Identifying vitamin A signaling by visualizing gene and protein activity, and by quantification of vitamin A metabolites

Abstract

Vitamin A (retinol) is an essential nutrient for embryonic development and adult homeostasis. Signaling by vitamin A is carried out by its active metabolite, retinoic acid (RA), following a two-step conversion. RA is a small, lipophilic molecule that can diffuse from its site of synthesis to neighboring RA-responsive cells where it binds retinoic acid receptors within RA response elements of target genes. It is critical that both vitamin A and RA are maintained within a tight physiological range to protect against developmental disorders and disease. Therefore, a series of compensatory mechanisms exist to ensure appropriate levels of each. This strict regulation is provided by a number synthesizing and metabolizing enzymes that facilitate the precise spatiotemporal control of vitamin A metabolism, and RA synthesis and signaling. In this chapter we describe protocols that (1) biochemically isolate and quantify vitamin A and its metabolites and (2) visualize the spatiotemporal activity of genes and proteins involved in the signaling pathway.

1. Introduction

1.1. Vitamin A and retinoic acid synthesis and signaling

Vitamin A (retinol) is an essential nutrient, critical for all stages of life from embryonic development through adult homeostasis. It is found in the diet in two primary forms, provitamin A carotenoids and preformed vitamin A (Green & Fascetti, 2016). Provitamin A carotenoids are derived from plant sources whereas preformed vitamin A comes largely from animal products such as liver, milk and eggs (Green & Fascetti, 2016). Once taken into the body vitamin A is metabolized to its active metabolite retinoic acid (RA), which directly regulates gene activity and function. It is critically important to maintain appropriate levels of vitamin A and RA throughout life as both excess and deficiency are associated with birth defects and adult disorders.

Vitamin A (retinol) enters the body as either preformed vitamin A or provitamin A carotenoids. Preformed vitamin A is hydrolyzed and taken up by enterocytes of the intestinal lumen (Fig. 1). Once inside the cell, it is esterified and packaged into chylomicrons that are secreted into circulation (Wongsiriroj et al., 2008). A large proportion of the retinol within these chylomicrons is acquired by liver hepatocytes and stored as retinyl esters in hepatic stellate cells (Moriwaki, Blaner, Piantedosi, & Goodman, 1988). Stores of retinyl esters can then be mobilized when needed via hydrolysis back to retinol, which is transported throughout the vasculature by retinol binding protein, RBP4 (Muenzner et al., 2013; Quadro et al., 2005). Target cells express the stimulated by retinoic acid 6 receptor (STRA6), which binds to circulating RBP4-retinol complexes, internalizing retinol for conversion to retinoic acid (Kawaguchi et al., 2007; Kelly, Widjaja-Adhi, Palczewski, & von Lintig, 2016).

Fig. 1.

Summary diagram of transport, signaling and degradation of vitamin A metabolism and RA synthesis. Retinol is transported throughout the vasculature and taken up by target cells where it is oxidized in a two-step reaction into retinoic acid (RA). β-carotene provides an alternative pathway by conversion into retinal by BCDO1 (b-carotene-15,15-dioxygenase). Once converted, RA can diffuse from the synthesizing cell and is either degraded by CYP26 enzymes or regulates cell signaling in target cells.

Once inside a cell, retinol is bound by cellular retinol binding proteins (CRBP) and then undergoes two consecutive oxidation reactions to become retinoic acid (Fig. 1). In the first reaction, retinol is oxidized to all-trans-retinal (also called retinaldehyde) by microsomal retinol dehydrogenase 10 (RDH10) or other short-chain retinol dehydrogenases that recognize CRBP-bound retinol as substrate (Napoli, 2012, 2017). RDH10 is the critical enzyme necessary for the regulation of this first oxidation step during embryonic development (Sandell et al., 2007) whereas other RDH enzymes contribute postnatally (Belyaeva, Adams, Popov, & Kedishvili, 2020; Napoli, 2012). In situations of vitamin A excess, where retinol exceeds the amount of CRBP protein, members of the cytosolic alcohol dehydrogenases (ADH) family may catalyze this reaction postnatally (Deltour, Foglio, & Duester, 1999; Kumar, Sandell, Trainor, Koentgen, & Duester, 2012). An important feature of this oxidation step is that it is reversible, allowing for the conversion of retinal back to retinol. The reduction of retinal to retinol is performed by the retinaldehyde reductase, dehydrogenase/reductase (SDR) member 3 (DHRS3, or retSDR1), which in doing so provides a mechanism for helping to regulate and balance appropriate levels of retinol, retinal and RA (Adams, Belyaeva, Wu, & Kedishvili, 2014; Billings et al., 2013; Feng, Hernandez, Waxman, Yelon, & Moens, 2010; Haeseleer, Huang, Lebioda, Saari, & Palczewski, 1998).

An alternative pathway to produce retinal is via conversion of β-carotene (Fig. 1), the principal source of which comes from the provitamin A carotenoids in various fruits and vegetables (Green & Fascetti, 2016). Similar to retinol, β-carotene is transported by chylomicrons, and converted into retinal in target cells by BCDO1 (b-carotene-15,15-dioxygenase) (Harrison, 2012; von Lintig & Vogt, 2000; Wyss et al., 2000). This secondary pathway may be used for RA synthesis, as well as vitamin A storage via conversion of retinal back to retinol.

The second oxidative step in retinol metabolism converts retinal to RA (Fig. 1). This irreversible reaction is carried out by retinaldehyde dehydrogenases including, ALDH1A1, ALDH1A2, and ALDH1A3 (previously RALDH1, RALDH2 and RALDH3) (Dupe et al., 2003; Fan et al., 2003; Niederreither et al., 2001). RA is then transported from the cytosol by cellular retinoic acid binding protein (CRABPII) to the nucleus where it binds to the retinoic acid receptors (RAR) RARα, RARβ and RARγ (Germain et al., 2006a). These RARs form heterodimers with the retinoid X receptors (RXR) RXRα, RXRβ or RXRγ (Germain et al., 2006b), which then regulate gene transcription through their binding to RA response elements (RAREs) of target genes. In the absence of RA, RAR-RXRs associate with RAREs and repress gene expression through recruitment of co-repressors that include nuclear receptor corepressor (NCoR), silencing mediator of RA and thyroid hormone receptor (SMRT), as well as histone deacetylases and methyltransferases. In contrast, when RA is present, it binds as a ligand to RAR-RXR and through direct association with RAREs, initiates transcription of target genes by disassociating the corepressors and recruiting co-activators that include steroid receptors (SRC-1, SRC-2 and SRC-3) (Al Tanoury, Piskunov, & Rochette-Egly, 2013).

RA is a small, lipophilic molecule and not only does it regulate gene activity within the cell in which it is produced, but it also acts as an autacoid acting in non-cell autonomous fashion on neighboring, RA-responsive cells. The distribution of RA creates spatial and temporal gradients that control signaling within target tissues (Shimozono, Iimura, Kitaguchi, Higashijima, & Miyawaki, 2013). However, the synthesis and diffusion of RA is constrained by catabolizing cytochrome p450 enzymes of the CYP26 family which degrade RA (Hernandez, Putzke, Myers, Margaretha, & Moens, 2007).

The ability to tightly regulate Vitamin A metabolism and RA synthesis and signaling is essential for proper embryogenesis and adult homeostasis. Cells and tissues therefore employ multiple feedback mechanisms to govern the levels of retinol, retinal and retinoic acid. When endogenous levels of RA are too high, Cyp26 genes are activated to help degrade excess RA. Furthermore, the enzymes responsible for oxidizing the first and second metabolic steps of vitamin A metabolism, such as Rdh10, and Aldh1a1, Aldh1a2, Aldh1a3, are downregulated. Conversely, Dhrs3 is upregulated to further convert the intermediate molecule retinal back to retinol (Billings et al., 2013; Elizondo, Corchero, Sterneck, & Gonzalez, 2000; Lee et al., 2012; Niederreither, McCaffery, Drager, Chambon, & Dolle, 1997; Zolfaghari, Chen, & Ross, 2012). In the opposite situation, whereby the levels of RA are too low, upregulation of Rdh10, Aldh1a1, Aldh1a2 and Aldh1a3, together with downregulation of the Cyp26 degradation enzymes (Sandell, Lynn, Inman, McDowell, & Trainor, 2012) can collectively lead to an increase in RA synthesis and maintenance. In addition, many of the proteins responsible for transporting retinol and RA are encoded by genes that contain RAREs (Balmer & Blomhoff, 2002, 2005; Rhinn & Dolle, 2012). This therefore provides additional opportunities for modulating vitamin A metabolism, and RA synthesis and signaling.

1.2. The role of vitamin A and retinoic acid signaling in development and disease

Vitamin A, and RA synthesis and signaling play vital roles during embryo development and throughout adult homeostasis. Vitamin A is required for proper bone development, protection of the skin and mucosa, immune system defense, epithelial integrity, and normal development and function of the reproductive organs, hair and teeth (Clagett-Dame & DeLuca, 2002; Clagett-Dame & Knutson, 2011; Conaway, Henning, & Lerner, 2013; Endo, Mikedis, Nicholls, Page, & de Rooij, 2019; Ross, 2012; Yokota et al., 2019). However, both excess and deficiency of Vitamin A or RA causes dysfunction, hence the need to maintain appropriate levels of vitamin A and RA throughout life. The deleterious effects of excess RA were first inferred in teratogenesis studies of embryo development, whereby an excess of RA produced congenital anomalies that included defects in the formation of the brain, nervous system, heart and limbs, together with malformed eyes, jaws and palate (Cohlan, 1954; Kalter & Warkany, 1961). Retinoids are synthetic forms or derivatives of vitamin A and are typically used to treat dermatological conditions. The most well-known is isotretinoin (13-cis-retinoic acid), which is marketed as Accutane, and used to treat severe acne. However, vitamin A is contraindicated during pregnancy and fetal retinoid syndrome, which is characterized by craniofacial, nervous system and cardiovascular anomalies, can occur as a consequence of a mother taking retinoids during pregnancy. Conversely, congenital abnormalities and even fetal death can result from a deficiency in vitamin A or RA. Major organ systems affected by a deficiency in vitamin A, and RA synthesis and signaling include the craniofacial, cardiovascular and ocular tissues, circulatory, respiratory and urogenital systems (Antipatis et al., 1998; Moreau, Vilar, Lelievre-Pegorier, Merlet-Benichou, & Gilbert, 1998; Zile, 1998).

Appropriate levels of vitamin A are also essential for the health of the mother and adults in general, not just during embryo development. In particular, vitamin A plays an essential role in vision which relies on the 11-cis-retinaldehyde metabolite as the light-absorbing chromophore of opsin proteins (Dowling & Wald, 1960; Wald, 1968). Through its effects on epithelial integrity and function, vitamin A is important in the prevention of xerophthalmia, which is defined by dryness of the conjunctiva and cornea of the eye. As xerophthalmia progresses, it eventually leads to blindness, thus making vitamin A deficiency the main cause of preventable blindness (WHO, 2009). Vitamin A deficiency is therefore still considered to be a major health issue, particularly in developing countries.

Interestingly, an excess of vitamin A or RA can result in a pattern of defects similar to those caused by a deficiency of RA. This is particularly true with respect to craniofacial anomalies such as cleft palate, and cardiovascular malformations. This phenomenon is thought to occur via overcompensation to excess vitamin A or RA. Under conditions of excess, the RDH and ALDH enzymes are shut down to prevent further synthesis of RA, while Cyp26 enzymes are upregulated to catabolize pre-existing RA. However, rather than reversing these actions when RA levels return to normal, the system remains off, leading to a prolonged period of RA deficiency and consequently developmental anomalies (Lee et al., 2012).

In summary, maintaining proper levels of vitamin A and RA are essential for embryo development and adult homeostasis. This is accomplished through a series of compensatory mechanisms that have evolved to maintain an appropriate balance of vitamin A and RA to protect against developmental disorders and disease. Differential and dynamic patterns of synthesizing and metabolizing enzymes facilitate the precise spatiotemporal control of vitamin A metabolism, and RA synthesis and signaling. In this chapter we describe protocols for visualizing the spatiotemporal activity of genes and proteins involved in this process as well as biochemically isolating and quantifying vitamin A and its metabolites.

1.3. Direct quantification of RA levels and retinoid homeostasis

Numerous analytical approaches have been developed to quantify retinoids including gas chromatography-mass spectroscopy (GC/MS) (Napoli, 1990; Napoli, Pramanik, Williams, Dawson, & Hobbs, 1985), liquid chromatography with ultraviolet absorption (LC-UV) (Kane, Folias, & Napoli, 2008; Kane & Napoli, 2010; Schmidt, Brouwer, & Nau, 2003; Wyss & Bucheli, 1997), liquid chromatography-electrochemical detection (LC/ECD) (Hagen, Washco, & Monnig, 1996; Sakhi, Gundersen, Ulven, Blomhoff, & Lundanes, 1998; Ulven et al., 2000), liquid chromatography-tandem mass spectroscopy (LC-MS/MS) (Arnold, Amory, Walsh, & Isoherranen, 2012; Kane, Chen, Sparks, & Napoli, 2005; Kane, Folias, Wang, & Napoli, 2008; Kane & Napoli, 2010), and liquid chromatography-multiple reaction monitoring cubed (LC-MRM³ also known as liquid chromatography-multistage tandem mass spectrometry) (Jones, Pierzchalski, Yu, & Kane, 2015). The most rigorously validated quantitative methodologies for determination of endogenous RA concentrations in cell systems and tissues as well as for in vitro cell metabolism assays are liquid chromatography-tandem mass spectrometry-based techniques (Arnold et al., 2012; Jones et al., 2015; Kane et al., 2005; Kane, Folias, Wang, et al., 2008; Kane & Napoli, 2010). Absolute quantification via LC-MS/MS-based approaches rely on a unique precursor to product ion m/z transition, termed multiple reaction monitoring (MRM) or selected reaction monitoring (SRM), to yield specificity (Kane et al., 2005; Kane, Folias, Wang, et al., 2008). Tandem quadrupole instruments yield the most robust and reproducible analytical detection with typical linear ranges of 3–5 orders of magnitude and typical instrument coefficients of variation of <5% (Kane et al., 2005; Kane, Folias, Wang, et al., 2008). Quantitation is derived from calibration curves constructed from authentic standards. Stable isotope-labeled retinoids or nonendogenous retinoid derivatives are typically used as internal standards to correct for extraction efficiency and variability during data acquisition (Kane & Napoli, 2010).

Recent work has shown that a variation of LC-MS/MS-based detection that includes an additional mass transition during detection yields additional specificity for RA quantification (Jones et al., 2015). This liquid chromatography-multistage tandem mass spectrometry methodology is also interchangeably known as LC-MS³ and LC-MRM³. Although MRM detection in LC-MS/MS methods is one of the most selective detection modalities, abundant species in extracts of complex matrices that coelute with the analyte of interest can interfere with quantification (Jones et al., 2015; Kane, 2012). Because most extraction methods for retinoids also extract (more abundant) lipids of similar nominal mass, the potential for interferences is significant and requires additional caution and careful method validation. Current strategies typically include the use of chromatographic separation to move interfering species to retention times away from RA and/or the inclusion of additional mass transitions to impart additional selectivity (Jones et al., 2015; Kane, 2012).

RA quantification poses several additional significant analytical challenges including low endogenous concentrations, often spatially localized occurrence, and existence of endogenous geometric isomers with distinct biological functions (Kane & Napoli, 2010). Isobaric geometric isomers present an additional analytical challenge as they must be chromatographically resolved before mass spectrometric detection. Additional experimental challenges that are critical to control include the susceptibility of retinoids to light-induced isomerization and oxidation, as well as adherence to plastic surfaces. Yellow or red laboratory lights (blocking UV wavelengths <~500nm) are recommended for experimental and sample preparation work in addition to efforts toward protecting samples from light by cover and/or amber vials in often non-UV blocking laboratory settings during analysis. Samples should be kept cold (on ice) during sample preparation and glass pipets, vials and containers, should be used as much as possible to prevent sample loss by adsorption to plastic (Kane & Napoli, 2010).

Typical detection limits for LC-MS/MS detection of RA are low fmol on column, which enables the determination of low nanomolar concentrations of endogenous RA in either cell systems or tissues. Typical linear ranges extend from low fmol to up to ~1–10pmol on column. Typical cell culture requirements range between 1 × 10⁵ and 1 × 10⁶ cells, depending on the cell type, level of RA present, and the lower limit of quantification (LLOQ) of the assay being used. Tissue requirements typically range from 1 to 100mg of tissue, with the minimum amount of tissue required depending on the LLOQ for the assay being used and the levels of RA present. If multiple assays are required, then tissue requirements can be higher. Minimum amounts of tissue required and efficiency of extraction from matrices should be evaluated before quantitative experiments are undertaken. The amount of RA is typically expressed as mol of RA per g tissue (requires accurate weight of tissue), mol of RA per g protein (requires a separate protein determination), mol of RA per million cells (requires accurate cell count), or mol RA per mL of fluid, such as media or plasma (requires an accurate volume determination) (Jones et al., 2015; Kane et al., 2005; Kane, Folias, Wang, et al., 2008; Kane & Napoli, 2010).

Quantification of retinoids that serve as the substrates for RA biosynthesis as well as vitamin A storage can inform the basis of homeostatic mechanisms (Kane, Folias, & Napoli, 2008; Wang, Yoo, Obrochta, Huang, & Napoli, 2015). The concentration of different retinoid species can differ by as much as six orders of magnitude, making methods that simultaneously detect all retinoids not technically practical. For example, retinyl ester levels can reach high micromolar levels whereas endogenous RA is typically low nanomolar in concentration. Quantification of RE and retinol can be accomplished by LC-UV (Kane, Folias, & Napoli, 2008; Kane & Napoli, 2010). These more abundant retinoids (typical concentrations in micromolar range) can be feasibly detected by UV detection due to their favorable molar absorptivity (typically ε = ~30,000–60,000M⁻¹ cm⁻¹), and unique absorption wavelengths, which are red-shifted from many other biological molecules (Kane, Folias, & Napoli, 2008; Kane & Napoli, 2010). LC-MS/MS methodology also exists for quantification of these more abundant RE and retinol species, which have less of a requirement for the sensitivity of LC-MS/MS-based approaches (Wang et al., 2015).

Retinal, or retinaldehyde, requires derivatization due to its reactive aldehyde group for accurate quantification. Retinaldehyde forms covalent Schiff base adducts with amine groups of proteins, phospholipids and other compounds, making it necessary to react the extract with hydroxylamine or O-ethylhydroxylamine to release the bound retinal and covert it to the respective syn and anti-retinaloxime adducts (Golczak, Bereta, Maeda, & Palczewski, 2010; Kane, Folias, & Napoli, 2008; Kane & Napoli, 2010; Wang et al., 2015). This is an important consideration for zebrafish, frog and avian models since retinaldehyde-vitellogenin Schiff-based adducts are the main storage form for retinoids in eggs and oocytes of oviparous animals (Costaridis, Horton, Zeitlinger, Holder, & Maden, 1996; Levi, Ziv, Admon, Levavi-Sivan, & Lubzens, 2012). Whereas, LC-UV methods have been popular, recent LC-MS/MS methodology provides more sensitive detection of retinal, which is important for samples of limited quantity that are often encountered in developmental studies (Golczak et al., 2010; Wang et al., 2015).

Additional, specialized analytical approaches for retinoids include tissues where cis-retinoid isomers have significant biological roles, such as the eye and pancreas. Methods for isomeric retinoid detection has been previously reviewed (Kane, 2012; Kane & Napoli, 2010). For the measurement of geometric isomers of retinoids found in the visual system, an isocratic or step-gradient normal-phase HPLC method has been developed to resolve 11-cis and all-trans-retinol and retinyl esters, as well as all-trans, 9-cis, and 11-cis-retinaloximes (Batten et al., 2004; Golczak et al., 2010). Chiral-HPLC can also be employed to resolve (13R)- and (13S)-enatiomers of all-trans-13, 14-dihydroretinol (Moise et al., 2008). For more specialized methods to measure cytotoxic lipofuscin chromophores formed in the eye such as pyridinium bisretinoids (N-retinyl-N-retinylidene ethanolamine or A2E) and retinal dimers, we refer the reader to several reviews (Bowrey et al., 2016; Crouch, Koutalos, Kono, Schey, & Ablonczy, 2015; Golczak et al., 2010).

1.4. Assessing retinoic acid signaling in cellular and animal models via reporter assays

Reporter screens can provide crucial information about the spatial resolution or extent of RA signaling within cells or tissues (reviewed in Table 1). These reporters are based on the expression of a readily assayable enzyme (beta-galactosidase or luciferase) or a fluorescent protein driven by a promoter controlled by RA signaling. Some RA signaling reporters are based on a RARE derived from the promoter of the Rarb gene consisting of a canonical GTTCAC direct repeat separated by 5 nucleotides (DR5) (Sucov, Murakami, & Evans, 1990). These reporters have been incorporated in cell models (Wagner, Han, & Jessell, 1992), as well as transgenic mice (Balkan et al., 1992; Rossant et al., 1991) and zebrafish (Perz-Edwards et al., 2001; Waxman & Yelon, 2011). However, a disadvantage of the use of DR5 based-reporters is that RA-signaling via other types of RAREs (Moutier et al., 2012) may not be detected. In addition, the DR5 enhancer is also recognized by other nuclear receptors such as COUP-TF (Cooney, Tsai, O’Malley, & Tsai, 1992).

Table 1.

Summary of retinoic acid signaling reporters.

Transgenic mouse lines	Transgene construct
RARE-LacZ (Rossant, Zirngibl, Cado, Shago, & Giguere, 1991) available from Jackson Labs Stock No: 008477	Express a beta-galactosidase reporter under the control of a minimal tk promoter containing 3 RARE derived from Rarb (Balkan, Colbert, Bock, & Linney, 1992)
RARE-hsp68LacZ	Express a beta-galactosidase reporter under the control of a minimal hsp68 promoter and three copies of RARE from Rarb
Feedback-inducible nuclear-receptor-driven (FIND) expression of GAL4-RAR/UAS-hsp-lacZ (Mata De Urquiza, Solomin, & Perlmann, 1999)	Express a chimeric receptor fusion of Gal4-DBD and RAR-LBD. A second transgene UAS-hsp-lacZ expresses a lacZ reporter driven by a minimal hsp68 promoter including four UAS. The expression of the chimeric receptor Gal4-RAR is itself autoregulated by incorporating 4 UAS in a minimal hsp68 promoter
Transgenic zebrafish lines	Transgene construct
Tg(3XRARE-tk-GFP)	Express GFP under the control of a minimal promoter (tk or gata-2) including three copies of RARE (Perz-Edwards, Hardison, & Linney, 2001)
Tg(3XRARE-gata-2-GFP)
Tg(12XRARE-ef1a:gfp)sk71	Express a GFP reporter driven by a promoter under the control of 12 concatenated RAREs (Waxman & Yelon, 2011)
Tg(β-actin:GDBD-RLBD); (UAS-reporter) or Tg(β-actin:VPBD-RLBD);(UAS:EGFP)	Express chimeric receptors based on Gal4-DBD or VP16-Gal4-DBD fused to the RARA-LBD. Ligand binding triggers receptor activation and is detected via an UAS-driven reporter (D’Aniello, Rydeen, Anderson, Mandal, & Waxman, 2013; Mandal et al, 2013)
Transgenic zebrafish expressing genetically encoded probes for RA(GEPRAs)	Created from chimeric receptors of RAR-LBD fused at the N and C-termini to CFP and YFP, respectively. Binding of RA causes a conformational change in the chimeric receptor detected via FRET (Shimozono et al., 2013)

Other reporters of RA signaling use a two-hybrid approach based on expression of a fusion protein of the ligand-binding domain (LBD) of RAR and the DNA-binding domain (DBD) of the GAL4 transcription factor. Expression of GAL4-nuclear receptor fusions has been a very valuable tool for high-throughput screening of compounds with agonist/antagonist activity against various nuclear receptors including RAR and RXR (Allenby et al., 1993; Grimaldi et al., 2015; Moise et al., 2009). In these systems binding of RA to the LBD of RAR induces expression of GAL4-regulated reporters (luciferase, GFP) controlled by upstream activation sequences (UAS). In comparison to DR5-based reporters, both binding of RA to the RAR-LBD and the induction of UAS-controlled reporters by GAL4-RAR chimeric receptors are very specific. Another advantage of the use of GAL4-RAR;UAS-reporter systems is that they do not rely on endogenous RAR receptors for reporter expression. However, the generation of compound GAL4-RAR;UAS-reporter expressing transgenic lines is cumbersome and time consuming since both the UAS-reporter and Gal4-RAR transgenes need to be present for activity. GAL4-RAR; UAS-reporter genes have also been introduced in mice and in zebrafish (D’Aniello et al., 2013; Mandal et al., 2013; Mata de Urquiza & Perlmann, 2003; Mata De Urquiza et al., 1999). Constitutive overexpression of chimeric receptors based on fusions of the Gal4-DBD to the RAR-LBD leads to phenotypic effects in mice. Therefore, a feedback-inducible nuclear-receptor-driven (FIND) expression system was generated to control the expression of the GAL-RAR chimeric receptor in mice (Mata de Urquiza & Perlmann, 2003; Mata De Urquiza et al., 1999). Expression of similar chimeric receptors in zebrafish encoding the Tg(β-actin:GDBD-RLBD); (UAS-reporter) or the Tg(β-actin:VPBD-RLBD);(UAS:EGFP) transgenes have not been reported to cause phenotypic effects (D’Aniello et al., 2013; Mandal et al., 2013).

Whereas reporter systems offer valuable spatial information and RA signaling read-outs, caution should be exercised in the interpretation of reporter assay response in terms of concentration and temporal relationships. It is important to stress that reporter-based methods are nonquantitative and do not provide a linear response to the level of RA in tissues and thus reflect a qualitative extent of RA signaling which is correlated with RA levels. And, whereas valuable qualitative relationships can be assessed under comparable conditions, specific, absolute quantitation for RA cannot be achieved via reporter assays. Some reporter assays have cited that in addition to all-trans-RA, other endogenous retinoids can produce signals to varying extents including 3,4-didehydro-RA, 9cRA, 4-oxo-RA, 4-hydroxy-RA, and 4-hydroxy-retinol (McCaffery & Drager, 1994; Wagner, 1997; Wagner et al., 1992; Zetterstrom et al., 1999). Temporal relationships should also be cautiously and carefully interpreted. Since reporter assays reflect RAR activation, they reveal the longer term consequences of receptor activation with a delay in the reporter signal appearance often hours after a change in RA concentration (McCaffery & Drager, 1994; Wagner, 1997; Wagner et al., 1992; Zetterstrom et al., 1999) (Figs. 2–7). Moreover, the half-life of the RAR reporters employed thus far generally outlasts the RA input. Thus, reporter assays may not accurately correlate with RA concentration via RA signaling in real-time. Temporal delays should be considered as well as the possibility that RA may even be significantly catabolized by the time the reporter signal is visualized. Lastly, experimental caution and consideration should be paid to reports that high levels of RA can turn off reporter response in some cases (McCaffery & Drager, 1994).

Fig. 2.

Example of retinoid absorbance spectra that are typically used for determining the concentration of retinol, RA and retinal. Reproduced from Kane, M. A., & Napoli, J. L. (2010). Quantification of endogenous retinoids. Methods in Molecular Biology, 652, 1–54. doi:10.1007/978-1-60327-325-1_1.

Fig. 7.

Comparison of representative reproducibility between the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter in HEK293 cells as compared to RA levels. Two sets of cells were treated with 5nM of all-trans retinoic acid (atRA or RA) and reporter response and RA levels were measured over time for 0–96h. (A) Two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter (B) RA in cell as determined by LC-MS/MS (C) RA in media as determined by LC-MS/MS. Cell culture for (A)–(C) and reporter assay conditions for (A) described in Section 4. LC-MS/MS quantitation for (B) and (C) as described in Section 1 and in Jones et al. Error bars represent standard deviation (n =3).

The most recent addition to the RA reporter systems allows for detection of RA in transgenic zebrafish expressing genetically encoded probes for RA(GEPRAs) (Shimozono et al., 2013). These reporters express the RAR-LBD flanked by Förster resonance energy transfer (FRET) partners CFP and YFP. Binding of RA causes a conformational change in the chimeric receptor which can be detected via FRET. Since this reporter does not require any additional substrates, it allows for the visualization of endogenous RA gradients in vivo in zebrafish embryos. This approach has great potential in studying the formation of RA in tissues in real-time, however, more studies are needed to establish the sensitivity of the GEPRAs-based reporter and its RA concentration-dependent correlation.

2. Direct quantification of retinoic acid production and endogenous retinoid levels in cells

Basal levels of RA can be determined in primary or cultured cells. If basal RA level determination is desired, skip to part 3 in Section 2.2 under “Protocols,” Retinoid Extraction. However, amounts of RA can be low in cellular systems necessitating large numbers of cells for analysis (e.g., 1 × 10⁶ cells or greater depending on the cell type). Alternatively, assessment of RA production is also a useful metric for evaluating RA homeostasis. RA production assays where the amount of RA produced from a given substrate over a defined time typically utilizes 1 × 10⁵ cells (or greater, if available) per replicate. For RA production evaluation, similar to standard enzyme activity assays, timepoint and concentration ranges should first be determined by performing a time-course experiment and a concentration-course experiment to determine conditions within the linear range (Kane & Napoli, 2010; Wang, Kane, & Napoli, 2011). The RA production assay is amenable to a wide variety of cell types and culture conditions.

2.1. Reagents

1. All-trans-Retinol (store at −20°C with desiccant)

2. All-trans-Retinoic acid (store at −20°C with desiccant)

3. All trans-Retinal (store at −20°C with desiccant)

4. Acetone (HPLC)

5. Saline (0.9% NaCl): 9g NaCl in 1L water

6. 0.025M KOH in 100% ethanol

7. 50mL 4M HCl

8. Hexanes (Certified or highest grade available)

2.2. Protocols

Part 1. Retinol solution preparation

1. Under yellow light or red light, bring the all-trans retinol (Sigma R7632) and all-trans-Retinoic Acid (Toronto Research Chemicals Cat# R250200) to room temperature.

2. Add 10mL ethanol to a clean borosilicate glass scintillation vial.

3. Use a clean disposable borosilicate glass Pasteur pipette to take up the desired amount of retinoid powder. The powder in the pipette tip should be about 1mm in diameter.

4. Put the pipette in the borosilicate glass scintillation vial containing 10mL ethanol.

5. Make sure the powder gets off the glass pipette tip.

6. Put the cap onto the vial and close the cap securely.

7. Vortex the solution for 20s.

8. Repeat step 7 two more times.

9. Measure the retinoid absorbance spectrum using a UV/Vis spectrophotometer by scanning wavelength from 250nm to 450nm.

10. Use ethanol to blank the spectrophotometer.

11. If the absorbance is too high (typically absorbance should be below 1 on most spectrophotometers), dilute the retinol solution with ethanol and repeat the measurement.

12. Make at least 3 measurements.

13. Calculate the retinol concentration based on Beer’s law: Absorbance=εLc (don’t forget the dilution factor if you dilute the solution).

Technical notes

• Work under yellow or red light.

• Always use borosilicate glass pipettes and containers. No plastic tubes or tips or pipettes. Retinoids stick to many plastics and variable loss of up to 40% can occur when pipetting retinoid solutions with regular (plastic) pipette tips. Loss due to surface adherence is especially pronounced at low concentrations.

• For determination of UV absorbance of retinoid solutions, cuvettes should be cleaned thoroughly before and after use with ethanol and/or acetone and then dried completely. If acetone is used, complete removal is particularly important as acetone will absorb significantly at low wavelengths. For more stringent cleaning, rinse cuvettes with concentrated nitric acid followed by water, then 100% ethanol and completely dry.

• λ_max of all-trans-retinol in ethanol is 325nm. Molar absorptivity (ε) is wavelength (λ) and solvent dependent. Use the appropriate solvent blank according to the solvent defining the ε value. Molar absorptivity of all-trans retinol in ethanol at 325nm is ε =52,770M⁻¹ cm⁻¹. λ_max of all-trans-retinoic acid in ethanol is 350nm. Molar absorptivity of all-trans retinoic acid at 350nm in ethanol ε =45,300M⁻¹ cm⁻¹. Path length L =1cm for standard cuvette.

• Calculate concentration using the following formula $c=\frac{A}{\epsilon L}$.

• Retinol solution preparation procedures are also applicable to other retinoids.

• Preparation of retinal can be accomplished with a similar procedure. All-trans-retinal has a λ_max of 338 and ε of 42,880 in ethanol or a λ_max of 368 and ε of 48,000 in hexane.

• The typical absorbance spectra of selected retinoids in ethanol is shown in Fig. 2.

Part 2. Treating cells with retinol

1. Follow existing protocols for culturing your cells of interest. Typically, the number of cells needed is at least 0.1 million live cells per extraction. If possible, use 0.5 or 1 million live cells per replicate. Prepare a minimum of three replicates. The culture volume should be at least 1mL. Adjust your culture vessel configuration if necessary. Typically, a culture of 0.1 million cells per well uses a 12-well plate with 1mL culture media. Determine the live/total cell ratio by Trypan Blue staining. The ratio should be at least 90% to be consistent with high viability and experimental conditions, which yields reproducible and representative results.

2. In most cases, cell culture media should be replaced with serum-free media immediately preceding the retinol treatment period. Serum contains retinol and will provide an additional (and possibly variable) amount or retinol as substrate. Serum-free media with added retinol (or other retinoid substrate) provides a defined substrate concentration. Most cell lines and types can tolerate serum-free conditions during the treatment period, which is typically on the order of hours.

3. If you expect some cell death during your treatment or transfection, try to get a live cell estimate at the time of assay.

4. Under yellow or red light, transfer desired amount of retinoid solution (described herein as retinol) to the culture using a Hamilton syringe. Typically, the final all-trans retinol concentration should be 2μM or another physiologically relevant concentration (typically 1–10μM). For example, if your culture volume is 2mL, and your fresh prepared retinol solution is 400μM, then you need add 10μL all trans retinol solution to the culture media. You do not have to remove 10μL culture media before adding all-trans retinol solution, but you need to add an equal volume of ethanol (or selected vehicle) to the control wells. You do not have to use Hamilton syringe for ethanol/vehicle controls.

5. After adding retinoid solution to the culture media, mix the media gently and return the culturing cells to the incubator for the desired period of time.

6. Collect both culture media samples and cell pellet samples under yellow light or red light.

7. Put the samples in −80°C immediately until extraction.

Technical notes

• For accurate delivery of a solution of defined retinoid concentration, proper cleaning of the Hamilton syringe used for delivery is a critical element. It is essential to clean the Hamilton syringe by flushing before and after each experiment by drawing up clean acetone 30 times. This will ensure there is no retinol carry-over. After cleaning the syringe, dry the syringe. Make sure both the glass barrel and the plunger are completely dry of residual acetone.

• RA production from retinal as a substrate can be performed with a similar procedure. Typical treatment concentrations range from 0.1 to 1μM.

• Catabolism of RA can be evaluated by determining the remaining RA over time and calculating the turnover of RA via the elimination half-life.

Part 3. Retinoid extraction and quantification

1. For the RA extraction, collect 800μL of media in a 16 × 125mm borosilicate glass disposable culture tube as previously described (Kane & Napoli, 2010).

2. Remove the remaining media and use a cell scraper to harvest the cells for LC-MS/MS-based analysis.

3. If immediate sample preparation is not possible, add 300μL of 0.9% Saline to each well to store the cells.

4. Cover collected cell and media samples in foil to protect from light and store at −80°C.

   RA can then be extracted from cells and media using a two-step liquid-liquid extraction, and quantified using liquid chromatography-multistage tandem mass spectrometry (Jones et al., 2015; Kane et al., 2005; Kane, Folias, Wang, et al., 2008; Kane & Napoli, 2010).

3. Direct quantification of RA in embryonic tissues

3.1. Reagents and materials

1. Acetone (HPLC)

2. Saline (0.9% NaCl)

3. 0.025M KOH in 100% ethanol

4. 4M HCl

5. Hexanes (Certified or highest grade available)

6. Internal standard(s) (choose according to desired analyses)

7. Glass homogenizer

8. Glass culture tubes and glass transfer pipets

3.2. Protocol

1. Tissue collection, homogenization, and extraction methodology has been described in detail previously (Kane & Napoli, 2010). Procedures and key points for analyses in developmental models are provided here.

2. Determine the minimum amount of tissue required for your RA assay by systematically testing a range of tissue quantities. Typical tissue amounts range from 1 to 100mg, depending on the RA content of the target tissue.

3. Collect tissue under yellow (or red) lights; if using a dissecting microscope use a yellow (or red) filter.

4. Freeze tissue upon collection in liquid nitrogen or on dry ice and store at −80°C until assay.

5. Determine an accurate tissue weight or determine the total protein content for normalization.

6. It is important to thaw tissue on ice and keep tissue on ice while homogenizing and extracting.

7. Homogenize tissue in saline (0.9% NaCl), typically 1mL saline. (Be sure to rinse all homogenizing glassware with water followed by ethanol and/or acetone before and after use to remove retinoid residue.)

8. Extract tissues using a two-step liquid-liquid extraction that allows for extraction of both neutral retinoids (retinol, retinyl esters) and polar retinoids (RA, RA metabolites) from the same sample (Kane & Napoli, 2010).

9. If retinaldehyde quantitation is desired, an aliquot of homogenate or a separate sample is required to allow for derivatization of the aldehyde group by O-ethylhydroxylamine (Kane, Folias, & Napoli, 2008; Kane & Napoli, 2010; Wang et al., 2015).

   Note: RA levels that have been reported in embryo and embryonic tissues are compiled in Table 2.

Table 2.

Select RA levels in mouse embryos and tissues.

Model	Type of sample	Day of assay	Reported value for RAa, b, c	RA Phenotypeb	Refb
Mouse: wild-type	Whole embryo	E12.5	20 pmol/g tissue	Normal	Wang et al. (2018)
Mouse: Dhrs3+/−	Whole embryo	E12.5	21 pmol/g tissue	Similar to normal	Wang et al. (2018)
Mouse: Dhrs3−/−	Whole embryo	E12.5	27 pmol/g tissue	Excess RA	Wang et al. (2018)
Mouse: wild-type	Whole embryo	E14.5	5.2 pmol/g tissue	Normal	Billings et al. (2013)
Mouse: Dhrs3+/−	Whole embryo	E14.5	5.2 pmol/g tissue	Similar to normal	Billings et al. (2013)
Mouse: Dhrs3−/−	Whole embryo	E14.5	7.7 pmol/g tissue	Excess RA	Billings et al. (2013)
Mouse: wild-type	Whole embryo	E10.5	350 pmol/g protein	Normal	Ashique et al. (2012)
Mouse: Rdh10m366Asp	Whole embryo	E10.5	275 pmol/g protein	Reduced RA	Ashique et al. (2012)
Mouse: wild-type	Whole embryo	E12.5	275 pmol/g protein	Normal	Ashique et al. (2012)
Mouse: Rdh10m366Asp	Whole embryo	E12.5	100 pmol/g protein	Reduced RA	Ashique et al. (2012)
Mouse: wild-type	Whole embryo	E14.5	210 pmol/g protein	Normal	Ashique et al. (2012)
Mouse: Rdh10m366Asp	Whole embryo	E14.5	110 pmol/g protein	Reduced RA	Ashique et al. (2012)
Mouse: Rbp4−/− VAS	Whole embryo	E12.5	120 pmol/g protein	Similar to normal	Fu et al. (2010)
Mouse: Rbp4−/− VAD	Whole embryo	E12.5	57 pmol/g protein	Reduced RA	Fu et al. (2010)
Mouse: Foxc1+/	Meninges	E14.5	740 pmol/g protein	Normal	Siegenthaler et al. (2009)
Mouse: Foxc1l/l (Foxc1-null)	Meninges	E14.5	590 pmol/g protein	Reduced RA	Siegenthaler et al. (2009)
Mouse: Foxc1+/	Cortex	E14.5	280 pmol/g protein	Normal	Siegenthaler et al. (2009)
Mouse: Foxc1l/l (Foxc1-null)	Cortex	E14.5	140 pmol/g protein	Reduced RA	Siegenthaler et al. (2009)
Mouse:wild-type	Hippocampus	E19	20 pmol/g tissue	Normal	Kane, Folias, Wang, and Napoli (2010)
Mouse: wild-type +ethanol, low BAC%	Hippocampus	E19	30 pmol/g tissue	Excess RA	Kane et al. (2010)
Mouse: wild-type +ethanol, high BAC%	hippocampus	E19	400 pmol/g tissue	Excess RA	Kane et al. (2010)
Mouse:wild-type	Cortex	E19	12 pmol/g tissue	Normal	Kane et al. (2010)
Mouse: wild-type +ethanol, low BAC%	Cortex	E19	24 pmol/g tissue	Excess RA	Kane et al. (2010)
Mouse: wild-type +ethanol, high BAC%	Cortex	E19	600 pmol/g tissue	Excess RA	Kane et al. (2010)

^aIn some cases, values have been rounded from reported data and/or estimated from graphical representations.

^bSee references indicated for specific RA data including error, statistical significance, and numbers of N as well as full description of accompanying phenotype.

^cNote that data expressed as per g protein is typically a ~25–250× greater value than per g tissue—depending on protein content of sample type.

3.3. Analysis

3.3.1. Quantification of RA

Quantify RA using an LC-MS/MS-based assay. Typical sample numbers range from 5 to 10 biological replicates. However, the sample numbers required to demonstrate statistical significance may be more or less depending on the normal variability of RA within the target tissue and the magnitude of change in RA observed in the experimental model.

3.3.2. Considerations for RA quantification in tissue

LC-MS/MS-based quantification in tissue is applicable to any tissue and yields a direct, absolute quantification of RA. It imparts temporal accuracy and represents the level of RA at the time of collection. However, LC-MS/MS-based quantification of RA is fairly time-intensive and requires specialized instrumentation. The spatial resolution of LC-MS/MS-based analysis, which relies on homogenization of tissue regions before analysis, is limited to the ability to dissect regions of interest and does not yield any intact visualization of RA. Additionally, the typical tissue requirements for RA analysis may necessitate pooling of samples.

4. Cell-based assays of RA signaling: F9 rare-LacZ reporter

4.1. Materials and reagents

1. 0.2% gelatin coated plates

2. Dulbecco’s Modified Eagle Medium:

   Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% Heat-Inactivated Fetal Bovine Serum (FBS)

3. 1× Penicillin-Streptomycin

4. 0.4 mg/mL G418, Geneticin

4.2. Reporter reagents (available upon request)

The RARE reporter cell line F9-RARE-lacZ (SIL15-RA) was a kind gift from Dr. Michael Wagner (SUNY Downstate Health Sciences University) and Dr. Peter McCaffery (University of Aberdeen). The RA-responsive F9 cell line expresses the E. coli lacZ gene under the control of a RARE element derived from the human retinoic acid receptor-β gene (RARβ) (Wagner et al., 1992).

4.3. Protocols

Culture of F9-RARE-LacZ cells

F9-RARE-LacZ cells can be cultured and maintained as previously described (Ababon, Li, Matteson, & Millonig, 2016; Wagner et al., 1992) using 0.2% gelatin coated plates and Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% Heat-Inactivated Fetal Bovine Serum (FBS), 1× Penicillin-Streptomycin, and 0.4mg/mL G418, Geneticin.

F9 RARE-LacZ assays of RA-signaling activity: concentration dependent response

1. Set up each each replicate of RA-treated F9 RARE-LacZ cells in duplicate assays to allow for the measurement of RA levels by LC-MS/MS in parallel with assessment of the RA-signaling activity observed at the same time point.

2. Cells should be minimally seeded at 5 × 10⁴ cells per well in a 0.2% Gelatin Coated 12-well cell culture plate.

3. Incubate cells with 1mL of media supplemented with either all-trans RA (final concentration 0.5, 5 or 50nM as in Fig. 3 or with desired concentration(s)), or unsupplemented culture media as a control.

Fig. 3.

Evaluating RA reporter response in the F9 RARE-LacZ stable cell line: β-Galactosidase reporter assay indicating RA-signaling activity as compared to RA levels. Cells were treated with 0–50nM of all-trans retinoic acid (atRA or RA) and reporter response and RA levels were measured over time for 0–96h. (A) The β-galactosidase reporter assay cell (B) RA in cell as determined by LC-MS/MS (C) RA in media as determined by LC-MS/MS. Cell culture for (A)–(C) and reporter assay conditions for (A) described in Section 3. LC-MS/MS quantitation for (B) and (C) as described in Section 1 and in Jones et al. Error bars represent standard deviation (n =3).

Determination of RA levels via LC-MS/MS in F9 RARE-LacZ stable cell line

1. At the desired time points (e.g., 0, 0.5, 1, 2, 3, 6, 12, 24, 48, 72, and 96h as in Fig. 3 or desired timepoint(s)) collect media aliquots.

2. Remove remaining culture media.

3. Use a cell scraper to harvest cells for LC-MS/MS analysis.

4. Media and cell pellets can be frozen at −80°C until sample extraction.

F9 RARE-LacZ stable cell line sample collection and β-galactosidase enzyme reporter assay

1. For the assessment of reporter response via β-Galactosidase, remove growth medium and wash cells twice with 200μL of PBS Buffer.

2. Add 200μL of Reporter Lysis Buffer to the cells and rock at room temperature for 15min.

3. Scrape off cells and transfer to a 1.5mL centrifuge tube.

4. Vortex for 10s, and centrifuge at 4°C for 2 min at top speed.

5. Transfer the supernatant into a 1.5mL DNA LoBind Tube (Eppendorf) and store at −80 °C until all required timepoints are collected.

6. A β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer (RLB) (Promega) kit can be used to assay the samples according to the manufacturer’s 96-well plate format. Absorbance is monitored at 420nm with a POLARstar Omega plate reader (BMG Labtech) or comparable plate reader.

4.4. Analysis

Evaluating RA reporter response in the F9 RARE-LacZ stable cell line: cellular assay of RA-signaling activity as compared to RA levels:

In Fig. 3, an example of data obtained from the F9 RARE-LacZ stable cell is shown in comparison to RA determined by LC-MS/MS-based detection. Cells were treated with varying concentrations of RA (0.5, 5, and 50nM RA) and reporter response via β-galactosidase staining as well as RA levels were assessed over time up to 96h. Fig. 3A, the β-galactosidase reporter assay showed an increase in signal starting at 3h, followed by a peak in signal at 24h, which subsequently decreased at 48h for all atRA concentrations. No difference was seen between the 5 and 50nM ATRA treatment. Fig. 3B and C shows the RA levels in the cell and media, respectively. Fig. 3B and C showed a sustained, concentration dependent level of RA through 12h before decreasing, likely due to metabolism of RA.

5. Cell-based assays of RA signaling: Two-hybrid Gal4-RAR;UAS-tk-gaussia luciferase reporter

5.1. Materials and reagents

1. 0.2% gelatin coated plates

2. Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)supplemented with 10% Heat-Inactivated Fetal Bovine Serum (FBS)

3. 1× Penicillin-Streptomycin

4. 0.4mg/mL G418, Geneticin

5.2. Reporter reagents (available upon request)

Human Embryonic Kidney (HEK293) cells were obtained ATCC (Graham, Smiley, Russell, & Nairn, 1977). A construct expressing a fusion of the DNA-binding domain of the GAL4 protein (aa 1–147) to the ligand binding domain (aa 156–457) of zebrafish RARαb under the control of a CMV promoter was obtained as a kind gift from Joshua Waxman (University of Cincinnati/Cincinnati Children’s Hospital) and has been previously described (D’Aniello et al., 2013; Mandal et al., 2013).

A construct expressing Gaussia luciferase under the control of the HSV thymidine kinase minimal promoter was generated by cloning 5 tandem copies of upstream activating sequences (UAS) in the Bgl2 site of the pGluc-mini-TK-2 plasmid (New England Biolabs).

5.3. Protocols

Culture HEK293 cells in Eagle’s Minimum Essential Media (MEM) with 10% FBS.

Two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter of RA presence reagents

1. Reverse co-transfect HEK293 cells with pGluc-mini-TK-2-UAS and pCS2p+Gal4-RAR reporter plasmids at 500ng each, per well, using Lipofectamine 2000 (ThermoFisher) in Opti-MEM (Invitrogen), and seed at 0.2 million cells per well in a 12-well cell culture plate.

2. After allowing the cells to attach, approximately 3h, add 1mL of Eagle’s MEM to each well and incubate overnight.

3. The next day, remove cell media and replace with 1mL of media supplemented with either all-trans Retinoic Acid (ATRA) (0.5, 5, 50nM as in Fig. 4 or desired concentration(s)), holo-Retinol Binding Protein 4 (RBP4) (0.4, 2, 10μM as in Fig. 5 or desired concentration(s)), or unsupplemented Eagle’s MEM, as a control at 0h.

4. Collect samples at 0, 0.5, 1, 2, 3, 6, 12, 24, 48, 72, and 96h as in Figs. 4 and 5 or desired timepoint(s).

Fig. 4.

Evaluating RA reporter response in the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter in HEK293 cells as compared to RA levels. Cells were treated with 0–50nM of all-trans retinoic acid (atRA or RA) and reporter response and RA levels were measured over time for 0–96h. (A) Two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter (B) RA in cell as determined by LC-MS/MS (C) RA in media as determined by LC-MS/MS. Cell culture for (A)–(C) and reporter assay conditions for (A) described in Section 4. LC-MS/MS quantitation for (B) and (C) as described in Section 1 and in Jones et al. Error bars represent standard deviation (n =3).

Fig. 5.

Evaluating RA reporter response in the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter in HEK293 cells as compared to RA levels produced from retinol treatment. Cells were treated with 0–10nM of holoRBP4-Retinol (RBP4-ROL) followed by quantification of reporter response and all-trans retinoic acid (RA) levels over time for 0–96h. (A) Two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter (B) RA in cell as determined by LC-MS/MS (C) RA in media as determined by LC-MS/MS. Cell culture for (A)–(C) and reporter assay conditions for (A) described in Section 4. Retinol treatment and LC-MS/MS quantitation for (B) and (C) as described in Section 1 and in Jones et al. Error bars represent standard deviation (n =3).

Determination of RA levels via LC-MS/MS in HEK293 cells expressing the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter

1. At the desired time points, collect media aliquots.

2. Remove the remaining culture media and use a cell scraper to harvest cells for LC-MS/MS analysis.

Gal4-RAR;UAS-tk-Gaussia luciferase expressing cells sample collection and Gaussia luciferase reporter assay

1. Collect 100μL of media in a 1.5mL DNA LoBind Tube (Eppendorf) for use in the reporter assay as previously performed (D’Aniello et al., 2013; Mandal et al., 2013).

2. A Gaussia Luciferase Flash Assay Kit (ThermoFisher) with a Corning 96-Well Solid White Polystyrene Microplate can then be used to determine luciferase activity according to the manufacturer’s instructions.

3. Luciferase activities [luminescence (relative light units/well)] should be monitored with a POLARstar Omega plate reader (BMG Labtech) or comparable plate reader.

Technical notes

• For detachment of cells use TrypLE Express Enzyme, no Phenol Red (1×) (Invitrogen).

• F9 RARE-LacZ Stable Cells should be passaged frequently and should not reach more than 80–90% confluency to prevent differentiation.

• For better transfection efficiency: (1) briefly vortex the Lipofectamine before use and (2) mix the Lipofectamine 2000 and plasmids in a flat dish, rather than a microcentrifuge tube, for optimal lipid formation.

• For LC-MS/MS-based quantitation, do not use enzyme-based detachment procedures (e.g., trypsin). Remove culture media and gently use a cell scraper to detach cells.

• All experiments should be conducted under yellow light to prevent light-induced isomerization and degradation of retinoids.

• Glass pipettes and glass tubes should always be used when handling retinoids.

• Retinoid samples can be stored in DMSO or DMF at 5–10mM at −80°C, but long term storage at any temperature should be avoided.

• The concentration of dissolved retinoids should be confirmed by measuring the absorption of a 1000-fold dilution of the stock solution in ethanol (or hexane for more apolar carotenoids or retinyl esters) using a UV-visual spectrophotometer and deriving the concentration via the Lambert-Beer law A_λ =(ε)(c) for a pathlength L =1cm (as described in Protocol I, Part 1). In the case of all-trans-RA dissolved in ethanol, the maximum absorption wavelength (λ_max) =350nm, and the molar extinction coefficient (ε_max)=45,300M⁻¹ cm⁻¹. For other retinoids spectral values and absorption coefficients can be found in Kane and Napoli (2010) and references therein. In addition, LC-UV can be used to assess the purity and isomerization state of various retinoid compounds employed.

5.4. Analysis

5.4.1. Evaluating RA reporter response in the two-hybrid Gal4-RAR; UAS-tk-Gaussia luciferase reporter as compared to RA levels

Fig. 4 shows an example of data obtained from the two-hybrid Gal4-RAR; UAS-tk-Gaussia luciferase reporter in HEK293 cells in comparison to RA determined by LC-MS/MS-based detection. Cells treated with varying concentrations of RA (0.5, 5, and 50nM RA) and reporter response via luciferase as well as RA levels were assessed over time up to 96h. Fig. 4A, the luciferase levels in the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter showed an increase in signal starting at around 12h with sustained signal through 96h. Fig. 4B and C shows RA levels in the cell and media, respectively. Fig. 4B and C shows a sustained, concentration dependent level of RA through 48h before declining, likely due to metabolism of RA.

Fig. 5 shows an example of the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter in HEK293 cells as used to evaluate reporter response from RA produced from (added) retinol substrate using the protocol described in Section 2. Similarly to Fig. 4A, the luciferase levels in the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter assay showed an increase in signal starting at around 12h and continuing through 96h. Fig. 5B and C shows the RA levels in the cell and media, respectively. Fig. 5B and C showed an increase in RA produced up until 12h with sustained, concentration dependent level of RA through 72h in both cells and media. Media maintains similar levels of RA at 96h, whereas cellular levels of RA decline at 96h, likely due to metabolism of RA.

5.5. Pros and cons

5.5.1. Considerations for various cellular assays

Each of the cellular assays has advantages and limitations to be considered when choosing an experimental approach as Summarized in Table 3. RA Extraction and Quantification via LC-MS/MS (part 3 of Section 2.2 “Protocols”) offers the advantage of direct, absolute quantitation of RA that is applicable to cell lines and tissues. The quantitation of RA has temporal accuracy and represents the levels of RA that are present at the time of collection (Fig. 6). Results are very reproducible as documented in methodology publications (Jones et al., 2015; Kane et al., 2005; Kane, Folias, Wang, et al., 2008) and reproducibility representative cellular assay data are shown in Fig. 7. Limitations of this approach include more experimental effort than reporter assays and the requirement for expensive, specialized instrumentation. For tissue quantitation in embryo, LC-MS/MS approaches do not provide intact spatial visualization, and measurement of specific spatial regions is limited by the ability to dissect those regions. Additionally, LC-MS/MS approaches have minimum tissue requirements that may necessitate pooling. The F9 RARE-LacZ Stable Cell Line β-Galactosidase Reporter (Section 4) offers the advantage of rapid results with no transfection required and reproducible results for comparable conditions. Limitations include the possibility that cells can differentiate if not maintained properly and the requirement for cell lysis. The F9 RARE-LacZ stable cell line β-galactosidase reporter is also limited by a response that saturates at moderate levels of RA (Fig. 3A), is nonquantitative, and displays a temporal delay post increase in RA accompanied by a transient signal response (Fig. 6). The two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter (Section 5) has the advantage that it can be applied to any cell line amenable to transfection and does not require cell lysis. The reporter offers rapid results and remains on once activated (illustrated in Figs. 4 and 6) thereby eliminating concerns of a transient response as is encountered in the F9 RARE-LacZ cell line. Results are very reproducible as shown in Fig. 7. Limitations of the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter include transfection difficulty in some cell lines, a non-quantitative response, and a temporal delay exposure to RA (Fig. 6).

Table 3.

Comparison of different reporters and quantification methods for cellular systems.

F9 RARE-LacZ stable cell line β-galactosidase reporter	Two-hybrid Gal4-RAR; UAS-tk-Gaussia luciferase reporter	RA extraction and quantification via LC-MS/MS
PROS	PROS	PROS
No transfection required Quick results (yes/no) Very reproducible	Can be used in multiple cell lines Reporter stays on once activated Does not require cell lysis Fastest results (yes/no) Very reproducible	Applicable to cell lines and tissues Direct, absolute RA quantification Temporal accuracy, represent levels at the time of collection Very reproducible
CONS	CONS	CONS
Cells can differentiate if not maintained properly Requires cell lysis Reporter response saturates at moderate levels of RA Nonquantitative Temporal delay then transient signal	Depending on cell line, transfection can be difficult Nonquantiative Temporal delay	More time intensive Expensive, specialized instrumentation required

Fig. 6.

Comparison of temporal responses of different cellular assays to determine RA levels or RA signaling. (A) RA reporter response after 50nM RA treatment in the F9 RARE-LacZ stable cell line via β-galactosidase reporter assay and RA levels measured via LC-MS/MS (B) RA reporter response after 50nM RA treatment in the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter in HEK293 cells as compared to RA levels (C) RA reporter response from RA produced after 10μM RBP4-ROL treatment in the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter in HEK293 cells as compared to RA levels. Reporter assays for (A) as in Section 3, reporter assays for (B) and (C) as in Section 4. LC-MS/MS quantitation as described in Section 1 and in Jones et al. Error bars represent standard deviation (n =3).

For RA production evaluation, similar to standard enzyme activity assays, the timepoint and concentration ranges for the retinoid substrate of interest should first be determined by performing a time-course experiment and a concentration-course experiment to determine conditions within the linear range (Kane & Napoli, 2010; Wang et al., 2011).

6. In situ hybridization can be used to visualize gene activity of vitamin A signaling components

The ability to accurately detect gene activity of components from the vitamin A pathway allows for the study of its role in development and disease. This section describes the use of in situ hybridization (ISH) as a sensitive method to identify spatiotemporal gene activity by localizing mRNA transcripts from a gene of interest (Fig. 8). To do this, ISH utilizes anti-sense RNA probes of varying length that are synthesized with nucleotides that contain a digoxigenin (DIG) moiety that can be bound by an enzyme linked anti-DIG antibody. Transcripts that are bound by the labeled riboprobe and antibody may be detected by a colorimetric reaction, whereby an NBT/BCIP substrate solution is metabolized to create a purple reaction product. Following the reaction, visualization of the bound mRNA transcripts can be detected in the regions of the purple color development and imaged by light microscopy.

Fig. 8.

Methods to identify components of vitamin A metabolism and RA synthesis. (A–C) Visualizing vitamin A metabolizing enzymes by in situ hybridization. (A) Raldh2 is expressed in the whole embryo at E9.5 in the foregut and caudal somites. Rdh10 (B) is expressed at E9.5 in the regions of the gut, forelimb and somites. In a transverse section (C), Rdh10 activity can be observed within the somites and forelimb. (D–F) The RARE-LacZ reporter mouse contains a RA response element under the control of a minimal promoter and is used to visualize RA signaling. LacZ staining of an E13.5 embryo (D) demonstrates the spatiotemporal activity of RA signaling throughout the head and trunk including the eye, neural tube, and limbs. High levels of RA signaling are present in many organs including the gut at E17.5 (E) as well as in tissue around the optic vesicle and telencephalon as evident in a transverse section of an E10.5 embryo (F). Immunostaining for Rdh10, a component of vitamin A metabolism, reveals expression the dorsal root ganglia, neural tube and motor nerve projection in a transverse section (G) and inset (G’) of an E10.5 embryo. Abbreviations: s, somite; nt, neural tube; fl, forelimb; ov, optic vesicle; t, telencephalon; drg, dorsal root ganglia.

6.1. Reagents

Note: All stock solutions should be treated with diethyl pyrocarbonate (DEPC) and autoclaved, or prepared with DEPC-H₂O.

1. 4% Paraformaldehyde in PBS

   Microwave to boiling 100mL of DEPC-PBS and then add 4g of paraformaldehyde powder (toxic) and stir to dissolve in a fume hood

2. Phosphate-Buffered Saline with Triton (0.1% Triton in PBS); 1mL Triton X-100 in 1L DEPC-PBS

3. Hybridization Buffer; 50% formamide, 5× SSC, 0.05M EDTA, 0.1% Triton X-100, 0.1% CHAPS, 0.05mg/mL Heparin, 50.0mg Yeast torula-RNA, 1.0g Boehringer blocking powder

4. Potassium-Tris Buffer with Triton (KTBT); 0.05M Tris/HCl (pH 7.5), 0.15M NaCl, 0.01M KCl, 1% Triton X-100

5. Alkaline Phosphatase Buffer; 0.01M Tris/HCl (pH 9.5), 0.005M NaCl, 0.005M MgCl₂, 0.01% Tween20

6. Maleic Acid Buffer with Triton (MABT); 100mM Maleic acid (pH 7.5; adjust with NaOH), 0.15M NaCl; 1% Triton X-100

6.2. Protocols

RNA probe synthesis

1. Mix the following at room temperature:

   Sterile distilled water (DEPC-H2O)	11.5 μL
   5 × transcription buffer	5.0 μL
   100 mM DTT	2.5 μL
   DIG 10 × nucleotide mix	2.5 μL
   Linearized plasmid (1 μg/μL)	1.5 μL
   Rnasin ribonuclease inhibitor	0.5 μL
   Polymerase (SP6, T3 or T7)	1.5 μL
   Total	25 μL

2. Incubate at 37 °C for 2h up to 6h.

3. Remove 1μL aliquot and run on 1% TAE gel to check synthesis. Expect to see RNA band 10-fold more intense than plasmid band, suggesting 10–15μg probe.

4. Add 2μL of DNAse1 (ribonuclease free) and incubate at 37 °C for 15min.

5. Add:

   50 μL	dH2O
   25 μL	10M ammonium acetate
   200 μL	100% ethanol

6. Mix and leave on dry ice for 30min or store at −80 °C overnight.

7. Spin in centrifuge at 4 °C for 20min at 13,000 to 15,000rpm.

8. Wash pellet in 50μL of 70% ethanol and spin at 4 °C for 5min at 13,000 to 15,000rpm.

9. Air dry pellet for 5–10min until obvious traces of ethanol have evaporated.

10. Redissolve pellet in 50μL hybridization solution and store at −20 °C. If a higher concentration of probe is needed the pellet may be resuspended in 30μL of hybridization solution.

In situ hybridization staining method

1. Dissect embryos in DEPC-PBS and fix in 4% paraformaldehyde for 2h to overnight (use scintillation vials).

2. Wash embryos in PBT at room temperature for 10min.

3. Dehydrate the embryos through a graded series of methanol diluted in PBT: wash in 25% methanol, 50% methanol, 75% methanol and 100% methanol for 10min each.

4. Embryos can be stored at −20 °C up to several weeks at this stage.

5. Rehydrate the embryos through a graded series of methanol diluted in PBT: wash in 75% methanol, 50% methanol, 25% methanol for 10min each.

6. Wash in PBT for 5min at room temperature.

7. Incubate in 10 μg/mL proteinase K (diluted in PBT) for 5–10min at room temperature. Incubation time will vary depending on the embryo’s stage.

   Incubation time by embryo stage:

   E6.5: 4min

   E7.5: 4–5min

   E8.5: 6min

   E9.5: 10min

   E10.5: 15mm

8. Stop the proteinase K reaction by washing with 2mg/mL glycine (diluted in PBT) for 5min at room temperature (make the same day of use).

9. Wash for 5min in PBT at room temperature.

10. Re-fix the embryos in 4% paraformaldehyde/0.25% glutaraldehyde for 20min at 4 °C.

11. Wash for 10min in PBT at room temperature.

12. Transfer the embryos to 2mL Eppendorf tube with a round bottom (up to 10 embryos can be processed in a single tube).

13. Incubate embryos for at least 1.5h in 1–2mL of pre-warmed hybridization buffer at 62 °C to 70 °C.

14. Incubate embryos with 2 μL of RNA probe per 1mL of hybridization buffer at 62 °C to 70 °C overnight in hybridization oven (1mL of buffer is enough for 10 mouse embryos at E10.5).

15. Wash embryos twice in 2mL of 2xSSC/0.1% chaps for 45min at 62 °C.

16. Wash embryos in 2mL of 0.2xSSC/0.1% chaps for 30min at 62 °C.

17. Wash in KTBT for 5min at room temperature.

18. Incubate the embryos in 2mL of 20% goat serum (or lamb serum) in Potassium-Tris Buffer with Triton (KTBT) for at least 1 to 1.5h at room temperature with continual rocking.

19. Replace blocking solution with fresh 20% goat serum in KTBT. Add 2 μL of DIG-alkaline phosphatase antibody and incubate at 4 °C overnight with continual rocking.

20. Rinse the embryos at least twice in KTBT.

21. Wash the embryos between 4 and 5 times in KTBT for 1h at room temperature with continual rocking.

22. Replace with 2mL of fresh KTBT and wash overnight at 4 °C with continual rocking.

23. In the morning, wash the embryos at least twice with KTBT for a combined time of 30min at 4 °C.

24. Wash the embryos in 1mL of alkaline phosphatase buffer for 10min at room temperature with continual rocking.

25. Add 3.375 μL of NBT (100mg/mL in DMF) and 3.5 μL of BCIP (50mg/mL in DMF) directly to the embryos in alkaline phosphatase buffer with shaking at room temperature in the dark (wrap the Eppendorf tube in foil or place in a covered container).

26. Stop the reaction once the desired color intensity is achieved by fixing the embryos in 4% paraformaldehyde. This can take from 15min to overnight, however 1 to 2h is more common. Continual observation of the color development is not advisable as the background will increase.

6.3. Safety considerations and standards

Paraformaldehyde is a toxic chemical and extreme caution should be used when handling. Be sure to always use proper personal protective equipment and handle under a fume hood.

6.4. Related techniques

In addition to the technique described above, other RNA in situ hybridization assays are available that include RNAscope, ViewRNA and SABER-FISH. An advantage of these techniques is their ability to use fluorescent probes which provide a multiplexed approach to characterize the activity of many genes within the same sample. The use of fluorescence additionally provides an option to quantify gene expression (Kishi et al., 2019; Kulikova, Franken, & Bisseling, 2018; Wang et al., 2012).

6.5. Alternative methods/procedures

6.5.1. Alternative posthybridization washes in maleic acid buffer with triton (MABT)

After hybridizing the embryos overnight at 62 °C in step 14, wash the embryos in a 1:1.5 mix of hybridization buffer and MABT (described in Section 2.1 “Materials, equipment and reagents”) at 65 °C for 1h. Then wash the embryos for 30min at 65 °C in MABT. For steps 17 through 23 replace KTBT with MABT.

6.5.2. Alternative color development

The color development described in step 25 of the protocol may also be carried out by using BMPurple solution in the place of NBT/BCIP. To do this, pre-warm BMPurple to 37 °C. Spin the tubes for 15–20s in micro-centrifuge to pellet precipitate. Place supernatant onto embryos that have already incubated in alkaline phosphatase buffer. Be sure to protect from light while color develops.

6.6. Troubleshooting and optimization

See Table 4.

Table 4.

Troubleshooting for common problems when using in situ hybridization.

Problem	Solution
Riboprobes with consistently high background staining	Wash the embryos in MABT instead of SSC/SDS as described in Section 6.5 “Alternative Methods/Procedures”
Riboprobes with consistently high background staining from probe trapping	To help alleviate probe trapping in the head in embryos at E9.5 and older, it is beneficial to poke the heads of the embryos during harvest to help clear riboprobe during washes
Poor staining quality from incomplete riboprobe penetration	To increase riboprobe penetration in whole embryos it is recommended to test increasing the incubation times with proteinase K listed in step 7
Poor staining quality from incomplete hybridization of the riboprobe	To optimize riboprobe hybridization, the temperature of hybridization in step 14 may be increased between 62 °C and 70 °C

6.7. Summary

In situ hybridization is a sensitive method to detect gene activity by localizing mRNA transcripts within a sample. Embryos are first harvested and fixed overnight in 4% PFA (it is important all solutions are DEPC treated to prevent ribonuclease contamination). The samples are then permeabilized using proteinase K, refixed with 4% PFA/0.25% glutaraldehyde and then hybridized with riboprobe overnight between 62 °C and 70 °C. The following day, samples are processed through a series of posthybridization washes, blocked in a 20% goat serum solution and incubated at 4 °C overnight with an anti-DIG-Alkaline Phosphatase antibody. Samples then undergo a series of washes and begin a colorimetric reaction in an NBT/BCIP substrate solution. Once color development is complete samples are postfixed in 4% PFA and can be imaged and/or stored at 4 °C.

7. Detection of RA signaling using a β-galactosidase assay with X-GAL

Retinoic acid signaling can be visualized in vivo using transgenic mouse lines containing a LacZ reporter. The method described in this section outlines the use of the transgenic RARE-LacZ mouse line to visualize retinoic acid (RA) activity by β-Galactosidase staining with X-gal (Fig. 8). To do this, the RARE-LacZ mouse line possesses a transgene with an RA response element (RARE) that drives expression of β-Galactosidase by RA binding to RA receptors on the RARE sequence (Rossant et al., 1991). The addition of the X-gal substrate, detailed in the below protocol, creates a purple product in the regions of RA activity, which provides an effective means to visualize the spatiotemporal activity of RA signaling. Further, other LacZ transgenic mouse lines, such as Rdh10^βgeo (Sandell et al., 2012), may be used to analyze the spatiotemporal activity of other components of the vitamin A pathway.

7.1. Reagents

1. Tissue Fixative: 0.2%glutalaldehyde/2%formalin

2. Wash Solution (to make 500mL)

   0.1 mL NP40 in 500 mL PBS

3. Stain Solution (to make 500mL)

   5.0 mL	0.5M K3Fe(CN)6
   5.0 mL	0.5M K4Fe(CN)6–3H2O
   1.0 mL	1.0M MgCl2
   5.0 mL	1% Sodium deoxycholate
   make up to 500 mL with Wash Solution
   Store in dark at 4 °C

4. Stock Substrate

   40mg/mL X-gal dissolved in DMF

   Store in dark at 4 °C

5. Working Stain Solution: add 1mL of X-gal stock to 39mL of stain solution. (store at 4 °C in the dark)

7.2. Protocol

1. Wash embryos in PBS at room temperature.

2. Fix embryos in cold 0.2%glutalaldehyde/2%formalin for 30 to 90min at 4 °C. Fixation time depends on the embryo stage.

   Fixation time by embryo stage:

   E7–8: 15min.

   E9–10: 30 min.

   E11–12: 1h.

   E13 and older: 1.5 h.

3. Wash embryos three times for 15min each in wash solution at room temperature with no rocking.

4. While the samples are washing, warm the stain solution at 37 °C in the dark. Then add the X-gal substrate to the pre-warmed stain solution and return to the dark at 37 °C until ready to begin step 5.

5. Replace the wash solution with the prewarmed working stain solution and incubate in the dark at 37 °C for up to 36h. Check the development periodically to determine when the stain is complete.

6. After staining, rinse the embryos thoroughly in PBS and store long-term in PBS/0.01% sodium azide at 4 °C.

7.3. Alternative methods/procedures

Alternatives to using X-gal in the stain solution in step 4 include Salmon-gal with tetrazolium salts, or S-gal with TNBT, which may provide a faster and more sensitive reaction in mouse embryos. It has been noted in comparative studies that Salmon-gal or S-gal were capable of detecting β-galactosidase activity with higher sensitivity than X-gal (Shen et al., 2017; Sundararajan, Wakamiya, Behringer, & Rivera-Perez, 2012).

7.4. Summary

The β-Galactosidase assay with X-gal is a straightforward method that can be used to detect gene activity of components of the vitamin A pathway by staining embryos from transgenic mouse lines, such as RARE-LacZ and Rdh10^βgeo (Sandell et al., 2012, 2007). In the six-step protocol, embryos are harvested and then fixed for a varying length of time depending on embryo stage. Once fixed, samples are washed and then incubated at 37 ° C in the dark with the working stain solution containing the X-gal substrate for up to 36h. When staining is complete the embryos can be imaged and/or stored at 4 °C with sodium azide.

8. Immunostaining can be used to visualize protein activity of vitamin A signaling components

An alternative method to study the vitamin A pathway is to detect protein activity of vitamin A signaling components. The immunostaining technique described in this section is a method to identify the spatiotemporal activity of proteins that are essential in the vitamin A pathway, such as the enzyme Rdh10 (Fig. 8). To do this, either whole embryo or sections are fixed to preserve the protein of interest and then permeabilized to allow penetration and binding of a primary antibody to the protein. Secondary antibodies, conjugated with either an enzyme or fluorophore, are then used to bind the primary antibody. Localization of the bound protein can be achieved by either a colorimetric reaction with an enzyme conjugated secondary antibody, or by fluorescent imaging of antibodies associated with a fluorophore. Detection by either method allows for the ability to visualize the spatiotemporal activity of the protein wherever it is bound within the sample.

8.1. Reagents

Dent’s Bleach; 100% MeOH: DMSO: 30% H₂O₂, 4:1:1

1. 4% Paraformaldehyde in PBS

   Microwave to boiling 100mL of PBS (DEPC treated) and then add 4g of paraformaldehyde powder (toxic) and stir to dissolve in a fume hood.

2. PBT (0.1% Triton in PBS): 1mL Triton X-100 in 1L PBS

3. BABB clearing solution; Benzyle benzoate: Benzyl alcohol, 2:1

8.2. Protocols

Section protocol

1. Section whole embryos by cryosectioning at 10μm (slides can be stored at −20 °C).

2. Let slides defrost for a few minutes at room temperature.

3. Heat-dry with a hair dryer or slide warmer for about 15–20s on the back of each slide.

4. Wash in PBT for 10min.

5. Prepare a slide holder for staining. Place a piece of tissue in each slot of that container that fits perfectly and soak with water.

6. Block the sections in 3% BSA (diluted in PBT) for 30min at room temperature (add about 200 μL of blocking solution on each slide and cover with a paraffin strip in the slide holder container).

7. Remove excess blocking solution and add about 150 μL of the primary antibody diluted in 3% BSA.

8. Cover each slide with a paraffin strip after adding the antibody solution to prevent the slides becoming dry.

9. Seal the container with a paraffin strip and store at 4 °C overnight.

10. Wash the sections three times for 5min with PBT at room temperature.

11. Add 150 μL of the secondary antibody (typically a 1:500 dilution) and cover with a paraffin strip.

12. Incubate at room temperature for 1h.

13. Wash the sections three times for 5min with PBT at room temperature.

14. Add DAPI at a 1:1000 dilution and incubate for 20min at room temperature (alternatively, DAPI can be added with the secondary antibody. Avoid coincubation if doing TUNEL or using a nuclear antibody).

15. Wash sections three times for 5min with PBT at room temperature.

16. Use vectashield to mount the slides (you can skip adding DAPI in the previous step and use vectashield with DAPI instead).

17. Store the slides in the dark at 4 °C for long-term storage.

Whole embryo protocol

1. Incubate embryos in Dent’s bleach for 2h at room temperature (this is a critical step that promotes antibody penetration and quenches auto-fluorescence).

2. Remove Dent’s bleach and replace with 100% methanol.

3. Equilibrate through a graded series of methanol diluted in PBT: wash in 100% methanol, 75% methanol, 50% methanol and 25% methanol for 10min each (the tissue will become sticky during this procedure so be aware not to suck it up into pipettes etc.).

4. Wash embryos in PBT for 10min.

5. Block in 3% BSA (diluted in PBT) for 2h at room temperature.

6. Apply the primary antibody diluted in 3% BSA and incubate overnight at 4 °C with gentle rocking (dilution varies per antibody, determine empirically for each).

7. Wash five times for 1h with PBT.

8. Apply the secondary antibody diluted in 3% BSA and incubate overnight at 4 °C with gentle rocking in the dark (wrap the Eppendorf tube in foil or place in a covered container).

9. Wash three times for 20min with PBT.

10. Incubate 20min in PBS with DAPI at a 1:1000 dilution (for small tissues 20min DAPI incubation is enough. To completely stain a whole E10.5 embryo, incubate overnight at 4 °C with gentle rocking).

11. Rinse embryos in PBT.

12. Dehydrate the embryos through a graded series of methanol diluted in PBT: wash in 25% methanol, 50% methanol, 75% methanol and 100% methanol for 10min each.

13. For long term storage, the tissue can be stored for weeks or months at 4 °C without loss of fluorescent intensity.

8.3. Safety considerations and standards

Paraformaldehyde is a toxic chemical and extreme caution should be used when handling. Be sure to always use proper personal protective equipment and handle under a fume hood.

If using the alternative procedure in Section 8.3 to clear whole embryos for imaging, it is important to use extra caution when handling benzyle alcohol-benzyl benzoate (BABB) as it is a powerful organic solvent that dissolves most plastics. Be sure to use and store BABB in a glass container.

8.4. Alternative methods/procedures

Prior to imaging, clearing stained embryos can help visualize internal structures in larger embryos. This can be achieved with benzyl alcohol-benzyl benzoate (BABB) by adding 1 part Benzyl alcohol to 2 parts Benzyl benzoate. Incubating the embryos for 10min should be enough to render the tissue almost completely transparent, but also fragile. Embryos can be imaged using a glass depression slide with a ring of silicon grease to hold a coverslip in place. A Teflon ring can be used between the grease ring and coverslip if additional space is needed for the sample. After imaging, the embryos can be transferred back to 100% methanol for long term storage at 4 °C.

8.5. Troubleshooting and Optimization

See Table 5

Table 5.

Troubleshooting for common problems when immunostaining.

Problem	Solution
High levels of autofluorescence	Test increasing incubation time with dents bleach to help reduce levels of autofluorescence. Additionally, the head and heart region can be poked for whole embryos at E9.5 and older to help prevent trapping by allowing the antibody to clear during washing steps
Consistently high levels of background staining	The blocking time in step 5 of the “whole embryo protocol” can be increased to help reduce high levels of background. Additionally, the number and duration of washes can be increased in step 7 after incubation with the primary antibody
Poor staining quality from incomplete penetration of the antibody	Test increasing incubation time with Dent’s bleach to help increase antibody penetration in whole embryos

8.6. Summary

Immunostaining can be used to detect protein activity within the vitamin A pathway by first harvesting samples and then fixing overnight in 4% PFA. Following fixation, the samples can either be sectioned or used for whole embryo staining. If using whole embryos, incubation with Dent’s bleach helps to promote antibody penetration and quench auto-fluorescence (Alanentalo et al., 2007). Both sections and whole embryos are blocked in a 3% BSA solution and incubated overnight with primary antibody followed by secondary antibody incubation the next day. Samples are then ready to be imaged and whole embryos can be cleared using benzyl alcohol-benzyl benzoate (BABB) to better visualize internal structures (Section 4.3).