIONS. EXPERIMENTAL DISRUPTION OF THE CCM PERTURBS EPITHELIAL DEVELOPMENT INTEGRITY AND POLARITY. OUR OBJECTIVE IS TO EXAMINE HOW AN ANCIENT ACTINMYOSIN SCAFFOLD COMMON TO ALL EUKARYOTES WAS RECRUITED BY THE MORE RECENTLY EVOLVED CCM TO COORDINATE CELL INTERACTIONS NECESSARY FOR MULTICELLULARITY AND TISSUE DIVERSITY. IMPORTANTLY THIS MULTI-FUNCTIONAL MODULE IS PRESENT IN ALL ANIMAL LINEAGES AND POTENTIALLY SATISFIES ALL REQUIREMENTS FOR MULTICELLULAR ADHESION AND MORPHOGENESIS - CADHERINS ENABLE ADHESION BETWEEN CELLS AND CATENINS BIND TO THE ACTIN SCAFFOLD TO ORGANIZE MULTICELLULAR ARCHITECTURE AND CAN SIGNAL TO THE NUCLEUS TO CHANGE GENE EXPRESSION. WE HAVE A DETAILED MECHANISTIC UNDERSTANDING OF THE COMPONENT PARTS AND FUNCTION OF THIS ADHESION MODULE IN BILATERIAN ANIMALS BUT THE EARLY EVOLUTION OF THIS MODULE IS FAR LESS CLEAR. OUR APPROACH IS TO EXAMINE THE FUNCTION OF CCM PROTEINS IN KEY NON-BILATERIAN ANIMAL LINEAGES INCLUDING CNIDARIANS CTENOPHORES AND SPONGES. LITTLE IS KNOWN ABOUT HOW THEY FUNCTION IN THESE ORGANISMS NOR HOW THEY EVOLVED TO TAKE ON NOVEL ROLES TO ENABLE MULTICELLULAR COMPLEXITY. WHAT IS THE MINIMAL SET OF ADHESION PROTEINS NECESSARY FOR THE COORDINATION OF CELL-CELL ADHESION AND LINKAGE TO THE ACTIN CYTOSKELETON AND ARE BASIC FUNCTIONS OF THIS SET SHARED BY ALL ANIMALS? THE CCM IS A LIKELY CANDIDATE AND WE WILL TEST THIS HYPOTHESIS BY OBSERVING CCM PROTEINS IN CELLS AND TISSUES OF NON-BILATERIAN METAZOANS DURING MORPHOGENESIS BY EXAMINING PHYSICAL INTERACTIONS BETWEEN THESE PUTATIVE EPITHELIAL PROTEINS WITH COMPARATIVE BIOCHEMISTRY AND USING FUNCTIONAL GENETICS TO DELETE AND REPLACE GENES PUTATIVELY INVOLVED IN TISSUE MORPHOGENESIS AND CELL-CELL ADHESION. OTHER POTENTIAL CADHERIN-RELATED PROTEINS SIGNALING AND ACTIN-BINDING PROTEINS WILL BE IDENTIFIED USING BIOINFORMATIC DISSECTION OF GENOMES. RESULTS WILL BE INTEGRATED WITH KNOWN PROPERTIES OF THE CADHERIN-CATENIN ADHESION MODULE IN BILATERIANS. OUR EXPERIMENTAL APPROACH IS UNUSUALLY COMPREHENSIVE AND INTERDISCIPLINARY FOR BROAD COMPARATIVE STUDIES AND INVOLVES A DIVERSE TEAM TO TACKLE THIS PROBLEM THAT COMPRISES EVOLUTIONARY-DEVELOPMENTAL BIOLOGISTS (LOWE/MARTINDALE) AN EVOLUTIONARY-CELL BIOLOGIST (NICHOLS) AND A BIOCHEMIST/STRUCTURAL BIOLOGIST (WEIS). THE CAPACITY TO DEVELOP ROBUST TISSUES THAT PROVIDE A PHYSICAL AND CHEMICAL BARRIER TO THE OUTSIDE ENVIRONMENT AND FACILITATE TISSUE SPECIALIZATION IS A BASIC CHARACTERISTIC OF COMPLEX LIFE. THE WORK OUTLINED IN THIS PROPOSAL ADDRESSES FUNDAMENTAL QUESTIONS ABOUT THE ASSEMBLY OF A KEY CELLULAR INNOVATION EARLY IN ANIMAL EVOLUTION EPITHELIA ARE AT THE CORE OF HYPOTHESES OF THE EVOLUTION OF MULTICELLULARITY AND ANIMAL BODY-PLAN COMPLEXITY AND THEREFORE CENTRAL TO THE NASA EXOBIOLOGY MISSION OF UNDERSTANDING ADVANCED LIFE.