Visualization of an endogenous retinoic acid gradient across embryonic development

Abstract

In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior-posterior axis of structures, including the hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded probes for RA). Using the principle of fluorescence resonance energy transfer we engineered the ligand-binding domains of RA receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor and acceptor, respectively, for the reliable detection of ambient free RA. We created three GEPRAs with different affinities for RA, enabling the quantitative measurement of physiological RA concentrations. Live imaging of zebrafish embryos at the gastrula and somitogenesis stages revealed a linear concentration gradient of endogenous RA in a two-tailed source-sink arrangement across the embryo. Modelling of the observed linear RA gradient suggests that the rate of RA diffusion exceeds the spatiotemporal dynamics of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients to tissue engineering and regenerative medicine.

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning

“…They participate in establishing and maintaining morphogen gradients that play central roles in establishing the position and identity of cells to create the architecture of developing tissues. [1][2][3][4] Gradients are also recognized determinants of events in adult organisms, although these have largely been explored on the level of particular cytokines. 5,6 Electrostatic interactions of cytokines with HSPGs restrict diffusion and permit gradients to persist, perhaps revealing why HSPG are uniformly present in all metazoa.…”

Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

“…[156][157][158] Insoluble ECM proteins, 1,138 such as collagens, noncollagenous glycoproteins (elastin, fibronectin, nephronectin, or laminin), and hydrophilic proteoglycans with large glycosaminoglycan FIG. 8.…”

Dual RXR Agonists and RAR Antagonists Based on the Stilbene Retinoid Scaffold

“…A ll-trans-retinoic acid 1, its isomer 9-cis-retinoic acid 2, and other natural retinoids are important signaling molecules that act in the embryo 1 and throughout adult life. 2−4 At the genomic level, retinoids bind the retinoid receptors (RARs, subtypes RARα,β,γ) 5 and retinoic X receptors (RXRs, subtypes RXRα,β,γ) 6 to regulate many important biological activities.…”