ANIMALS CAN BE DIVIDED INTO TWO BROAD CLADES BASED ON THEIR BODY PLANS. OVER 99% OF EXTANT ANIMALS BELONG TO A SINGLE CLADE OF COMPLEX BILATERALLY SYMMETRICAL ANIMALS CALLED THE BILATERIA AND 4 REMAINING EARLY EMERGING CLADES ARE CALLED NON-BILATERIANS. THE EVOLUTION OF COMPLEX ANIMALS REMAINS AN ENDURING UNSOLVED QUESTION IN BIOLOGY UNLIKELY TO BE SOLVED BY THEORIES INVOKING ENVIRONMENTAL CHANGES IN EARTH'S HISTORY AND ITS RESOLUTION IS CRITICAL TO UNDERSTANDING THE EXISTENCE OF COMPLEX LIFE IN THE UNIVERSE. THE DEVELOPMENTAL ORIGINS OF BILATERALITY NOR INCREASES IN GENOMIC COMPLEXITY CANNOT BE THE DEFINITIVE EVENT THAT DROVE THE DRAMATICALLY SUCCESSFUL RADIATION OF COMPLEX ANIMALS BECAUSE THESE TRAITS PREDATED THE ORIGIN OF BILATERIA. RATHER EMBRYOLOGICAL AND MOLECULAR STUDIES IN BILATERIANS AND NON-BILATERIANS INDICATE THAT THE CRITICAL DEVELOPMENTAL CONSTRAINT TO BILATERIAN RADIATION WAS A SHIFT IN THE SITE OF GASTRULATION FROM ONE EMBRYONIC POLE IN PRE-BILATERIANS (E.G. CTENOPHORES AND CNIDARIANS) TO THE OPPOSITE POLE IN VIRTUALLY ALL OF BILATERIA. GASTRULATION WAS CRUCIAL FOR EVOLUTIONARY RADIATION OF ANIMALS BECAUSE IT IS DURING THIS PROCESS THAT EMBRYOS ACQUIRE CELLULAR AND MORPHOLOGICAL COMPLEXITY DURING ONTOGENY. GASTRULATION REQUIRES COMPLEX MOLECULAR INTERACTIONS BETWEEN CELLS FROM DIFFERENT EMBRYONIC REGIONS LEADING TO ACTIVATION OF SPATIALLY RESTRICTED GENE REGULATORY NETWORKS (GRN). THESE GRNS IN TURN INDUCE ORGANIZED TISSUES WITH DISTINCT DIFFERENTIATED CELL TYPES THAT HAVE BEEN CRUCIAL FOR THE SUCCESS OF BILATERIA. IN MOST ANIMALS THE LOCALIZED SIGNAL FOR ASYMMETRIC ACTIVATION OF GRNS THAT INDUCE GASTRULATION CAN BE TRACED BACK TO THE ANIMAL-VEGETAL (AV) AXIS A POLARITY ESTABLISHED IN THE OVUM DURING OOGENESIS. WE PROPOSE THAT A SHIFT IN THE SITE OF ACTIVATION OF A SUBSET OF MOLECULAR SIGNALING PATHWAYS FROM THE ANIMAL TO THE VEGETAL POLE TRIGGERED AN AXIAL REORGANIZATION THAT RELIEVED THE DEVELOPMENTAL CONSTRAINT OF GENERATING MULTIPLE GERM LAYER CELL TYPES FROM THE SAME SPATIAL LOCATION (ANIMAL POLE) THAT DROVE BODY PLAN EVOLUTION OF BILATERIA. OUR GOAL IS TO EXPERIMENTALLY RECONSTRUCT THE MECHANISM FOR THIS AXIS REORGANIZATION IN EMBRYOS OF THREE PHYLOGENETICALLY PIVOTAL EXPERIMENTAL SYSTEMS. OUR CENTRAL HYPOTHESIS IS THAT CRITICAL UPSTREAM REGULATORS OF CELL SIGNALING PATHWAYS FOCUSED AT THE ANIMAL POLE OF PRE-BILATERIANS LED TO GASTRULATION AT THAT POLE AND THAT A SHIFT OF A SUBSET OF THESE REGULATORS TO THE VEGETAL POLE LED TO GASTRULATION AT THAT POLE IN BILATERIA. THE CENTRAL OBJECTIVES OF THIS PROPOSAL ARE TO: 1) TO IDENTIFY AND FUNCTIONALLY CHARACTERIZE NOVEL MEDIATORS OF ANIMALVEGETAL AXIS POLARITY IN NON-BILATERIANS 2) TO IDENTIFY GENES THAT ARE DIRECTLY ACTIVATED OR REPRESSED BY BETA-CATENIN IN SEA URCHINS NEMATOSTELLA AND MNEMIOPSIS TO DETERMINE THE UNDERLYING CIS-REGULATORY MECHANISMS 3) TO EXPERIMENTALLY RE-ENGINEER THE PRIMARY EGG AXIS AND GASTRULATION SITE BY MANIPULATING THE SPATIAL ACTIVATION OF SIGNALING PATHWAYS. OUR RESEARCH WILL COMBINE EMBRYO MANIPULATION WITH MODULATION OF GENE EXPRESSION USING MRNA INJECTION MORPHOLINOS TRANSGENESIS AND CRISPR/CAS9 IN SEA URCHINS (BILATERIA) AND TWO PREBILATERIANS (CNIDARIANS AND CTENOPHORES). WE WILL ALSO USE CO- IMMUNOPRECIPITATION TO IDENTIFY UPSTREAM REGULATORS OF THESE SIGNALING PATHWAYS AND CELL SORTING TO IDENTIFY NOVEL MODIFICATIONS OF GRNS IN EMBRYOS WITH RE-ENGINEERED PRIMARY EGG AXES. WE AIM TO EXPERIMENTALLY DISSECT THE CELLULAR AND MOLECULAR CONSTRAINTS TO RAPID ANIMAL BODY PLAN DIVERSIFICATION WHICH DIRECTLY ADDRESSES THE GOALS OF SECTION 3.14.IV OF THE 2015 ASTROBIOLOGY ROADMAP ASSOCIATED WITH THE EVOLUTION OF ADVANCED LIFE THE ORIGIN AND EARLY EVOLUTION OF THOSE BIOLOGICAL FACTORS THAT ARE ESSENTIAL TO MULTICELLULAR LIFE SUCH AS DEVELOPMENTAL PROGRAMS INTERCELLULAR SIGNALING PROGRAMMED CELL DEATH THE CYTOSKELETON CELLULAR ADHESION CONTROL AND DIFFERENTIATION IN THE CONTEXT OF THE ORIGIN OF ADVANCED LIFE.