Development 103 Supplement, 31-40 (1988)Printed in Great Britain © The Company of Biologists Limited L988

31

Pattern formation in the facial primordia

S. E. WEDDEN*, J. R. RALPHS and C. TICKLE

Department of Anatomy and Developmental Biology, University College & Middlesex School of Medicine, Windeyer Building, ClevelandSt, London W1P6DB, UK

* Present address: Department of Physiology and Biophysics, Harvard Medical School, Boston, USA

Summary

Pattern formation is the developmental process thatleads to the spatial ordering of cell differentiation. Wehave explored the problem of pattern formation in thedevelopment of the face of chick embryos. At earlystages, the developing face consists of a series of smallbuds of tissue, the facial primordia that encircle theprimitive mouth. The concepts of positional infor-mation provide a framework for considering how thepatterns of differentiated cells are generated in theface. We suggest that the cranial neural crest cellsmust first be informed to which facial primordiumthey belong and then of their position within thatprimordium.

The cells of the early primordia appear indis-tinguishable. However, when the mesenchyme cellsare placed in high-density culture, cartilage differen-tiates. The extent and pattern of cartilage differen-tiation is characteristic for the cell population of eachfacial primordium. Myogenic cells also differentiate inthe cultures, but the proportion of myogenic cells isindependent of the extent of chondrogenesis. Withinthe facial primordia, a set of epithelial-mesenchymal

interactions appears to be required for outgrowth andpattern formation along the proximodistal axis of thechick beaks. In culture, face epithelium locally inhibitscartilage differentiation and suggests that another setof epithelial-mesenchymal interactions may be in-volved in cell patterning. The mechanisms involved inspecifying the mediolateral axis of the face, forexample, the midpoint of the upper beak, are notknown.

Vitamin A derivatives, collectively known as reti-noids, affect the development of the face of chickembryos and lead to a specific facial defect. Upperbeak development is inhibited but the lower beakdevelops normally. The response to retinoids could berelated to the specification of cells to belong to thefacial primordium that will form the upper beak.Alternatively, retinoids may interfere with positionalcues that operate to inform cells of their positionwithin that primordium.

Key words: neural crest, frontonasal mass,chondrogenesis, morphogen, chicken embryo, retinoids.

Introduction

At early stages in embryonic development, the ver-tebrate face has a common plan. A series of smallbuds of tissue, the facial primordia, forms around theprimitive mouth (Fig. 1). The upper jaw developsfrom five main buds of tissue: a central primordium,the frontonasal mass (sometimes present as themedian nasal processes), the two lateral nasal pro-cesses on either side and flanking these, the twomaxillae. The lower jaw develops from the pairedmandibular primordia. The same plan of facial pri-mordia is found in the embryos of both birds and

mammals. However, most of the information abouthow the face develops is based on work carried out inchickens because the embryos are readily accessiblefor experimental manipulations.

The facial primordia are made up mainly of neuralcrest cells that have migrated from the cranial crestand settle to form the facial primordia (Noden, 1975).The neural crest cells give rise to the connectivetissues of the face. The myogenic cells of the facialmuscles constitute a separate cell lineage. The myo-genic cells originate from the paraxial mesoderm andalso migrate into the facial primordia (Noden, 1983a;see also Noden, this volume).

32 S. E. Wedden, J. R. Ralphs and C. Tickle

Fig. 1. Face of stage-24 chicken embryo showing plan ofprimordia. fnm, frontonasal mass; /, lateral nasal process;mx, maxilla; md, mandible. Bar, lmm.

The development of the facial primordia involvesthe four fundamental processes that underlie all ofembryonic development. These processes aregrowth, morphogenesis, cell differentiation and pat-tern formation. Here, we will consider pattern forma-tion - the process that leads to the spatial ordering ofcell differentiation. The relationship between celldifferentiation and pattern formation is illustrated bythe development of the upper and lower beaks ofchicken embryos. The same cell types, cartilage,muscle, bone and other connective tissues differen-tiate in both beaks but are arranged in differentpatterns (Fig. 2). We wish to understand how the

patterns of differentiated cells are generated. Whatcontrols cell differentiation in the facial primordia togive the typical skeleton and associated muscles in thelower beak but a different pattern of tissues in theupper beak?

Positional information and pattern formation in theheadPattern formation can be considered from the view-point of positional information (Wolpert, 1969; 1981).According to this view, cells are informed of theirposition within the embryo and acquire a positionalvalue. Positional values are ultimately interpreted interms of spatially appropriate cytodifferentiation.Development proceeds in a step-wise fashion becausepositional cues can only operate over small distances(Crick, 1970). Positional values acquired at an earlystage in development affect the interpretation ofpositional values given later. For example, in chicklimb development, cells must acquire positionalvalues at an early stage that dictate whether theybelong to a wing or a leg. Interactions within thedeveloping limb bud determine which part of the limbthey will form. If these same principles are applied tochick face development, the cranial neural crest cellsthat give rise to the upper and lower beaks must firstbe informed to which facial primordia they belongand then of their position within that facial primor-dium.

How are cranial neural crest cells informed towhich facial primordium they belong? In general, themesoderm of the embryo appears to act as thetemplate that carries positional information alongthe head-to-tail axis of the embryo. For example, it is

Fig. 2. (A) Side view of cleared whole mount of the bill of a stage-36 chicken embryo stained with alcian green to showthe pattern of cartilage differentiation in upper and lower beaks, e, egg tooth. Bar, 1 mm. (B) Face of stage-34 chickenembryo: section taken close to the midline showing the patterns of tissues in upper and lower beaks, c, cartilage; musclearrowed. Bar, 1 mm.

Pattern formation in the facial primordia 33

the mesoderm that determines whether a chick limbbud is a leg bud or a wing bud. In addition, theinformation carried by the mesoderm appears todictate the regional differentiation of the ectodermand the endoderm. For example, the specific patternof the feather tracts is determined by the mesoderm(Sengel, 1975). The appropriate spatial differen-tiation of migratory neural crest cells in the trunkcould also be controlled by the mesoderm. The trunkneural crest cells differentiate according to their finalposition rather than their origin (see for example LeDouarin, 1980). However, in the head where neuralcrest cells settle to form the facial primordia there isvirtually no mesoderm to convey positional infor-mation and the cells must be informed to whichprimordium they belong by some other means.

One possibility is that the neural crest cells in thehead are informed to which facial primordium theybelong, prior to emigration. The individual facialprimordia are populated by neural crest cell popu-lations that arise in different regions of the headneural folds. The origins and pathways of thesecranial crest cells have been extensively investigatedin chick embryos. Recently, the remarkable feat ofmapping cranial crest migration in a mammalianembryo has been accomplished (Tan & Morriss-Kay,1986). In chick embryos, the neural crest cells thatsettle to form the frontonasal mass first migrate fromthe prosencephalic region (reviewed Le Douarin,1982) and are joined by cells migrating later mainlyfrom the anterior mesencephalic region (Noden,1978). The cells in the maxillae come from theposterior mesencephalic region (Noden, 1975),whereas the cells in the mandibular primordia comemainly from the region of the anterior rhombence-phalon but there is also a contribution from cells thatarise in the posterior mesencephalon (Noden, 1975).In the trunk, exchanges between different regions ofthe neural crest almost invariably lead to normaldevelopment (Le Douarin, 1980a; reviewed LeDouarin, 1982). This suggests that trunk neural crestcells are initially equivalent and that their subsequentbehaviour depends on where they settle (see also Bee& Newgreen, this volume). Until recently, the sameappeared to be true of cranial neural crest (see, forexample Noden, 1978). However, it has now beenshown, in a series of transplantations of crest fromforebrain and midbrain regions to the hindbrain, thatectopic beak-like structures can develop. Forexample, an upper 'beak' can form in the neck and aset of mandibular structures can arise in addition tothe normal set, in the arch below (Noden, 19836).The formation of ectopic beak structures strongly

suggests that, in the head, the neural crest cells arealready programmed before emigration.

A second proposal recently put forward by Thoro-good (1987) is that cranial epithelium acts as atemplate to inform cells of their position in the head.For example, in the development of the tissuessurrounding the chick eye, transient synthesis of typeII collagen at the mesenchymal-epithelial interfaceof the periocular mesenchyme and the pigmentedretina, correlates with the time at which neural crestcells become committed to form the scleral cartilage(Thorogood, Bee & Von der Mark, 1986). Specifi-cation of cranial neural crest cells that form the facialprimordia could also involve cues provided by epi-thelial interfaces encountered during migration. Theneural crest cells of each facial primordium havefollowed different routes to reach their destinations.The neural crest cells that settle in the frontonasalmass move at first rostrally above the prosencephalonand then around the front of the developing brainwhereas the cells that settle in the maxillae andmandibles migrate laterally from the neural crest (seeJohnston, 1966; Noden, 1975). Transient synthesis oftype II collagen can be detected at mesenchymal-epithelial interfaces on some of these pathways(Thorogood et at. 1986). This may be involved inpreparing the neural crest cells to subsequently differ-entiate as cartilage in the facial primordia but it is notclear whether this could characterize cells as belong-ing to, say, a frontonasal mass as opposed to amandibular primordium.

A problem for the idea that positional informationis conveyed during the migration of neural crest cellsis the development of ectopic beak-like structuresthat result in certain cranial crest transplantationexperiments (see above) since the routes taken by thecrest cells must clearly be abnormal. It thereforeappears most likely that the cues that inform neuralcrest cells to which facial primordium they belongoperate before the cells start migrating.

Cell populations in the facial primordiaThe facial primordia of chick embryos contain appar-ently homogeneous populations of undifferentiatedcells (Fig. 3) The cells in frontonasal mass primordiaappear virtually indistinguishable from those in theprimordia. However, when the mesenchyme cellpopulations from each of the facial primordia areplaced in high-density (micromass) cultures, there aredistinct differences in the extent and pattern ofchondrogenesis (Wedden, Lewin-Smith & Tickle,1986). (Micromass cultures have been extensivelyused to study the chondrogenic potential of cellpopulations in developing limbs (Ahrens, Solursh &Reiter, 1977).) At early stages (stage-20 to -24chicken embryos), the mesenchyme cell population

M S. E. Wedden, J, R. Ralphs and C. Tickle

f $&&f&&

Fig. 3. (A) Face of stage-28 chicken embryo: sectionclose to midline. fnm, frontonasal mass, md, mandible.Bar, 0-2 mm. (B,C) Enlargements of boxed areas on A offrontonasal mass and mandible respectively showingapparently homogeneous populations of undifferentiatedcells. Bars, 50;«n.

4A

B

of the frontonasal mass undergoes extensive chondro-genesis and forms an almost continuous sheet ofcartilage; in mandibular cultures, cartilage differen-tiation is less extensive and discrete nodules form;and in cultures of maxillae cells, virtually no chondro-genesis occurs at all (Fig. 4). Therefore, it is clear thatwith respect to cartilage differentiation in culture,there are distinct differences in the mesenchyme cellpopulations of the frontonasal mass, mandibles andmaxillae at an early stage in the development of thefacial primordia.

It is possible that interactions within the facialprimordia have already taken place in chick embryosby stage 20 and that these have differentially modifiedthe cell populations. For example, it may be that thefrontonasal mass and mandibles at first contain equiv-alent cell populations but interactions within themandible have led to some cells no longer having thepotential to form cartilage. However, cultures that

c

Fig. 4. Micromass cultures of cells from facial primordiaof stage-24 chicken embryos. Cultures fixed at 72 h,stained with alcian blue to show cartilage matrix andcounterstained with eosin. Edge of cultures indicated byarrows. Bars, 1 mm. (A) Frontonasal mass cultures:extensive chondrogenesis in a sheet. (B) Mandiblecultures: less cartilage differentiation in a nodularpattern. (C) Maxillae cultures: virtually no cartilagedifferentiation.

Pattern formation in the facial primordia 35

have been established with cells from the mandiblesof embryos at stages 17/18 have a sparse nodularpattern of chondrogenesis and in cultures from evenearlier mandibles (from stage 16/17 chick embryos),hardly any cartilage differentiates at all. Cultures offrontonasal mass cells from stage-17 chick embryosstill form a sheet of cartilage (J. R. Ralphs, unpub-lished observations).

The pattern of chondrogenesis in micromass cul-tures of the individual facial primordia could beinterpreted as reflecting differences in the neural crestcell populations. However, the facial primordia con-tain, in addition to neural crest cells, cells of mesoder-mal origin that will give rise to the myogenic cells ofthe muscles (Noden, 1983a). Therefore, the differ-ences in the extent of cartilage differentiation in thecultures may reflect, not characteristic populations ofneural crest cells, but different proportions of pre-sumptive myogenic cells. The effects of myogeniccells on the extent and pattern of chondrogenesis incultures of chick limb bud cells have recently beendemonstrated by Cottrill, Archer, Hornbruch & Wol-pert (1986). They irradiated the somites at the levelwhere the wing buds will develop to eliminate themyogenic cell lineage. In cultures of cells from thesubdistal regions of muscle-less limb buds, a sheet ofcartilage forms rather than the nodular pattern that isnormally obtained. However, it should be noted thatin cultures from different regions of chick limb buds,there appears to be no correlation between the extentof chondrogenesis and the number of muscle cellsthat differentiate (Swalla & Solursh, 1986).

Recently, we have investigated the possibility thatthe proportion of myogenic and potentially myogeniccells can account for the different patterns in chon-drogenesis in cultures of chick facial primordia(Ralphs, Dhoot & Tickle, 1988). In the intact face, nodifferentiated myogenic cells can be detected in anyof the primordia in chick embryos at stages 20 and 24.However, when the cells are placed in micromassculture, myogenic cells differentiate and this revealsthe presence of potentially myogenic cells in theprimordia. The myogenic cells are recognized byantibodies to the heavy chains of skeletal musclemyosin (Dhoot, 1986) and desmin, the intermediatefilament protein characteristic of muscle (Osborn,Geisler, Shaw, Sharp & Weber, 1981).

The number of muscle cells that differentiate in thecultures can be compared with the extent and patternof chondrogenesis. With cells from stage-20 embryos,cultures of all the facial primordia contain about thesame number of myogenic cells despite the distinctdifferences in cartilage differentiation. With cellsfrom embryos at stage 24, cultures of the frontonasalmass and the mandible now both contain an increasednumber of myogenic cells compared with the cultures

from the earlier embryos. However, the same num-ber of myogenic cells differentiate in cultures of bothprimordia. In the frontonasal mass cultures, themyogenic cells are distributed more or less singlythroughout the sheet of cartilage (Fig. 5A), whereas,in the mandibular cultures, the myogenic cells areclustered between the nodules (Fig. 5B). In maxillaecultures, very few myogenic or chondrogenic cellsdifferentiate (Fig. 5C). Therefore we conclude thatthe distinct patterns of chondrogenesis in micromasscultures of cells from chick facial primordia are not areflection of dilution of the neural crest cell popu-lations with different proportions of potentiallymyogenic cells. Instead, there appear to be realdifferences in the neural crest cell populations in eachfacial primordium in terms of the ability of the cells todifferentiate into cartilage when placed in high-density culture.

Interactions in chick facial primordiaOnce the facial primordia have been established, cellsmust be informed of their position within the facialprimordium to which they belong. The laying down ofthe pattern of cellular differentiation along theproximodistal axes of the beaks (this axis runs fromthe base to the tip) appears to involve a set ofepithelial-mesenchymal interactions. This has beenshown by Wedden (1987) in a series of transplantationexperiments. She grafted fragments of facial primor-dia with and without their associated epithelium toholes cut in wing buds and assayed their develop-ment. With just the mesenchyme from the frontona-sal mass or mandibular primordia, outgrowth andpattern formation is inhibited. In control fragmentswith intact epithelium considerable outgrowth occursand beak-like structures are formed.

Pattern formation along the proximodistal axis ofthe developing limb is also coupled with outgrowth.A pronounced thickening in the epithelium at the tipof the limb bud, the apical ectodermal ridge(Saunders, 1948; Summerbell, 1974) is required foroutgrowth and patterning. When the apical ectoder-mal ridge is removed from a limb bud, furtheroutgrowth is inhibited. The limb that develops istruncated and lacks distal structures. In the limb, theapical ectodermal ridge maintains a region of undif-ferentiated mesenchyme at the tip of the bud as itelongates. This region has been called the progresszone. The progress zone model suggests that patternalong the proximodistal axis of the limb may bespecified by the length of time cells spend at the tip ofthe limb (Summerbell, Lewis & Wolpert, 1973). Cellsthat leave the progress zone early form proximalstructures whereas cells that leave later form distalones such as digits. It is not clear whether the

M S. E. Wedden, J. R. Ralphs and C. Tickle

Fig. 5. Central regions of micromass cultures of cellsfrom facial primordia of stage-24 chicken embryos.Cultures fixed at 72 h, labelled with an antibody toskeletal muscle myosins to detect differentiated myogeniccells and counterstained with haematoxylin. Bars, 50urn.(A) Frontonasal mass cultures: single myogenic cells havedifferentiated throughout the cartilage sheet.(B) Mandible cultures: differentiated myogenic cellsclustered between cartilage nodules (n). (C) Maxillaecultures: a typical region in which no myogenic cells arevisible.

epithelial-mesenchymal interactions in the facial pro-cesses also involve an apical ectodermal ridge and aprogress zone mechanism.

A second set of epithelial-mesenchymal interac-tions that may be involved in the patterning ofcellular differentiation during development of thefacial primordia is suggested by experiments in cul-ture. Using micromass cultures of chick facial primor-dia, we have shown that face epithelium locallyinhibits cartilage differentiation in these cultures(Wedden et al. 1986). Epithelium from either man-dibular or frontonasal mass primordia is inhibitorywhen tested on cultures of frontonasal mass mesen-chyme. Non-ridge epithelium from the limb bud alsoinhibits cartilage differentiation in its immediatevicinity in micromass cultures of limb bud cells(Solursh, Singley & Reiter, 198J). These interactionsbetween epithelium and mesenchyme demonstratedin micromass cultures could serve to confine cartilagedifferentiation to the core of a developing limb bud(Solursh, 1984) or facial primordium. However, re-cent experiments in which chick limb buds have beenpermanently denuded or dorsal epithelium show thatthis, surprisingly, has no effect on the cartilagepattern that develops (Martin & Lewis, 1986).

The inhibition of cartilage differentiation inducedby epithelium in the cultures of postmigratory neuralcrest cells should be contrasted with the epithelialstimulation of chondrogenesis of premigratory cranialcrest cells in culture (Bee & Thorogood, 1980).However, it should be noted that in these organcultures of premigratory crest cells and epithelium,cells differentiate into cartilage within the explant anda rim of nonchondrogenic tissue develops immedi-ately below the epithelium.

Finally, we should consider the mechanisms thatlead to specification of the mediolateral axis of theface. For example, cartilage differentiation is con-fined to the centre of the frontonasal mass in chickembryos to give the prenasal cartilage and the mid-point of the upper beak is also defined by differen-tiation of the epithelium to form an egg tooth. Theformation of the egg tooth by the epithelium requiresa signal from the mesenchyme (Tonegawa, 1973).How are cells informed of their position with respectto this mediolateral axis?

To explore the problem of specification of themidline of the chick upper beak, Wedden has isolatedfragments from the frontonasal mass and assayedtheir development when grafted to limb buds. Infragments taken both from the midline and extremelateral regions of the frontonasal mass of embryos atearly stages (18-21), a cartilage rod and an egg toothcan develop. Therefore, from one frontonasal mass itis possible to obtain at least three cartilage rods andegg teeth. After stage 21, only central regions of the

Pattern formation in the facial primordia 37

Fig. 6. Cleared whole mount of graft of half of thefrontonasal mass of a stage-24 chicken embryo. Graftfixed at 6 days and stained with alcian green to showcartilage differentiation. Two prenasal cartilages havedeveloped from the single fragment of tissue. Theepithelium at the tip of each cartilage rod has'differentiated into an egg tooth. There is also a third eggtooth (arrowed) that has differentiated in the epitheliumover a small lateral outgrowth. In this case, themesenchyme of the frontonasal mass was separated fromthe epithelium and then reannealed but the same resultcan also be obtained with intact fragments. Bar, 1 mm.

frontonasal mass give rise to midline structures. Thissuggests that the signals that confine cartilage differ-entiation to the centre of the frontonasal mass beginoperating at stage 21 (Wedden & Tickle, 1986a).

The basis of the regulatory behaviour of lateralfragments of the frontonasal mass at early stages thatresults in the formation of midline structures is notclear. The new midpoint could be formed by cells inlateral positions now taking on central characteristicsfollowing removal of the native midpoint. Alterna-tively, cell proliferation could generate cells to reformmidpoint structures. The frontonasal mass can appar-ently regulate in the intact face. In a series ofexperiments to explore the effects of the growing eyeson interorbital structures in chick embryos, both opticvesicles were removed and in one case a fragment ofamnion was also inserted into the anterior neuropore.This resulted in formation of a bifid upper beak(Silver, 1962).

Another interesting feature of the behaviour ofgrafts of the frontonasal mass primordium is thedevelopment of duplicated prenasal cartilages andegg teeth from a single fragment (Fig. 6). This occurswith fragments taken from a range of developmentalstages when the primordium is divided into either twoor three pieces (S. E. Wedden: unpublished obser-vations). At present, the basis of these duplications isnot known.

Effects of vitamin A derivatives (retinoids) on thedevelopment of the faceWhen all-ftww-retinoic acid is applied to chick em-bryos at early stages in the development of the facialprimordia, facial defects result (Tamarin, Crawley,Lee & Tickle, 1984). The upper beak is missing butthe lower beak develops normally (Fig. 7). Thefrontonasal mass appears to be specifically affected.Pattern formation and outgrowth of this primordiumis inhibited. Fusion with the maxillae fails to occurleading to clefting of the primary palate (Tamarin etal. 1984). The full defect is produced when chickembryos are treated between stages 18 and 21 (Wed-den & Tickle, 19866) and can also be caused byapplying a synthetic analogue of retinoic acid, (E)-4-2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphaenyl)-l-propenyl) benzoic acid (TTNPB: Loeliger,Bollag & Mayer, 1980), which is metabolically ratherstable. In mammalian embryos, 13-ds-retinoic acidmay have a similar effect: applied when the facialprimordia are populated with neural crest cells,development of the upper jaw is affected and mediancleft lip can result (Goulding & Pratt, 1986).

Our working hypothesis for pattern formation inthe face is that cells first acquire a positional valuethat informs them to which facial primordium theybelong and then are informed of their position withinthat primordium. This framework may help interpret-ation of the specific defect. One possibility is that theresponse to retinoids is determined by the positionalvalues that characterize the cells of the frontonasalmass. A second possibility is that retinoids interferewith the positional cues that inform cells of theirposition within the frontonasal mass and operatespecifically within this primordium.

We can investigate how retinoids affect cells fromthe facial primordia in culture and find out whetherthe origin of the cells affects the response. We havecompared the effects of retinoids (all-fra/u-retinoicacid and TTNPB) when added to the medium of themicromass cultures of cells from either the frontona-sal mass or mandible. The retinoids inhibit cartilagedifferentiation in the cultures. There is a doseresponse. Cartilage differentiation in cultures offrontonasal mass and mandible cells shows the samesensitivity to each retinoid. Either lxlO~6M-retinoicacid or 1 x 1CT8

M-TTNPB abolishes chondrogenesis(Wedden, Lewin-Smith & Tickle, 1987). Therefore,in culture, the response of frontonasal mass andmandible cells to the addition of retinoids appearsvery similar. However, there is a subtle differencewhen the amount of matrix secreted and the area ofcartilage is examined in the retinoid-treated cultures.In mandible cell cultures, the dose response toretinoids involves a progressive reduction in bothparameters whereas in frontonasal mass cultures, the

S. E. Wedden, J. R. Ralphs and C. Tickle

Fig. 7. Defective facial development in chick embryos following application of all-Zra/w-retinoic acid. (A) Whole mountof the head of a 10-day chicken embryo which was treated in vivo with a\\-trans-retino\c acid. The treatment involvedimplanting a bead presoaked in lOmgrnT1 retinoic acid in the right wing bud of a stage-20 embryo. (B) Section of ahead from an embryo treated as above. Note that the defect involves failure of upper beak development whereas thelower beak appears to develop normally. Bar, 1 mm.

area of cartilage-producing cells is much less sensitiveto increasing doses of retinoid than the amount ofmatrix secreted.

In the intact primordia in the embryo following atreatment with TTNPB that would lead to the specificdefect, the concentration of the retinoid in thefrontonasal mass is 5-5X10~8M and in the mandible4-6X10~8M (Wedden etal. 1987). This concentrationof TTNPB applied to the mandible cells in culturewould be sufficient to markedly reduce chondro-genesis. Therefore, the puzzle is why the mandiblesof treated embryos nevertheless develop normally.

Retinoids could interfere with positional cues thatoperate as the facial primordia develop. The trun-cated upper beaks that result from retinoid treatmentare reminiscent of the effect of removing the epi-thelium from transplants of the frontonasal mass.Wedden (1987) has investigated whether the epi-thelium is the target of retinoid action by makingcombinations between the epithelium and mesen-chyme of facial primordia from treated and untreatedchick embryos, and assaying the development of therecombined tissues. In combinations of frontonasalmass tissues, outgrowth and pattern formation isinhibited when the mesenchyme is taken from treatedembryos, whereas from combinations of treated epi-thelium and untreated mesenchyme, upper beak-likestructures develop. She therefore concluded that it isthe mesenchyme of the frontonasal mass that isaffected by retinoids and not the epithelium. It isinteresting that retinoid treatment of either the mes-enchyme or epithelium of mandibular primordiareduces the extent of outgrowth. In combinations of

mandibular tissues from treated and untreated em-bryos, lower beak structures develop but these areshorter than controls.

In chick limb development, retinoids may act asmorphogens to spatially control the pattern of celldifferentiation. When retinoic acid is locally appliedto the anterior margin of a chick limb bud, a gradientof retinoic acid is established across the bud (Tickle,Lee & Eichele, 1985). This signal leads to theformation of a duplicate set of digits that develops inmirror-image symmetry with the normal set. Thesignal generated by local application of retinoic acidmimics the effect of grafting an additional polarizingregion. The polarizing region is a signalling regionconsisting of a small group of cells found at theposterior margin of the bud (Saunders & Gasseling,1968; Tickle, Summerbell & Wolpert, 1975) andcontrols the pattern of structures that develop acrossthe anteroposterior axis of the limb. Recently, en-dogenous retinoic acid has been demonstrated indeveloping chick wing buds (Thaller & Eichele,1987). The concentration of this endogenous retinoidis similar to that which experimentally brings aboutpattern changes. Furthermore, the posterior half ofthe wing bud where the polarizing region is locatedcontains more retinoic acid than the anterior half.

These experiments with chick limbs may be rel-evant to the effects of retinoids in the face. Theconcentration of TTNPB in the frontonasal mass oftreated embryos is 5-5X10~8M. A similar retinoidconcentration, lOx 10~8

M-TTNPB, has been found tobring about pattern changes in developing chick limbbuds (Eichele, Tickle & Alberts, 1985). Therefore, it

Pattern formation in the facial primordia 39

is tempting to speculate that retinoids may act assignalling substances in pattern formation in thefrontonasal mass. The facial defect might thus arisebecause the applied retinoid distorts the normalretinoid distribution that signals the pattern of struc-tures that develop in the frontonasal mass.

S.E.W. carried out the work reported here during thecourse of an MRC studentship. The research of C.T. andJ.R.R. is supported by the MRC. C.T. thanks Dr A.Tamarin for introducing her to the face. We thank Pro-fessor L. Wolpert, J. Richman and S. Croucher for readingthis manuscript.

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