Search

A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 forelimb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Internal N 6 -methyladenosine (m 6 A) modification is widespread in messenger RNAs (mRNAs) and is catalyzed by heterodimers of methyltransferase-like protein 3 (Mettl3) and Mettl14. To understand the role of m 6 A in development, we deleted Mettl14 in embryonic neural stem cells (NSCs) in a mouse model. Phenotypically, NSCs lacking Mettl14 displayed markedly decreased proliferation and premature differentiation, suggesting that m 6 A modification enhances NSC self-renewal. Decreases in the NSC pool led to a decreased number of late-born neurons during cortical neurogenesis. Mechanistically, we discovered a genome-wide increase in specific histone modifications in Mettl14 knockout versus control NSCs. These changes correlated with altered gene expression and observed cellular phenotypes, suggesting functional significance of altered histone modifications in knockout cells. Finally, we found that m 6 A regulates histone modification in part by destabilizing transcripts that encode histone-modifying enzymes. Our results suggest an essential role for m 6 A in development and reveal m 6 A-regulated histone modifications as a previously unknown mechanism of gene regulation in mammalian cells.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications., (© 2017 Cunningham et al.; Published by Cold Spring Harbor Laboratory Press.)

Retinoic acid (RA) is a biologically active metabolite of vitamin A (retinol) that serves as an important signaling molecule in orchestrating diverse developmental processes including multiple roles during ocular development. Loss-of-function studies using gene knockouts of RA-synthesizing enzymes encoded by Aldh1a1, Aldh1a2, and Aldh1a3 (also known as Raldh1, Raldh2, and Raldh3) have provided valuable insight into how RA controls eye morphogenesis including corneal development. However, it is unclear whether endogenous RA is required for maintenance and regeneration of adult cornea. Here, we investigated the role of Aldh1a genes in the adult cornea using a novel conditional Aldh1a1,2,3-flox/flox;Rosa26-CreERT2 loss-of-function mouse model to determine the biological function of RA. Our findings indicate that loss of RA synthesis results in corneal thinning characterized by reduced thickness of the stromal layer, impaired corneal epithelial cell proliferation, and increased apoptosis. Corneal thinning in Aldh1a-deficient mice was significantly rescued by RA administration, indicating an important role of endogenous RA signaling in adult corneal homeostasis and regeneration. Thus, Aldh1a1,2,3-flox/flox;Rosa26-CreERT2 mice provide a useful model for investigating the mechanistic role of RA signaling in adult corneal maintenance and could provide new insights into therapeutic approaches for controlling corneal repair to prevent vision loss., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bipotent neuromesodermal progenitors (NMPs) residing in the caudal epiblast drive coordinated body axis extension by generating both posterior neuroectoderm and presomitic mesoderm. Retinoic acid (RA) is required for body axis extension, however the early molecular response to RA signaling is poorly defined, as is its relationship to NMP biology. As endogenous RA is first seen near the time when NMPs appear, we used WNT/FGF agonists to differentiate embryonic stem cells to NMPs which were then treated with a short 2-h pulse of 25 nM RA or 1 µM RA followed by RNA-seq transcriptome analysis. Differential expression analysis of this dataset indicated that treatment with 25 nM RA, but not 1 µM RA, provided physiologically relevant findings. The 25 nM RA dataset yielded a cohort of previously known caudal RA target genes including Fgf8 (repressed) and Sox2 (activated), plus novel early RA signaling targets with nearby conserved RA response elements. Importantly, validation of top-ranked genes in vivo using RA-deficient Raldh2 -/- embryos identified novel examples of RA activation (Nkx1-2, Zfp503, Zfp703, Gbx2, Fgf15, Nt5e) or RA repression (Id1) of genes expressed in the NMP niche or progeny. These findings provide evidence for early instructive and permissive roles of RA in controlling differentiation of NMPs to neural and mesodermal lineages., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Retinoic acid (RA) repression of Fgf8 is required for many different aspects of organogenesis, however relatively little is known about how endogenous RA controls gene repression as opposed to gene activation. Here, we show that nuclear receptor corepressors NCOR1 and NCOR2 (SMRT) redundantly mediate the ability of RA to repress Fgf8. Ncor1;Ncor2 double mutants generated by CRISPR/Cas9 gene editing exhibited a small somite and distended heart phenotype similar to that of RA-deficient Raldh2-/- embryos, associated with increased Fgf8 expression and FGF signaling in caudal progenitors and heart progenitors. Embryo chromatin immunoprecipitation studies revealed that NCOR1/2 but not coactivators are recruited to the Fgf8 RA response element (RARE) in an RA-dependent manner, whereas coactivators but not NCOR1/2 are recruited RA-dependently to a RARE near Rarb that is activated by RA. CRISPR/Cas9-mediated genomic deletion of the Fgf8 RARE in mouse embryos often resulted in a small somite defect with Fgf8 derepression caudally, but no defect was observed in heart development or heart Fgf8 expression. This suggests the existence of another DNA element whose function overlaps with the Fgf8 RARE to mediate Fgf8 repression by RA and NCOR1/2. Our studies support a model in which NCOR1/2 mediates direct RA-dependent repression of Fgf8 in caudal progenitors in order to control somitogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.

Background: Vertebrate body axis extension occurs in a head-to-tail direction from a caudal progenitor zone that responds to interacting signals. Wnt/β-catenin signaling is critical for generation of paraxial mesoderm, somite formation, and maintenance of the axial stem cell pool. Body axis extension requires Wnt8a in lower vertebrates, but in mammals Wnt3a is required, although the anterior trunk develops in the absence of Wnt3a., Results: We examined mouse Wnt8a(-/-) and Wnt3a(-/-) single and double mutants to explore whether mammalian Wnt8a contributes to body axis extension and to determine whether a posterior growth function for Wnt8a is conserved throughout the vertebrate lineage. We find that caudal Wnt8a is expressed only during early somite stages and is required for normal development of the anterior trunk in the absence of Wnt3a. During this time, we show that Wnt8a and Wnt3a cooperate to maintain Fgf8 expression and prevent premature Sox2 up-regulation in the axial stem cell niche, critical for posterior growth. Similar to Fgf8, Wnt8a requires retinoic acid (RA) signaling to restrict its caudal expression boundary and possesses an upstream RA response element that binds RA receptors., Conclusions: These findings provide new insight into interaction of caudal Wnt-FGF-RA signals required for body axis extension., (© 2015 Wiley Periodicals, Inc.)

Background: The bat has strikingly divergent forelimbs (long digits supporting wing membranes) and hindlimbs (short, typically free digits) due to the distinct requirements of both aerial and terrestrial locomotion. During embryonic development, the morphology of the bat forelimb deviates dramatically from the mouse and chick, offering an alternative paradigm for identifying genes that play an important role in limb patterning., Results: Using transcriptome analysis of developing Natal long-fingered bat (Miniopterus natalensis) fore- and hindlimbs, we demonstrate that the transcription factor Meis2 has a significantly higher expression in bat forelimb autopods compared to hindlimbs. Validation by reverse transcriptase and quantitative polymerase chain reaction (RT-qPCR) and whole mount in situ hybridisation shows that Meis2, conventionally known as a marker of the early proximal limb bud, is upregulated in the bat forelimb autopod from CS16. Meis2 expression is localised to the expanding interdigital webbing and the membranes linking the wing to the hindlimb and tail. In mice, Meis2 is also expressed in the interdigital region prior to tissue regression. This interdigital Meis2 expression is not activated by retinoic acid (RA) signalling as it is present in the retained interdigital tissue of Rdh10 (trex/trex) mice, which lack RA. Additionally, genes encoding RA-synthesising enzymes, Rdh10 and Aldh1a2, and the RA nuclear receptor Rarβ are robustly expressed in bat fore- and hindlimb interdigital tissues indicating that the mechanism that retains interdigital tissue in bats also occurs independently of RA signalling., Conclusions: Mammalian interdigital Meis2 expression, and upregulation in the interdigital webbing of bat wings, suggests an important role for Meis2 in autopod development. Interdigital Meis2 expression is RA-independent, and retention of interdigital webbing in bat wings is not due to the suppression of RA-induced cell death. Rather, RA signalling may play a role in the thinning (rather than complete loss) of the interdigital tissue in the bat forelimb, while Meis2 may interact with other factors during both bat and mouse autopod development to maintain a pool of interdigital cells that contribute to digit patterning and growth.

Retinoic acid (RA) signalling has a central role during vertebrate development. RA synthesized in specific locations regulates transcription by interacting with nuclear RA receptors (RARs) bound to RA response elements (RAREs) near target genes. RA was first implicated in signalling on the basis of its teratogenic effects on limb development. Genetic studies later revealed that endogenous RA promotes forelimb initiation by repressing fibroblast growth factor 8 (Fgf8). Insights into RA function in the limb serve as a paradigm for understanding how RA regulates other developmental processes. In vivo studies have identified RAREs that control repression of Fgf8 during body axis extension or activation of homeobox (Hox) genes and other key regulators during neuronal differentiation and organogenesis.

Although many regulatory networks involved in defining definitive endoderm have been identified, the mechanisms through which these networks interact to pattern the endoderm are less well understood. To explore the mechanisms involved in midgut patterning, we dissected the transcriptional regulatory elements of nephrocan (Nepn), the earliest known midgut specific gene in mice. We observed that Nepn expression is dramatically reduced in Sox17(-/-) and Raldh2(-/-) embryos compared with wild-type embryos. We further show that Nepn is directly regulated by Sox17 and the retinoic acid (RA) receptor via two enhancer elements located upstream of the gene. Moreover, Nepn expression is modulated by Activin signaling, with high levels inhibiting and low levels enhancing RA-dependent expression. In Foxh1(-/-) embryos in which Nodal signaling is reduced, the Nepn expression domain is expanded into the anterior gut region, confirming that Nodal signaling can modulate its expression in vivo. Together, Sox17 is required for Nepn expression in the definitive endoderm, while RA signaling restricts expression to the midgut region. A balance of Nodal/Activin signaling regulates the anterior boundary of the midgut expression domain., (© 2014. Published by The Company of Biologists Ltd.)

Unlabelled: The important roles of retinols and their metabolites have recently been emphasized in the interactions between hepatic stellate cells (HSCs) and natural killer (NK) cells. Nevertheless, the expression and role of retinol metabolizing enzyme in both cell types have yet to be clarified. Thus, we investigated the expression of retinol metabolizing enzyme and its role in liver fibrosis. Among several retinol metabolizing enzymes, only alcohol dehydrogenase (ADH) 3 expression was detected in isolated HSCs and NK cells, whereas hepatocytes express all of them. In vitro treatment with 4-methylpyrazole (4-MP), a broad ADH inhibitor, or depletion of the ADH3 gene down-regulated collagen and transforming growth factor-β1 (TGF-β1) gene expression, but did not affect α-smooth muscle actin gene expression in cultured HSCs. Additionally, in vitro, treatments with retinol suppressed NK cell activities, whereas inhibition of ADH3 enhanced interferon-γ (IFN-γ) production and cytotoxicity of NK cells against HSCs. In vivo, genetic depletion of the ADH3 gene ameliorated bile duct ligation- and carbon tetrachloride-induced liver fibrosis, in which a higher number of apoptotic HSCs and an enhanced activation of NK cells were detected. Freshly isolated HSCs from ADH3-deficient mice showed reduced expression of collagen and TGF-β1, but enhanced expression of IFN-γ was detected in NK cells from these mice compared with those of control mice. Using reciprocal bone marrow transplantation of wild-type and ADH3-deficient mice, we demonstrated that ADH3 deficiency in both HSCs and NK cells contributed to the suppressed liver fibrosis., Conclusion: ADH3 plays important roles in promoting liver fibrosis by enhancing HSC activation and inhibiting NK cell activity, and could be used as a potential therapeutic target for the treatment of liver fibrosis., (© 2014 by the American Association for the Study of Liver Diseases.)

Retinoic acid (RA) generated in the mesoderm of vertebrate embryos controls body axis extension by downregulating Fgf8 expression in cells exiting the caudal progenitor zone. RA activates transcription by binding to nuclear RA receptors (RARs) at RA response elements (RAREs), but it is unknown whether RA can directly repress transcription. Here, we analyzed a conserved RARE upstream of Fgf8 that binds RAR isoforms in mouse embryos. Transgenic embryos carrying Fgf8 fused to lacZ exhibited expression similar to caudal Fgf8, but deletion of the RARE resulted in ectopic trunk expression extending into somites and neuroectoderm. Epigenetic analysis using chromatin immunoprecipitation of trunk tissues from E8.25 wild-type and Raldh2(-/-) embryos lacking RA synthesis revealed RA-dependent recruitment of the repressive histone marker H3K27me3 and polycomb repressive complex 2 (PRC2) near the Fgf8 RARE. The co-regulator RERE, the loss of which results in ectopic Fgf8 expression and somite defects, was recruited near the RARb RARE by RA, but was released from the Fgf8 RARE by RA. Our findings demonstrate that RA directly represses Fgf8 through a RARE-mediated mechanism that promotes repressive chromatin, thus providing valuable insight into the mechanism of RA-FGF antagonism during progenitor cell differentiation., (© 2014. Published by The Company of Biologists Ltd.)

Background: The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family., Results: Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not present in amphibians and reptiles., Conclusions: The study of the ancient forms of ADH2 and ADH7 sheds new light on the evolution of the vertebrate ADH system, whereas the special features showed by the novel forms point to the acquisition of new functions following the ADH gene family expansion which occurred in amphibians.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The vitamin A metabolite retinoic acid (RA) serves as a ligand for nuclear RA receptors that control differentiation of progenitor cells important for vertebrate development. Genetic studies in mouse embryos deficient for RA-generating enzymes have been invaluable for deciphering RA function. RA first begins to act during early organogenesis when RA generated in trunk mesoderm begins to function as a diffusible signal controlling progenitor cell differentiation. In neuroectoderm, RA functions as an instructive signal to stimulate neuronal differentiation of progenitor cells in the hindbrain and spinal cord. RA is not required for early neuronal differentiation of the forebrain, but at later stages RA stimulates neuronal differentiation in forebrain basal ganglia. RA also acts as a permissive signal for differentiation by repressing fibroblast growth factor (FGF) signaling in differentiated cells as they emerge from progenitor populations in the caudal progenitor zone and second heart field. In addition, RA signaling stimulates differentiation of spermatogonial germ cells and induces meiosis in male but not female gonads. A more complete understanding of the normal functions of RA signaling during development will guide efforts to use RA as a differentiation agent for therapeutic purposes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The enteric nervous system (ENS) forms from the neural crest-derived precursors that colonize the bowel before differentiating into a network of neurons and glia that control intestinal function. Retinoids are essential for normal ENS development, but the role of retinoic acid (RA) metabolism in development remains incompletely understood. Because RA is produced locally in the tissues where it acts by stimulating RAR and RXR receptors, RA signaling during development is absolutely dependent on the rate of RA synthesis and degradation. RA is produced by three different enzymes called retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3) that are all expressed in the developing bowel. To determine the relative importance of these enzymes for ENS development, we analyzed whole mount preparations of adult (8-12-week old) myenteric and submucosal plexus stained with NADPH diaphorase (neurons and neurites), anti-TuJ1 (neurons and neurites), anti-HuC/HuD (neurons), and anti-S100β (glia) in an allelic series of mice with mutations in Raldh1, Raldh2, and Raldh3. We found that Raldh1-/-, Raldh2+/-, Raldh3+/- (R1(KO)R2(Het)R3(Het)) mutant mice had a reduced colon myenteric neuron density, reduced colon myenteric neuron to glia ratio, reduced colon submucosal neuron density, and increased colon myenteric fibers per neuron when compared to the wild type (WT; Raldh1WT, Raldh2WT, Raldh3WT) mice. These defects are unlikely to be due to defective ENS precursor migration since R1(KO)R2(Het)R3(KO) mice had increased enteric neuron progenitor migration into the distal colon compared to WT during development. RALDH mutant mice also have reduced contractility in the colon compared to WT mice. These data suggest that RALDH1, RALDH2 and RALDH3 each contribute to ENS development and function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Retinoic acid (RA) signaling controls patterning and neuronal differentiation within the hindbrain, but forebrain RA function remains controversial. RA is produced from metabolism of retinol to retinaldehyde by retinol dehydrogenase (RDH), followed by metabolism of retinaldehyde to RA by retinaldehyde dehydrogenase (RALDH). Previous studies on Raldh2-/- and Raldh3-/- mice demonstrated an RA requirement for γ-aminobutyric acid (GABA)ergic and dopaminergic differentiation in forebrain basal ganglia, but no RA requirement was observed during early forebrain patterning or subsequent forebrain cortical expansion. However, other studies suggested that RA controls forebrain patterning, and analysis of ethylnitrosourea-induced Rdh10 mutants suggested that RA synthesized in the meninges stimulates forebrain cortical expansion., Results: We generated Rdh10-/- mouse embryos that lack RA activity early in the head and later in the meninges. We observed defects in hindbrain patterning and eye RA signaling, but early forebrain patterning was unaffected. Retinaldehyde treatment of Rdh10-/- embryos from E7-E9 rescues a cranial skeletal defect, resulting in E14.5 embryos lacking meningeal RA activity but maintaining normal forebrain shape and cortical expansion., Conclusions: Rdh10-/- embryos demonstrate that RA controls hindbrain but not early forebrain patterning, while studies on retinaldehyde-rescued Rdh10-/- embryos show that meningeal RA synthesis is unnecessary to stimulate forebrain cortical expansion., (Copyright © 2013 Wiley Periodicals, Inc.)

In mammalian species, the process of meiosis, in which genes are randomly assorted between parental chromosomes during formation of egg and sperm cells, occurs prenatally in females but postnatally in males. To understand sex-specific differences in signaling mechanisms that underlie fertility, many studies have focused on identifying factors that control meiotic induction. Studies in mice using genetic knockout of the transcriptional regulator Polycomb repressive complex-1 (PRC1) and pharmacological inhibition of retinoic acid (RA) signaling suggest that PRC1 prevents female meiotic induction until release of PRC1 repression by increased RA signaling in the ovary. However, genetic studies with mice lacking RA synthesis in reproductive tissues indicate that RA is required for male but not female meiosis, suggesting that RA functions as a male-specific inducer of meiosis and that another factor releases PRC1 repression to initiate female meiosis. Correct resolution of the molecular events governing female and male meiosis is important for treating infertility and devising improved birth control strategies.

The vitamin A metabolite retinoic acid (RA) provides patterning information during vertebrate embryogenesis, but the mechanism through which RA influences limb development is unclear. During patterning of the limb proximodistal axis (upper limb to digits), avian studies suggest that a proximal RA signal generated in the trunk antagonizes a distal fibroblast growth factor (FGF) signal. However, mouse and zebrafish genetic studies suggest that loss of RA suppresses forelimb initiation. Here, using genetic and pharmacological approaches, we demonstrate that limb proximodistal patterning is not RA dependent, thus indicating that RA-FGF antagonism does not occur along the proximodistal axis of the limb. Instead, our studies show that RA-FGF antagonism acts prior to limb budding along the anteroposterior axis of the trunk lateral plate mesoderm to provide a patterning cue that guides formation of the forelimb field. These findings reconcile disparate ideas regarding RA-FGF antagonism and provide insight into how endogenous RA programs the early embryo., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1(-/-) mice, but not males, were resistant to high-fat (HF) diet-induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1(-/-) mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet-induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.

Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.

Unlabelled: Clinical trials and animal models suggest that infusion of bone marrow cells (BMCs) is effective therapy for liver fibrosis, but the underlying mechanisms are obscure, especially those associated with early effects of BMCs. Here, we analyzed the early impact of BMC infusion and identified the subsets of BMCs showing antifibrotic effects in mice with carbon tetrachloride-induced liver fibrosis. An interaction between BMCs and activated hepatic stellate cells (HSCs) was investigated using an in vitro coculturing system. Within 24 hours, infused BMCs were in close contact with activated HSCs, which was associated with reduced liver fibrosis, enhanced hepatic expression of interleukin (IL)-10, and expanded regulatory T cells but decreased macrophage infiltration in the liver at 24 hours after BMC infusion. In contrast, IL-10-deficient (IL-10(-/-) ) BMCs failed to reproduce these effects in fibrotic livers. Intriguingly, in isolated cells, CD11b(+) Gr1(high) F4/80(-) and CD11b(+) Gr1(+) F4/80(+) BMCs expressed more IL-10 after coculturing with activated HSCs, leading to suppressed expression of collagen and α-smooth muscle actin in HSCs. Moreover, these effects were either enhanced or abrogated, respectively, when BMCs were cocultured with IL-6(-/-) and retinaldehyde dehydrogenase 1(-/-) HSCs. Similar to murine data, human BMCs expressed more IL-10 after coculturing with human HSC lines (LX-2 or hTERT), and serum IL-10 levels were significantly elevated in patients with liver cirrhosis after autologous BMC infusion., Conclusion: Activated HSCs increase IL-10 expression in BMCs (CD11b(+) Gr1(high) F4/80(-) and CD11b(+) Gr1(+) F4/80(+) cells), which in turn ameliorates liver fibrosis. Our findings could enhance the design of BMC therapy for liver fibrosis., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Although alginate-poly-L-lysine (AP(L)) encapsulation of cells producing bioactive peptides has been widely tested, it is unknown whether AP(L) supports lasting catabolic functions of encapsulated cells in adipose tissue, which are required for obesity reduction. We tested functions of AP(L)-encapsulated fibroblasts isolated from wild-type (WT) and aldehyde dehydrogenase 1a1 knockout mice (KO), which resist obesity on a high-fat (HF) diet, have a higher metabolic rate, and express increased levels of thermogenic uncoupling protein-1 (Ucp1) in their deleterious visceral fat depots compared to WT mice. To enable in vivo detection and quantification, fibroblasts were stably transfected with green-fluorescent protein. WT- or KO-containing microcapsules were injected into two visceral depots of WT mice fed an HF diet. Eighty days after transplantation, microcapsules were located in vivo using magnetic resonance imaging. KO microcapsules prevented weight gain in obese WT mice compared to a mock- and WT capsule-injected groups on an HF diet. The weight loss in KO-treated mice corresponded to lipid reduction and induction of thermogenesis in the injected visceral fat. The non-treated subcutaneous fat was not altered. Our data suggest that the AP(L) polymer supports long-term catabolic functions of genetically-modified fibroblasts, which can be potentially used for depot-specific obesity treatment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Recent data link vitamin A and its retinoid metabolites to the regulation of adipogenesis, insulin sensitivity, and glucose homeostasis. Retinoid metabolism is tightly controlled by an enzymatic network in which retinaldehyde dehydrogenases (Aldh1-3) are the rate-limiting enzymes that convert retinaldehyde to retinoic acid. Aldh1a1-deficient mice are protected from diet-induced obesity and hence diabetes. Here we investigated whether Aldh1a1 and the retinoid axis regulate hepatic glucose and lipid metabolism independent of adiposity. The impact of Aldh1a1 and the retinoid pathway on glucose homeostasis and lipid metabolism was analyzed in hepatocytes in vitro and in chow-fed, weight-matched Aldh1a1-deficient vs. wild-type (WT) mice in vivo. Aldh1a1-deficient mice displayed significantly decreased fasting glucose concentrations compared with WT controls as a result of attenuated hepatic glucose production. Expression of key gluconeogenic enzymes as well as the activity of Forkhead box O1 was decreased in Aldh1a1-deficient vs. WT livers. In vitro, retinoid or cAMP agonist stimulation markedly induced gluconeogenesis in WT but not Aldh1a1-deficient primary hepatocytes. Aldh1a1 deficiency increased AMP-activated protein kinase α activity, decreased expression of lipogenic targets of AMP-activated protein kinase α and significantly attenuated hepatic triacylglycerol synthesis. In metabolic cage studies, lean Aldh1a1-deficient mice manifested enhanced oxygen consumption and reduced respiratory quotient vs. WT controls, consistent with increased expression of fatty acid oxidation markers in skeletal muscle. Taken together, this work establishes a role for retinoid metabolism in glucose homeostasis in vivo and for Aldh1a1 as a novel determinant of gluconeogenesis and lipid metabolism independent of adiposity.

High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Alcohol dehydrogenases (ADH) catalyze the reversible metabolism of many types of alcohols and aldehydes to prevent the possible toxic accumulation of these compounds. ADHs are of interest in Parkinson's disease (PD) since these compounds can be harmful to dopamine (DA) neurons. Genetic variants in ADH1C and ADH4 have been found to associate with PD and lack of Adh4 gene activity in a mouse model has recently been reported to induce changes in the DA system. Adh1 knockout (Adh1-/-) and Adh1/4 double knockout (Adh1/4-/-) mice were investigated for possible changes in DA system related activity, biochemical parameters and olfactory function compared to wild-type (WT) mice. Locomotor activity was tested at ∼7 (adult) and >15 months of age to mimic the late onset of PD. Adh1-/- and Adh1/4-/- mice displayed a significantly higher spontaneous locomotor activity than WT littermates. Both apomorphine and d-amphetamine increased total distance activity in Adh1-/- mice at both age intervals and in Adh1/4-/- mice at 7 months of age compared to WT mice. No significant changes were found regarding olfactory function, however biochemical data showed decreased 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in the olfactory bulb and decreased homovanillic acid (HVA)/DA ratios in the olfactory bulb, frontal cortex and striatum of Adh1/4-/- mice compared to WT mice. Our results suggest that lack of Adh1 alone or Adh1 and Adh4 together lead to changes in DA system related behavior, and that these knockout mice might be possible rodent models to study presymptomatic PD., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Retinoic acid (RA) is the active metabolite of vitamin A (retinol) that controls growth and development. The first step of RA synthesis is controlled by enzymes of the alcohol dehydrogenase (ADH) and retinol dehydrogenase (RDH) families that catalyze oxidation of retinol to retinaldehyde. The second step of RA synthesis is controlled by members of the aldehyde dehydrogenase (ALDH) family also known as retinaldehyde dehydrogenase (RALDH) that further oxidize retinaldehyde to produce RA. RA functions as a ligand for DNA-binding RA receptors that directly regulate transcription of specific target genes. Elucidation of the vitamin A metabolic pathway and investigation of the endogenous function of vitamin A metabolites has been greatly improved by development of mouse ADH, RDH, and RALDH loss-of-function models. ADH knockouts have demonstrated a postnatal role for this enzyme family in clearance of excess retinol to prevent vitamin A toxicity and in generation of RA for postnatal survival during vitamin A deficiency. A point mutation in Rdh10 generated by ethylnitrosourea has demonstrated that RDH10 generates much of the retinaldehyde needed for RA synthesis during embryonic development. Raldh1, Raldh2, and Raldh3 knockouts have demonstrated that RALDH1, RALDH2, and RALDH3 generate most of the RA needed during embryogenesis. These mouse models serve as instrumental tools for providing new insight into retinoid function. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism., (© 2011 Elsevier B.V. All rights reserved.)

The ganglionic eminence contributes cells to several forebrain structures including the cerebral cortex, for which it provides GABAergic interneurons. Migration of neuronal precursors from the retinoic-acid rich embryonic ganglionic eminence to the cerebral cortex is known to be regulated by several factors, but retinoic acid has not been previously implicated. We found retinoic acid to potently inhibit cell migration in slice preparations of embryonic mouse forebrains, which was reversed by an antagonist of the dopamine-D(2) receptor, whose gene is transcriptionally regulated by retinoic acid. Histone-deacetylase inhibitors, which amplify nuclear receptor-mediated transcription, potentiated the inhibitory effect of retinoic acid. Surprisingly, when retinoic acid signalling was completely blocked with a pan-retinoic acid receptor antagonist, this also decreased cell migration into the cortex, implying that a minimal level of endogenous retinoic acid is necessary for tangential migration. Given these opposing effects of retinoic acid in vitro, the in vivo contribution of retinoic acid to migration was tested by counting GABAergic interneurons in cortices of adult mice with experimental reductions in retinoic acid signalling: a range of perturbations resulted in significant reductions in the numerical density of some GABAergic interneuron subpopulations. These observations suggest functions of retinoic acid in interneuron diversity and organization of cortical excitatory-inhibitory balance., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Aldehyde dehydrogenase 1A1 (ALDH1A1) is a member of a superfamily of detoxification enzymes found in various tissues that participate in the oxidation of both aliphatic and aromatic aldehydes. In the brain, ALDH1A1 participates in the metabolism of catecholamines including dopamine (DA) and norepinephrine, but is uniquely expressed in a subset of dopaminergic (DAergic) neurons in the ventral mesencephalon where it converts 3,4-dihydroxyphenylacetaldehyde, a potentially toxic aldehyde, to 3,4-dihydroxyphenylacetic acid, a non toxic metabolite. Therefore, loss of ALDH1A1 expression could be predicted to alter DA metabolism and potentially increase neurotoxicity in ventral mesencephalic DA neurons. Recent reports of reduced levels of expression of both Aldh1a1 mRNA and protein in the substantia nigra (SN) of Parkinson's disease patients suggest possible involvement of ALDH1A1 in this progressive neurodegenerative disease. The present study used an Aldh1a1 null mouse to assess the influence of ALDH1A1 on the function and maintenance of the DAergic system. Results indicate that the absence of Aldh1a1 did not negatively affect growth and development of SN DA neurons nor alter protein expression levels of tyrosine hydroxylase, the DA transporter or vesicular monoamine transporter 2. However, absence of Aldh1a1 significantly increased basal extracellular DA levels, decreased KCl and amphetamine stimulated DA release and decreased DA re-uptake and resulted in more tyrosine hydroxylase expressing neurons in the SN than in wildtype animals. These data suggest that in young adult animals with deletion of the Aldh1a1 gene there is altered DA metabolism and dysfunction of the DA transporter and DA release mechanisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Vitamin A metabolite retinoic acid (RA) regulates life-sustaining differentiation processes and metabolic homeostasis. The aldehyde dehydrogenase-1 (Aldh1) family of enzymes (Aldh1a1, a2, and a3) catalyzes RA production from retinaldehyde and thereby controls concentrations of this transcriptionally active metabolite. The hierarchy of Aldh1 functions in adipose tissue has not been elucidated. We hypothesized that Aldh1 enzymes produce endogenous RA and regulate adipogenesis and fat formation in a fat depot-specific manner. We demonstrate that adipogenesis in vitro is accompanied by RA production generated primarily by Aldh1a1. In Aldh1a1-deficient adipocytes, adipogenesis is impaired compared with wild-type adipocytes due to markedly reduced expression of PPARγ regulated through zinc-finger protein 423 (ZFP423)-dependent mechanisms. These effects were recovered to some extent either by RA stimulation or overexpression of any of the Aldh1 enzymes in Aldh1a1(-/-) cells arguing that Aldh1a1 plays a dominant role in autocrine RA production. In vivo studies in C57/BL6 and Aldh1a1(-/-) mice on a regular diet revealed that multiple Aldh1 enzymes regulate differences in the formation of sc and visceral fat. In Aldh1a1(-/-) mice, visceral fat essentially lacked all Aldh1 expression. This loss of RA-producing enzymes was accompanied by 70% decreased expression of ZFP423, PPARγ, and Fabp4 in visceral fat of Aldh1a1(-/-) vs. wild-type mice and by the predominant loss of visceral fat. Subcutaneous fat of Aldh1a1(-/-) mice expressed Aldh1a3 for RA production that was sufficient to maintain expression of ZFP423 and PPARγ and sc fat mass. Our data suggest a paradigm for regulation of fat depots through the concerted action of Aldh1 enzymes that establish RA-dependent tandem regulation of transcription factors ZFP423 and PPARγ in a depot-specific manner.

Retinoic acid (RA) is purported to be required for expression of genes controlling proximodistal (Meis2) or anteroposterior (Shh) limb patterning. Embryos lacking RDH10, the primary enzyme synthesizing retinaldehyde during mouse development, survive until E14.5 with stunted forelimbs but apparently normal hindlimbs. Using embryos carrying the RARE-lacZ RA-reporter transgene, we show that endogenous RA activity in Rdh10(trex/trex) mutants is detected in neuroectoderm but not limbs during initiation and patterning. Treatment of Rdh10 mutants with 25 nM RA restores RARE-lacZ activity to limb mesoderm, validating RARE-lacZ and verifying that RA is absent in mutant limbs. In Rdh10 mutants, hindlimbs exhibit normal Meis2/Shh expression and skeletal patterning, and although forelimbs are growth-retarded their Meis2 expression remains normal. Later in development, Rdh10 mutants lack interdigital RA activity and accordingly fail to exhibit normal loss of interdigital mesenchyme. These findings demonstrate that RA is unnecessary for limb patterning but required later for interdigital tissue loss., (Copyright © 2011 Wiley-Liss, Inc.)

Although retinoic acid (RA) has been implicated as an extrinsic signal regulating forebrain neurogenesis, the processes regulated by RA signaling remain unclear. Here, analysis of retinaldehyde dehydrogenase mutant mouse embryos lacking RA synthesis demonstrates that RA generated by Raldh3 in the subventricular zone of the basal ganglia is required for GABAergic differentiation, whereas RA generated by Raldh2 in the meninges is unnecessary for development of the adjacent cortex. Neurospheres generated from the lateral ganglionic eminence (LGE), where Raldh3 is highly expressed, produce endogenous RA, which is required for differentiation to GABAergic neurons. In Raldh3⁻/⁻ embryos, LGE progenitors fail to differentiate into either GABAergic striatal projection neurons or GABAergic interneurons migrating to the olfactory bulb and cortex. We describe conditions for RA treatment of human embryonic stem cells that result in efficient differentiation to a heterogeneous population of GABAergic interneurons without the appearance of GABAergic striatal projection neurons, thus providing an in vitro method for generation of GABAergic interneurons for further study. Our observation that endogenous RA is required for generation of LGE-derived GABAergic neurons in the basal ganglia establishes a key role for RA signaling in development of the forebrain., Competing Interests: The authors have declared that no competing interests exist.

Class IV alcohol dehydrogenase (ADH4) efficiently reduces aldehydes produced during lipid peroxidation, and may thus serve to protect from toxic effects of aldehydes e.g. on neurons. We hypothesized that ADH4 dysfunction may increase risk for Parkinson's disease (PD) and previously reported association of an ADH4 allele with PD. We found that a promoter polymorphism in this allele induced a 25-30% reduction of transcriptional activity. Based on these findings, we have now investigated whether Adh4 homo- (Adh4-/-) or heterozygous (Adh4+/-) knockout mice display any dopamine system-related changes in behavior, biochemical parameters or olfaction compared to wild-type mice. The spontaneous locomotor activity was found to be similar in the three groups, whereas administration of d-amphetamine or apomorphine induced a significant increase in horizontal activity in the Adh4-/- mice compared to wild-type mice. We measured levels of monoamines and their metabolites in striatum, frontal cortex and substantia nigra and found increased levels of dopamine and DOPAC in substantia nigra of Adh4-/- mice. Investigation of olfactory function revealed a reduced sense of smell in Adh4-/- mice accompanied by alterations in dopamine metabolite levels in the olfactory bulb. Taken together, our results suggest that lack of Adh4 gene activity induces changes in the function of the dopamine system, findings which are compatible with a role of loss-of-function mutations in ADH4 as possible risk factors for PD., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Sex-specific initiation of meiosis in the fetal ovary has been suggested to require retinoic acid (RA) for induction of Stra8, with expression of the RA-degrading enzyme Cyp26b1 in fetal testis delaying meiosis until postnatal development. In this study, we investigate Raldh2(-/-) mice lacking RA synthesis and signalling in mesonephros and adjacent gonad and reveal that Stra8 expression in the fetal ovary does not require RA signalling. In contrast to previous observations, we find that Stra8 is expressed in the absence of physiologically detectable levels of RA. Ketoconazole inhibition of Cyp26b1 in Raldh2(-/-) testis allows RA-independent induction of Stra8, but only when the mesonephros remains attached, pointing to a non-RA signal from the mesonephros that induces Stra8 in the adjacent gonad. These findings demonstrate that Cyp26b1 prevents the onset of meiosis by metabolizing a substrate other than RA that controls Stra8 expression, thus changing the paradigm for how studies on Cyp26 function are conducted.

Epicardial signaling and Rxra are required for expansion of the ventricular myocardial compact zone. Here, we examine Raldh2(-/-) and Rxra(-/-) mouse embryos to investigate the role of retinoic acid (RA) signaling in this developmental process. The heart phenotypes of Raldh2 and Rxra mutants are very similar and are characterized by a prominent defect in ventricular compact zone growth. Although RA activity is completely lost in Raldh2(-/-) epicardium and the adjacent myocardium, RA activity is not lost in Rxra(-/-) hearts, suggesting that RA signaling in the epicardium/myocardium is not required for myocardial compact zone formation. We explored the possibility that RA-mediated target gene transcription in non-cardiac tissues is required for this process. We found that hepatic expression of erythropoietin (EPO), a secreted factor implicated in myocardial expansion, is dependent on both Raldh2 and Rxra. Chromatin immunoprecipitation studies support Epo as a direct target of RA signaling in embryonic liver. Treatment of an epicardial cell line with EPO, but not RA, upregulates Igf2. Furthermore, both Raldh2(-/-) and Rxra(-/-) hearts exhibit downregulation of Igf2 mRNA in the epicardium. EPO treatment of cultured Raldh2(-/-) hearts restores epicardial Igf2 expression and rescues ventricular cardiomyocyte proliferation. We propose a new model for the mechanism of RA-mediated myocardial expansion in which RA directly induces hepatic Epo resulting in activation of epicardial Igf2 that stimulates compact zone growth. This RA-EPO-IGF2 signaling axis coordinates liver hematopoiesis with heart development.

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB(1) cannabinoid receptors (CB(1)R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB(1)R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB(1)R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227-235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB(1)R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB(1)R mRNA and protein, the most efficacious being the RARgamma agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB(1)R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARgamma to the CB(1)R gene 5' upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB(1)R expression was attenuated by small interfering RNA knockdown of RARgamma in hepatocytes. We conclude that RARgamma regulates CB(1)R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals., (Copyright 2010 Elsevier Inc. All rights reserved.)

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/beta-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/beta-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Previous studies on retinoic acid receptor (RAR) mutants suggested that retinoic acid (RA) is required for loss of interdigital mesenchyme during digit formation. Here, we report that the RA-generating enzyme retinaldehyde dehydrogenase-2 (Raldh2) is expressed in the interdigital mesenchyme whereas Cyp26b1, controlling RA degradation, is expressed in digits, limiting autopodal RA action to the interdigital zones. Embryonic day 13.5 Raldh2-/- mouse embryos lose expression of the RARE-lacZ RA-reporter transgene and matrix metalloproteinase-11 (Mmp11) throughout the interdigital mesenchyme, while expression of RARb, Fgf18, and high mobility group N1 (Hmgn1) is lost at the digit-interdigit junction. Raldh2-/- autopods exhibit reduced interdigital apoptosis associated with loss of Bmp7 expression, but Bmp2, Bmp4, Msx2, and Fgf8 were unaffected. Although interdigital expression of Hmgn1 was greatly down-regulated in Raldh2-/- autopods, complementary expression of Sox9 in digit cartilage was unaffected. Regulation of Hmgn1 and Fgf18 at the digit-interdigit junction suggests RA controls tissue remodeling as well as apoptosis.

In humans and mice, mutations in the Ret gene result in Hirschsprung's disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.

Cell-cell signaling regulated by retinoic acid (RA), Wnt/beta-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/beta-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2(-/-) mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2(-/-) mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/beta-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/beta-catenin signaling as the body axis extends.

Retinoic acid (RA) is thought to be a key signaling molecule involved in limb bud patterning along the proximodistal or anteroposterior axes functioning through induction of Meis2 and Shh, respectively. Here, we utilize Raldh2-/- and Raldh3-/- mouse embryos lacking RA synthesis to demonstrate that RA signaling is not required for limb expression of Shh and Meis2. We demonstrate that RA action is required outside of the limb field in the body axis during forelimb induction but that RA is unnecessary at later stages when hindlimb budding and patterning occur. We provide evidence for a model of trunk mesodermal RA action in which forelimb induction requires RA repression of Fgf8 in the developing trunk similar to how RA controls somitogenesis and heart development. We demonstrate that pectoral fin development in RA-deficient zebrafish embryos can be rescued by an FGF receptor antagonist SU5402. In addition, embryo ChIP assays demonstrate that RA receptors bind the Fgf8 promoter in vivo. Our findings suggest that RA signaling is not required for limb proximodistal or anteroposterior patterning but that RA inhibition of FGF8 signaling during the early stages of body axis extension provides an environment permissive for induction of forelimb buds.

Retinoic acid is a metabolic derivative of vitamin A that plays an essential function in cell-cell signaling by serving as a ligand for nuclear receptors that directly regulate gene expression. The final step in the conversion of retinol to retinoic acid is carried out by three retinaldehyde dehydrogenases encoded by Raldh1 (Aldh1a1), Raldh2 (Aldh1a2), and Raldh3 (Aldh1a3). Mouse Raldh gene knockout studies have been instrumental in understanding the mechanism of retinoic acid action during eye development. Retinoic acid signaling in the developing eye is particularly complex as all three Raldh genes contribute to retinoic acid synthesis in non-overlapping locations. During optic cup formation Raldh2 is first expressed transiently in perioptic mesenchyme, then later Raldh1 and Raldh3 expression begins in the dorsal and ventral retina, respectively, and these sources of retinoic acid are maintained in the fetus. Retinoic acid is not required for dorsoventral patterning of the retina as originally thought, but it is required for morphogenetic movements that form the optic cup, ventral retina, cornea, and eyelids. These findings will help guide future studies designed to identify retinoic acid target genes during eye organogenesis.

High levels of aldehyde dehydrogenase (ALDH) activity have been proposed to be a common feature of stem cells. Adult hematopoietic, neural, and cancer stem cells have all been reported to have high ALDH activity, detected using Aldefluor, a fluorogenic substrate for ALDH. This activity has been attributed to Aldh1a1, an enzyme that is expressed at high levels in stem cells and that has been suggested to regulate stem cell function. Nonetheless, Aldh1a1 function in stem cells has never been tested genetically. We observed that Aldh1a1 was preferentially expressed in mouse hematopoietic stem cells (HSCs) and expression increased with age. Hematopoietic cells from Aldh1a1-deficient mice exhibited increased sensitivity to cyclophosphamide in a non-cell-autonomous manner, consistent with its role in cyclophosphamide metabolism in the liver. However, Aldh1a1 deficiency did not affect hematopoiesis, HSC function, or the capacity to reconstitute irradiated recipients in young or old adult mice. Aldh1a1 deficiency also did not affect Aldefluor staining of hematopoietic cells. Finally, Aldh1a1 deficiency did not affect the function of stem cells from the adult central or peripheral nervous systems. Aldh1a1 is not a critical regulator of adult stem cell function or Aldefluor staining in mice.

The alcohol dehydrogenase enzymes in mice and humans are encoded by a linked group of genes in the same transcriptional orientation. The enzymes play important roles in alcohol metabolism and retinoid signaling and homeostasis. The expression patterns at the mRNA level of the mouse Adh4 (class II) gene and the recently identified Adh6a and Adh6b genes (class V) are now reported to complete this analysis for the entire family. Adh4 is expressed at high levels in liver and is detectable in small intestine and testes. Adh6b is expressed in liver but Adh6a is not. Adh6a is expressed at high levels in small intestine while Adh6b is not. Adh6a expression is detectable in the female adrenal and not at all in the male adrenal, but Adh6b is expressed at moderate levels in both sexes. Although Adh6a and Adh6b have expression patterns different from each other, neither expresses like any other gene in the complex, suggesting different control mechanisms and possibly different functions.