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Home , Engrailed (gene), Homeotic gene, Ligand (biochemistry), Pax genes

Development ID, 1435-1449 (1991) 1435 Printed in Great Britain © The Company of Biologists Limited 1991

Pax-6, a murine paired box , is expressed in the developing CNS

CLAUDIA WALTHER and PETER GRUSS

Max Planck Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Gtittingen, Germany

Summary

A multigene family of paired-box-containing (Pax of the forebrain and the hindbrain. In the neural tube, genes) has been identified in the mouse. In this report, expression is mainly confined to mitotic active cells in the we describe the expression pattern of Pax-6 during ventral ventricular zone along the entire anteroposterior embryogenesis and the isolation of cDNA clones span- axis starting at day 8.5 of development. Pax-6 is also ning the entire coding region. The Pax-6 consists expressed in the developing eye, the pituitary and the of 422 amino acids as deduced from the longest open nasal epithelium. reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with Key words: paired box, , Pax, CNS day 8 of gestation, Pax-6 is expressed in discrete regions development, eye development, embryogenesis.

Introduction gsb-d paired box, a murine multigene family of paired- box-containing genes () of to date eight The fruit fly Drosophila has proved a powerful model members has been isolated (Deutsch et al. 1988; for deciphering the complex genetic program underly- Dressier et al. 1990; Plachov et al. 1990; Jostes et al. ing development. A large number of developmental 1991; Goulding et al. 1991; Walther et al. 1991). All Pax control genes of Drosophila have been isolated and genes described to date exhibit a temporally and genetically analyzed. These studies have revealed an spatially restricted expression pattern during embryo- array of hierachical and combinatorial interactions of genesis, compatible with a regulatory role in vertebrate their gene products that are required for controlling development. The Pax genes, with the exception of and (for reviews see Pax-1, are expressed in the developing nervous system. Akam (1987); Scott and Carroll (1987); Ingham (1988)). Expression is detected along the entire anteroposterior Many of these genes contain motifs axis of the neural tube and the hindbrain where it is such as homeobox, -finger motif, helix-loop-helix confined in the transverse plane to distinct regions. Pax- motif and paired box and these in turn have been used 3 transcripts are already detected at day 8.5 of gestation to isolate potential developmental control genes from a and are restricted to mitotic cells in the dorsal half of variety of vertebrate species (for review see Scott et al. the neural tube, the alar and roof plate (Goulding et al. (1989); Kessel and Gruss (1990)). Like their Drosophila 1991). Pax-7 shows a similar expression pattern except counterparts, many of these vertebrate genes appear to that it is not expressed in the most dorsal part of the play a role in controlling pattern formation and neural tube (Jostes et al. 1991). Pax-2 and Pax-8 are morphogenesis during development (Kessel et al. 1990; expressed later in neurogenesis beginning with day 10 Wright etal. 1989). and day 11 of gestation, respectively. Transcripts are The paired box was originally discovered in the detected in discrete subpopulations of postmitotic cells Drosophila genes paired, gooseberry- within the alar and basal plate (Nornes et al. 1990; distal (gsb-d) and gooseberry-proximal (gsb-p) (Bopp et Plachov et al. 1990). al. 1986; Baumgartner et al. 1987) and subsequently in The Pax genes were cloned on the basis of their two developmental^ regulated tissue-specific genes of to a sequence motif found in segmentation Drosophila, Pox neuro and Pox meso (Bopp et al. genes of Drosophila, which are involved in building the 1989). The paired box has been highly conserved during metameric . Segmentation is also an import- evolution, being present in such divergent organisms as ant feature of vertebrate embryogenesis (Hogan et al. nematodes, zebrafish, Xenopus, chicken and man 1985) and all the Pax genes described to date are (Dressier et al. 1988; Burn et al. 1989). It encodes a expressed in segmented structures such as the differen- protein domain of 128 amino acids which has been tiating somites or the developing excretory system. In shown to be a novel DNA-binding motif (Treisman et the mouse, however, paired-box-containing genes do al. 1991; Chalepakis etal. 1991; Goulding etal. 1991). not seem to be involved in the generation of segmental Based on sequence homology to the Drosophila structures but rather in their differentiation since 1436 C. Walther and P. Gruss expression begins only after these structures have DNA sequence analysis already formed (Deutsch et al. 1988; Dressier et al. Overlapping subclones of the Pax-6 cDNA clones were 1990; Plachov et al. 1990; Jostes et al. 1991; Goulding et generated in M13 mpl8 and mpl9 vectors. Single-stranded al. 1991). DNA was prepared and sequenced with the dideoxy method The potential developmental importance of members (Sanger et al. 1977) using either Sequenase (US Biochemicals) of the Pax gene family is further highlighted by the or T7 kits (Pharmacia). Computer analysis of the sequence correlation of a Pax gene with the developmental was performed using the GCG program package (Devereux et al. 1984). mutant undulated (un). Beginning with day 9 of gestation, Pax-1 is expressed in the sclerotome of the differentiating somites and at later stages in the Northern analysis intervertebral discs anlagen of the developing vertebral Embryos were obtained from natural matings of female column. A point in the Pax-1 gene causing a NMRI and male C57BL/6 mice and the day of the vaginal reduction of DNA-binding affinity (Chalepakis et al. plug was designated day 0.5. Tissues were isolated from adult female NMRI mice. Embryos and tissues were frozen in liquid 1991) disturbs the normal development of the vertebral nitrogen, homogenized in guanidinium (Chirgwin column and was shown to be the cause for the un et al. J;979)'ahd poly(A)+ RNA was prepared using oligo(dT)- (Balling et al. 1988). cellulose columns according to Ausubel et al. (1987). Samples In this report, we present the isolation of cDNAs containing approximately 10 ug of denatured poly(A)+ RNA encompassing the complete coding region of the Pax-6 were electrophoresed on 1% agarose/formaldehyde gels, protein and a detailed analysis of the expression pattern transferred to Hybond-N membranes using IOXSSC and of Pax-6 during embryogenesis. Pax-6 is another crosslinked under UV light. The filters were hybridized with the random oligo-labeled (Feinberg and Vogelstein, 1983) member of the murine paired-box-containing gene genomic HindUI fragment of Pax-6 (see screening of cDNA family, which however encodes a rather divergent libraries) under the following conditions: 7.5% dextran paired domain and paired-type homeodomain. The sulfate, 5xSSC, 5xDenhardts, 50% formamide (Fluka), 1 % expression pattern of Pax-6 also differs in several SDS, 10mM Tris-HCl pH7.5, 0.1 mM sodium pyrophosphate, aspects. Unlike other Pax genes, Pax-6 is not expressed 0.1 mM ATP and 0.1 mgml"1 denatured salmon sperm DNA in segmented mesodermal structures. Pax-6 also is the at 65°C for at least 16h. Filters were washed in 2xSSC, 1 % first Pax gene described to be expressed in the ventral SDS at 65°C, followed by two washes in O.lxSSC, 0.1 % SDS neural tube before neural differentiation starts. The at 65°Cfor30min. expression pattern of Pax-6 in the neural tube is compatible with a role for Pax-6 in the regional In situ hybridization specification of cells in the neural tube with respect to Radioactive RNA probes were generated by in vitro the dorsal-ventral axis. Transcripts of Pax-6 are also transcription from a linearized plasmid clone containing an present in distinct regions of the developing brain, but •EcoRl-AT/eZ-cDNA-fragment encoding most of the 3' part in contrast to Pax-3 and Pax-7, the overall spatial of the Pax-6 paired box (see Fig. 1) using 100 /*Ci 35S-CTP and distribution of transcripts remains essentially the same T3 or T7 RNA polymerases (Pharmacia). After DNAase throughout development. Furthermore, Pax-6 is ex- digestion, probes were extracted with phenol and chloro- pressed in the developing pituitary, the olfactory form/isoamylalcohol and precipitated 3x with ammonium epithelium and in the developing eye in a pattern acetate. The probes were resuspended in 50% formamide, 10 mM DTT. suggestive of a regulatory role for Pax-6 in the Sections were prepared and hybridized essentially as development of the main structures of the eye. described (Hogan et al. 1986; Dony and Gruss, 1987) with some minor modifications. Sections (8/xm) were cut with a cryostat at -20°C and transferred onto slides subbed with gelatine and chrome alum. Sections were dried at 50°C, fixed in 4% paraformaldehyde (PFA) for 20min, rinsed with PBS Materials and methods and dehydrated in a graded ethanol series. Slides were air dried and stored at — 20°C until hybridized. Prior to Screening of cDNA libraries hybridization, sections were rehydrated in distilled water and Approximately 1.5 xlO6 clones of a AgtlO cDNA library incubated at 70 °C in 2xSSC for 30min. The following steps prepared from 8.5 day p.c. mouse embryos (Fahrner et al. were done at room temperature. After a second rinse with 1987) and 8X105 clones of a AgtlO cDNA library prepared water, sections were digested with 0.125 mg ml""1 pronase for from 11.5 day embryos (Clontech) were transferred to lOmin. The digestion was stopped in 0.2% for 30s. Hybond-N membranes and hybridized under high-stringency Slides were rinsed in phosphate-buffered saline (PBS), conditions with a random oligo-labeled (Feinberg and refixed in 4% PFA for 20min, again rinsed with PBS and Vogelstein, 1983) genomic 640bp HindUI fragment containing acetylated for lOmin in 0.1M triethanolamine with 1/400 the second of the Pax-6 paired box and flanking intron volume acetic anhydride. After rinsing with PBS, sections sequences (for description of the isolation of genomic clones were dehydrated and dried. The probe was diluted to of Pax-6 see Walther et al. (1991)). Hybridization was done SxKf'ctsmin"1 fA~l in a buffer containing 50% formamide, overnight at 65°C, in 4xSSC, 0.5% SDS, 5xDenhardts, 2 x SSC, 10 mM Tris, 10 mM NaPO4 pH 6.8, 5 mM EDTA, 10 % O.lmgml"1 denatured salmon sperm DNA, followed by a dextran sulfate, 150^/g tRNAml"1, 150/igml"1 denatured wash with 2xSSC, 0.1% SDS for 30min at 65°C and two salmon sperm DNA, 0.1 mM UTP, 10 mM S-ATP, lmM more washes with O.lxSSC, 0.1% SDS for 30min at 65°C. ADP/3S, 50 mM DTT and 10 mM 0-mercaptoethanol. The Positive clones were plaque purified and inserts were hybridization mix was boiled for 2min, directly applied onto subcloned into the Bluescript KS+ vector (Stratagene). the sections and covered with siliconized coverslips. Hybridiz- Pax-6, isolation and expression 1437

ation was done overnight in a humid chamber at 50°C, Pax-6 mRNA. It is also possible that this clone lacks followed by a wash in washing buffer (50% formamide, part of the 5' untranslated region, since a transcript of 2xSSC, 10 nun /S-mercaptoethanol) at 37 °C for 3h and a about 3kb is detected in northern blot analysis of RNAase digestion at 37°C for 15min. A second wash in embryonic mouse RNA (Fig. 4). washing buffer was done overnight followed by dehydration in graded ethanol. Slides were dipped in Kodak NTB-2 emulsion (diluted 1:1 with water) and exposed for 6-7 days at The deduced Pax-6 protein 4°C. Developing was done at room temperature in Kodak D- The two longest cDNA clones, cp64 and cp611, contain 19 for 3 min, followed by 1 min in 1 % acetic acid and 3 min in an open reading frame encoding a protein of 422 amino 30% sodium thiosulfate. After extensive rinses with water, acids or 436 amino acids, with the insertion, respect- sections were stained with Giemsa, dried and coverslips were ively. The predicted protein contains a paired domain applied. Photomicrographs were taken with a Leitz Labovert and a paired-type homeodomain (Fig. 2). The open bright-field/dark-field microscope. reading frame starts at position 46 and continues until a stop codon is encountered at position 1470 in Fig. 2. Only one in-frame translational start site, Results at position 163, is present between a stop codon at the 5' end of this reading frame and the paired box. The Isolation and structure of the Pax-6 cDNAs sequence surrounding this AUG (CCAGCAUGC) Initially Pax-6 was isolated by screening a genomic agTees well with the consensus sequence derived from library under low-stringency conditions with paired box eukaryotic translational initiation sites (CCPuC- probes of Pax-1, Pax-2, Pax-3 and gsb-d (Walther et al. CAUGG; (Kozak, 1986, 1987). Since this 5' region is 1991). Subsequently four overlapping cDNA clones colinear in three independent cDNA clones (cp61, cp64, cp61, cp611 and cp6111 (Fig. 1) were isolated cp611, cp64) and was also analysed in genomic clones from a 8.5 and a 11.5 (cp6111) day embryonic cDNA (not shown) a cDNA artefact can be excluded and this library using part of the Pax-6 paired box as a probe. AUG can be considered to represent the translational Of these clones, cp61 may represent a partially start site of the Pax-6 protein. This places the paired unspliced Pax-6 mRNA. Intron sequences are present domain very close to the amino terminus of the Pax-6 at the 5' untranslated end of this clone, as indicated by a protein, as seen with all other Pax . splice site consensus sequence and colinearity with the Comparison of the sequence of Pax-6 with other genomic sequence that is spliced out in the other clones published paired domains shows that the paired domain (not shown). At present, however, it cannot be of Pax-6 represents a rather divergent new class of excluded that clone cp61 could also represent part of an paired domain (Walther et al. 1991). The alternative splice product. Clone cp611 contains an identity ranges from 66 % with Pax-3 to 75 % with Pax- insertion of 42bp in the paired box, after nucleotide 2, which is significantly lower than between members of position 303. This insertion represents an additional the same paired domain class (Burri et al. 1989; exon as confirmed by analysis of the genomic sequence Goulding et al. 1991; Walther et al. 1991). Moreover, (not shown). Clone cp611 therefore represents an the sequence shows nonconservative amino acid alternative splice product of Pax-6. Otherwise, the four changes at a number of positions that are conserved in cDNA clones are completely colinear. The longest all other paired domains (Walther et al. 1991). cDNA clone, cp611 spans 2481bp of the Pax-6 mRNA. Remarkably, the additional 14 amino acids present in The complete nucleotide sequence of cp611 is shown in clone cp611 are inserted into the most conserved region Fig. 2. No putative polyadenylation signal is present at of the paired domain. the 3' end of this clone, which also lacks a poly(A) tract. The Pax-6 protein contains in addition to the paired Thus, clone cp611 does not contain the 3' end of the domain, a paired-type homeodomain, as seen with prd, gsb-d, gsb-p, Pax-3 and Pax-7 (Bopp et al. 1986; Goulding et al. 1991; Jostes et al. 1991). Again the homeodomain of Pax-6 is rather divergent (Fig. 3). Only 54-61 % of the amino acid sequence of Pax-6 is q>«4 identical to the Drosophila and the other murine paired-type homeodomains. In comparison, the amino cp«l acid identity between all other paired-type homeo- 31 domains ranges from 72 % (Pax-7/gsb-p) to 95 % (Pax-

303 3/Pax-7). The sequence of the predicted second helix of 24(1 the helix-turn-helix motif (HTH), the recognition helix, Fig. 1. The structure of the overlapping Pax-6 cDNA is highly conserved in all paired-type homeodomains, clones is outlined. The coding region is represented by the including Pax-6. Pax-6, however, shows a number of open box, the stippled area marks the paired domain and nonconservative amino acid changes in the first helix of the hatched area the paired-type homeodomain. The the HTH motif at positions that are conserved in all dashed line represents the region of clone cp61 5' from other paired-type homeodomains (Fig. 3). nucleotide position 31 in Fig. 2 that is not colinear with the other cDNA clones. The insertion in the paired box of The sequence homology between the Drosophila clone cp611 is indicated by a triangle. Restriction sites are: proteins and Pax-3 and Pax-7, is extended by 18 B, flg/II; E, EcoRl; N, Nhel. aminoacids at the amino terminal end of the homeo- 1438 C. Walther and P. Gruss

1 ACAACGACGAAAGAGAS^TGCCTCTTAAAGGCAGAAGACTTTAACC^ 90

91 GACGTATATCCCAGTTCTCAGAGCCCCGTATTCGAGCC^^ MSTCACAGC 180 1 MetGlnAsn 3erHisSer 6

181 270 GlyValAsnGlnLeuGlyGlyValPheValAsnGlyArgProLeuProAspSerThrArgGlnLysIleValGluLeuAlaHisSerGly 36

271 360 37 AlaArgProCysAspIleSerArgllGLeuGlnThrHisAlaAspAlaLysValGlnValLeuAspAsnGluAsnValSerAsnGlyCys 66

361 450 67 ValSerLysIleLeuGlyArgTyrTyrGluThrGlySerlleArgProArgAlalleGlyGlySerLysProArgValAlaThrProGlu 96

451 540 97 ValValSerLysIleAlaGlnTyrLysArgGluCysProSerllePheAlaTrpGluIleArgAspArgLeuLeuSerGluGlyValCys 126

541 630 127 ThrAsnAspAsnlleProSerValSerSerlleAsnArgValLeuArgAsnLeuAl, SerGluLysGlnGlnMetGlyAlaAspGlyMet 156

157 TyrAspLysLeuArgMetLeuAsnGlyGlnThrGlySerTrpGlyThrArgProGlyTrpTyrProGlyThrSerValProGlyGlnPro 186

187 ThrGlnAspGlyCysGlnGlnGlnGluGlyGlyGlyGluAsnThrAsnSerlleSerSerAsnGlyGluAspSerAspGluAlaGlnMet 216

811 CGACTTCAGCTGAAGCGGAACiriXX^^GAAATAGAACATCTTTT^ 900 217 ArgLeuGlnLeuLysArgLysLeuGlnArgAsnArgThrSerPheThrGlnGluGlnlleGluAlaLeuGluLysGluPheGluArgThr 246

901 CATTATCCAGATGTGTTTGCCCGGGAAAGACTAGCAGCCAAAATAGATCrACCTGAAGCAA& 990 247 HlsTyrProAspValPheAIaArgGluArgLeuAIaAIaLysIleAspLeuProGlviAlaArglleGlnValTrpPheSerAsnArgArg 276

991 GCCAAATGGAGAAGAGAAGAGAA? :TGAGGAACCAGAGAAGACAGGCCAGCAACACTCCTAGTCACATTCX;TATCAGCAGCAGCTTCAGT 1080 277 AlaLysTrpArgArgGluGluLys LeuArgAsnGlnArgArgGlnAlaSerAsnThrProSerHisIleProIleSerSerSerPheSer 306

307 ThrSerValTyrGlnProIleProGlnProThrThrProValSerSerPheThrSerGlySerMetLeuGlyArgThrAspThrAlaLeu 336

337 ThrAsnThrTyrSerAlaLeuProProMetProSerPheThrMetAlaAsnAsnLeuProMetGlnProProValProSerGlnThrSer 366

367 SerTyrSerCysMetLeuProThrSerProSerValAsnGlyArgSerTyrAspThrTyrThrProProHlsMetGlnThrHlsMetAsn 396

397 SerGlnProMetGlyThrSerGlyThrThrSerThrGlyLeuIleSerProGlyValSerValProValGlnValProGlySerGluPro 426

427 AspMetSerGlnTyrTrpProArgLeuGln 436

1621 GGGAATGGACTAGAACCAAGGACCTTGCGTACAGAAGGCAa3GTATCAGTTGGAA<^AAIXrr^^ 1710 1711 TTTGGTGTATTATTTGTAAATGGGCATTGGTATCTTATAATGA^ 1800 1801 GTGGTTGTTTAAAGGAAACCATGATCGACAACATTTGCCATGGAT^ 1890

2071 GGTAGAGTGTGTCTTCGATATAATACAATTTGTTTTATGTCAAAATGTAAGTATTTGTCTTCCCTAGAAATC^^ 2160

2251 CAAAAGTTAATTGTTCTTAGATATAGCTGTATTACTXnTCACAGTCCAATCATTTTGTGC^ 2340

24 31 GCCAAAGCCTTTGTATTTTCTTTATTACCCTTGACCGTAAGACATGAATTC 2481 Pax-6, isolation and expression 1439 Fig. 2. The DNA sequence and the deduced amino acid encompassing the more divergent 3' part of the paired sequence of the longest cDNA clone of Pax-6, cp611, are box. depicted. Paired domain and paired-type homeodomain are A single Pax-6 transcript of about 3kb is detected boxed. Stop codons and translation start site are from day 10.5 to day 15.5 of mouse embryogenesis underlined. The 42 nucleotide insertion, present only in this clone, is double underlined. (Fig. 4). Using the amount of Hras transcript (Leon et al. 1987) for standardization, Pax-6 mRNA is most abundant at day 10.5 p.c., with decreasing levels thereafter (Fig. 4). In adult brain, a faint signal, domain. In Pax-6, this extended homology is reduced to corresponding to a mRNA of about the same size as in six amino acids situated immediately adjacent to the the embryo, could be detected whereas adult , amino terminus of the homeodomain. Sequence com- heart, intestine, liver, lung, spleen, testis and uterus parison to Pax gene products that do not contain a show no Pax-6 expression even after prolonged paired-type homeodomain revealed that Pax-2 and Pax- exposure (not shown). 8 contain downstream of the paired domain a region The precise spatial and temporal expression pattern that is homologous to sequences in the first part of the during embryogenesis was analyzed by in situ hybridiz- Pax-6 homeodomain. This region includes the first hehx ation to sections from day 6.5 p.c. to day 13.5 p.c. as of the homeodomain. Within a region of 24 amino acids well as day 15.5 p.c, 16.5 p.c. and 18.5 p.c. embryos. (delineated by arrows in Fig. 3), 15 amino acids are Hybridization was performed with both sense and identical between Pax-6 and Pax-2 and 13 amino acids antisense RNA probes derived from the 3' part of the are identical between Pax-6 and Pax-8 (Fig. 3). If paired box. These probes had already been shown to be conservative amino acid exchanges are included the specific for Pax-6 in northern blot analysis. Moreover, it homology increases to 19/24 and 17/24 amino acids, has been shown that paired box probes even from respectively. highly homologous Pax genes do not cross hybridize A third conserved sequence motif, the octapeptide, is under the conditions employed (Plachov et al. 1990; usually present downstream of the paired domain Jostes etal. 1991). (Burn et al. 1989; Goulding et al. 1991; Jostes et al. No Pax-6 transcripts were detected in day 6.5 p.c. 1991). Pax-6, like prd, does not contain this motif. and day 7.5 p.c. embryos (not shown). Expression was As with all murine paired domain proteins analyzed first seen at day 8.0 of gestation and was confined to to date, the carboxy terminal region of the predicted ectodermal-derived structures throughout develop- Pax-6 protein is very rich in , and ment, the only exception being , where Pax-6 (45 %) (Chalepakis et al. 1991; Dressier et al. transcripts are seen in a small subset of cells (not 1990; Goulding et al. 1991; Plachov et al. 1990). A shown). search of the NBRF protein sequence bank with the regions outside of the two conserved domains revealed Expression in the central nervous system no significant homology to any known protein. Pax-6 expression was first detected in the presumptive forebrain and hindbrain at day 8.0 of gestation; Pax-6 is expressed during embryogenesis however, transcripts were absent from the presumptive In order to determine the expression pattern of Pax-6, met- and mesencephalon (Fig. 5B,D,F). This overall Poly(A)+ RNA from various adult tissues and from distribution of Pax-6 transcripts is maintained during embryos of day 10.5 to day 15.5 of gestation was development with the exception of the metencephalon examined by northern blot analysis, using a probe where expression is detected from day 15.5 of gestation

C Helix 1 Helix 2 J C Helix 3 )

ISSNGEDSDEAQMRLQLKRKLQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQVWFSNRRAKWRREEK Pax-6 PQ-DEGSDIDSEPD-P QR-S—T--A--L-E--RA IYT—E—QRAK-T V R—KQAG Pax-3 NRLDEGSGV-SEPD-P QR-S--T—A—L-E A IYT—E--QRTK-T—FV R—KQAG Pax-7 P-DEDISDC-SEPGIA QR-C—T-SAS-LDE—RA Q IYT--E--QRTN-T RL-KQHT prd GRDSDISDT-SEPGIP QR-S—T—A—L RA-S—Q YT—E—QTTA-T RL-KHSG gsb-p VG-ED-SE-D-EPSV QR-S--T-SND—D RI-A—Q YT—E--QTTA-T V RL-KQLN gab-d RKHLRADT Q-L DRV PS QASEHIKSEQ Pax-2 RKHLRTDT-S-HHL CP Q EAY-SPSHTKGEQ Pax-8

Fig. 3. Alignment of the amino acid sequences of all paired-type homeodomains described to date. The depicted partial sequences of Pax-2 and Pax-8 are located in the carboxyterminal region of the predicted protein sequence (Dressier et al. 1990; Plachov et al. 1990). The sequences shown include 20 amino acids aminoterminal of the homeodomain to illustrate the extended homologies. Dashes indicate amino acids that are identical with the amino acid of Pax-6 at this position. The ohelixes are illustrated at the top. The serine at position 9 of the recognition helix which is important for the sequence specificity of the DNA binding of the paired protein (Treisman et al. 1989) is marked by an asterix. Pax-3 (Goulding et al. 1991); Pax-7 (Jostes et al. 1990); gsb-d, gsb-p and prd (Bopp et al. 1986). 1440 C. Walther and P. Gruss

>r> sections of day 9.0 p.c. embryos were analyzed to © -H r-i f*5 ^f determine the onset of Pax-6 expression in the neural •G T3 "O "O T3 "O epithelium. As seen in day 8.5 p.c. embryos, Pax-6 is expressed in the closed neural tube but not in the adjacent mesoderm and the neural plate (Fig. 6A,B,C,D). Initially, at day 8.5 p.c, Pax-6 transcripts are present in a broad band of cells in the transverse plane of the neural tube, being absent only from the most dorsal and ventral cells (Fig. 6A,B). Beginning with day 9.0 of gestation cells in the dorsal neural tube appear to down-regulate Pax-6 since expression is confined to the ventral part of the neural 3 kb - tube, the basal plate (Fig. 6C,D). While high levels of Pax-6 transcripts are found in the basal plate, no sharp dorsal boundary of expression can be defined that would coincide with the dorsal border of the basal plate. Rather the signal extends dorsally into the alar plate where it is however weaker than in the basal plate (Fig. 6E-N). Floorplate and the cells directly adjacent to the floorplate do not express Pax-6 at any of the developmental stages examined (Fig. 6A-P). Fig. 6E,F also illustrate that Pax-6 expression in the basal plate starts to regress dorsally at day 10.5 p.c. when the ventral horns begin to form and before the ventricular zone (see below) starts to regress. At these early stages, the neural tube consists of a single layer of pseudostrati- fied mitotically active cells, the ventricular zone, which is radially arranged around the lumen (Sidman et al. 1959; Sauer, 1959). Between day 11 and 12 a second cell Hras layer, the intermediate zone, has formed lateral to the ventricular zone. It consists of differentiating postmi- ^ff ' • '- totic cells which migrate radially from the ventricular zone. In sections through thoracic and lumbar levels of Fig. 4. Northern blot analysis of Pax-6 expression during mouse development. Approximately 10 mg of the neural tube at day 11.5 p.c, Pax-6 transcripts are polyadenylated RNA of the embryonic stages indicated restricted to the ventricular zone and the expression were hybridized with a genomic Hindlll fragment pattern within this zone is identical to the previous day containing the second exon of the Pax-6 paired box (Fig. 6G,H). In cross sections from the cervical level of encoding amino acids 45-116 of the paired domain. The day 11.5 p.c. and in sections from day 12.5 p.c. control hybridization with a Hras probe is shown at the embryos, a second signal appears in the ventral bottom. intermediate zone, spreading radially from the ven- tricular zone (Fig. 6I,J, arrows). The signal corresponds to the spotted band of expression seen in the close-up of onwards. In the developing spinal cord, expression was a sagittal section of a day 12.5 p.c. embryo in Fig. 6K,L first seen coincident with neural tube closure (see (marked by arrows). Pax-6 expression in the basal plate below). No transcripts were detected in the neural has further regressed concordant with the reduction of groove (Fig. 5C,D). Pax-6 expression in the neural tube the ventricular zone. The same pattern is seen at day extends along the entire anteroposterior axis up to the 13.5 p.c. (Fig. 6M,N). Pax-6 expressing cells are still rhombencephalic isthmus (Fig. 5G,H and Fig. 7A,B) found at day 15.5 of gestation (Fig. 6O,P). By this time, and is mainly restricted to mitotically active cells in the virtually all neurons have been born (Nornes and Carry, ventral ventricular zone. These expression patterns will 1978) and regression of the ventricular zone is virtually be described in detail in the following sections. complete. Transcripts of Pax-6 are present in a subset of ependymal cells around the wider part of the residual Expression in the developing neural tube lumen of the neural tube, the neural canal, but not in The distribution of Pax-6 transcripts in the developing the ependymal layer lining the rest of the lumen. spinal cord was analyzed by examining cross sections of Another low signal is observed ventrally in a subset of day 8.5 p.c. to day 15.5 p.c. embryos. Pax-6 expression postmitotic cells (Fig. 60,P). was first observed in the closed neural tube at day 8.5 of gestation (Fig. 6A,B)- In more caudal regions of the embryo, no signal could be detected in the neural groove. Since the developing spinal cord shows a Expression in the developing brain rostral-caudal gradient of maturation (Nornes and The earliest expression of Pax-6 was observed at day 8.0 Das, 1974; Nornes and Carry, 1978), serial cross of gestation in the neuroepithelium of forebrain and Pax-6, isolation and expression 1441

Fig. 5. In situ expression analysis of Pax-6 at day 8.0, day 8.5 and day 10.5 of gestation. Pictures at the left (A,C,E, G) represent bright-field images of the embryos, pictures at the right (B,D,F,H) represent the corresponding dark-field images. (A) Transverse section of a day 8.0 p. c. embryo showing forebrain, midbrain and allantois (see Fig. 51). (B) Dark-field image of A. (C) Transverse section of the same embryo at a lower level, showing hindbrain and neural groove (see Fig. 51). (D) Dark-field image of C. (E) Parasagittal section of a day 8.5 p.c. embryo, anterior is to the left, posterior is to the right. (F) Dark-field image of E. The apparent gap of the hybridization signal between hindbrain and neural tube is due to the plane of the section and not to a gap of expression. (G) Parasagittal section of a day 10.5 p.c. embryo. (H) Dark-field image of G. The large arrow marks the limit of Pax-6 expression between the diencephalon and the mesencephalon. The small arrow indicates the expression of Pax-6 in the nasal pit. (I) Schematic representation of the levels of the sections in Fig. 5A-D. Abbreviations: al, allantois; f, forebrain; d, diencephalon; h, hb, hindbrain; m, midbrain; ms, mesencephalon, mt, metencephalon; my, myelencephalon; ng, neural groove; np, nasal pit; nt, neural tube; pr, preoptic recess; t, telencephalon.

5A.B

5C,D

hindbrain (Fig. 5A-D). This overall spatial expression pression in the metencephalon is first observed at day pattern is maintained during brain development from 15.5 p.c. of development (Fig. 7E,F). day 8.0 p.c. to day 18.5 p.c; Pax-6 transcripts are In the telencephalon, from day 10.5 p.c. to 18.5 p.c., present in the forebrain, the tel- and diencephalon, and high levels of transcript are detected in the lateral and in the myelencephalon, but not in the roof of the dorsal neural epithelium but not in the basal telen- mesencephalon (Fig. 5A-D and Fig. 7A-J). Ex- cephalon. The signal remains restricted to the ventricu- Fig. 6. Expression of Pax-6 in the developing neural tube analyzed by in situ hybridization. Photographs A,C,E, G,I,M,O show transverse sections of the neural tube at consecutively later stages of neural tube development, photographs B,D,F,H,J,N,P represent the corresponding dark-field images, respectively. (A,B) Day 8.5 p.c, (C,D) day 9.0, (E,F) day 10.5, (G,H) day 11.5, (I,J) day 12.5. (K,L) Close-up of a sagittal section of a day 12.5 p.c. embryo. Arrows point to the signal in postmitotic cells corresponding to the signal marked by arrows in the transverse section of a day 12.5 p.c. embryo in I,J. (M,N) Day 13.5, (O,P) day 15.5. Abbreviations: ap, alar plate; bp, basal plate; e, ependymal layer; dh, dorsal horns; fp, floorplate; i, intermediate zone; nt, neural tube; rf, roof plate; s, somite; v, ventricular zone; vh, ventral horns. Pax-6, isolation and expression 1443 lar zone throughout development (Fig. 5G,H and and the expression decreases together with the re- Fig. 7A-J). The olfactory bulbs develop as protrusions gression of the ventricular zone (Fig. 7A-J). from the rostro-ventral telencephalon and also express high levels of Pax-6 transcript (Fig. 7E-J). Pax-6 is expressed in the developing eye The expression domain extends from the telencepha- Pax-6 expression in the developing eye was first lon into the diencephalon up to a sharp caudal border at detected at day 8.5 p.c. in the optic sulcus, a lateral the posterior commissure, which delineates the bound- evagination at the base of the forebrain (not shown). By ary between diencephalon and mesencephalon. This day 9.5 p.c, this evagination has formed the optic caudal limit of expression persists through all develop- vesicle which approaches the surface ectoderm. Tran- mental stages analyzed (Fig. 5G,H; Fig. 7C-J; large scripts of Pax-6 are present in the epithelial layer of the filled arrows). A second boundary lies within the vesicle, the optic stalk and the overlying surface diencephalon and coincides with the sulcus diencephali- ectoderm, which will give rise to the lens (Fig. 8A-D). cus medius (Niimi et al. 1962) which separates ventral Between day 10 and day 12, the optic vesicle and dorsal thalamus. Beginning with day 10.5 p.c. (not invaginates, forming the optic cup, while the lens forms shown) before the intermediate and marginal zones by indentation of the thickened surface ectoderm, have formed, a strong hybridization signal is seen in the which eventually detaches from the surface. The ventral thalamus (Fig. 7A-D). By day 15.5 p.c, the invaginated neuroectoderm (inner layer and presump- size of the ventral thalamus and its germinal zone have tive neural retina), a mitotic highly active layer is become reduced and nucleus reticularis, zona incerta comparable in organization to the ventricular zone of and ventral lateral geniculate are differentiating in the the neural tube (Miale and Sidman, 1961; Pei and intermediate zone. Pax-6 is now expressed in a smaller Rhodin, 1970). Pax-6 is strongly expressed in the inner area probably corresponding to the presumptive zona layer of the cup and in the lens, whereas the outer layer incerta (Fig. 7E,F), where it is still detected at day 18.5 (presumptive pigmental retina), a zone of low mitotic p.c. (Fig. 7G-J), when neurogenesis has essentially activity, shows only a low level of Pax-6 expression been completed. A sharp drop in signal intensity is (Fig. 8E-L). In addition, Pax-6 transcripts are detected observed dorsal to the sulcus diencephalicus medius, in the overlying surface ectoderm which is part of the i.e., the dorsal thalamus shows a distinctly lower level future cornea (Fig. 8G-J, arrows). At day 12.5 p.c. the of expression (Fig. 7C,D; open arrow). This weaker hybridization pattern is essentially the same as on the expression seen in earlier stages (day 10.5 to day 12.5 previous day. The now differentiating lens fiber cells in p.c.) is no longer detected at day 15.5 p.c. (Fig. 7E,F). the posterior wall of the lens are still expressing Pax-6 Ventrally, a signal extending into the anterior hypo- and transcripts are still detected in a narrow ventricular thalamus in the region of the preoptic recess of the third zone extending along the optic stalk (Fig. 8K,L). By ventricle is observed at day 10.5 p.c. to day 12.5 p.c. day 15.5 p.c., most of the structures of the adult eye are (Fig. 7A-D). Pax-6 mRNA is also present in the present, the lids have developed as has the cornea, the epithalamus before the intermediate zone is formed at lens and the retina continue to differentiate. By this day 13 (Fig. 7A-D) and at later stages in differentiating time two layers have developed in the neural retina and structures such as the nuclei habenulae (Fig. 7E,F,I,J). can be clearly distinguished, an outer layer of high cell The pineal gland, an evagination of the epithalamus, density and mitotic active cells and an inner layer of expresses Pax-6 at all stages examined (Fig. 7E,F,I,J). lower cell density containing mainly postmitotic ganglion cells. Both layers express Pax-6 at about equal The metencephalon does not express Pax-6 at early amounts (Fig. 8M,N). Expression is also observed in stages (Fig. 7A-D). Expression is however seen at day the anterior epithelium of the cornea and in the lens 15.5 p.c. to 18.5 p.c. in the external granular layer (Fig. 8M,N). (EGL) and in cells distributed in the dorsal cerebellum (Fig. 7E,F). The EGL represents a second germinal Expression in the developing pituitary and the zone unique for the cerebellum, where mitotic activity olfactory epithelium increases while it is decreasing in the ventricular zone The anterior pituitary develops from an outpocketing of between day 13 and 15 of gestation (Miale and Sidman, the roof ectoderm of the stomodeum (Rathke's pouch) 1961). It is formed by cells that migrated from the in close association with the infundibulum. The rostral part of the rhombic lip of the myelencephalon to infundibulum is formed from an evagination of the floor the surface of the cerebellum. Cells of the EGL will give of the diencephalon which will give rise to the posterior rise to the granular layer of the adult cerebellum, where lobe (pars neuralis) of the mature pituitary. Beginning Pax-6 is expressed at low levels (not shown). Pontine with day 11.5 of gestation, Pax-6 transcripts are clearly nuclei, originating from cells from the more caudal part detectable in the developing adenohypophysis (anterior of the rhombic lip also show expression of Pax-6 pituitary). Expression of Pax-6 is restricted to Rathke's (Fig. 7E-J), as well as inferior olivary nuclei (not pouch and its derivatives, the pars intermedia and shown). Both structures relay information to the distalis. No transcripts could be detected in the cerebellum. infundibulum and the differentiating pars neuralis in the The expression pattern of Pax-6 in the myelencepha- developmental stages examined (day 10.5 p.c. to day lon is essentially the same as in the spinal cord. 18.5 p.c; Fig. 7C-L). Peak levels of Pax-6 expression Transcripts are most abundant in the ventricular zone occur at day 12.5 p.c. with decreasing signal intensity 1444 C. Walther and P. Gruss

Fig. 7. Expression of Pax-6 in the developing brain. The left panel of photographs shows the bright-field image, the right panel the corresponding dark-field image of the head region of a (A,B) day 11.5 p.c. embryo; (C,D) day 12.5 p.c. embryo, the open arrow marks the sulcus diencephalicus medius; (E,F) day 15.5 p.c. embryo; (G,H) day 16.5 p.c. embryo; (I,J) day 18.5 p.c. embryo. (K,L) Qose up of a sagittal section through the head of a day 11.5 p.c. embryo. Abbreviations: c, cerebellum; d, diencephalon; dt, dorsal thalamus; h, nuclei habenulae; i, infundibulum; e, epiphysis (pineal gland); egl, external granular layer; et, epithalamus; ms, mesencephalon; mt, metencephalon; my, myelencephalon; n, pontine nuclei; nc, nasal cavity; o, olfactory lobe; p, anterior pituitary; pr, preoptic recess; t, telencephalon; vt, ventral thalamus. thereafter (Fig. 7C-L). By day 15.5 of gestation, still observed in these structures at day 18.5/?.c. shortly before all three parts begin differentiating, weak (Fig. 71 J). labelling remains in the epithelium lining the remaining As evident from Fig. 5G,H and Fig. 7A-J, Pax-6 is lumen of Rathke's pouch and in the anterior lobe also expressed in the developing olfactory epithelium of (Fig. 7E,F). Expression just above background level is the nose from the relative primitive olfactory pit stage Pax-6, isolation and expression 1445

at day 10.5 to the adult-like nasal structure at day 18.5 suggests that Pax-6 acts as a transcriptional regulator by (Fig. 5G,H; Fig. 71,J). binding to DNA. Sequence comparison of the Pax-6 paired domain and paired-type homeodomain with other known domains of these types reveals that both Discussion are rather divergent. They show nonconservative amino acid exchanges at a number of positions which are The Pax-6 gene is a divergent member of the murine conserved in all other paired domains or paired-type paired box gene family (Walther et al. 1991). It encodes homeodomains. Pax-6, however, contains a glycine at a paired-type homeodomain in addition to the paired position 15 of the paired domain, like all other paired domain. Hence the Pax-6 protein contains two DNA- domains described to date. Replacement of this glycine binding domains as seen with Pax-3, Pax-7, prd, gsb-p to a serine as seen with Pax-1 in the mouse mutant un, and gsb-d (Goulding et al. 1991; Jostese/a/. 1991; Bopp results in a dramatic decrease in DNA-binding affinity, et al. 1986). The presence of these DNA-binding motifs concommitant with a reduced transcriptional activation 1446 C. Walther and P. Gruss

Fig. 8. Expression of fta-<5 in the developing eye. Close-ups of sections through embryos at: (A,B) day 9.0 p.c., arrow points to signal in the surface ectoderm; (C,D) day 9.5 p.c, (E,F) day 10.5 p.c, (G, H) day 11.5 p.c, arrow points to signal in the surface ectoderm; (I,J) day 11.5 p.c; (K,L) day 12.5 p.c; (M,N) day 15.5 p.c, the signal in the pigment layer of the retina is an artefact, it is also seen with the sense probe; (O,P) Close-up of a horizontal section of a day 15.5 p.c. embryo, showing the nasal structure; a, anterior; p, posterior. Abbreviations: a, anterior; ac, anterior epithelium of the cornea; f, forebrain; il, inner layer of the neural retina; le, lens; li, lid; nr, neural retina; oc, optic cup; ol, outer layer of the neural retina; os, optic stalk; ov, optic vesicle; p, posterior; pr, pigment layer of the retina; oe, olfactory epithelium. ability (Chalepakis et al. 1991). The serine at position 9 many positions from the other paired domain proteins of the recognition helix of the homeodomain, which has (Walther et al. 1991), suggesting that Pax-6 might have a been shown to be important for the binding specificity different DNA-binding specificity to the other paired of the paired protein (Treisman et al. 1989), is also domain proteins. This is supported by our preliminary conserved in Pax-6. Experiments using deletion mu- experiments (C.W., unpublished). tants or mutated paired domains have demonstrated Pax-6 is also expressed in a variant form which that the first part of the paired domain is required for contains an insertion of fourteen amino acids in the DNA binding and is sufficient for conferring DNA- most highly conserved region of the paired domain. The binding specificity (Treisman et al. 1991; Chalepakis et potential importance of this alternative form of the Pax- al. 1991). The sequence of Pax-6 in this region differs at 6 protein is highlighted by its conservation during Pax-6, isolation and expression 1447 evolution, being also found as a product of the zebrafish polarizing effect on the neural tube, by controlling at pax-6 gene (A. Piischel, pers. communication). At least in part the neuronal differentiation pattern along present the function of this insertion is unclear. In the the D-V axis (van Straaten et al. 1989; Placzek et al. context of the DNA-binding ability of the paired 1990; Hirano et al. 1991; Yamada et al. 1991). The domain, it is, however, tempting to speculate that this mechanisms by which floorplate and notochord exert alternative form might change the DNA-binding their polarizing activity is yet unclear. Possible candi- properties of Pax-6. dates for morphogenetic signals emanating from these To date no other murine gene has been isolated that structures are , which are possibly present in shows such extensive sequence homology to Pax-6, as notochord (Hornbruch and Wolpert, 1986) and floor- seen between the paralogous genes Pax-3/Pax-7 and plate (Wagner et al. 1990) or growth factors like stem Pax-21Pax-5/Pax-8 which are likely to have been arisen cell (SCF) shown to be expressed in the by duplication. Nevertheless, Pax-4 shows the same floorplate (Matsui etal. 1990; Keshet etal. 1991). Since genomic structure as Pax-6 (as analyzed so far) and Pax-6 is expressed early in neural tube development contains a number of amino acids that are unique for before neural differentiation begins, Pax-6 could be Pax-6 and Pax-4 (Walther etal. 1991). In addition, Pax- involved in interpreting such a signal into positional 4 encodes a homeodomain that at least in part is highly information within the neural tube, thereby establishing homologous to the homeodomain of Pax-6 (M. Asano the ventral character of the neural tube and being and P. Gruss, unpublished results). Pax-6 and Pax-4 involved in the generation of motorneurons. Ex- therefore, might have been derived from the same pression could be regulated in a position- and/or ancestral gene by duplication early in evolution which concentration-dependant manner, the threshold level could account for the high degTee of sequence of required for transcription of Pax-6 divergence of their paired boxes. roughly coinciding with the position of the border Sequence comparison of the entire Pax-6 protein with between basal and alar plate. This is at least partly all other Pax proteins revealed that sequences in Pax-2 supported by recent experiments which have demon- and Pax-8 downstream of the paired domain are strated that the notochord influences the expression of homologous to the first part of the Pax-6 homeodomain Pax-6. In the chicken, transplantation of an additional including the first helix. This raises the intriguing notochord lateral to the neural tube changes the possibility that Pax-2 and Pax-8 have a rudimentary expression domain of the chicken homolog of Pax-6 (M. homeodomain which has lost the helix-turn-helix motif. Goulding, A. Lumbsden and P. Gruss, unpublished This part of the homeodomain might have been deleted results). Another murine Pax gene, Pax-3, is expressed in the duplication or exon shuffling events which in the dorsal part of the neural tube beginning with day generated the Pax genes or alternatively was randomly 8.5 of gestation and expression of the chicken homolog mutated during evolution. In any case, it will be of Pax-3 is also influenced by the notochord. (Goulding interesting to see which function this truncated homeo- etal. 1991) (M. Goulding, A. Lumbsden and P. Gruss, domain has aquired or retained. unpublished results). The expression domain of Pax-6 extends into the alar plate, which generates a zone at The expression pattern of Pax-6 during embryogen- the border of alar and basal plate where the expression esis was examined by northern and in situ analysis. Pax- of Pax-6 and Pax-3 overlaps. This raises the possibility 6 is expressed in distinct regions of the brain beginning that the Pax-3 gene product could act directly or at day 8 of gestation, in the neural tube, in the indirectly on Pax-6 and/or the Pax-6 gene product on developing eye, in the pituitary, the olfactory bulbs and Pax-3, thereby probably defining ventral and dorsal of in the olfactory epithelium. the neural tube. A third Pax gene, Pax-7, shows a In the developing neural tube, Pax-6 transcripts are similar expression pattern in the developing nervous detected in the ventral ventricular zone but not in floor system as Pax-3 (Jostes et al. 1991) and might therefore plate cells and cells immediately adjacent to the floor be also involved in the generation of the ventral-dorsal plate. Expression is detected prior to neural differen- polarity of the neural tube. In contrast, Pax-2 and Pax-8 tiation and declines with the regression of the ventricu- begin to be expressed at later stages of neural tube lar zone. At early stages in development, the spinal development in discrete subsets of postmitotic cells and cord represents a pseudostratified epithelium of mitoti- thus are not involved in these early steps of neural tube cally active cells which are radially arranged around the development. lumen (Sidman etal. 1959; Sauer, 1959). Cells that have undergone their final mitosis and start to differentiate In the brain, Pax-6 expression extends from the most migrate radially out of the ventricular zone and settle in rostral part of the CNS, the telencephalon, to the the adjacent intermediate zone. A dorsal-ventral myelencephalon, sparing the roof of the mesencepha- (D-V) polarity within the neural tube becomes evident lon and the early metencephalon. This overall spatial during this process since functionally different neurons pattern of expression of Pax-6 remains essentially the originate from distinct regions (Jacobson, 1985). Motor same during brain development with the exception of neurons for example arise from the ventral part the metencephalon where expression is detected from whereas interneurons of the dorsal horns and neurons day 15.5 p.c. onwards. Pax-6 is expressed in the of the sensory pathway originate primarily from the forebrain and the hindbrain at day 8.0 p.c. and dorsal part of the neural tube (Altman and Bayer, continues to be expressed in the telencephalon and the 1984). Notochord and floorplate were shown to have a diencephalon which develop from the forebrain, and in 1448 C. Walther and P. Gruss the myelencephalon which develops from the hind- 1989). Thus the instructive inductive events (Gurdon, brain. Especially striking is the sharp limit of expression 1987) act before the optic vesicle is formed, which then at the border between diencephalon and mesencepha- induces the determined lens ectoderm to become lens. lon corresponding to a well-studied boundary between In addition, the retina seems to be important for these two regions of the brain (Vaage, 1969; Keyser, growth, differentiation and maintenance of the lens. A 1972; Puelles et al. 1987). Within the diencephalon, the similar multistep process seems to be required for expression domain of Pax-6 matches another regional cornea formation where in addition the lens is necessary boundary, the zona limitans intrathalamica which during a certain period in development (Zak and delineates the border between ventral and dorsal Linsenmayer, 1985) and at later stages for maintenance thalamus (Niimi et al. 1962) and which corresponds to of the cornea (Hay and Revel, 1969). Pax-6 is first the interparencephalic neuromeric boundary of Puelles expressed at day 8.0 of gestation, i.e. not during the et al. (1987) and the reticular protuberance of Altaian early stages of gastrulation and neurulation, making it and Bayer (1986). Interestingly, the murine Distal-less unlikely that Pax-6 is involved in the initial steps of related gene, Dlx (Price et al. 1991), and two members induction. The expression pattern rather suggests that of the Wnt family, Wnt-3 and Wnt-3A (Roelink and Pax-6 is involved in the regulation of the following Nusse, 1991), show also a restricted expression pattern inductive events. In this context, it is noteworthy that with respect to this boundary. A number of other genes Pax-6 is expressed in the inducing as well as in the is also expressed in distinct regions of the brain during responding tissue. In addition, Pax-6 might be required development, e.g. the Hox genes in the hindbrain (for for growth, differentiation and maintenance of the lens review see Kessel and Gruss (1990)), En-1 and En-2 in and the cornea. the midbrain-hindbrain region (Davis and Joyner, In summary, Pax-6 represents a rather divergent 1988; Davis et al. 1988; Davidson et al. 1988) and Wnt- member of the paired box gene family, encoding a 3A also in the telencephalon (Roelink and Nusse, potential . Pax-6 is the earliest 1991). Two other Pax genes, Pax-3 and Pax-7, show expressed Pax gene described so far and is in contrast to expression in all brain regions at early stages in all other Pax genes not expressed in segmented development (day 8.5 p.c), the expression, however, structures during murine embryogenesis. The ex- regresses posteriorly during development. Thus, Pax-6 pression pattern suggests a regulatory role for Pax-6 in in combination with other genes generates overlapping the development of the CNS and the eye. or complementary expression domains in the brain in a similar way as in the transverse plane of the neural tube We thank K. Fahrner and B. Hogan for their generous gift or as the Hox genes along the anteroposterior axis. The of the cDNA library and R. AltschSffel for photograghic combination of the gene products may define subdiv- work. We are grateful to Dr Riekmann for his help with the isions in the brain at early stages and might later be anatomy of the brain and we would also like to thank M. required for refinement and differentiation of these Goulding and particularly R. Fritsch for helpful discussions subdivisions. As in the spinal cord Pax-6 expression is and comments on the manuscript. We thank A. Ptischel for not exclusively restricted to mitotic active cells in the communication of unpublished results and continous dis- ventricular zone. Expression is still detected at day 18.5 cussion. C.W. is supported by a Boehringer Ingelheim Fond Fellowship. This project was supported by the Max Planck in the ventral thalamus, epithalamus and myelencepha- Society and Deutsche Forschungsgemeinschaft grant No. En lon when mitosis has ceased (Gardette et al. 1982). Thus 84/18-4. certain cell populations retain or reinitiate expression of Pax-6 after their final mitosis. References Pax-6 expression in the eye was examined from day 8.5 to day 15.5 of gestation. During this developmental AKAM, M. (1987). The molecular basis for metameric pattern in period, the main structures of the eye are formed. the Drosophila embryo. Development 101, 1-22. Expression of Pax-6 is first detected at day 8.5 in the ALTMAN, J. AND BAYER, S. A. (1984). The development of the rat spinal cord. 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