Project Grant R01HL175982

THE ELECTROPHYSIOLOGICAL PROPERTIES OF EMBRYOS - PROJECT SUMMARY CONGENITAL HEART DISEASE (CHD) LEADS TO SEVERE MORBIDITY AND MORTALITY IN CHILDREN IN THE US AND WORLDWIDE. DESPITE THIS IMPACT ON CHILD HEALTH, WE SIMPLY DO NOT UNDERSTAND THE GENETIC CAUSES OF CHD. INDEED, WE RECENTLY SHOWED THAT ION CHANNELS PLAY A CRITICAL ROLE IN CHD, LR PATTERNING, AND EARLY GERM LAYER SPECIFICATION. WE PREVIOUSLY OUTLINED AN ION CHANNEL SIGNALING NETWORK WHERE ERG CHANNELS SUPPRESS VOLTAGE-GATED CALCIUM CHANNELS AND MTOR SIGNALING ENABLING THE EXIT FROM PLURIPOTENCY TO GERM LAYER DIFFERENTIATION. HOWEVER, THIS STUDY RAISED SEVERAL ADDITIONAL QUESTIONS. FOR EXAMPLE, DO THE GERM LAYERS HAVE DIFFERENT ELECTROPHYSIOLOGICAL PROPERTIES? WHAT ARE THE CALCIUM-RESPONSIVE TRANSCRIPTION FACTORS THAT ACTIVATE MTOR SIGNALING? THE GOAL OF THIS PROPOSAL IS TO ANSWER THESE QUESTIONS. WE PROPOSE, AND OUR PRELIMINARY DATA SUPPORT, THAT ION CHANNELS DEFINE A NEW PARADIGM FOR CELL SIGNALING IN EARLY EMBRYONIC CELLS. OUR DATA SUPPORT AN ELECTROPHYSIOLOGICAL MODEL WHERE SPECIFIC GERM LAYER FATES ARE DEPENDENT ON AN ION CHANNEL NETWORK. OUR OVERARCHING HYPOTHESIS IS THAT ION CHANNELS DEFINE ELECTRICAL MEMBRANE POTENTIAL AND REGULATE VOLTAGE-GATED CA2+ CHANNELS THAT ESTABLISH AN EXIT FROM PLURIPOTENCY TOWARDS SPECIFIC CELL FATES, GASTRULATION, AND LR PATTERNING PROVIDING A PLAUSIBLE MECHANISM FOR OUR PATIENTS WITH HTX AND CHD. OUR ELECTROPHYSIOLOGICAL PATHWAY THEN INTEGRATES WITH BIOCHEMICAL SIGNALING PATHWAYS THAT DEFINE SPECIFIC CELL FATES IN THE EMBRYO. IN THIS PROPOSAL, WE WILL FOCUS ON UNDERSTANDING THE DIFFERENCES IN ELECTRICAL PROPERTIES OF THE DIFFERENT GERM LAYERS USING BOTH ELECTROPHYSIOLOGICAL MEASUREMENTS AND GENE KNOCKDOWN IN XENOPUS. WE WILL THEN ALSO IDENTIFY THE TRANSCRIPTION FACTORS THAT REGULATE MTOR SIGNALING IN THE EARLY EMBRYO. OUR PUBLISHED RESULTS STRONGLY POINT TOWARDS ETS AND CRE TRANSCRIPTION FACTORS, AND WE WILL IDENTIFY PRECISELY WHICH TRANSCRIPTION FACTORS PLAY A KEY ROLE IN MTOR SIGNALING AND PLURIPOTENCY. A MAJOR STRENGTH OF OUR PROPOSAL IS OUR EXPERTISE; WE HAVE FORGED A COLLABORATION BETWEEN XENOPUS DEVELOPMENTAL BIOLOGISTS AND ELECTROPHYSIOLOGISTS THAT WILL ALLOW US TO RIGOROUSLY INVESTIGATE MEMBRANE POTENTIAL AS AN EMBRYONIC PATTERNING MECHANISM.