Development 109, 75-80 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

75

Retinoid-binding protein distribution in the developing mammalian nervous

system

M. MADEN1'*, D. E. ONG2 and F. CHYTIL2

'Anatomy and Human Biology Group, King's College London, Strand, London WC2R 2LS, UK2Department of Biochemistry, University of Vanderbilt Medical School, Nashville, Tennessee, 37323 USA

• Author for correspondence

Summary

We have analysed the distribution of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) in the day 8.5-day 12 mouseand rat embryo. CRBP is localised in the heart, gutepithelium, notochord, otic vesicle, sympathetic ganglia,lamina terminalis of the brain, and, most strikingly, in aventral stripe across the developing neural tube in thefuture motor neuron region. This immunoreactivityremains in motor neurons and, at later stages, motoraxons are labelled in contrast to unlabelled sensoryaxons. CRABP is localised to the neural crest cells,which are particularly noticeable streaming into thebranchial arches. At later stages, neural crest deriva-tives such as Schwann cells, cells in the gut wall andsympathetic ganglia are immunoreactive. An additional

area of CRABP-positive cells are neuroblasts in themantle layer of the neural tube, which subsequentlyappear to be the axons and cell bodies of the commissu-ral system. Since retinol and retinoic acid are theendogenous ligands for these binding proteins, we pro-pose that retinoids may play a role in the developmentand differentiation of the mammalian nervous systemand may interact with certain homoeobox genes whosetranscripts have also been localised within the nervoussystem.

Key words: retinoic acid, retinol, cellular retinoic acid-binding protein, cellular retinol-binding protein, neuraltube, notochord, motor nerves, sensory nerves, laminaterminalis, neural crest, commissural neurons.

Introduction

It is becoming increasingly apparent that retinoic acid(RA), the most biologically active naturally occurringform of vitamin A, may play an important role in themorphogenesis of the embryo. In particular, the limband the nervous system seem to employ RA duringcrucial phases of their development. When it is adminis-tered to the developing chick limb bud (Tickle et al.1982; Summerbell, 1983) or the regenerating amphibianlimb (Maden, 1982) it has the remarkable ability tostimulate the production of extra limbs. What is more,endogenous RA has been identified in the chick limbbud (Thaller and Eichele, 1987) and the regeneratingamphibian limb (Maden, Summerbell and Waterson,unpublished results). In the chick, endogenous RA isdifferentially distributed across the anteroposterior axisof the limb bud, whereas its metabolic precursor,retinol, is not. This is precisely the behaviour one wouldexpect of RA if it was acting as a morphogen.

In the developing nervous system of Xenopus, theadministration of RA causes anteroposterior trans-formations such that the forebrain and midbrain arereduced or absent and the hindbrain and spinal cord are

correspondingly exaggerated (Durston et al. 1989). En-dogenous RA is present in whole Xenopus embryos atthis stage. RA also promotes neurite outgrowth inexplanted amphibian spinal cord and is present en-dogenously in this tissue (Hunter etai, unpublished).These results suggest that RA may be involved invarious aspects of the developing nervous system aswell as the limb. It is therefore a matter of considerableinterest to uncover the mechanism of action of RAwithin the cells of the embryo.

It has been known for some time that, at least inembryonal carcinoma cells, RA causes a change in thepattern of gene activity and thus can act on the nucleus(Roberts and Sporn, 1984). Two types of protein arethought to mediate its action between the cell mem-brane and nucleus, namely binding proteins and recep-tors. Cellular retinoic acid-binding protein (CRABP) isa 15.6X103 MT protein with a high degree of ligandspecificity for RA (Chytil and Ong, 1984). CRABP isfound in the cytoplasm of a variety of cell types in theadult rat, particularly those that require RA for theirnormal function, such as skin and testis (Ong et al.1982). The distribution of this protein in mammalianembryonic tissues has not previously been described,

76 M. Maden, D. E. Ong and F. Chytil

although it is now known that the CRABP gene istranscribed in the limb bud (Dolle et al. 1989) and indiscrete populations of neuroblasts in the brain andspinal cord of early mouse embryos (Vaessen etal.1989; Perez-Castro etal. 1989). Thus there may be anassociation between developmental systems that areaffected by RA and the presence of CRABP and in thework reported here we confirm this by looking in detailat the distribution of the protein in the mouse embryo.

A closely related protein, cellular retinol-bindingprotein (CRBP), has a ligand specificity for retinol(Chytil and Ong, 1984), the metabolic precursor of RA,and a wide distribution in the cytoplasm of the cells ofthe adult rat (Ong et al. 1982). Again, its distribution inthe mammalian embryo has not yet been investigated,although the CRBP gene is transcribed in the brain andspinal cord amongst other organs such as the gut, liverand heart (Perez-Castro etal. 1989).

In the chick embryo CRABP is indeed concentratedin precisely those systems whose development is underthe influence of RA. It is expressed at highest levels inthe limb buds, followed by the spinal cord and brain andabsent in the rest of the trunk (Momoi et al. 1988). Inthe limb bud, CRABP was localised by immunocyto-chemistry to the rapidly dividing, RA-responsive cellsat the distal tip of the bud (Maden etal. 1988). In thenervous system, CRABP is found in the neural crestcells, sensory axons, and the axons and cell bodies ofthe commissural system (Maden etal. 1989a). Thus,there is a clear association in the chick embryo betweenthe presence of CRABP in a developing organ and theinvolvement of RA in its development.

The receptors for RA have recently been discovered(Petkovich etal. 1987; Giguere etal. 1987) and thesehave a structure, including ligand-binding and DNA-binding domains similar to steroid receptors. There areat least three types of receptor in the mouse, a, fi and y(Zelent etal. 1989), and an additional one in the newtlimb, termed 8 (Ragsdale etal. 1989). The a and 0receptors seem to have a widespread distribution intissues of the adult mouse whereas the y receptor ispredominantly expressed in skin (Zelent et al. 1989). Inthe mouse limb bud, the a and y receptors are ex-pressed uniformly and the (3 receptor is not expressed atall at early stages, in contrast to the graded distributionof CRABP transcripts (Dolle et al. 1989). The receptorsidentified in the newt are expressed in the regeneratinglimb (Giguere etal. 1989; Ragsdale etal. 1989), which,as described above, is a developing system responsiveto the profound pattern changes induced by RA.

Thus in the mechanism of action of RA the receptorsare the component that interacts with the DNA to alterthe pattern of gene activity in the cell, but the functionof the binding proteins is not clear. They may beresponsible for regulating the intercellular level of freeRA that is available for binding to the receptors(Maden etal. 1988); they may define populations ofcells that are responsive to RA (Maden et al. 19896) orthey may be involved in the metabolism of retinoids.Another possible function involving homoeobox tran-scripts is considered in the Discussion. To help resolve

these issues, we have investigated by immunocytochem-istry the distribution of CRBP and CRABP at variousstages of mouse and rat embryogenesis. We find thatCRBP is localised in several differentiating organs ofthe early embryo, but, most interestingly, as a stripeacross the ventral neural tube where motoneuronsform. Later staining is found in the motor axons, thesympathetic ganglia and in discrete regions of the brain.CRABP, on the other hand, seems to be specific for theneural crest and the axons and cell bodies of thecommissural system. The possible relationships be-tween CRBP, CRABP, their ligands and the localis-ations of some relevant homoeobox genes is discussed.

Materials and methods

Crossbred strains of mice and rats were used and theirembryos staged according to Rugh (1968). Embryos werefixed in Perfix (Fisher Scientific, New Jersey) for 3h, dehy-drated, cleared in xylene and embedded in wax. 7̂ /m sectionswere cut. CRBP was immunolocalised with an affinity-purified anti-rat CRBP rabbit lgG and CRABP was immuno-localised with an affinity-purified anti-rat CRABP rabbit IgG.The technique has been described previously (Porter et al.1985) and was used with only one modification, namely thatthe dilution buffer for all immunochemical reagents wasphosphate-buffered saline containing 1 % normal goat serumand 0.1 % crystalline bovine serum albumin. The anti-CRBPIgG was diluted to an absorbance at 280 nin of 0.35 and theanti-CRABP IgG was diluted to an absorbance at 280 nM of0.01. Colour was developed by the avidin-biotinylated per-oxidase complex with a kit from Vector Laboratories (Burl-ingame, California).

For the Western blot, several day 10 mouse embryos werehomogenised in solubilisation buffer for SDS-polyacrylamidegel electrophoresis according to Laemmli (1970) and centri-fuged to remove debris. Aliquots of the extract (100-200/.ig),pure rat CRBP (100 ng) and pure rat CRABP (80 ng) wereelectrophoresed on an 11 % SDS-polyacrylamide gel and theproteins transferred to nitrocellulose paper. Immunoreac-tivity was detected using affinity-purified IgG fractions fromrabbit antisera to rat CRBP and rat CRABP and radio-iodinated protein A as previously described (Porter et al.1985).

Results

The experiments described here were conducted bothon rat and mouse embryos with identical results.Originally rat embryos were used because we knew thatour affinity-purified antibodies reacted with a singleprotein of the correct molecular weight on a Westernblot of rat tissues (Porter etal. 1985). We then foundthat sections of mouse embryos gave the same stainingpatterns as sections of rat embryos and the descriptionsbelow apply equally to each species. To confirm that theaffinity-purified antibodies only reacted with a singleband of the appropriate molecular weight in mouseembryos, homogenates of day 10 mouse embryos wereblotted. In Fig. 1, it can be seen that both CRBP andCRABP antibodies identify such a band. In addition tothese Western blots, we have also shown that sections

CRBP and CRABP in mammalian embryo 77

I

Fig. 1. Lanes a and b - Western blot of pure rat CRBP(lane a) and day 10 mouse embryo (lane b) reacted withaffinity-purified antibody to CRBP demonstrating thepresence of a single band at the appropriate molecularweight in mouse embryos. Lanes c and d - blot of pure ratCRABP (lane c) and day 10 mouse embryo (lane d) reactedwith affinity-purified antibody to CRABP demonstrating thepresence of a single band of the appropriate molecularweight in mouse embryos.

treated with antibody that had been preincubated withpurified CRBP or CRABP gave no staining as dosections treated with preimmune serum (Fig. 7). Thesecontrols confirm the specificity of the immunoreactivitydescribed below.

CRBPOn day 8.5 of development, several areas of the embryoshowed immunoreactivity to CRBP. The strongest andmost obvious staining was found in the myocardium ofthe heart. The epithelium of the gut was also intenselystained, especially in the mid- and hindgut region(Fig. 2) and less so in the foregut. A weaker, butsignificant, level of reactivity was detected in thenotochord (Fig. 3) and this showed a greater intensityat more rostral levels suggesting that CRBP is associ-ated with some differentiative event appearing in ananterior-to-posterior sequence during development.The only other structure showing any reactivity in thesesections was the otic vesicle, which develops adjacent tothe neural tube of the hindbrain. Sections at this levelshowed a clear contrast between positive reactivity ofthe otic vesicle and the absence of reactivity in thehindbrain.

On subsequent days, certain staining patterns such asthe myocardium and gut lining remained a constantfeature of the embryos (e.g. Fig. 6). The notochordincreased its immunoreactivity and by day 12 wasphysically well-separated from the ventral floor plate ofthe neural tube (cf. Fig. 3) to remain an island ofintense staining in the unstained sclerotome (Fig. 5).

In other organ systems, however, new patterns ofimmunoreactivity emerged and this was most striking inthe nervous system. On day 10, the neural tube showeda clear band of staining in the future motor neuron

region (Fig. 4). The band extended from the lumen tothe periphery of the neural tube thus including bothproliferating and differentiating cells. As a stripe it didnot extend ventrally all the way to the floor plate on firstappearance (Fig. 4), but did so as motor neuron differ-entiation was established (Figs 5, 6, 8). This staininghad begun very faintly on day 9.5 in the forelimb region(later in the hindlimb region) and, by day 10.5 whenmotor neurons had extended axons, these were alsoimmunoreactive in clear contrast to the sensory axons.This differentiation between CRBP positive motoraxons and CRBP negative sensory axons had become soclear by day 12 that, at the brachial plexus where thetwo types of nerve mix (Fig. 8), stripes of labelled andunlabelled axons could be seen (Fig. 9).

One other component of the nervous system thatbecame intensely labelled upon differentiation was thesympathetic ganglia, which is in contrast to the lack ofreactivity of the dorsal root ganglia (Fig. 8). Thedeveloping brain also showed localisations in two re-gions. Neuroblasts in the floor of the hindbrain wereheavily labelled as the ventrally located immunoreac-tivity of the motor neurons in the spinal cord continuedanteriorly into the hindbrain. No areas of labelling wereobvious in the mid-brain, but in the forebrain thelamina terminalis was an area of intensity surroundedby unlabelled regions (Fig. 10).

CRABPThere were far fewer cell types that were immuno-reactive to CRABP than to CRBP. In fact, only twosubsets of neural cells and their descendents werelabelled to any great extent and, furthermore, theywere a different class of neural cells from those de-scribed above.

In transverse sections through the trunk region of day8.5 embryos, individual CRABP-positive cells couldeasily be identified, although there were not many ofthem. They were outside the neural tube and in twotracts, one underneath the epidermis and the othermore medial and ventral around the dorsal aorta. Insections through the hindbrain, by contrast, there werelarge numbers of immunoreactive cells concentrated inthe newly formed cephalic ganglia and streaming outfrom them into the branchial arches (Fig. 11). At thisstage, no cells within the neural tube were labelledalthough in some sections individual cells that had justdetached or were in the process of detaching from thedorsal neural tube were labelled. This distribution andbehaviour suggested that these CRABP-positive cellswere the neural crest and that the commencement ofmigration was related to the first appearance of (orincrease in levels of) CRABP.

By day 9.5, the period of extensive neural crestmigration had terminated and a second population ofimmunoreactive cells became apparent. These were aproportion of cells in the mantle layer of the neural tube(Fig. 12). One day later both CRABP-positive popu-lations were clearly distinguishable in the same section(Fig. 13). Descendants of migrating neural crest cellsremained immunoreactive and were located in the wall

78 M. Maden, D. E. Ong and F. Chytil

of the developing gut (Fig. 14), in the sympatheticganglia, scattered throughout the dorsal root ganglia(Fig. 13) and distributed along the nerve fibres(Fig. 15). The cells in the gut wall are likely to bedeveloping into enteric ganglia and the scattered cellsalong nerve fibres and in dorsal root ganglia are mostlikely Schwann cells since each of these cell types isknown to be derived from neural crest, at least in thechick (Le Douarin, 1982). The tracts of immunoreactiveneurons and axons in the neural tube are in thelocations expected for the commissural and funicularcells, which project contralaterally and ipsilaterally asrelay neurons (Altman and Bayer, 1984).

In the developing brain of a day 12 embryo, CRABPwas present only in the hindbrain and midbrain. In thehindbrain, the labelled neuroblasts were extensivelydistributed through the thickness of the hindbrainepithelium. In the midbrain, labelled neuroblasts weremore restricted, being present only in the peripheralcell layers (Fig. 16). At the midbrain-forebrain bound-ary, CRABP immunolabelling suddenly ceased.

Limb budsUsing the same CRABP antibody, we have previouslydescribed a distinct pattern of immunoreactivity in thedeveloping chick limb bud where the most intenselylabelled regions are at the distal tip in rapidly dividing,undifferentiated cells and in proximal regions wheredermis, connective tissue and muscle are differentiating(Maden etal. 1988). We therefore expected to see asimilar localisation in mouse and rat limb buds and weresurprised to observe the complete absence of anyimmunoreactivity in the limb buds (not shown). This isdespite the demonstrated existence of high levels ofCRABP in mouse limb buds by sucrose gradientcentrifugation (Kwarta etal. 1985; Maden, unpub-lished). The following explanation of this paradoxseems possible. Recently it has been shown that thereare in fact two CRABPs in the chick embryo (Kitamotoetal. 1988) and the rat (Bailey and Siu, 1988) and thefirst 25 amino acids of the N-terminus have beensequenced in each case. Chick CRABP I has anidentical sequence to rat CRABP I (Eriksson etal.1981). Chick CRABP I differs from chick CRABP II in3 positions whereas chick CRABP I (and rat CRABP I)differs from rat CRABP II in 7 positions. Thus it islikely that our antibody (which was made against apurified rat protein rather than a peptide sequence)does not distinguish between CRABP I and II in thechick, but does distinguish between CRABP I and II inthe rat because they are more dissimilar. This suggeststhat the two proteins are tissue specific, CRABP I beingrestricted to the nervous system and CRABP II to thelimb. It is known that rat CRABP I and CRABP II havea tenfold difference in dissociation constant and differ-ent ligand specificities (Bailey and Siu, 1988). Thus,even before RA gets to the multiple retinoic acidreceptors (see Introduction), there may be an initialdegree of tissue specificity in their interaction withCRABP.

Figs 2-10. Sections of mouse and rat embryos treated withaffinity-purified anti-rat CRBP antibody.Fig. 2. Day 8.5 mouse embryo showing pale staining of thenotochord (n) and more intense staining of the epitheliumof the developing gut (g). nt=neural tube. Bar=25 j*m.Fig. 3. Higher power view of Fig. 2 to show reactivity inthe notochord (arrow). Bar=10/<m.Fig. 4. Day 10 mouse embryo showing immunoreactivity ina band across the ventral neural tube (arrowhead) markingthe position of the future ventral motor horns and also inthe epithelium of the developing gut (arrow). Bar=100^m.Fig. 5. High power view of the notochord of a day 12 ratembryo showing high levels of immunoreactivity(arrowhead). Bar=10j/m.Fig. 6. Day 12 rat embryo at the level of the forelimb budsshowing immunoreactivity in the ventral neural tube andmotor horns (m), motor nerves (mn) (see Fig. 8) andmyocardium (h). Bar=100jan.Fig. 7. Adjacent section to Fig. 5 in which the antibody hadfirst been incubated with pure rat CRABP. The absence ofimmunoreactivity here demonstrates the specificity of thestaining patterns in the other sections.Fig. 8. Higher power view of Fig. 6 to show the presence ofstaining in the ventral horn and motor nerves (mn) and theabsence of staining in the dorsal root ganglion (dr) andsensory nerves (sn). Labelled cells of the developingsympathetic ganglion can also be seen (sy). Bar=100;«n.Fig. 9. Adjacent section to Fig. 8 showing unlabelledsensory and labelled motor nerves mixing at the brachialplexus and forming stripes. Bar=10/im.Fig. 10. Day 12 rat forebrain showing the labelled laminaterminalis (arrow) surrounded by unlabelled areas of theforebrain. Bar=200/«n.

Discussion

Using affinity-purified antibodies to CRBP andCRABP, we have identified groups of cells in the earlymouse and rat embryo that are immunoreactive foreach of these binding-proteins. There was a greaternumber of CRBP-positive than CRABP-positive celltypes and, furthermore, they were non-overlapping.Thus CRBP was present in the developing heart, gutepithelium, notochord, otic vesicle, sympatheticganglia and lamina terminalis of the brain. Most signifi-cantly, CRBP immunoreactivity identified future motorneurons in the early ventral spinal cord by appearing asa stripe across the neural tube. CRABP was present inthe mantle layer of the spinal cord in the locationexpected for commissural and funicular cells and exter-nal to the neural tube in what we presume to be neuralcrest cells. A recent in situ hybridisation study hasshown a similar distribution of CRBP and CRABPmRNA transcripts (Perez-Castro et al. 1989) suggestingthat the protein distribution we have observed iscontrolled at the level of transcription. On the basis ofthese patterns and the fact that the natural ligands forthese binding-proteins are retinol (CRBP) and retinoicacid (CRABP), we propose that retinoids play a role inthe development of the early mammalian nervoussystem. In particular, retinol may play a role in the

11 12

41

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Figs 11-16. Sections of mouse and rat embryos treated with affinity-purified anti-rat CRABP antibody.Fig. 11. Slightly oblique section through a day 8.5 mouse embryo showing immunoreactive cells emerging from the cephalicganglion (c) adjacent to the neural tube and streaming into the branchial arch (ba). Bar=50//m.Fig. 12. Day 9.5 mouse embryo showing labelled cells in the mantle layer of the neural tube (arrows) as well as individualcells in the sclerotome (arrowheads) that are likely to be of neural crest origin as they are in the correct location to becomeincorporated into the dorsal root ganglia and peripheral nerves (see Fig. 13). Bar=50;an.Fig. 13. Day 10.5 rat embryo. Two populations of labelled cells are present, those in the mantle layer of the neural tube(arrow) and those in the dorsal root ganglia (dr) and peripheral nerves (pn). Bar=100;un.Fig. 14. Section through the developing gut of a 10.5 day rat embryo. Labelled cells are present in the wall of the gut(arrowheads) and are likely to be neural crest cells that will coalesce to form the enteric ganglia. Bar=40jum.Fig. IS. High power view of the nerves in the brachial plexus region showing labelled cells (arrowheads) that are likely to beSchwann cells along its length. mh=motor horn. Bar=25/nn.Fig. 16. Section through the posterior mid-brain of a day 12 rat embryo showing intense labelling of peripheral neuroblasts.v=ventricle. Bar=50jim.

CRBP and CRABP in mammalian embryo 79

decision of cells to differentiate as motoneurons ratherthan any other type of neuron and RA may influencethe neural crest either in its differentiation or mi-gration.

With regard to the neural crest, the presence of highlevels of CRABP is very pertinent since retinoids arepotent teratogens in mammalian development. A majortarget of their teratogenic effect is the branchial arches(e.g. Sulik etal. 1988) and it is interesting to note thatwe observed what appeared to be a massive ingressionof CRABP-positive neural crest cells precisely intothose structures. Retinoids are known to inhibit themigration of neural crest cells in vitro (Thorogood et al.1982; Smith-Thomas et al. 1987) and in vivo (Pratt et al.1987). Thus it is reasonable to propose that neural crestcells are targets for retinoid teratogenicity because theycontain high levels of CRABP and that once disturbedin their behaviour the branchial arches are the struc-tures that suffer the most because they do not receivetheir normally large contribution of neural crest cells.

The localisation of CRBP immunoreactivity in aventral stripe in the early neural tube is, as far as we areaware, the earliest onset of differentiation that has beenobserved. In recent years, most attention has been paidto the localisation of homoeobox gene transcripts in thedeveloping CNS, detected by in situ hybridisation.These studies have been primarily concerned with theanteroposterior distribution of transcripts along thespinal cord rather than in the transverse axis of the cordand are generally conducted on older embryos than day10 (e.g. Graham etal. 1989). However, some geneshave been studied in transverse sections of the neuraltube. For example, Hox 3.1 shows a ventral-to-dorsalgradient in day 12.5 embryos although the ependymallayer does not label (Breier et al. 1988). Hox 1.4 showsthe converse pattern with a higher grain density in thedorsal portion of the neural tube than the ventralalthough, again, the ependymal layer did not label(Toth et al. 1987). A comparative study of many of thegenes in the Hox 2 cluster shows that they also have asharp dorsal restriction in the neural tube at day 12.5(Graham and Krumlauf, pers. comm.). Mostinterestingly, a detailed study using alternate serialsections labelled either with Hox 2.5 or CRBP hasrevealed that the dorsal boundary of CRBP immuno-reactivity is the same as the ventral boundary of Hox 2.5expression (Graham etal. 1990).

This type of analysis in combination with the knowl-edge that retinoids can influence the expression of awide variety of homoeobox genes in embryonal carci-noma cells (Mavilio etal. 1988; Papalopulu pers.comm.) naturally leads to the suggestion that homoeo-box genes may be a target of retinoid action in theembryo. Further evidence for this assertion and particu-larly relevant to the above relationship between RA,CRABP and the branchial arches is the observationthat ectopic expression of Hox 1.1 in transgenic miceleads to head and neck deformities that resemble thoseseen in retinoic acid embryopathy (Balling et al. 1989).These authors suggest that excess RA may act byinducing the ectopic expression of Hox 1.1., although

this is not the only homoeobox to be expressed in thebranchial arches (Hill etal. 1989; Robert etal. 1989).

Indeed the correlation between RA activity andhomoeobox gene expression is also consistent with theother two developing systems that retinoids affect. Avariety of homoeobox genes are expressed in thedeveloping limb buds of amphibians, chicks and mice(Oliver et al. 1989; Dolle and Duboule, 1989; Hill et al.1989; Robert etal. 1989; Wedden etal. 1989) and RAaffects limb buds both positively (see Introduction) andnegatively (Kochaar, 1973). Xhox 3 is involved inestablishing the anteroposterior axis of the Xenopusembryo (Ruiz i Altaba and Melton, 1989) and RAcauses transformations in this axis (Durston et al. 1989).So it is logical to conclude that retinoids and homoeo-box genes may be intimately related in the developmentof pattern in various systems. Determining exactly whatthe nature of this relationship is and how the retinoid-binding proteins and receptors fit into the scheme is amajor task for the future, but it will surely lead tocrucial insights into the molecular basis of vertebratepattern formation.

We thank Drs Jack Price, Anthony Graham, Robb Krum-lauf and Nancy Papalopulu for very valuable comments anddiscussions. This work was supported in part by United StatesPublic Health Service Grants HD-09195 and CA-20850 andthe General Foods Corp.

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(Accepted 12 February 1990)