structural and gene expression abnormalities induced by retinoic acid in the forebrain

ELSEVIER

Reproductive Toxicology, Vol. 12, No. 2, pp. 169-176, 1998 0 1998 Elsevier Science Inc.

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PI1 SO890-6238(97)00151-2

STRUCTURAL AND GENE EXPRESSION ABNORMALITIES INDUCED BY RETINOIC ACID IN THE FOREBRAIN

FREDERIC CLOTMAN, GENEVIEVE VAN MAELE-FABRY, LIU CHU-WU,

and JACQUES J. PICARD Laboratory of Developmental Genetics, Catholic University of Louvain, Louvain-la-Neuve, Belgium

INTRODUCTION

Retinoids exert essential functions during embryonic

development (reviewed in Reference 1) and have been shown for a long lime to be potent teratogens in laboratory animals (reviewed in Reference 2). However, these molecules are particularly effective in the treatment

of various dermatologic disorders (3,4). Therefore, 13- cis-retinoic acid (isotretinoin, 13cRA) was marketed in 1982. Unfortunately, a characteristic pattern of congenital malformations was rapidly identified in infants who had been exposed to 13cRA during the first trimester of pregnancy (5-8). These malformations affected mainly the central nervous system (CNS), the craniofacial structures, the heart, and the thymus (reviewed in Reference 7,9). The teratogenic activity of 13cRA is currently thought to be mainly mediated by all-truns-RA (AT- RA), a stereoisomer produced by spontaneous isomer-

ization of 13cRA. Indeed, AT-RA is transferred prefer-

entially from the mother to the embryo when compared to 13cRA (10-13). In addition, 13cRA induces the same pattern of congenital malformations as AT-RA but re- quires higher concentrations to affect development to the same extent (14). Despite intense research in this field, the molecular and cellular mechanisms that cause the

observed malformations are still unknown. In addition to several more caudal structures, the

forebrain, the most rostral part of the CNS, constitutes one of the main targets of the retinoids. The forebrain neuroep- itbelium can be morphologically and functionally affected by an excess of retinoids. In addition, the eyes and other

derivatives of the forebrain neuroepithelium are affected frequently (Table 1). The aim of this review is to focus on the structural malformations and changes in developmental

Presented as part of Ihe Vth International Symposium on Verte- brate Whole Embryo Cultllre: Clinical and Genetic Implications, held in Jerusalem, Israel, l-4 April, 1997.

Address correspondence to Jacques J. Picard, Laboratory of Developmental Genetics, Place Croix du Sud 5 Bte 3, 1348 Louvain- la-Neuve, Belgium. E-mail: [email protected]

Present address of Liu Chu-Wu: Department of Aquaculture, Zhanjiang Ocean University, Zhanjiang, P.R. China.

169

gene expression induced by an excess of AT-RA in the

forebrain of mouse embryos. As a first step, we will review

the malformations of rostral structures observed in infants

and laboratory animals exposed prenatally to retinoids. As a

second step, we will detail the modifications induced by

AT-FM in the expression of forebrain developmental genes.

Finally, we will discuss models proposed to explain these

modifications and the induced morphologic abnormalities

to shed light on the mechanisms involved in the induction

of forebrain abnormalities.

STRUCTURAL ABNORMALITIES INDUCED BY RETINOIDS IN THE FOREBRAIN AND ITS

DERIVATIVES

Human teratogenicity

The abnormalities detected after prenatal exposure to

retinoids in forebrain-related structures of human embryos

(2,5,7,8) are listed in Table 1. The CNS malformations

included ventriculomegaly and hydrocephaly, microcephaly, cortical blindness, and structural malformations of the

cerebral cortex. Follow-up of the children revealed abnor-

mal neurologic signs and functional deficits of the visual

system. In addition, at 5 years, 52% of the children had an

intellectual deficit, and among the latter, 38% had no major

malformations (7). The eyes, which partly arise from the

forebrain, could also be morphologically disturbed (mi-

crophtalmia). Other craniofacial structures were affected

similarly by retinoid exposure. Facial asymmetry, malde-

velopment of cranial bones, maxillary hypoplasia, and cleft

palate were reported. The latter affected structures arise

mainly from the development of neural crest cells (NCCs)

that originate from the neuroepithelium. Forebrain and

midbrain NCCs give rise to the frontonasal (upper midface)

and first arch (maxillomandibular) mesenchyme. The frontonasal and visceral arch mesenchyme subsequently form

most of the facial and anterior neck skeletal and connective

tissues. Therefore, structural abnormalities of many craniofacial structures can be attributed to defects in forebrain

NCC development.

170 Reproductive Toxicology Volume 12, Number 2, 1998

Table 1. Abnormalities of the forebrain derivatives induced by human exposure to 13cRA and laboratory animal

exposure to AT-RA

Human exposure to 13cRA Rodent exposure to retinoids

0 Microcephaly l Ventriculomegaly 0 Hydrocephaly

aqueductal stenosis communicating fourth

ventricle cyst Hydranencephaly

0 Cortical blindness 0 Cortex malformations

Cortical agenesis Focal agyria Heterotopias Calcifications

0 Eye defects: Microphthalmia

0 Facial assymetry 0 Hypertelorism 0 Maldevelopment of the

midfacial bones

0 Anencephaly 0 Exencephaly l Encephalocoele 0 Holoprosencephaly 0 Microcephaly

0 Hydrocephaly

0 Eye defects: Microphthalmia Exophthalmia Coloboma

0 Cranial abnormalities Cleft palate Cleft lip Maxillary hypoplasia

From References 2,5,7,8,41,53

LABORATORY ANIMAL TERATOGENICITY

In rodent embryos, retinoids induce the same pattern of abnormalities as in the human species (Table 1). Anencephaly, exencephaly and encephalocoele, holoprosencephaly, microcephaly, hydrocephaly, eye defects, and craniofacial abnormalities were observed after in vivo or in vitro exposure to retinoids (recently reviewed in Reference 2). Abnormalities observed in rodents but

not reported in humans correspond probably to early defects that are too severe to allow survival. Rodents exposed prenatally to retinoids also presented behav- ioural dysfunctions (15). Therefore, the pattern of rodent

abnormalities matches the human syndrome remarkably. Recently, exposure of other laboratory animals has

provided new information regarding the mechanisms of action of retinoids. In the ascidian Halocynthia roretzi,

exposure of the embryos to AT-RA induced a larval phenotype with elimination of the rostra1 structures, the papillae (16). The amphioxus larvae failed to form the mouth, the ciliated pit, and/or the gill slits, all rostra1 structures, after exposure to the same drug (17). In Xenopus, 1 PM AT-RA inhibited the formation of the cement gland, an organ that forms from the rostral-most ectoderm (18), whereas 10 PM AT-RA completely inhibited the development of the head (19,20). Finally, implantation of a bead soaked with RA in the anterior side of a chick embryo induced a reduction of the

forebrain (21). Taken together, these data show that retinoids can inhibit the development of rostra1 structures in many different species. The underlying mechanisms

are not well understood and may correspond to inhibition of the development of rostra1 structures or to a repatterning of these rostra1 structures to more caudal ones. The unilateral microinjection of a droplet of RA in the presumptive head of Xenopus embryos showed that the drug inhibits the differentiation of rostra1 structures instead of rostrally repatterning the structures according to the rostrocaudal axis (22). This finding suggests that retinoids may act by inhibiting the development of the

rostra1 portion of the embryo. Other mechanisms related to retinoid teratogenicity

have been investigated. Several studies demonstrated that excessive cell death in regions of programmed cell

death could be involved in the development of craniofacial malformations (23-25), although this hypothesis remains controversial (26). Frontonasal defects and me- dian and lateral cleft lips observed after retinoid administration have been attributed to decreases in the proliferation of neural crest-derived mesenchyme cells located in the frontonasal region, which subsequently may also result in clefts of the primary palate (27,28). Retinol, AT-RA, and 13cRA can affect the survival and/or proliferation of the cranial NCCs (29-32) and inhibit their migration (29,30,33-35). The latter effect could be

mediated by modifications of the components of the extracellular matrix (35,36). These data confirm that a significant part of the malformations induced by retinoids could arise from perturbations in the development or migratory ability of the NCCs.

MODIFICATIONS INDUCED BY RETINOIDS IN DEVELOPMENTAL GENE EXPRESSION

Modifications induced by RA in the expression level or in the expression pattern of forebrain-expressed developmental genes have been investigated. We will

review the modifications detected in the expression domains of Otx2 and several other genes expressed in this part of the CNS. We will show that a model proposed recently cannot explain all the observed modifications. We will summarize some observations we made recently after the exposure of early somite embryos to AT-RA, propose an alternative model to explain the modifications in gene expression, and correlate these modifications with the structural abnormalities reviewed above.

AN EXCESS OF AT-RA AFFECTS THE EXPRESSION OF 0TX2

The 0tx2 gene has been studied because it is essential for the early development of the head. 0tx2 is a

AT-RA-induced forebrain abnormalities l F. CLOTMAN ET AL. 171

\ II “Deletion”

model

Fig. 1. Two hypothetical models proposed to explain the alterations induced by AT-RA in the forebrain. In each case, a schematic representation of the CNS is displayed on the left, and the rostro-caudal extension of the expression domains of rostra1 CNS-expressed developmental genes are represented as lines on the right (adapted from Reference 44). A, repatteming model proposed by Avantaggiato et al. (44). AT-RA induces an ordered repatteming of the whole CNS, resulting in the shortening of the forebrain and the enlargement of the hindbrain. Subsequently, the expression of Dlxl, Emxl, and Emx2 is lost. The arrows indicate the direction and the extent of the repatteming. B, expression domains of rostra1 CNS-expressed developmental genes in control embryos. C, deletion model proposed as an alternative to the repatteming model. AT-RA induces a deletion of the most rostra1 part of the CNS, resulting in a shortening of the forebrain and the loss of the Dlxl, Eml, and Emx2 expression domains. The arrow indicates the extend of the deletion. In the hindbrain, which is not enlarged, AT-RA induces a caudal repatteming indicated by the rostra1 shifts in the expression domains of Wnt-I, Hoxb-I, and in the caudal domain of Pux-2. The data are from References 41 and 44 (embryos exposed at E7.4), and from Reference 52 (embryos exposed at E8.2). for., forebrain; mid., midbrain; hind., hindbrain.

homeobox-containing gene homologous to the orthoden-

title gene of Drosophila. In the mouse, it is expressed in the three germ layers from gastrulation onwards. Subse- quently, expression is restricted progressively to limited regions of the brain (37). Inactivation of Otx2 results in defects during gastrulation and in the formation of the axial mesoderm (38). Those abnormalities impair the development of the fore- and midbrain (38-40). Thus, the expression of Otx.! is required for the normal devel-

opment of the rostra1 part of the CNS. AT-RA can repress the expression level of 0tx2 in

embryonal carcinoma cells (41). In addition, exogenous AT-RA modifies the expression pattern of Otx2. Expo-

sure of Xenopus embryos to 1 PM AT-RA abolished the expression of the 01~2 homologous gene (18,20). In chick embryos cultumd in vitro, 5 PM AT-RA had no effect on cOtx2 expression. Increasing concentrations led to a gradual repression of the expression in progressively more numerous cellular compartments. The expression was abolished in the ectoderm and lateral plate mesoderm after exposure -to 15 PM. In addition, expression was repressed in the prechordal and chordal mesend-

oderm after treatment with 30 or 45 PM AT-RA. At all concentrations, the expression was retained in the neuroepithelium. However, exposure to 30 or 45 PM AT-RA induced a shortening of the 0tx2 expression domain according to the rostrocaudal axis of the embryo (Figure 1; Reference 42). Similar observations were

made after maternal administration of AT-RA in the

mouse. Embryos exposed between Embryonic Day 7 (E7) and E7.6 showed a repression of 0tx2 expression in the rostra1 embryonic mesoderm, prechordal plate, ven- tral mesoderm, endoderm adjacent to the prechordal plate, and in the foregut pocket (41). The expression was retained in the neuroectoderm, but the 0tx2 expression domain was shortened according to the rostrocaudal axis

(Figure 1; References 41,43,44). Similar results were obtained in our laboratory. E7

mouse embryos were exposed to AT-RA for 12 h at the EB (early allantoic bud) and LB (late allantoic bud) stage. Whole mount in situ hybridization (ISH) was

carried out with digoxigenin-labeled 0tx2 antisense RNA probe, and ISH on histologic sections was performed with a 35S-labeled 0tx2 antisense RNA probe. After whole mount ISH, AT-RA at a concentration of 1 I_LM had no effect on the morphology of both EB- and LB-stage embryos nor on the expression pattern of 0tx2

(Table 2). When the embryos were treated with 4 PM AT-RA at both stages, the formation of the foregut was inhibited or reduced, the headfolds of some treated embryos were less prominent than in the control embryos, and the neural groove appeared curved (Table 2; Figure 2). The Otx2 expression domain in the EB-stage embryos treated with 4 PM AT-RA was shortened, and the signal was very faint in 29% of the embryos (Figure 2). No change in the pattern of 0tx2 expression was seen


Table 2. Development and expression of Otx2 in embryos explanted at presomitic stages and cultured for 12 h with AT-RA

Stage at explant

Treat. (PM)

No. of embryos

Stage after 12 h culture dysmorphogenesis

1-3 4-6 Neural LHF somite somite Foregut Headfold groove

EB 0 50 17 (34) 31 (62) 0 0 0 0 1 23 8 (35) 15 (65) 0 0 0 0 4 44 3 (7) 35 (79) 0 32 (73) 20 (45) 21 (48%)

LB 0 52 4 (8) 3 (6) 43 (83) 0 0 0 1 20 0 4 (20) 16 (80) 0 0 0 4 48 0 2 (4) 45 (94) 25 (52) 10 (20) 8 (17%)

Treat; AT-RA treatment (concentration); LHF, late headfold stage; EB, early allantoic bud stage; LB, late allantoic bud stage. Numbers in parentheses: percentages

in the LB-stage embryos, but in a few of these embryos (14%) its expression became faint (not shown). Similar results were obtained after ISH on histologic sections performed on 12 EB-stage embryos exposed for 12 h to 4 PM AT-RA. In the control embryos, the expression of 0tx2 extended over the whole cranial region of the neuroepithelium from a sharp caudal border to the cranial tip of the neural plate. The expression domain of Otx2 was shorter in treated embryos than in the control embryos (Figure 3). In addition, the intensity of the

labeling was much lower, as observed in sections hybrid- ized simultaneously under the same conditions with the

OED NG OED NG

Fig. 2. Modifications in the expression domain of 0tx2 in EB-stage embryos exposed for 12 h to 4 PM AT-RA. Whole- mount ISH for Otx2. The extra-embryonic membranes have been removed, and the embryos are viewed dorsally. In the control embryos (A), the signal was detected in the rostra1 part of the CNS. In treated embryos (B), the expression was restricted to the most rostra1 tip of the CNS and was very faint. C and D are schematic representations of the Otx2 expression in A and B, respectively. OED, Otx2 expression domain; NC, neural groove. Scale bar is 100 pm.

same probe and with the emulsion exposed for the same time (Figure 3). To evaluate the reduction of the Otx2 labeling in treated embryos, we used an estimation of the mean gray level of a defined area as an indicator of the ISH labeling intensity (Vidas Rel. 2.1 software, Kontron Elektronic GMBH). The mean intensity of 0tx2 expression estimated in 12 control embryos was 3.9, whereas the mean intensity in 12 treated embryos was 2.2.

Taken together, these observations suggest that an excess of AT-RA can repress and/or displace the expres-

sion of Otx2. The sensitivity of Otx2 to AT-RA seems to be very variable between species, as 1 PM is sufficient to modify Otx2 expression in Xenopus, whereas 15 FM is required in chick embryos. The 0tx2 expression level could be repressed by an excess of AT-RA, both in cultured cells and in whole embryo, through the activity of &-acting element(s) (likely retinoic acid response elements or RAREs) known to be located directly up-

Fig. 3. Expression of 0tx2 in 12 h to 4 PM AT-RA analyzed by ISH on sections. Sagittal sections in a control (A) and a treated (B) embryo. The expression of 0~x2 was shortened after AT-RA exposure (arrows). In addition, the intensity of the labeling was weaker in the treated embryos, although the sections were processed in the same conditions. a, amnion; h, anlage of the heart; nf, neural folds; np, neural plate; pf, pocket of the foregut; v, vitelline vesicle. Scale bar is 100 pm.

AT&%-induced forebrain abnormalities 0 F. CLOTMAN ET AL. 173

stream from the 0tx2 coding sequence (41). The spatial regulation of 0tx2 expression was also disturbed. How-

ever, the interpretatiasn of these observations is difficult

because treated embryos presented important morphologic abnormalities. The Xenopus embryos displayed deletions of rostra1 br,ain regions and of the cement gland

where Otx2 is normally expressed. In chick embryos, RA induced deficiencies of rostra1 neural structures, including the loss of the forebrain. The mouse embryos presented extended morphologic alterations of the brain. In particular, embryos exposed between E7 and E7.6 displayed an “atelenl:ephalic microcephaly” phenotype characterized by a partial to complete absence of the rostra1 structures of the body, including the brain, where

0tx2 is normally expressed (41). Thus, both the expression of Otx2 and the tissues where the gene is normally expressed are affecte.d by RA exposure, preventing the determination of what is the cause and what is the

consequence among these abnormalities.

AT-RA MODIFIES THE EXPRESSION PATTERN OF OTHER BRAIN-SPECIFIC

DEVELlDPMENTAL GENES

To correctly interpret the modifications induced by

RA in the expression pattern of Otx2, they were compared with modifications in the domains of other genes expressed in the fore-, mid-, and hindbrain (Figure 1).

The expression domain of Otxl, which is almost identi- cal to that of Otx2, was also shortened after exposure to AT-RA. Emx2, Emx.1, and Dlxl are normally expressed

in restricted regions of the forebrain. After exposure to the drug, expression of these genes was lost or detected in domains smaller a:nd located closer to the rostra1 tip of the CNS than in the control embryos (41). The rostra1 expression border of Wnt-1, which is normally located in the midbrain, was closer to or matched the rostra1 tip of the CNS. En-2, which is normally expressed around the hindbrain/midbrain border, was missing or had its expression domain close to the rostra1 tip of the CNS. The

expression boundaries of En-l and of the caudal expression domain of Pax-2 were also shifted rostrally com-

pared to the control embryos (44). Finally, the rostra1

expression border of Hoxb-1 was comparable to the caudal border of the 0tx2 domain. In control embryos, a gap normally separates these two expression domains. This gap was abohshed in embryos exposed to RA (Figure 1; Reference 41).

A FIRST MODEL: ORDERED REPATTERNING OF THE ROSTRAL CNS

RA is likely to be directly involved in the establish- ment or maintenance of the expression of some brain- specific developmental genes (45,46). However, because

its direct involvement in the regulation of the genes affected by an excess of AT-RA has not been demon-

strated, the observed modifications led Avantaggiato et

al. (44) to propose an indirect explanatory model that we will call the “repatteming” model (Figure 1A). Accord- ing to this model, AT-RA administration between E7 and

E7.6 to the pregnant mouse induces a coordinate anteriorization of the expression domains concomitant with an

ordered repatteming of the rostral CNS. RA may inhibit the expression of rostral markers and allow the abnormal expression of caudal markers in rostra1 locations. It may thereby partly or completely transform the identity of forebrain into midbrain and midbrain into hindbrain. This would result in an enlargement of the hindbrain and a partial or complete loss of the forebrain.

This model is in good agreement with the modifications observed in the expression domains of most of the studied genes (Figure 1A). However, some charac-

teristics of the model do not match the experimental data. First, an ordered repatteming of the CNS implies that the

relative location of the expression borders of different genes are conserved. This is not the case for Hoxb-I,

because its rostra1 expression border was displaced relative to the expression domains of Otx, En, or Wnt-1

genes. In particular, the gap between the expression domains of Hoxb-1 and 0tx2 disappeared (Figure 1A).

Hoxb-1 has been reported previously to be ectopically expressed after RA exposure (References 47-51; F. Clotman, 1997, unpublished data). Its expression border

was displaced from the boundary between rhombomeres 3 and 4 toward a more rostra1 position located in the rostra1 hindbrain. The loss of the gap between Hoxb-I

and Otx2 suggests that RA can induce ectopic expression of Hoxb-I without affecting the location of the caudal expression border of Otx2. This finding would imply that the rostra1 part of the hinbrain is repattemed, whereas the caudal part of the midbrain is not. Therefore, RA could disturb differentially the different parts of the brain. Secondly, these perturbations are also different according to the developmental stage of the exposed embryos.

According to the repatteming model, exposure between E7 and E7.6 repattems the CNS. Later exposure did not repattem the fore- and midbrain (41). However, rhom-

bomere transformation and caudal repatteming in the hindbrain are well documented at this developmental stage (References 47-51; F. Clotman, 1997, unpublished data). Therefore, the mechanisms involved in the repatteming of the hindbrain and of the fore- and midbrain could be different. Alternatively, the fore- and midbrain could loose the competence to be repattemed after E7.6, whereas the hindbrain would keep it. This shows that the repatteming model is not consistent for the whole CNS at all stages of development. Third, the repatteming model is not consistent with morphologic abnormalities re-

Volume 12, Number 2, 1998

Fig. 4. Three-dimensional reconstructions of the expression domains of 0tx2 and Otxl in a control embryo (A) and a 3 to 4 somite-stage embryo exposed for 8 h to 1 FM AT-RA (B). Dorsal view of the prospective fore- and midbrain. The contours of the embryo are figured by a thin white line. Rostra1 is to the top, caudal is to the bottom. The small part of the embryo on top devoid of any signal is the heart. The uppermost part of the Otx2 signal (yellow) is the rostra1 tip of the CNS. The lower part of this signal is at the mesencephalic-rhomben- cephalic boundary. Comparison of the expression domain of Otx2 (yellow) with that of Otxl (red). In treated embryos, the neural folds were narrower than in control embryos. The relative location of the caudal expression borders was not modified after treatment with AT-RA. However, the rostra1 triangular portion of the CNS expressing Otx2 but not Otxl (white arrows) in the control embryo (A), was missing in the treated embryo (B). Scale bar is 100 pm.

ported after localized microinjection of Xenopus embryos with RA. In this species, RA induced a loss of rostra1 structures, whereas anteriorization of caudal structures was not observed (see above). Therefore, these abnormalities were interpreted as deletions instead of repatteming in the CNS.

AN ALTERNATIVE MODEL: AT-RA INDUCES A DELETION OF THE ROSTRAL PART OF

THE CNS

To detect subtle modifications in the forebrain of

mouse embryos exposed later than the critical E7 to E7.6 period, we compared the expression patterns of Otx2, Otxl, Pax-6, and Emx2 in early somite-stage embryos (E8.2) exposed for 8 h to 1 ~_LM AT-RA in a whole embryo culture system (52). Our observations showed that the most rostra1 portion of the prospective forebrain was lost in embryos exposed to RA. The caudal expression borders of the studied genes were not modified relative to each other by the treatment. The relative cranial expression boundaries of Otxl, Emx2, and Pax-6 were also normal. However, the most rostra1 portion of the neural plate expressing 0tx2 but not Otxl nor Emx2 and Pax-6 was missing (Figure 4). Therefore, under these conditions, AT-RA induced a deletion of the most rostra1 part of the CNS. This conclusion led us to propose

an alternative to the repatteming model, which will be

referred to as the “deletion” model (Figure lC), to explain abnormalities induced by RA. According to the

deletion model, RA can induce deletion of structures located in or derived from the most rostra1 part of the CNS (Figure 1C).

In the mouse, a deletion of the most rostra1 part of the forebrain is consistent with morphologic abnormalities of the forebrain and the eyes observed in embryos exposed in vitro at E8.2 to AT-RA (52). It is also

consistent with the brain (41), eye, and crania-facial phenotypes (53) reported after in vivo exposure between E6.2 and E8.2. In particular, the atelencephalic micro-

cephaly and anencephaly phenotypes described by Sime- one et al. (41) probably result from deletions of the fore-

and/or midbrain. The deletion model also corresponds to morphologic abnormalities reported after exposure of prochordates, Xenopus, and chick embryos to AT-RA

(see above). It can also explain forebrain-derived NCCs, eye, and rostra1 CNS abnormalities observed in human embryos exposed prenatally to retinoids. In addition, this model is consistent with several features that do not fit in the repatteming model. First, it explains the relative

displacement of the expression border of Hoxb-1 when compared with Otx, En, and Wnt-1 genes. In the hind-

brain, RA induces ectopic expression of Hox genes in more rostra1 domains (References 47-51; F. Clotman, 1997, unpublished data). The deletion model does not

require any modification of the location of the caudal expression border of the Otx, En, or Wnt-1 genes. Therefore, it explains why the rostra1 expression border of Hoxb-I is rostrally displaced when compared with the expression borders of those genes. Second, this model is consistent with the differences in the fore/midbrain and in the hindbrain observed after exposure at different

developmental stages. If RA induces rostralization and possible repatteming in the hindbrain and induces deletions in the forebrain, the timing of sensitivity of the two

compartments can be different. This difference may be related to the rostrocaudal RA gradient naturally present in the embryo. Indeed, RA seems to be absent in the

rostra1 regions of the embryo, and therefore could have no function in these regions, whereas the increasing concentrations detected from the hindbrain to the caudal regions are likely to be involved in the rostrocaudal patterning of the hindbrain and spinal cord (54-56). The deletion model can thus explain all the structural abnormalities and modifications in gene expression observed after exposure of embryos to retinoids.

CONCLUSIONS

The repatteming and deletion models arose from observations performed in different experimental condi-

AT-RA-induced forebrain abnormalities l F. CLOTMAN ET AL. 175

tions. However, as all the reported observations fit in the

deletion model, we assume that this latter can apply in all

the experimental conditions. In our experimental condi-

tions, this deletion was restricted to the rostra1 tip of the forebrain (52). In ex:periments reported previously, the alteration of rostra1 structures extended to the whole forebrain and into rhe midbrain. This suggests that increasingly severe conditions of retinoid exposure can induce increasingly extended deletions of rostra1 structures.

Acknowledgments-We .are indebted to E. Boncinelli for kindly providing the Otx2 probe. We thank A. Bastin and N. Pacico for excellent technical assista.nce with the ISH, J. Lepage for animal husbandry, and F. Desneux for photographic reproductions. This work was supported in part by EEC contract BI02-CT93-0107, by the F.R.S.M. contract 3.4588.92, by the Region Wallonne and by the Fonds du Developpement Scientihque of the Catholic University at Louvain- la-Neuve, Belgium. F.C. holds a fellowship from the F.R.I.A. (Bel- gium), and G.V.M.F. is a fellow of the EC.

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structural and gene expression abnormalities induced by retinoic acid in the forebrain

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