Retinoic acid modulates attachment of mouse fibroblasts to laminin substrates

The effect of retinoic acid treatment on cell attachment to plastic substrates precoated with fibronectin, gelatin, laminin, and type IV collagen was investigated. Both retinoic acid-treated and control cells attached efficiently to fibronectin or gelatin substrates without any significant difference. In contrast, retinoic acid-treated cells attached to laminin or type IV collagen substrates, while control cells showed little or no attachment. The minimal effective concentration of retinoic acid for pretreatment to yield a significant increase in the attachment assay was higher than 10(-8) M. The attachment of retinoic acid-treated cells to laminin substrates reached a maximum at 60 min, while that to type IV collagen substrates had a time lag and did not reach a maximum by 60 min. The effect of retinoic acid treatment reached a maximum at 2 days and was partly reversible. These results suggest that retinoic acid may increase NIH/3T3 cell adhesion through an effect on laminin receptors. Other mouse fibroblast lines, 3T3-Swiss, 3T6-Swiss, Balb/3T3, and Balb/3T12-3 (spontaneously transformed Balb/3T3), responded to retinoic acid treatment in a manner similar to that of NIH/3T3 cells. However, the virus-transformed Balb/3T3 lines, SV-T2 and M-MSV, showed significant attachment to laminin substrates without retinoic acid treatment, and retinoic acid did not affect or slightly decreased the cell attachment to laminin substrates.     

Retinoic acid inhibits migration of cranial neural crest cells in the cultured mouse embryo

Clinical observations have demonstrated that isotretinoin (13-cis-retinoic acid; cis-RA) is a human teratogen causing primarily heart and craniofacial malformations. Isotretinoin exposure to the early postimplantation mouse embryo in culture results in specific defects in craniofacial development that may be due to an interference in the early migration of cranial neural crest (CNC) cells [Goulding and Pratt, 1986]. The present study was designed to test this hypothesis by examining the migration of these cells in whole embryo culture. Day 8 CD-1 mouse embryos were cultured for 6-48 hr in the presence or absence of cis-RA at 2 X 10(-6) to 2 X 10(-5) M. Embryos either were fixed for light microscopy using Nichols' method for localization of CNC cells or were processed for scanning and transmission electron microscopy. At the light microscopic level, CNC cells in the mid-brain region of control embryos had migrated to the region of the first and second visceral arches after 6 hr in culture. Cis-RA interfered with this migration; CNC cells in treated embryos either did not leave the neuroepithelium (NE) or were aggregated near the NE. Autoradiographic studies indicated that cis-RA did not affect the overall viability or DNA synthesis of the CNC cells. However, at the TEM level, there was a dramatic increase in the number of cellular blebs in the CNC cells. Our results demonstrate a direct effect of 13-cis-RA on the CNC cells and suggest that this effect is due to alterations in the cell surface.     

Retinoic acid modulates the retinoblastoma protein during adipocyte terminal differentiation

Terminal differentiation is characterized by a permanent withdrawal of cells from the cell cycle. Retinoblastoma protein (RB) has been involved in cell cycle progression. Accumulating evidence also implicates RB in the promotion of differentiation of many cell types. We present new insights into the role of RB and other cell cycle regulatory proteins in adipocyte differentiation and on the role of retinoic acid (RA) in the regulation of the latter process. It is shown that RA reduces RB expression and enhances RB phosphorylation by a mechanism that involves down-regulation of the cyclin-dependent kinase inhibitor (CKI) p21(Cip1), having this fact as important consequences for both the cell cycle progression and the adipocyte differentiation process. The effects of RA result in the blockage of adipogenesis, but may also favor the retention of a pool of adipose cells able to re-enter the cell cycle, which may be important for the developmental dynamics of adipose tissue in vivo. In addition, these results reinforce the idea that there is a cross-talk between the cell cycle machinery and the adipocyte differentiation machinery that can be modulated by external signals, including nutrients.     

Retinoic acid signalling specifies intermediate character in the developing telencephalon

The organisation of the telencephalon into its major structures depends on its early regionalisation along the dorsoventral axis. Previous studies have provided evidence that sonic hedgehog (SHH) is required for the generation of telencephalic cells of ventral character, and that sequential WNT and fibroblast growth factor (FGF) signalling specifies cells of dorsal telencephalic character. However, the signalling mechanisms that specify telencephalic cells of an intermediate character remain to be defined. We provide evidence here that retinoic acid has a crucial role in specifying telencephalic progenitor cells of intermediate character.     

Retinoic acid promotes proliferation and chondrogenesis in the distal mesodermal cells of chick limb bud

Distal and proximal mesoderm of chick limb bud was respectively dissociated and cultured in the medium containing various concentrations of retinoic acid (RA). At low concentrations (5-50 ng/ml), RA promoted proliferation and chondrogenesis in the distal mesodermal cells. The distal cells of stage 20-24 limb buds were responsive to RA, although those of stages 25-27 were unresponsive. Both the cells of anterior and posterior regions of the distal mesoderm were responsive to RA, while the cells of proximal mesoderm were unresponsive. At higher concentrations, the growth-promoting effect of RA was reduced and chondrogenesis in the distal cells was rather inhibited. These results were discussed in relation to the role of RA as the morphogen in normal limb development and experimental duplicate formation.     

Retinoic acid synthesis and hindbrain patterning in the mouse embryo

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.     

Retinoic acid modulates dome formation by MDCK cells in defined medium

Retinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K-1). After 4-5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7 days with 3 x 10(-7) M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 x 10(-3) microns 2 in area, whereas in control medium the majority of the domes were between 0 and 9 x 10(-3) microns 2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K-1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1-independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K-1 lacking PGE1. This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1-independent MDCK cells. Dibutyryl cAMP-resistant MDCK cells, which normally do not form domes in medium K-1, were also studied. Remarkably, the dibutyryl cAMP-resistant MDCK cells were observed to form domes at a significant frequency when medium K-1 was supplemented with retinoic acid. However in medium K-1 lacking PGE1, an effect of retinoic acid on dome formation by dibutyryl cAMP-resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP-resistant MDCK cells to form domes in medium K-1 has previously been attributed to their decreased cAMP-dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP-dependent protein kinase or activation of a separate pathway.     

Retinoic acid-dependent signaling pathways and lineage events in the developing mouse spinal cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells.     

Retinoic acid increases the expression of NGF gene in mouse L cells

Retinoic acid (RA), the acid form of vitamin A, is shown to enhance the synthesis of nerve growth factor (NGF) in cultures of mouse L cells. Maximal stimulation was observed in cells growing in a serum-free medium supplemented with 10(-6)M RA during 48 h. The drug increased both the level of NGF mRNA and the amount of mature NGF protein secreted by the cells. RA was previously reported to increase the number of NGF receptors on some neuroblastoma cells (Haskell et al., 1987 Cell and Tiss. Res., 247, 67-73). It seems, therefore, that RA may influence nerve cell differentiation by promoting both the synthesis of the neurotrophic factor and the responsiveness of target cells.     

Retinoic acid modulates the pattern of cell division in embryos ofLymnaea stagnalis (Mollusca)

All-trans retinoic acid is well known as a modulator of positional specification in vertebrate development. A similar mechanism may operate in molluscan development. Molluscan development is characterized by an invariant pattern of cell divisions, which allows the study of individual cells in the developing organism. Low concentrations of exogenous retinoic acid applied during gastrulation affect the cell division pattern in the early larval stage of the molluscLymnaea stagnalis. A few cells from the apical plate, a larval organ consisting of seven large cleavage-arrested cells, were induced by retinoic acid to resume cell division. They typically formed an area of proliferating small cells that resembles the adjacent areas of precursor cells of adult ectoderm. The identification of individual cells that are transformed by retinoic acid may provide new insights into the mechanisms underlying positional specification within the embryo.     

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N-cadherin and its associated proteins

Retinoic acid (RA) is a well-known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal-regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N-cadherin expression. However, RA treatment caused sustained expression of N-cadherin and its associated proteins including alpha- and beta-catenin suggesting that modulation of expression of these molecules is associated with RA-induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA-induced sustained expression of cell adhesion molecules and overcomes RA-induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell-to-cell interactions at the post-precartilage condensation stage.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.     

Retinoic acid synthesis and metabolism are concurrent in the mouse uterus during peri-implantation

Vitamin A (retinol) and its active metabolite, retinoic acid (RA), serve dual roles in the female reproductive tract. Cytochrome P450 26A1 (Cyp26a1), an RA-metabolizing enzyme, is involved in mammalian early pregnancy. In order to investigate the role of RA synthesis and metabolism during embryo implantation, we first investigated the spatiotemporal expression of RA-signal in the mouse uterus during the peri-implantation period. RA-signal-related molecules, including binding proteins, synthesizing enzymes, catabolizing enzymes and receptors, were all expressed in the mouse uterus during embryo implantation. The locations of the RA synthetic system (Aldh1a1, Aldh1a2, CRBP1) and catabolizing enzyme (Cyp26a1) were distinctive in the mouse uterus during the peri-implantation period. Aldh1a1 was located in the gland epithelium, whereas Aldh1a2 and CRBP1 were located in the stroma and Cyp26a1 was expressed in the luminal and glandular epithelium. These results demonstrate that RA synthesis occurs in the stroma, whereas RA metabolism takes place in the endometrial epithelium. When endometrial epithelial cells were isolated on day 4.5 of pregnancy and treated with E₂ (17beta-estradiol) or a combination of E₂ and progesterone, all-trans-RA (10???M) significantly down-regulated the expression of LIF, HB-EF and CSF-1 in these cells in vitro. Taken together, these results suggest that the accumulation of RA in the stroma during mouse embryo implantation has an inhibitory effect on the expression of the three implantation-essential genes, LIF, HB-EGF and CSF-1. Therefore, the expression of Cyp26a1 in luminal and glandular epithelium might block the adverse effect of RA in order to promote successful embryo implantation.     

Retinoic acid derived from the fetal ovary initiates meiosis in mouse germ cells

Meiotic initiation of germ cells at 13.5 dpc (days post‐coitus) indicates female sex determination in mice. Recent studies reveal that mesonephroi‐derived retinoic acid (RA) is the key signal for induction of meiosis. However, whether the mesonephroi is dispensable for meiosis is unclear and the role of the ovary in this meiotic process remains to be clarified. This study provides data that RA derived from fetal ovaries is sufficient to induce germ cell meiosis in a fetal ovary culture system. When fetal ovaries were collected from 11.5 to 13.5 dpc fetuses, isolated and cultured in vitro, germ cells enter meiosis in the absence of mesonephroi. To exclude RA sourcing from mesonephroi, 11.5 dpc urogenital ridges (UGRs; mesonephroi and ovary complexes) were treated with diethylaminobenzaldehyde (DEAB) to block retinaldehyde dehydrogenase (RALDH) activity in the mesonephros and the ovary. Meiosis occurred when DEAB was withdrawn and the mesonephros was removed 2 days later. Furthermore, RALDH1, rather than RALDH2, serves as the major RA synthetase in UGRs from 12.5 to 15.5 dpc. DEAB treatment to the ovary alone was able to block germ cell meiotic entry. We also found that exogenously supplied RA dose‐dependently reduced germ cell numbers in ovaries by accelerating the entry into meiosis. These results suggest that ovary‐derived RA is responsible for meiosis initiation. J. Cell. Physiol. 228: 627–639, 2013. © 2012 Wiley Periodicals, Inc.     

Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos.

Considerable evidence now suggests that retinoic acid (RA) is an important modulator of patterning events in early neuronal development in vertebrates. In this paper, we describe the effects of exogenously applied RA on early neural development in the zebrafish embryo. Neural anatomy is assessed by immunocytochemical and histochemical analysis of the developing embryo in whole mounts at 24 h post-fertilization. RA was applied for one hour at concentrations ranging from 10(-9) to 10(-6) M to embryos at 50% epiboly, the midgastrula stage, and at 10(-7) M to embryos at early and late gastrula stages. The neuroanatomical analysis shows that 10(-7) M RA causes a defined lesion to the developing central nervous system which corresponds to a loss of a region of the brain in the caudal midbrain-rostral hindbrain area, the precursor of the cerebellum and associated neural structures. The region that fails to develop corresponds to the cranial expression domain of the engrailed protein as assessed by the monoclonal antibody 4D9 (Patel et al. 1989: Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58, 955-968). Structures caudal to rhombomere 4 are unaffected by 10(-7) M RA, as are the cranial midbrain and forebrain: 10(-7) M RA also affects the development of cranial ganglia, principally the Vth, anterior lateral line and VIIIth ganglia, suggesting that RA affects normal development of the cranial neural crest. Effects of RA at stages immediately prior to and after gastrulation show some similar and some distinct features. Results are discussed in terms of the possible role of RA as an endogenous moderator of normal head development.