Mount Desert Island Biological Laboratory

COMPARATIVE FUNCTIONAL GENOMICS INBRE IN MAINE

COMPARATIVE BIOLOGY OF TISSUE REPAIR, REGENERATION AND AGING

COMPARATIVE FUNCTIONAL GENOMICS INBRE IN MAINE

EXTRAMURAL RESEARCH FACILITIES CONSTRUCTION CONSTRUCTIO*

THE FUNCTION OF GERM GRANULES IN MAINTAINING PLURIPOTENCY IN THE C. ELEGANS GERMLINE

IDENTIFYING PATHWAYS REQUIRED FOR INTEGRATION OF KIDNEY ORGANOID AND HOST EPITHELIA

THE MDIBL CENTER FOR COMPARATIVE BIOLOGY OF TISSUE REPAIR, REGENERATION AND AGING - OVERALL PROJECT SUMMARY LIMITED REGENERATION OF DAMAGED ORGANS AND TISSUES IN HUMANS LEADS TO CHRONIC DISEASE AND DEGENERATION, WHILE IN MANY INVERTEBRATES AND LOWER VERTEBRATES, CELLULAR REGENERATION REPAIRS AND PRESERVES TISSUE FUNCTION THROUGHOUT THE LIFESPAN. COBRE PHASES I AND II COMPARATIVE BIOLOGY OF TISSUE REPAIR, REGENERATION AND AGING, PLAYED A CENTRAL ROLE IN ESTABLISHING THE KATHRYN W. DAVIS CENTER FOR REGENERATIVE BIOLOGY AND AGING (DAVIS CENTER) AT THE MDI BIOLOGICAL LABORATORY (MDIBL) AS A LEADING INSTITUTE IN COMPARATIVE BIOLOGY. THE DAVIS CENTER WAS FOUNDED ON THE PRINCIPLE THAT COMPARATIVE STUDIES OF DIVERSE ANIMAL MODELS WOULD LEAD TO A MECHANISTIC UNDERSTANDING OF TISSUE AND ORGAN REGENERATION AND IDENTIFY KEY POINTS OF INTERVENTION TO IMPROVE REGENERATION IN HUMAN TISSUES. IN ADDITION, DAVIS CENTER RESEARCH HAS FOCUSED ON MECHANISMS OF AGING USING MULTIPLE ORGANISMS TO LEARN HOW CELLULAR ANABOLIC AND CATABOLIC PATHWAYS MAY BE LINKED TO EXTENSION OF HEALTHSPAN. COMPARATIVE STUDIES SUCH AS THESE PROVIDE A SCREENING PLATFORM AND RATIONAL FOUNDATION FOR DEVELOPMENT OF REGENERATIVE MEDICINE THERAPIES, INCLUDING SMALL MOLECULE DRUG CANDIDATES CAPABLE OF STIMULATING TISSUE REGENERATION AND SLOWING OR REVERSING AGING-INDUCED DEGENERATIVE CHANGES IN PATIENTS. COBRE PHASES I AND II SUPPORTED EIGHT EARLY-CAREER PROJECT LEADERS AND ONE MID-CAREER PROJECT LEADER. SIX PROJECT LEADERS GRADUATED WITH MAJOR INDEPENDENT GRANT SUPPORT AND THE MOST RECENT COBRE RECRUIT, PRAYAG MURAWALA, HAS ACHIEVED SIGNIFICANT PROFESSIONAL SUCCESS AND IS ON A PATH TO INDEPENDENT R01 SUPPORT. PAST AND CURRENT COBRE PROJECT LEADERS HAVE ACHIEVED MULTIPLE PROFESSIONAL MILESTONES IN PUBLICATIONS, GRANTS, PATENTS, NEW DISEASE MODELS AND RESEARCH TOOLS, AND FORMATION OF AN IDEA PROGRAM/MAINE STATE GOVERNMENT PARTNERSHIP THAT ALLOWED MDIBL TO OBTAIN $3M IN VOTER-APPROVED STATE BOND FUNDING TO EXPAND RESEARCH INFRASTRUCTURE. COBRE PHASE III WILL CONTINUE TO BUILD THE RESEARCH INFRASTRUCTURE OF THE DAVIS CENTER BY ESTABLISHING SELF- SUSTAINING CORE FACILITIES AND FUNDING PILOT PROGRAMS IN AGING AND REGENERATION RESEARCH. ESSENTIAL CORE SERVICES WILL BE PROVIDED TO COBRE GRADUATES, MDIBL INVESTIGATORS, VISITING SCIENTISTS, PILOT PROJECT AWARDEES, MDIBL COURSE PARTICIPANTS FROM INBRE NETWORK INSTITUTIONS, AND INVESTIGATORS IN NORTHEAST REGION (NER) IDEA STATES. PILOT PROJECT FUNDING AND IMAGING TECHNOLOGY ACCESS GRANTS WILL BE MADE AVAILABLE TO ALL INVESTIGATORS IN NER IDEA STATES. COBRE PHASE III WILL GREATLY ENHANCE THE DEVELOPMENT OF THE DAVIS CENTER AND MDIBL, WHICH IN TURN WILL ENSURE THE CONTINUED ENHANCEMENT OF THE BIOMEDICAL RESEARCH ENVIRONMENT IN MAINE.

MOLECULAR MECHANISMS OF OSMOSENSING AND SIGNAL TRANSDUCTION IN C ELEGANS

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION - MDI BIOLOGICAL LABORATORY HAS LAUNCHED A NEW INITIATIVE, MDI BIOSCIENCE, THAT LEVERAGES THE LAB’S INTERNATIONALLY RECOGNIZED EXPERTISE IN COMPARATIVE AND MOLECULAR BIOLOGY TO DISCOVER NEW MEDICINES WITH GREATER EFFICIENCY AND EFFICACY THAN IS CURRENTLY POSSIBLE WITH EXISTING APPROACHES. THE PURPOSE OF THIS PROJECT IS TO FURTHER EXPAND OUR CAPACITY BY CREATING A STATE-OF-THE-ART RESEARCH FACILITY FOCUSED ON DEVELOPING NOVEL MODELS FOR DRUG DISCOVERY. THE NEW RESEARCH FACILITY WILL PLAY A KEY ROLE IN SPEEDING DRUG DISCOVERY BY STRENGTHENING PARTNERSHIPS WITH PHARMACEUTICAL COMPANIES, FOSTERING GREATER COLLABORATIONS WITH MAINE HEALTH, THE ROUX INSTITUTE AND THE JACKSON LABORATORY, AS WELL AS EXPANDING BIOENTREPRENEURSHIP TRAINING OPPORTUNITIES FOR MAINE STUDENTS.

ORIGINS OF RENAL PHYSIOLOGY

DATA TO ACTION: A SECONDARY SCHOOL-BASED CITIZEN SCIENCE PROJECT TO ADDRESS ARSENIC CONTAMINATION OF WELL WATER

CENTER FOR REGENERATIVE BIOLOGY AND MEDICINE AT MOUNT DESERT ISLAND BIOLOGICAL LABORATORY

MECHANISMS OF TUBULE INTERCONNECTION

MODELING DR AND MRNA TRANSLATION TO UNDERSTAND ADAPTIVE MECHANISMS THAT PROMOTE HEALTH

THE FUNCTION OF GERM GRANULE PROCESSES THAT MAINTAIN PLURIPOTENCY IN THE C. ELEGANS GERMLINE - PROJECT SUMMARY THE PLURIPOTENT POTENTIAL OF A CELL IS GENERALLY ASSUMED TO BE A PRODUCT OF ITS NUCLEUS, REGULATED BY GENE EXPRESSION AND CHROMATIN STATE. THIS VIEW INFORMS MOST APPROACHES TO DRUG DISCOVERY, WHERE AN IDEAL THERAPEUTIC WOULD TARGET NUCLEAR PROCESSES TO INDUCE PLURIPOTENCY AND PROLIFERATION TO HEAL A WOUND OR INHIBIT PLURIPOTENCY AND PROLIFERATION IN CANCER. OFTEN OVERLOOKED ARE SOMATIC CELL NUCLEAR TRANSFER EXPERIMENTS THAT DEFINITIVELY SHOW CELLULAR POTENTIAL IS ALSO A PRODUCT OF THE CYTOPLASM, SPECIFICALLY, THE CYTOPLASM OF GERM CELLS. THE LONG-TERM OBJECTIVES OF THIS RESEARCH ARE TO UNDERSTAND HOW CELLULAR PLURIPOTENCY AND REGENERATIVE CAPACITY ARE RETAINED IN GERM CELLS THROUGH THEIR SPECIALIZED CYTOPLASM. WE EXPECT THESE FINDINGS TO INFORM AND EXPAND APPROACHES TO DRUG DISCOVERY AND LEAD TO THERAPEUTICS THAT FUNCTION BY TARGETING THE CYTOPLASMIC MILIEU. CYTOPLASMIC PROCESSES UNIQUE TO GERM CELLS OFTEN OCCUR IN PROTEIN/RNA ASSEMBLIES THAT RESIDE ON OR NEAR THE NUCLEAR PERIPHERY. THESE ASSEMBLIES ARE COLLECTIVELY CALLED GERM GRANULES. BECAUSE CORE GERM-GRANULE COMPOSITION IS CONSERVED FROM NEMATODES TO HUMANS, WE CAN USE C. ELEGANS NEMATODES TO VISUALIZE GERM GRANULES IN VIVO AND TO EXPEDITE STUDIES INTO THEIR FUNCTION. MUTATIONS THAT DISPLACE OR SEVERELY DISRUPT GERM GRANULES IN C. ELEGANS CAUSE STERILITY AND GERMLINE-TO-SOMATIC REPROGRAMMING. IN THIS PROPOSAL, WE LOOK AT THE EARLIEST EVENTS OF SOMATIC REPROGRAMMING TO DETERMINE HOW PROCESSES WITHIN GERM GRANULES ANTAGONIZE THIS FATE. WE INVESTIGATE THE CONTRIBUTION OF GLYCINE-RICH REPEAT MOTIFS TO THE SPECIALIZED GERM-GRANULE MICROENVIRONMENT. WE THEN DESCRIBE TWO NOVEL INTERACTIONS WITH A CORE GERM-GRANULE PROTEIN CALLED GLH-1 (DDX4 IN HUMANS). ONE OF THESE INTERACTIONS WITH GLH-1 LIKELY FACILITATES THE HAND-OFF OF MRNAS FROM GERM GRANULES TO INITIATE TRANSLATION. THE OTHER IS A DYNAMIC INTERACTION WITH GLH-1 THAT ACTIVATES A CONSERVED CELLULAR PATHWAY TO TRIGGER ENGULFMENT BY SURROUNDING SOMA. LASTLY, WE FOLLOW UP ON ANOTHER CORE GERM-GRANULE PROTEIN THAT WE NAMED LOTR-1 (TDRD5/7 IN HUMANS) AND ITS LOTUS-DOMAIN-DEPENDENT INTERACTION WITH A 3’UTR BINDING COMPLEX. TOGETHER, THESE FINDINGS WILL PROVIDE AN UNDERSTANDING OF CYTOPLASMIC PROCESSES WITHIN GERM GRANULES THAT ENSURE THE PLURIPOTENT POTENTIAL OF GERM CELLS.

PROMOTING ENVIRONMENTAL HEALTH LITERACY THROUGH SCIENTIFIC COMMUNICATION AND INTERGENERATIONAL LEARNING IN A K-12 SAFE DRINKING WATER CITIZEN SCIENCE PROJECT (COMMUNICATING DATA) - ABSTRACT/SUMMARY (30 LINES): THE COMMUNICATING DATA PROJECT ENGAGES SECONDARY SCHOOL TEACHERS AND STUDENTS FROM BOTH RURAL AND URBAN AREAS IN MAINE AND NEW HAMPSHIRE AS CITIZEN SCIENTISTS IN COLLECTING BOTH PRIVATE AND PUBLIC DRINKING WATER SAMPLES FOR HEAVY METAL ANALYSIS AND COMMUNICATING THEIR FINDINGS WITH A GOAL OF IMPROVING PUBLIC HEALTH IN THEIR COMMUNITIES. THIS NEW PROJECT WILL BUILD ON OUR CURRENT SEPA PROJECT WHICH HAS A FOCUS ON ARSENIC IN PRIVATE WELL WATER IN RURAL COMMUNITIES. BASED ON WHAT WE ARE LEARNING IN OUR FIRST PROJECT, THAT NEARLY ALL COMMUNITIES HAVE A MIX OF SOME PUBLIC WATER SYSTEMS AS WELL AS PRIVATE WELLS, WE WILL PROVIDE A MORE INCLUSIVE EXPERIENCE FOR STUDENTS BY HAVING THEM COLLECT HOME DRINKING WATER SAMPLES FOR ANALYSIS, NO MATTER WHAT THE SOURCE. WE WILL PROVIDE CURRICULUM ON DRINKING WATER SYSTEMS SO THAT STUDENTS CAN IDENTIFY THEIR SOURCE OF DRINKING WATER. WHILE OUR FIRST PROJECT FOCUSED ON ARSENIC IN WELLS, MANY SCHOOLS FOUND THAT URANIUM AND LEAD AND OTHER CONTAMINANTS WERE ALSO ISSUES THAT NEED ATTENTION. SO, WE WILL DEVELOP CURRICULUM THAT ADDRESSES MULTIPLE DRINKING WATER CONTAMINANTS AND PROVIDE TRAINING FOR TEACHERS IN THESE AREAS. WE HAVE ALREADY DEVELOPED DATA LITERACY TOOLS AND CURRICULUM THAT WILL SERVE TEACHERS AND STUDENTS IN THIS NEW PROJECT. WITH THESE SUPPORTS IN PLACE, OUR NEW SEPA PROJECT WILL FOCUS ON HELPING STUDENTS CREATE DATA VISUALIZATIONS SO THAT THEY CAN MORE EFFECTIVELY COMMUNICATE THEIR FINDINGS TO DIVERSE AUDIENCES. WE KNOW FROM SURVEYS OF HOMEOWNERS WHO CONTRIBUTED WELL WATER SAMPLES IN THE FIRST YEARS OF OUR CURRENT PROJECT, THAT THE LIKELIHOOD OF MITIGATING ARSENIC IN DRINKING WATER WAS RELATED TO THE EXTENT OF PARENT-CHILD INTERACTIONS IN THE HOME. TO FURTHER SUPPORT THESE INTERACTIONS, WE WILL ALSO FOCUS ON INTERGENERATIONAL LEARNING. IN GUIDED FOCUS SESSIONS WITH PARENTS, WE WILL GET THEIR INPUT ON DEVELOPING APPROPRIATE AT-HOME CONVERSATION PROMPTS AND SINK-SIDE ACTIVITIES TO ENGAGE FAMILIES MORE ACTIVELY IN UNDERSTANDING THE IMPORTANCE OF HEALTHY DRINKING WATER. WE ANTICIPATE THAT THIS APPROACH, COMBINED WITH OUR EXPANDED FOCUS ON MULTIPLE CONTAMINANTS IN DRINKING WATER FROM HOMES DEPENDENT ON EITHER PRIVATE OR PUBLIC DRINKING WATER SOURCES IN EITHER URBAN OR RURAL ENVIRONMENTS WILL ENABLE US TO MORE BROADLY DISSEMINATE OUR PROJECT AND FACILITATE ITS REPLICABILITY IN OTHER GEOGRAPHIC AREAS. .

LIFESPAN EXTENSION BY DIFFERENTIAL TRANSLATION MEDIATED BY EIF-4G IN C. ELEGANS

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION

REU SITE: RESEARCH EXPERIENCES IN MARINE MOLECULAR PHYSIOLOGY AND ENVIRONMENTAL STRESS

CONTROL OF CELL PROLIFERATION BY RUNX PROTEINS

OSCILLATORY CA2 SIGNALING IN THE C. ELEGANS INTESTINE

BIOINFORMATICS "TRAIN THE TRAINER" (T3): THE INTEGRATION OF BIOINFORMATICS INTO AN UNDERGRADUATE BIOLOGY CURRICULUM

IMPROVING MARINE AND FRESHWATER ANIMAL FACILITIES AND ENVIRONMENT AT MDIBL

REU SITE: COMPARATIVE ANIMAL MODEL APPROACHES TO REGENERATION AND AGING -THIS REU SITE AWARD TO THE MDI BIOLOGICAL LABORATORY, LOCATED IN BAR HARBOR, ME, WILL SUPPORT THE TRAINING OF 10 STUDENTS FOR 10 WEEKS DURING THE SUMMERS OF 2026?2028. EACH SUMMER, UNDERGRADUATE STUDENTS WILL CONDUCT INDEPENDENT, MENTORED RESEARCH PROJECTS FOCUSED ON UNDERSTANDING HOW LIVING THINGS REPAIR AND RENEW THEMSELVES AFTER INJURY AND AS THEY AGE. STUDENTS WILL WORK ALONGSIDE EXPERIENCED SCIENTISTS USING A DIVERSE SET OF ANIMAL MODELS. COMPARITIVE STUDIES USING THESE ANIMALS PROVIDE STUDENTS WITH A UNIQUELY BROAD PERSPECTIVE ON THE BIOLOGY OF HEALING AND AGING ACROSS THE ANIMAL KINGDOM. THE PROGRAM PLACES SPECIAL EMPHASIS ON RECRUITING STUDENTS FROM PRIMARILY UNDERGRADUATE INSTITUTIONS, COMMUNITY COLLEGES, AND CAMPUSES WITH LIMITED RESEARCH INFRASTRUCTURE, BROADENING PARTICIPATION IN SCIENCE AND HELPING BUILD AND STRENGTHEN THE STEM WORKFORCE. IN ADDITION TO THEIR LABORATORY RESEARCH, STUDENTS PARTICIPATE IN WORKSHOPS ON BIOINFORMATICS, ADVANCED MICROSCOPY, AND SCIENCE COMMUNICATION, AS WELL AS A CAREER SNAPSHOT SERIES THAT EXPOSES THEM TO A WIDE RANGE OF SCIENTIFIC CAREER PATHS IN ACADEMIA, INDUSTRY, POLICY, AND BEYOND. STUDENTS WILL LEARN HOW RESEARCH IS CONDUCTED, AND PRESENT THE RESULTS AT A STUDENT SYMPOSIUM. ASSESSMENT OF THIS PROGRAM WILL BE CONDUCTED THROUGH PRE- AND POST-PROGRAM SURVEYS BASED ON THE BIO REU URSSA COMMON ASSESSMENT TEMPLATE, ALONG WITH LONG-TERM TRACKING OF STUDENT CAREER OUTCOMES. STUDENTS SHOULD APPLY TO THE REU SITE USING NSF ETAP (EDUCATION AND TRAINING APPLICATION: HTTPS://ETAP.NSF.GOV). THE TRAINING STUDENTS WILL RECEIVE IS ALIGNED WITH THE NSF PRIORITY IN BIOTECHNOLOGY. THE SCIENTIFIC FOCUS OF THIS REU SITE IS COMPARATIVE REGENERATIVE AND AGING BIOLOGY, DRAWING ON THE COMPLEMENTARY STRENGTHS OF NON-MAMMALIAN ANIMAL MODEL SYSTEMS. RESEARCH GROUPS AT MDIBL INVESTIGATE QUESTIONS SPANNING KIDNEY REGENERATION IN ZEBRAFISH, LIMB AND HEART REGENERATION IN AXOLOTLS, STEM CELL REGULATION AND AGING IN KILLIFISH, NEURONAL REGENERATION IN ZEBRAFISH, NUCLEOLAR ORGANIZATION IN NEURONS USING C. ELEGANS, TISSUE REPAIR MEDIATED BY MACROPHAGES IN SALAMANDERS, MICRORNA REGULATION OF STEM CELL FATE IN DROSOPHILA, AND THE ROLE OF CYTOPLASMIC GERM GRANULES IN GERMLINE IMMORTALITY AND PLURIPOTENCY. STUDENTS RECEIVE INDIVIDUALIZED MENTORSHIP FROM THIRTEEN FACULTY INVESTIGATORS AND TWO CORE FACILITY DIRECTORS, WITH EACH PROJECT LEVERAGING MDIBL'S STATE-OF-THE-ART LIGHT MICROSCOPY FACILITY ? EQUIPPED WITH TWO-PHOTON CONFOCAL, SPINNING DISK, SUPER-RESOLUTION, AND LIGHTSHEET MICROSCOPES ? AND THE COMPARATIVE GENOMICS AND DATA SCIENCE CORE FOR BIOINFORMATICS SUPPORT. PROFESSIONAL DEVELOPMENT IS WOVEN THROUGHOUT THE PROGRAM VIA THE COMMUNICATING SCIENCE COURSE, WHICH COVERS SCIENTIFIC WRITING, POSTER AND ORAL PRESENTATION SKILLS, AND OUTREACH COMMUNICATION, AND VIA A RESPONSIBLE CONDUCT OF RESEARCH WORKSHOP COVERING DATA MANAGEMENT, ETHICAL AUTHORSHIP, AND RESEARCH INTEGRITY. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

EXPLORING TISSUE-SPECIFIC MECHANISMS AND REQUIREMENTS FOR NUCLEOLIN ORGANIZATION IN VIVO - PROJECT SUMMARY PRECISE ORGANIZATION OF CELLULAR SPACE IS A FUNDAMENTAL THEME OF EUKARYOTIC CELL BIOLOGY AND IS ESSENTIAL FOR CONTROL OVER COMPLEX BIOLOGICAL REACTIONS. SUBCELLULAR ORGANIZATION OF RNA BINDING PROTEINS ENABLES TARGETED REGULATION OF RNA METABOLISM, AND THEIR DISORGANIZATION IS A HALLMARK OF HUMAN DISEASE. FOR EXAMPLE, THE RNA BINDING PROTEIN, NUCLEOLIN (NCL), DISPLAYS PRECISE ORGANIZATION WITHIN THE NUCLEOLUS. THIS ORGANIZATION IS CONSIDERED CRITICAL FOR RIBOSOME BIOGENESIS AND, CONSEQUENTLY, IS DISRUPTED IN SEVERAL FORMS OF DISEASE. BECAUSE NCL IS AN ESSENTIAL GENE IN VERTEBRATE ANIMAL MODELS AND NO CLEAR HOMOLOG EXISTS IN DROSOPHILA, MOST STUDIES OF NCL ORGANIZATION USE IN VITRO MODELS. HOWEVER, THE NUCLEOLUS IS EXQUISITELY SENSITIVE TO ENVIRONMENT AND IN VITRO CONDITIONS CAN INFLUENCE NUCLEOLAR DYNAMICS. AS A RESULT, THE BIOLOGICAL MECHANISMS THAT REGULATE NCL ORGANIZATION ARE UNCLEAR. WE SURMOUNT THESE CHALLENGES WITH OUR DISCOVERY OF THE C. ELEGANS HOMOLOG OF NCL, NAMED NUCL-1. DELETING ENDOGENOUS NUCL-1 RESULTS IN VIABLE WORMS, PROVIDING THE OPPORTUNITY TO FILL A LARGE GAP IN KNOWLEDGE AND EXPLORE MECHANISMS OF NCL ORGANIZATION IN A LIVING ANIMAL, TO BETTER UNDERSTAND ITS ROLE IN DISEASE. C. ELEGANS ARE SHORT-LIVED, OPTICALLY CLEAR ANIMALS WITH FACILE GENETICS, MAKING THEM THE IDEAL MODEL FOR OUR PROPOSED IN VIVO STUDIES. WE RECENTLY USED CRISPR/CAS9 GENETIC ENGINEERING TO DEVELOP AN IN VIVO, SPLIT- FLUOR SYSTEM TO VISUALIZE NUCL-1 IN GERM CELL NUCLEOLI. COMBINING THIS SYSTEM WITH SUPER-RESOLUTION (AIRYSCAN) IMAGING, WE WERE THE FIRST TO MAP THE INTRICATE SUB-NUCLEOLAR ORGANIZATION OF GERM CELLS IN LIVING C. ELEGANS. PRECISE SUB-NUCLEOLAR ORGANIZATION IS THOUGHT TO BE ESSENTIAL FOR RIBOSOME BIOGENESIS IN ALL EUKARYOTIC CELLS, BUT OUR IN VIVO WORK CHALLENGES THIS HYPOTHESIS. FULL DELETION OF THE NUCL-1 GENE CAUSES IMPAIRED FERTILITY, DELAYED DEVELOPMENT, EXTENDED LIFESPAN, AND SMALLER BODY SIZE, ALL PHENOTYPES LINKED TO IMPAIRED RIBOSOME BIOGENESIS. IN CONTRAST, DELETING THE ARGININE-GLYCINE (RG) REPEAT DOMAIN FROM ENDOGENOUS NUCL-1 DISRUPTS SUB-NUCLEOLAR ORGANIZATION, BUT WORMS ARE HEALTHY AND FERTILE WITH MOTOR HYPERACTIVITY. THESE RESULTS DECOUPLE PRECISE SUB- NUCLEOLAR ORGANIZATION FROM PHENOTYPES ASSOCIATED WITH RIBOSOME BIOGENESIS AND LEAD TO THE HYPOTHESIS THAT THERE ARE TISSUE-SPECIFIC REQUIREMENTS FOR SUB-NUCLEOLAR ORGANIZATION. OVER THE NEXT FIVE YEARS, WE WILL EXPAND OUR IN VIVO, SPLIT-FLUOR SYSTEM INTO A MODULAR GENETIC TOOLKIT WITH WHICH TO EXTEND OUR STUDY OF NUCL-1 ORGANIZATION TO ALL MAJOR SOMATIC TISSUES. USING THIS TOOLKIT WE WILL ADDRESS THE FOLLOWING KNOWLEDGE GAPS: (1) WHAT IS THE EXTENT OF DIVERSITY IN SUB-NUCLEOLAR ORGANIZATION ACROSS SOMATIC TISSUES AND HOW DOES THE NUCL-1 RG REPEAT DOMAIN CONTRIBUTE TO THAT ORGANIZATION, AND (2) WHICH SOMATIC TISSUES REQUIRE PRECISE SUB-NUCLEOLAR ORGANIZATION FOR THEIR NORMAL FUNCTION? THE PROPOSED RESEARCH WILL EXPLORE TISSUE-SPECIFIC PATTERNS, MECHANISMS, AND FUNCTIONS OF SUB-NUCLEOLAR ORGANIZATION AND WILL LAY THE GROUNDWORK FOR UNDERSTANDING THE ROLE OF NCL ORGANIZATION IN HUMAN DISEASE.

DEVELOPMENT AND VALIDATION OF GENETIC CELL ABLATION SYSTEMS IN AXOLOTL - ABSTRACT: THE AXOLOTL IS A CLASSICAL AND ROBUST MODEL OF TISSUE REGENERATION. AXOLOTLS ARE WELL KNOWN FOR STUDIES OF LIMB REGENERATION BUT ALSO REGENERATE THEIR EYES, LUNGS, HEART, KIDNEY, SPINAL CORD, BRAIN AND PROVIDE A SYSTEM TO STUDY IMMUNE REGULATION OF REGENERATION OF ALL BODY TISSUES AND ORGANS. STUDYING MECHANISMS OF REGENERATION IN AXOLOTL PROVIDES A ROADMAP FOR ENGINEERING SIMILAR REGENERATION MECHANISMS IN MAMMALS AND ULTIMATELY HUMANS. TWO KEY QUESTIONS IN REGENERATION BIOLOGY THAT REMAIN INCOMPLETELY UNDERSTOOD ARE: (1). WHAT ARE THE SOURCES OF REPLACEMENT CELLS DURING INJURY REPAIR? AND (2). HOW DO CELLS INTERACT DURING REGENERATION TO CREATE COMPLEX TISSUES THAT FUNCTION AS WELL AS THE ORIGINAL ORGANS? OUR WORK ON GENERATING TRANSGENIC AXOLOTL HAS BROUGHT THIS CLASSICAL SYSTEM INTO THE MODERN GENOMIC ERA WHERE WE CAN NOW LINEAGE TRACE CELLS THAT CONTRIBUTE TO REGENERATION AND START TO TEASE APART SPECIFIC REGENERATION MECHANISMS IN DIFFERENT ORGANS. HOWEVER, TO FULLY UNDERSTAND THE CONTRIBUTION OF DIFFERENT CELL TYPES AND LINEAGES TO REGENERATION, CELL ABLATION APPROACHES ARE REQUIRED. CELL ABLATION ALLOWS US TO ASK QUESTIONS SUCH AS: WHAT IS THE ROLE OF A SPECIFIC CELL TYPE IN A COMPLEX MIXTURE OF CELLS DURING REGENERATION? IS A CERTAIN CELL TYPE NECESSARY FOR ORGAN/APPENDAGE REGENERATION? CAN OTHER CELLS ACT AS A RESERVE POOL WHEN THE CELL TYPE THAT NORMALLY PARTICIPATES IN THE REGENERATIVE PROCESS IS MISSING? HOW DO DIFFERENT CELL TYPES CONTRIBUTE TO THE OVERALL PATTERNING OF THE ORGAN OR TISSUE REGENERATE? TO ANSWER THESE IMPORTANT QUESTIONS, WE PROPOSE GENERATING BROADLY APPLICABLE TRANSGENIC AXOLOTL TOOLS FOR LINEAGE SPECIFIC CELL ABLATION STUDIES. THESE TOOLS EITHER UTILIZE ENZYMES THAT PRODUCES TOXINS OR MAKE USE OF PROTEINS/TOXINS THAT ACTIVATE/INDUCE CELL APOPTOSIS. SPECIFICALLY, WE WILL GENERATE NITROREDUCTASE (NTR) OR DIPHTHERIA TOXIN RECEPTOR (DTR) TRANSGENIC AXOLOTLS UNDER LOXP CONTROL AND CROSS THEM WITH TISSUE SPECIFIC INDUCIBLE CRE LINES THAT ARE ALREADY AVAILABLE IN OUR LABORATORY TO SPECIFICALLY EXPRESS DTR OR NTR IN THE TISSUE OF INTEREST. THIS WILL PROVIDE US SPATIAL CONTROL OVER CELL ABLATION. FURTHER BY PROVIDING METRONIDAZOLE (DRUG) OR DIPHTHERIA (TOXIN) RESPECTIVELY TO NTR AND DTR LINES, WE WILL BE ABLE TO ACHIEVE TEMPORAL CONTROL. SUCH SPATIOTEMPORAL CONTROL OF CELL ABLATION CAN BE ACHIEVED OVER ANY CELL TYPE IF AN INDUCIBLE CRE LINE IS AVAILABLE FOR THE TISSUE OF INTEREST. HENCE, WE BELIEVE THAT THE DEVELOPMENT OF GENETIC CELL ABLATION SYSTEMS IN AXOLOTL WILL OPEN DOORS FOR STUDYING A WIDE VARIETY OF ORGANS AND APPENDAGES FOR EXAMPLE, LIMB, TAIL, EYE, LUNG, HEART, KIDNEY, SPINAL CORD, BRAIN AND THE ROLE OF IMMUNE CELLS IN AXOLOTL TISSUE REGENERATION.

LONG-TERM RISK FOR PSYCHIATRIC DISORDERS FOLLOWING EARLY LIFE EXPOSURE TO ENVIRONMENTAL ARSENIC - PROJECT SUMMARY / ABSTRACT ARSENIC EXPOSURE FROM WELL WATER AND FOOD IS A MAJOR PUBLIC HEALTH CONCERN. EARLY LIFE (E.G., PRENATAL) EXPOSURE TO LOW LEVEL ARSENIC INCREASES RISK OF DEVELOPING PSYCHIATRIC DISORDERS SUCH AS DEPRESSION AND ANXIETY IN ADULTHOOD. ARSENIC IS AN ENDOCRINE DISRUPTOR AND AVAILABLE EVIDENCE SUGGESTS THAT ITS PSYCHOPATHOLOGICAL EFFECTS STEM FROM INTERFERENCE WITH GLUCOCORTICOID RECEPTOR (GR) SIGNALING; HOWEVER, GAPS REMAIN IN OUR UNDERSTANDING OF THE UNDERLYING MECHANISMS. KRÜPPEL-LIKE FACTOR 9 (KLF9) IS A TRANSCRIPTION FACTOR IMPORTANT FOR NEUROGENESIS AND METABOLIC REGULATION AND IS A REGULATORY HUB AT THE JUNCTION OF MULTIPLE ENDOCRINE AND CELLULAR STRESS SIGNALING PATHWAYS. KLF9 HAS BEEN IMPLICATED IN STRESS-INDUCED PSYCHOPATHOLOGY AND NEURODEGENERATION. WE AND OTHERS HAVE SHOWN THAT KLF9 IS A FEEDFORWARD REGULATOR OF GR SIGNALING. WE HAVE OBTAINED PRELIMINARY DATA INDICATING THAT THE GR-KLF9 GENE REGULATORY PATHWAY IS PERTURBED IN ZEBRAFISH EMBRYOS EXPOSED TO LOW-LEVEL ARSENIC, THIS EXPLORATORY/DEVELOPMENTAL RESEARCH PROJECT WILL USE ZEBRAFISH AS A MODEL SYSTEM TO TEST THE HYPOTHESIS THAT CHRONIC EARLY LIFE EXPOSURE TO LOW LEVELS OF ARSENIC INCREASES RISK OF DEPRESSION AND/OR OTHER PSYCHIATRIC DISORDERS BY DEVELOPMENTALLY ALTERING HOMEOSTATIC SETPOINTS TO PERSISTENTLY IMPEDE GR- AND/OR KLF9-DEPENDENT STRESS RESPONSIVITY, RESULTING IN LONG-TERM LOSS OF PLASTICITY AND DYSFUNCTION. ZEBRAFISH ARE AN IDEAL MODEL SYSTEM FOR THE PROPOSED STUDIES AS THEY SHARE ALL THE RELEVANT GENES AND NEUROENDOCRINE SIGNALING PATHWAYS WITH HUMANS, AND ARE SOCIAL, DIURNAL ANIMALS WITH COMPLEX BEHAVIORS THAT CAN BE MEASURED AND RELATED TO HUMAN PSYCHOLOGY AND STRESS-RESPONSIVE BEHAVIOR. MOREOVER, THE LARVAE PROVIDE UNSURPASSED ACCESS FOR EXPERIMENTAL MANIPULATION AND OBSERVATION, AND TRANSGENIC REPORTER LINES FOR MONITORING GR AND BRAIN ACTIVITY BY FLUORESCENCE MICROSCOPY. THE PROPOSED RESEARCH WILL USE THOSE TOOLS, AS WELL AS A KLF9 AND GR-KNOCKOUT LINES THAT WE RECENTLY CREATED USING CRISPR, TO ACCOMPLISH TWO SPECIFIC AIMS. THE FIRST IS TO DETERMINE IF CHRONIC EXPOSURE TO ARSENIC RESULTS IN LONG-TERM GLUCOCORTICOID RESISTANCE AND INCREASED NEUROINFLAMMATION. TO THAT END WE WILL ASSESS THE EFFECTS OF CHRONIC ARSENIC EXPOSURE DURING EARLY DEVELOPMENT ON BASAL AND STRESS-INDUCED ACTIVITY OF GR TARGET GENES AND INFLAMMATION MARKERS IN LARVAE AND ADULT BRAIN TISSUE, AS WELL AS ON LARVAL AND ADULT CORTISOL LEVELS. THE SECOND SPECIFIC AIM IS TO DETERMINE IF CHRONIC DEVELOPMENTAL EXPOSURE TO LOW LEVEL ARSENIC RESULTS IN ABERRANT BRAIN ACTIVITY AND BEHAVIORS. TO ACCOMPLISH THIS, WE WILL USE FLUORESCENCE MICROSCOPY AND AUTOMATED MOVEMENT TRACKING TO ASK HOW CHRONIC DEVELOPMENTAL EXPOSURE TO LOW-LEVEL ARSENIC AFFECT BRAIN DEVELOPMENT, BASAL AND STRESS-RESPONSIVE NEURONAL ACTIVITY IN PARTS OF THE LARVAL BRAIN THAT REGULATE NEUROENDOCRINE STRESS SIGNALING, AND BEHAVIOR OF THE EXPOSED LARVAE ACROSS THE LIFESPAN. THE PROJECT WILL OPEN A NEW AVENUE FOR FUTURE RESEARCH FOCUSED ON THE MECHANISMS UNDERLYING THE THE PATHOPHYSIOLOGICAL EFFECTS OF EARLY LIFE ARSENIC EXPOSURE ON THE NEUROENDOCRINE STRESS SYSTEM.

NONSENSE MEDIATED DECAY AND ALTERNATIVE SPLICING IN LIFESPAN EXTENSION FROM DIETARY RESTRICTION IN C. ELEGANS.

THE ROLE OF FOXQ1 IN TCDD-INDUCED JAW MALFORMATIONS

REDOX-SENSITIVE DEVELOPMENTAL PATHWAYS AND GENE REGULATORY NETWORKS

ENVIRONMENTAL ARSENIC, IMMUNOREGULATION, AND VIRAL DISEASE RISK - PROJECT SUMMARY / ABSTRACT ARSENIC EXPOSURE FROM WELL WATER AND FOOD IS A MAJOR PUBLIC HEALTH CONCERN AND IS ASSOCIATED WITH INCREASED MORBIDITY AND MORTALITY FROM VIRAL INFECTIONS. INFLUENZA A VIRUS (IAV) ANNUALLY INFECTS ~5 MILLION PEOPLE CAUSING ACUTE RESPIRATORY SYMPTOMS AND UP TO 646,000 DEATHS. THE SEVERITY OF VIRAL DISEASE VARIES BETWEEN INDIVIDUALS AND IS LIKELY TO INVOLVE BOTH GENETIC AND ENVIRONMENTAL EFFECTS ON IMMUNOREGULATION. EPIDEMIOLOGICAL AND ANIMAL MODEL STUDIES SUGGEST THAT EARLY LIFE EXPOSURE TO ARSENIC ALTERS THE IMMUNE RESPONSE TO PATHOGENS, RESULTING IN PROLONGED OR EXCESSIVE INFLAMMATION. FURTHERMORE, AVAILABLE EVIDENCE INDICATES THAT ARSENIC IS AN ENDOCRINE DISRUPTOR THAT INTERFERES WITH THE GLUCOCORTICOID RECEPTOR (GR) SIGNALING PATHWAY, A CRITICAL REGULATOR OF INFLAMMATION, POSSIBLY WITH INTERGENERATIONAL EPIGENETIC EFFECTS. HOWEVER, SIGNIFICANT GAPS REMAIN IN OUR UNDERSTANDING OF HOW ARSENIC DISRUPTS GR SIGNALING AND PROMOTES INFLAMMATION. THIS EXPLORATORY/ DEVELOPMENTAL RESEARCH PROJECT WILL USE ZEBRAFISH AS A MODEL SYSTEM TO TEST THE NOVEL HYPOTHESIS THAT ARSENIC EXACERBATES VIRAL DISEASE BY DOWNREGULATING THE KLF9-DEPENDENT ANTI-INFLAMMATORY GR SIGNALING PATHWAY. PRELIMINARY STUDIES INDICATE THAT KLF9 IS A GR-RESPONSIVE NEGATIVE REGULATOR OF PROINFLAMMATORY GENES, AND THAT BASAL AND CORTISOL- INDUCED KLF9 ACTIVITY IS SUPPRESSED IN ZEBRAFISH EMBRYOS EXPOSED TO VERY LOW LEVELS OF ARSENIC. ZEBRAFISH LARVAE HAVE A FUNCTIONAL INNATE IMMUNE SYSTEM AND ARE A POWERFUL MODEL TO STUDY HOST-PATHOGEN INTERACTIONS DURING SYSTEMIC OR LOCALIZED INFLUENZA (IAV) INFECTION, AS TRAFFICKING OF MACROPHAGES AND NEUTROPHILS TO THE SITE OF IAV INFECTION CAN BE VISUALIZED USING LIVE IMAGING WITH TRANSGENIC LINES WITH FLUORESCENTLY LABELED LEUKOCYTES AS WELL AS FLUORESCENTLY LABELED VIRUSES. THE PROPOSED RESEARCH WILL USE THOSE TOOLS, AS WELL AS GR AMD KLF9 KNOCKOUT LINES THAT WE RECENTLY CREATED USING CRISPR, TO ACCOMPLISH TWO SPECIFIC AIMS. THE FIRST IS TO DETERMINE IF ARSENIC DYSREGULATES THE INFLAMMATORY RESPONSE TO IAV INFECTION BY SUPPRESSING THE ANTI-INFLAMMATORY GR-KLF9 SIGNALING PATHWAY, LEADING TO EXCESSIVE AN/OR PROLONGED PRO-INFLAMMATORY GENE EXPRESSION AND FAILURE TO RESOLVE THE RESPONSE. THIS WILL BE ACCOMPLISHED BY ASKING HOW TREATMENT OF ZEBRAFISH LARVAE WITH ARSENIC AFFECTS EXPRESSION OF KLF9 AND DOWNSTREAM PROINFLAMMATORY GENES THAT WE HAVE IDENTIFIED AS PUTATIVE TARGETS OF KLF9-MEDIATED REPRESSION, AND ASSESSING THE EFFECTS OF ARSENIC AND KLF9 DOSAGE ON THE RESPONSE DYNAMICS OF INFLAMMATORY CELLS (NEUTROPHILS AND MACROPHAGES) AND NF-KB ACTIVITY FOLLOWING IAV INFECTION. THE SECOND SPECIFIC AIM IS TO DETERMINE IF ARSENIC EXPOSURE HAS INTERGENERATIONAL EFFECTS ON THE INNATE IMMUNE RESPONSE TO IAV INFECTION THAT CORRELATE WITH ABERRANT ACTIVITY OF THE GR-KLF9 IMMUNOREGULATORY PATHWAY. TO ACCOMPLISH THIS, F0 ARSENIC- OR VEHICLE-EXPOSED WILD-TYPE LARVAE WILL BE RAISED TO ADULTHOOD AND INBRED THROUGH 2 GENERATIONS WITHOUT FURTHER EXPOSURE. IN LARVAE FROM EACH GENERATION (F1 AND F2) WE WILL ASSESS IMMUNOREGULATORY GENE EXPRESSION AND LARVAL SURVIVAL AFTER IAV INFECTION. THE PROJECT WILL ELUCIDATE A NOVEL ANTI-VIRAL IMMUNOREGULATORY PATHWAY IMPACTED BY ARSENIC, OPENING AN AVENUE FOR FUTURE RESEARCH FOCUSED ON FURTHER ELUCIDATING THE UNDERLYING MECHANISMS.

EMERGING LEADERS IN REGENERATIVE BIOLOGY AND DEVELOPMENT (REBILD) - ENTER THE TEXT HERE THAT IS THE NEW ABSTRACT INFORMATION FOR YOUR APPLICATION. PROJECT SUMMARY/ABSTRACT THE LONG-TERM GOAL OF THE PROPOSED “EMERGING LEADERS IN REGENERATIVE BIOLOGY AND DEVELOPMENT (REBILD) PROGRAM” AT MDI BIOLOGICAL LABORATORY (MDIBL) IS TO MEET THE NEED FOR A WORKFORCE SKILLED IN REGENERATIVE AND DEVELOPMENTAL BIOLOGY RESEARCH. EVERY HUMAN SUFFERS FIBROTIC SCARRING IN THEIR LIFETIME, AND FIBROSIS ACCOUNTS FOR OVER 45% OF HUMAN DEATHS, DUE TO ORGAN FAILURE. THUS, THERE IS A GREAT NEED FOR THERAPIES THAT IMPROVE WOUND HEALING AND TISSUE REGENERATION. WHILE THE REGENERATIVE-BIOLOGY FIELD HAS GROWN EXPONENTIALLY OVER RECENT DECADES, LARGELY DUE TO TECHNICAL ADVANCES SUCH AS CRISPR AND NEXT-GENERATION SEQUENCING, THIS GROWTH HAS NOT SEEN CORRESPONDING GROWTH IN THE WORKFORCE. WE BELIEVE THAT THE BEST APPROACH TO DEVELOP SUCH A WORKFORCE IS TO TRAIN SCIENTISTS USING A CROSS-SPECIES APPROACH, ENABLING THEM TO UNDERSTAND REGENERATION BY STUDYING ORGANISMS THAT DIFFER IN REGENERATION CAPACITY TO IDENTIFY COMMON AND DISTINCT TISSUE-REGENERATION PROCESSES. A KNOWLEDGE OF REGENERATION ALSO REQUIRES AN UNDERSTANDING OF THE UNIQUE DEVELOPMENTAL BIOLOGY ACROSS THESE ORGANISMS. OUR PROGRAM IS DESIGNED FOR GRADUATE STUDENTS, POSTDOCTORAL RESEARCHERS, AND EARLY-CAREER SCIENTISTS. WE WILL IMPLEMENT OUR PROGRAM VIA THREE AIMS. IN AIM 1 WE WILL OFFER AN ANNUAL TEN-DAY RESEARCH TRAINING COURSE AND NETWORKING SYMPOSIUM FOCUSED ON THE INTER-RELATIONSHIP BETWEEN REGENERATIVE AND DEVELOPMENTAL-BIOLOGY RESEARCH. THE COURSE WILL INCLUDE HANDS-ON LABORATORY TRAINING USING ANIMAL MODELS THAT DIFFER IN REGENERATION CAPACITY; KEYNOTE LECTURES AND OTHER ACTIVITIES DESIGNED TO PROMOTE INTERACTIONS AMONG PROGRAM PARTICIPANTS; BIOINFORMATICS, MICROSCOPY, AND RESPONSIBLE CONDUCT OF RESEARCH TRAINING; AND A HALF-DAY WEEKEND SYMPOSIUM TO PROVIDE COURSE PARTICIPANTS AND REBILD PROGRAM ALUMNI WITH OPPORTUNITIES TO NETWORK AND PRESENT THEIR RESEARCH. IN AIM 2 WE WILL ESTABLISH A YEAR-ROUND VISITING SCIENTIST PROGRAM ENABLING COURSE ALUMNI AND OTHER RESEARCHERS TO SPEND UP TO FOUR WEEKS AT MDIBL AND WORK ALONGSIDE OUR LAB MEMBERS TO RECEIVE ADDITIONAL, FOCUSED RESEARCH TRAINING USING ADVANCED METHODOLOGIES SUCH AS TRANSGENESIS ON ONE OR MORE OF THE MODEL ORGANISMS USED IN THE 10-DAY COURSE. IN AIM 3 WE WILL ESTABLISH A SUSTAINABLE COMMUNITY OF INTERDISCIPLINARY REBILD RESEARCHERS, BY ESTABLISHING STRONG MENTORING FOR ALL PARTICIPANTS AND CREATING AN ONLINE NETWORKING FORUM THAT WILL PROVIDE MULTIPLE RESOURCES SUCH AS COURSE MATERIALS TO REBILD PROGRAM STUDENTS, FACULTY, INVITED SPEAKERS, AND OTHER REGENERATION/DEVELOPMENTAL BIOLOGY RESEARCHERS, AND WILL CREATE AN ONLINE NETWORKING AND DISCUSSION FORUM FOR LONG-TERM, POST-PROGRAM INTERACTIONS, INCLUDING MENTORING, AND FOR FURTHER EXPANDING THIS COMMUNITY. OUR PROGRAM WILL ALSO INCLUDE ANNUAL MID-YEAR CAREER-DEVELOPMENT AND GRANT-WRITING WORKSHOPS; A SEMINAR SERIES IN REGENERATION AND DEVELOPMENT; AND AN OUTCOME-ASSESSMENT PLAN. TOGETHER, THE ELEMENTS OF OUR REBILD PROGRAM WILL CREATE A WORKFORCE WELL-POSITIONED TO MAKE DISCOVERIES IN THEIR OWN LABORATORIES AND TO FURTHER EXPAND THIS RESEARCH COMMUNITY.

MOLECULAR MECHANISMS OF LIMB AND TAIL REGENERATION IN AXOLOTL - PROJECT SUMMARY AXOLOTLS, A TYPE OF SALAMANDER, ARE REMARKABLE IN THEIR ABILITY TO REGENERATE, AS ADULTS, THEIR LIMBS AND TAIL UPON AMPUTATION. MY LAB STUDIES AXOLOTL LIMB AND TAIL REGENERATION WITH THE BROAD GOAL OF DELINEATING THE UNDERLYING BIOLOGICAL PROCESSES IN ORDER TO ENABLE DEVELOPMENT OF NOVEL STRATEGIES FOR SCARLESS TISSUE REGENERATION IN HUMANS. IN MAMMALS, A WOUND IS QUICKLY COVERED BY AN EPIDERMAL LAYER FOLLOWED BY FORMATION OF A FIBROTIC SCAR (FIBROSIS). HOWEVER, IN AXOLOTLS, A WOUND DOES NOT HEAL WITH A SCAR; RATHER, A POOL OF PROGENITOR CELLS, TERMED THE BLASTEMA, FORMS BENEATH THE WOUND EPIDERMIS, AND THESE CELLS GIVE RISE TO ALL OF THE CELL TYPES REQUIRED FOR REGROWTH OF LIMBS AND TAILS. WHEN I ENTERED THE FIELD OF AXOLOTL RESEARCH ~15 YEARS AGO, I RECOGNIZED THAT MODERN RESEARCH TOOLS AVAILABLE FOR ANALYSIS OF OTHER ORGANISMS SUCH AS MICE WERE LACKING IN AXOLOTL RESEARCH, HINDERING RESEARCH PROGRESS. TO HELP FILL THIS GAP, I PLAYED KEY ROLES IN DEVELOPING NEW TECHNOLOGIES, INCLUDING APPROACHES FOR GENERATING TRANSGENIC AXOLOTLS, AND USING ADVANCED APPROACHES FOR GENERATING AND ANALYZING LARGE DATASETS ON ENTIRE BIOLOGICAL SYSTEMS, TO ANSWER FUNDAMENTAL QUESTIONS ON REGENERATION. RECENTLY, I HAVE BEEN A LEADER IN DEVELOPING CUTTING-EDGE APPROACHES FOR IMAGING AT A SUBCELLULAR RESOLUTION IN AXOLOTLS. USING THESE ADVANCED TOOLS, MY LABORATORY HAS MADE SIGNIFICANT DISCOVERIES ON AXOLOTL LIMB AND TAIL REGENERATION. FOR EXAMPLE, I DISCOVERED THAT DEDIFFERENTIATION OF FIBROBLASTS IS THE KEY FOR SUCCESSFUL LIMB REGENERATION IN AXOLOTL. IN A SUBSEQUENT STUDY, OUR TEAM SHOWED THAT POOR DEDIFFERENTIATION OF FIBROBLASTS IN XENOPUS FROGS IS THE UNDERLYING REASON FOR POOR LIMB REGENERATION IN THESE FROGS. SIMILARLY, THE UNIQUE REGENERATIVE CAPACITY OF FIBROBLASTS IN AXOLOTLS IS NOT OBSERVED IN MAMMALIAN FIBROBLASTS, LIKELY LEADING TO FIBROSIS IN MAMMALS. IN A RECENT STUDY, WE DISCOVERED A NOVEL POPULATION OF PROGENITOR CELLS IN THE AXOLOTL TAIL THAT ARE CAPABLE OF REGENERATING ALL CELL TYPES REQUIRED FOR TAIL REGENERATION, INCLUDING FIBROBLASTS, MUSCLES, AND VERTEBRAE. IDENTIFICATION OF THESE NOVEL PROGENITORS IS THE FIRST EXAMPLE OF SUCH CELLS IN ANY ORGANISM, AND THEIR ABSENCE IN MAMMALS MAY HELP EXPLAIN MAMMALS’ POOR REGENERATIVE ABILITIES. MANY FUNDAMENTAL QUESTIONS ABOUT REGENERATION IN AXOLOTL REMAIN, INCLUDING: HOW ARE CELLS ACTIVATED TO ACQUIRE THE CHARACTERISTICS OF A BLASTEMA CELL? WHICH CELL TYPES ARE ESSENTIAL FOR REGENERATION, AND WHICH ARE DISPENSABLE? OVER THE NEXT FIVE YEARS, I WILL FOCUS ON TWO BROAD QUESTIONS: 1) UNDERSTANDING HOW FIBROBLAST FUNCTION IN LIMBS IS AFFECTED BY THE ACTIVITIES OF OTHER CELL TYPES; AND 2) DETERMINING HOW THE NOVEL PROGENITOR CELLS IN TAILS GIVE RISE TO THE DIVERSE CELL TYPES AND ACTIVATE PATTERNING MECHANISMS. COMPARISON OF MY FINDINGS IN LIMB VERSUS TAIL WILL IDENTIFY REGENERATION PATHWAYS THAT ARE FUNDAMENTAL TO BOTH BODY PARTS AS WELL AS PATHWAYS THAT MAY BE SPECIFIC TO EACH CELLULAR CONTEXT. TOGETHER, MY RESULTS WILL HAVE POTENTIAL TO LEAD TO DESIGN OF APPROACHES TO SUCCESSFULLY PROMOTE REGENERATION OF INJURED OR DISEASED MUSCULOSKELETAL STRUCTURES, INCLUDING LIMBS, IN HUMANS.

REU SITE: COMPARATIVE ANIMAL MODEL APPROACHES TO REGENERATION AND AGING -THIS REU SITE AWARD TO THE MOUNT DESERT ISLAND BIOLOGICAL LABORATORY (MDIBL), LOCATED IN BAR HARBOR, ME, WILL SUPPORT THE TRAINING OF 10 STUDENTS FOR 10 WEEKS DURING THE SUMMERS OF 2023-2025. IT IS ANTICIPATED THAT A TOTAL OF 30 STUDENTS, PRIMARILY FROM SCHOOLS WITH LIMITED RESEARCH OPPORTUNITIES OR FROM UNDERREPRESENTED GROUPS, WILL BE TRAINED IN THE PROGRAM. STUDENTS WILL BE AT ALL STAGES OF THEIR UNDERGRADUATE STUDIES, AND SPECIAL CONSIDERATION WILL BE GIVEN TO APPLICANTS INTERESTED IN MICROSCOPY AND COMPARATIVE ANIMAL MODEL RESEARCH ON REGENERATION AND AGING. STUDENTS WILL LEARN HOW RESEARCH IS CONDUCTED, AND MANY WILL PRESENT THE RESULTS OF THEIR WORK AT SCIENTIFIC CONFERENCES. ASSESSMENT OF THE PROGRAM WILL BE DONE THROUGH THE ONLINE SALG URSSA TOOL. STUDENTS WILL BE REQUIRED TO REGISTER IN THE NSF ETAP SYSTEM (ETAP.NSF.GOV) AND TRACKED AFTER THE PROGRAM TO DETERMINE THEIR CAREER PATHS. THIS REU PROGRAM MDIBL WILL EXPOSE STUDENTS TO COMPARATIVE ANIMAL MODEL APPROACHES USED TO UNDERSTAND THE BIOLOGY OF REGENERATION AND AGING. LABS AT THIS REU SITE ARE INTERESTED IN HOW CELLS, TISSUES, ORGANS, AND LIMBS ARE REPAIRED OR REPLENISHED AFTER INJURY OR DURING THE ORDINARY COURSE OF DEVELOPMENT AND THE MECHANISMS RETAINED OR REEMPLOYED TO ENSURE AN ORGANISM THRIVES ONCE DEVELOPMENT IS COMPLETE. TO ANSWER THESE QUESTIONS, RESEARCHERS AT MDIBL USE VARIOUS ANIMAL MODELS. THESE ANIMALS INCLUDE ZEBRAFISH, KILLIFISH, AXOLOTL, FRUIT FLIES (DROSOPHILA), AND NEMATODES (CAENORHABDITIS ELEGANS), WHICH CAN BE USED TO COMPARE THE GENETIC SIMILARITIES AND DIFFERENCES IN AGING AND REGENERATION STRATEGIES ACROSS SPECIES. STUDENTS WILL BE FULLY INTEGRATED INTO THE SCIENTIFIC CULTURE OF MDIBL THROUGH SYMPOSIA, SEMINARS, ACTIVITIES, RESPONSIBLE CONDUCT OF RESEARCH TRAINING, AND WEEKLY CAREER DISCUSSIONS. STUDENTS MORE INFORMATION ABOUT THE PROGRAM IS AVAILABLE IN THE NSF ETAP REGISTRATION SYSTEM AND BY VISITING HTTPS://MDIBL.ORG/EDUCATION/STUDENT-FELLOWSHIPS/REU-SUMMER-FELLOWSHIPS/, OR BY CONTACTING THE PI (DR. UPDIKE AT DUPDIKE@MDIBL.ORG) OR THE CO-PI (DR. DISNEY AT JDISNEY@MDIBL.ORG). THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

REPRODUCIBLE AND FAIR BIOINFORMATICS ANALYSIS OF OMICS DATA - PROJECT SUMMARY/ABSTRACT MODERN BIOMEDICAL RESEARCH IS INCREASINGLY QUANTITATIVE. THE NEXT GENERATION OF RESEARCHERS WILL NEED AN ENTIRELY NEW SET OF QUANTITATIVE SKILLS TO FULLY TAKE ADVANTAGE OF THE DATA THEY CREATE. IN RESPONSE TO THIS NEED, THE GOAL OF THE CURRENT R25 PROPOSAL IS TO TRANSFORM AN EXISTING, 5-DAY BIOINFORMATICS TECHNIQUES COURSE INTO A NEW, TWO-WEEK SHORT COURSE, REPRODUCIBLE AND FAIR BIOINFORMATICS ANALYSIS OF OMICS DATA, TO PROVIDE A UNIQUE EDUCATIONAL OPPORTUNITY FOR BIOMEDICAL RESEARCH SCIENTISTS-IN-TRAINING TO BEGIN TO DEVELOP CORE COMPETENCIES IN BIOINFORMATICS AND BIOSTATISTICAL ANALYSES OF LARGE DATASETS. THE NEW COURSE WILL ALSO ADDRESS NIH PRIORITIES INCLUDING RIGOR AND REPRODUCIBILITY AND FINDABLE, ACCESSIBLE, INTEROPERABLE AND REUSABLE (FAIR) DATA PRINCIPLES. DURING THE LAST FIVE YEARS, THE DARTMOUTH FACULTY WHO ARE SERVING AS PRINCIPAL INVESTIGATORS HAVE TAUGHT A 5-DAY COURSE AT THE MDI BIOLOGICAL LABORATORY ON BIOINFORMATICS AND BIOSTATISTICS TO ~40 TRAINEES/YEAR (202 TOTAL). BASED ON OVERWHELMINGLY POSITIVE FEEDBACK, THE CURRENT PROJECT WILL EXTEND THIS 5-DAY COURSE INTO A LONGER, TWO-WEEK SHORT COURSE AT THE MDI BIOLOGICAL LABORATORY THAT WILL FEATURE A LOW STUDENT-TO- INSTRUCTOR RATIO (5:1), MORE HANDS-ON EXPERIENTIAL LEARNING, AND EXCEPTIONAL FACULTY WHO ARE HIGHLY EXPERIENCED IN TEACHING AND PERFORMING BIG-DATA ANALYSES. THE NEW COURSE IS DESIGNED TO ACCOMMODATE ~35 TRAINEES PER YEAR (175 TOTAL OVER THE FIVE-YEAR R25 PROJECT). IT WILL INCORPORATE MODULES ON BIOSTATISTICS, SCIENTIFIC RIGOR AND REPRODUCIBILITY, AND FAIR DATA PRINCIPLES. THE COURSE DESIGN WILL ALSO INVOLVE A SHORT CONCEPTUAL PRESENTATION, FOLLOWED BY AN EXERCISE IN WHICH STUDENTS WILL GAIN CONFIDENCE BY APPLYING A NEW SKILL. EACH ACTIVE-LEARNING SESSION WILL HAVE THREE LEVELS OF DIFFICULTY (BEGINNER, INTERMEDIATE, AND ADVANCED) TO ALLOW EACH STUDENT TO PROGRESS AT THEIR OWN PACE. THE LOW STUDENT-TO-FACULTY RATIO WILL ALLOW COURSE FACILITATORS TO GUIDE PARTICIPANTS THROUGH REALISTIC CHALLENGES WITHOUT CAUSING FRUSTRATION. THE SPECIFIC AIMS OF THE PROPOSED COURSE INCLUDE: SPECIFIC AIM 1. DEVELOP A TWO-WEEK SHORT COURSE PRIMARILY FOR POSTDOCTORAL FELLOWS AND GRADUATE STUDENTS THAT IMPROVES THEIR ABILITY TO DESIGN AND ANALYZE OMICS EXPERIMENTS SUCH AS RNA-SEQ, 16S (MICROBIOME), METAGENOMICS, AND SC-RNA-SEQ DATA; SPECIFIC AIM 2. ENHANCE THE IMPACT OF RESEARCH BY BIOMEDICAL SCIENTISTS BY TEACHING THEM THE RESPONSIBLE CONDUCT OF RESEARCH, THE SECURE AND ETHICAL USE OF DATA, AS WELL AS RIGOR AND REPRODUCIBILITY AND FAIR DATA PRINCIPLES; SPECIFIC AIM 3. DISSEMINATE THE TRAINING CURRICULUM TO A BROAD AUDIENCE; AND SPECIFIC AIM 4. EVALUATE THE SHORT- AND LONG-TERM IMPACTS OF THE COURSE ON STUDENTS, INCLUDING A LONG-TERM FOLLOW-UP TO DETERMINE STUDENTS’ CONFIDENCE IN AND ACTUAL INTEGRATION OF BIOINFORMATICS, BIOSTATISTICS, AND FAIR DATA PRINCIPLES INTO THEIR RESEARCH, AND THE REPORTED IMPACT OF THIS COURSE ON THEIR CAREER TRAJECTORY AND COMPETITIVENESS IN THE JOB MARKET. IN SUMMARY, THE PROPOSED COURSE WILL PROVIDE A UNIQUE CROSS- TRAINING, EDUCATIONAL OPPORTUNITY FOR BIOMEDICAL RESEARCH SCIENTISTS-IN-TRAINING TO BEGIN TO DEVELOP CORE COMPETENCY IN BIOINFORMATICS AND BIOSTATISTICAL ANALYSES OF LARGE DATA SETS.

ESTABLISHING A NETWORK OF SKILLED BD2K PRACTITIONERS: THE SUMMER WORKSHOP ON POPULATION-SCALE GENOMIC STUDIES OF ENVIRONMENTAL STRESS

REU SITE: EXPLORING 21ST CENTURY CAREERS IN THE BIOLOGICAL SCIENCES: A COMPARATIVE REGENERATIVE BIOLOGY APPROACH

REU SITE: RESEARCH EXPERIENCES IN MARINE COMPARATIVE FUNCTIONAL GENOMICS

PATHWAY TO BIOTRAILS: DNA-ASSISTED SPECIES IDENTIFICATION FOR CITIZEN SCIENCE

ANALYZING PACLITAXEL-INDUCED CHANGES IN THE SKIN AS POSSIBLE CAUSE OF CIPN

MDIBL STEER: PATHWAYS OF CHEMICAL ACTION IN HUMAN DISEASE

REU SITE: COMPARATIVE APPROACHES IN CELLULAR, MOLECULAR AND ENVIRONMENTAL BIOLOGY

THE GOAL OF THIS PROJECT IS TO CREATE AND PILOT A NATIONAL MODEL OF ENVIRONMENTAL EDUCATION THAT FACILITATES SCHOOLS AND COMMUNITY ORGANIZATIONS WORK

A HIGH THROUGHPUT SCREEN FOR INHIBITORS OF NEMATODE DETOXIFICATION GENES

GENE REGULATORY CIRCUITRY UNDERLYING THE DYNAMIC CONTROL OF GLUCOCORTICOID SIGNALING

UNDERSTANDING HOW VASA PROTEINS PROMOTE CELLULAR PLURIPOTENCY IN THE C. ELEGANS GERMLINE - PROJECT SUMMARY/ABSTRACT VASA DEAD-BOX RNA HELICASES REGULATE GERMLINE PLURIPOTENCY IN ANIMALS RANGING FROM C. ELEGANS TO HUMANS. VASA PROTEINS ARE FREQUENTLY REPURPOSED AS TRANSIENT PLURIPOTENCY FACTORS DURING DEVELOPMENT, REGENERATION, AND TUMORIGENESIS. THE MOLECULAR MECHANISMS BY WHICH VASA DRIVES PLURIPOTENCY ARE UNCLEAR. INITIAL STUDIES IN DROSOPHILA SUPPORTED A ROLE FOR VASA IN TRANSLATION, BUT MORE RECENT WORK HAS FOCUSED ON ITS FUNCTIONS IN SMALL RNA BIOGENESIS AND AS AN RNA SOLVENT. THE LONG-TERM GOAL OF THIS PROJECT IS TO DETERMINE HOW VASA PROTEINS PROMOTE CELLULAR PLURIPOTENCY IN ORDER TO MANIPULATE ITS FUNCTION TO ENHANCE REGENERATION OR SLOW TUMORIGENESIS. THE IMMEDIATE OBJECTIVE IS TO TEST THE HYPOTHESIS THAT THE C. ELEGANS VASA HOMOLOG, GLH-1, REGULATES ER-DIRECTED TRANSLATION. IN C. ELEGANS, THE GLH FAMILY OF VASA HOMOLOGS FUNCTION REDUNDANTLY, ALLOWING FOR THE STUDY OF MUTANTS WITHOUT COMPROMISED FERTILITY. TO IDENTIFY GLH-1 BINDING PARTNERS IN VIVO WE HAVE USED QUANTITATIVE MASS SPECTROMETRY TO ISOLATE PROTEINS FROM WHOLE WORM LYSATE THAT CO-IMMUNOPRECIPITATE WITH GLH-1. THE RESULTS EMPHASIZE ASSOCIATIONS BETWEEN GLH-1 AND TRNA SYNTHETASES, TRANSLATION FACTORS, AND MULTIPLE COMPONENTS OF THE ER TRANSLOCON. COMPARING THESE ASSOCIATIONS TO THOSE IN WORMS WITH PRECISE GLH-1 MUTATIONS REVEALS THAT TRANSLOCON INTERACTIONS ARE MEDIATED BY A ZINC FINGER DOMAIN UNIQUE TO VASA PROTEINS. IN AIM 1 WE WILL DETERMINE GLH-1’S SPATIOTEMPORAL ASSOCIATION WITH THE TRANSLOCON USING AN IN VIVO SPLIT-SUPERFOLDER GFP STRATEGY. IN AIM 2 WE WILL TEST FOR A FUNCTIONAL INTERACTION BY INTRODUCING PRECISE MUTATIONS INTO THE TRANSLOCON AND MEASURING GLH-1’S MODULATION OF RESULTING ER DYSFUNCTION. IN AIM 3 WE WILL TEST THE HYPOTHESIS THAT GLH-1 REGULATES THE TRANSLATION OF ER-DIRECTED TRANSCRIPTS BY PERFORMING POLYSOME PROFILING OF WILD-TYPE AND GLH-1 DELETION WORMS. THE PROPOSED EXPERIMENTS WILL REVEAL A NEW ASPECT OF GERM CELL BIOLOGY AND A NOVEL POST-TRANSCRIPTIONAL MECHANISM BY WHICH VASA FUNCTIONS IN THE GERMLINE. THEIR COMPLETION WILL RESULT IN IDENTIFICATION OF TRANSCRIPTS UNDER VASA POST-TRANSCRIPTIONAL CONTROL, WHICH WILL REPRESENT A NEW SET OF THERAPEUTIC TARGETS TO ENHANCE REGENERATION AND SLOW TUMORIGENESIS. THE APPLICANT, DR. EMILY SPAULDING, WILL PERFORM THE RESEARCH PROJECT AT MOUNT DESERT ISLAND BIOLOGICAL LABORATORY (MDIBL) IN BAR HARBOR, MAINE. MDIBL HAS A 122-YEAR HISTORY OF BIOMEDICAL RESEARCH AND IS AN NIH CENTER FOR BIOMEDICAL RESEARCH EXCELLENCE IN REGENERATIVE BIOLOGY AND MEDICINE. THE APPLICANT’S SPONSOR, DR. DUSTIN UPDIKE, IS AN NIH-FUNDED ASSOCIATE PROFESSOR AND HAS A WELL-DOCUMENTED RECORD OF SUCCESSFUL MENTORSHIP AND EXCELLENCE IN C. ELEGANS GERM CELL BIOLOGY. EXECUTION OF DR. SPAULDING’S TRAINING PLAN WILL ADD EXPERTISE IN C. ELEGANS RESEARCH AND GERM CELL BIOLOGY TO HER BACKGROUND IN TRANSLATIONAL REGULATION IN MICE. THE PROPOSED TRAINING SUPPORTS DR. SPAULDING’S GOAL OF RUNNING A LABORATORY THAT LEVERAGES THE ADVANTAGES OF BOTH C. ELEGANS AND THE MOUSE TO INVESTIGATE HOW DISTINCT CELL TYPES METABOLIZE RNA IN UNIQUE WAYS AND HOW PERTURBATION OF THESE MECHANISMS CAN LEAD TO HUMAN DISEASE.

UNDERSTANDING GERM CELL PLURIPOTENCY THROUGH AN INDUCIBLE P-GRANULE DEGRADATION SYSTEM

REGULATION OF A CELL VOLUME SENSITIVE CHANNEL BY STE20 KINASE SIGNALING

THIS 2 YEAR PROJECT EDUCATES TWO, INLAND MAINE HIGH SCHOOLS' (BANGOR AND WATERVILLE SENIOR HIGH SCHOOL) ABOUT MARINE VEGETATIVE ENVIRONMENTS THROUGH

MDIBL SYMPOSIUM ON STEM CELLS AND AGING

THIS PROJECT CREATES AN ECOLOGIST-IN-RESIDENCE PROGRAM IN 5 MOUNT DESERT ISLAND ELEMENTARY SCHOOLS. THE PROJECT IS AN EDUCATION PROGRAM ON EELGRASS

MOUNT DESERT ISLAND STEM CELL SYMPOSIUM

ORIGINS OF RENAL PHYSIOLOGY

MOUNT DESERT ISLAND STEM CELL SYMPOSIUM

ORIGINS OF RENAL PHYSIOLOGY

ORIGINS OF RENAL PHYSIOLOGY

MOUNT DESERT ISLAND STEM CELL SYMPOSIUM

WORKSHOP: EARLY WARNING AND INTEGRATED MANAGEMENT OF NATURAL TOXIN EVENT MT. DESERT ISLAND MARINE BIOL. LAB. SALISBURY COVE ME