Biomedical subjects
Publications and source records attributed to M Maden.
Recent studies on the regenerating newt limb, using cells transfected with chimeric retinoic acid receptors that can be activated by thyroid hormone, have provided unique insights into the function of specific retinoic acid receptor isoforms.
BACKGROUND: Retinoic acid (RA) is a morphogenetically active signalling molecule thought to be involved in the development of severely 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. CONCLUSIONS: These results demonstrate at least three roles for RA in central nervous system development: neural crest survival, neurite outgrowth and hindbrain patterning.
Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic development. In the work reported here we have determined which retinoids are present in zebrafish embryos and how their levels change throughout development and into adulthood. All-trans-RA is present and its level does not change significantly during embryogenesis. We failed to detect other retinoic acid isomers such as 9-cis-RA and 4-oxo-RA, but we did observe a rapid rise in the level of didehydroretinol after gastrulation. The most striking result is that the zebrafish embryo, like Xenopus and tunicates, contains a vast excess of t-retinal whereas the embryos of higher vertebrates have an excess of t-retinol. However, as the zebrafish grows, the levels of t-retinol rise so that by adulthood t-retinol and t-retinal concentrations are more equivalent, indicating a changing pattern of retinoid metabolism with growth. To examine the significance of the use of t-retinal as a precursor of t-RA we treated embryos with disulphiram, an inhibitor of retinaldehyde dehydrogenase. This resulted in embryos with an undulating notochord and correspondingly abnormal somites and ventral floor plate. In contrast to this effect, 4-methylpyrazole, which inhibits alcohol dehydrogenases, had no effect on development. This effect of disulphiram suggests that t-RA may be involved in the establishment of the anteroposterior axis of the embryo.
We provide here further data on the dramatic homeotic transformation of tails into limbs which is induced by retinoids during frog tadpole tail regeneration. The effect can still be produced up to nine days after tail amputation by which time tail regeneration has essentially been completed. Complete tail amputation is needed for the effects to be manifest, partial damage of various sorts to the tail is not enough. We show that as well as retinyl palmitate, other retinoids such as all-trans-retinoic acid and TTNPB, which is a RAR specific retinoid, can induce the homeotic transformation. TTNPB has a 300 x greater potency than retinoic acid. Prolactin, which inhibits thyroid hormone production, prevents the appearance of limbs on the tail from which we conclude that thyroid hormone is needed. We present preliminary evidence from RT-PCR that all six retinoid receptors, the three retinoic acid receptors (RARs), and the three retinoid X receptors (RXRs), are present in the normal tail blastema and that after retinoid treatment RAR alpha, RXR alpha, and RXR beta may be up-regulated. Finally, we show that when RA synthesis is inhibited, normal tail regeneration is inhibited. We conclude that tail regeneration depends upon a particular endogenous level of RA, but that when this level is raised by external administration and thyroid hormone receptors are present the up-regulation of certain retinoid receptors allows novel nuclear receptor interactions which results in the induction of limb-specific genes leading to the appearance of limbs on the tail.
We exposed st.10 chicks to retinoic acid (RA), both globally, and locally to individual rhombomeres, to look at its role in specification of various aspects of hindbrain derived morphology. Previous studies have looked at RA exposure at earlier stages, during axial specification. Stage 10 is the time of morphological segmentation of the hindbrain and is just prior to neural crest migration. Rhombomere 4 localised RA injections result in specific alterations of pathways some crest cells that normally migrate to sites of differentiation of neurogenic derivatives. The r4 crest cells that give rise to mesenchymal derivatives are unaffected. In addition, r4 gene expression is also partially altered by RA; within 6 hours of r4 exposure to RA, ectopic expression of Krox-20 is seen in r4 and Hoxb-1 expression is lost while Hoxa-2 expression continues normally. When we examined these RA-treated animals later in development, they showed an anterior displacement of the facial ganglion in addition to a mis-direction of the extensions of its distal axons and a dramatic decrease in the number of contralateral vestibuloacoustic neurons normally seen in r4. Only this r4-specific neuronal type is affected in r4; the motor neuron projections seem normal in experimental animals. The specificity of this result, combined with the loss of Hoxb-1 expression in r4 and the work by Krumlauf and co-workers showing gain of contralateral neurons co-localised with ectopic Hoxb-1 expression, indicates a role for Hoxb-1 and RA in the specification of this cell type in normal development. These results suggest that RA, at st.10, is able to affect some aspects of segment identity while leaving others unchanged.
We have analysed the endogenous retinoids present in whole mouse embryos from day 9 to day 14 of development and in individual components of the embryo at two stages, day 10.5 and day 13, by HPLC. We can only detect two retinoids, all-trans-RA (tRA) and all-trans-retinol (t-retinol), and t-retinol is 5-10-fold in excess over tRA. We cannot detect 9-cis-RA or any didehydroretinoids; thus mammalian embryos seem to differ in their retinoid content from other embryos such as chick, Xenopus, and fish. The levels of tRA do not change significantly over the 6 days of development analysed, whereas t-retinol rises sharply as the liver develops. Within the embryo, tRA is present at high levels in the developing spinal cord and at very low levels in the forebrain; indeed there is a gradient of endogenous tRA from the forebrain to the spinal cord. Other parts of the embryo had intermediate levels of tRA. When a teratogenic dose of RA was administered to day 10.5 embryos, the levels of tRA present in individual tissues of the embryo rose dramatically--from 175-fold to 1,400-fold--and the levels rose in all tissues not in any exclusive areas. We then determined which areas of the embryo were malformed by such a teratogenic dose. The lower jaw, palate, vertebrae, tail, and limbs were consistently abnormal, and since these areas received a dose of tRA no higher than any other it was concluded that cell-specific factors must determine the teratogenic response of these tissues. We then considered whether cellular retinoic acid-binding protein I or II (CRABP I or II) played any role in this response by determining their relative levels in each of the tissues analysed. There was no correlation between the presence of CRABP I and II and the distribution of administered RA. Neither was there a clear correlation in detail between the presence of CRABP I and II and the sites of teratogenesis. We therefore conclude that other factors, for example, nuclear factors, must be responsible for the teratogenic response to RA.
We have investigated the role that cellular retinoic acid binding protein I (CRABP-I) may play in the development of the murine hindbrain. Since the central nervous system (CNS) represents a major site of the teratogenic action of retinoic acid (RA), we have also determined the effects of exposure of high levels of RA on CRABP-I expression within the CNS. Expression of CRABP-I can first be detected within the presumptive hindbrain of presomitic mouse embryos and later also appears in neural crest cells and neural crest derivatives; it is thus tissue specific at these early stages. Exposure of 7.75-day mouse embryos to RA induces two phenotypes: one is externally normal and the other is exencephalic. In the exencephalic embryos we show that there is abnormal crest migration, a fusion of the trigeminal and facial-acoustic ganglia, a rostral and lateral shift of the otic vesicle, and a loss of hindbrain rhombomeres. Furthermore, and in contrast to in vitro studies, we demonstrate that CRABP-I appears to be up-regulated in both phenotypes of mouse embryos treated with RA and that this up-regulation is accompanied by an anteriorization of its expression within the nervous system. This new CRABP-I expression domain thus retains its tissue specificity. The role that CRABP-I may play in normal development of the hindbrain and in teratogenesis and the similarity of these results to those obtained with various Hox genes are discussed.
The distribution of cellular retinoic acid-binding protein (CRABPI) and cellular retinol binding protein (CRBPI) was studied in a series of prenatal and early postnatal rats, covering the main stages of development and eruption of the molar teeth. CRABPI positive cells were found in the mesenchymal cells of the dental follicle from the cap stage and in the dental papilla from the early bell stage. In the dental papilla, CRABPI positive cells were situated adjacent to the enamel organ in the cervical loop region and in the subodontoblastic region. Newly formed odontoblasts were CRABPI positive for a short period of time. The enamel organ was CRBPI and CRABPI negative, except for the presence of CRABPI positive cells in the internal enamel epithelium over the tip of cusps and in parts of the stratum intermedium. During root formation, CRABPI positive cells were found in the developing periodontal ligament, in the dental papilla adjacent to the epithelial root sheath and in the subodontoblastic zone. During crown formation, CRBPI positive cells were mainly localized to the mesenchymal cells of the dental papilla during the cap stage of crown development. The periosteum of the developing mandible contained CRABPI positive cells while some osteoclasts appeared to show a weak but positive reaction to CRBPI. The findings were considered in terms of the possible significance of retinoid-binding proteins during tooth and bone development.
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By using biolistics to transfect the regenerating amphibian limb with cDNAs encoding chimeric receptors, the functions of individual retinoic acid receptors have been pinpointed.
The distribution of cellular retinoic acid-binding proteins I and II (CRABP I and II) during the first 6 days of chick development has been investigated using immunoblotting. Since retinoic acid (RA) is teratogenic to some parts of the embryo, stimulatory to other parts, and has no effect on others it may be that the distribution of cytoplasmic proteins such as CRABP I and II plays some role in this differential activity. Neither protein is expressed in the day 2 embryo, but from day 3 onwards both proteins are expressed and CRABP I is in considerable excess over CRABP II. Within the day 4 embryo there is some significant variation in the distribution according to tissue type. Neural tissues, neural crest derivatives, and limb buds most strongly express CRABP I whilst other tissues contain only moderate levels, and heart and epidermis do not express CRABP I at all. CRABP II has a widespread distribution, although at a lower level than CRABP I, with the exception of somites and ectoderm which do not express it at all. In the limb buds, there is a significant variation in CRABP I levels across the anteroposterior axis which suggests that these two CRABPs may have different functions during development. The relationship of these distributions in the embryo to the role of endogenous RA and the teratogenic effects of RA is discussed.
Retinoids have been implicated in pattern formation processes in both developing chick limbs and in the regenerating limbs of urodele amphibians as well as in other aspects of embryonic development. Since chick wing buds have been shown to have a higher concentration of all-trans-retinoic acid (RA) in the posterior region than in the anterior region, we set out to look for a gradient of RA in the regenerating limb of the axolotl, Ambystoma mexicanum. We used high-performance liquid chromatography to separate, identify, and measure the concentration of retinoids present in the tissues. Our results show that the concentration of RA is about five times higher in posterior quarters of the limb regeneration blastema compared to anterior quarters. In addition, levels of RA are about two and a half times higher in blastemas from the radius-ulna level of the limb compared to those from the humerus level. By contrast, the limb regenerates of the African clawed frog, Xenopus laevis (which produce a regenerative outgrowth which is pattern-deficient), do not exhibit an anteroposterior gradient of any of the retinoids investigated. This paper also reports on the levels of retinoids in a number of other tissues as well. This anteroposterior gradient of RA in the axolotl limb regeneration blastema has significant implications for pattern formation during limb development and regeneration.
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kreisler is a recessive mutation resulting in gross malformation of the inner ear of homozygous mice. The defects in the inner ear are related to abnormalities in the hindbrain of the embryo, adjacent to the ear rudiments. At E9.5, the neural tube posterior to the boundary between the third and fourth rhombomeres, r3 and r4, appears unsegmented, and the region that would normally correspond to r4 is unusually thick-walled and contains many dying cells. The absence of morphological segmentation in the posterior hindbrain corresponds to an altered pattern of gene expression in that region, with major abnormalities posterior to the r4/5 boundary and minor abnormalities anterior to it. From the expression patterns at E9.5 of Krox-20, Hoxb-1 (Hox 2.9), Hoxb-2 (Hox 2.8), Hoxa-3 (Hox 1.5), Hoxd-4 (Hox 4.2) and cellular retinoic-acid binding protein I (CRABP I), it appears that the fundamental defect is a loss of r5 and r6. Correspondingly, the glossopharyngeal ganglion and nerve, associated with r6 are missing and the abducens nerve, which originates from r5 and r6, is also absent. Examination of Krox-20 expression at stages as early as E8.5 indicates that Krox-20 fails ever to be expressed in its r5 domain in the homozygous kreisler mutant. The abnormal amount of cell death is seen only later. An interpretation is that the cells that would normally become specified at an early stage as r5 and r6 adopt an r4 character instead, producing an excess of r4 cells that is disposed of subsequently by cell death.
The most remarkable of all the effects of retinoids on embryonic systems is the homeotic transformation of tails into legs which was recently reported using an Indian species of frog. Since then several attempts have been made to repeat these results on other species, notably Xenopus, with no success. Here I report the successful repetition of this homeotic transformation using Rana temporaria tadpoles treated with retinyl palmitate. The phenomenon is concentration-dependent, time-dependent, and stage-dependent. There is some difference in effect according to the tail amputation level. The limbs induced are always hindlimbs and there can be between 1 and 9 of them. There is a tendency to induce limbs in pairs so that even numbered groups are produced in considerable excess over odd numbered groups. As assessed by cartilage staining the majority of the limbs are normal in the proximodistal and anteroposterior axes. The other types of outgrowths induced are double-posterior limbs, posterior half-limbs (usually defective in the proximodistal axis), and spikes. As assessed by the anatomy of the muscle patterns some of these apparently normal limbs are duplicated in the dorsoventral axis. The stage and species dependencies of this phenomenon suggest that it may involve thyroid hormone receptors whose levels rise leading up to metamorphosis and whose interactions with retinoic acid receptors have recently been described.
Retinoids are low molecular weight, lipophilic derivatives of vitamin A which have a profound effect upon the development of a diverse array of animals. Here, I review these effects on Invertebrates: a colonial hydroid, a colonial ascidian, and Vertebrates: the regenerating amphibian limb, the developing chick limb bud, the regenerating amphibian tail, the anteroposterior axis of the early embryo, the developing chick embryo skin. There is a striking uniformity of effect of retinoids on pattern formation when applied to these diverse organisms. The majority react by being posteriorized in their development, although additional effects can also be seen. Several hypotheses which can explain these results are discussed along with the deduction that they lead to: retinoids may be components of a universal developmental mechanism or they may simply act in a similar way to alter a universal developmental mechanism. In either case the experimental analysis of retinoid effects on development has important implications for the evolution of developmental mechanisms.
Retinoids are important molecules in various aspects of embryological development. Here the distribution of cellular retinoic acid-binding protein I (CRABPI) was studied in the continuously growing incisor of adult rats using an affinity-purified rabbit polyclonal antibody. CRABPI was present throughout the presecretory and secretory ameloblast layer. The protein disappeared from that layer during its maturation phase. The adjacent dental mesenchyme of the developing pulp stained positively for CRABPI, especially in the layer immediately beneath the fully differentiated odontoblasts. Little CRABPI was present in the odontoblast layer itself. The distribution of CRABPI, both in the undifferentiated basal region of the incisor tooth and associated with the cells during hard-tissue formation, suggests a role for this molecule during differentiation and hard-tissue genesis.
As retinoic acid is an important signaling molecule during embryological development. Since periodontal tissues are thought to have fetal-like properties, the present study was to determine the presence and distribution of cellular retinoic acid-binding protein I(CRABPI) in the periodontal tissues of the rat. Following demineralization, wax sections of the molar teeth of four adult male rats were cut in a plane parallel to the molar tooth row. Five representative sections from each jaw were then processed and stained to reveal the presence of CRABP. This protein was identified by immunochemistry utilizing an affinity-purified rabbit polyclonal antibody. The IgG antibody was used at a dilution of 1 microgram ml-1 and immunoreactivity detected with a kit according to protocol. Immunoreactivity was detected with diaminobenzidine and sections counterstained with hematoxylin. Results showed that virtually all the cells of periodontal ligament, the vast majority representing fibroblasts, contained CRABP. Fibroblasts in the gingiva above the level of the alveolar crest also stained positively for CRABP. The periodontal tissues of all three molar teeth were similarly affected. Fibroblasts in other regions of the oral cavity, such as the dermis of the skin of the cheek or the lamina propria of the buccal mucosa or of the diastema region, exhibited little or no staining for CRABP. Because of its properties as a signaling molecule and its ability to interact with other regulatory molecules, retinoic acid and cellular retinoic acid-binding protein may play an important role in the biology of the periodontal tissues.