Decoding Developmental Signaling in Vertebrate Embryos

We developed and characterized a suite of zebrafish-optimized optogenetic tools to reversibly activate three key developmental signaling pathways: FGF, BMP, and Nodal (Figure 1). This toolkit enables tunable, wavelength-dependent, spatially localized signaling activation in zebrafish embryos. We are also developing transgenic zebrafish lines expressing these signaling activators. We use this powerful optogenetic platform to investigate the developmental biology questions outlined in the following sections.

Figure 1. Zebrafish-optimized optogenetic signaling activator toolkit

Gastrulating zebrafish embryos expressing optogenetic activators of FGF, BMP, and Nodal signaling were exposed to blue light (455 nm) for 30 min (bottom row) or maintained in dark conditions (top row). Immunofluorescence staining for phosphorylated signaling effectors demonstrates blue light–mediated signaling activation in vivo.

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Click image to enlarge.

Figure 1. Zebrafish-optimized optogenetic signaling activator toolkit

Gastrulating zebrafish embryos expressing optogenetic activators of FGF, BMP, and Nodal signaling were exposed to blue light (455 nm) for 30 min (bottom row) or maintained in dark conditions (top row). Immunofluorescence staining for phosphorylated signaling effectors demonstrates blue light–mediated signaling activation in vivo.

The advancement of molecular optogenetics has already yielded important insights in developmental biology and has promising applications in other fields, including regeneration and tissue engineering. However, there is currently a relative dearth of molecular optogenetic technology available in zebrafish, despite this translucent vertebrate model’s suitability for these approaches. The UDS is developing novel molecular optogenetic strategies to expand the repertoire of these valuable tools. We are working to build optogenetic technology to activate additional signaling pathways, to create reversible signaling inhibitors, and to develop tools responding to different wavelengths for orthogonal manipulation. To increase the reproducibility and ease of optogenetic experiments in zebrafish, we are continuing to generate transgenic lines expressing these tools.

Developmental patterning relies on spatial signaling gradients. 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 may 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 signaling gradients during patterning, we will eliminate endogenous signaling and use our optogenetic toolkit to impose custom signaling distributions in zebrafish embryos (Figure 2). By assessing patterning consequences, such an approach will provide insights into the information content contained in developmental signaling gradients.

Diverse spatio-temporal gene expression is necessary for healthy embryonic development (Figure 3). Gene expression is regulated by signaling, which is itself spatio-temporally dynamic. Each gene is thought to uniquely interpret signaling inputs including amplitude, duration, and combinations, leading to distinct expression profiles. This interpretation ultimately takes place at the level of DNA, through differential interactions with transcriptional regulators and chromatin changes. Yet the gene-specific input/output relationships between signaling, chromatin accessibility, and gene expression have not been systematically characterized during vertebrate development. Using our optogenetic toolkit, we are working to determine the mechanisms by which developmental genes decode signaling dynamics and combinations by optogenetically manipulating these inputs and profiling DNA– and RNA–level responses. Our in vivo signaling manipulations enable a direct link between signaling input and responses, and will help explain how diverse spatio-temporal gene expression profiles are generated in developing vertebrates.