Inhibition Of Retinoic Acid Signaling Research Articles

The Utilization of PRAME in the Diagnosis, Prognosis, and Treatment of Melanoma.

Melanoma, a deadly form of skin cancer, has seen improved survival rates due to advances in diagnosis and treatment, yet the need for further improvement remains critical. Tumor-associated antigens, such as PRAME (Preferentially Expressed Antigen in Melanoma), offer promising avenues for enhanced diagnostic precision, prognostic assessment, and targeted immunotherapy. PRAME, a cancer testis antigen, is selectively expressed in various cancers, including melanoma, and plays a key role in promoting tumorigenesis through inhibition of retinoic acid signaling, epithelial-to-mesenchymal transition, and immune evasion. This review explores the diagnostic utility of PRAME in distinguishing melanoma from benign nevi, its prognostic value in aggressive melanoma subtypes, and its potential as a therapeutic target in cancer vaccines and adoptive T-cell therapies. While PRAME-targeted therapies face challenges such as tumor heterogeneity and immune suppression, ongoing research aims to overcome these barriers, offering hope for more effective melanoma treatments.

Osteopetrosis-like disorders induced by osteoblast-specific retinoic acid signaling inhibition in mice

Osteopetrosis is an inherited metabolic disease, characterized by increased bone density and narrow marrow cavity. Patients with severe osteopetrosis exhibit abnormal bone brittleness, anemia, and infection complications, which commonly cause death within the first decade of life. Pathologically, osteopetrosis impairs not only the skeletal system, but also the hemopoietic and immune systems during development, while the underlying osteoimmunological mechanisms remain unclear. Osteoclastic mutations are regarded as the major causes of osteopetrosis, while osteoclast non-autonomous theories have been proposed in recent years with unclear underlying mechanisms. Retinoic acid (RA), the metabolite of Vitamin A, is an essential requirement for skeletal and hematopoietic development, through the activation of retinoic acid signaling. RA can relieve osteopetrosis symptoms in some animal models, while its effect on bone health is still controversial and the underlying mechanisms remain unclear. In this study, we constructed an osteoblast-specific inhibitory retinoic acid signaling mouse model and surprisingly found it mimicked the symptoms of osteopetrosis found in clinical cases: dwarfism, increased imperfectly-formed trabecular bone deposition with a reduced marrow cavity, thin cortical bone with a brittle skeleton, and hematopoietic and immune dysfunction. Micro-CT, the three-point bending test, and histological analysis drew a landscape of poor bone quality. Single-cell RNA sequencing (scRNA-seq) of the femur and RNA-seq of osteoblasts uncovered an atlas of pathological skeletal metabolism dysfunction in the mutant mice showing that osteogenesis was impaired in a cell-autonomous manner and osteoclastogenesis was impaired via osteoblast-osteoclast crosstalk. Moreover, scRNA-seq of bone marrow and flow cytometry of peripheral blood, spleen, and bone marrow uncovered pathology in the hematopoietic and immune systems in the mutant mice, mimicking human osteopetrosis. Results showed that hematopoietic progenitors and B lymphocyte differentiation were affected and the osteoblast-dominated cell crosstalk was impaired, which may result from transcriptional impairment of the ligands Pdgfd and Sema4d. In summary, we uncovered previously unreported pathogenesis of osteopetrosis-like disorder in mice with skeletal, hematopoietic, and immune system dysfunction, which was induced by the inhibition of retinoic acid signaling in osteoblasts, and sheds new insights into a potential treatment for osteopetrosis.

Inhibition of Retinoic Acid Signaling in Proximal Tubules Protects Against AKI by Enhancing Kim1-Dependent Efferocytosis

Inhibition of Retinoic Acid Signaling in Proximal Tubules Protects Against AKI by Enhancing Kim1-Dependent Efferocytosis

Inhibition of retinoic acid signaling in proximal tubular epithelial cells protects against acute kidney injury.

Retinoic acid receptor (RAR) signaling is essential for mammalian kidney development but, in the adult kidney, is restricted to occasional collecting duct epithelial cells. We now show that there is widespread reactivation of RAR signaling in proximal tubular epithelial cells (PTECs) in human sepsis-associated acute kidney injury (AKI) and in mouse models of AKI. Genetic inhibition of RAR signaling in PTECs protected against experimental AKI but was unexpectedly associated with increased expression of the PTEC injury marker Kim1. However, the protective effects of inhibiting PTEC RAR signaling were associated with increased Kim1-dependent apoptotic cell clearance, or efferocytosis, and this was associated with dedifferentiation, proliferation, and metabolic reprogramming of PTECs. These data demonstrate the functional role that reactivation of RAR signaling plays in regulating PTEC differentiation and function in human and experimental AKI.

CNKSR2, a downstream mediator of retinoic acid signaling, modulates the Ras/Raf/MEK pathway to regulate patterning and invagination of the chick forebrain roof plate.

During embryonic development, the forebrain roof plate undergoes invagination, leading to separation of the cerebral hemispheres. Any defects in this process, in humans, lead to middle interhemispheric holoprosencephaly (MIH-HPE). In this study, we have identified a previously unreported downstream mediator of retinoic acid (RA) signaling, CNKSR2, which is expressed in the forebrain roof plate in the chick embryo. Knockdown of CNKSR2 affects invagination, cell proliferation and patterning of the roof plate, similar to the phenotypes observed upon inhibition of RA signaling. We further demonstrate that CNKSR2 functions by modulating the Ras/Raf/MEK signaling. This appears to be crucial for patterning of the forebrain roof plate and its subsequent invagination, leading to the formation of the cerebral hemispheres. Thus, a set of novel molecular players have been identified that regulate the morphogenesis of the avian forebrain.

The inhibition of retinoic acid signaling in innate immune cells paradoxically suppresses respiratory syncytial virus infection through enhanced antiviral immunity

Respiratory syncytial virus (RSV) infection is a major cause of morbidity and mortality in children. It is also associated with enhanced Th2 immunity and asthma. Vitamin A deficiency weakens mucosal immunity because of the central role of retinoic acid (RA) in regulating different immune cell functions. We therefore sought to evaluate the effect of RA signaling deficiency on immune responses to RSV infection.

Disruptions in network plasticity precede deficits in memory following inhibition of retinoid signaling.

Retinoic acid, the active metabolite of vitamin A, is important for vertebrate cognition and hippocampal plasticity, but few studies have examined its role in invertebrate learning and memory, and its actions in the invertebrate central nervous system are currently unknown. Using the mollusc Lymnaea stagnalis, we examined operant conditioning of the respiratory behavior, controlled by a well-defined central pattern generator (CPG), and used citral to inhibit retinoic acid signaling. Both citral- and vehicle-treated animals showed normal learning, but citral-treated animals failed to exhibit long-term memory at 24 h. Cohorts of citral- or vehicle-treated animals were dissected into semi-intact preparations, either 1 h after training, or after the memory test 24 h later. Simultaneous electrophysiological recordings from the CPG pacemaker cell (right pedal dorsal 1; RPeD1) and an identified motorneuron (VI) were made while monitoring respiratory activity (pneumostome opening). Activity of the CPG pneumostome opener interneuron (input 3 interneuron; IP3) was also monitored indirectly. Vehicle-treated conditioned preparations showed significant changes in network parameters immediately after learning, such as reduced motorneuron bursting activity (from IP3 input), delayed pneumostome opening, and decoupling of coincident IP3 input within the network. However, citral-treated preparations failed to exhibit these network changes and more closely resembled naïve preparations. Importantly, these citral-induced differences were manifested immediately after training and before any overt changes in the behavioral response (memory impairment). These studies shed light on where and when retinoid signaling might affect a central pattern-generating network to promote memory formation during conditioning of a homeostatic behavior.NEW & NOTEWORTHY We provide novel evidence for how conditioning-induced changes in a CPG network are disrupted when retinoid signaling is inhibited. Inhibition of retinoic acid signaling prevents long-term memory formation following operant conditioning, but has no effect on learning. Simultaneous electrophysiological and behavioral analyses indicate network changes immediately following learning, but these changes are prevented with inhibition of retinoid signaling, before any overt changes in behavior. These data suggest sites for retinoid actions during memory formation.

Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling.

Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

2046-P: Retinoic Acid Signaling in Pancreatic Endocrine Cells Regulates Islet Cell Specification

Diabetes is characterized by β cell death and/or dysfunction. Currently, one promising treatment option for patients with diabetes involves replenishing the body with β cells to restore glucose homeostasis. Despite great progress using in vitro differentiation protocols to generate human β cells from stem cell populations, there are still challenges associated with generating fully functional β cells. To improve this therapeutic strategy, my studies are focusing on defining the role of cell signaling pathways in pancreatic islet cell specification. Genetic studies in fish, mice, and humans have demonstrated that retinoic acid (RA) is broadly required for pancreas development; however, the role of RA signaling in pancreatic endocrine cell specification remains unclear. By examining the role of RA using in vivo endocrine-specific inhibition of RA signaling at critical time points during pancreas development, we demonstrate that inhibition of RA signaling in the endocrine progenitor population results in the generation fewer β and increased Δ cell genes by embryonic day (e)16.5. These defects persist after birth and are accompanied by overt hyperglycemia in the adult. Whole transcriptome analysis further indicates that cross-talk between RA signaling and the Wnt pathway is important for islet cell specification. In addition, we show that RA signaling is also important for human β cell specification. Together, these data demonstrate the importance of RA signaling in the endocrine progenitor population and provide a framework to more clearly define the mechanisms by which RA signaling directs endocrine cell fate in mice and humans. Future studies on the role of RA and its interacting pathways during pancreas development will better inform strategies to treat diabetes using cell replacement therapies. Disclosure D. Lorberbaum: None. L. Sussel: None. Funding American Diabetes Association (1-18-PDF-107 to D.L.)

Early Inhibition of Retinoic Acid Signaling Rapidly Generates Cardiomyocytes Expressing Ventricular Markers from Human Induced Pluripotent Stem Cells

Current protocols for the differentiation of cardiomyocytes from human induced pluripotent stem cells (iPSCs) generally require prolonged time in culture and result in heterogeneous cellular populations. We present a method for the generation of beating cardiomyocytes expressing specific ventricular markers after just 14 days. Addition of the pan-retinoic acid receptor inverse agonist BMS 493 to human iPSCs for the first 8 days of differentiation resulted in increased protein expression of the ventricular isoform of myosin regulatory light chain (MLC2V) from 18.7% ± 1.72% to 55.8% ± 11.4% (p <0.0001) in cells co-expressing the cardiac muscle protein troponin T (TNNT2). Increased MLC2V expression was also accompanied by a slower beating rate (49.4 ± 1.53 vs. 93.0 ± 2.81 beats per minute, p <0.0001) and increased contraction amplitude (201% ± 8.33% vs. 100% ± 10.85%, p <0.0001) compared to untreated cells. Improved directed differentiation will improve in vitro cardiac modeling.

Inhibition of retinoic acid signaling induces aberrant pericyte coverage and differentiation resulting in vascular defects in congenital diaphragmatic hernia.

The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.

A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.

Retinoic acid signaling maintains epithelial and mesenchymal progenitors in the developing mouse ureter

The differentiated cell types of the mature ureter arise from the distal ureteric bud epithelium and its surrounding mesenchyme. Uncommitted epithelial cells first become intermediate cells from which both basal and superficial cells develop. Mesenchymal progenitors give rise to separated layers of adventitial fibrocytes, smooth muscle cells and lamina propria fibrocytes. How progenitor expansion and differentiation are balanced is poorly understood. Here, we addressed the role of retinoic acid (RA) signaling in these programs. Using expression analysis of components and target genes, we show that pathway activity is restricted to the mesenchymal and epithelial progenitor pools. Inhibition of RA signaling in ureter explant cultures resulted in tissue hypoplasia with a relative expansion of smooth muscle cells at the expense of lamina propria fibroblasts in the mesenchyme, and of superficial cells at the expense of intermediate cells in the ureteric epithelium. Administration of RA led to a slight reduction of smooth muscle cells, and almost completely prevented differentiation of intermediate cells into basal and superficial cells. We identified cellular programs and transcriptional targets of RA signaling that may account for this activity. We conclude that RA signaling is required and sufficient to maintain mesenchymal and epithelial progenitors in early ureter development.

Abstract 2506: Citral reduces breast tumor growth by inhibiting cancer stem cell marker ALDH1A3

Abstract Breast cancer stem cells (CSCs) can be identified by increased Aldefluor activity which is primarily due to aldehyde dehydrogenase 1A3 (ALDH1A3) expression. In addition to being a CSC marker, ALDH1A3 regulates genes expression via inducing retinoic acid (RA) signaling and plays an important role in mediating progression of cancers, including breast, melanoma, lung and glioblastoma. Therefore, ALDH1A3 represents a novel druggable anti-cancer target of interest. There are no currently characterized inhibitors of the ALDH1A3 isoform, so twelve general ALDH inhibitors were tested for their effectiveness in targeting ALDH1A3 activity. Ability to directly inhibit ALDH activity was quantified with the Aldefluor assay on a patient tumor xenograft and on breast cancer cell lines with known ALDH isoform activity. Inhibition of RA signaling was quantified by measuring expression of the RA-inducible genes RARRES1 and RARβ. To investigate the anti-cancer properties of these compounds, apoptosis was quantified with the annexin V assay. Citral significantly reduced ALDH1A3- and ALDH2-associated Aldefluor activity in breast cancer cell lines and also reduced Aldefluor activity of a patient-derived xenograft. Citral reduced expression of RA-inducible genes through reducing ALDH1A3 activity. Citral induced apoptosis in vitro in an ALDH-independent manner in breast cancer cell lines and reduced proliferation in the MDA-MB-231 breast cancer cell line. Nanoparticle (NP)-encapsulated citral was generated for in vivo use and administered intravenously to female NOD/SCID mice harbouring MDA-MB-231 ALDH1A3-overexpression tumors. 10mg/kg NP-citral significantly decreased the growth of ALDH1A3-driven MDA-MB-231 tumors. Nanoparticle-encapsulated citral shows promise as an adjuvant therapy for patients with tumors that have a large population of high-ALDH1A3 CSCs. Citation Format: Margaret L. Thomas, Roberto De Antueno, Krysta Coyle, Brianne Cruickshank, Michael Giacomantonio, Roy Duncan, Carman Giacomantonio, Paola Marcato. Citral reduces breast tumor growth by inhibiting cancer stem cell marker ALDH1A3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2506.

Abstract B23: Citral reduces ALDH1A3 activity in breast cancer: Potential applications in targeting breast cancer stem cells

Abstract Breast tumors contain a subpopulation of cells known as cancer stem cells (CSCs) which are highly tumorigenic, resistant to typical chemo- and radiotherapy, and express high levels of aldehyde dehydrogenase (ALDH) isoforms ALDH1A3 and ALDH1A1. These enzymes initiate retinoic acid (RA) signaling and potentially contribute to detoxification of chemotherapy. A compound that inhibits these enzymes could be used as an adjuvant therapy to target breast CSCs and/or re-sensitize this population to chemotherapy. In particular, there are no currently characterized inhibitors of the ALDH1A3 isoform, so twelve general ALDH inhibitors were tested for their effectiveness in targeting ALDH1A3 activity. Ability to directly inhibit ALDH activity was quantified with the Aldefluor assay on a patient tumor xenograft and on breast cancer cell lines MDA-MB-231, MDA-MB-468, and SKBR3. Inhibition of RA signaling was quantified by measuring RA-inducible gene expression after 24 hours treatment with ALDH inhibitor. To investigate the anti-cancer properties of these compounds, apoptosis was quantified with the annexin V assay after 24 hours treatment. To determine if increased ALDH1A3 expression is involved in chemoresistance, MDA-MB-231 cells overexpressing ALDH1A3 and MDA-MB-468 cells with ALDH1A3 shRNA knockdown were treated for 72 hours with doxorubicin, 4-hydroperoxycyclophosphamide, or paclitaxel. Increased ALDH1A3 expression did not affect the amount of chemotherapy-induced apoptosis or cell proliferation rates. Citral significantly reduced ALDH1A3-mediated expression of RA-inducible genes and significantly reduced the ALDH activity of the patient-derived xenograft as well as ALDH1A3-overexpression MDA-MB-231 cells. Citral induced apoptosis in an ALDH1A3-dependant manner in breast cancer cell lines, and its ability to reduce MDA-MB-231 in vivo growth was investigated by implanting MDA-MB-231 cells in female NOD/SCID mice and injecting with 0.1mg/kg citral. Tumor size was not affected by citral injections, however recent data suggests that micelle-encapsulated citral has the potential to reduce tumor bulk. Should citral prove to be an effective breast cancer and ALDH1A3 inhibitor in future tumor xenograft experiments, this compound could potentially be used as adjuvant therapy for breast cancer patients. Citation Format: Margaret L. Thomas, Krysta M. Coyle, Brianne Cruickshank, Michael Giacomantonio, Melissa Wallace, Carman Giacomantonio, Paola Marcato. Citral reduces ALDH1A3 activity in breast cancer: Potential applications in targeting breast cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B23.

Retinoic acid plays an evolutionarily conserved and biphasic role in pancreas development

As the developing zebrafish pancreas matures, hormone-producing endocrine cells differentiate from pancreatic Notch-responsive cells (PNCs) that reside within the ducts. These new endocrine cells form small clusters known as secondary (2°) islets. We use the formation of 2° islets in the pancreatic tail of the larval zebrafish as a model of β-cell neogenesis. Pharmacological inhibition of Notch signaling leads to precocious endocrine differentiation and the early appearance of 2° islets in the tail of the pancreas. Following a chemical screen, we discovered that blocking the retinoic acid (RA)-signaling pathway also leads to the induction of 2° islets. Conversely, the addition of exogenous RA blocks the differentiation caused by Notch inhibition. In this report we characterize the interaction of these two pathways. We first verified that signaling via both RA and Notch ligands act together to regulate pancreatic progenitor differentiation. We produced a transgenic RA reporter, which demonstrated that PNCs directly respond to RA signaling through the canonical transcriptional pathway. Next, using a genetic lineage tracing approach, we demonstrated these progenitors produce endocrine cells following inhibition of RA signaling. Lastly, inhibition of RA signaling using a cell-type specific inducible cre/lox system revealed that RA signaling acts cell-autonomously in PNCs to regulate their differentiation. Importantly, the action of RA inhibition on endocrine formation is evolutionarily conserved, as shown by the differentiation of human embryonic stem cells in a model of human pancreas development. Together, these results revealed a biphasic function for RA in pancreatogenesis. As previously shown by others, RA initially plays an essential role during embryogenesis as it patterns the endoderm and specifies the pancreatic field. We reveal here that later in development RA is involved in negatively regulating the further differentiation of pancreatic progenitors and expands upon the developmental mechanisms by which this occurs.

Retinoic Acid-Activated Ndrg1a Represses Wnt/β-catenin Signaling to Allow Xenopus Pancreas, Oesophagus, Stomach, and Duodenum Specification

How cells integrate multiple patterning signals to achieve early endoderm regionalization remains largely unknown. Between gastrulation and neurulation, retinoic acid (RA) signaling is required, while Wnt/β-catenin signaling has to be repressed for the specification of the pancreas, oesophagus, stomach, and duodenum primordia in Xenopus embryos. In attempt to screen for RA regulated genes in Xenopus endoderm, we identified a direct RA target gene, N-myc downstream regulated gene 1a (ndrg1a) that showed expression early in the archenteron roof endoderm and late in the developing pancreas, oesophagus, stomach, and duodenum. Both antisense morpholino oligonucleotide mediated knockdown of ndrg1a in Xenopus laevis and the transcription activator-like effector nucleases (TALEN) mediated disruption of ndrg1 in Xenopus tropicalis demonstrate that like RA signaling, Ndrg1a is specifically required for the specification of Xenopus pancreas, oesophagus, stomach, and duodenum primordia. Immunofluorescence data suggest that RA-activated Ndrg1a suppresses Wnt/β-catenin signaling in Xenopus archenteron roof endoderm cells. Blocking Wnt/β-catenin signaling rescued Ndrg1a knockdown phenotype. Furthermore, overexpression of the putative Wnt/β-catenin target gene Atf3 phenocopied knockdown of Ndrg1a or inhibition of RA signaling, while Atf3 knockdown can rescue Ndrg1a knockdown phenotype. Lastly, the pancreas/stomach/duodenum transcription factor Pdx1 was able to rescue Atf3 overexpression or Ndrg1a knockdown phenotype. Together, we conclude that RA activated Ndrg1a represses Wnt/β-catenin signaling to allow the specification of pancreas, oesophagus, stomach, and duodenum progenitor cells in Xenopus embryos.

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice.

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.