The Carnegie stage 9 (CS9) embryo is a pivotal phase signifying the conclusion of gastrulation and the onset of early organogenesis, crucial for initiating major organ system development. Utilizing spatial transcriptomics, we analyzed an intact CS9 human embryo in a spatially detailed manner. Through the examination of 75 transverse cryosections, we digitally reconstructed a 3D model, allowing us to identify diverse cell types, including those from brain and spine regions, the primitive gut tube, distinct somite formation stages, somatic mesoderm, splanchnic mesoderm, etc. Notably, we observed two distinct trajectories of hindbrain development, pinpointed the isthmic organizer at the midbrain-hindbrain boundary, delineated the bi-layered structure of neuromesodermal progenitor (NMP) cells, and described the early aorta formation and primordial germ cells (PGCs) presence in the aorta-gonad-mesonephros (AGM) region. This study provides key insights into the transcriptomic and spatial intricacies shaping the human body plan.

Genetic studies in mice have demonstrated that retinoic acid receptor alpha (RARα) deficiency leads to male infertility without affecting overall viability, suggesting that pharmacological inhibition of this receptor could be a viable contraceptive strategy. This review describes the use of experimental approaches to develop RARα-selective antagonists for male contraception. Initial studies with BMS-189453, a pan-RAR antagonist, showed significant testicular degeneration and reversible infertility in mice. The search for RARα-specific antagonists led to the development of YCT-529, a potent and selective RARα antagonist with favorable pharmacokinetics. YCT-529 demonstrated excellent in vivo efficacy in inhibiting spermatogenesis and inducing infertility in mice, with fertility recovery following drug discontinuation. YCT-529 is now in clinical development as a candidate for male contraception.

In the complex process of skeletal development, the significance of m6A RNA methylation-a predominant form of RNA modification-has not been fully explored. This review discuss how m6A RNA methylation plays an important, though not yet fully understood, role in regulating skeletal formation. It examines how m6A influences key signaling pathways essential for skeletal development and homeostasis, suggesting various possible interactions between m6A methylation and these critical pathways. While the exact mechanisms for many of these interactions remain to be elucidated, m6A RNA methylation is anticipated to be a key emerging regulator in skeletal structure development across vertebrates. Highlighting the need for further research, this overview provides an in-depth look at the potential regulatory interactions of m6A RNA methylation within skeletal system. Uniquely, this review is the most comprehensive compilation of evidence linking components of m6A RNA methylation to signaling pathways involved in skeletogenesis.

Inner ear organoid development-from germ layer to otocyst formation-relies on timed chemical cues to recapitulate major signals in vivo. In contrast, later stages of differentiation-from otic vesicle (OV) to organoid formation-are self-guided, even though these stages are modulated by several key morphogens in vivo. We sought to elucidate additional morphogens that might improve culture efficiency and influence cell fate decisions. Using a whole-transcriptomic approach, we identified major differences in native and stem cell-derived OVs related to anterior-posterior patterning and retinoic acid (RA) signaling. Increasing the level of RA during OV formation in these cultures modulated organoid efficiency, increased nonsensory markers, decreased sensory markers, and decreased hair cell production. The organoid culture platform mimics the exquisite RA sensitivity found in normal inner ear development and may help identify RA-responsive genes driving organogenesis and cell fate specification.

Large-scale GWAS studies have uncovered hundreds of genomic loci linked to facial and brain shape variation, but only tens associated with cranial vault shape, a largely overlooked aspect of the craniofacial complex. Surrounding the neocortex, the cranial vault plays a central role during craniofacial development and understanding its genetics are pivotal for understanding craniofacial conditions. Experimental biology and prior genetic studies have generated a wealth of knowledge that presents opportunities to aid further genetic discovery efforts. Here, we use the conditional FDR method to leverage GWAS data of facial shape, brain shape, and bone mineral density to enhance SNP discovery for cranial vault shape. This approach identified 120 independent genomic loci at 1% FDR, nearly tripling the number discovered through unconditioned analysis and implicating crucial craniofacial transcription factors and signaling pathways. These results significantly advance our genetic understanding of cranial vault shape and craniofacial development more broadly.

Vitamin A and its primary active derivative, all-trans retinoic acid (RA), are endogenous signaling molecules essential for numerous biological processes, including cell proliferation, differentiation, and immune modulation. Owing to its differentiation-inducing effect, RA was the first differentiating agent approved for the clinical treatment of acute myeloid leukemia. While the classical mechanisms of RA signaling involve nuclear receptors, such as retinoic acid receptors (RARs), emerging evidence suggests that RA also engages in non-covalent and covalent interactions with a broader range of proteins. However, tools for thoroughly characterizing these interactions have been lacking, and a comprehensive understanding of the landscape of RA-modified and RA-interacting proteins remains limited. Here, we report the development of two RA-based chemical reporters, RA-yne and RA-diazyne, to profile RA-modified and RA-interacting proteins, respectively, in live cells. RA-yne features a clickable alkyne group for metabolic labeling of RA-modified proteins, while RA-diazyne incorporates a photoactivatable diazirine and an alkyne handle for crosslinking and capturing RA-interacting proteins. Using quantitative proteomics, we demonstrate the high-throughput identification of these proteins, revealing that non-covalent interactions are more prevalent than covalent modifications. Our global profiling also uncovers a large number of RA-interacting proteins mainly enriched in pathways related to mitochondrial processes, ER homeostasis, and lipid metabolism. Overall, this work introduces new RA-derived chemical reporters, expands the resource for studying RA biology, and enhances our understanding of RA-associated pathways in health and disease.

Vitamin A is an essential nutrient crucial to ensuring proper mammalian embryonic development. β-Carotene is the most prevalent form of vitamin A in food that, when transferred in its intact form from mother to the developing tissues, can serve as an in situ source of retinoic acid, the active form of vitamin A. We have previously provided evidence that the maternal-fetal transfer of β-carotene across the placenta is mediated by lipoproteins and that β-carotene itself regulates placenta lipoprotein biogenesis by means of its derivatives β-apo-10'-carotenoids and retinoic acid. These metabolites exert antagonistic transcriptional activity on placental microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB), two key players of lipoprotein biosynthesis. Here, we analyzed the time-dependency of this regulation over the course of 24 h upon a single maternal administration of β-carotene. We also tested the hypothesis that the transcriptional repressor intestine-specific homeobox (ISX) plays a role in the regulation of Mttp in placenta. We observed that ISX is expressed in placenta of mouse dams and is regulated by β-carotene availability. Furthermore, we demonstrated that the absence of Isx disrupts the β-carotene-mediated regulation of placental MTP. We also showed that this mechanism is organ-specific, as it was not observed in enterocytes of the intestine, a major place of Isx expression. Therefore, we identified ISX as a "master" regulator of a placental β-carotene-dependent transcriptional regulatory cascade that fine-tunes the flux of provitamin A carotenoid towards the developing fetus.

Inhibiting the androgen receptor (AR) is effective for treatment of advanced prostate cancers because of their AR-dependent luminal epithelial cell identity. Tumors progress during therapy to castration-resistant prostate cancer (CRPC) by restoring AR signaling and maintaining luminal identity or by converting through lineage plasticity to a neuroendocrine (NE) identity or double-negative CRPC (DNPC) lacking luminal or NE identities. Here, we show that DNPC cells express genes defining basal, club, and hillock epithelial cells from benign prostate. We identified KLF5 as a regulator of genes defining this mixed basal, club, and hillock cell identity in DNPC models. KLF5-mediated upregulation of RARG uncovered a DNPC sensitivity to growth inhibition by retinoic acid receptor agonists, which down-regulated KLF5 and up-regulated AR. These findings offer CRPC classifications based on prostate epithelial cell identities and nominate KLF5 and RARG as therapeutic targets for CRPC displaying a mixed basal, club, and hillock identity.

Myopia, or near-sightedness, is rapidly growing in prevalence, with significant long-term implications for ocular health. There is thus great impetus to better understand molecular signaling pathways leading to myopia. We and others have reported that all-trans retinoic acid (atRA) is involved in myopigenic signaling, yet the understanding of how atRA is transported and exerts a myopigenic influence is poor. Here we measured the concentrations of atRA in the serum in wild-type C57BL/6 mice under control conditions and after atRA feeding, previously shown to induce myopia. We also developed a mathematical model that describes fluid fluxes and the advective-diffusive transport of atRA in choroid and sclera, including atRA synthesis in the choriocapillaris, atRA degradation by scleral cells, and binding of atRA to the carrier protein serum albumin. This model, developed for both mice and humans, showed that atRA produced in the choriocapillaris was able to permeate well into the sclera in both mice and humans at biologically-relevant concentrations, and that atRA feeding greatly increased tissue levels of atRA across both the choroid and sclera. We were also able to identify which parameters most influence atRA concentration in ocular tissues, guiding future experimental work. Our findings support atRA's role in myopigenic signaling.

Palatogenesis is a complex developmental process requiring temporospatially coordinated cellular and molecular events. The following review focuses on genetic, epigenetic, and environmental aspects directing palatal formation and their implication in orofacial clefting genesis. Essential for palatal shelf development and elevation (TGF-β, BMP, FGF, and WNT), the subsequent processes of fusion (SHH) and proliferation, migration, differentiation, and apoptosis of neural crest-derived cells are controlled through signaling pathways. Interruptions to these processes may result in the birth defect cleft lip and/or palate (CL/P), which happens in approximately 1 in every 700 live births worldwide. Recent progress has emphasized epigenetic regulations via the class of non-coding RNAs with microRNAs based on critically important biological processes, such as proliferation, apoptosis, and epithelial-mesenchymal transition. These environmental risks (maternal smoking, alcohol, retinoic acid, and folate deficiency) interact with genetic and epigenetic factors during palatogenesis, while teratogens like dexamethasone and TCDD inhibit palatal fusion. In orofacial cleft, genetic, epigenetic, and environmental impact on the complex epidemiology. This is an extensive review, offering current perspectives on gene-environment interactions, as well as non-coding RNAs, in palatogenesis and emphasizing open questions regarding these interactions in palatal development.

The enteric nervous system (ENS) is formed from vagal neural crest cells (NCC), which generate most of the neurons and glia that regulate gastrointestinal function. Defects in the migration or differentiation of NCC in the gut can result in gastrointestinal disorders such as Hirschsprung disease (HSCR). Although mutations in many genes have been associated with the etiology of HSCR, a significant proportion of affected individuals have an undetermined genetic diagnosis. Therefore, it's important to identify new genes, modifiers and environmental factors that regulate ENS development and disease. Rdh10 catalyzes the first oxidative step in the metabolism of vitamin A to its active metabolite, RA, and is therefore a central regulator of vitamin A metabolism and retinoic acid (RA) synthesis during embryogenesis. We discovered that retinol dehydrogenase 10 (Rdh10) loss-of-function mouse embryos exhibit intestinal aganglionosis, characteristic of HSCR. Vagal NCC form and migrate in Rdh10 mutant embryos but fail to invade the foregut. Rdh10 is highly expressed in the mesenchyme surrounding the entrance to the foregut and is essential between E7.5-E9.5 for NCC invasion into the gut. Comparative RNA-sequencing revealed downregulation of the Ret-Gdnf-Gfrα1 gene signaling network in Rdh10 mutants, which is critical for vagal NCC chemotaxis. Furthermore, the composition of the extracellular matrix through which NCC migrate is also altered, in part by increased collagen deposition. Collectively this restricts NCC entry into the gut, demonstrating that Rdh10-mediated vitamin A metabolism and RA signaling pleiotropically regulates the NCC microenvironment during ENS formation and in the pathogenesis of intestinal aganglionosis.

Tooth/skeletal dysplasia, such as hypophosphatasia (HPP), has been extensively studied. However, there are few definitive treatments for these diseases owing to the lack of an in vitro disease model. Cells differentiated from patient-derived induced pluripotent stem cells (iPSCs) demonstrate a pathological phenotype. This study aimed to establish a method for fabricating hard tissue-forming cells derived from human iPSCs (hiPSCs) for the pathological analysis of tooth/skeletal dysplasia. Healthy (HLTH) adult-derived hiPSCs were cultured in a hard tissue induction medium (HM) with or without retinoic acid (RA) under 3D culture conditions, and mineralization and expression of dentinogenesis- and osteogenesis-related markers in 3D hiPSC constructs were evaluated. hiPSCs derived from patients with hypophosphatasia were also cultured in HM with RA. HLTH-derived hiPSCs formed mineralized 3D constructs and showed increased expression of dentinogenesis- and osteogenesis-related markers; addition of RA promoted the expression of these markers in hiPSC constructs. HPP-derived hiPSC constructs showed lower mineralization and expression of dentinogenesis- and osteogenesis-related markers than HLTH-derived hiPSCs, indicating an impaired ability to differentiate into odontoblasts and osteoblasts. This method for fabricating 3D hiPSC constructs allows for simultaneous assessment of dentinogenesis and osteogenesis, with HPP-derived hiPSC constructs recapitulating pathological phenotypes.

Retinoids are metabolic derivatives of vitamin A and play crucial roles in the regulation of various tissues and organs during prenatal and postnatal development. Active retinoids, like all-trans-retinoic acid, are synthesized in the cytoplasm and subsequently interact with nuclear retinoic acid receptors (RARα, RARβ, and RARγ) to enhance transcription of specific genes. In the absence of retinoids, RARs can still bind to response elements of target genes but repress their transcription. Chondrogenic cell differentiation and cartilage maturation in the growth plate require the absence of retinoid signaling and transcriptional repression by unliganded RARs. This led to the hypothesis that synthetic retinoid agonists may be pharmacological agents to inhibit those cellular processes and counter the excessive formation of cartilage and bone in conditions like heterotopic ossification (HO). HO can be instigated by diverse culprits including trauma, invasive surgeries, inflammatory disorders, or genetic conditions. One such genetic disease is fibrodysplasia ossificans progressiva (FOP), a rare disorder driven by activating mutations in the ACVR1 gene. Patients with FOP have severe and progressive HO formation in soft tissues, leading to extensive permanent loss of mobility and increased mortality. Synthetic retinoid agonists selective for RARα or RARγ showed efficacy against injury-induced and genetic HO in mouse models. The RARγ agonists showed the highest effectiveness, with palovarotene being selected for clinical trials in patients with FOP. Post hoc analyses of phase II and phase III clinical trials showed that palovarotene has significant disease-modifying effects for FOP, but with significant risks such as premature growth plate closure in some younger subjects. This review provides an overview of retinoid and RAR roles in skeletal development and discusses the identification of palovarotene as a potential FOP therapy, the clinical data supporting its regulatory approval in some countries, and the potential applications of this drug for other relevant disorders besides FOP.

Cardiac development is a complex and intricate process involving numerous molecular signals and pathways. Researchers have explored cardiac development through a long journey, starting with early studies observing morphological changes and progressing to the exploration of molecular mechanisms using various molecular biology methods. Currently, advancements in stem cell technology and sequencing technology, such as the generation of human pluripotent stem cells and cardiac organoids, multi-omics sequencing, and artificial intelligence (AI) technology, have enabled researchers to understand the molecular mechanisms of cardiac development better. Many molecular signals regulate cardiac development, including various growth and transcription factors and signaling pathways, such as WNT signaling, retinoic acid signaling, and Notch signaling pathways. In addition, cilia, the extracellular matrix, epigenetic modifications, and hypoxia conditions also play important roles in cardiac development. These factors play crucial roles at one or even multiple stages of cardiac development. Recent studies have also identified roles for autophagy, metabolic transition, and macrophages in cardiac development. Deficiencies or abnormal expression of these factors can lead to various types of cardiac development abnormalities. Nowadays, congenital heart disease (CHD) management requires lifelong care, primarily involving surgical and pharmacological treatments. Advances in surgical techniques and the development of clinical genetic testing have enabled earlier diagnosis and treatment of CHD. However, these technologies still have significant limitations. The development of new technologies, such as sequencing and AI technologies, will help us better understand the molecular mechanisms of cardiac development and promote earlier prevention and treatment of CHD in the future.

Recent research efforts in endocrine disruption have focused on evaluating non-EATS (estrogen, androgen, thyroid, and steroidogenesis) pathways. Retinoid signaling disruption is noteworthy because of its teratogenic effects and environmental relevance. However, current environmental risk assessments are limited in their ability to evaluate impacts on individuals and populations. This study characterizes an Adverse Outcome Pathway (AOP) network linking retinoid signaling disruption to teratogenicity and survival in zebrafish. We identified Retinoic Acid Receptor (RAR) overactivation as the molecular initiating event leading to key events including craniofacial (CFM) and tail (TM) malformations, posterior swim bladder (SB) non-inflation, impaired swimming performance, and reduced feeding, ultimately resulting in decreased survival. Our study (1) determines critical sensitivity windows for CFM, posterior SB non-inflation, and TM, (2) provides quantitative measurements for CFM and TM, and (3) defines impacts on higher biological levels including food ingestion, swimming, and survival. Results show that all-trans retinoic acid (ATRA) induces strong teratogenic effects with sensitivity windows between 4 and 48 h post fertilization (hpf) for CFM, TM, and posterior SB non-inflation. TM is the most sensitive indicator, with EC50 of 0.2 - 0.26 µg/L across exposure windows 4-48, 4-72, 4-96, and 4-120 hpf. Besides inducing known malformations, ATRA impaired posterior SB inflation with EC50 of 1 - 1.21 µg/L across the same exposure windows. ATRA exposure (1 µg/L) resulted in 50 % food ingestion inhibition at 7 days post fertilization (dpf) and 10 % survival at 14 dpf. This study provides a regulatory-relevant framework linking developmental effects to population outcomes, highlighting ecological risks and needs for improved risk assessments.

Aquatic environments, including marine and freshwater ecosystems, are vital for ecological balance and biodiversity. The rising global demand for aquaculture products necessitates increased production, with intensified aquaculture practices posing significant environmental risks. This review explores the pathways through which chemical pollutants, heavy metals, pharmaceuticals, and environmental stressors induce teratogenic effects in aquatic species. The review highlights the impact of pesticide include triazine herbicides, organophosphate and organochlorine insecticides, and carbamates on aquatic life, emphasizing their interference with endocrine systems and developmental processes. Heavy metals like mercury, lead, cadmium, arsenic, and chromium are noted for their persistence and bioaccumulative properties, disrupting cellular and hormonal functions. Pharmaceuticals, including NSAIDs, antibiotics, and chemotherapeutic agents, exert teratogenic effects by disrupting physiological and developmental pathways. Environmental stressors includes temperature fluctuations, salinity variations, pH changes, and oxygen level imbalances exacerbate the teratogenic impact of pollutants. This review highlights the importance of comprehensive environmental management and understanding these complex interactions is essential for formulating efficient strategies to safeguard the effective measures to protect aquatic ecosystems and the biodiversity.

During embryogenesis, homogenous groups of cells self-organize into stereotypic spatial and temporal patterns that make up tissues and organs. These emergent patterns are controlled by transcription factors and secreted signals that regulate cellular fate and behaviors through intracellular regulatory circuits and cell-cell communication circuits. However, the principles of these circuits and how their properties are combined to provide the spatio-temporal properties of tissues remain unclear. Here we develop a framework to explore building-block circuits of developmental programs. We use single-cell gene expression data across developmental stages of the human intestine to infer the key intra- and inter-cellular circuits that control developmental programs. We study how these circuits are joined into higher-level hyper-motif circuits and explore their emergent dynamical properties. This framework uncovers design principles of developmental programs and reveals the rules that allow cells to develop robust and diverse patterns.

All-trans retinoic acid (ATRA) signaling is essential in numerous different biological contexts. This review highlights the diverse roles of ATRA during development, function, and diseases of the pancreas. ATRA is essential to specify pancreatic progenitors from gut tube endoderm, endocrine and exocrine differentiation, and adult islet function. ATRA concentration must be carefully regulated during the derivation of islet-like cells from human pluripotent stem cells (hPSCs) to optimize the expression of key pancreatic transcription factors while mitigating adverse and unwanted cell-types in these cultures. The ATRA pathway is integral to the pancreas and here we will present selected studies from decades of research that has laid the essential groundwork for ongoing projects dedicated to unraveling the complexities of ATRA signaling in the pancreas.

Nr2f transcription factors (TFs) are conserved regulators of vertebrate atrial cardiomyocyte (AC) differentiation. However, little is known about the mechanisms directing Nr2f expression in ACs. Here, we identified a conserved enhancer 3' to the nr2f1a locus, which we call 3'reg1-nr2f1a (3'reg1), that can promote Nr2f1a expression in ACs. Sequence analysis of the enhancer identified putative Lef/Tcf and Foxf TF binding sites. Mutation of the Lef/Tcf sites within the 3'reg1 reporter, knockdown of Tcf7l1a, and manipulation of canonical Wnt signaling support that Tcf7l1a is derepressed via Wnt signaling to activate the transgenic enhancer and promote AC differentiation. Similarly, mutation of the Foxf binding sites in the 3'reg1 reporter, coupled with gain- and loss-of-function analysis supported that Foxf1 promotes expression of the enhancer and AC differentiation. Functionally, we find that Wnt signaling acts downstream of Foxf1 to promote expression of the 3'reg1 reporter within ACs and, importantly, both Foxf1 and Wnt signaling require Nr2f1a to promote a surplus of differentiated ACs. CRISPR-mediated deletion of the endogenous 3'reg1 abrogates the ability of Foxf1 and Wnt signaling to produce surplus ACs in zebrafish embryos. Together, our data support that downstream members of a conserved regulatory network involving Wnt signaling and Foxf1 function on a nr2f1a enhancer to promote AC differentiation in the zebrafish heart.

In mammals, differentiation of germ cells is crucial for sexual reproduction, involving complex signaling pathways and environmental cues defined by the somatic cells of the gonads. This review examines the long-standing model positing that all-trans retinoic acid (ATRA) acts as a meiosis-inducing substance (MIS) in the fetal ovary by inducing expression of STRA8 in female germ cells, while CYP26B1 serves as a meiosis-preventing substance (MPS) in the fetal testis by degrading ATRA and preventing STRA8 expression in the male germ cells until postnatal development. Recent genetic studies in the mouse challenge this paradigm, revealing that meiosis initiation in female germ cells can occur independently of ATRA signaling, with key roles played by other intrinsic factors like DAZL and DMRT1, and extrinsic signals such as BMPs and vitamin C. Thus, ATRA can no longer be considered as 'the' long-searched MIS. Furthermore, evidence indicates that CYP26B1 does not prevent meiosis by degrading ATRA in the fetal testis, but acts by degrading an unidentified MIS or synthesizing an equally unknown MPS. By emphasizing the necessity of genetic loss-of-function approaches to accurately delineate the roles of signaling molecules such ATRA in vivo, this chapter calls for a reevaluation of the mechanisms instructing and preventing meiosis initiation in the fetal ovary and testis, respectively. It highlights the need for further research into the molecular identities of the signals involved in these processes.

Chiari malformation type 1 (CM1) is a structural defect that involves the herniation of the cerebellar tonsils through the foramen magnum, causing mild to severe neurological symptoms. Little is known about the molecular and developmental mechanisms leading to its pathogenesis, prompting current efforts to elucidate genetic drivers. Inherited genetic disorders are reported in 2-3% of CM1 patients; however, CM1, including familial forms, is predominantly non-syndromic. Recent work has focused on identifying CM1-asscoiated variants through the study of both familial cases and de novo mutations using exome sequencing. This article aims to review the current understanding of the genetics of CM1. We discuss three broad classes of CM1 based on anatomy and link them with genetic lesions, including posterior fossa-linked, macrocephaly-linked, and connective tissue disorder-linked CM1. Although the genetics of CM1 are only beginning to be understood, we anticipate that additional studies with diverse patient populations, tissue types, and profiling technologies will reveal new insights in the coming years.

Retinoic acid receptors (RARs) are known as crucial endocrine receptors that could mediate a broad diversity of biological processes. However, the data on endocrine disrupting effects of emerging chemicals by targeting RAR (ant)agonism are far from sufficient. Herein, we investigated the RARα agonistic or antagonistic activities for 75 emerging chemicals of concern, and explored their interactions with this receptor. A recombinant two-hybrid yeast assay was used to examine the RARα activities of the test chemicals, wherein 7 showed effects of RARα agonism and 54 exerted potentials of RARα antagonism. The representative chemicals with RARα agonistic activities, i.e. 4-hydroxylphenol (4-HP) and bisphenol AF (BPAF), significantly increased the mRNA levels of CRABP2 and CYP26A1, while 4 select chemicals with RARα antagonistic potentials, including bisphenol A (BPA), tetrabromobisphenol A (TBBPA), 4-tert-octylphenol (4-t-OP), and 4-n-nonylphenol (4-n-NP), conversely decreased the transcriptional levels of the test genes. The in silico molecular docking analysis using 3 different approaches further confirmed the substantial binding between the chemicals with RARα activities and this nuclear receptor protein. This work highlights the promising strategy for screening endocrine-disrupting effects of emerging chemicals of concern by targeting RARα (ant)agonism.

Uterine fibroids, the most common nonmalignant tumors affecting the female genital tract, are a significant medical challenge. This article focuses on the most recent studies that attempted to identify novel non-hormonal therapeutic targets and strategies in UF therapy.

This review covers the analysis of the pharmacological and biological mechanisms of the action of natural substances and the role of the microbiome in reference to UFs. This study aimed to determine the potential role of these compounds in UF prevention and therapy.

While there are numerous approaches for treating UFs, available drug therapies for disease control have not been optimized yet. This review highlights the biological potential of vitamin D, EGCG and other natural compounds, as well as the microbiome, as promising alternatives in UF management and prevention. Although these substances have been quite well analyzed in this area, we still recommend conducting further studies, particularly randomized ones, in the field of therapy with these compounds or probiotics. Alternatively, as the quality of data continues to improve, we propose the consideration of their integration into clinical practice, in alignment with the patient's preferences and consent.

Nuclear hormone receptors (NHRs) are a family of ligand-regulated transcription factors that control key aspects of development and physiology. The regulation of NHRs by ligands derived from metabolism or diet makes them excellent pharmacological targets, and the mechanistic understanding of how NHRs interact with their ligands to regulate downstream gene networks, along with the identification of ligands for orphan NHRs, could enable innovative approaches for cellular engineering, disease modeling and regenerative medicine. We review recent discoveries in the identification of physiologic ligands for NHRs. We propose new models of ligand-receptor co-evolution, the emergence of hormonal function and models of regulation of NHR specificity and activity via one-ligand and two-ligand models as well as feedback loops. Lastly, we discuss limitations on the processes for the identification of physiologic NHR ligands and emerging new methodologies that could be used to identify the natural ligands for the remaining 17 orphan NHRs in the human genome.

Bronchopulmonary dysplasia (BPD) is a common morbidity affecting preterm infants and is associated with substantial long-term disabilities. The pathogenesis of BPD is multifactorial, and the clinical phenotype is variable. Extensive research has improved the current understanding of the factors contributing to BPD pathogenesis. However, effectively preventing and managing BPD remains a challenge. This review aims to provide an overview of the current evidence regarding the prevention of BPD in preterm infants, offering practical insights for clinicians.

Vitamin A is an important nutrient for multiple physiological functions. To elucidate the role of vitamin A in vivo, vitamin A-deficient diets have been often used in mice to establish a vitamin A-deficiency model. However, the information on the appropriate feeding periods and time course of changes in vitamin A content in organs after the start of vitamin A-deficient diet feeding is lacking. This study aimed to assess the retinoids levels in liver and white adipose tissue in mice fed a vitamin A-deficient diet for ≤8 weeks. High-performance liquid chromatography was used to measure the retinoids levels in liver and white adipose tissue every 2 weeks for ≤8 weeks. Vitamin A-deficient diet feeding significantly decreased retinol in the liver over 6 weeks, but retinyl palmitate, a main storage form of vitamin A, was not changed over 8 weeks. The plasma retinol level remained constant throughout the experiment. In white adipose tissue, retinyl palmitate gradually decreased over 8 weeks. These results indicate that vitamin A-deficient diet feeding longer than 6 weeks reduced retinol in liver and retinyl palmitate in white adipose tissue over 8 weeks, although it is not enough for the induction of a whole-body vitamin A deficiency.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Currently, there is a dearth of in-depth analysis and research on the impact of canthaxanthin on the production performance, egg quality, physical characteristics, and offspring health of laying hens. Furthermore, the metabolic mechanism of cantharidin in the body remains unclear. Therefore, to solve the above issues in detail, our study was conducted with a control group (C group), a low-dose canthaxanthin group (L group), and a high-dose canthaxanthin group (H group), each fed for a period of 40 days. Production performance was monitored during the experiment, in which L and H groups showed a significant increase in ADFI. Eggs were collected for quality analysis, revealing no significant differences in qualities except for yolk color (YC). The YC of the C group almost did not change, ranging from 6.08 to 6.20; however, the trend in YC change in other groups showed an initial intense increase, followed by a decrease, and eventually reached dynamic equilibrium. By detecting the content of canthaxanthin in the yolk, the YC change trend was found to be correlated with canthaxanthin levels in the yolk. The content of unsaturated fatty acid increased slightly in L and H groups. Following the incubation period, the physical characteristics and blood biochemical indices of chicks were evaluated. It was observed that the shank color of chicks in the L and H groups was significantly higher than that in the C group at birth. However, by the 35th day, there were no significant differences in shank color among the three groups. Further investigation into the metabolic mechanism involving canthaxanthin revealed that the substance underwent incomplete metabolism upon entering the body, resulting in its accumulation as well as metabolic by-product accumulation in the yolk. In summary, this study highlighted the importance of understanding canthaxanthin's role in production performance, egg quality, and offspring health, providing valuable insights for breeders to optimize feeding strategies.

Spermatogenesis in mammalian testes is essential for male fertility, ensuring a continuous supply of mature sperm. The testicular microenvironment finely tunes this process, with retinoic acid, an active metabolite of vitamin A, serving a pivotal role. Retinoic acid is critical for various stages, including the differentiation of spermatogonia, meiosis in spermatogenic cells, and the production of mature spermatozoa. Vitamin A deficiency halts spermatogenesis, leading to the degeneration of numerous germ cells, a condition reversible with retinoic acid supplementation. Although retinoic acid can restore fertility in some males with reproductive disorders, it does not work universally. Furthermore, high doses may adversely affect reproduction. The inconsistent outcomes of retinoid treatments in addressing infertility are linked to the incomplete understanding of the molecular mechanisms through which retinoid signaling governs spermatogenesis. In addition to the treatment of male reproductive disorders, the role of retinoic acid in spermatogenesis also provides new ideas for the development of male non-hormone contraceptives. This paper will explore three facets: the synthesis and breakdown of retinoic acid in the testes, its role in spermatogenesis, and its application in male reproduction. Our discussion aims to provide a comprehensive reference for studying the regulatory effects of retinoic acid signaling on spermatogenesis and offer insights into its use in treating male reproductive issues.

Germ cells (GCs) serve as indispensable carriers in both animals and plants, ensuring genetic continuity across generations. While it is generally acknowledged that the timing of germline segregation differs significantly between animals and plants, ongoing debates persist as new evidence continues to emerge. In this review, we delve into studies focusing on male germ cell specifications in plants, and we summarize the core gene regulatory circuits in germ cell specification, which show remarkable parallels to those governing meristem homeostasis. The similarity in germline establishment between animals and plants is also discussed.

The members retinoic acid receptors (RARs) (α, β, and γ) and retinoid X receptors (RXRs) (α, β, and γ) belong to the retinoid receptor family. They regulate the biological action of classical retinoids through nuclear retinoid receptors, a transcription factor that is regulated by ligands. Through the binding of particular retinoic acid-responsive elements (RAREs) located in target gene promoters, RARs and members of the RXRs form heterodimers. By binding to its nuclear receptors and triggering the transcription of the target genes downstream, retinoic acid (RA) mediates the expression of certain genes. Retinoids so mainly control gene expression to carry out their biological actions. RARs are essential for many biological processes, such as development, immunity, reproduction, organogenesis, and homeostasis. Apart from their physiological functions, RARs are also linked to pathologies and tumors due to mutations, protein fusions, changes in expression levels, or abnormal post-translational changes that lead to aberrant functions and homeostasis breakdown. The oncogenic development of animal tissues or cultured cells is linked to altered expression of retinoid receptors. The RAR-α is over-expressed in several malignancies. Increased invasion and migration in several cancer forms, including HNSC carcinoma, pediatric low-grade gliomas, lung adenocarcinoma, and breast cancer, have been linked to its upregulated expression. Numerous approved therapeutic regimens targeting RAR-α have been developed, improving patient survival rates.

This study's main objective was to identify novel RAR-α-targeting drugs and evaluate the expression patterns of RAR-α in breast cancer patients.

In-silico investigation using a variety of bioinformatics tools like UALCAN, TISCH, TIMER 2.0, ENRICHR, and others were employed to examine the expression of RAR-α. Further we evaluated in-silico inhibition of RAR-α with trifarotene and also tested the cytotoxicity of trifarotene in breast cancer cells.

Our research indicates that RAR-α is upregulated in several malignancies including Breast Cancer. It regulates granulocyte differentiation and has an association with the retinoic acid receptor signaling pathway and cellular response to estrogen stimulus. Furthermore, trifarotene was found as a potential synthetic compound that targets RAR-α through in silico and in-vitro study.

Overall, this research indicates that elevated expression of RAR-α enhances the onset of breast cancer. Using trifarotene medication to target RAR-α will significantly boost the response of breast cancer individuals to treatment and delay the development of resistance to drugs.

Fetal Alcohol Spectrum Disorder (FASD) is a common neurodevelopmental disorder that affects an estimated 2-5% of North Americans. FASD is induced by prenatal alcohol exposure (PAE) during pregnancy and while there is a clear genetic contribution, few genetic factors are currently identified or understood. In this study, using a candidate gene approach, we performed a genetic variant analysis of retinoic acid (RA) metabolic and developmental signaling pathway genes on whole exome sequencing data of 23 FASD-diagnosed individuals. We found risk and resilience alleles in ADH and ALDH genes known to normally be involved in alcohol detoxification at the expense of RA production, causing RA deficiency, following PAE. Risk and resilience variants were also identified in RA-regulated developmental pathway genes, especially in SHH and WNT pathways. Notably, we also identified significant variants in the causative genes of rare neurodevelopmental disorders sharing comorbidities with FASD, including STRA6 (Matthew-Wood), SOX9 (Campomelic Dysplasia), FDG1 (Aarskog), and 22q11.2 deletion syndrome (TBX1). Although this is a small exploratory study, the findings support PAE-induced RA deficiency as a major etiology underlying FASD and suggest risk and resilience variants may be suitable biomarkers to determine the risk of FASD outcomes following PAE.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.

Vitamins are essential micronutrients that play key roles in many biological pathways associated with sepsis. The gut microbiome plays a pivotal role in the progression of sepsis and may contribute to the onset of multi-organ dysfunction syndrome (MODS). The aim of this study was to investigate the changes in serum vitamins, and their correlation with intestinal flora and metabolomic profiles in patients with sepsis.

The serum levels of vitamins were determined by Ultra Performance Liquid Chromatography (UPLC). 16S rRNA gene sequencing and Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) targeted metabolomics were used for microbiome and metabolome analysis.

In the training cohort: After univariate, multivariate (OPLS-DA) and Spearman analyses, it was concluded that vitamin levels of 25 (OH) VD3 and (VD2 + VD3), as well as vitamins A and B9, differed significantly among healthy controls (HC), non-septic critical patients (NS), and sepsis patients (SS) (P < 0.05). The validation cohort confirmed the differential vitamin findings from the training cohort. Moreover, analyses of gut flora and metabolites in septic patients and healthy individuals revealed differential flora, metabolites, and metabolic pathways that were linked to alterations in serum vitamin levels. We found for the first time that vitamin B9 was negatively correlated with g_Sellimonas.

Sepsis patients exhibited significantly lower levels of 25 (OH) VD3 and (VD2 + VD3), vitamins A and B9, which hold potential as predictive markers for sepsis prognosis. The changes in these vitamins may be associated with inflammatory factors, oxidative stress, and changes in gut flora.

Nr2f transcription factors (TFs) are conserved regulators of vertebrate atrial cardiomyocyte (AC) differentiation. However, little is known about the mechanisms directing Nr2f expression in ACs. Here, we identified a conserved enhancer 3' to the nr2f1a locus, which we call 3'reg1-nr2f1a (3'reg1), that can promote Nr2f1a expression in ACs. Sequence analysis of the enhancer identified putative Lef/Tcf and Foxf TF binding sites. Mutation of the Lef/Tcf sites within the 3'reg1 reporter, knockdown of Tcf7l1a, and manipulation of canonical Wnt signaling support that Tcf7l1a is derepressed via Wnt signaling to activate the transgenic enhancer and promote AC differentiation. Similarly, mutation of the Foxf binding sites in the 3'reg1 reporter, coupled with gain- and loss-of-function analysis supported that Foxf1 promotes expression of the enhancer and AC differentiation. Functionally, we find that Wnt signaling acts downstream of Foxf1 to promote expression of the 3'reg1 reporter within ACs and, importantly, both Foxf1 and Wnt signaling require Nr2f1a to promote a surplus of differentiated ACs. CRISPR-mediated deletion of the endogenous 3'reg1 abrogates the ability of Foxf1 and Wnt signaling to produce surplus ACs in zebrafish embryos. Together, our data support that downstream members of a conserved regulatory network involving Wnt signaling and Foxf1 function on a nr2f1a enhancer to promote AC differentiation in the zebrafish heart.

Recent advances have brought forth the complex interplay between tumor cell plasticity and its consequential impact on drug resistance and tumor recurrence, both of which are critical determinants of neoplastic progression and therapeutic efficacy. Various forms of tumor cell plasticity, instrumental in facilitating neoplastic cells to develop drug resistance, include epithelial-mesenchymal transition (EMT) alternatively termed epithelial-mesenchymal plasticity, the acquisition of cancer stem cell (CSC) attributes, and transdifferentiation into diverse cell lineages. Nuclear receptors (NRs) are a superfamily of transcription factors (TFs) that play an essential role in regulating a multitude of cellular processes, including cell proliferation, differentiation, and apoptosis. NRs have been implicated to play a critical role in modulating gene expression associated with tumor cell plasticity and drug resistance. This review aims to provide a comprehensive overview of the current understanding of how NRs regulate these key aspects of cancer biology. We discuss the diverse mechanisms through which NRs influence tumor cell plasticity, including EMT, stemness, and metastasis. Further, we explore the intricate relationship between NRs and drug resistance, highlighting the impact of NR signaling on chemotherapy, radiotherapy and targeted therapies. We also discuss the emerging therapeutic strategies targeting NRs to overcome tumor cell plasticity and drug resistance. This review also provides valuable insights into the current clinical trials that involve agonists or antagonists of NRs modulating various aspects of tumor cell plasticity, thereby delineating the potential of NRs as therapeutic targets for improved cancer treatment outcomes.

PRAME constitutes one of the largest multi-copy gene families in Eutherians, encoding cancer-testis antigens (CTAs) with leucine-rich repeats (LRR) domains, highly expressed in cancer cells and gametogenic germ cells. This study aims to elucidate genetic interactions between two members, Pramex1 and Pramel1, in the mouse Prame family during gametogenesis using a gene knockout approach.

Single-gene knockout (sKO) of either Pramex1 or Pramel1 resulted in approximately 7% of abnormal seminiferous tubules, characterized by a Sertoli-cell only (SCO) phenotype, impacting sperm count and fecundity significantly. Remarkably, sKO female mice displayed normal reproductive functions. In contrast, Pramex1/Pramel1 double knockout (dKO) mice exhibited reduced fecundity in both sexes. In dKO females, ovarian primary follicle count decreased by 50% compared to sKO and WT mice, correlating with a 50% fecundity decrease. This suggested compensatory roles during oogenesis in Pramex1 or Pramel1 sKO females. Conversely, dKO males showed an 18% frequency of SCO tubules, increased apoptotic germ cells, and decreased undifferentiated spermatogonia compared to sKO and WT testes. Western blot analysis with PRAMEX1- or PRAMEL1-specific antibodies on sKO testes revealed compensatory upregulation of each protein (30-50%) in response to the other gene's deletion. Double KO males exhibited more severe defects in sperm count and litter size, surpassing Pramex1 and Pramel1 sKO accumulative effects, indicating a synergistic enhancement interaction during spermatogenesis. Additional experiments administering trans-retinoic acid (RA) and its inhibitor (WIN18,446) in sKO, dKO, and WT mice suggested that PRAMEX1 and PRAMEL1 synergistically repress the RA signaling pathway during spermatogenesis.

Data from sKO and dKO mice unveil a synergistic interaction via the RA signaling pathway between Pramex1 and Pramel1 genes during gametogenesis. This discovery sets the stage for investigating interactions among other members within the Prame family, advancing our understanding of multi-copy gene families involved in germ cell formation and function.

Retinoic acid (RA) is involved in antero-posterior patterning of the chordate body axis and, in jawed vertebrates, has been shown to play a major role at multiple levels of the gene regulatory network (GRN) regulating hindbrain segmentation. Knowing when and how RA became coupled to the core hindbrain GRN is important for understanding how ancient signaling pathways and patterning genes can evolve and generate diversity. Hence, we investigated the link between RA signaling and hindbrain segmentation in the sea lamprey Petromyzon marinus, an important jawless vertebrate model providing clues to decipher ancestral vertebrate features. Combining genomics, gene expression, and functional analyses of major components involved in RA synthesis (Aldh1as) and degradation (Cyp26s), we demonstrate that RA signaling is coupled to hindbrain segmentation in lamprey. Thus, the link between RA signaling and hindbrain segmentation is a pan vertebrate feature of the hindbrain and likely evolved at the base of vertebrates.