Exogenous Retinoic Acid Research Articles

Retinoic Acid Signaling Modulates Recipient Gut Barrier Integrity and Microbiota After Allogeneic Hematopoietic Stem Cell Transplantation in Mice.

Graft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). An impaired intestinal epithelial barrier is an important component of GVHD pathogenesis. However, contributing host factors that modulate mucosal barrier integrity during GVHD are poorly defined. We hypothesized that vitamin A and retinoic acid (RA) exert positive impacts on maintaining intestinal barrier function after HSCT, thus preventing or dampening GVHD severity. Unexpectedly, we found that exogenous RA increased intestinal permeability of recipient mice after allogeneic HSCT. Serum bacterial endotoxin levels were significantly higher in GVHD mice fed a vitamin A-high (VAH) diet compared to those fed a vitamin A-normal (VAN) diet, indicating a more compromised intestinal barrier function. Furthermore, VAH mice showed more severe lung GVHD with increased donor T cell infiltration in this tissue and died significantly faster than VAN recipients. 16S rRNA sequencing of fecal samples revealed significant differences in the diversity and composition of gut microbiota between VAN and VAH transplant recipients. Collectively, we show that retinoic acid signaling may negatively impact intestinal barrier function during GVHD. Mild vitamin A supplementation is associated with increased lung GVHD and more profound gut dysbiosis. Micronutrients such as vitamin A could modulate complications of allogeneic HSCT, which may be mediated by shaping gut microbiota.

Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells

The vitamin A derivative, retinoid acid (RA) is key player in guiding adaptive mucosal immune responses. However, data on the uptake and metabolism of vitamin A within human immune cells has remained largely elusive because retinoids are small, lipophilic molecules which are difficult to detect. To overcome this problem and to be able to study the effect of vitamin A metabolism in human immune cell subsets, we have synthesized novel bio-orthogonal retinoid-based probes (clickable probes), which are structurally and functionally indistinguishable from vitamin A. The probes contain a functional group (an alkyne) to conjugate to a fluorogenic dye to monitor retinoid molecules in real-time in immune cells. We demonstrate, by using flow cytometry and microscopy, that multiple immune cells have the capacity to internalize retinoids to varying degrees, including human monocyte-derived dendritic cells (DCs) and naïve B lymphocytes. We observed that naïve B cells lack the enzymatic machinery to produce RA, but use exogenous retinoic acid to enhance CD38 expression. Furthermore, we showed that human DCs metabolize retinal into retinoic acid, which in co-culture with naïve B cells led to of the induction of CD38 expression. These data demonstrate that in humans, DCs can serve as an exogenous source of RA for naïve B cells. Taken together, through the use of clickable vitamins our data provide valuable insight in the mechanism of vitamin A metabolism and its importance for human adaptive immunity.

Short Wavelength (Blue) Light Is Protective for Lens-Induced Myopia in Guinea Pigs Potentially Through a Retinoic Acid-Related Mechanism.

PurposeTo investigate the effect of short-wavelength light (SL) on guinea pigs with lens-induced myopia (LIM) and the possible retinoic acid (RA)–related mechanisms.MethodsTwo-week-old guinea pigs (n = 60) with monocular −5D lenses were reared under white light (WL, 580 lux) or SL (440 nm, 500 lux). The left eyes were uncovered as control. Refractive error (RE) and axial length (AL) were measured at baseline, one week, two weeks, and four weeks after intervention. Retinal RA was measured from four guinea pigs after two and four weeks of treatment with HPLC. Two-week-old guinea pigs (n = 52) with monocular −5D lens were fed with either RA or its synthesis inhibitor citral every third day in the morning, and half from each group were reared under WL or SL conditions. RE and AL were recorded at baseline and two and four weeks after intervention. Retinal RA was measured after four weeks of intervention.ResultsAt the end of treatment, guinea pigs exposed to SL were less myopic than to WL (2.06 ± 1.69D vs. −1.00 ± 1.88D), accompanied with shorter AL (P = 0.01) and less retinal RA (P = 0.02). SL reduced retinal RA even after exogenous RA supplementation (P = 0.02) and decelerated LIM compared to WL (1.66 ± 1.03D vs. −3.53 ± 0.90D). Citral slowed ocular growth, leading to similar RE in W+CI and S+CI groups (3.39 ± 1.65D vs. 5.25 ± 0.80D).ConclusionsOverall, SL reduced LIM in guinea pigs, even in those supplemented with oral RA, accompanied by reduced retinal RA levels. Oral RA accelerated eye elongation, but citral equally decelerated eye elongation under SL and WL with no significant retinal RA reduction.

Crosstalk Between Retinoic Acid and Sex-Related Genes Controls Germ Cell Fate and Gametogenesis in Medaka.

Sex determination (SD) is a highly diverse and complex mechanism. In vertebrates, one of the first morphological differences between the sexes is the timing of initiation of the first meiosis, where its initiation occurs first in female and later in male. Thus, SD is intimately related to the responsiveness of the germ cells to undergo meiosis in a sex-specific manner. In some vertebrates, it has been reported that the timing for meiosis entry would be under control of retinoic acid (RA), through activation of Stra8. In this study, we used a fish model species for sex determination and lacking the stra8 gene, the Japanese medaka (Oryzias latipes), to investigate the connection between RA and the sex determination pathway. Exogenous RA treatments act as a stress factor inhibiting germ cell differentiation probably by activation of dmrt1a and amh. Disruption of the RA degrading enzyme gene cyp26a1 induced precocious meiosis and oogenesis in embryos/hatchlings of female and even some males. Transcriptome analyzes of cyp26a1–/–adult gonads revealed upregulation of genes related to germ cell differentiation and meiosis, in both ovaries and testes. Our findings show that germ cells respond to RA in a stra8 independent model species. The responsiveness to RA is conferred by sex-related genes, restricting its action to the sex differentiation period in both sexes.

Retinoic Acid Synthesis Deficiency Fosters the Generation of Polymorphonuclear Myeloid-Derived Suppressor Cells in Colorectal Cancer.

Metabolism is reprogrammed in cancer to fulfill the demands of malignant cells for cancer initiation and progression. Apart from its effects within cancer cells, little is known about whether and how reprogramed metabolism regulates the surrounding tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSC) are key regulators of the TME and greatly affect tumor progression and therapeutic responses. In this study, our results revealed that retinol metabolism-related genes and enzymes were significantly downregulated in human colorectal cancer compared with adjacent colonic tissues, and tumors exhibited a defect in retinoic acid (RA) synthesis. Reduced ADH1-mediated retinol metabolism was associated with attenuated RA signaling and accumulated MDSCs in colorectal cancer tumors. Using an in vitro model, generating MDSCs from CD34+ myeloid precursors, we found that exogenous RA could abrogate the generation of polymorphonuclear MDSCs (PMN-MDSC) with negligible impact on myeloid differentiation. Mechanistically, RA could restrain the glycolytic capacity of myeloid cells, which in turn activated the AMP-activated protein kinase (AMPK) pathway, further impairing the suppressive capacity of myeloid cells. Supplementation with RA could significantly delay tumor growth, with reduced arginase-1-expressing myeloid cells and increased CD8+ and granzyme B+ T cells in both colitis-associated and implanted MC38 mouse colorectal cancer models. Our results indicated that the defect in ADH1-mediated RA synthesis could provide a possible mechanism that fosters the generation of PMN-MDSCs in colorectal cancer and that restoring RA signaling in the TME could serve as a promising therapeutic strategy to abrogate the generation of PMN-MDSCs.

APC mutations in human colon lead to decreased neuroendocrine maturation of ALDH+ stem cells that alters GLP-2 and SST feedback signaling: Clue to a link between WNT and retinoic acid signalling in colon cancer development.

APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC-zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC-mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn't explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis is developed that human CRC evolves due to an imbalance between WNT and RA signaling.

Postnatal loss of Cyp26a1 and Cyp26b1 in mice causes impaired retinoid homeostasis in multiple organs

Retinoic acid (RA), the active metabolite of vitamin A (retinol), is essential to various biological processes such as maintenance of epithelia and regulation of cell division, proliferation and differentiation. Cyp26a1 and Cyp26b1 enzymes catalyze hydroxylation of RA and are believed to be responsible for RA clearance in all chordates. Global knockout of either Cyp26a1 or Cyp26b1 is embryonic lethal, demonstrating a critical role of Cyp26 enzymes in regulating RA signaling during fetal development. However, whether Cyp26 activity is required in postnatal life to maintain vitamin A homeostasis is still not clear. We hypothesized that postnatal loss of Cyp26a1 and Cyp26b1 would decrease RA clearance, and consequently alter tissue retinoid concentrations and cause retinoid toxicity. A tamoxifen‐inducible Cre‐loxP system was used to induce a conditional global knockout of Cyp26a1 and Cyp26b1 (double‐knockout; DKO). The knock‐out was induced in female and male mice with tamoxifen injections at 21 days postpartum and then these animals were monitored (body weight and condition) biweekly for 8 weeks. Animals were euthanized if they received a body condition score ≤ 2 or had lost > 15% of their pre‐tamoxifen injection body weight. Tissues were harvested for histological analysis and retinoid measurements. All WT animals were healthy and euthanized after the 8‐week follow‐up but 45% of male and 41% of female DKO animals were euthanized before the completion of the 8‐week follow‐up according to the criteria described above. Compared to WT mice, DKO animals showed significant decrease in weight gain. DKO mice had moderate to severe blepharitis and dermatitis with skin thickening and inflammation. Spleens of DKO mice were significantly enlarged and showed extramedullary hematopoiesis. Cell proliferation in the epidermis and in the epithelium of nonglandular stomach was increased in DKO animals. In general, retinoid homeostasis was altered in DKO animals. In comparison to WT animals, skin, spleen and liver RA concentrations were significantly increased in DKO mice while tissue retinol and retinyl ester concentrations were unchanged. In contrast, serum retinol and retinol binding protein 4 (RBP4) concentrations were significantly decreased in DKO mice with no change in RA concentrations. When DKO animals were dosed with exogenous RA (1mg/kg; i.p.), animals showed signs of toxicity and the exposure to RA was significantly greater in DKO animals than in WT animals. Collectively these data show that Cyp26a1 and Cyp26b1 are required for maintaining vitamin A homeostasis and retinoid signaling also in postnatal life.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides

Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa, dazl, sycp3 and rec8, which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen.

Retinoic Acid Signaling Regulates the Metamorphosis of Feather Stars (Crinoidea, Echinodermata): Insight into the Evolution of the Animal Life Cycle.

Many marine invertebrates have a life cycle with planktonic larvae, although the evolution of this type of life cycle remains enigmatic. We recently proposed that the regulatory mechanism of life cycle transition is conserved between jellyfish (Cnidaria) and starfish (Echinoderm); retinoic acid (RA) signaling regulates strobilation and metamorphosis, respectively. However, the function of RA signaling in other animal groups is poorly understood in this context. Here, to determine the ancestral function of RA signaling in echinoderms, we investigated the role of RA signaling during the metamorphosis of the feather star, Antedon serrata (Crinoidea, Echinodermata). Although feather stars have different larval forms from starfish, we found that exogenous RA treatment on doliolaria larvae induced metamorphosis, like in starfish. Furthermore, blocking RA synthesis or binding to the RA receptor suppressed metamorphosis. These results suggested that RA signaling functions as a regulator of metamorphosis in the ancestor of echinoderms. Our data provides insight into the evolution of the animal life cycle from the viewpoint of RA signaling.

Structure, development, and functional morphology of the cement gland of the giant danio, Devario malabaricus.

Aquatic species in several clades possess cement glands producing adhesive secretions of various strengths. In vertebrates, transient adhesive organs have been extensively studied in Xenopus laevis, other anurans, and in several fish species. However, the development of these structures is not fully understood. Here, we report on the development and functional morphology of the adhesive gland of a giant danio species, Devario malabaricus. We found that the gland is localized on the larval head, is composed of goblet-like secretory cells framed by basal, bordering, and intercalated apical epithelial cells, and is innervated by the trigeminal ganglion. The gland allows nonswimming larvae to adhere to various substrates. Its secretory cells differentiate by 12 hours postfertilization and begin to disappear in the second week of life. Exogenous retinoic acid disrupts the gland's patterning. More importantly, the single mature gland emerges from fusion of two differentiated secretory cells fields; this fusion is dependent on nonmuscle myosin II function. Taken together, our studies provide the first documentation of the embryonic development, structure, and function of the adhesive apparatus of a danioninae. To our knowledge, this is also the first report of a cement gland arising from convergence of two bilateral fields.

Novel Markers of the Metabolic Impact of Exogenous Retinoic Acid with A Focus on Acylcarnitines and Amino Acids.

Treatment with all-trans retinoic acid (ATRA), the carboxylic form of vitamin A, lowers body weight in rodents by promoting oxidative metabolism in multiple tissues including white and brown adipose tissues. We aimed to identify novel markers of the metabolic impact of ATRA through targeted blood metabolomics analyses, with a focus on acylcarnitines and amino acids. Blood was obtained from mice treated with a high ATRA dose (50 mg/kg body weight/day, subcutaneous injection) or placebo (controls) during the 4 days preceding collection. LC-MS/MS analyses with a focus on acylcarnitines and amino acids were conducted on plasma and PBMC. Main results showed that, relative to controls, ATRA-treated mice had in plasma: increased levels of carnitine, acetylcarnitine, and longer acylcarnitine species; decreased levels of citrulline, and increased global arginine bioavailability ratio for nitric oxide synthesis; increased levels of creatine, taurine and docosahexaenoic acid; and a decreased n-6/n-3 polyunsaturated fatty acids ratio. While some of these features likely reflect the stimulation of lipid mobilization and oxidation promoted by ATRA treatment systemically, other may also play a causal role underlying ATRA actions. The results connect ATRA to specific nutrition-modulated biochemical pathways, and suggest novel mechanisms of action of vitamin A-derived retinoic acid on metabolic health.

Competing mechanisms of plasticity impair compensatory responses to repetitive apnoea.

Intermittent reductions in respiratory neural activity, a characteristic of many ventilatory disorders, leads to inadequate ventilation and arterial hypoxia. Both intermittent reductions in respiratory neural activity and intermittent hypoxia trigger compensatory enhancements in inspiratory output when experienced separately, forms of plasticity called inactivity-induced inspiratory motor facilitation (iMF) and long-term facilitation (LTF), respectively. Reductions in respiratory neural activity that lead to moderate, but not mild, arterial hypoxia occludes plasticity expression, indicating that concurrent induction of iMF and LTF impairs plasticity through cross-talk inhibition of their respective signalling pathways. Moderate hypoxia undermines iMF by enhancing NR2B-containing NMDA receptor signalling, which can be rescued by exogenous retinoic acid, a molecule necessary for iMF. These data suggest that in ventilatory disorders characterized by reduced inspiratory motor output, such as sleep apnoea, endogenous mechanisms of compensatory plasticity may be impaired, and that exogenously activating respiratory plasticity may be a novel strategy to improve breathing. Many forms of sleep apnoea are characterized by recurrent reductions in respiratory neural activity, which leads to inadequate ventilation and arterial hypoxia. Both recurrent reductions in respiratory neural activity and hypoxia activate mechanisms of compensatory plasticity that augment inspiratory output and lower the threshold for apnoea, inactivity-induced inspiratory motor facilitation (iMF) and long-term facilitation (LTF), respectively. However, despite frequent concurrence of reduced respiratory neural activity and hypoxia, mechanisms that induce and regulate iMF and LTF have only been studied separately. Here, we demonstrate that recurrent reductions in respiratory neural activity ('neural apnoea') accompanied by cessations in ventilation that result in moderate (but not mild) hypoxaemia do not elicit increased inspiratory output, suggesting that concurrent induction of iMF and LTF occludes plasticity. A key role for NMDA receptor activation in impairing plasticity following concurrent neural apnoea and hypoxia is indicated since recurrent hypoxic neural apnoeas triggered increased phrenic inspiratory output in rats in which spinal NR2B-containing NMDA receptors were inhibited. Spinal application of retinoic acid, a key molecule necessary for iMF, bypasses NMDA receptor-mediated constraints, thereby rescuing plasticity following hypoxic neural apnoeas. These studies raise the intriguing possibility that endogenous mechanisms of compensatory plasticity may be impaired in some individuals with sleep apnoea, and that exogenously activating pathways giving rise to respiratory plasticity may be a novel pharmacological strategy to improve breathing.

FGF, BMP, and RA signaling are sufficient for the induction of complete limb regeneration from non-regenerating wounds on Ambystoma mexicanum limbs

Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.

Transcriptomic responses of regenerating earthworms (Eisenia foetida) to retinoic acid reveals the role of pluripotency genes

Exogenous retinoic acid (RA) delays and disturbs the regeneration of Eisenia foetida and inhibits the expression of pluripotent gene Sox2. However, studies of E. foetida conducted at the molecular level have been unable to elucidate its regeneration and mechanisms of RA effects on its regeneration. We merged existing transcriptomic data for E. foetida to generate a high-confidence set of transcriptomes. The de novo assembly of transcriptomes was performed by using the Trinity method, and functional annotations were analysed. We performed RNA-seq on four samples of regenerating tail fragments, three across a time-course (0, 3 and 7 days post amputation) and the fourth sample exposed to RA (7 days post amputation). E. foetida regeneration genes underwent significant upregulation and downregulation over the examined time periods, which may have been caused by a shared regulatory programme controlled by multiple gene families. The inhibition of RA against earthworm regeneration is likely related to the expression of these genes. Using annotation data and clustering, we also identified specific transcripts of 6 gene superfamilies enriched among genes exhibiting differential expression during regeneration periods and exhibiting the same expression patterns as those of the Sox2 gene. The regeneration transcriptome of tail fragment regeneration serves as a strong resource for investigating global expression changes that occur during regeneration and the toxicity of RA. This study offers insight for better understanding the regeneration of lower animals and molecular mechanisms of RA toxicity in invertebrates.

Retinoic Acid Receptor RARα-Dependent Synaptic Signaling Mediates Homeostatic Synaptic Plasticity at the Inhibitory Synapses of Mouse Visual Cortex.

Homeostatic synaptic plasticity is a synaptic mechanism through which the nervous system adjusts synaptic excitation and inhibition to maintain network stability. Retinoic acid (RA) and its receptor RARα have been established as critical mediators of homeostatic synaptic plasticity. In vitro studies reveal that RA signaling enhances excitatory synaptic strength and decreases inhibitory synaptic strength. However, it is unclear whether RA-mediated homeostatic synaptic plasticity occurs in vivo, and if so, whether it operates at specific types of synapses. Here, we examine the impact of RA/RARα signaling in the monocular zone of primary visual cortex (V1m) in mice of either sex. Exogenous RA treatment in acute cortical slices resulted in a reduction in mIPSCs of layer 2/3 pyramidal neurons, an effect mimicked by visual deprivation induced by binocular enucleation in postcritical period animals. Postnatal deletion of RARα blocked RA's effect on mIPSCs. Cell type-specific deletion of RARα revealed that RA acted specifically on parvalbumin (PV)-expressing interneurons. RARα deletion in PV+ interneurons blocked visual deprivation-induced changes in mIPSCs, demonstrating the critical involvement of RA signaling in PV+ interneurons in vivo Moreover, visual deprivation- or RA-induced downregulation of synaptic inhibition was absent in the visual cortical circuit of constitutive and PV-specific Fmr1 KO mice, strongly suggesting a functional interaction between fragile X mental retardation protein and RA signaling pathways. Together, our results demonstrate that RA/RARα signaling acts as a key component for homeostatic regulation of synaptic transmission at the inhibitory synapses of the visual cortex.SIGNIFICANCE STATEMENTIn vitro studies established that retinoic acid (RA) and its receptor RARα play key roles in homeostatic synaptic plasticity, a mechanism by which synaptic excitation/inhibition balance and network stability are maintained. However, whether synaptic RA signaling operates in vivo remains undetermined. Here, using a conditional RARα KO mouse and cell type-specific Cre-driver lines, we showed that RARα signaling in parvalbumin-expressing interneurons is crucial for visual deprivation-induced homeostatic synaptic plasticity at inhibitory synapses in visual cortical circuits. Importantly, this form of synaptic plasticity is absent when fragile X mental retardation protein is selectively deleted in parvalbumin-expressing interneurons, suggesting a functional connection between RARα and fragile X mental retardation protein signaling pathways in vivo Thus, dysfunction of RA-dependent homeostatic plasticity may contribute to cortical circuit abnormalities in fragile X syndrome.

All-Trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. I: Altered Seminiferous Cord Maturation and Testicular Cell Fate.

Exposure to excess retinoic acid (RA) disrupts the development of the mammalian testicular seminiferous cord. However, the molecular events surrounding RA-driven loss of cord structure have not previously been examined. To investigate the mechanisms associated with this adverse developmental effect, fetal rat testes were isolated on gestational day 15, after testis determination and the initiation of cord development, and cultured in media containing all-trans RA (ATRA; 10-8 to 10-6 M) or vehicle for 3 days. ATRA exposure resulted in a concentration-dependent decrease in the number of seminiferous cords per testis section and number of germ cells, assessed by histopathology and immunohistochemistry. Following 1 day of culture, genome-wide expression profiling by microarray demonstrated that ATRA exposure altered biological processes related to retinoid metabolism and gonadal sex determination. Real-time RT-PCR analysis confirmed that ATRA enhanced the expression of the key ovarian development gene Wnt4 and the antitestis gene Nr0b1 in a concentration-dependent manner. After 3 days of culture, ATRA-treated testes contained both immunohistochemically DMRT1-positive and FOXL2-positive somatic cells, providing evidence of disrupted testicular cell fate maintenance following ATRA exposure. We conclude that exogenous RA disrupts seminiferous cord development in ex vivo cultured fetal rat testes, resulting in a reduction in seminiferous cord number, and interferes with maintenance of somatic cell fate by enhancing expression of factors that promote ovarian development.

Exogenous Vitamin D signaling alters skeletal patterning, differentiation, and tissue integration during limb regeneration in the axolotl

Urodele amphibians such as the axolotl regenerate complete limbs as adults, and understanding how the “blueprint”, or pattern, of the regenerate is established and manipulated are areas of intense interest. Nutrient signaling plays an important role in pattern formation during regeneration. Retinoic acid signaling is the most characterized pathway during this process. Exogenous retinoic acid (RA) reprograms the pattern information in regenerating cells to a more posterior, ventral, and proximal identity. Vitamin D signaling shares several molecular similarities with RA and has been shown to alter pattern formation during zebrafish pectoral fin regeneration. To determine if exogenous Vitamin D signaling is capable of reprograming pattern in the axolotl limb blastema, we treated regenerating limbs with a potent Vitamin D agonist. Under the studied conditions, exogenous Vitamin D did not act in a manner similar to RA and failed to proximalize the pattern of the resulting regenerates. The Vitamin D treatment did result in several skeletal defects during regeneration, including carpal fusions along the A/P axis; failure to integrate the newly regenerated tissue with the existing tissue, formation of ectopic nodules of cartilage at the site of amputation, and altered bone morphology in uninjured skeletal tissue.

A Single-Cell Transcriptomic Atlas of Thymus Organogenesis Resolves Cell Types and Developmental Maturation

Thymus development is critical to the adaptive immune system, yet a comprehensive transcriptional framework capturing thymus organogenesis at single-cell resolution is still needed. We applied single-cell RNA sequencing (RNA-seq) to capture 8days of thymus development, perturbations of Tcell receptor rearrangement, and invitro organ cultures, producing profiles of 24,279 cells. We resolved transcriptional heterogeneity of developing lymphocytes, and genetic perturbation confirmed Tcell identity of conventional and non-conventional lymphocytes. We characterized maturation dynamics of thymic epithelial cells invivo, classified cell maturation state in a thymic organ culture, and revealed the intrinsic capacity of thymic epithelium to preserve transcriptional regularity despite exposure to exogenous retinoic acid. Finally, by integrating the cell atlas with human genome-wide association study (GWAS) data and autoimmune-disease-related genes, we implicated embryonic thymus-resident cells as possible participants in autoimmune disease etiologies. This resource provides a single-cell transcriptional framework for biological discovery and molecular analysis of thymus organogenesis.

The role of retinoic acid signaling in starfish metamorphosis

BackgroundAlthough retinoic acid (RA) signaling plays a crucial role in the body patterning of chordates, its function in non-chordate invertebrates, other than its mediation of environmental cues triggering metamorphosis in cnidarians, is largely unknown. We investigated the role of RA signaling in the metamorphosis of starfish (Echinodermata).ResultsWe found that exogenous RA treatment induced metamorphosis in starfish larvae. In contrast, inhibitors of RA synthesis and RA receptors suppressed metamorphosis triggered by attachment to a substrate. Gene expressions of the RA signaling component were detected in competent larvae.ConclusionsThis study provides insight into the ancestral function of RA signaling, which is conserved in the metamorphosis of cnidarians and starfish.

Retinoic Acid Maintains Function of Neural Crest-Derived Ocular and Craniofacial Structures in Adult Zebrafish.

PurposeRetinoic acid (RA) is required for embryonic formation of the anterior segment of the eye and craniofacial structures. The present study further investigated the role of RA in maintaining the function of these neural crest–derived structures in adult zebrafish.MethodsMorphology and histology were analyzed by using live imaging, methylacrylate sections, and TUNEL assay. Functional analysis of vision and aqueous humor outflow were assayed with real-time imaging.ResultsBoth decreased and increased RA signaling altered craniofacial and ocular structures in adult zebrafish. Exogenous treatment with all-trans RA for 5 days resulted in a prognathic jaw, while inhibition of endogenous RA synthesis through treatment with 4-diethylaminobenzaldehyde (DEAB) decreased head height. In adult eyes, RA activity was localized to the retinal pigment epithelium, photoreceptors, outer plexiform layer, inner plexiform layer, iris stroma, and ventral canalicular network. Exogenous RA increased apoptosis in the iris stroma and canalicular network in the ventral iridocorneal angle, resulting in the loss of these structures and decreased aqueous outflow. DEAB, which decreased RA activity throughout the eye, induced widespread apoptosis, resulting in corneal edema, cataracts, retinal atrophy, and loss of iridocorneal angle structures. DEAB-treated fish were blind with no optokinetic response and no aqueous outflow from the anterior chamber.ConclusionsTight control of RA levels is required for normal structure and function of the adult anterior segment. These studies demonstrated that RA plays an important role in maintaining ocular and craniofacial structures in adult zebrafish.