Expression of cellular retinoic acid binding protein II (chick-CRABP II) in the chick embryo

We previously demonstrated the presence of cellular retinoic acid binding protein II, chick-CRABP II, in chick embryos. In the present study, we investigated the distribution of chick-CRABP II in 14-day chick embryos by means of immunoblot analysis. Chick-CRABP II was expressed in skin, muscle, bone with tendon of the embryos, but not expressed in the nervous system. In adult chick tissues, chick-CRABP II was not detected on immunoblotting; Chick-CRABP II in adults amounts to less than 10 ng/mg soluble protein. These observations suggest that chick- CRABP II is an embryonic protein involved in the development of specific tissues, such as bone, muscle and skin.     

NMR study of the binding of all-trans-retinoic acid to type II human cellular retinoic acid binding protein.

Cellular RA binding proteins are thought to play important roles in the (RA), a hormonally active metabolite of vitamin A that has profound effects on cell growth, + differentiation and morphogenesis. Binding of RA to type II human cellular RA binding proteins (CRABPII) has been investigated by NMR spectroscopy. The sequential resonance assignments of +CRABPII in the presence of RA were established by heteronuclear three-dimensional NMR at pH 7.3. The resonance assignments of the bound RA were achieved by homonucl NMR. The secondary structures of holo-CRABPII determined by NMR were ess as revealed by the crystal structure of holo-CRABPII. Most of the nuclear Overhauser effects (NOEs) between CRABPII and the bound RA were consistent with those predicted crystal structure of holo-CRABPII. The results suggested that the conformations in solution and in the crystalline state are highly similar. Compared to the ligand binding pocket, especially the ligand entrance, was stabilize Ser12-Leu18, one of the structure elements that constitute the ligand binding pocket, became more mobile upon binding of RA. Intramolecular NOEs between protons of the bo the carboxylate end of the bound RA is well fixed but the β-ionone     

Cellular retinoic acid-binding protein and the role of retinoic acid in the development of the chick embryo

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.     

Differential expression of chicken CYP26 in anterior versus posterior limb bud in response to retinoic acid

Multiple studies indicate that quantitative control of the levels of all-trans-retinoic acid (RA) in the vertebrate embryo is necessary for correct development. The function of RA in cells is regulated by a number of coordinated mechanisms. One of those mechanisms involves controls on the rate of RA catabolism. Recently, enzymes capable of catabolizing RA were found to constitute a new family, called CYP26, within the cytochrome P450 superfamily. CYP26 homologues have been isolated from human, mouse, zebra fish, and recently from the chick. In this study, we examined the regulation of chicken CYP26 (cCYP26) expression by RA during the early phase of chick limb outgrowth. In the anterior limb mesenchyme and apical ectodermal ridge (AER), cCYP26 expression was induced in a concentration dependent manner by implanting beads soaked in 0.1, 1, and 5 mg/ml RA. The RA-induced expression of cCYP26 in anterior limb mesenchyme and the AER was detected as early as 1 hr after treatment and was not affected by the presence of cycloheximide. In contrast to the anterior limb, the induction of cCYP26 was dramatically reduced (or absent) when RA beads were implanted in the posterior limb mesenchyme. Furthermore, induction of cCYP26 expression in the anterior mesenchyme was inhibited by transplantations of the zone of polarizing activity (ZPA) and by Shh-soaked beads. Our data suggest that different mechanisms regulate retinoid homeostasis in the AER and mesenchyme during limb bud outgrowth. J. Exp. Zool. 290:136-147, 2001.     

Identification of the cellular retinoic acid binding protein (cRABP) within the embryonic mouse (CD-1) limb bud

Retinoic acid, a physiologically active metabolite of vitamin A, is known animal teratogen. Among other malformations, limb abnormalities are produced and are attributed to a selective inhibition of differentiating prechondrogenic mesenchyme resulting in reduced or absent cartilage elements. Evidence is available that the cellular retinoic acid binding protein (cRABP) may be important in mediating the biological effects of retinoic acid. In this study, the cRABP has been identified by sucrose gradient sedimentation analysis in the gestation day 10 (Theiler stages 16-17) mouse forelimb bud, which contains retinoic-acid-sensitive prechondrogenic mesenchyme. Saturation analysis demonstrated values for the apparent dissociation constant (Kd) of 2.0 and 2.2 X 10(-9)M and for the total specific binding capacity for [3H]-trans-retinoic acid of 24.5 and 25.6 pmoles per mg cytosolic protein. The binding specificity of the forelimb bud cRABP for all-trans-retinoic acid was demonstrated in competition assays using all-trans-retinol, all-trans-retinal, and 13-cis-retinoic acid. In addition, 13-cis-retinoic acid was demonstrated to have a lower affinity for the cRABP than all-trans-retinoic acid, a result which may be related to the lower teratogenic potency of the 13-cis-retinoic acid. Thus, the cRABP was demonstrated in the mouse forelimb bud at a time of susceptibility for the production of limb malformations by retinoic acid. The role of the cRABP in the mechanism of retinoic acid teratogenicity remains to be delineated.     

Cellular retinoic acid binding protein: detection and quantitation in regenerating axolotl limbs

The concentrations of apo (unoccupied), holo (occupied), and total cellular retinoic acid binding protein (CRABP) were measured at various stages of axolotl limb regeneration. The ratio of apo-CRABP to holo-CRABP declined with advancing regenerate stage until the CRABP was all in the holo form. The increase in holo-CRABP is correlated with a stage-dependent shift in the effect of exogenous retinoic acid on regenerate pattern, from pattern duplication to inhibition of regeneration. The data suggest, though they do not prove, that these different morphological effects could be due to a shift from a CRABP-dependent to a CRABP-independent mechanism of exogenous retinoic acid (RA) action that is related to stage-specific variations in endogenous RA levels.     

Cell populations synthesizing cartilage proteoglycan core protein in the early chick limb bud.

Cartilage specific macromolecules are known to be synthesized in the mesenchyme of the embryonic chick limb bud, especially in areas of prechondrogenic condensations (Shinomura et al, 1984). Even though the mesenchyme seems homogeneous according to histological criteria, studies in the past have suggested the presence of different cell populations with different chondrogenic potential (Solursh et al, 1982; Swalla et al, 1984). In this study we have investigated by means of flow cytometry, the synthesis of proteoglycan core protein during early development of the chick limb bud in order to identify the different chondrocyte progenitor cells. We were able to identify by virtue of different size and density a cell population which synthesizes core protein extensively at stage 24 and stage 25 of development. This cell population synthesizes core protein predominantly at the proximal half of the limb bud at stage 24. However at stage 25 the same population synthesizes core protein predominantly at the distal half of the limb bud. These observations indicate that the distal half of stage 25 limb bud is mostly homogeneous with prechondrogenic cells and is in agreement with in vitro experiments that show high chondrogenic potential of the mesenchymal cells from this stage.     

Mouse limb bud cells respond to retinoic acid in vitro with reduced growth.

Retinoic acid (RA) has dramatic effects on the pattern of developing and regenerating vertebrate limbs. These effects are considered to result from RA-induced changes in the positional identity of limb cells, and involve the formation of extra structures. Whether the growth required to form the supernumerary parts of the pattern is a primary effect of RA treatment or a secondary effect that follows after a change in positional identity is not at present known. In this paper we have investigated the effects of RA treatment on the growth of cells from anterior and posterior halves of mouse limb buds in vitro. We observed that under our culture conditions, limb bud cells treated with 1 nM to 1 microM RA (0.3 ng/ml to 300 ng/ml) continue to grow but do so at a significantly slower rate than control cultures. There is a maximum inhibition of growth (50% of controls) between 10 nM and 100 nM RA, which corresponds to the measured range of concentrations of RA in vivo. Our observation of a significant decrease in growth rate over a wide range of RA concentrations is consistent with comparable reports of growth inhibition for a large number of other cell types in vitro as well as with the observation that exogenous RA inhibits blastemal growth in amphibians during the period of exposure to RA. We propose that the effects of RA on growth, either enhancement in vivo or reduction in vitro, can be seen as consequences of the ability of RA to alter positional identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Appearance of myosin in the chick limb bud

Quantitative microcomplement fixation has been used to detect the appearance of myosin in the chick embryonic limb bud. It has been shown that myosin or a myosinlike molecule is present by stage 23, before muscle can be distinguished histologically.     

Direct effects of retinoic acid on the development of the tail bud in chick embryos

Retinoic acid (RA) has been reported to induce vascular lesions and haematoma formation in the vicinity of the tail bud during the critical period for inducing abnormalities of tail bud development in hamsters (Wiley, '83; Tibbles and Wiley, '88), mice (Tibbles and Wiley, '88) and chicken embryos (Jelinek and Kistler, '81). Experiments were conducted to determine whether or not these vascular lesions were the primary cause of the malformations which they accompanied. Chick embryos were exposed for varying lengths of time to several dosages of RA. Primitive streaks or tail buds from treated embryos were then excised prior to vascularization and transplanted to the coelomic walls of untreated host embryos. The grafts were harvested at 3 or 6 days after grafting and processed for histological examination. Observations of serial sections of controls showed that the primitive streak and early (stage 13-14) tail bud were able to form neural tubes and a variety of other structures including ganglia, nerve fibres, and kidney tubules. Treatment of donor embryos with RA prior to grafting, however, affected the frequency and characteristics of the neural tubes and other tissues developing in the grafts. The effects of RA on development were correlated with both the dosage and length of exposure to the teratogen prior to grafting. Since the grafts were made before the appearance of blood vessels in the tail buds, we have concluded that the effects of RA on the development of tail bud tissues, and especially the secondary neural tube, are direct and are not mediated solely through the disruptive effects of vascular lesions seen in intact embryos.     

Ectodermal influence on physiological cell death in the posterior necrotic zone of the chick wing bud

The phenomenon of "programmed cell death" in the posterior necrotic zone (PNZ) of the chick wing bud was reexamined. Prospective PNZs (pPNZs) were excised from stage 18-21 donor wings and observed for signs of necrosis in vitro. Cell death was quantified by a chromium-51 release assay. Prospective PNZs from the youngest donors (stage 18) showed no signs of death above control levels, while necrosis increased in vitro with increasing donor age. Cell death in the PNZ at stage 24 could be inhibited by removing the overlying ridge at stage 20 or 21. These results suggest that cell death in the PNZ is not rigidly determined early in development as previous studies suggest, but remains responsive to the cellular environment until shortly before the cells die.     

Cellular retinoic acid binding protein I mediates rapid non-canonical activation of ERK1/2 by all-trans retinoic acid

All-trans retinoic acid (atRA), one of the active ingredients of vitamin A, exerts canonical activities to regulate gene expression mediated by nuclear RA receptors (RARs). AtRA could also elicit certain non-canonical activities including, mostly, rapid activation of extracellular signal regulated kinase 1/2 (ERK1/2); but the mechanism was unclear. In this study, we have found that cellular retinoic acid binding protein I (CRABPI) mediates the non-canonical, RAR- and membrane signal-independent activation of ERK1/2 by atRA in various cellular backgrounds. In the context of embryonic stem cells (ESCs), atRA/CRABPI-dependent ERK1/2 activation rapidly affects ESC cell cycle, specifically to expand the G1 phase. This is mediated by ERK stimulation resulting in dephosphorylation of nuclear p27, which elevates nuclear p27 protein levels to block G1 progression to S phase. This is the first study to identify CRABPI as the mediator for non-canonical activation of ERK1/2 by atRA, and demonstrate a new functional role for CRABPI in modulating ESC cell cycle progression.     

Nerve-dependent and -independent tenascin expression in the developing chick limb bud.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.     

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.