Prenatal retinoic acid exposure reveals candidate genes for craniofacial disorders

Abstract

Syndromes that display craniofacial anomalies comprise a major class of birth defects. Both genetic and environmental factors, including prenatal retinoic acid (RA) exposure, have been associated with these syndromes. While next generation sequencing has allowed the discovery of new genes implicated in these syndromes, some are still poorly characterized such as Oculo-Auriculo-Vertebral Spectrum (OAVS). Due to the lack of clear diagnosis for patients, developing new strategies to identify novel genes involved in these syndromes is warranted. Thus, our study aimed to explore the link between genetic and environmental factors. Owing to a similar phenotype of OAVS reported after gestational RA exposures in humans and animals, we explored RA targets in a craniofacial developmental context to reveal new candidate genes for these related disorders. Using a proteomics approach, we detected 553 dysregulated proteins in the head region of mouse embryos following their exposure to prenatal RA treatment. This novel proteomic approach implicates changes in proteins that are critical for cell survival/apoptosis and cellular metabolism which could ultimately lead to the observed phenotype. We also identified potential molecular links between three major environmental factors known to contribute to craniofacial defects including maternal diabetes, prenatal hypoxia and RA exposure. Understanding these links could help reveal common key pathogenic mechanisms leading to craniofacial disorders. Using both in vitro and in vivo approaches, this work identified two new RA targets, Gnai3 and Eftud2, proteins known to be involved in craniofacial disorders, highlighting the power of this proteomic approach to uncover new genes whose dysregulation leads to craniofacial defects.

Systemic vitamin intake impacting tissue proteomes

“…Proteomic studies have established dietary vitamin status as a regulator of tissue protein abundances. The regulatory feedback between vitamin status and protein expression is highlighted by findings where the abundances of proteins directly related to the vitamin are impacted by systemic intake of that vitamin, including: abundances of proteins related to craniofacial development and neural crest processes are impacted in an established maternal retinoic acid toxicity-driven model of craniofacial birth defects [30], deficiency in their riboflavin cofactor is accompanied by reduced abundances of acyl-coenzyme A dehydrogenases and accumulation of the enzymes' substrates [51], treatment with the active form of vitamin D is accompanied by increased expression of calcium binding proteins [184], and vitamin E supplementation impacts proteins related to redox processes [206] (Table 1). However, the literature in this field is sparse and, in all likelihood, the vast majority of vitamin-status to protein abundance relationships are undescribed; especially considering the void in the literature for several vitamins.…”

“…An iTRAQ or TMT set is a pool of samples that are run on the LC-MS/MS concurrently. Because the samples in a set are all analyzed simultaneously, a set's contribution to the total number of unique proteins is similar to that of a single sample Study Year Platform Tissue # Proteins Identified # Samples [ 30 ] 2018 Orbitrap mouse embryo heads group 1: 313 total group 2: 372 total 2 groups [ 31 ] 2018 Triple-TOF gerbil plasma 109 total 30 [ 32 , 185 , 187 ] 2015 Orbitrap human plasma 4,705 total 589/set 72 iTRAQ sets [ 42 ] 2018 QTOF rat thalami 1,440 total 6 x 3 tech. reps. [ 51 ] 2017 QTOF duck livers 1,749 total 3 iTRAQ sets [ 52 ] 2019 QTOF fetal duck livers 3,801 total 1 iTRAQ set [ 91 ] 2019 Triple-TOF rat hippocampi 4,807 total 2 iTRAQ sets [ 120 ] 2015 Orbitrap human follicular fluid 227 total 1 TMT set [ 121 ] 2014 QTOF mouse intestine 2,039 total 10 [ 156 ] 2019 Orbitrap pig plasma 534 total 45 ...…”

“…Vitamin A Creation of a model of craniofacial disorders induced by prenatal retinoic acid exposure is reported to impact protein abundances whose functions are associated with neural crest processes [30]. Plasma carotenoids abundances are reported to be associated with plasma proteins of diverse functions in Nepalese children, potentiating development of inexpensive assays to predict carotenoids deficiency [32].…”

“…In an orbitrap-based study of mouse embryo heads, toxic levels of prenatal retinoic acid exposure intended to model an established risk factor for craniofacial birth defects are reported to induce abundance alterations in proteins associated with craniofacial development and neural crest processes [ 30 ]. In a parallel triple-TOF-based study of gerbil plasma and 2DGE-MS-based study of gerbil liver and white adipose tissue, a few dozen protein abundances linked to a handful of biological processes are reported to respond to dietary retinol, β-carotene, lutein, or lycopene; though process or pathway enrichment analyses are not reported.…”

Role of Retinoic Acid Signaling, FGF Signaling and Meis Genes in Control of Limb Development

“…In the Cyp26b1 knockout, Meis1/2 expression expands into the distal limb, similar to chick RA-treatment studies, but forelimb and hindlimb buds are truncated along the entire proximodistal axis, which is inconsistent with RA functioning to induce proximal identity [ 51 ]. In addition, in Cyp26b1 knockouts, the presence of endogenous RA in distal limbs where it should not normally exist results in a phenotype similar to exogenous RA teratogenesis with increased apoptosis and a block in chondrogenic differentiation, particularly to form the intricate skeletal structures of distal tissues such as hand/foot [ 53 , 54 ] or craniofacial structures [ 55 ]. Thus, the presence of endogenous RA in the proximal limb does not necessarily correlate with a designed function in proximodistal patterning of Meis1/2 expression, but instead may simply indicate diffusion overflow from the trunk where RA is required for body axis formation and forelimb initiation, with this RA being neither necessary nor harmful to proximal limb development.…”