Genetic control of brain morphogenesis through Otx gene dosage requirement

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1 Development 124, (1997) Printed in Great Britain The Company of Biologists Limited 1997 DEV Genetic control of brain morphogenesis through Otx gene dosage requirement Dario Acampora, Virginia Avantaggiato, Francesca Tuorto and Antonio Simeone* International Institute of Genetics and Biophysics, CNR, Via G. Marconi, 12, Naples, Italy *Author for correspondence ( Should be considered as equal first author SUMMARY Understanding the genetic mechanisms that control patterning of the vertebrate brain represents a major challenge for developmental neurobiology. Previous data suggest that Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, might both contribute to brain morphogenesis. To gain insight into this possibility, the level of OTX proteins was modified by altering in vivo the Otx gene dosage. Here we report that Otx genes may cooperate in brain morphogenesis and that a minimal level of OTX proteins, corresponding either to one copy each of Otx1 and Otx2, or to only two copies of Otx2, is required for proper regionalization and subsequent patterning of the developing brain. Thus, as revealed by anatomical and molecular analyses, only Otx1 / ; Otx2 +/ embryos lacked mesencephalon, pretectal area, dorsal thalamus and showed an heavy reduction of the Ammon s horn, while the metencephalon was dramatically enlarged occupying the mesencencephalic area. In 8.5 days post coitum (d.p.c.) Otx1 / ; Otx2 +/ embryos, the expression patterns of mesencephalic-metencephalic (mes-met) markers such as En-1 and Wnt-1 confirmed the early presence of the area fated to give rise to mesencephalon and metencephalon while Fgf-8 transcripts were improperly localized in a broader domain. Thus, in Otx1 / ; Otx2 +/ embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mes-met primitive neuroepithelia that triggers the following repatterning involving the transformation of mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic expression of Wnt-1 and En-2. Taken together these findings support the existence of a molecular mechanism depending on a precise threshold of OTX proteins that is required to specify early regional diversity between adjacent mes-met territories and, in turn, to allow the correct positioning of the isthmic organizer. Key words: Otx genes, brain patterning, isthmic organizer, FGF8, ZLI, mouse INTRODUCTION Morphogenesis of the central nervous system (CNS) and differentiation of the neural structures are highly complex processes. When induced by an organizer (Spemann and Mangold, 1924), responding ectoderm tissue undergoes morphogenetic changes and becomes subdivided into broad regions corresponding to the forebrain, midbrain and hindbrain (Gallera, 1971; Storey et al., 1992; Ruiz i Altaba, 1994). Anatomical and histological studies postulate the existence of genetic fate determinants that subdivide the large neural regions into smaller longitudinal and transverse domains (Vaage, 1969; Altman and Bayer, 1988; Figdor and Stern, 1993; Rubenstein et al., 1994). Some of the patterning events along the anterior-posterior axis may require the presence of transverse rings of neuroepithelia that possess inductive and boundary properties. It has been suggested that the isthmus at the mesencephalic-metencephalic (mes-met) boundary and the zona limitans intrathalamica (ZLI) at the prosomere 2-prosomere 3 (p2-p3) boundary might be neuroepithelial organizers (Martinez et al., 1991; Marin and Puelles, 1994). It has been proposed that organizing centers are generated at the boundary between juxtaposed differently specified territories where cooperative interactions result in the production of signalling molecules with inducing properties (Meinhardt, 1983). According to this model, primarily supported by studies on Drosophila segmentation (Ingham and Martinez Arias, 1992; Perrimon, 1994), signalling molecules such as FGF8, Wnt-1 and Sonic hedgehog (Shh) are sharply expressed at the isthmus and ZLI, respectively (Bally-Cuif and Wassef, 1995). Recently, FGF8-inducing properties at the isthmic organizer of chick embryos have been demonstrated (Crossley et al., 1996). FGF8 and isthmic morphogenetic activities are able to organize ectopic midbrain development and induce the expression of mes-met genes such as Engrailed (En) and Wnt-1 (Ang, 1996; Crossley et al., 1996; Joyner, 1996). Moreover, recently it has been shown that the Fgf-8 molecule is also involved in the establishment of the anteroposterior polarity and growth of the mesencephalon (Lee et al., 1997). However, the molecular mechanisms defining the threshold of diversity necessary to distinguish and specify adjacent territories, thus allowing the correct

2 3640 D. Acampora and others positioning of an organizing boundary, have not yet been elucidated. Gene candidates for the specification of brain regions have been isolated (Rubenstein et al., 1994), most of which are homologs of Drosophila genes controlling head development (Finkelstein and Perrimon, 1990; Cohen and Jürgens, 1991; Rubenstein et al., 1994). Among them, Otx1 and Otx2 from 0- to 3-somite stage, during regionalization and patterning of the developing brain, begin to be coexpressed along the prosencephalon and the mesencephalon with a posterior border, which is gradually restricted to the mesencephalic side of the isthmic constriction (Simeone et al., 1992a; Ang, 1996). Similarly, in the diencephalon, their expression becomes localized to the dorsal thalamus, pretectal area and ZLI (Simeone et al., 1993). In vivo retinoic acid treatment and transplantation experiments in mouse and chick embryos, respectively, suggest that Otx genes might contribute to regional specification of the developing brain (Simeone et al., 1995; Avantaggiato et al., 1996; Millet et al., 1996). Otx1 and Otx2 null mice have been generated to elucidate their role. Otx1 / mice showed spontaneous epileptic seizures and multiple subtle abnormalities affecting the dorsal telencephalic cortex, mesencephalon and cerebellum (Acampora et al., 1996). Otx2 +/ newborns revealed variable penetrance of craniofacial and brain malformations strictly depending on their genetic background (Matsuo et al., 1995), while Otx2 null mice died early during embryonic development, thus showing that proper development of the epiblast and specification of the rostral neuroectoderm fated to become fore-midbrain were strongly impaired (Acampora et al., 1995; Ang et al., 1996). Thus, Otx1 does not seem to be required for regionalization and subsequent patterning of the rostral neuroectoderm. However, such a role cannot be ruled out for Otx2, due to the early embryonic lethality of Otx2 / embryos (Acampora et al., 1995; Ang et al., 1996). It is likely that mechanisms underlying the regionalization and patterning of the rostral neural tube might require an appropriate threshold of OTX proteins, as recently demonstrated for the otd gene product in the formation of specific subdomains of the Drosophila head (Royet and Finkelstein, 1995). To gain insight into this possibility, we modified the in vivo level of OTX proteins by altering the Otx gene dosage. Here we report that Otx genes might cooperate and that a critical threshold of OTX proteins is required for regionalization and subsequent patterning of the developing brain. We also provide strong evidence that Otx gene dosage is required in controlling a morphogenetic boundary with organizing properties such as the isthmus. Thus, our data reveal a previously unsuspected mechanism, depending on a precise Otx genedosage that is required for brain morphogenesis. MATERIALS AND METHODS Generation and genotyping of mice Mice and embryos were generated by crossing Otx1 +/ ; Otx2 +/ males with Otx1 +/ ; Otx2 +/+ females in a BL6/DBA2 background. DNA extraction from yolk sac or tail tip was performed as described (Acampora et al., 1995). Genotypes were identified by using appropriate combinations of oligonucleotides as described previously (Acampora et al., 1995, 1996). Genetic and phenotypic analyses All the embryos were collected, fixed in 4% paraformaldehyde, stored in 70% ethanol and genotyped (Acampora et al., 1995, 1996). For histology and anatomy, fifteen Otx1 +/+ ; Otx2 +/, thirty three Otx1 +/ ; Otx2 +/, thirteen Otx1 / ; Otx2 +/ (Table 1) and only five Otx1 / ; Otx2 +/+ ; four Otx1 +/ ; Otx2 +/+ and four wild-type brains were analyzed at 19 days post coitum (d.p.c.) as follows: brains were dissected, photographed, sectioned and stained with Cresil-Violet (Acampora et al., 1996). A detailed analysis of the Otx1 / ; Otx2 +/+ and Otx1 +/ ; Otx2 +/+ brains had been previously described (Acampora et al., 1996). Probes and in situ hybridization In situ hybridization experiments on sections and whole embryos were performed as previously described (Wilkinson, 1992; Hogan et al., 1994) using 35 S-labelled and digoxigenin-labelled RNA probes, respectively. The Otx2 functional allele was monitored using the Otx2- del probe (Acampora et al., 1995). The Wnt-1, Pax-2 and En-2 probes were the same as previously described (Avantaggiato et al., 1996). The Pax-6 probe was a 428 bp PCR product corresponding to the region between amino acids 293 and 436 (Walther and Gruss, 1991). The Fgf-8 probe was a 712 bp PCR product corresponding to the AIGF1 variant (Tanaka et al., 1992) and containing the coding region from amino acid 1 (Crossley et al., 1996) plus 103 bp of 3 UT. The Emx- 2 probe was the same as previously described (Simeone et al., 1992b). The Gbx2 probe was kindly provided by Dr G. Martin. The Shh and T-brain-1 probes were PCR products spanning the regions between amino acid 109 and 177 (Echelard, 1993) and amino acids 599 and 680 (Bulfone et al., 1995), respectively. RESULTS Genetic analysis and anatomy of Otx1 / ; Otx2 +/ brains Mice carrying different combinations of functional copies of Otx1 and Otx2 genes were generated by crossing preselected Otx1 +/ ; Otx2 +/ double heterozygous males with Otx1 +/ ; Otx2 +/+ females. All the mice are derived from the original backcross of chimaeric males to BL6/DBA2 F 1 females (Acampora et al., 1995, 1996) and kept in a mixed background (129/Sv, BL6 and DBA2), which is particularly useful for our genetic analysis since the Otx1 +/+ ; Otx2 +/ genotype gives reduced penetrance of mild craniofacial and brain malformations as compared to other backgrounds (Acampora et al., 1995; Matsuo et al., 1995). Progeny were scored at late gestation (19 d.p.c.) to verify Mendelian inheritance of genotypes and to analyze exclusively macroscopic brain patterning abnormalities such as regional deletions and/or transformations. All genotypes were transmitted at the expected Mendelian frequency (Table 1) and, interestingly, only Otx1 / ; Otx2 +/ showed 100% of macroscopic brain patterning abnormalities (Table 1). The observation that 100% of Otx1 / ; Otx2 +/ embryos showed severe brain malformations (Fig. 1D- F), whereas a similar phenotype was never observed either in the parental Otx1 +/ ; Otx2 +/ animals or in other genotypes (Table 1), suggests that in mice carrying a single copy of Otx2, Otx1 is able to compensate for the Otx2 requirement, leading to a normal brain morphogenesis. Therefore, a minimal threshold of OTX proteins, corresponding to either one copy each of Otx1 and Otx2 (Otx1 +/ ; Otx2 +/ ) or to only two copies of Otx2 (Otx1 / ; Otx2 +/+ ) (Table 1), is required for the gross development of the brain.

3 Otx requirement in brain patterning 3641 Table 1. Otx gene dosage effects on brain morphogenesis Prenatal analysis at 19 d.p.c.* Number Expected frequency Observed frequency Macroscopic brain Genotypes of embryos of genotypes of genotypes patterning abnormalities (%) Otx1 +/+ Otx2 +/ Otx1 +/ Otx2 +/ Otx1 / Otx2 +/ Otx1 +/+ Otx2 +/ Otx1 +/ Otx2 +/ Otx1 / Otx2 +/ TOTAL 131 Macroscopic brain patterning abnormalities included only deletions and/or transformations of regional identities. *Number of litters = 15. Number of embryos per litter = 7. One out of 14 Otx1 / ; Otx2+/ embryos showed complete acephaly. To characterize the nature of abnormalities of Otx1 / ; Otx2 +/ brains, a detailed histological and anatomical examination was performed at 19 d.p.c. All the brains were normal in their general brain patterning (Fig. 1A-C) except Otx1 / ; Otx2 +/ brains (Fig. 1D-F). The Otx1 / ; Otx2 +/ brains lacked the mesencephalon, pretectal area (p1) and dorsal thalamus (p2) while the hippocampus showed a heavy reduction of the Ammon s horn and disappearence of the fimbria but retained a well-identified dentate gyrus (Fig. 1E,F and see below). On the contrary, the metencephalon (cerebellum and pons) was dramatically enlarged and protruded rostrally to occupy the regions in which the mesencephalon and the caudal diencephalon would normally be found (Fig. 1D,E). The enlarged cerebellar structure appeared to substitute for all of the dorsal Fig. 1. Comparison at 19 d.p.c. between Otx1 +/ ; Otx2 +/ and Otx1 / ; Otx2 +/ brains. (A-F) Dorsal view (A,D), sagittal (B,E) and frontal (C,F) sections of Otx1 +/ ; Otx2 +/ (A-C) and Otx1 / ; Otx2 +/ (D-F) brains showing that, in Otx1 / ; Otx2 +/, the mesencephalon disappears and is substituted by an enlarged cerebellum (D,E) and that the pretectum, the dorsal thalamus and the Ammon s horn, but not the dentate gyrus of the hippocampus, are not identified (open arrows in E,F). Abbreviations: Te, telencephalon; Ms, mesencephalon; Cb, cerebellum; Hi, hippocampus; hy, hypothalamus; dg, dentate gyrus; dt, dorsal thalamus; pt, pretectum. Otx1 +/ ; Otx2 +/ and Otx1 / ; Otx2 +/ whole brains and sections are photographed at the same magnification.

4 3642 D. Acampora and others Fig. 2. Expression domains of Pax-6, Otp, Pax-2, Emx-2 and Gbx2 in wild-type and Otx1 / ; Otx2 +/ brains at 19 and 12.5 d.p.c. (A-H) Cytoarchitecture (A,E ) and Pax-6 expression (A,E) in the presumptive external granular layer of 19 d.p.c. cerebella of wild-type (A,A ) and Otx1 / ; Otx2 +/ (E,E ); Otp (B,F), Pax-2 (C,G) and Emx2 (D,H) expression patterns in 12.5 d.p.c. wild-type (B-D) and Otx1 / ; Otx2 +/ (F-H) embryos confirm that the presumptive ventral mesencephalon acquires ventral metencephalic markers (arrows in B,C and F,G) and loses the mesencephalic expression of Emx2 (arrows in D and H). (I-L ) Gbx2 (I,K) and Emx2 (J,L) expression, respectively, in the dorsal thalamus and hippocampus of 19 d.p.c. wild type (I,J) and Otx1 / ; Otx2 +/ (K,L) confirm the lack of dorsal thalamus (compare I,I to K,K ) and the heavy reduction of the Ammon s horn with disappearence of the fimbria but not of the dentate gyrus (compare J,J to L,L ). Bright fields of the corresponding sections are labelled with a prime( ). PCL, layer of Purkinje cells; zi, zona incerta; vt, ventral thalamus; fi, fimbria (other abbreviations as in the previous figure). mesencephalon as inferred from histological analysis in frontal and sagittal sections (Fig. 1 and data not shown). In addition, the dorsal pretectal area appeared to be absent and, in its presumptive position, a thin tissue layer fused the cerebellar structure directly to the telencephalon and precisely to the area corresponding to the dentate gyrus (Fig. 1E and data not shown). This anatomical-histological analysis was supported and confirmed by in situ hybridization experiments probing at 19, 16.5 and 12.5 d.p.c. with a number of regionally restricted genes such as Pax-2, Pax-6, Otp, Gbx2, T-brain-1 and Emx2 (Simeone et al., 1992b, 1994; Puelles and Rubenstein, 1993; Stoykova and Gruss, 1994; Bulfone et al., 1995). Thus, at 19 d.p.c. the Pax-6 expression was restricted to the presumptive external granular layer of both the wild-type (Fig. 2A,A ) and Otx1 / ; Otx2 +/ cerebella (Fig. 2E,E ), confirming that, accordingly to the histology, the dorsal mesencephalon of the Otx1 / ; Otx2 +/ brains was replaced by an enlarged cerebellar structure.

5 Otx requirement in brain patterning 3643 At 12.5 d.p.c. in wild-type embryos, Otp and Pax-2 were expressed along the metencephalon with a rostral border very close or coincident to the isthmus (Fig. 2B,C) while Emx2 was also transcribed along the mesencephalon at this stage (Fig. 2D) (Simeone et al., 1992b). In 12.5 d.p.c. Otx1 / ; Otx2 +/ embryos both Otp and Pax-2 expression patterns were anteriorized up to the presumptive caudal diencephalon (Fig. 2F,G) while Emx2 transcripts disappeared from the presumptive mesencephalon (Fig. 2H), confirming that also the ventral mesencephalon was replaced by an enlarged metencephalon (pons). At 19 d.p.c., the Gbx2 expression was restricted to the dorsal thalamus in the wild type (Fig. 2I,I ) but according to anatomical inspection was undetectable in the Otx1 / ; Otx2 +/ diencephalon (Fig. 2K,K ); an identical result was observed at earlier stages (data not shown). Emx2 was highly expressed in the dentate gyrus (Fig. 2J,J ) and recent findings indicate that it is functionally involved in the establishment of this structure (Pellegrini et al., 1996; Yoshida et al., 1997). According to the histology, Emx2 was still expressed in the presumptive dentate gyrus of Otx1 / ; Otx2 +/ brain, confirming that only the Ammon s horn was heavily reduced or partially missing (Fig. 2L,L ). Therefore genetic, anatomical and molecular findings indicate that a critical threshold of Otx gene products is required for brain morphogenesis. Molecular events underlying brain patterning abnormalities To investigate the molecular events underlying the establishment of this abnormal phenotype, we studied the expression pattern of Otx2, Fgf-8, En-2, Wnt-1 and Pax-2 (Simeone et al., 1992a, 1993; Crossley and Martin, 1995; Ang, 1996; Joyner, 1996) at 10.5 and 12.5 d.p.c. in relation to the embryo morphology. At 10.5 d.p.c., as compared to the wild type (Fig. 3B- F), their expression domains appeared coordinately anteriorized in Otx1 / ; Otx2 +/ embryos (Fig. 3B -F ), suggesting that, consistent with the morphological changes (Fig. 3A ), the isthmus was rostrally shifted to the area corresponding to the Fig. 3. Head morphology at 10.5 d.p.c. and expression domains of Otx2, Fgf-8, Wnt-1, En-2 and Pax-2 in wild-type and Otx1 / ; Otx2 +/ embryos at 10.5 d.p.c. (A,A ) Comparing wild-type (A) and Otx1 / ; Otx2 +/ (A ) head morphology, the mesencephalon, pretectum and dorsal thalamus are not detected in the mutant where the metencephalon extends rostrally to occupy the mesencephalic and caudal diencephalic regions. (B-F) In wild-type embryos, Otx2 (B), Fgf-8 (C), Wnt-1 (D), En-2 (E) and Pax-2 (F) identify mes-met domains and define a molecular code at the isthmus. (B -F ) In Otx1 / ; Otx2 +/ embryos, Otx2 (B ), Fgf-8 (C ), Wnt-1 (D ), En-2 (E ) and Pax-2 (F ) are coordinately anteriorized, confirming that an isthmic-like structure has been generated more rostrally. However, neither Wnt-1 nor En-2 appear activated in the rostral prosencephalon (D,E ). Bright fields of the corresponding sections are labelled with a double prime( ). Di, diencephalon; is, isthmus; Mt, metencephalon (other abbreviations as in the previous figures). The filled arrows in C-F point to the posterior border of Otx2 in wild-type and Otx1 / ; Otx2 +/ embryos.

6 3644 D. Acampora and others caudal diencephalon (p1 and p2). When compared to the wild type (Fig. 3A), the mesencephalon, pretectum and dorsal thalamus could not be identified in Otx1 / ; Otx2 +/ embryos (Fig. 3A ). One might expect that repatterning of the territory rostral to the presumptive isthmic organizer would result in the acquisition of mesencephalic features by that tissue. However, in Otx1 / ; Otx2 +/ embryos at 10.5 d.p.c., neither Wnt-1 nor En-2 were expressed in prosencephalic domain rostral to the isthmic-like structure (Fig. 3D,E ). Several hypotheses could explain this unexpected observation: (i) limited competence of rostral prosencephalic territory to respond to isthmic-like inductive signals (Martinez et al., 1991; Marin and Puelles, 1994; Bally-Cuif and Wassef, 1995; Crossley et al., 1996), (ii) a temporal delay in the response of target tissues to the inductive signals or (iii) impaired signalling properties of the isthmic-like structure. To investigate these possibilities, in situ hybridization experiments probing wild-type and Otx1 / ; Otx2 +/ embryos at 12.5 d.p.c. with Otx2, Fgf-8, Wnt-1 and En-2 genes were performed (Simeone et al., 1993; Crossley and Martin, 1995; Joyner, 1996). The resulting data support the second hypothesis, that the response to the inductive signals was delayed. In fact, at 12.5 d.p.c., the molecular code defined at the isthmus by the expression patterns of Otx2, Fgf-8, Wnt-1 and En-2 in the wild type (Fig. 4A-D) was stably retained at the isthmic-like structure in the Otx1 / ; Otx2 +/ embryos (Fig. 4A -D ) and Wnt-1 and En-2 were also found to be expressed in the presumptive commissural plate of the telencephalon (Fig. 4E,E ) and along the dorsal telencephalic cortex (Fig. 4D ), respectively. Furthermore, at 12.5 d.p.c., Otx1 / ; Otx2 +/ embryos still retained the expression of telencephalic genes such as T- brain-1 (Fig. 4F,F ) and Emx-2 (Fig. 2H) (Simeone et al., 1992b; Bulfone et al., 1995). Taken together, these data indicate that the telencephalon had partially transformed its molecular identity, and simultaneously expressed mesencephalic and telencephalic genes. Transplantation experiments suggested that the failure of grafts of both isthmic tissue and beads soaked in FGF8 to induce mesencephalic phenotype in the rostral prosencephalon could be due to the lack of competence and/or to the presence of a morphogenetic barrier that blocks the rostral transmission of inductive signals. The ZLI might play this antagonistic role and, in turn, could possess organizing properties similar to those of the isthmic constriction (Martinez et al., 1991; Figdor and Stern, 1993; Marin and Puelles, 1994; Rubenstein et al., 1994; Bally-Cuif and Wassef, 1995; Crossley et al., 1996). The signalling molecule Shh is normally expressed at the ZLI (Echelard et al., 1993; Bally-Cuif and Wassef, 1995) (Fig. 4G,H) together with Otx1 and Otx2 (Simeone et al., 1993). In situ hybridization experiments showed that, at 10.5 and 12.5 d.p.c., Shh was not detected in the presumptive ZLI of Otx1 / ; Otx2 +/ embryos (Fig. 4G,H ), indicating that the ZLI was either absent or strongly impaired. Early altered expression of Fgf-8 in Otx1 / ; Otx2 +/ embryos triggers a repatterning process At 8.5 d.p.c., the morphology of Otx1 / ; Otx2 +/ embryos could not be distinguished from that of wild type (Fig. 5A-C and data not shown). At this stage, Wnt-1, En-1 and Fgf-8 expression patterns of wild type were compared to those of Otx1 / ; Otx2 +/ embryos. Wnt-1 and En-1 were expressed in a similar domain both in wild-type (Fig. 5A,B) and Otx1 / ; Otx2 +/ (Fig. 5A,B ) embryos, indicating that the mes-met territory was correctly identified by these genes at this stage. Nevertheless, in 8.5 d.p.c. Otx1 / ; Otx2 +/ embryos, Fgf-8 was improperly activated in a broader domain (Fig. 5C ). At 9 d.p.c., Fgf-8 did not present its characteristic ring at the isthmus and was clearly misexpressed along the presumptive mesencephalon (compare Fig. 5F to F ). In addition, Wnt-1 expression failed to be restricted to its posterior ring of expression close to the mes-met boundary (compare Fig. 5D to D ), but was still transcribed along the roof of the presumptive mesencephalon (Fig. 5D ). En-1 was still detected in a continuous domain, partially overlapping that of Wnt-1, but slightly anteriorized as compared to the wild type (Fig. 5E,E ). Therefore, in Otx1 / ; Otx2 +/ embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mesencephalic and metencephalic primitive neuroepithelia rather than of a premature failure in the establishment of the mesencephalic domain. Dynamic expression of mes-metencephalic genes between 9 and 10.5 d.p.c. in Otx1 / ; Otx2 +/ embryos To investigate how head morphology and expression patterns described at 10.5 d.p.c. were established, we analyzed the expression of Fgf-8, Wnt-1, Otx2, En-1, Otp and Gbx2 at different developmental stages between 9 and 10.5 d.p.c. in Otx1 / ; Otx2 +/ embryos. At 9.5 d.p.c. the Otx2 expression was regressing from posterior to anterior (Fig. 6A) and Fgf-8 was expressed in the presumptive telencephalic commissural plate and all along the presumptive midbrain and caudal diencephalon (Fig. 6B), while Wnt-1 was still transcribed along the presumptive midbrain and caudal diencephalon (p2, p3) (Fig. 6C). However, like Fgf-8, Wnt-1 did not define any stripe in the presumptive mes-metencephalic area (Figs 5D,F, 6B,C). At 9.75 d.p.c., while Otx2 increased its posterior retraction (Fig. 6D), Fgf-8 and Wnt-1 expression began to be posteriorly repressed (Fig. 6E,F) and the Wnt-1 domain was restricted to a small area where Otx2 and Fgf-8 were also transcribed (Fig. 6E). It is worth noting that, in Otx1 / ; Otx2 +/ embryos at 9.75 d.p.c., the posterior borders of Otx2 and Wnt-1 were coincident (Fig. 6D,F), while that of Fgf-8 was slightly more posterior (Fig. 6E). According to the dynamic expression patterns observed, the area immediately posterior to that expressing Wnt-1 should be destined to acquire a metencephalic identity as previously shown at 10.5 and 12.5 d.p.c. Therefore, to define how this identity was acquired, we hybridized Otx1 / ; Otx2 +/ embryos at 9.75 d.p.c. with En-1, and at 10 d.p.c. with Otp and Gbx2. In wild-type embryos, En-1 is expressed in a mes-metencephalic domain centered on the isthmus (Figs 3E, 5B,E), while Gbx2 and Otp are transcribed at 10.5 d.p.c. in the hindbrain (Fig. 6J,K), where the rostral expression of Gbx2 is coincident with the isthmic expression of Fgf-8 (Fig. 6I,J), and that of Otp is located slightly posteriorly (Fig. 6I,K). In Otx1 / ; Otx2 +/ embryos, En-1 at 9.75 d.p.c. overlapped both Wnt-1 and Fgf-8 domains and included the territory posterior to Wnt-1 (Fig. 6G). Furthermore, at 10 d.p.c., both Gbx2 (Fig. 6N) and Otp (Fig. 6O) domains resulted coordinately anteriorized, thus maintaining correct spatial relationships with Fgf-8 expression (compare Fig. 6I,J,K to M,N,O). Thus, as supported by these findings and those previously

7 Otx requirement in brain patterning 3645 shown, the area posterior to Wnt-1 acquired a metencephalic identity. It is also to note that, at 10 d.p.c., the morphology and relative size of rhombomeres posterior to the metencephalon (Fig. 6P ) as well as the expression of a typical marker for rhombomere 4 such as Hoxb1 appeared unaltered (Fig. 6P and data not shown). Therefore, the establishment of the molecular identities described at 10.5 and 12.5 d.p.c. is outlined between 9 and 10 d.p.c. by dynamic changes in the expression patterns of genes restricted to or expressed also in the mes-metencephalic areas. Since most of these genes are functionally involved in the establishment of these territories, the alterations in their expression patterns might contribute to the morphological changes observed in Otx1 / ; Otx2 +/ embryos. DISCUSSION It has been proposed that Otx1 and Otx2 are involved in different aspects of brain development (Simeone et al., 1992a, Fig. 4. Otx2, Fgf-8, Wnt-1, En-2, T-brain-1 and Shh expression at 12.5 d.p.c. and for Shh also at 10.5 d.p.c. in wild-type and Otx1 / ; Otx2 +/ embryos. (A-E ) At 12.5 d.p.c. in wild-type embryos (A-D), Otx2 (A), Fgf-8 (B), Wnt-1 (C) and En-2 (D), sharply identify the mes-met territory and the isthmus, while in Otx1 / ; Otx2 +/ (A -E ) Otx2 (A ), Fgf-8 (B ), Wnt-1 (C ) and En-2 (D ) expression patterns confirm that the mesencephalon and caudal diencephalon acquire mes-met molecular markers and that En-2 (D ) and Wnt-1 (E) are both activated in the telencephalon where En-2 is now expressed along the presumptive cortex (D ) and Wnt-1 in the commissural plate (E,E ). (F-F ) Expression of T-brain-1 both in wild-type (F) and Otx1 / ; Otx2 +/ (F ) embryos. (G-H ) In wild-type embryos at 10.5 d.p.c. (G) and 12.5 d.p.c. (H), Shh expression marks the ZLI while in Otx1 / ; Otx2 +/ (G,H ) embryos, Shh is not expressed in the area corresponding to the presumptive ZLI suggesting that the latter is strongly impaired or completely absent. ZLI, zona limitans intrathalamica; cp, commissural plate; np, neurohypophysis; or, ocular recess (other abbreviations as in the previous figures). The open arrows in B-D, B -D point to the posterior border of Otx2 in wild-type (B-D) and Otx1 / ; Otx2 +/ (B -D ) embryos. (A-F ) and (G-H ) Sagittal and frontal sections, respectively; (E ) bright field of E.

8 3646 D. Acampora and others

9 Otx requirement in brain patterning 3647 Fig. 5. Expression domains of Wnt-1, En-1 and Fgf-8 in wild-type and Otx1 / ; Otx2 +/ embryos at 8.5 and 9 d.p.c. (A-C ) At 8.5 d.p.c. Wnt-1 (A,A ) and En-1 (B,B ) identify presumptive mes-met territory both in wild-type (A,B) and Otx1 / ; Otx2 +/ (A,B ) embryos while Fgf-8 (C,C ), as compared to the wild-type (C) embryos, is improperly transcribed in a broader domain in Otx1 / ; Otx2 +/ (arrowheads in C ). (D-F ) At 9 d.p.c. the expression domains of Wnt- 1 (D,D ), En-1 (E,E ) and Fgf-8 (F,F ) show that in Otx1 / ; Otx2 +/ (D -F ), as compared to the wild type (D-F), Wnt-1 (D ) and Fgf-8 (F ) fail to be restricted in their characteristic narrow stripe at the mes-met boundary while En-1 (E ) is transcribed in a domain similar to that of wild type (E). Embryos at 8.5 d.p.c. show 5 to 8 somites and those at 9 d.p.c. 13 to 17 somites. Pros, prosencephalon (other abbreviations as in the previous figures). 1993, 1995; Acampora et al., 1995; 1996; Matsuo et al., 1995; Ang et al., 1996; Avantaggiato et al., 1996; Millet et al., 1996). Null mice for Otx1 and Otx2 reveal quite different roles for these genes despite their relatively high homology. Otx1 seems to be involved in terminal events of corticogenesis (Acampora et al., 1996), while Otx2 is required for the very early specification of domains destined to give rise to fore-mid and rostral hindbrain (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., 1996), thus making it impossible to decifer its role in the subsequent steps of brain development. The differences between Otx1 / and Otx2 / phenotypes could be due at least in part (to be proven) to the fact that Otx1 is activated once gastrulation is over, whilst Otx2 is expressed before the onset of gastrulation. Their expression patterns during early regionalization and subsequent patterning of the rostral CNS suggest that they play an important role in signalling regional subdivisions of brain territories and defining morphogenetic boundaries (Simeone et al., 1992a). Recently, it has been shown that Fig. 6. Expression patterns of mes-metencephalic genes at 9.5, 9.75 and 10 d.p.c. in Otx1 / ; Otx2 +/ and at 10.5 d.p.c. in wild-type embryos. (A-C) At 9.5 d.p.c. in Otx1 / ; Otx2 +/ embryos Otx2 (A) appears repressed in the posterior midbrain while Fgf-8 (B) is expressed all along the presumptive midbrain, in the caudal diencephalon and telencephalic commissural plate and Wnt-1 (C) spans the midbrain and the caudal diencephalon. (D-G) At 9.75 in mutant embryos, Otx2 (D) is furtherly repressed in a more expanded region of the posterior presumptive midbrain; Fgf-8 (E) and Wnt-1 (F) expression domains begin to be retracted from posterior showing that Otx2 and Wnt-1 now share a common posterior border different from that of Fgf-8; at the same stage, the En-1 (G) domain includes those of Wnt-1 and Fgf-8 and the territory immediately posterior. At 10.5 d.p.c. in wild-type embryos, Otx2 (H) and Fgf-8 (I) are restricted to the mesencephalic and metencephalic side of the isthmic constriction, respectively, and both Gbx2 (J) and Otp (K) are expressed in the rostral metencephalon where Otp is slightly posterior to, and Gbx2 coincident with, the isthmic expression of Fgf-8. At 10 d.p.c. in Otx1 / ; Otx2 +/ embryos, as the posterior repression of both Otx2 (L) and Fgf-8 (M) increases, both Gbx2 (N) and Otp (O) expression domains become anteriorized. (P-P ) Hoxb1 expression (P) in the rhombomere 4 and histology (P ) of the hindbrain showing that rhombomeres posterior to the metencephalon at 10.5 d.p.c. in Otx1 / ; Otx2 +/ embryos appear normal. Bright fields of the corresponding sections are labelled with a prime( ). r2, r4 and r6, rhombomeres 2, 4 and 6, respectively (other abbreviations as in the previous figures). The arrowheads point to the positions corresponding to the posterior border of Otx2 and the arrows in (P ) point to the rhombomeric boundaries.

10 3648 D. Acampora and others in Drosophila the otd gene product is expressed in a graded distribution and that different otd levels are required for the development of specific head subdomains (Royet and Finkelstein, 1995). Therefore, it is conceivable that early patterning of the developing vertebrate brain could require a similar mechanism (Hirth et al., 1995; Thor, 1995). In this case, highly evolutionary conserved sets of genes could imply similar strategy(ies) in brain development to generate regionally restricted fates. Thus, in order to test this possibility, the level of OTX proteins was modified by altering the Otx gene dosage in vivo. A detailed genetic analysis was performed (Table 1). Interestingly, only Otx1 / ; Otx2 +/ embryos showed 100% of macroscopic brain malformations. The presence of an additional functional copy either of Otx2 (Otx1 / ; Otx2 +/+ ) or Otx1 (Otx1 +/ ; Otx2 +/ ) rescued the abnormal phenotype completely, indicating that a critical threshold of Otx gene product is required for brain morphogenesis: this supports the existence of a conserved mechanism defined, at least in part, by critical levels of Otx gene products. Moreover, the genetic analysis indicates that Otx1 and Otx2, at least in a genetic combination (Otx1 +/ ; Otx2 +/ ), cooperate to specify correct brain development. It is interesting that a similar genetic analysis has been recently reported (Suda et al., 1996). In this analysis, Suda et al. (1996) described phenotypic impairements in Otx1 +/ ; Otx2 +/ mice, whereas regional brain patterning abnormalities were apparent in Otx1 / ; Otx2 +/ embryos (Table 1; Fig. 1). It has been proposed that organizing centres are generated at the boundary between differently specified juxtaposed territories where cooperative interactions result in the production of signalling molecules with inducing properties (Meinhardt, 1983; Ingham and Martinez Arias, 1992; Perrimon, 1994). Transplantation experiments have provided evidence supporting the presence of either genetic determinants imposing territorial competence or morphogenetic signals emitted by regions with organizing properties (e.g. isthmic organizer) (Martinez et al., 1991; Marin and Puelles, 1994; Bally-Cuif and Wassef, 1995; Joyner, 1996). Thus, rostral midbrain transplants in a more anterior region are influenced by adjacent territory to acquire host fate, while transplants of the mes-met junction maintain their identity and induce surrounding tissue to acquire a mes-met fate. On the contrary, forebrain territories rostral to the ZLI (ventral thalamus and secondary prosencephalon) never change their fate in response to signal(s) emitted by the mes-met junction. Interestingly, the host response to the morphogenetic signal is restricted to a single prosomere. Therefore, there is a specific response of different brain regions to the same signal and neuromeric boundaries might act either as morphogenetic sources or as morphogenetic barriers that block the spreading of the signal. According both to theory (Meinhardt, 1983) and embryological findings (Marin and Puelles, 1994; Martinez et al., 1991), FGF-8-inducing properties at the isthmic organizer of chick embryos have recently been demonstrated (Crossley et al., 1996). Thus, the secreted factor FGF-8 is expressed at the right time and in the right position to be involved in development of the organizer. In fact, it shows midbrain-inducing property being able to change the fate of the caudal diencephalon (Crossley and Martin, 1995; Crossley et al., 1996). Similarly, in the forebrain, the signalling molecule Shh is expressed at the ZLI, the boundary separating dorsal (p2) and ventral (p3) thalamus (Echelard et al., 1993; Puelles and Rubenstein, 1993; Bally-Cuif and Wassef, 1995). Molecular, anatomical and embryological observations suggest that this boundary could also have morphogenetic property(ies), thereby playing an organizing role of prosencephalic territories and/or an antagonistic effect on the rostral transmission of inductive signals, thus representing a sort of morphogenetic barrier (Martinez et al., 1991; Figdor and Stern, 1993; Marin and Puelles, 1994; Rubenstein et al., 1994; Bally- Cuif and Wassef, 1995; Crossley et al., 1996). Therefore, an essential point is to determine the molecular mechanism(s) defining the regional diversity necessary to specify adjacent territories with different identity (e.g. midbrain and hindbrain) and, in turn, to allow the correct positioning and/or the establishment of an organizer (e.g. isthmus). Previous data suggest that Otx genes might contribute to the specification of regional diversity between adjacent territories as well as in the positioning and/or establishment of morphogenetic boundaries. In fact, (i) Otx genes are expressed when early regionalization takes place (Simeone et al., 1992a), (ii) Otx2 null mice do not develop neuroectoderm rostral to the rhombomere 3 (Acampora et al., 1995), (iii) the caudal limit of Otx2 expression identifies the mid-hindbrain boundary at the isthmic constriction (Millet et al., 1996), (iv) Otx genes are both expressed in close proximity to the ZLI (Simeone et al., 1993) and (v) retinoic-acid-induced phenocopies show an early ordered anteroposterior repatterning of the brain, correlating with the posterior repression of Otx2 (Simeone et al., 1995; Avantaggiato et al., 1996). Here we report that mice carrying only one functional copy of Otx2 show dramatic brain malformations. As inferred by molecular and anatomical-histological analyses, the nature of these abnormalities indicates a profound perturbation of morphogenetic mechanisms involved in specifying the correct identity of regional domains belonging to the forebrain, midbrain and rostral hindbrain. Thus, the resulting phenotype seems to be the consequence of a repatterning process, involving either the partial transformation of the dorsal telencephalon into a mesencephalon, or a more complete transformation of the caudal diencephalon and mesencephalon into an enlarged metencephalon (cerebellum and pons). Alternatively, the observed phenotype could be due to a failure in the establishment of both the caudal diencephalon and mesencephalon followed by overgrowth of the metencephalon. Thus, the rostral hindbrain expands and the isthmus abuts the telencephalon. To assess this crucial point, we studied early molecular events conferring identity to the broad anteroposterior domains within the rostral CNS. In particular, Wnt-1, En genes and members of the Pax family have provided strong evidence that they are key elements of a genetic cascade leading to mes-met development (Thomas and Capecchi, 1990; McMahon et al., 1992; Bally- Cuif and Wassef, 1995; Rowitch and McMahon, 1995; Danielian and McMahon, 1996; Joyner, 1996). Initially Wnt-1 and En-1 expression domains overlap in the presumptive midbrain region. In 8.5 d.p.c. Otx1 / ; Otx2 +/ embryos, Wnt- 1 and En-1 expression patterns are essentially maintained confirming the early presence of the area destined to give rise to mesencephalon and metencephalon. Conversely, at the same stage, Fgf-8 transcripts were improperly localized in a broader domain, invading adjacent rostral territory and at 9 d.p.c. Wnt-1 and Fgf-8 fail to form their narrow stripe at the mes-met boundary.

11 Otx requirement in brain patterning 3649 Later, at d.p.c., following a series of morphological and molecular dynamic changes (see Results section), the repatterning process begins to be evident and involves the transformation of the mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic acquisition of mesencephalic features such as the expression of En-2 and Wnt-1 genes. Thus, considering the molecular events underlying the repatterning observed in Otx1 / ; Otx2 +/ embryos, we conclude that the isthmus is rostrally shifted to the presumptive caudal diencephalon and not created ex novo. In addition, in Otx1 / ; Otx2 +/ mice, the rostral expression of Fgf-8 in the presumptive mesencephalic area appears to trigger a repatterning process rather than an overgrowth of mesencephalon and caudal diencephalon (Lee et al., 1997). This might be explained considering that: (i) the Fgf-8 ectopic expression along the whole mesencephalon of Otx1 / ; Otx2 +/ embryos is a transient phenomenon observed only between 8.5 and 9.5 d.p.c. and (ii) the increased size of midbrain in embryos ectopically expressing Fgf-8 under the Wnt-1 control (Lee et al., 1997) was described in embryos carrying a normal Otx gene dosage. Therefore, altogether these findings support the existence of a previously unsuspected mechanism depending on a precise threshold of OTX proteins that is strictly required to distinguish adjacent territories with different fates such as mesencephalon and metencephalon, rather than in their early establishment. Thus, in Otx1 +/ ; Otx2 +/ or in Otx1 / ; Otx2 +/+ embryos, the threshold of Otx gene products is able to confer to the mesencephalic field a sufficient level of specification to allow the correct positioning of the Fgf-8-inducing properties at the isthmic organizer whereas, only in Otx1 / ; Otx2 +/ embryos, is an insufficient level likely to be responsible for the Fgf-8 misexpression that triggers the following repatterning process. Furthermore, the finding that, in Otx1 /, Otx2 +/ embryos, the dorsal telencephalon acquires mesencephalic molecular features and the ZLI is absent as revealed by anatomical inspection and the molecular loss of Shh expression, suggest that the telencephalic competence to express mesencephalic genes could be directly related to the loss of the ZLI and/or Shh-mediated signalling. 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