Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Abstract

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly ImprovesIn VitroHematopoietic Stem/Progenitor Cell Expansion

“… 23 Similarly, culture of CB cells on flat surfaces of PDMS reduced CD38 surface expression, suggesting that material absorption of some factor by the PDMS indirectly contributes to the change in CD38 expression. Retinoids are notoriously challenging to quantify, 72 and it is probable that this medium contains some retinoids at concentrations below our detection limits. Whether depletion of trace quantities of ATRA from medium by PDMS is sufficient to modify CD38 expression or whether other factors in the media may be augmented by PDMS remains unknown.…”

CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development

“…, and Chd7 -/-mouse embryos together with F9 cells expressing RARE-lacZ (Supplemental Figure 4) (34). This assay showed no measurable differences in RA activity across the three genotypes.…”

“…Cells were then incubated with anti-Tuj1 antibody (1:100, catalog MRB-435P, Berkeley Antibody Company) overnight at 4°C, washed assays, 1 day prior to coculture, F9 cells (10 5 cells/well) were plated on 0.2% gelatin-coated (MilliporeSigma) 96-well plates in culture media without G418. Separate wells were plated in triplicate for standard ATRA concentration response curves (0, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM), as previously described (34). ATRA was dissolved in DMSO.…”

Generating retinoic acid gradients by local degradation during craniofacial development: One cell's cue is another cell's poison

“…Given the importance of RA in establishing unique fate specification gradients, it is imperative to quantify the endogenous levels of RA existing in each specific biological system within an organism. RA cannot be directly visualized histologically, given its non‐peptidic, amphipathic nature, and diffusibility (Ababon, Li, Matteson, & Millonig, ). Traditional methods to quantify endogenous RA levels have relied on reporter‐based assays, where the RARE of a gene of interest is fused to either a LacZ or a luciferase construct.…”