Absence Of Retinoic Acid Research Articles

Ligand-free RAR can interact with the RNA polymerase II subunit hsRPB7 and repress transcription.

Upon binding retinoic acid (RA), the retinoic acid receptors (RARs) are able to positively and negatively regulate transcription. It has been shown that the DNA-binding domain and carboxy terminus of RARs are necessary for the ligand-dependent ability of the receptor to repress AP-1 transcriptional activity. A fusion of these two regions, shown to constitutively inhibit AP-1 activity, was used in a yeast two-hybrid screen to identify a novel hRARalpha-interacting protein. This protein, hsRPB7, a subunit of RNA polymerase II, interacts with hRARalpha in the absence of RA and addition of RA disrupts the interaction. Truncation analysis indicates that hsRPB7 specifically interacts with the hRARalpha DNA-binding domain. This interaction appears to compromise transcription, since overexpressed hRARalpha, in the absence of RA, is able to repress the activity of several RNA polymerase II-dependent activators, including AP-1 and the glucocorticoid receptor. This repression is relieved by transfected hsRPB7, strongly suggesting that ligand-free hRARalpha can block AP-1 activity by sequestering hsRPB7. The repression is dependent on the integrity of the hRARalpha DBD, since a mutation within the DBD blocks both the hRARalpha-hsRPB7 interaction and ligand-free hRARalpha repression of AP-1. These results provide evidence that non-liganded hRARalpha can regulate transcription by directly interacting with RNA polymerase II, and thus suggest a novel pathway by which hRARalpha can cross-talk with AP-1 and perhaps other families of transcriptional activators.

Mild vitamin A deficiency leads to inborn nephron deficit in the rat

Vitamin A plays a critical role in fetal organogenesis, and its severe deficiency during pregnancy is known to result in malformations of several organs, including the kidney. However, the consequences of mild vitamin A deficiency (VAD) has received little attention. In the present study, we examined the effect of in utero exposure to mild VAD on renal organogenesis. A rat model of mild VAD compatible with normal gestation was developed. Plasma retinol was determined by reverse phase HPLC in mothers and fetuses. Nephron counting was performed in kidneys of fetuses and pups issued from control and VAD mothers. Metanephroi explanted from 14-day-old fetuses from both groups were cultured in the presence or absence of retinoic acid (RA), and growth and differentiation were assessed. c-ret expression was analyzed from fetuses exposed in utero to VAD or to normal vitamin A status and also in metanephroi grown in culture with or without RA using RT-PCR. The 50% reduction in circulating vitamin A levels induced by vitamin A deprivation in pregnant rats did not affect the overall fetal development. However, the number of nephrons was reduced by 20% in 21-day-old VAD fetuses. The number of nephrons was closely correlated with circulating vitamin A level in both VAD and control fetuses. Metanephroi taken from VAD fetuses developed to a lesser extent in vitro, but their capacity to respond to exogenous retinoic acid was not altered. Finally, we found that the expression of the proto-oncogene c-ret was modulated according to the retinoid environment. We conclude that vitamin A supply to the fetus is critical in determining the number of nephrons. Data available thus far on the frequency of mild VAD during pregnancy and on the long-term consequences of inborn nephron deficit highlight the clinical relevance of the present study.

Differentiation dependency of eicosanoid enzyme expression in human tracheobronchial cells

The expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase-2 (PGHS-2) was studied in retinoic acid (RA) sufficient and RA-deficient normal human tracheobronchial epithelial (NHTBE) cell cultures. In the absence of RA, in which the cultures became squamous metaplastic, neither 15-LO nor PGHS-2 were expressed. In RA-sufficient cultures, which differentiated into a mucociliary epithelium, both enzymes were expressed: PGHS-2 during early phases and 15-LO during late stages of differentiation. Depending on the stage of differentiation, the RA-sufficient cultures produced PGE 2 and in the presence of exogenous linoleic acid (LA) 13-HODE. Experiments are underway to examine the effects of inflammatory cytokines on eicosanoid metabolism and the role these metabolites play in airway diseases.

Vitamin A Is Required for Regulation of Polymeric Immunoglobulin Receptor (pIgR) Expression by Interleukin-4 and Interferon-γ in a Human Intestinal Epithelial Cell Line

The secretory immunoglobulin A (IgA) antibody response to infections of mucosal surfaces requires transport of IgA from the basal to apical surface of mucosal epithelial cells by a specific transport protein, the polymeric immunoglobulin receptor (pIgR). We have tested the hypothesis that the vitamin A metabolite all-trans retinoic acid (RA) is required for the regulation of pIgR expression by the cytokines interleukin-4 (IL-4) and interferon-γ (IFN-γ) in HT-29 cells, a well-differentiated human epithelial cell line derived from a colonic carcinoma. pIgR expression is upregulated by IFN-γ and IL-4 when HT-29 cells are grown in normal media, but this upregulation was significantly lower when cells were grown in vitamin A–depleted media. Treatment with RA at concentrations from 10−9 to 10−5 mol/L restored normal levels of pIgR expression. The percentages of cells expressing cell-surface pIgR after 24, 48 and 72 h of treatment with RA, IL-4 and IFN-γ were 66 ± 10, 90 ± 5 and 92 ± 1, respectively, significantly higher than the percentages seen without RA treatment, which were 32 ± 2.3, 72 ± 1.2 and 30 ± 7, respectively. In addition, the intensity of fluorescence of pIgR-positive cells was significantly higher in the RA-treated cultures than in the cultures without RA treatment. Similarly, pIgR mRNA levels (adjusted for β-actin mRNA levels) in RA-supplemented cultures were 404, 105 and 949% higher at 24, 48 and 72 h, respectively, than were pIgR mRNA levels in identical cultures grown in the absence of RA. These data indicate that RA strongly interacts with IL-4 and IFN-γ to regulate pIgR expression in HT-29 cells, suggesting that vitamin A may be required for proper in vivo regulation of IgA transport in response to mucosal infections.

Ligand-dependent regulation of retinoic acid receptor alpha in rat testis: in vivo response to depletion and repletion of vitamin A.

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-alpha (RAR alpha). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RAR alpha messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RAR alpha messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RAR alpha levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RAR alpha, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RAR alpha levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RAR alpha is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Ligand-Dependent Regulation of Retinoic Acid Receptor in Rat Testis: In Vivo Response to Depletion and Repletion of Vitamin A

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-α (RARα). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RARα messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RARα messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RARα levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RARα, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RARα levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RARα is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Retinoic acid induces apoptosis-associated neural differentiation of a murine teratocarcinoma cell line.

Incubation with all-trans retinoic acid (RA) induces PCC7-Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue-like pattern of neuronal, astroglial, and fibroblast-like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA-induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan-caspase inhibitor zVAD-fmk (benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10(-7) M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron-specific protein kinase C substrate GAP-43. RA-induced apoptosis increased with the plating density of PCC7-Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA-damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle-arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis-inhibiting factors by the cultured cells is indicated. Neither ApoI/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl-2 proto-oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13-dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long-term treatment with phorbol 12,13-dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of apoptosis of PCC7-Mz1 cells. We conclude that the apoptotic activity following RA treatment of cultured PCC7-Mz1 cells probably is controlled by the same cascade of gene regulatory events that govern the early cell lineage determinations in this in vitro model system of neural development.

Effects of retinoic acid and TGF-ß1 on the proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

PURPOSE. The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) and transforming growth factor-ß1 (TGF-ß1) on proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. METHODS. Chick RPE cells were cultured in the presence or absence of RA (10 -5 to 10 -7 M) or TGF-ß1 (1 to 100 U/ml) for a 10-day period. The effects of RA and TGF-ß1 on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content in the cells. RESULTS. RA significantly inhibited RPE cell proliferation in a time-dependent manner. The most marked inhibition was observed on day 10 of culture, and the most effective concen-tration of RA was 10 -6 M. Melanin synthesis was increased on day 10, and the most effective concentration of RA was also 10 - 6 M. TGF-ß1 also inhibited RPE cell proliferation and increased melanin synthesis. CONCLUSIONS. These findings indicate that RA and TGF-b1 have regulatory effects on the proliferation and differentiation of chick RPE cells in vitro.

Expression of Retinoid X Receptors in Human Dermal Fibroblasts

Retinoic acid (RA) is known to exert profound effects on growth and differentiation in human dermal fibroblasts. In the observations presented here, we examined the regulation of expression of members of the RXR multigene family in human dermal fibroblasts. We showed that the messenger RNAs for both RXRα and RXRβ are expressed in human fibroblasts, but that the messenger RNA for RXRγ is not detectable in these cells. Electrophoretic mobility shift studies of binding to the β2RARE in human dermal fibroblasts demonstrated that a single complex binds to β2RARE in the absence of RA. Stimulating cells with all-transRA induced a second complex. An antibody to the RXRβ protein supershifted both complexes, while an antibody to the RXRα S/B protein had no effect on the binding. These data demonstrate that RXRβ plays an important role in retinoid-regulated signal transduction pathways in human dermal fibroblasts and the regulation of expression of the RXR gene family is different from that of the RAR gene family.

Gα12 and Gα13 Mediate Differentiation of P19 Mouse Embryonal Carcinoma Cells in Response to Retinoic Acid

P19 mouse embryonal carcinoma cells can be stimulated to differentiate into endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens. At low concentrations, retinoic acid stimulates P19 embryonal cells to differentiate to cells displaying an endodermal phenotype, whereas at higher concentrations it stimulates differentiation to neuroectoderm. The Galpha12 and Galpha13 subunits of heterotrimeric G-proteins are expressed in the embryonal P19 cells and stimulated in response to retinoic acid as the cells differentiate to endodermal or neuroectodermal phenotypes. Suppression of the expression of either Galpha12 or Galpha13 by antisense RNA is shown to promote cell detachment from substratum and apoptosis. Expression of the constitutively active, mutant form of Galpha12 (Q229L), in contrast, stimulates loss of the embryonal phenotype. Expression of the constitutively active form of Galpha13 (Q226L) stimulates differentiation of the cells from embryonal to endodermal, in the absence of retinoic acid. Thus, both Galpha12 and Galpha13 are essential to stimulation of cell differentiation by retinoic acid. Deficiency of either Galpha12 or Galpha13 increases programmed cell death.

Activation of phosphatidylinositol 3'-kinase by insulin-like growth factor-I rescues promyeloid cells from apoptosis and permits their differentiation into granulocytes.

Insulin-like growth factor-I (IGF-I) promotes cell division and prevents programmed cell death in hemopoietic progenitors. Human HL-60 promyeloid cells differentiate toward the granulocytic lineage when stimulated with retinoic acid (RA) in serum-containing medium. When deprived of serum, however, we found that these cells differentiate poorly in the presence of RA, as assessed by expression of the alpha subunit of the beta2 integrin heterodimer, CD11b/CD18. However, when IGF-I is added to RA-treated cells, the proportion of CD11b-positive cells increases to a level similar to that in RA-treated cells cultured in serum-containing medium. Cells treated with RA alone not only differentiate poorly but also undergo apoptosis, as assessed by flow cytometry using propidium iodide and HO33342. In serum-free medium, one-third of RA-treated cells become apoptotic compared with only 5% apoptotic cells in the absence of RA. However, addition of IGF-I to RA-treated cells prevents the appearance of this apoptotic population and increases phosphatidylinositol 3'-kinase (PI 3-kinase) activity by fivefold. Wortmannin, a PI 3-kinase inhibitor, potently decreases this IGF-I-induced lipid kinase activity, blocks the ability of IGF-I to prevent apoptosis, and inhibits IGF-I-enhanced CD11b expression. These data demonstrate that IGF-I acts on RA-treated progenitors to promote their differentiation along the granulocytic lineage. IGF-I acts by rescuing these cells from apoptotic cell death via a downstream pathway that is dependent upon PI 3-kinase.

A Role forIndian hedgehogin Extraembryonic Endoderm Differentiation in F9 Cells and the Early Mouse Embryo

Hedgehoggenes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression ofIndian hedgehogmRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulatesIndian hedgehogexpression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or noIndian hedgehogmessage is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells.In situhybridization analysis forIndian hedgehogmRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mountin situhybridization analysis of Day 7.5 and 8.5 embryos indicates thatIndian hedgehogexpression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full lengthIndian hedgehogcDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest thatIndian hedgehogis involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis.

The Interconversion of Protein Phosphatase 2A between PP2A 1and PP2A 0during Retinoic Acid-Induced Granulocytic Differentiation and a Modification on the Catalytic Subunit in S Phase of HL-60 Cells

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-induced differentiation of HL-60 cells have been investigated. PP2A activity of HL-60 cells for phosphorylated myelin basic protein showed a sharp and transient increase after 18-h treatment with 1 μ mretinoic acid, which corresponded to G1/S boundary of the cell cycle. This PP2A of the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 mNaCl, while PP2A from control cells was eluted with 0.23 mNaCl. The phosphorylase phosphatase activity of PP2A in the 0.13 meluate was greatly enhanced in the presence of protamine compared with that of the later eluting PP2A. Immunoblot analyses with antisera against B′ and Bα subunits showed that the PP2A in the 0.13 mNaCl eluate from 18-h retinoic acid-treated cells was PP2A 0(AC-B′), whereas the PP2A eluted with 0.23 mNaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A 1(AC-Bα). These results strongly suggest that PP2A undergoes a transient and reversible interconversion of holoenzyme forms during the initial stage of retinoic acid-induced granulocytic differentiation. PP2A activity assayed after dissociation of the catalytic subunit, for phosphorylase as substrate, showed a sharp and transient decrease in S phase of HL-60 cells irrespective of the presence or absence of retinoic acid. Immunoblot analyses with antisera against C-terminus and N-terminus of the catalytic subunit of PP2A suggested that a modification at the C-terminus is responsible for the decrease in PP2A activity. Immunoreactivity to the C-terminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group from the C-terminus. These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells.

Constitutive expression of prostaglandin endoperoxide G/H synthetase (PGHS)-2 but not PGHS-1 in hum an tracheal epithelial cells in vitro

Primary cultures of human tracheal epithelial (HTE) cells cultured in vitro, in defined serum-free media, express prostaglandin endoperoxide G/H synthase (PGHS) activity and produce prostaglandin E 2 (PGE 2). In contrast to every other cell type studied to date, HTE cells appear to constitutively express PGHS-2, the ‘inducible’ form of the enzyme, while expressing little or no PGHS-1, the ‘housekeeping’ isoenzyme in vitro. Prostaglandin synthesis in HTE cells was reduced by a selective PGHS-2 inhibitor, N-(2-cyclohexyloyl-4-nitrophenyl] methane-sulfonamide (NS398), with an IC 50 of approximately 1 μM. Immunoblotting and immunoprecipitation of enzymatic activity with isozyme-specific antisera revealed only the PGHS-2 isoform. Full length human cDNA probes detected only PGHS-2 message in Northern blots. Neither PGHS-2 activity nor mRNA levels were dependent on, nor stimulated by peptide growth factors present in the defined serum-free growth medium, or by serum. Prolonged maintenance in the absence of retinoic acid, however, lead to a decline in PGHS activity. Phorbol-myristate acetate (PMA) induced PGHS-2 activity and mRNA and neither PMA-induced, nor constitutive PGHS-2 expression was suppressed by corticosteroids. Actinomycin D-treatment for six hours reduced the PGHS-2 activity and mRNA to only 50% that of untreated cells, suggesting that PGHS-2 mRNA is extremely stable in these cells. HTE cells, at least in vitro, appear unique among prostaglandin-producing cells in that they express PGHS-2, constitutively, independent of regulation by growth factors, serum, or corticosteroids and fail to express PGHS-1 under any culture condition studied.

Retinoic acid in development and regeneration

Retinoids are low molecular weight, lipophilic derivatives of vitamin A which have profound effects upon the development of various embryonic systems. Here I review the effects on developing and regenerating limbs, regenerating amphibian tails and the developing central nervous system (CNS). In the regenerating amphibian limb, retinoids can proximalize, posteriorize and ventralize the axes of the blastema. In the chick limb bud retinoids can only posteriorize the tissue. In the regenerating amphibian tail retinoids can homeotically transform tail tissue into hindlimb tissue. In the developing and regenerating limb retinoic acid has been detected endogenously, confirming that this molecule plays a role in the generation of pattern and we have shown that limbs cannot develop in the absence of retinoic acid. In the developing CNS retinoic acid specifically affects the hindbrain where it causes a transformation of anterior rhombomeres into more posterior ones. Again, endogenous retinoic acid has been detected in the CNS and in the absence of retinoids the posterior hindbrain has been found to be affected. The effects of retinoids on the CNS are most likely to be mediated via theHox genes acting in the mesoderm after gastrulation. It has also been proposed that the establishment of the head-to-tail axis in the mesoderm is established by retinoic acid. These data show that retinoids play an important role in both the development and regeneration of various systems in the embryo and post-embryonically

Retinoid-induced differentiation regulates prostaglandin H synthase and cPLA2 expression in tracheal epithelium.

Rat tracheal epithelial cells cultured in vitro at an air-liquid interface can differentiate into a mucociliary or squamous phenotype depending on the presence or absence of retinoic acid (RA). The airway epithelium is known to produce a number of eicosanoids. We propose that eicosanoid biosynthesis is dependent on the differentiation status of the epithelium. Therefore, prostaglandin production and the expression of cytosolic phospholipase A2 (cPLA2) and the prostaglandin H synthase (PGHS) isoforms were investigated during differentiation to these two phenotypes. The major eicosanoid produced by both phenotypes was prostaglandin E2 (PGE2). Proliferating undifferentiated cultures produced low levels of PGE2 regardless of retinoid status. Differentiated mucociliary cultures produced high levels of PGE2 (50 ng/10(6) cells), whereas differentiated squamous cultures produced low levels of PGE2 (< 5 ng/10(6) cells). Mucociliary cultures expressed high levels of cPLA2 and PGHS-2 isoform mRNA and protein. In contrast squamous cultures expressed very low levels of cPLA2 and PGHS-2 transcript and protein. The PGHS-1 isoform was expressed in squamous but not in mucociliary cultures. We investigated changes in expression of these enzymes during retinoid treatment of established squamous cultures. Treatment with RA resulted in a rapid (24 h) downregulation of PGHS-1 mRNA expression. However, the cPLA2 and PGHS-2 genes were expressed in squamous cultures only after 3 days of RA treatment coincident with redifferentiation of the culture to a mucociliary phenotype. These studies reveal that retinoid-induced differentiation of airway epithelium into either a mucociliary or squamous phenotype results in profound changes in the expression of cPLA2 and PGHS isozymes that regulate prostaglandin formation.

Vitamin A-deficient quail embryos have half a hindbrain and other neural defects

Background: Retinoic acid (RA) is a morphogenetically active signalling molecule thought to be involved in the development of several embryonic systems (based on its effect when applied in excess and the fact that it can be detected endogenously in embryos). Here, we adopt a novel approach and use the vitamin A-deficient (A−) quail embryo to ask what defects these embryos show when they develop in the absence of RA, with particular reference to the nervous system.Results We have examined the anatomy, the expression domains of a variety of genes and the immunoreactivity to several antibodies in these A− embryos. In addition to the previously documented cardiovascular abnormalities, we find that the somites are smaller in A− embryos, otic vesicle development is abnormal and the somites continue up to and underneath the otic vesicle. In the central nervous system, we find that neural crest cells need RA for normal development and survival, and the neural tube fails to extend any neurites into the periphery. Using general hindbrain morphology and the expression patterns of Hoxa-2, Hoxb-1, Hoxb-4, Krox-20 and FGF-3 as markers, we conclude that segmentation in the myelencephalon (rhombomeres 4–8) is disrupted. In contrast, the dorsoventral axis of the neural tube using Shh, islet-1 and Pax-3 as markers is normal.Conclusion These results demonstrate at least three roles for RA in central nervous system development: neural crest survival, neurite outgrowth and hindbrain patterning.

Morphogen-induced Decline in G Triggers F9 Teratocarcinoma Stem Cell Progression via Phospholipase C and Mitogen-activated Protein Kinase

The linkage between Gialpha2 and morphogen-induced promotion of F9 embryonic teratocarcinoma stem (F9 stem) cells to primitive endoderm was explored using probes of the mitogen-activated protein (MAP) kinase network. The morphogen-induced decline in Gialpha2 is shown to trigger activation of phospholipase C, thereby activating protein kinase C, MAP kinase, and cell progression to primitive endoderm. In the absence of retinoic acid, reduction-of-function mutants (Gialpha2-deficient) display the effects of morphogen, i.e. activation of phospholipase C, protein kinase C, MAP kinase, and progression to primitive endoderm. Gain-of-function mutants (expressing the Q205L activating-mutation of Gialpha2) displayed no activation of phospholipase C, protein kinase C, MAP kinase and no progression to primitive endoderm, even in the presence of retinoic acid. Selective inhibitors of protein kinase C, like the gain-of-function mutations, effectively block morphogen-induced progression to primitive endoderm. Morphogen triggers F9 stem cell progression by triggering Gialpha2 loss and thereby activation of downstream elements, including protein kinase C and MAP kinase.

Retinoic acid suppresses in-vitro decidualization of human endometrial stromal cells.

All-trans retinoic acid (RA) has potent effects on cell differentiation and gene expression. Previous studies have demonstrated that human endometrial stromal cells express mRNA for retinoic acid receptors (RARs) and cellular RA-binding protein-II (CRABP-II). We examined whether RA regulates stromal cell differentiation (decidualization), a critical process in preparation of the uterus for blastocyst implantation. Decidualization was induced by incubating cultured stromal cells with medroxyprogesterone acetate (MPA) and oestradiol. Decidualization was defined by the induction of prolactin, insulin-like growth factor binding protein-1 (IGFBP-1), appearance of a differentiated phenotype and changes in fibronectin expression. RA treatment significantly (P < 0.05) suppressed prolactin and IGFBP-1 production associated with stromal cells decidualization. The formation of differentiated cells was inhibited by RA, and consistent with maintenance of the undifferentiated phenotype, fibronectin mRNA content was approximately 3.5-times greater than in the absence of RA. Upon induction of decidualization, the expression of mRNA for the major RA receptor sub-types (RAR-alpha, -beta and -gamma) was maintained while the relative amounts of CRABP-II mRNA progressively decreased with differentiation. With RA treatment, RAR-alpha and RAR-gamma mRNA concentrations were approximately 70 and 25% respectively of those in cells decidualized in the absence of RA. The effects of RA appear to be partially mediated by inhibition of cAMP action. RA suppressed intracellular cAMP concentrations induced by MPA and oestradiol to approximately 35% of those in cells without RA. Addition of 50 microM dibutyryl cAMP to stromal cells treated with MPA and oestradiol only partially reversed the suppression of decidualization and prolactin release by RA. In summary, we have demonstrated that in-vitro decidualization of human endometrial stromal cells induced by MPA and oestradiol treatment is suppressed by RA.

Role of retinoic acid in mouse neural crest cell development in vitro.

This study examines the role of retinoic acid (RA) in mouse neural crest cell development in culture. We have compared the effects of RA on the developmental behavior of mouse neural crest cells from different axial levels, that is the mesencephalic (cranial) and sympathoadrenal (trunk) levels by colony assay using antibodies against cell-type-specific markers. In control cultures in the absence of RA, the efficiency of colony formation by cranial neural crest cells was considerably lower than in colony cultures of trunk neural crest cells. Pulse-labelling experiments using 5-bromo-2'-deoxyuridine (BrdU) showed that the proportion of neural crest cells that incorporated BrdU was significantly smaller in day 5 cultures of cranial neural crest cells in the absence of RA compared to cultures from the trunk level. However, BrdU-incorporation matched the labelling observed in trunk crest cell cultures when RA was added to the culture medium. The efficiency of colony formation and the proportion of BrdU-incorporated cells in trunk crest cell cultures was similar in the presence and absence of RA. The results suggest that in the early stages of in vitro development, RA has a larger impact on proliferation and/or survival of cranial neural crest cells than of trunk neural crest cells. Moreover, the data indicate that RA significantly affects melanogenesis and the differentiation of serotonergic neurons in mouse neural crest cell cultures from both axial levels. Whereas melanogenesis was suppressed by RA treatment, serotonergic neurogenesis was promoted. Double-labelling experiments with antibodies against BrdU and serotonin (5-HT) indicated that RA selectively promoted proliferation of these neurons at a later stage of in vitro development. Furthermore, RA acted upon both committed cells and multipotent cells, Based on the results, we conclude that RA plays multiple critical roles in mouse neural crest cell development.