Retinoic acid, RARs and early development

in Journal of Molecular Endocrinology
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Marie BerenguerDevelopment, Aging, and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA

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Gregg DuesterDevelopment, Aging, and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA

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https://orcid.org/0000-0003-4335-3650

Correspondence should be addressed to G Duester: duester@SBPdiscovery.org

This paper forms part of a special issue marking 35 Years Since the Discovery of the Retinoic Acid Receptor. The guest editors for this section were Simak Ali and Vincent Giguère.

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Vitamin A (retinol) is an important nutrient for embryonic development and adult health. Early studies identified retinoic acid (RA) as a metabolite of retinol, however, its importance was not apparent. Later, it was observed that RA treatment of vertebrate embryos had teratogenic effects on limb development. Subsequently, the discovery of nuclear RA receptors (RARs) revealed that RA controls gene expression directly at the transcriptional level through a process referred to as RA signaling. This important discovery led to further studies demonstrating that RA and RARs are required for normal embryonic development. The determination of RA function during normal development has been challenging as RA gain-of-function studies often lead to conclusions about normal development that conflict with RAR or RA loss-of-function studies. However, genetic loss-of-function studies have identified direct target genes of endogenous RA/RAR that are required for normal development of specific tissues. Thus, genetic loss-of-function studies that eliminate RARs or RA-generating enzymes have been instrumental in revealing that RA signaling is required for normal early development of many organs and tissues, including the hindbrain, posterior body axis, somites, spinal cord, forelimbs, heart, and eye.

Abstract

Vitamin A (retinol) is an important nutrient for embryonic development and adult health. Early studies identified retinoic acid (RA) as a metabolite of retinol, however, its importance was not apparent. Later, it was observed that RA treatment of vertebrate embryos had teratogenic effects on limb development. Subsequently, the discovery of nuclear RA receptors (RARs) revealed that RA controls gene expression directly at the transcriptional level through a process referred to as RA signaling. This important discovery led to further studies demonstrating that RA and RARs are required for normal embryonic development. The determination of RA function during normal development has been challenging as RA gain-of-function studies often lead to conclusions about normal development that conflict with RAR or RA loss-of-function studies. However, genetic loss-of-function studies have identified direct target genes of endogenous RA/RAR that are required for normal development of specific tissues. Thus, genetic loss-of-function studies that eliminate RARs or RA-generating enzymes have been instrumental in revealing that RA signaling is required for normal early development of many organs and tissues, including the hindbrain, posterior body axis, somites, spinal cord, forelimbs, heart, and eye.

Introduction

The history of retinoic acid (RA) signaling began with the discovery that its precursor vitamin A (also called retinol) is essential for embryonic growth and development as shown by vitamin A deficiency studies (Wilson et al. 1953). Other early studies demonstrated that RA is a naturally occurring metabolite of retinol that can be generated by alcohol dehydrogenase that metabolizes retinol to retinaldehyde (Bliss 1951), followed by aldehyde dehydrogenase metabolizing retinaldehyde to RA (Elder & Topper 1962). Although a normal function for RA remained elusive for many years, it was observed in the 1970s and early 1980s that vertebrate embryos treated with RA suffered limb teratogenesis (Kochhar 1973, Maden 1982, Tickle et al. 1982), suggesting that RA may be important for limb development.

A major breakthrough in how RA controls development occurred upon discovery of nuclear RA receptors (RARs), which use RA as a ligand to control transcription of key genes (Giguère et al. 1987, Petkovich et al. 1987). Humans and mice possess three RARs (RARA, RARB, and RARG) that are required for many developmental processes as shown by RAR double knockout studies which were needed to overcome redundant functions among the three RARs (Lohnes et al. 1994, Mendelsohn et al. 1994). RARs bind to RA target genes as a heterodimer complex with one of the three retinoid X receptors (RXRA, RXRB, or RXRG) (Kastner et al. 1995). Binding of RAR/RXR occurs at what is known as an RA response element (RARE) that consists of two 6 bp repeated DNA sequences (Hoffmann et al. 1990). RAR/RXR bound to a RARE can recruit nuclear receptor coactivators (NCOA) or nuclear receptor corepressors (NCOR) depending upon binding of RA to the RAR portion of the heterodimer, thus directly activating or repressing transcription of nearby genes (Kumar et al. 2016). A ligand for RXR is not required for RA signaling during the early stages of embryogenesis (Mic et al. 2003).

Discovery of the requirement for RARs in normal development stimulated studies designed to identify enzymes needed to generate RA from its natural source, vitamin A (retinol) (Fig. 1). RA is produced from retinol in a two-step process as alluded to above in early metabolic studies. In embryos, RA synthesis was found to be initiated by retinol dehydrogenase-10 (RDH10) that converts retinol to retinaldehyde in specific tissues (Sandell et al. 2007). To prevent excessive production of retinaldehyde, reverse metabolism of retinaldehyde back to retinol is performed by dehydrogenase/reductase-SDR family member 3 (DHRS3) (Feng et al. 2010, Billings et al. 2013); interestingly, DHRS3 physically interacts with RDH10 to form a complex that regulates the rate of RA synthesis by modifying the retinol/retinaldehyde ratio (Belyaeva et al. 2017). In the second step of RA synthesis, retinaldehyde is metabolized to RA by three retinaldehyde dehydrogenases expressed in specific tissues, i.e., ALDH1A1, ALDH1A2, and ALDH1A3 (also known as RALDH1, RALDH2, and RALDH3) (Niederreither et al. 1999, Mic et al. 2002, Dupé et al. 2003, Fan et al. 2003). Conversion of retinaldehyde to RA is irreversible. Thus, in order to eliminate an excessive amount of RA, three P450 family enzymes (CYP26A1, CYP26B1, and CYP26C1) degrade RA in specific tissues; this results in a short (~1 h) half-life for RA (Hernandez et al. 2007, Pennimpede et al. 2010). It is important to note that although retinol is available in all cells of the embryo, RA synthesis and RA degradation occur only in specific tissues to enable tissue-specific RA signaling. For instance, ALDH1A2 first appears in development at E7.5 expressed only in presomitic mesoderm, but at E8.5 ALDH1A2 is also expressed in the optic vesicles and ALDH1A3 begins expression in the optic vesicles at E8.5, with ALDH1A1 beginning expression in the optic vesicles at E9.5 (Sirbu et al. 2005, Molotkov et al. 2006). Also, CYP26 enzymes are expressed in specific locations, such as certain hindbrain rhombomeres, the caudal progenitor region, and the distal limb, resulting in the degradation of RA in specific locations (Abu-Abed et al. 2002). Thus, during early development, RA is generated in specific tissues, diffuses to nearby tissues, and is degraded in specific tissues, resulting in the presence of RA only in certain tissues and hence the ability of RA signaling to control the development of specific tissues.

Figure 1
Figure 1

Mechanism of retinoic acid (RA) synthesis, RA degradation, and RA signaling. Retinol is transported in the blood via retinol-binding protein-4 (RBP4) which makes it available to all cells of the body. In embryos, retinol is metabolized to the intermediate metabolite retinaldehyde by retinol dehydrogenase-10 (RDH10) and then retinaldehyde is metabolized to RA by one of three aldehyde dehydrogenase-1A (ALDH1A) enzymes. Retinaldehyde can also be converted back to retinol by dehydrogenase/reductase-SDR family member 3 (DHRS3). RA can be degraded by one of three CYP26 enzymes to limit RA activity. RA can act locally or be released from cells in which it is generated and travel as a signal to other cells. RA enters the nucleus and binds to one of three RA receptors (RARs) that form a heterodimer complex with one of three retinoid X receptors (RXRs). RAR/RXR heterodimers bind a repeated DNA sequence known as an RA response element (RARE) whose consensus sequence is AGGTCA (N1,2,5) AGGTCA where N refers to 1, 2, or 5 random nucleotides. RA directly controls transcription by serving as a ligand for RARs. Binding of RA to RARs results in a conformational shift in RAR that alters its ability to bind nuclear receptor coactivators (NCOA) or nuclear receptor corepressors (NCOR) that control further downstream events in transcriptional activation or repression.

Citation: Journal of Molecular Endocrinology 69, 4; 10.1530/JME-22-0041

Genetic loss-of-function studies have been instrumental in determining the normal functions of RA signaling during development (Rhinn & Dolle 2012, Cunningham & Duester 2015, Ghyselinck & Duester 2019). RAR double knockout studies revealed many functions of RA signaling during the late stages of embryogenesis, but these studies did not reveal the early embryonic functions of RA signaling due to the presence of one remaining RAR (Lohnes et al. 1994, Mendelsohn et al. 1994). Fortunately, Aldh1a2 single knockouts and Rdh10 single knockouts have provided a wealth of information on the earliest functions of RA signaling during embryogenesis. Also, Cyp26 knockouts revealed abnormal development due to the presence of RA signaling in locations that do not normally have such signaling (Pennimpede et al. 2010). Studies on these knockouts revealed that embryonic RA signaling begins just a few hours before the formation of the first somite (vertebral progenitor) and is limited to the developing eye (optic vesicle) and posterior body axis (hindbrain, spinal cord, and trunk) (Sirbu et al. 2005, Cunningham et al. 2015) (Fig. 2). This early somite stage is just a snapshot of what is happening around E8.5. As development proceeds to later stages, the locations of RA signaling change as the locations of Aldh1a and Cyp26 expression change. Here, we will focus on the early functions of RA signaling during development.

Figure 2
Figure 2

Retinoic acid (RA) signaling target tissues during early embryogenesis. Early mouse embryos that have 1–15 somites exhibit RA signaling activity (shown in green) in the developing eye (optic vesicle) and throughout the posterior body axis (developing trunk); the anterior border of RA signaling activity in the trunk occurs in the middle of the hindbrain, and the posterior border occurs at the junction with the caudal epiblast that contains neuromesodermal progenitors (NMPs) that differentiate to form either spinal cord or somites; the diagram shows a dorsal view of a 7-somite mouse embryo. Tissues that require RA signaling for normal development include the optic vesicle, posterior hindbrain, somites, spinal cord, forelimbs, NMPs, and heart that is located underneath on the ventral side of the embryo.

Citation: Journal of Molecular Endocrinology 69, 4; 10.1530/JME-22-0041

Role of RA during limb development

As previously mentioned, one of the earliest reported actions of RA is its teratogenic ability to cause defects in limb anteroposterior patterning (thumb to little finger) and proximodistal limb patterning (upper arm to hand) when vertebrate embryos are treated with pharmacological levels of RA (Maden 1982, Tickle et al. 1982). Subsequent studies showed that RA treatment of distal limb tissue alters limb proximodistal patterning by causing distal expression of normally proximal-specific homeobox genes Meis1 and Meis2 which inhibits distal limb development normally controlled by distal Fgf8 expression (Mercader et al. 2000); these studies also showed that FGF8 treatment of proximal limb tissue downregulates Meis1/Meis2. Later studies demonstrated that RA normally generated in the trunk by Aldh1a2 diffuses into proximal limb tissue (Niederreither et al. 1999, Mic et al. 2002). Additionally, Cyp26b1 is expressed in distal limb tissue and degrades distal RA for normal limb proximodistal patterning (Yashiro et al. 2004), plus Fgf8 was shown to be required for Cyp26b1 expression in distal limb (Probst et al. 2011). Together, these findings suggested that a proximal-high gradient of RA signaling is needed to activate proximal Meis1/Meis2 expression, along with a distal-high gradient of FGF signaling that is needed to repress Meis1/Meis2 distally to enable normal limb proximodistal patterning, thus a two-signal model. Fgf8 knockout studies supported the hypothesis that FGF8 signaling is required for limb proximodistal patterning (Mariani et al. 2008). However, the hypothesis that RA signaling is required for limb proximodistal patterning (and Meis1/Meis2 expression) was not supported by loss of RA signaling in proximal limb tissue performed either with the Aldh1a2 knockout eliminating the second step of RA synthesis (Zhao et al. 2009) or the Rdh10 knockout eliminating the first step of RA synthesis (Cunningham et al. 2013); also, these knockouts demonstrated that RA signaling is not required for limb anteroposterior patterning. Instead, the Aldh1a2 and Rdh10 knockouts demonstrated that RA signaling is required for initiation of forelimb (but not hindlimb) budding; see below for description of this function. Together, all of these studies are the best demonstration that one cannot determine the normal functions of endogenous RA by using RA treatment (gain-of-function) studies, thus pointing out the necessity of performing RA loss-of-function studies (preferably using genetic knockouts) to determine the normal functions of endogenous RA signaling.

The disparate conclusions obtained with RA treatment vs RA synthesis knockout can be explained by side effects due to the high amount of administered RA that ectopically activates Meis1/Meis2 in distal limb (Cunningham & Duester 2015). Based upon the knockout studies one can visualize a one-signal model for limb proximodistal patterning that is driven by distal FGF8 that activates genes needed for limb outgrowth in a proximal to distal direction and that activates Cyp26b1 distally to eliminate RA in distal limb which might activate unwanted distal Meis1/Meis2 expression. Interestingly, although Meis1/Meis2 expression in proximal limb does not require RA, it has been observed that Meis1/Meis2 do have nearby RAREs and their expression in the trunk does require RA for a normal level of expression (Berenguer et al. 2020). These observations suggest that RA treatment can force a gene to be expressed in an ectopic location by swamping its associated RAREs with RAR/RXR bound to RA, thus activating the gene in a tissue that is not normally exposed to endogenous RA.

Role of RA signaling during forelimb initiation

As mentioned above, genetic loss of Aldh1a2 or Rdh10 in mouse does not alter limb proximodistal or anteroposterior patterning, but loss of RA signaling with these mutants prevents initiation of forelimb development, although hindlimb development is not affected. Aldh1a2 mutants lack expression of Tbx5 in trunk lateral plate mesoderm that generates the forelimb field (Zhao et al. 2009), and Rdh10 mutants exhibit greatly reduced Tbx5 expression in the forelimb field (Cunningham et al. 2013). As Tbx5 is essential for forelimb initiation (Agarwal et al. 2003, Rallis et al. 2003), RA functions upstream of this important regulator of forelimb bud initiation. Other studies support a requirement for RA in forelimb bud initiation, i.e., treatment of chick embryos with an RA synthesis inhibitor (Stratford et al. 1996) and vitamin A-deficient rat embryos (White et al. 1998).

Comparison of the forelimb defects in mouse Aldh1a2 and Rdh10 knockouts revealed that RA signaling throughout the trunk anteroposterior axis is required to restrict Fgf8 expression to two domains on either side of the forelimb field (Cunningham & Duester 2015). One domain is located in the anterior trunk where the heart is forming, and another domain is located in the posterior-most region of the body axis (caudal epiblast or tailbud). Loss of RA synthesis results in ectopic Fgf8 expression that stretches posteriorly from the heart into the forelimb field and anteriorly from the caudal epiblast into the forelimb field, associated with loss of forelimb field Tbx5 expression. These findings, plus the observation that WT mouse embryos treated with FGF8 fail to activate forelimb Tbx5, demonstrate that the forelimb bud initiation defects in RA-deficient mutants are due to excessive FGF8 activity in the trunk leading to a disruption of forelimb Tbx5 activation (Cunningham et al. 2013). This permissive model of RA action is supported by studies in zebrafish showing that forelimb bud initiation can be rescued in aldh1a2 mutants by introducing a heat-shock inducible dominant-negative FGF receptor (Cunningham et al. 2013). It is possible that RA may also function instructively to activate Tbx5 or some other gene in the forelimb field, but there are currently no genetic loss-of-function studies that support this model.

RA controls posterior body axis extension and somitogenesis

Bipotential neuromesodermal progenitors (NMPs) are cells in the caudal epiblast of vertebrate embryos that express both T (Brachyury) and Sox2; these progenitor cells undergo balanced differentiation to either presomitic mesoderm (expressing T and Tbx6) or spinal cord neuroectoderm (expressing Sox2 and Sox1) (Henrique et al. 2015). Although RA is not required for NMP establishment (Cunningham et al. 2016), loss of RA results in altered NMP differentiation with decreased Sox1 and Sox2 expression in cells that remain in the epithelial layer (prospective neural progenitors); loss of RA signaling also leads to increased Tbx6 expression in cells that migrate through the primitive streak via the gastrulation process to form mesodermal progenitors resulting in a small-somite defect (Cunningham et al. 2015).

In Aldh1a2 knockout embryos that completely lack RA activity, Tbx6 expression in presomitic mesoderm is increased; however, trunk somites are approximately half of their normal size, suggesting RA is required for proper condensation of migratory fibroblast-like presomitic mesodermal cells to form epithelial somites (Cunningham et al. 2015). Treatment of mouse Aldh1a2 knockouts with the FGF inhibitor SU5402 rescues trunk somite size, suggesting that the mechanism of RA action during somitogenesis is similar to that during forelimb initiation in which RA signaling represses caudal Fgf8; thus, excessive caudal FGF8 signaling evidently interferes with somitogenesis when Fgf8 is expressed too far anteriorly (Cunningham et al. 2015).

The mechanism of caudal Fgf8 repression requires a RARE located 4.1 kb upstream of Fgf8 that is required for caudal Fgf8 repression; this RARE is conserved in amniote embryos such as human, mouse, and chick but is not present near zebrafish fgf8 (Kumar & Duester 2014, Kumar et al. 2016). Interestingly, zebrafish embryos do not require RA for NMP differentiation or caudal fgf8 repression as loss of RA signaling does not affect body axis extension or somitogenesis (Berenguer et al. 2018).

The Fgf8 RARE functions in an RAR/RXR- and RA-dependent manner to recruit NCORs (NCOR1 and NCOR2) and polycomb repressive complex 2 (PRC2) that stimulate deposition of the repressive H3K27me3 chromatin mark near Fgf8 (Kumar & Duester 2014, Kumar et al. 2016). These observations provided the first in vivo evidence that RA signaling can directly repress a gene through a RARE silencer, plus these studies also demonstrated that NCOR can function ligand-dependently for gene repression.

RA regulates early heart patterning

In vertebrates, loss of RA results in a failure of heart tube looping resulting in defective formation of ventricular (anterior) and atrial (posterior) chambers (Niederreither et al. 2001, Keegan et al. 2005). The mechanism of RA signaling in the early heart involves RA-FGF8 antagonism as described above for somitogenesis and forelimb initiation (Ryckebusch et al. 2008, Sirbu et al. 2008, Sorrell & Waxman 2011). FGF8 signaling located in the anterior heart field is needed to establish ventricular identity (Pradhan et al. 2017). As loss of RA signaling results in posterior expansion of heat Fgf8 expression, RA represses Fgf8 to prevent expansion of anterior ventricular progenitors into the posterior region of the heart where atria develop. Thus, proper formation of forelimbs, somites, and heart all require RA repression of Fgf8, pointing out a major function of RA signaling during development (Cunningham & Duester 2015).

RA is required for hindbrain anteroposterior patterning

In mouse embryos undergoing the late stage of gastrulation, RA synthesis begins upon initial expression of Rdh10 and Aldh1a2 in the presomitic mesoderm; RA generated in the presomitic mesoderm is secreted and diffuses into the developing hindbrain as far anteriorly as rhombomere 3 (r3); the hindbrain initially forms eight rhombomeres (r1–r8) located along the anteroposterior axis (Sirbu et al. 2005). In the hindbrain, RA activates expression of the 3’-Hox genes (Hox1-4 groups) that are known to be essential for rhombomere formation and identity (Krumlauf 1993, Maden et al. 1996, Niederreither et al. 2000, Begemann et al. 2001). In the case of Hoxb1, RA signaling directly regulates expression through two RAREs, one that functions as an enhancer to activate expression in r4 and another RARE that functions as a silencer to repress expression in r3 and r5 (Marshall et al. 1994, Studer et al. 1994). Hnf1b (vhnf1), a repressor of Hoxb1, is activated by RA signaling in posterior hindbrain (as far anterior as r5) and spinal cord which also functions to repress Hoxb1 posterior to r4 (Hernandez et al. 2004, Sirbu et al. 2005).

RA control of spinal cord development

As mentioned above, bipotential NMPs undergo balanced differentiation to either spinal cord neuroectoderm or presomitic mesoderm in vertebrate embryos. Loss of RA in mouse or chick embryos results in decreased expression of Sox1/Sox2-expressing neural progenitors which delays differentiation of NMPs to spinal cord neuroectoderm (neural plate) (Cunningham et al. 2015, Henrique et al. 2015).

As the spinal cord further develops, RA also plays a role in differentiation of specific neural progenitors to motor neurons. After neural progenitors emerge from the caudal epiblast to form the spinal cord, they are exposed to RA derived from adjacent somites as well as Sonic hedgehog (SHH) generated in the floor plate of the ventral spinal cord. Together, RA and SHH activate Pax6 and Olig2 expression in ventral spinal cord progenitors to stimulate a motor neuron fate (Diez del Corral et al. 2003, Novitch et al. 2003, Molotkova et al. 2005, England et al. 2011). ChIP-seq studies comparing WT and Aldh1a2 knockouts have shown that no RARE is associated with activation of Pax6; instead, the requirement of RA signaling for Pax6 activation may proceed through several indirect mechanisms due to the ability of RA to directly activate Sox2 and Cdx1, both required for Pax6 expression, along with the ability of RA to directly repress caudal Fgf8 that prevents Pax6 expression in the caudal epiblast (Berenguer et al. 2020).

Function of RA during eye development

Eye development begins when the optic vesicles develop as out-pocketings of the forebrain, and soon afterward, the optic vesicles invaginate to form optic cups with a connection to the forebrain known as the optic stalk that will later form the optic nerve. Early RAR double knockout studies showed that although the optic vesicle forms and undergoes optic cup formation, RA is required afterward for further optic cup morphogenesis and proper formation of anterior eye structures adjacent to the optic cup such as cornea and eyelids (Lohnes et al. 1994); this topic is discussed in more detail below. In order to determine whether RA plays an earlier role in optic cup formation, RAR triple knockout studies could be performed (not yet reported), but for now one can rely on targeting RA-generating enzymes. During the optic vesicle stage, all three ALDH1A RA-generating enzymes are expressed (Molotkov et al. 2006). This triple redundancy initially hampered genetic loss-of-function studies to interrogate eye RA function; however, the discovery that Aldh1a1−/− mice survive as adults and are fertile (Fan et al. 2003) enabled the generation of Aldh1a1/Aldh1a2/Aldh1a3 triple knockout embryos that lack all RA signaling activity in the early optic field. Analysis of such triple knockouts revealed that although the optic vesicle forms, it does not properly invaginate to form the optic cup resulting in loss of the ventral portion of the optic cup (Molotkov et al. 2006).

Subsequent to optic cup formation, RA signaling is also required for eye morphogenetic processes that result in proper formation of anterior eye structures (cornea and eyelids) adjacent to the optic cup (retina). This later function of eye RA signaling was initially discovered by examination of RAR double knockouts (Lohnes et al. 1994) and also by analysis of the Rara/Rxra double knockout, thus revealing a requirement for RXR in eye morphogenesis (Kastner et al. 1994). During the late stage of eye RA signaling, RA is generated only by Aldh1a1 (optic cup dorsal retina) and Aldh1a3 (optic cup ventral retina) since expression of Aldh1a2 has ceased in the optic field (Molotkov et al. 2006). Although Aldh1a1 and Aldh1a3 single knockouts revealed no obvious eye defects due to redundancy, Aldh1a1/Aldh1a3 double knockouts exhibit severe eye defects during the late stage of morphogenesis (Matt et al. 2005, Molotkov et al. 2006). Aldh1a1/Aldh1a3 double knockouts demonstrated that RA generated in the optic cup is not required locally for development of the retina, nor for eye dorsoventral patterning as previously suggested from RA gain-of-function studies, but instead, RA generated in the dorsal and ventral retina diffuses outside of the optic cup to surrounding perioptic mesenchyme to regulate its migration around the optic cup during anterior eye formation (Matt et al. 2005, Molotkov et al. 2006); in the absence of RA signaling, there is excessive migration of perioptic mesenchyme resulting in ectopic corneal and eyelid tissue that secondarily exerts a mechanical force that distorts optic cup morphology. This late loss of RA signaling in the eye decreases expression of Pitx2 and Foxc1 in perioptic mesenchyme, which are known regulators of eye morphogenesis (Matt et al. 2005). Further studies revealed that after RA activates Pitx2 expression in the perioptic mesenchyme, Pitx2 then activates Dkk2 which functions to inhibit WNT signaling that limits migration of the perioptic mesenchyme (Kumar & Duester 2010).

Perspectives for future studies

Great progress has been made in understanding RA signaling; however, a reproducibility crisis exists (Duester 2017). As mentioned above, particularly with regard to the role of RA signaling during limb development, functions for RA based upon gain-of-function studies are often not supported by in vivo genetic loss-of-function studies likely due to off-target effects induced by RA treatment. Another issue is whether RA signaling may also act nongenomically to control processes other than nuclear gene transcription using a mechanism that involves cytoplasmic RARs that control mRNA translation or other nongenomic processes (Liou et al. 2005, Aoto et al. 2008). However, the nongenomic hypothesis has not been supported by genetic loss-of-function studies that remove endogenous RA, suggesting that pharmacological levels of RA may lead to nongenomic effects while normal levels of endogenous RA do not function nongenomically (Duester 2022). Thus, a focus on in vivo genetic loss-of-function studies is needed to bring consensus on how RA signaling normally functions during development or in adult tissues. Such studies have identified many tissues that require endogenous RA signaling for normal development as described above (Fig. 2). In reality, even though sometimes very difficult, genetic loss-of-function studies are needed to determine the normal function of any gene, protein, or molecule as gain-of-function studies (treatments with molecules/proteins or overexpression of genes) cannot provide this answer.

For future studies, genomic and epigenetic methods will be more effective than analyzing one gene at a time, thus allowing a global identification of RA-regulated genes and associated RAREs required for developmental processes. Identification of RA direct target genes can be achieved by comparing WT and RA knockout embryos (or specific tissues) using RNA-seq and ChIP-seq to identify genes with significant decreases or increases in expression that also have RA-regulated changes in deposition of the gene activation mark H3K27ac or the gene repression mark H3K27me3 located near RAREs (Berenguer et al. 2020). Direct RA target genes identified in this manner can be subjected to knockout studies to determine if they are required for specific developmental processes, and many times such genes will be found to be essential for mediating the pathways used by RA signaling to control development. This basic knowledge will improve strategies to prevent or treat developmental defects.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This work was funded by the National Institutes of Health (National Eye Institute) grant R01 EY031745 (G D) and the National Institutes of Health (National Institute of Arthritis and Musculoskeletal and Skin Diseases) grant R56 AR067731 (G D).

Data availability statement

No data is provided in this report.

Acknowledgements

A special thanks to Pierre.

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    Figure 1

    Mechanism of retinoic acid (RA) synthesis, RA degradation, and RA signaling. Retinol is transported in the blood via retinol-binding protein-4 (RBP4) which makes it available to all cells of the body. In embryos, retinol is metabolized to the intermediate metabolite retinaldehyde by retinol dehydrogenase-10 (RDH10) and then retinaldehyde is metabolized to RA by one of three aldehyde dehydrogenase-1A (ALDH1A) enzymes. Retinaldehyde can also be converted back to retinol by dehydrogenase/reductase-SDR family member 3 (DHRS3). RA can be degraded by one of three CYP26 enzymes to limit RA activity. RA can act locally or be released from cells in which it is generated and travel as a signal to other cells. RA enters the nucleus and binds to one of three RA receptors (RARs) that form a heterodimer complex with one of three retinoid X receptors (RXRs). RAR/RXR heterodimers bind a repeated DNA sequence known as an RA response element (RARE) whose consensus sequence is AGGTCA (N1,2,5) AGGTCA where N refers to 1, 2, or 5 random nucleotides. RA directly controls transcription by serving as a ligand for RARs. Binding of RA to RARs results in a conformational shift in RAR that alters its ability to bind nuclear receptor coactivators (NCOA) or nuclear receptor corepressors (NCOR) that control further downstream events in transcriptional activation or repression.

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    Figure 2

    Retinoic acid (RA) signaling target tissues during early embryogenesis. Early mouse embryos that have 1–15 somites exhibit RA signaling activity (shown in green) in the developing eye (optic vesicle) and throughout the posterior body axis (developing trunk); the anterior border of RA signaling activity in the trunk occurs in the middle of the hindbrain, and the posterior border occurs at the junction with the caudal epiblast that contains neuromesodermal progenitors (NMPs) that differentiate to form either spinal cord or somites; the diagram shows a dorsal view of a 7-somite mouse embryo. Tissues that require RA signaling for normal development include the optic vesicle, posterior hindbrain, somites, spinal cord, forelimbs, NMPs, and heart that is located underneath on the ventral side of the embryo.

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