Project abstract. This is an application for the Administrative Supplement to the currently active R01 GM141546- 02 entitled “Role of retinoid oxidoreductase complex in controlling the embryonic development”. The major goal of the parent grant is to understand how retinoic acid (RA) is produced in a quickly changing spatiotemporal pattern to control the expression of precise sets of genes at different developmental stages. Critical RA-sensitive processes during development are RA- concentration dependent, which underscores the importance of the precise control over RA synthesis in a strictly defined and rapidly regulated manner. The central hypothesis of the project is that retinoid oxidoreductase complex comprised of the two enzymes, RDH10 and DHRS3, represents a previously unrecognized universally conserved mechanism that can both provide the RA synthesis with robustness (Aim 1) and enable the dynamic changes in RA spatiotemporal pattern by regulating the levels of RA precursor (Aim 2). The hypothesis will be tested using a zebrafish embryogenesis model to take advantage of external fertilization and transparency of zebrafish for intra-vital visualization of RA synthesis and formation of the complex. In ongoing studies, we have established unique zebrafish knock-in lines expressing fluorescently-tagged enzymes, which in combination with knockout lines and RA-reporter lines will be used to visualize the expression of the complex components and changes in the spatiotemporal pattern of RA synthesis. The imaging studies are essential component of both Aims. The Administrative Supplement will allow us to purchase BioTek Cytation C10 Confocal Imaging Reader. Cytation C10 is an imaging system which combines a laser confocal microscope, widefield fluorescence microscope and a multimode reader. The acquisition of this imaging system will facilitate and streamline the workflow in the PI laboratory, because this instrument can be operated in several imaging modes, widefield for imaging of whole zebrafish embryos, widefield fluorescence for imaging of the fluorescently-tagged enzymes and their colocalization, and laser confocal mode for imaging at the subcellular level. The advanced Gen5 software will enhance our analysis capabilities and facilitate preparation of publications thus supporting success and continuation of the studies under the parent grant.