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CRABP and the teratogenic effects of retinoids
IOMMENT
CRABPand the terat0genic effects of retin0ids R. BALLING
MAXPLANER INSTITVTEOF BIOPHYSICALCHEMISTRY, DEPARTMENT OF MOLECULARCELL BIOLOGY,
3400 GO~NGEN,FRG. High doses of vitamin A or its derivatives (retinoids) induce malformations in developing embryosL Because retinoic acid seems to be the biologically m~st active metabolite, this syndrome is known as retinoic acid embryopathy 2. Prominent features of this syndrome include limb abnormalities and craniofacial defects. The teratogenic activity of retinoids has been known for a long time, but until recently a molecular mechanism has not been in sight. Recently, increasing attention has been paid towards the role of retinoids in normal embryogenesis ~-5. There is growing evidence that retinoic acid is a morphogen, and as such is responsible for specifying positional information. A morphogenetic function for retinoic acid is suggested by the observation that in the developing limb bud, retinoic acid is distributed in a gradient, with the highest concentration in the posterior region 6. A mirror image duplication of the digits results if this distribution is perturbed by local application of retinoic acid to the anterior part of the limb bud. Retinoic acid therefore mimics the action of the zone of polarizing activity found at the posterior end of the limb bud 7. In addition to the limb bud, the anterior part of the primitive streak (Hensen's node) also has polarizing activity, as do two midline structures that are derived from the regressing node: the floorplate of the neural tube and the noLochord 8,9. Since this polarizing activity probably reflects in part the action of retinoic acid, a spatially controlled distribution of retinoids could be a primary morphogenetic determinant in the developing embryo: cells might receive their positional identity by reading concentration values of retinoids. What is the link between the teratological and developmental actions of retinoic acid? Recent results from four different labs have
provided some interesting insights into this question. They report that the cellular retinoic acid-binding protein (CRABP) is expressed predominantly in those structures that are affected by application of high doses of retinoic acid during embryogenesis. By use of in situ hybridizationm, H, immunohistochemica112,13 and direct retinoid-binding studies 12 of mouse and chick embryos, expression of CRABP was localized to specific areas of the central nervous system, the craniofacial mesenchyme, the visceral arches, ganglia of the peripheral nervous system and the limb bud. Many of these tissues are neural crest derivatives, tissues that are particularly affected in retinoic acid embryopathy. Similarly, expression of CRABP in the limb bud 10,14 points to another site in which abnormalities can be induced by exposure to high doses of retinoic acid. The authors suggest that one of the reasons for these tissues being the major target in retinoic acid embryopathy is that they express high levels of the CRABP. CRABP belongs to a family of low molecular weight proteins that are all apparently involved in binding hydrophobic ligands is. These include the fatty acid-binding proteins (FABPs), an adipocyte-binding protein (aPs), the P2 protein of peripheral nerve myelin, two cellular retinol-binding proteins (CRBPs) and CRABP. CRABP is found in the cytoplasm and displays a high degree of ligand specificity. Although we know that CRABP can bind retinoic acid, its role in normal embryogenesis is not known. It has been suggested that CRABP could act by soaking up free retinoic acid, thereby decreasing its free concentration ~7. Interestingly, two groups have reported that CRABP is found in the limb bud with a distribution antiparallel to retinoic acid, the highest concentration of CRABP being found at the anterior limb bud margin l°,l~. T1G FEBRUARY-199] VOL. "7 NO. 2
:¢?1991 Else~ic'rScience Publisher>Ltd I[KI 0168 9',7991 $02.00
m
This could act as an amplification mechanism to enhance a specific ligand gradient. The gradient for retinoic acid reported in the limb bud is not high, concentrations ranging from about 20 nm towards the posterior to about 50 nM at the anterior6. High levels of CRABP at sites of low concentrations of retinoic acid and low levels of CRABP at sites of high concentrations of retinoic acid would steepen a gradient of retinoic acid across the anteroposterior axis of the limb bud. Eventually retinoic acid ends up in the nucleus where it is bound by the retinoic acid receptors (reviewed in Ref. 18). Here the retinoic acid response pathway connects with the transcriptional machinery of the cell. Do the cytoplasmic binding proteins merely fulfil a transport function, carrying retinoic acid from the cytoplasm to the nucleus? Or could they be much more intimately inw)lved in the metabolism of retinoids, connecting individual steps of a 'retinoid metabolic chain'? The analysis of retinoid synthesis and degradation pathways, in conjunction with determination of the subcellular localization of the various retinoid-binding proteins, may help us to answer some of these questions. As nuclear receptors are apparently not distributed as a gradient, the position of the primal' morphogenetic signal v,Athin the regulatory cascade in the embryo should be upstream of the nuclear receptors. CRABP could be inw)lved in helping to create a gradient of retinoic acid that is then translated into a corresponding degree of receptor occupancy and a concomitant change of receptormediated gene activation. The highly restricted spatial expression pattern of CRABP in the developing embryo supports this view. However, things might be more complicated. First of all there is apparently more than one
~]OMMENT CRABP 19,20. An anti-CRABP antibody detected an anteroposterior gradient of CRABP in the chick but no staining was seen in the mouse limb bud~3. The authors suggest that the antibody distinguishes between two CRABPs in the mouse, but not in the chick, and that the two proteins are tissue specific, one CRABP being specific for the nervous system, the other for the limb bud. CRABP I and CRABP II share extensive sequence homology, but their dissociation constants for retinoic acid differ by about 15fold 19,2~. It remains to be shown whether CRABP 1 and II are encoded by different genes or are splice variants of the same locus. The cellular retinol-binding protein (CRBP) also comes in two forms, one of which shows a distinctive, spatially restricted expression pattern in the developing embryom, ~6. The expression pattern of CRBP does not overlap with CRABR As more morphogenetically active metabolites of vitamin A are identified, we might expect additional cytoplasmic retinoid-binding proteins, each displaying its own ligand specificity. Finding a correlation between the sites of CRABP expression and the tissues that are affected by teratogenic doses of retinoic acid is no proof that this effect is mediated by CRABP. However, a closer look at the phenotype might help us to understand some of the basic biological principles of h o w positional identity is established (and possibly disturbed). So far, the phenotypic description of the retinoic acid embryopathy has been rather confusing and has not offered any insight into the role of retinoic acid in normal development. This may change as a result of some recent work dealing with genes from the homeobox gene (Hox) family. Ectopic expression of a murine Hox gene in transgenic mice resulted in a phenotype remarkably similar to the retinoic acid embryopathy syndrome 21. In addition to craniofacial malformations, these mice had vertebral column abnormalities that were interpreted as homeotic transformations 2z. Since retinoic acid can activate or suppress H o x genes 23, teratogenic concentrations of retinoic acid in the developing embryo may lead to aberrant H o x
gene expression. Consequently, teratogenic concentrations of retinoic acid and ectopic H o x gene expression, for example in transgenic mice, may point to the same biological event: h o w cells receive positional information. Within the framework of h o m e o b o x gene function, one might take a fresh look at the phenotype of retinoic acid embryopathy. Experiments by Michael Kessel suggest that a number of homeotic transformations along the vertebral column can be induced by retinoic acid (pers. commun.). It is tempting to consider the deregulation of murine H o x genes by retinoic acid as the molecular basis for these transformations. Where does CRABP fit into this picture? A crucial event in specifying positional identity of cells in the embryo seems to be the concentration of morphogens. CRABP is a good candidate for playing a major role in targeting retinoic acid to the right place at the right time in the right concentration. The next experiments will have to be genetic. Deleting, mutating or inappropriately expressing CRABPs or CRBPs should show whether these proteins are active participants in establishing positional information or whether they play some other role in this borderland between embryology and teratology.
References I
Kochhar, D.M. (1967) Acta Pathol. Microbiol. Scand. 70, 398404 2 Lammer, E.J. et al. (1985) New Engl. J. Med. 313, 837-841 3 Summerbell, D. and Maden, M. (1990) Trends Neurosci. 13, 142-147
TIG FEBRUARY1991 VOL. 7 NO. 2
m
4 Eichele, G. (1989) Trends Genet. 5, 246-251 5 Brockes, J.P. (1989) Neuron 2, 1285-1294 6 Thaller, C. and Eichele, G. (1987) Nature 327, 625-628 7 Saunders, J.W. and Gasseling, M.T. (1968) in Epithelial-Mesencbymal Interactions (Fleischmajer, R. and Billingham, R.E., eds), pp. 78--97, Williams & Wilkins 8 Wagner, M., Thaller, C., Jessell, T. and Eichele, G. (1990) Nature 345, 819-822 9 Hombruch, A. and Wolpert, L. (1986) J. Embo'ol. F~cp.MorphoL 94, 257-265 10 Perez-Castro, A.V., Toth-Rogter, L.E., Wei, L-N. and Nguyen-Huu, M.C. (1989) Dev. Biol. 86, 8813---8817 11 Vaessen, M-J., Meijers, J.H.C., Bootsma, D. and van Kessel, A.D. (1990) Development 110, 371-378 12 Dencker, L., Annarwall, E., Busch, C. and Eriksson, U. (1990) Development 110, 343-352 13 Maden, M., Ong, D.E. and Chytil, F. (1990) Development 109, 75-80 14 Maden, M. (1988) Nature 335, 733-735 15 Sundelin, J. et al. (1985) J. Biol. Chem. 260, 6494-6499 16 Demmer, L.A. et al. (1987) J. Biol. Chem. 262, 2458-2467 17 Maden, M., Ong, D.E., Summerbell, D. and Chytil, F. (1989) Development (Suppl.), 109-119 18 Evans, R. (1988) Science 240, 889-894 19 Kitamato, T. et al. (1989) Biochem. Biophys. Res. Commun. 164, 531-536 20 Bailey, J. and Siu, C.H. (1988) J. Biol. Chem. 263, 9326-9332 21 Bailing, R., Mutter, G., Gruss, P. and Kessel, M. (1989) Cell 58, 337-347 22 Kessel, M., Bailing, R. and Gruss, R (1990) Cell 61,301-308 23 Simeone, A. et al. (1990) Nature 346, 763-766
CRABPand the terat0genic effects of retin0ids R. BALLING
MAXPLANER INSTITVTEOF BIOPHYSICALCHEMISTRY, DEPARTMENT OF MOLECULARCELL BIOLOGY,
3400 GO~NGEN,FRG. High doses of vitamin A or its derivatives (retinoids) induce malformations in developing embryosL Because retinoic acid seems to be the biologically m~st active metabolite, this syndrome is known as retinoic acid embryopathy 2. Prominent features of this syndrome include limb abnormalities and craniofacial defects. The teratogenic activity of retinoids has been known for a long time, but until recently a molecular mechanism has not been in sight. Recently, increasing attention has been paid towards the role of retinoids in normal embryogenesis ~-5. There is growing evidence that retinoic acid is a morphogen, and as such is responsible for specifying positional information. A morphogenetic function for retinoic acid is suggested by the observation that in the developing limb bud, retinoic acid is distributed in a gradient, with the highest concentration in the posterior region 6. A mirror image duplication of the digits results if this distribution is perturbed by local application of retinoic acid to the anterior part of the limb bud. Retinoic acid therefore mimics the action of the zone of polarizing activity found at the posterior end of the limb bud 7. In addition to the limb bud, the anterior part of the primitive streak (Hensen's node) also has polarizing activity, as do two midline structures that are derived from the regressing node: the floorplate of the neural tube and the noLochord 8,9. Since this polarizing activity probably reflects in part the action of retinoic acid, a spatially controlled distribution of retinoids could be a primary morphogenetic determinant in the developing embryo: cells might receive their positional identity by reading concentration values of retinoids. What is the link between the teratological and developmental actions of retinoic acid? Recent results from four different labs have
provided some interesting insights into this question. They report that the cellular retinoic acid-binding protein (CRABP) is expressed predominantly in those structures that are affected by application of high doses of retinoic acid during embryogenesis. By use of in situ hybridizationm, H, immunohistochemica112,13 and direct retinoid-binding studies 12 of mouse and chick embryos, expression of CRABP was localized to specific areas of the central nervous system, the craniofacial mesenchyme, the visceral arches, ganglia of the peripheral nervous system and the limb bud. Many of these tissues are neural crest derivatives, tissues that are particularly affected in retinoic acid embryopathy. Similarly, expression of CRABP in the limb bud 10,14 points to another site in which abnormalities can be induced by exposure to high doses of retinoic acid. The authors suggest that one of the reasons for these tissues being the major target in retinoic acid embryopathy is that they express high levels of the CRABP. CRABP belongs to a family of low molecular weight proteins that are all apparently involved in binding hydrophobic ligands is. These include the fatty acid-binding proteins (FABPs), an adipocyte-binding protein (aPs), the P2 protein of peripheral nerve myelin, two cellular retinol-binding proteins (CRBPs) and CRABP. CRABP is found in the cytoplasm and displays a high degree of ligand specificity. Although we know that CRABP can bind retinoic acid, its role in normal embryogenesis is not known. It has been suggested that CRABP could act by soaking up free retinoic acid, thereby decreasing its free concentration ~7. Interestingly, two groups have reported that CRABP is found in the limb bud with a distribution antiparallel to retinoic acid, the highest concentration of CRABP being found at the anterior limb bud margin l°,l~. T1G FEBRUARY-199] VOL. "7 NO. 2
:¢?1991 Else~ic'rScience Publisher>Ltd I[KI 0168 9',7991 $02.00
m
This could act as an amplification mechanism to enhance a specific ligand gradient. The gradient for retinoic acid reported in the limb bud is not high, concentrations ranging from about 20 nm towards the posterior to about 50 nM at the anterior6. High levels of CRABP at sites of low concentrations of retinoic acid and low levels of CRABP at sites of high concentrations of retinoic acid would steepen a gradient of retinoic acid across the anteroposterior axis of the limb bud. Eventually retinoic acid ends up in the nucleus where it is bound by the retinoic acid receptors (reviewed in Ref. 18). Here the retinoic acid response pathway connects with the transcriptional machinery of the cell. Do the cytoplasmic binding proteins merely fulfil a transport function, carrying retinoic acid from the cytoplasm to the nucleus? Or could they be much more intimately inw)lved in the metabolism of retinoids, connecting individual steps of a 'retinoid metabolic chain'? The analysis of retinoid synthesis and degradation pathways, in conjunction with determination of the subcellular localization of the various retinoid-binding proteins, may help us to answer some of these questions. As nuclear receptors are apparently not distributed as a gradient, the position of the primal' morphogenetic signal v,Athin the regulatory cascade in the embryo should be upstream of the nuclear receptors. CRABP could be inw)lved in helping to create a gradient of retinoic acid that is then translated into a corresponding degree of receptor occupancy and a concomitant change of receptormediated gene activation. The highly restricted spatial expression pattern of CRABP in the developing embryo supports this view. However, things might be more complicated. First of all there is apparently more than one
~]OMMENT CRABP 19,20. An anti-CRABP antibody detected an anteroposterior gradient of CRABP in the chick but no staining was seen in the mouse limb bud~3. The authors suggest that the antibody distinguishes between two CRABPs in the mouse, but not in the chick, and that the two proteins are tissue specific, one CRABP being specific for the nervous system, the other for the limb bud. CRABP I and CRABP II share extensive sequence homology, but their dissociation constants for retinoic acid differ by about 15fold 19,2~. It remains to be shown whether CRABP 1 and II are encoded by different genes or are splice variants of the same locus. The cellular retinol-binding protein (CRBP) also comes in two forms, one of which shows a distinctive, spatially restricted expression pattern in the developing embryom, ~6. The expression pattern of CRBP does not overlap with CRABR As more morphogenetically active metabolites of vitamin A are identified, we might expect additional cytoplasmic retinoid-binding proteins, each displaying its own ligand specificity. Finding a correlation between the sites of CRABP expression and the tissues that are affected by teratogenic doses of retinoic acid is no proof that this effect is mediated by CRABP. However, a closer look at the phenotype might help us to understand some of the basic biological principles of h o w positional identity is established (and possibly disturbed). So far, the phenotypic description of the retinoic acid embryopathy has been rather confusing and has not offered any insight into the role of retinoic acid in normal development. This may change as a result of some recent work dealing with genes from the homeobox gene (Hox) family. Ectopic expression of a murine Hox gene in transgenic mice resulted in a phenotype remarkably similar to the retinoic acid embryopathy syndrome 21. In addition to craniofacial malformations, these mice had vertebral column abnormalities that were interpreted as homeotic transformations 2z. Since retinoic acid can activate or suppress H o x genes 23, teratogenic concentrations of retinoic acid in the developing embryo may lead to aberrant H o x
gene expression. Consequently, teratogenic concentrations of retinoic acid and ectopic H o x gene expression, for example in transgenic mice, may point to the same biological event: h o w cells receive positional information. Within the framework of h o m e o b o x gene function, one might take a fresh look at the phenotype of retinoic acid embryopathy. Experiments by Michael Kessel suggest that a number of homeotic transformations along the vertebral column can be induced by retinoic acid (pers. commun.). It is tempting to consider the deregulation of murine H o x genes by retinoic acid as the molecular basis for these transformations. Where does CRABP fit into this picture? A crucial event in specifying positional identity of cells in the embryo seems to be the concentration of morphogens. CRABP is a good candidate for playing a major role in targeting retinoic acid to the right place at the right time in the right concentration. The next experiments will have to be genetic. Deleting, mutating or inappropriately expressing CRABPs or CRBPs should show whether these proteins are active participants in establishing positional information or whether they play some other role in this borderland between embryology and teratology.
References I
Kochhar, D.M. (1967) Acta Pathol. Microbiol. Scand. 70, 398404 2 Lammer, E.J. et al. (1985) New Engl. J. Med. 313, 837-841 3 Summerbell, D. and Maden, M. (1990) Trends Neurosci. 13, 142-147
TIG FEBRUARY1991 VOL. 7 NO. 2
m
4 Eichele, G. (1989) Trends Genet. 5, 246-251 5 Brockes, J.P. (1989) Neuron 2, 1285-1294 6 Thaller, C. and Eichele, G. (1987) Nature 327, 625-628 7 Saunders, J.W. and Gasseling, M.T. (1968) in Epithelial-Mesencbymal Interactions (Fleischmajer, R. and Billingham, R.E., eds), pp. 78--97, Williams & Wilkins 8 Wagner, M., Thaller, C., Jessell, T. and Eichele, G. (1990) Nature 345, 819-822 9 Hombruch, A. and Wolpert, L. (1986) J. Embo'ol. F~cp.MorphoL 94, 257-265 10 Perez-Castro, A.V., Toth-Rogter, L.E., Wei, L-N. and Nguyen-Huu, M.C. (1989) Dev. Biol. 86, 8813---8817 11 Vaessen, M-J., Meijers, J.H.C., Bootsma, D. and van Kessel, A.D. (1990) Development 110, 371-378 12 Dencker, L., Annarwall, E., Busch, C. and Eriksson, U. (1990) Development 110, 343-352 13 Maden, M., Ong, D.E. and Chytil, F. (1990) Development 109, 75-80 14 Maden, M. (1988) Nature 335, 733-735 15 Sundelin, J. et al. (1985) J. Biol. Chem. 260, 6494-6499 16 Demmer, L.A. et al. (1987) J. Biol. Chem. 262, 2458-2467 17 Maden, M., Ong, D.E., Summerbell, D. and Chytil, F. (1989) Development (Suppl.), 109-119 18 Evans, R. (1988) Science 240, 889-894 19 Kitamato, T. et al. (1989) Biochem. Biophys. Res. Commun. 164, 531-536 20 Bailey, J. and Siu, C.H. (1988) J. Biol. Chem. 263, 9326-9332 21 Bailing, R., Mutter, G., Gruss, P. and Kessel, M. (1989) Cell 58, 337-347 22 Kessel, M., Bailing, R. and Gruss, R (1990) Cell 61,301-308 23 Simeone, A. et al. (1990) Nature 346, 763-766