Biomedical subjects
Publications and source records attributed to G Eichele.
It has long been suggested that pattern formation depends in part on signalling molecules known as 'morphogens', diffusible substances that determine cell fate in a concentration-dependent way. Retinoic acid, a small hydrophobic molecule that binds to nuclear receptors, is a candidate morphogen for specifying the anteroposterior pattern of vertebrate limbs.
We have isolated, sequenced and examined the expression pattern of two tandemly arranged homeobox-containing genes from the chicken. The predicted amino acid sequences of the homeodomain and the adjacent carboxyterminal portion of the protein of the first gene is virtually identical (99%) to that of murine homeobox 2.1 and hence we refer to it as Ghox 2.1 (Gallus homeobox). The closest mouse homologue of the second homeodomain is Hox 2.2 (95% identical within the homeobox), and hence referred to as Ghox 2.2. Northern analysis of embryonic RNA reveals major transcripts of 2 kb for Ghox 2.1 and 1.7 kb for Ghox 2.2. To investigate the transcript pattern, embryos of various stages were dissected into heads, trunks and limb buds and the RNA was analysed by Northern blotting and RNase protection assays. Ghox 2.1 transcripts are present in all three regions. Ghox 2.2 RNA is found in trunks and limb buds, but it is strikingly absent from the developing head. In situ hybridization with 35S-labelled antisense riboprobes derived from Ghox 2.1 demonstrates that this gene is expressed at high levels in spinal chord, myelencephalon and mesonephros. Dorsal root ganglia and the lung rudiment also contain Ghox 2.1 message, but in somewhat lower amounts. Mid- and forebrain, the heart, presomitic mesenchyme and notochord do not contain detectable levels of Ghox 2.1 mRNA. Of particular interest is the expression of Ghox 2.1 in a well-defined patch of mesenchymal tissue situated in an anterioproximal region of the limb bud.
Wing buds whose posterior half is excised, develop into wings lacking distal structures. However, such experimentally generated preaxial half wing buds can be rescued by implanting a retinoic-acid-releasing bead at their anterior margin. The polarity of the pattern that originates from preaxial half wing buds is reversed. For example, instead of a 234 digit pattern typical for normal wings, the order of digits is 432. This result implies that retinoic acid has the capacity to reprogram anterior limb bud tissue, and that the resulting change in cell fate does not depend on the presence of posterior tissue regions such as the zone of polarizing activity (ZPA).
It has long been proposed that concentration gradients of morphogens provide cues to specify cell fate in embryonic fields. Recent work in a variety of vertebrate systems give bona fide evidence that retinoic acid, the biologically active form of vitamin A, is a candidate for such a morphogen. In the developing chick wing, for example, locally applied retinoic acid triggers striking changes in the pattern along the anteroposterior axis. Instead of giving rise to a wing with the normal 234 digit pattern, wing buds treated with retinoic acid develop a 432234 mirror-image symmetrical digit pattern. For this review, we focus on three aspects of limb morphogenesis. (1) We summarize the experimental evidence supporting the notion that retinoic acid is a candidate morphogen. (2) Limb buds contain high levels of cellular retinoic-acid-binding protein (CRABP). Using order of magnitude calculations, we evaluate how the concentration of CRABP might affect the occupancy state of the retinoic acid receptor. (3) We discuss the spatio-temporal expression pattern of homeobox-containing genes in the developing limb and speculate about the possibility that retinoic acid influences the pattern of expression of homeobox genes.
Retinoids (vitamin A derivatives) have been shown to have striking effects on developing and regenerating vertebrate limbs. In the developing chick limb, retinoic acid is a candidate morphogen that may coordinate the pattern of cellular differentiation along the anteroposterior limb axis. We describe a series of investigations of the metabolic pathway of retinoids in the chick limb bud system. To study retinoid metabolism in the bud, all-trans-[3H]retinol, all-trans-[3H]retinal and all-trans-[3H]retinoic acid were released into the posterior region of the limb anlage, the area that contains the zone of polarizing activity, a tissue possibly involved in limb pattern formation. We found that the locally applied [3H]retinol is primarily converted to [3H]retinal, [3H]retinoic acid and a yet unidentified metabolite. When [3H]retinal is locally applied, it is either oxidized to [3H]retinoic acid or reduced to [3H]retinol. In contrast, local delivery of retinoic acid to the bud yields neither retinal nor retinol nor the unknown metabolite. This flow of metabolites agrees with the biochemical pathway of retinoids that has previously been elucidated in a number of other animal systems. To find out whether metabolism takes place directly in the treated limb bud, we have compared the amount of [3H]retinoid present in each of the four limb anlagen following local treatment of the right wing bud. The data suggest that retinoid metabolism takes place mostly in the treated limb bud. This local metabolism could provide a simple mechanism to generate in a controlled fashion the biologically active all-trans-retinoic acid from its abundant biosynthetic precursor retinol. In addition, local metabolism supports the hypothesis that retinoids are local chemical mediators involved in pattern formation.
It has long been suggested that the generation of biological patterns depends in part on gradients of diffusible substances. In an attempt to bridge the gap between this largely theoretical concept and experimental embryology, we have examined the physiology of diffusion gradients in an actual embryonic field. In particular, we have generated in the chick wing bud concentration gradients of the morphogenetically active retinoid TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl] benzoic acid, a synthetic vitamin A compound. Upon local application of TTNPB the normal 234 digit pattern is duplicated in a way that correlates with the geometry of the underlying TTNPB gradient; low doses of TTNPB lead to a shallow gradient and an additional digit 2, whereas higher doses result in a steep, far-reaching gradient and patterns with additional digits 3 and 4. The experimentally measured TTNPB distribution along the anteroposterior axis, can be modeled by a local source and a dispersed sink. This model correctly predicts the site of specification of digit 2, and provides an empirical estimate of the diffusion coefficient (D) of retinoids in embryonic limb tissue. The numerical value of approximately 10(-7) cm2s-1 for D suggests that retinoids are not freely diffusible in the limb rudiment, but interact with the previously identified cellular retinoic acid binding protein. In addition, D affords an estimate of the time required to establish a diffusion gradient as 3 to 4 h. This time span is in a range compatible with the time scale of pattern specification in developing vertebrate limbs. Our studies support the view that diffusion of morphogenetic substances is a plausible mechanism of pattern formation in secondary embryonic fields.
Small, positively charged beads that slowly release known amounts of all-trans-retinoic acid have been implanted below the apical ectodermal ridge at the anterior margin (opposite somite 16) of wing buds of 3 1/2 day-old chick embryos. The continuous release of retinoic acid is shown to create an anteroposterior concentration gradient of retinoic acid in the limb field that is stable with time, despite the fact that this compound is metabolized by the limb tissue. With beads that release increasing amounts of retinoic acid, the normal 234 digit pattern is progressively altered to a 2234, to a 32234, and then to a 432234 pattern. The tissue concentrations of all-trans-retinoic acid required to change the digit pattern in this way range between 1 and 25 nM. When the same amounts of retinoic acid are released from posteriorly implanted beads (placed below the apical ectodermal ridge opposite somite border 19/20 or somite 20), the normal digit pattern is unaffected. Implantations of beads that release all-trans-retinoic acid are thus identical in their effect to grafts of cells from the limb polarizing region, which cause similar dose-dependent changes in the digit pattern when grafted to the anterior margin of the bud (but not when grafted opposite somites 19 or 20). Because of the low concentrations of retinoic acid required for its biological effect, the graded response observed, and the fact that a concentration gradient is established across the limb field, all-trans-retinoic acid closely mimics the putative morphogen that has been postulated to be emitted by polarizing region cells during normal development.
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All-trans-retinoic acid causes striking digit pattern changes when it is continuously released from a bead implanted in the anterior margin of an early chick wing bud. In addition to the normal set of digits (234), extra digits form in a mirror-symmetrical arrangement, creating digit patterns such as a 432234. These retinoic acid-induced pattern duplications closely mimic those found after grafts of polarizing region cells to the same positions with regard to dose-response, timing, and positional effects. To elucidate the mechanism by which retinoic acid induces these pattern duplications, we have studied the temporal and spatial distribution of all-trans-retinoic acid and its potent analogue TTNPB in these limb buds. We find that the induction process is biphasic: there is an 8-h lag phase followed by a 6-h duplication phase, during which additional digits are irreversibly specified in the sequence digit 2, digit 3, digit 4. On average, formation of each digit seems to require between 1 and 2 h. The tissue concentrations, metabolic pattern, and spatial distribution of all-trans-retinoic acid and TTNPB in the limb rapidly reach a steady state, in which the continuous release of the retinoid is balanced by loss from metabolism and blood circulation. Pulse-chase experiments reveal that the half-time of clearance from the bud is 20 min for all-trans-retinoic acid and 80 min for TTNPB. Manipulations that change the experimentally induced steep concentration gradient of TTNPB suggest that a graded distribution of retinoid concentrations across the limb is required during the duplication phase to induce changes in the digit pattern. The extensive similarities between results obtained with retinoids and with polarizing region grafts raise the possibility that retinoic acid serves as a natural "morphogen" in the limb.
A method of controlled release that allows the continuous local application of retinoids (vitamin A derivatives) in living tissues has been developed. Several biocompatible 200-microns-diameter polymeric beads have been tested as possible carriers. Each type of bead was loaded by soaking in an isotopically labeled retinoid solution, washed, and then transferred into tissue culture medium for quantitative release measurements. Positively-charged ion-exchange resins of the Dowex 1 type were found to be the most suitable for the controlled release of retinoic acid, a negatively charged compound. For the controlled release of uncharged retinoids such as retinyl acetate, uncharged acrylic ester polymer beads are preferred; these beads can also be used to release the negatively charged compounds retinoic acid and prostaglandin E1. In all cases, a prolonged release is obtained that persists for more than a day. During this interval, the release is diffusion-controlled, and the total amount of compound released is directly proportional to the amount of the compound that the bead is exposed to during the initial loading step. High-performance liquid chromatography has been used to analyze the nature of the released retinoid. When the positively charged beads are loaded with all-trans-retinoic acid, there is a time-dependent decrease in the proportion of the all-trans isomer released which is due to an increased release of two cis isomers. This isomerization reaction occurs at a considerably slower rate when the uncharged beads are used as carriers. To mimic the conditions under which the local release of retinoic acid causes striking pattern duplications in developing chick wings, beads loaded with isotopically labeled retinoids were manually implanted into a slit cut into wing buds of stage-20 chick embryos. The release rate obtained was comparable to that found in vitro, and a time-dependent accumulation of the released radioactive compound was measured that was confined to the tissue near the site of implantation. All of the beads tested were readily accommodated by the tissue and could be easily removed at any time to terminate the treatment. It is believed that the controlled release of chemicals from such tiny biocompatible implants has a wide potential range of applications in biology.
Aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme that catalyses the transamination reaction: L-aspartate + 2-oxoglutarate----oxaloacetate + L-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. This paper proposes a mechanism of action that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 A resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogues, as well as on model building studies. The enzyme is composed of two identical subunits, which consist of two domains. The coenzyme is bound to the larger domain and is situated in a pocket near the subunit interface. The proximal and distal carboxylate group of dicarboxylic substrates are bound to Arg386 and Arg292 , respectively, the latter residue belonging to the adjacent subunit. These interactions largely determine the substrate specificity of the enzyme. They not only position the substrate efficient catalysis but also bring about a bulk movement of the small domain that closes the active site crevice and moves Arg386 about 3 A closer to the coenzyme. The replacement of the epsilon-amino group of Lys258 by the alpha-amino group of the substrate in the aldimine bond to pyridoxal phosphate is accompanied by a tilting of the coenzyme by approximately 30 degrees. The released epsilon-amino group of Lys258 serves as a proton acceptor/donor in the 1,3- prototropic shift producing the ketimine intermediate. At this stage, or after hydrolysis of the ketimine bond, the coenzyme rotates back to an orientation between that in the "external" aldimine intermediate and that in the pyridoxal form. Throughout this process, the protonated pyridine nitrogen atom maintains a hydrogen bond to the beta-carboxylate group of Asp222 . Upon formation of the pyridoxamine form, the small domain moves back to its original position. The proposed mechanism is compatible with the known kinetic and stereochemical features of enzymic transamination.
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X-ray diffraction studies to 2.8-A resolution have yielded the three-dimensional structure of mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an isologous alpha 2 dimer (Mr = 2 x 45,000). The subunits are rich in secondary structure and contain two domains, one of which anchors the coenzyme, pyridoxal 5'-phosphate. Each active site lies between the subunits and is composed of residues from both of them.
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