Decoding developmental signaling during zebrafish embryogenesis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Define DNA-level mechanisms of signaling interpretation: Healthy embryonic development depends on the establishment of unique spatiotemporal gene expression patterns that give rise to stereotyped cell fate decisions. Diverse gene expression patterns are regulated by signaling. Using complimentary sequencing approaches, we seek to determine the mechanisms by which developmental genes decode signaling amplitude, duration, and combinations by optogenetically manipulating these inputs and profiling DNA- and RNA-level responses. We are working to experimentally introduce defined signaling pulses and correlate directly with changes in chromatin accessibility and transcript levels, linking signaling input and responses in vivo. Our work will help explain how diverse spatiotemporal gene expression profiles are generated in developing vertebrate embryos. Decode spatial signaling gradients: Developmental patterning relies on spatial gradients of signaling molecules. Classic models postulate that cells read out signaling molecule concentration to make fate decisions, implying that specific spatial distributions are important for normal pattering. However, contemporary work suggests a more nuanced strategy in which gradients instead function by engaging a patterning network, requiring only spatial signaling asymmetry instead of a precise gradient shape. It is also possible that both mechanisms—or others—are used depending on the patterning context. To determine the “error tolerance” of the BMP gradient during dorsoventral patterning, we are working to eliminate endogenous BMP signaling and use optogenetics to impose custom BMP signaling distributions in zebrafish embryos. Developmental consequences of distribution variants such as step functions will be determined by assessing morphology, gene expression, and signaling. Expand optogenetic signaling technology in zebrafish: We recently developed a novel zebrafish-optimized optogenetic FGF signaling activator and systematically characterized its in vivo properties, along with those of optogenetic BMP and Nodal activators. Together, these comprise a powerful and well-characterized signaling manipulation toolkit available to the scientific community. Our work and that of others demonstrates the value of cutting-edge optogenetic strategies to enable new lines of inquiry by providing unprecedented experimental control. Toward this goal, and to continue creating new technology for the UDS to pursue future lines of investigation into signaling-mediated patterning, we are working to to expand the available toolkit of molecular optogenetic signaling regulators in zebrafish to 1) activate additional pathways, 2) reversibly inhibit signaling, and 3) activate signaling with different light wavelengths to enable orthogonal experiments. Finally, we will continue generating transgenic zebrafish expressing optogenetic tools to enhance their utility.