Search PubMed⌕ Search

Biomedical subjects

M Maden

Publications and source records attributed to M Maden.

At least 37 records · Page 2Linked to original sources
PubMed1999

Embryonal carcinoma cell lines stably transfected with mRARbeta2-lacZ: sensitive system for measuring levels of active retinoids.

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.

Animals↗
PubMed1999

Heads or tails? Retinoic acid will decide.

A recent study (Niederreither et al. Nat Genet 1999;21:444-448 [Ref. 1]) describes the phenotype of a gene knockout for an enzyme, retinaldehyde dehydrogenase 2 (RALDH-2), that synthesizes retinoic acid (RA) in the early embryo. The effects generated by this single enzyme mutation are remarkably similar to those previously described in vitamin A-deprivation studies and compound retinoic acid receptor knockouts, which involve multiple systems of the embryo. With other data on the distribution of RA, its role in axial specification of the early embryo is considerably clarified. Surprisingly, it seems that head development is unaffected in these RALDH-2 knockout embryos; thus, the anterior of the embryo does not require RA, despite the observations that the hindbrain seems exquisitely sensitive to RA perturbation. Head development may be realised by a cytochrome P450 enzyme (CYP26), which has been described recently. Between these two opposing forces, the hindbrain develops.

Aldehyde Oxidoreductases↗
PubMed1999

Mesencephalic innervation of the vibrissal follicle-sinus complex in the mouse embryo.

Peripheral projections of neurones whose cell bodies lie in the mesencephalic nucleus of the fifth cranial nerve, situated between the central grey and mesencephalic reticular formation, were studied in mouse embryos aged between day 9 and 15 and in postnatal day 1 mice. Nonspecific neural antibody staining allowed visualisation of the developing cranial nerves, in particular the descending mesencephalic tract. This facilitated successful dissection of the descending mesencephalic tract and trigeminal ganglion in the heads of fresh mouse embryos and postnatal mice. The fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil), was injected into the descending mesencephalic tract in mouse embryos aged 12.5, 13.5 and 15 days of gestation and also into postnatal day 1 mice. Following a period of incubation, 100 microm sections were viewed under visible light and episcopic fluorescence. Mesencephalic neurones were observed to pass superiorly over the trigeminal ganglion and enter the maxillary division to innervate vibrissal follicle-sinus complexes, whilst none was observed innervating mandibular and maxillary intraoral structures. There was no fluorescent labelling in non-Dil injected control specimens. Using a highly specific neuronal tracer, this study shows that mesencephalic neurones in the periphery project exclusively to follicle sinus complexes in the developing mouse embryo and remain at least until postnatal day 1. These observations, contrary to those made in other animals, indicate a species specificity of mesencephalic peripheral projections.

Animals↗
PubMed1999

Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.

We describe here how the early limb bud of the quail embryo develops in the absence of retinoids, including retinoic acid. Retinoid-deficient embryos develop to about stage 20/21, thus allowing patterns of early gene activity in the limb bud to be readily examined. Genes representing different aspects of limb polarity were analysed. Concerning the anteroposterior axis, Hoxb-8 was up-regulated and its border was shifted anteriorly whereas shh and the mesodermal expression of bmp-2 were down-regulated in the absence of retinoids. Concerning the apical ectodermal genes, fgf-4 was down-regulated whereas fgf-8 and the ectodermal domain of bmp-2 were unaffected. Genes involved in dorsoventral polarity were all disrupted. Wnt-7a, normally confined to the dorsal ectoderm, was ectopically expressed in the ventral ectoderm and the corresponding dorsal mesodermal gene Lmx-1 spread into the ventral mesoderm. En-1 was partially or completely absent from the ventral ectoderm. These dorsoventral patterns of expression resemble those seen in En-1 knockout mouse limb buds. Overall, the patterns of gene expression are also similar to the Japanese limbless mutant. These experiments demonstrate that the retinoid-deficient embryo is a valuable tool for dissecting pathways of gene activity in the limb bud and reveal for the first time a role for retinoic acid in the organisation of the dorsoventral axis.

Animals↗
PubMed1999

Hindbrain respecification in the retinoid-deficient quail.

We report here the development and rescue of the truncated hindbrain of retinoid-deprived quail embryos. The embryo is completely rescued by an injection of retinol into the egg; this confirms retinol, or a related retinoid, as a required molecule in hindbrain development. Staging the retinoid replacement enabled us to determine that the 3-4 somite stage is the period when retinoids are required for normal development. Analysis of the development of the retinoid-deprived hindbrain phenotype through somitogenesis has revealed a pathway of retinoid action in early hindbrain regionalization. The hindbrain of the retinoid-deprived embryo is normal in size, during early somitogenesis, but has a respecified pattern of Krox-20 expression. From the earliest expression of Krox-20, at the 5 somite stage, the rhombomere 3 stripe fills the caudal third of the developing hindbrain to the level of the first somite. Morphologically only 2, instead of the normal 5, rhombomere bulges form. These 2 bulges express genes and, later, develop morphology characteristic of rhombomeres 1 and 2 and rhombomere 3. Posterior hindbrain specific genes, Hoxb-1, Fgf3, MafB, and the rhombomere 5 stripe of Krox-20 are never expressed in the head neuroepithelium of these embryos. From the initial formation of the neural plate, there is no evidence of rhombomere 4-7 specific characteristics. These results indicate the specification of the posterior hindbrain is lost and its cells participate in the formation of an enlarged anterior hindbrain. In our previous study, we reported the absence of the posterior hindbrain in retinoid-deprived quails (Maden, M., Gale, E., Kostetskii, I., Zile, M., 1996. Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 6, 417-426). Here, we show this phenotype to be the result of respecification of the hindbrain cells. This provides evidence for a region specific response to a single stimulus, retinol, which suggests a pre-rhombomeric regionalization of the hindbrain.

Animals↗
PubMed1999

Axolotl/newt.

Explore the source record for details and available documents.

Ambystoma↗
PubMed1998

Vertebrate development: a nervous vitamin.

The vitamin A-related retinoids have recently been shown to be involved in the development of the central nervous system, both in specifying position along the head-to-tail axis and in the development of a specific subset of motor neurons.

Animals↗
PubMed1998

Retinoic acid as a chemotactic molecule in neuronal development.

We describe the results of experiments on dissociated cultures of embryonic chick neural tissue which were designed to investigate further the role of all-trans-retinoic acid (tRA) on neurite outgrowth and, by inference, on the developing nervous system in vivo. We show that tRA increases both the number of neurites and the length of neurites extending from these chick neural tube cells at nM concentrations. Secondly, using the newly designed Dunn chamber in which stable gradients of compounds can be generated, we show that neurites respond to a gradient of tRA by growing up the gradient. These observations indicate a role for RA in vivo, not only in the initial outgrowth of neurites, but also in their guidance to the appropriate targets.

Animals↗
PubMed1998

The role of vitamin A in the development of the central nervous system.

We describe here the defects that arise in the central nervous system (CNS) of quail embryos when they develop in the absence of vitamin A. It has been assumed that because of the effects of excess vitamin A and its metabolites, particularly retinoic acid (RA), on the CNS they are involved in various aspects of CNS development. We show that this is indeed the case, because these deficient quail embryos have three defects in their CNS. First, the posterior hindbrain fails to develop because the cells fated to form this part of the CNS in the very early embryo die by apoptosis. Second, the neural tube fails to extend neurites into the periphery both in vivo and in vitro. Third, the neural crest cells throughout the embryo die by apoptosis. These results demonstrate a crucial requirement for vitamin A in CNS development.

Animals↗
PubMed1998

The distribution of endogenous retinoic acid in the chick embryo: implications for developmental mechanisms.

The aim of these experiments was to determine the endogenous distribution of retinoic acid (RA) across a wide range of embryonic stages in the chick embryo. By high pressure liquid chromatography, it was revealed that didehydroRA is the most prevalent retinoic acid in the chick embryo and that the tissues of the stage 24 embryo differed widely in their total RA content (didehydroRA + all-trans-RA). Some tissues such as the heart had very little RA and some such as the neural tube had very high levels, the total variation between these two being 29-fold. We showed that these tissues also synthesised RA and released it into the medium, thus validating the use of the F9 reporter cell system for further analyses of younger staged embryos. With these F9 cells, we showed that, at stage 4, the posterior end of the embryo had barely detectably higher levels of RA than the anterior end, but that a significant level of RA generation was detected as soon as somitogenesis began. Then a sharp on/off boundary of RA was present at the level of the first somite. We could find no evidence for a posterior-to-anterior gradient of RA. Throughout further development, various consistent observations were made: the developing brain did not generate RA, but the spinal part of the neural tube generated it at very high levels so there must be a sharp on/off boundary in the region of the hindbrain/spinal cord junction; the mesenchyme surrounding the hindbrain generated RA whereas the hindbrain itself did not; there was a variation in RA levels from the midline outwards with the highest levels of RA in the spinal neural tube followed by lower levels in the somites followed by lower levels in the lateral plate; the posterior half of the limb bud generated higher levels than the anterior half. With these observations, we were able to draw maps of endogenous RA throughout these early stages of chick embryogenesis and the developmental implications of these results are discussed.

Animals↗
PubMed1998

Retinoids are endogenous to the porcine blastocyst and secreted by trophectoderm cells at functionally-active levels.

Retinoic acid and its isomers are the major morphogens in vertebrate development. For mammals, it was previously considered that circulating retinoic acid was recruited from the uterine environment, to influence embryonic differentiation, morphogenesis and development. Here we report that retinoic acid is endogenous to the blastocyst of the domestic pig, Sus scrofa, as detected by high pressure liquid chromatography. Furthermore, using a continuous, normal line of porcine trophectoderm cells, TE1, we have identified the trophoblast as a major source of retinoids. Endogenous retinoic acid was found at a concentration of approximately 35 nM in extracts of entire blastocysts, and at a similar concentration in extracts of TE1 cells. Retinoids in explants of blastocysts and in conditioned medium from the TE1 cell line were found to be functionally-active, inducing gene expression from a retinoic-acid-responsive enhancer element in an in vitro assay system. In conclusion, we propose that there is a morphogenetic role for endogenous, and trophoblast-derived, retinoids in the early development of the pre-gastrulation porcine embryo; and that the TE1 cell line therefore provides a useful in vitro system for the study of retinoid metabolism. Furthermore, an implication of this study is that endogenous retinoids may play an active role in the pre-implantation embryology of other species, such as the human.

Animals↗
PubMed1997

Retinoic acid and its receptors in limb regeneration.

The effects of retinoids on the regenerating amphibian limb are described: the mesenchymal cells of the blastema can be proximalized, posteriorized and ventralized. Ectopic limbs are also induced after retinoid treatment of regenerating tails, but not during limb development unless the limb bud is damaged. The cellular and molecular alterations induced by retinoids are reported as well as experiments which have revealed the importance of endogenous retinoids for normal limb regeneration. Various retinoic acid receptors are expressed in the regeneration blastema and the experiments which have revealed functions for individual isoforms are described. These experiments reveal that retinoids are a crucial component of the normal, regenerating limb and demonstrate the value of the regenerating limb as an experimental system for providing functional data on individual retinoic acid receptors.

Journal Article↗
PubMed1997

Positional apoptosis during vertebrate CNS development in the absence of endogenous retinoids.

We have previously shown that quail embryos that develop in the absence of vitamin A have severe defects in their central nervous system. One defect is a completely missing posterior hindbrain. Here we have studied how this comes about by examining cell death using a wholemount technique. In these A- embryos we observe two narrow bands of ectopic apoptosis. One is in the mesenchyme in the region of the first somite and occurs at the 4-6 somite stage, before neural tube closure. The second band follows immediately afterwards and occurs in the neuroepithelium of the presumptive posterior hindbrain at the 6-8 somite stage. Electron microscopy shows that the dying neuroepithelial cells exhibit the characteristics of apoptosis. Rescuing the embryos by injecting retinol before gastrulation completely prevents these apoptotic events. In an effort to identify some of the genes that may be involved in the apoptotic pathway we show that Msx-2 is upregulated in the apoptotic neuroepithelium and thus may be involved, whereas Bmp-4 is not altered and thus presumably not involved. Since these apoptotic event take place at the time of specification of axial identity and segmentation in the mesenchyme and neuroepithelium we conclude that these cells die because they are wrongly specified in terms of their rostrocaudal position, a novel phenomenon which we refer to as positional apoptosis.

Age Factors↗
PubMed1997

Hoxb-8 has a role in establishing early anterior-posterior polarity in chick forelimb but not hindlimb.

The distribution of Hoxb-8 transcripts through the chick flank and early forelimb mirrors the distribution of polarizing activity in the flank at these early stages. Polarizing activity displayed by Hoxb-8-expressing tissue is only realised when placed adjacent to the AER and appears to be mediated through Shh induction, suggesting that Hoxb-8 may lie genetically upstream of Shh. Accordingly, Hoxb-8 expression is rapidly induced by retinoic acid (RA) treatment in the anterior of the forelimb in a spatial and temporal manner that is consistent with the induction of Shh and formation of the ZPA. Furthermore, inhibition of RA synthesis in the flank downregulates the expression of endogenous Hoxb-8 and results in the loss of Shh expression. However, once the ZPA has become established the posterior limb mesoderm displays resistance to the induction of Hoxb-8 expression. Grafting of ZPA cells to the anterior of a host limb renders the host anterior tissue resistant to RA-induced Hoxb-8 expression. These results indicate that Hoxb-8 expression may be regulated by the established ZPA through a negative feedback loop. The anterior AER also secretes an inhibitory factor, preventing RA-induced or already established Hoxb-8 expression in the cells immediately underneath the AER. Consistent with a role for Hoxb-8 in positioning of the forelimb ZPA, Hoxb-8 expression is not seen in RA-induced duplications at the anterior of the hindlimb. However, grafting of Hoxb-8-expressing tissue to the hindlimb can lead to Shh expression and similar duplications, suggesting that factors mediating ZPA formation are very similar in both wing and leg.

Animals↗
PubMed1996

Retinoic acid is required for the initiation of outgrowth in the chick limb bud.

BACKGROUND: Retinoic acid (RA) is present in the chick limb bud, and excess RA induces limb duplications. Here, we have investigated the role of endogenous RA during chick limb development by preventing the synthesis of RA and testing the effect on various genes expressed during limb initiation and outgrowth. RESULTS: We demonstrate that the stage 20/21 limb bud synthesizes didehydroretinoic acid (ddRA), and that the posterior half of the limb bud synthesizes ddRA at a higher rate than the anterior half. Disulphiram inhibits this synthesis at micromolar concentrations. Administering disulphiram to embryos prior to limb bud outgrowth (stages 12-18) abolishes outgrowth, and no limb develops in the majority of cases. Disulphiram treatment also prevents the expression of Sonic hedgehog (Shh), but the expression of the fibroblast growth factor-8 gene (Fgf-8) appears as normal in the ectoderm over the prospective limb bud. The application of a bead soaked in RA can rescue Shh expression. Disulphiram treatment of later limb buds (stages 20-23) similarly down-regulates Shh, and also Fgf-4, expression, whereas the expression of Fgf-8, as at earlier stages, is initially unaffected. Again, RA can rescue the expression of Shh in these limb buds. CONCLUSIONS: RA, in conjunction with Fgf-8, may be needed for the induction of the chick limb bud and the induction of Shh and Fgf-4 expression. The expression of Shh and Fgf-4 remains dependent upon the continued synthesis of RA within the limb bud. Didehydroretinoic acid is the major active retinoid in the stage 20 chick limb bud.

Animals↗