Retinoic acid and mammalian craniofacial morphogenesis

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1 J. Biosci., Vol. 21, Number 3, May 1996, pp Printed in India. Retinoic acid and mammalian craniofacial morphogenesis NORIKO OSUMI-YAMASHITA* Department of Developmental Biology, Division of Life Science of Maxillo-Facial Systems, Graduate School of Dentistry, Tokyo Medical and Dental University, , Yushima, Bunkyo-ku, Tokyo 113, Japan MS received 13 September 1995 Abstract. Retinoic acid is a morphogenetic signalling molecule in vertebrate embryos, one being known to perform a specific function in organizing the body pattern along the anteroposterior axis. This molecule has especially attracted research attention because retinoic acid treatment will also induce abnormal morphogenesis, particularly in the craniofacial structures. The present review discusses recent molecular insights revealing how the retinoic acid signal is transduced within a cell, specifically focusing on the involvement of cranial neural crest cells in retinoic acid-induced abnormal morphogenesis in the mammalian head. Keywords. Retinoic acid (RA); mammalian embryos; craniofacial morphogenesis; neural crest cells; facial primordia; retinoic acid receptors; retinoid X receptors; cellular retinoid binding proteins; Hox genes. 1. Introduction Retinoic acid (RA), a natural metabolite of vitamin A, is essential for normal mammalian development. Early in 1930s, the developmental importance of retinoids was recognized by Hale (1937) who first observed that ocular malformations were caused by vitamin A deficiency in pigs. Mammalian embryos exhibit anomalies also by excess vitamin A (Cohlan 1953). Other natural and synthesized retinoids including RA are also known as teratogens to induce abnormalities in various organs (Shnefelt 1972; Geelen 1979). Since synthetic retinoids are applied to pregnant human mothers for the therapeutic treatment of severe forms of cystic acne, a number of human congenital anomalies have been recognized as caused by RA (Lammer et al 1985). Limb- and craniofacial development have been subjects of experimental investigations on retinoids for over 25 years (e.g., Morriss 1972; Tickle et al 1975; Niazi and Saxena 1978; Maden 1982). Nowadays, several lines of evidences suggest that RA is a morphogenetic signaling molecule in vertebrate embryos and, in particular, has a role in organizing the body pattern along the anteroposterior axis. There have already been several reviews on functions of RA in vertebrate development (Eichele 1989; Tabin 1991; Morriss-Kay 1992a, b, 1993; Hoffmann and Eichele 1994), including several articles in this issue related to limb development and regeneration. Here, I introduce the mammalian craniofacial morphogenesis, review the recent studies of molecular aspects in RA signaling, discuss the RA's effects on mammalian craniofacial development and gene expression, and consider the perspectives for future studies in this field. *Fax ; , noriko.dev@dent.tmd.ac.jp. 313

2 314 Noriko Osumi-Yamashita 2. Mammalian craniofacial morphogenesis Craniofacial morphogenesis starts with formation of the head folds at the most rostral portion of the neural plate, the source of neural crest cells. Subsequently, four morphological units, i.e., the forebrain, midbrain/prorhombomere A (prorha, rostral hindbrain), prorhb (preotic hindbrain), and prorhc (caudal hindbrain), are observed Figure 1. Schematic illustration showing migration patterns of cranial crest cells in mouse embryos. (A), (B) Lateral and views at the 5-6 somite stage. (C) Dorsal view at 8 somite stage. (D) Lateral view at the pharyngula stage. (A) and (D) are illustrated based on DiI labeling results by Osumi-Yamashita et al (1994, 1996). (B) and (C) are basically modified from figure 6 in Tan and Morriss-Kay (1985) who observed rat crest cell emigration by scanning electron microscopy. (A) Showing prorhombomeres A (prorha), prorhb, prorhc, and presumptive prorhd. Preotic sulcus (PO S) is an obvious landmark in the hindbrain. ProRhA develops into rhombomere 1 and 2 (rl and r2), prorhc gives rise to r3 and r4, prorhc forms r5 and r6, and prorhd becomes r7 (or r7 and r8). Regions to which Dif labelling of crest cells was performed are shown by different colors. (B) At 5-6 somite stage, crest cells start to migrate from the forebrain, midbrain/prorha region. (C) At the 8 somite stage, three streams of crest cells are seen to migrate from prorha, prorhb and prorhc/d. Note that there are crest free zones at the boundaries between prorha/prorhb and prorhb/prorhc. Forebrain is not seen in dorsal view. (D) Mapping of cranial crest cells. Both forebrain and anterior midbrain crest cells populate the frontonsal mass. At the more later stage, midbrain crest cells occupy the lateral nasal prominence (unpublished results), Crest cells originating from prorha (r1 + r2) populate the first pharyngeal arch and the trigeminal ganglion. Those from prorhb (r3 + r4) migrate rostrally to the otic vesicle to populate the acousticofacial ganglia and the second arch. Cells originating from presumptive r5 + r6 (pro RhC) migrate along the caudal aspects of the otic vesicle and populate the third pharyngeal arch and IXth ganglion, while those from presumptive r7 (prorhd) migrate to the fourth arch and the Xth ganglion. Notably, single prorhombomeres produced crest cells populating in single pharyngeal arches. Mx, maxillary prominence; Md, mandibular prominence; TG, trigeminal ganglion; a2,-a4, the second to the fourth pharyngeal arches; OP, olfactory placode; OV, otic vesicle.

3 RA and mammalian craniofacial morphogenesis 315 from anterior to posterior in the rostral neural plate (figure 1; Adelman 1925; Bartelmez 1923; Bartelmez and Evans 1925). In mammals, neurulation does not occur simply in anteroposterior sequence: closure of the neuroepithelium gradually proceeds both anteriorly and posteriorly from the fore-midbrain boundary and anteriorly from the posterior region of the prorhc. In this process, the rostral neural tube, or the forebrain, further differentiates the telencephalon and diencephalon, and the prorhombomeres are subdivided to form rhombomeres 1-7 (r1-7), in such a way that a single prorhombomere produces an odd and even pair or rhombomeres (Trainer and Tam 1995; Osumi-Yamashita et al 1996). Simultaneously, neural crest is formed at the junction between the neuroepithelium and surface ectoderm, and crest cells subsequently start to migrate into the embryonic body. In contrast to trunk crest cells or crest cells in other animals, mammalian cranial crest cells emigrate from the head neuroepithelium before its closure (Nichols 1981, 1986; Tan and Morriss-Kay 1985). These cranial crest cells are the source of various craniofacial structures. At the time when migration of cranial crest cells nearly ceases, several facial primordia are formed in the head (figure 1). The most anteriorly situated is the frontonasal prominence, where the lateral and medial nasal prominences subsequently protrude flanking the nasal pit. The lateral and nasal prominences are thought to contribute to the nasal capsule and nasal septum, respectively (N Osumi-Yamashita, unpublished results). Caudally, the first pharyngeal (branchial) arch later develops into the maxillary and mandibular prominences. These are the primordia of upper and lower jaws. Farther caudally, the second (hyoid), third, and fourth (and sixth, thought not clearly identified) pharyngeal arches follow. These posterior arches contribute the formation of several pharyngeal organs, such as the parathyroid gland and thymus. Within the facial primordia, cranial crest cells differentiate into various types of mesenchymal as well as neural tissues, such as bones, cartilage, teeth, cranial sensory ganglia, and melanocytes (for review, see Morriss-Kay and Tan 1987; Osumi- Yamashita and Eto 1990). At the level of the posterior pharyngeal region, vagal (postotic) crest cells participate in formation of the heart, especially of the aorticopulmonary septum (Kirby 1987; Fukiishi and Morriss-Kay 1992). In the frontonasal region, the mesenchyme has dual origin of crest cells, i.e., both forebrain and midbrain contribute to the production of crest cells which occupy the frontonasal mesenchyme (Matsuo et al 1993; Osumi-Yamashita et al 1994). Specifically, midbrain crest cells pass through the area between the presumptive lens placode and the optic cup to reach the frontonasal region just beneath the presumptive nasal placode where they form the lateral nasal prominence (figure 1; N Osumi-Yamashita et al, unpublished results). The forebrain crest cells, on the other hand, seem to be predominantly destined to the medial nasal prominence (N Osumi-Yamashita, unpublished results). In the pharyngeal region, the crest cell migration is segmentaly organized especially in the preotic area (figure 1, Serbedzija et al 1992; Matsuo et al 1993; Osumi-Yamashita et al 1994, 1996; Trainer and Tam 1995). The segmental distribution of the crest cells primarily follows the segmentation of the hindbrain, i.e., prorhombomeres. Crest cells from the midbrain and prorha (future r1 and r2), migrate toward the first pharyngeal arch. From prorhb (r3 and r4) crest cells migrate to the second (hyoid) arch. ProRhC (r5-7), which is the most caudal prorhombomere, produces crest cells populating the third and more posterior arches. In case of mammals, such segmental distribution appears to be accomplished by existence of the crest-free zones at the boundaries of

4 316 Noriko Osumi-Yamashita prorha/b (the preotic sulcus) and of prorhb/c (figure 1, Tan and Morriss-Kay 1985). These hindbrain crest cells later attach to even-numbered rhombomeres, where cranial nerve roots form (Adelman 1925; Bartelmez 1923; Bartelmez and Evans 1925). In the mouse, Hox genes have spatially restricted patterns of expression that appear before the formation of rhombomeric morphology and later map to specific rhombomeric boundaries (see review in Krumlauf 1993; and references therein). The Hox paralogues have similar anterior boundaries of expression, and they are expressed mostly in a tworhombomere periodicity. Hox genes are also expressed in pharyngeal arches. Since the Hox genes are originally cloned as homologues of Drosophila homeotic genes that are involved in specification of individual segments, the expression pattern of the genes, or Hox code, is implied to be related to the pre-patterned information contained in hindbrain crest cells (reviewed in Wilkinson 1990; Hunt and Krumlauf 1991; Krumlauf 1993; see also Noden 1983, for pre-patterning of the cranial crest). As described above, one prorhombomere gives rise to two successive rhombomerers that are associated with a single pharyngeal arch. Therefore, the nested expression pattern of Hox code may be already prefigured at the state of prorhombomeres (see figure 9 in Osumi-Yamashita et al 1996). For example, prorha gives rise to rl + r2 and produces crest cells populating the first arch, both of which have no Hox code, prorhb gives rise to r3 + r4 and produces crest cells populating the second arch, expressing pb paralogue Hox genes (Hoxa-2 and b-2) in common, the anterior portion of prorhc gives rise to r5 + r6 and the third arch, which have pb + Zen/pb paralogue Hox code (Hoxa-2 and b-2 + Hoxa-3, b-3 and d-3), and finaly the posterior portion of prorhc gives rise to r7 and the fourth arch, which have pb + Zen/pb + Dfd paralogue Hox code (Hoxa-2 and b-2 + Hoxa-3, b-3 and d-3 + Hoxa-4, b-4 and d-4). Thus, prorhombomeres may possibly represent basic metamerical units in mammalian craniofacial development. As mentioned above, both excess RA and vitamin A deficiency cause developmental abnormalities in craniofacial structures (including the heat in this review). This implies that RA plays crucial roles in morphogenesis of the head. Before entering into RA's functions on craniofacial morhogenesis, I will briefly survey molecular mechanisms involved in RA signaling pathway. 3. Retinoic acid receptors and retinoid X receptors The biological effects of RA are known to be mediated by two classes of nuclear RA-binding receptors, the retinoic acid receptors (RARs) and the retinoid 'X' receptors (RXRs). Both of these receptor families belong to the steroid/thyroid hormone receptor superfamily, with highly conserved 'zinc-finger' DNA-binding domains (Evans 1988; Green and Chambon 1988; Leid et al 1992; Mangelsdorf et al 1994). The RARs and RXRs share overlapping ligand specificity; 9-cis RA is a common ligand for both receptors with high affinity, whereas all-trans RA binds only to RARs (Heyman et al 1992; Levin et al 1992; Mangelfdorf et al 1992). RARs form heterodimers with RXRs, while RXRs can bind to DNA as homodimers (Yu et al 1991; Kliewer et al 1992a, b; Leid et al 1992). Of importance, various combinations of RAR/RXR heterodimers are shown to act as transcription factors by binding to a common specific responsive element (RARE). [For detailed biochemical character of these receptors and molecular mechanisms of activation of specific target genes, see Leid et al (1992), Mangelsdorf et al (1994) and also Umesono in this issue].

5 RA and mammalian craniofacial morphogenesis 317 Figure 2. Specific expression of RAR ß (left) and y (right) genes in day 10 mouse embryos. RAR ß expression is predominantly observed in the lateral nasal prominence (L), while RAR γ transcripts are distributed both in the lateral and medial (M) nasal prominences as well as maxillary (Mx) and mandibular (Md) prominences. In order to understand functions of RA in morphogenesis, it is necessary to know the spatiotemporal patterns of expression of RAR and RXR genes. Mammalian RARs are composed of three subtypes, a, and y, each of which has several isomers. Each of these RAR subtypes shows very specific spatiotemporal patterns of expression during development, with the exception of RAR α that is almost ubiquitously expressed in embryo. In the craniofacial primordia of day 10 mouse embryos, RAR y is expressed both in the medial and lateral nasal prominences, while RAR ß expression is restricted to the lateral nasal prominence (figure 2, also see Osumi-Yamashita et al 1990, 1992). Differential expression patterns of RARs in the lateral and medial nasal prominences apparently coincide with different chondrogenic potentials of these mesenchymal cells as observed in vitro. For example, mesenchymal cells derived from the day 11 lateral nasal prominence show higher cartilage formation that those from the medial nasal prominence (Motoyama et al 1994). However, this interpretation is superficially inconsistent with RAR γ expression in later stages; expression of RAR γ is associated with pre-cartilagenous mesenchymal condensations and with cartilage itself, but disappears with onset of ossification (Osumi-Yamashita et al 1990; Ruberte et al 1990). Antisense oligonucleotide against RAR γ1 was reported to enhance chondrogenesis of mouse limb bud mesenchyme in vitro (Motoyama and Eto 1994). Thus, RAR γ expression per se is not likely o be related to chondrogenesis. Expression domains of RAR ß and RAR γ are exclusive from each other; RAR ß is expressed in the hindbrain at the occipital level and throughout the posterior neural tube, while RAR γ is expressed in the open neural epithelium around the caudal neuropore (Ruberte et al 1991). In day mouse embryos RAR ß is expressed in the

6 318 Noriko Osumi-Yamashita mesenchyme surrounding the olfactory epithelium, from which RAR γ message is absent. RAR γ is expressed in the condensed mesenchyme which will form the nasal septum and capsule (Osumi-Yamashita et al 1990). These specific expression patterns in developing embryos lead us to expect that RAR ß and γ may have specific developmental and morphogenetic functions. RXRs (RXR α, ß, and γ) show less specific patterns of expression than RAR subtypes (Mangelsdorf et al 1992). Mouse RXR γ expression, for example, is high in the skeletal and tongue muscle, pituitary, and in parts of the brain, while RXR α expression is high in the skin and small intestine, and RXR ß shows an ubiquitous expression pattern. The chick homologue of RXR γ has been shown to be expressed in the developing peripheral nervous system derived from the neural crest (Rowe et al 1991). RXR γ and RAR ß are co-expressed in spinal motor neurons, and RXRα and RAR γ are both abundantly expressed in the epidermis, suggesting a selective co-expression of these two receptor systems. In order to analyse molecular mechanisms involved in RA signaling pathways, many of retinoid receptor genes have been targeted so far by several laboratories. Despite the expectation from their expression patterns, mice carrying the disrupted RAR α (Li et al 1993; Lufkin et al 1993), ß2 (Mendelsohn et al 1994), or γ 2 (Lohhnes et al 1993) did not show any severe malformations, leaving the possibility of considerable functional redundancy among members of the RAR family. A series of "double knockout" mice were subsequently generated and they showed various craniofacial (mainly eye and heart malformations) and skeletal abnormalities (Lohhnes et al 1993; Mendelsohn et al 1994). Especially, the phenotypes of RAR α/7 double-mutants presented some striking similarities with human congenital disease recognized as neurocristophathy in which abnormal behaviour of cranial crest cells are thought to be involved (Siebert et al 1985). Moreover, when RXR α is targeted alone or together with RAR γ, myocardial and/or ocular malformations were seen again (Sucov et al 1994; Kastner et al 1994). Both of these malformations belong to the vitamin A deficient syndrome, providing the evidence of specific requirement for RXR in RA signaling. RAR α1/rxr cc and RAR α 1/RAR ß double mutants also result in cardiac defects as persistent truncus arteriosus and aortic arch abnormalities as well as other defects in the eye, thymus, skeletal bones etc. (Henry Sucov, personal communication; Luo et al 1996), reminiscent of human Di George syndrome (Huber et al 1967). Now it is clear that functions of retinoid receptors are not always seen in regions showing high expression level of the receptors. For example, RXR α isruption (Sucov et al 1994, 1995; Kastner et al 1994) causes cardiac malformations, although specific expression of the gene is not detected in the heart primordia. RAR ß (Luo et al 1995) or RAR ß2 (Mendelsohn et al 1994) null mutants conversely give no phenotypes, despite their predominant expression in the highly restricted regions such as the lateral nasal prominen ces (figure 2). The same may also be true for RAR γ Lohhnes et al 1993). It is-therefore not always easy to interpret these contradictory results, e.g., functional redundancy, background levels of gene expression, and other co-regulating factors to select specific organs where the gene product should be functional, would possibly be involved. 4. Cellular retinoid binding proteins The maintenance of physiological levels of RA is crucial for normal embryonic development. It is generally accepted that the retinoid binding proteins perform such

7 RA and mammalian craniofacial morphogenesis 319 functions by controlling the quantitative availability of RA to the nuclear receptors. There are four closely related retinoid binding proteins in embryos: two cytoplasmic retinol binding proteins (CRBP I and II), and two cytoplasmic RA binding proteins (CRABP I and II). [For detailed biochemical information, see Ong et al (1994)]. These proteins also exhibit unique spatiotemporal patterns of expression during morphogenesis; suggesting different roles of these molecules. High levels of CRBP I expression, for example, is seen in the developing eye and heart (Dolle et al 1990); both tissues are vulnerable for vitamin A deficiency. It is thus speculated that nuclear RA levels are highest in cells expressing CRBP I where RA is thought to be synthesized. In fact, there is an evidence that RA is synthesized in mouse Hensen's node (Hogan et al 1992), where transcripts of CRBP I are specifically observed (Ruberte et al 1991). Interestingly, in the craniofacial region, CRBP I protein is distributed in the endoderm and surface ectoderm but not in the mesenchyme, while CRABP I protein distribution is observed in the mesenchyme but not in the epithelium (Gustafson et al 1993). Such complimentary fashion of CRBP I and CRABP I distribution patterns is also encountered by the developing limb (Gustafson et al 1993). These data suggest involvement of retinoid metabolism in epithelial-mesenchymal interactions during pattern formation of these structures. Transcripts and proteins of CRABP I are also observed in the early hindbrain and in the cranial neural crest (Ruberte et al 1991, 1992; Maden et al 1992). Since admistrated RA is accumulated in embryonic cells expressing CRABP I (Denker et al 1987, 1990, 1991), these cells have been proposed to require high levels of RA for normal development. In other words, CRABPs may function to shuttle RA to the nucleus. Alternatively, because the structures exhibiting high expression levels of CRABP genes are vulnerable for RA excess, CRABPs would rather capture RA and thereby reduce the effective concentration of RA in cells that express the gene. Expression of CRABP II overlaps with that of CRABP I and RAR ß to some extent, but is also excluded from the domain where CRABP I is expressed, e.g., different expression patterns of CRABP I and II genes observed in rhombomeres (Ruberte et al 1992). Recently, CRABP II null mutant mice have been generated showing minor developmental defects as an additional postaxial digit in the forelimb, but abnormalities in other organs are not reported (Fawcett et al 1995). Therefore, CRABPs may have little function in embryrogenesis. 5. Effects of RA on craniofacial development Exogenous RA induces various types of malformations in craniofacial structures according to different treatments (adminstration procedures, doses and developmental stages at the exposure). Most recently, Simeone et al (1995) performed detailed time-course analyses of the effect of maternal RA treatments on the development of mouse CNS from the beginning of gastrulation throughout induction and patterning of the neural tube. The RA treatments on 6. 5 day or earlier have little or no effects on morphology (Simeone et al 1995). At later stages, RA induces dramatic changes in the anterior-posterior patterning of the craniofacial region. When administrated on days, RA produces atelencephalic microcephaly with the loss of anterior sense organs, and consequently causes posteriorization (Simeone et al 1995). Similar posteriorized phenotypes are also reported in other studies (Conlon and Rossant 1992; Marshall et al 1992). In particular, Marshall et al (1992) described that the identity of

8 320 Noriko Osumi-Yamashita the trigeminal nerve is changed into that of the facial nerve in RA-treated mouse embryos at 7. 5 day from the observation of patterns of motor neuron distribution in rhombomeres. It was interpreted that RA induced "posterior transformation" on the hindbrain identity. Similar phenotypes were also induced in rat embryos. Lee et al (1995) treated 9. 0 day (7. 5 day in mouse) and 9. 5 day rat embryos in vitro with all-trans-ra for 6 h and further cultured up to the stage corresponding to 11 5 day in vivo embryos. The former treatment induced posteriorization in the CNS and pharyngeal arches, while the latter treatment resulted in fusion of the first and second arches as well as that of the trigeminal and acousticofacial ganglia (figure 3). Such fused arches are also reported in cultured mouse embryos treated with all-trans or 13-cis RA (Goulding and Pratt 1986; Webster et al 1986; Brown et al 1992) as well as in those treated in utero (Leonard et al 1995). Interestingly, DiI labelling study revealed that hindbrain crest cells derived from prorha and prorhb do not mix within the fused arch. It supports the idea that differential identities of the crest cell populations are still maintained (figure 3C; Lee et al 1995). In contrast, crest cells from prorha ectopically migrate into the second arch in cultured rat embryos treated at 9. 0 day, suggesting that RA affected not only the identity of the hindbrain neuroectoderm but also that of the crest cells derived from the prorhombomeres (figure 3B; Lee et al 1995). Another effect of RA is observed in segmentation of the head region. Two forms of segmentation are seen in vertebrate embryos: (i) mesodermal somites in the trunk and occipital region of the head and (ii) rhombomeric divisions in the hindbrain with Figure 3. Schematic illustration of migration patterns of rat hindbrain crest cells in normal development (A) and in embryos treated with RA at the early (B) and late (C) neural plate stage. Neural crest cells from the midbrain/prorha are shown as open dots and those from prlrhb as closed dots. RA stage-dependently alters the identity of hindbrain crest cells from a standpoint of cell lineage. (A) In control embryos, hindbrain crest cells are segmentally distributed; i.e., crest cells from the midbrain/prorha migrate to the first branchial arch (a1), including the maxillary and mandibullar prominences, and the trigeminal ganglion; and those from prorhb migrate to the second branchial arch (a2) and acousticofacial ganglion. (B) In the early-stage treated embryos, the migration patterns of the hindbrain crest cells are changed since some of the midbrain/prorha crest cells alter their migration pathway posteriorly to the second branchial arch and acousticofacial ganglion. (C) In late-stage treated embryos, even though the FBA and fusion of trigeminal and acousticofacial ganglia are present, the segmental migration pattern of neural crest cells remains the same as in the control embryos, namely, the labelled cells from the midbrain/prorha migrate to the anterior part of the FBA including the trigeminal ganglion, while those from prorhb migrate to the posterior part including the acousticofacial ganglion.

9 RA and mammalian craniofacial morphogenesis 321 associated metamerism of the adjacent pharyngeal arches. High levels of RA given at day 8 0 suppress rhombomeric segmentation in the mouse (Morriss-Kay et al 1991; Wood et al 1994). Therefore, localized control of RA levels appears to be essential for segmentation of the hind brain. RA has apparently a reverse effect on the cranial mesoderm. In normal development, the cranial mesoderm is never epithelialized and only incompletely segmented as somitomeres (Meier and Tam 1982). When embryos are treated with RA, somite-like structures form in the cranial mesoderm anterior to the native somites (Morriss-Kay et al 1991; Sundin et al 1993). This ectopic (anteriorly shifted) somitogenesis can be viewed as RA posteriorized the cranial paraxial mesoderm that would normally develop into somitomeres. At the later stages when migration of cranial crest cells ceases RA treatment induces another type of the craniofacial malformation. Admistration of RA of day 9 5 mouse embryos results in craniofacial abnormalities such as small jaws, cleft palate, and abnormal external ears, which are similar to mandibulofacial dysostosis (Treacher- Collins syndrome) in human (Osumi-Yamashita et al 1992). These deflects were attributed to the region specific cell death in the first pharyngeal arch, dorsal aspects of the maxillary and mandibular prominences. From this teratological study it can be drawn that there are domains involved in the formation of specific head structures and have different sensitivities for excess RA. 6. RA and developmental control genes Exogenous RA affects expression of its own nuclear receptors and cellular binding proteins in different ways. Exression of CRABP II that has two RAREs is actually activated in the presence of RA (Durand et al 1992; Harnish et al 1992). CRABP I is also anteriorly upregulated in the mouse CNS in response to exogenous RA (Leonard et al 1995). However, no clear correlation was observed between the presence of CRABP I and II and the sites of teratogenesis (Horton and Maden 1995). RAR ß is induced ectopically in the mouse embryo treated with RA, while expression patterns of RAR α and γ genes are not substantially changed (Osumi-Yamashita et al 1992). Such a rapid response of RAR ß is also observed at the protein level in cultured mesenchymal cells derived from the mouse facial primordia (Motoyama et al 1994). Mice carrying reporter constructs (RARE with/without RAR ß promoter followed by lacz gene) also show an anteriorly expanded expression domain of the reporter gene after exposure to RA in utero (Rossant et al 1991; Balkan et al 1992; Mendelsohn et al 1994). Interestingly, Zimmer and Zimmer (1992) observed that RAR ß2-lacZ transgene was induced segmentally (like a pair-rule genes in Drosophila.) in the anterior CNS by maternal admistration of RA at day to This may imply that anterior CNS is segmented in such a way that reflects the different induction pattern of RAR b2-lacz. Yet ' it is not clear so far whether such ectopic induction of RAR ß is really involved in RA-induced teratogenesis; RAR ß has RARE on its own promoter and can be autoregulated (de The et al 1990; Sucov et al 1990). Again, it must be noticed that activation of RAR ß promoter is apparently stage-specific (Rossant et al 1991; Conlon and Rossant 1992; Zimmer and Zimmer 1992). This can reflect altered expression of other genes in a time-dependent manner. Transcription of many developmental genes are known to be responsive to RA. Of particular interest is the clustered Hox genes because those towards the 3' side of the

10 322 Noriko Osumi-Yamashita cluster are expressed earlier in development with more anterior boundaries of expression along the body axis than those at the 5' end (McGinnis and Krumlauf 1992). It is also reported that RA induces both Hoxb-1 and Hoxa-1 (3' side) genes through the RARE elements (Marshall et al 1994; Studer et al 1994). Moreover, 3' genes respond rapidly to RA, while sequential activation of 5' genes involves time related factors (Simeone et al 1990, 1991; Papalopulu et al 1991; Arcioni et al 1992). These evidences altogether explain the developmental mechanism that the initial gradient of RA in the body axis influences on expression of clustered Hox genes, by which identities of individual segments may be established. Hogan et al (1992) have put forth the possibility that such anteroposterior patterning occurs during and soon after gastrulation based on their findings that RA is probably synthesized in the mouse node at a certain point of gastrulation onwards, which is consistent with the level at which RAR ß starts to be expressed in the body axis, Obviously, differential activation of clustered Hox genes also implies the mechanism of the posteriorization caused by excess RA: maternal RA treatments of mouse embryos at the neural plate stage shift the expression domains of Hox genes anteriorly, giving the more posterior identity to the domain of expression than in the normal state (Morriss-Kay et al 1991; Conlon and Rossant 1992; Marshall et al 1992; Simeone et al 1995). On the contrary, expression of the fore- and midbrain specific genes such as Otx2, Otx1, Emx2, Emx1,and Dix1 are generally repressed by RA treatment (Simeone et al 1995), consistent with the RA-induced rostral truncation. In turn, this may be interpreted as follows: such rostral CNS-specific genes require low levels or RA for their normal expression. This is exactly what is observed in the mouse embryo since endogenous RA is present at very low levels in the developing forebrain and at high levels in the spinal cord (Horton and Maden 1995). Another gene that may be involved in RA signaling pathway during the morphogenesis is COUP-TFs (chicken ovalubmin upstream promoter-transcription factors) belonging to the nuclear receptor superfamily, though their ligand is not yet known. Cell culture studies have shown that COUP-TF I is a potent inhibitor of transcriptional activation by RARs, RXRs, the vitamin D receptor, and the thyroid hormone receptor (Kliewer et al 1992c; Cooney et al 1992). Notably, COUP-TF I is expressed in distinct areas within the anterior CNS during mouse development (Qiu et al 1994). The recent report using Xenopus embryo explants further suggests that COUP-TF I is a potent inhibitor of RA-induced gene expression (Schuh and Kimelman 1992). These findings well support the idea that COUP-TF I plays an active role in the patterning of anterior neural tissue in vertebrate embryos, possibly by regulating cellular responses to retinoids. 7. Perspectives Developmental roles of the retinoid pathway in craniofacial development is still largely in vague. Combination of molecular, cellular and morphogenetic approaches will be required further to provide insights into its fundamental mechanisms. Large portion of the craniofacial structures are derivatives of the pharyngeal arch system. However, little is known about how each pharyngeal arch skeleton is patterned during development. Noden (1983) raised, from the transplantation experiments using chick and quail embryos, the possibility that each region of cranial crest is precommitted, i.e., the crest cells carry the morphological identities before their emigration. Genetic disruption of

11 RA and mammalian craniofacial morphogenesis 323 Hoxa-2 gene resulted in a mirror-image duplication of the first pharyngeal arch skeleton within the second arch (Rijli et al 1993), providing possible molecular evidence for Noden's hypothesis. Moreover, reciprocal interactions between the epithelium and mesenchyme can be involved in formation of craniofacial structures (Thorogood 1988; Wedden et al 1988). Yet, we do not know whether there are any so called "organizerlike" areas, such as ZPA or AER in the limb bud, in the pharyngeal region. Detailed analyses of expression of possibly candidate molecules, e.g., sonic hedgehog or goosecoid genes would provide useful information to elucidate this important issue. Acknowledgements The author gratefully acknowledge Dr Kazuhiro Eto for his continuous support and encouragement and Dr Youichirou Ninomiya for his excellent technical assistance. Sincere gratitude is extended to Dr Sumihare Noji, Dr Gillian Morriss-Kay for discussions, Dr Henry Sucov for helpful comments and providing his unpublished results, and to Dr Shigeru Kuratani for suggestions and critical reading of the manuscript. This work was partly supported by a Grant-in Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (No , ). References Arcioni L, Simeone A, Guazzii S, Zappavigna V, Boncinelli E and Mavilio F 1992 The upstream region of the human homeobox gene HOX3D is a target for regulation by retinoic acid and HOX homeoproteins; EMBO J Adelman H B 1925 The development of the neural folds and cranial ganglia of the rat; J. Comp. Neural Balkan W, Colbert M, Brock C and Linney E 1992 Transgenic indicator mice for studying activated retinoic acid receptors during development; Proc. Natil. Acad. Sci. USA Bartelmez G W 1923 The subdivisions of the neural folds in man; J. Comp. Neural Bartelmez G W and Evans H M 1925 Development of the human embryo during the period of somite formation, including embryos with 2 to 16 pairs of somites; Contrib. Embryol Brown N A, Hunt P and Krumlauf R 1992 Craniofacial developmental abnormalities induced by triazoles a comparison of homeobox gene expression and stage-specificity with retinoic acid-induced defects; Teratology Cohlan S Q 1953 Excessive intake of vitamin A as a cause of congenital anomalies in the rat; Science Conlon R A and Rossant J 1992 Exogenous retinoic acid rapidly induces anterior ectopic expression of murin Hox-2 genes in vivo; Development Cooney A J, Tsai S Y, O'Malley B W and Tsai M J 1992 Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP- TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors; Mol. Cell. Biol Dencker L, Annerwell E, Busch C and Eriksson U 1990 Localization of specific retinoid-binding sites and expression of cellular retinoic acid-binding protein (CRABP) in the early mouse embryo; Development Dencker L, D'Argy R, Danielsson B R G, Ghantous H and Sperber G Saturable accumulation of retinoic acid in neural and neural crest derived cells in early embryonic development; Dec. Pharmacol. Ther Dencker L, Gustafson A L, Annerwell E, Bush C and Eriksson U 1991 Retinoid-binding proteins in craniofacial development; J. Craniof. Genet. Dev. Biol

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