Retinoid signaling in skeletal development: Scoping the system for predictive toxicology

Abstract

All-trans retinoic acid (ATRA), the biologically active form of vitamin A, is instrumental in regulating the patterning and specification of the vertebrate embryo. Various animal models demonstrate adverse developmental phenotypes following experimental retinoid depletion or excess during pregnancy. Windows of vulnerability for altered skeletal patterning coincide with early specification of the body plan (gastrulation) and regional specification of precursor cell populations forming the facial skeleton (cranial neural crest), vertebral column (somites), and limbs (lateral plate mesoderm) during organogenesis. A common theme in physiological roles of ATRA signaling is mutual antagonism with FGF signaling. Consequences of genetic errors or environmental disruption of retinoid signaling include stage- and region-specific homeotic transformations to severe deficiencies for various skeletal elements. This review derives from an annex in Detailed Review Paper (DRP) of the OECD Test Guidelines Programme (Project 4.97) to support recommendations regarding assay development for the retinoid system and the use of resulting data in a regulatory context for developmental and reproductive toxicity (DART) testing.

Keywords: Developmental toxicity; Retinoid signaling; Skeletal development.

Figure 1.. Retinoid signaling during morphogenesis of the craniofacial skeleton.

Functional inactivation of Rdh10 in mouse manifests in severe anterior defects (facial malformation, ear and eye deficiency, and loss of forelimb) due to inability of the embryo to metabolically convert retinol → retinoic acid (Rhinn, Schuhbaur et al. 2011). ATRA and FGF8 signals depicted by red and green bars, respectively. Endogenous ATRA is required, but at different threshold levels, for normal development of the midface and branchial arches. Positional information of premigratory hindbrain CNC cells is determined by threshold ATRA levels coming from the paraxial mesoderm/occipital somites. ATRA signaling through RAR/RXR 'posteriorizes' the hindbrain and is essential for specification of rhombomeres r5–r8 (5- to 11-somite stage). ATRA also specifies pharyngeal endoderm, which in turn secretes factors that create an environment permissive to (but not required for) postotic CNC migration. Abbreviations: ov, otic vesicle; os, occipital somite 1–5; cs1, first cervical somite; r1–r8, rhombomeres 1–8; FNP, frontonasal process; BA, branchial arches 1–6; PS, primitive streak. Dotted lines indicate midbrain/hindbrain and hindbrain/spinal cord junctions.

Figure 2.. Retinoid signaling in metameric organization of the paraxial mesoderm during somitogenesis.

A molecular oscillator (clock) delivers a periodic signal controlling somite production from the presomitic mesoderm (PSM). During axis elongation the signal is displaced posteriorly by a system of traveling signaling gradients (wavefront) that depends on RALDH2 in newly formed somites (rostral) and FGF8 coming posteriorly (Iimura, Denans et al. 2009, Rhinn and Dolle 2012, Shimozono, Iimura et al. 2013). ATRA antagonizes the FGF8-mediated growth front (based on (Strate, Min et al. 2009). Cyp26a1 expression is highest posteriorly; functional inactivation manifests as severe posterior defects in the mouse (loss of hindlimb, caudal regression) due to premature cessation of posterior elongation (Rhinn and Dolle 2012). ATRA signaling modulates somite size in the trunk region, but not the tail region. ATRA thresholds control bilateral symmetry of the left and right somite columns and determine vertebral identity through RARE-dependent Hox genes (gastrulation). Retinoid excess disrupts PSM growth and caudal extension in the posterior region.

Figure 3.. Ontogeny of Hox-mediated axial patterning and its regulation by retinoid signaling.

Hox-patterning is decoded during somitogenesis in spatial and temporal waves of transcription to determine relative positions in the vertebral column at which the paralogs are expressed during development [from (Luo, Rhie et al. 2019). Emergence of the somitic column is depicted from PSM based on the ‘posterior dominance’ model [redrawn from (Iimura and Pourquie 2007). Somite colors reflect chromosomally linked Hox genes (simple representation of only 3 genes) in temporal colinear expression. Newly formed somites are morphologically similar across the trunk but are fixed with regards to future vertebral identity. Normal patterning (left) and phenotype with Hox gene inactivation (right) and potential consequences of ATRA excess imposed during late gastrulation (GD 7.3 mouse) and ATRA deficiency.

Figure 4.. ATRA signaling during development of the fetal appendicular skeleton.

TOP: Mouse forelimb from early outgrowth (A, GD 9.5) to precartilage induction (B, GD10.5) to precartilage pattern (C, GD 11.5); corresponding stages in the hindlimb are delayed by a half day. The precartilage pattern is laid down in proximo-distal fashion for the stylopod (humerus, femur), zeugopod (radius-ulna, tibia-fibula), and autopod (digits of fore- and hind paw). BOTTOM: permissive ATRA signaling on proximo-distal patterning (from Uzkudun, Marcon et al. 2015). ATRA (from RALDH2) enters the proximal limb-bud and is degraded distally by CYP26B1 induced by FGF8; cells leaving the ATRA-free distal mesenchyme have positional values determining regional identity for stylopod (Meis), zeugopod (Hoxa11), and autopod (Hoxa13). Gradients represent two-signal model for ATRA and FGF8 signal inputs; a one-signal model (not shown) was also simulated wherein the time exposed to FGF8 alone determined regional identity.

Figure 5.. Framework and examples of potential AOPs for skeletal dysmorphogenesis linked to disruption of retinoid signaling.

MIE, Molecular Initiating Event; KE, Key Events upstream to downstream; AO, Adverse Outcome. MIEs may include, for example: vitamin A deficiency, chemical inhibitors of RALDH2 or CYP26 enzymatic activity/expression; pharmacological agonists/antagonists of RAR or partner receptor signaling pathways (e.g., RXR/PPARγ); agents that modulate RAR/RXR binding to RARE sites. Subcellular KEs may be reflected in critical imbalances to local ATRA concentration or threshold responses leading to changes in Hox patterning and other molecular determinants of cell lineages. Cellular KEs entail developmental programming of undifferentiated progenitors of body axis determinants, depending on the stage of gestation and region of the embryo affected. Tissue KEs reflect the collective behavior of target cells directed by heterotypic interactions for rudimentary organs. AOs reflect the phenotypes’ resulting from stage and positional alterations in the fetal skeleton.

Figure 6.. Case examples for Class Distribution.

Distribution from chemical hits (n=261) having AC50 < 2 μM in one or more of the 8 ToxCast assays (Baker, Boobis et al. 2018). Each assay target is indicated with the number of chemical his registered in the EPA CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard, last accessed October, 2020). CYP1A1: NVS_ADME_hCYP1A1. RARs: ATG_RARa_TRANS_up, ATG_RARb_TRANS_up, ATG_RARg_TRANS_up. RXRs: ATG_RXRa_TRANS_up, ATG_RXRb_TRANS_up, ATG_RXRg_TRANS_up. DR5: ATG_DR5_CIS_up. As might be expected the DR5 lights up several potential RAR/RXR combinations. First group: vitamin A (retinol) and retinoid ligands (ATRA/RAR, Bexarotene/RXR) references. Second group: Triazole effects on biochemical activity of CYP1A1 as a surrogate for CYP26 isoforms; malformations, vertebral transformations, and caudal regression are linked to CYP26 inhibition (Menegola, Broccia et al. 2001, Kamata, Shiraishi et al. 2008, Tonk, Pennings et al. 2015). Third group: several organochlorine pesticides of a persistent nature have weak RARg-agonist activity and can transactivate retinoid-responsive genes (e.g., CYP26A1) via RARE (Lemaire, Balaguer et al. 2005, Kamata, Shiraishi et al. 2008). Fourth group: several organotin biocides are known to preferentially bind RXRs with nM affinity, but forms a non-permissive RAR/RXR heterodimer (Grun, Watanabe et al. 2006, Brtko and Dvorak 2015).