Pax-6, a murine paired box gene, is expressed in the ... › content › develop › 113 › 4 › 1435...Pax-6, isolation and expression 1437 ation was done overnight in a humid chamber

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

1435

Pax-6, a murine paired box gene, 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 genes (Paxgenes) has been identified in the mouse. In this report,we describe the expression pattern of Pax-6 duringembryogenesis and the isolation of cDNA clones span-ning the entire coding region. The Pax-6 protein consistsof 422 amino acids as deduced from the longest openreading frame and contains, in addition to the paireddomain, a paired-type homeodomain. Beginning withday 8 of gestation, Pax-6 is expressed in discrete regions

of the forebrain and the hindbrain. In the neural tube,expression is mainly confined to mitotic active cells in theventral ventricular zone along the entire anteroposterioraxis starting at day 8.5 of development. Pax-6 is alsoexpressed in the developing eye, the pituitary and thenasal epithelium.

Key words: paired box, homeobox, Pax, CNSdevelopment, eye development, embryogenesis.

Introduction

The fruit fly Drosophila has proved a powerful modelfor deciphering the complex genetic program underly-ing development. A large number of developmentalcontrol genes of Drosophila have been isolated andgenetically analyzed. These studies have revealed anarray of hierachical and combinatorial interactions oftheir gene products that are required for controllingpattern formation and morphogenesis (for reviews seeAkam (1987); Scott and Carroll (1987); Ingham (1988)).Many of these genes contain conserved sequence motifssuch as homeobox, zinc-finger motif, helix-loop-helixmotif and paired box and these in turn have been usedto isolate potential developmental control genes from avariety of vertebrate species (for review see Scott et al.(1989); Kessel and Gruss (1990)). Like their Drosophilacounterparts, many of these vertebrate genes appear toplay a role in controlling pattern formation andmorphogenesis during development (Kessel et al. 1990;Wright etal. 1989).

The paired box was originally discovered in theDrosophila segmentation genes paired, gooseberry-distal (gsb-d) and gooseberry-proximal (gsb-p) (Bopp etal. 1986; Baumgartner et al. 1987) and subsequently intwo developmental^ regulated tissue-specific genes ofDrosophila, Pox neuro and Pox meso (Bopp et al.1989). The paired box has been highly conserved duringevolution, being present in such divergent organisms asnematodes, zebrafish, Xenopus, chicken and man(Dressier et al. 1988; Burn et al. 1989). It encodes aprotein domain of 128 amino acids which has beenshown to be a novel DNA-binding motif (Treisman etal. 1991; Chalepakis etal. 1991; Goulding etal. 1991).

Based on sequence homology to the Drosophila

gsb-d paired box, a murine multigene family of paired-box-containing genes (Pax genes) of to date eightmembers has been isolated (Deutsch et al. 1988;Dressier et al. 1990; Plachov et al. 1990; Jostes et al.1991; Goulding et al. 1991; Walther et al. 1991). All Paxgenes described to date exhibit a temporally andspatially restricted expression pattern during embryo-genesis, compatible with a regulatory role in vertebratedevelopment. The Pax genes, with the exception ofPax-1, are expressed in the developing nervous system.Expression is detected along the entire anteroposterioraxis of the neural tube and the hindbrain where it isconfined in the transverse plane to distinct regions. Pax-3 transcripts are already detected at day 8.5 of gestationand are restricted to mitotic cells in the dorsal half ofthe neural tube, the alar and roof plate (Goulding et al.1991). Pax-7 shows a similar expression pattern exceptthat it is not expressed in the most dorsal part of theneural tube (Jostes et al. 1991). Pax-2 and Pax-8 areexpressed later in neurogenesis beginning with day 10and day 11 of gestation, respectively. Transcripts aredetected in discrete subpopulations of postmitotic cellswithin the alar and basal plate (Nornes et al. 1990;Plachov et al. 1990).

The Pax genes were cloned on the basis of theirhomology to a sequence motif found in segmentationgenes of Drosophila, which are involved in building themetameric body plan. Segmentation is also an import-ant feature of vertebrate embryogenesis (Hogan et al.1985) and all the Pax genes described to date areexpressed in segmented structures such as the differen-tiating somites or the developing excretory system. Inthe mouse, however, paired-box-containing genes donot seem to be involved in the generation of segmentalstructures but rather in their differentiation since

1436 C. Walther and P. Gruss

expression begins only after these structures havealready formed (Deutsch et al. 1988; Dressier et al.1990; Plachov et al. 1990; Jostes et al. 1991; Goulding etal. 1991).

The potential developmental importance of membersof the Pax gene family is further highlighted by thecorrelation of a Pax gene with the developmentalmutant undulated (un). Beginning with day 9 ofgestation, Pax-1 is expressed in the sclerotome of thedifferentiating somites and at later stages in theintervertebral discs anlagen of the developing vertebralcolumn. A point mutation in the Pax-1 gene causing areduction of DNA-binding affinity (Chalepakis et al.1991) disturbs the normal development of the vertebralcolumn and was shown to be the cause for the unphenotype (Balling et al. 1988).

In this report, we present the isolation of cDNAsencompassing the complete coding region of the Pax-6protein and a detailed analysis of the expression patternof Pax-6 during embryogenesis. Pax-6 is anothermember of the murine paired-box-containing genefamily, which however encodes a rather divergentpaired domain and paired-type homeodomain. Theexpression pattern of Pax-6 also differs in severalaspects. Unlike other Pax genes, Pax-6 is not expressedin segmented mesodermal structures. Pax-6 also is thefirst Pax gene described to be expressed in the ventralneural tube before neural differentiation starts. Theexpression pattern of Pax-6 in the neural tube iscompatible with a role for Pax-6 in the regionalspecification of cells in the neural tube with respect tothe dorsal-ventral axis. Transcripts of Pax-6 are alsopresent in distinct regions of the developing brain, butin contrast to Pax-3 and Pax-7, the overall spatialdistribution of transcripts remains essentially the samethroughout development. Furthermore, Pax-6 is ex-pressed in the developing pituitary, the olfactoryepithelium and in the developing eye in a patternsuggestive of a regulatory role for Pax-6 in thedevelopment of the main structures of the eye.

Materials and methods

Screening of cDNA librariesApproximately 1.5 xlO6 clones of a AgtlO cDNA libraryprepared from 8.5 day p.c. mouse embryos (Fahrner et al.1987) and 8X105 clones of a AgtlO cDNA library preparedfrom 11.5 day embryos (Clontech) were transferred toHybond-N membranes and hybridized under high-stringencyconditions with a random oligo-labeled (Feinberg andVogelstein, 1983) genomic 640bp HindUI fragment containingthe second exon of the Pax-6 paired box and flanking intronsequences (for description of the isolation of genomic clonesof Pax-6 see Walther et al. (1991)). Hybridization was doneovernight at 65°C, in 4xSSC, 0.5% SDS, 5xDenhardts,O.lmgml"1 denatured salmon sperm DNA, followed by awash with 2xSSC, 0.1% SDS for 30min at 65°C and twomore washes with O.lxSSC, 0.1% SDS for 30min at 65°C.Positive clones were plaque purified and inserts weresubcloned into the Bluescript KS+ vector (Stratagene).

DNA sequence analysisOverlapping subclones of the Pax-6 cDNA clones weregenerated in M13 mpl8 and mpl9 vectors. Single-strandedDNA was prepared and sequenced with the dideoxy method(Sanger et al. 1977) using either Sequenase (US Biochemicals)or T7 kits (Pharmacia). Computer analysis of the sequencewas performed using the GCG program package (Devereux etal. 1984).

Northern analysisEmbryos were obtained from natural matings of femaleNMRI and male C57BL/6 mice and the day of the vaginalplug was designated day 0.5. Tissues were isolated from adultfemale NMRI mice. Embryos and tissues were frozen in liquidnitrogen, homogenized in guanidinium thiocyanate (Chirgwinet al. J;979)'ahd poly(A)+ RNA was prepared using oligo(dT)-cellulose columns according to Ausubel et al. (1987). Samplescontaining approximately 10 ug of denatured poly(A)+ RNAwere electrophoresed on 1% agarose/formaldehyde gels,transferred to Hybond-N membranes using IOXSSC andcrosslinked under UV light. The filters were hybridized withthe random oligo-labeled (Feinberg and Vogelstein, 1983)genomic HindUI fragment of Pax-6 (see screening of cDNAlibraries) under the following conditions: 7.5% dextransulfate, 5xSSC, 5xDenhardts, 50% formamide (Fluka), 1 %SDS, 10mM Tris-HCl pH7.5, 0.1 mM sodium pyrophosphate,0.1 mM ATP and 0.1 mgml"1 denatured salmon sperm DNAat 65°C for at least 16h. Filters were washed in 2xSSC, 1 %SDS at 65°C, followed by two washes in O.lxSSC, 0.1 % SDSat 65°Cfor30min.

In situ hybridizationRadioactive RNA probes were generated by in vitrotranscription from a linearized plasmid clone containing an•EcoRl-AT/eZ-cDNA-fragment encoding most of the 3' partof the Pax-6 paired box (see Fig. 1) using 100 /*Ci 35S-CTP andT3 or T7 RNA polymerases (Pharmacia). After DNAasedigestion, probes were extracted with phenol and chloro-form/isoamylalcohol and precipitated 3x with ammoniumacetate. The probes were resuspended in 50% formamide,10 mM DTT.

Sections were prepared and hybridized essentially asdescribed (Hogan et al. 1986; Dony and Gruss, 1987) withsome minor modifications. Sections (8/xm) were cut with acryostat at -20°C and transferred onto slides subbed withgelatine and chrome alum. Sections were dried at 50°C, fixedin 4% paraformaldehyde (PFA) for 20min, rinsed with PBSand dehydrated in a graded ethanol series. Slides were airdried and stored at — 20°C until hybridized. Prior tohybridization, sections were rehydrated in distilled water andincubated at 70 °C in 2xSSC for 30min. The following stepswere done at room temperature. After a second rinse withwater, sections were digested with 0.125 mg ml""1 pronase forlOmin. The digestion was stopped in 0.2% glycine for 30s.Slides were rinsed in phosphate-buffered saline (PBS),refixed in 4% PFA for 20min, again rinsed with PBS andacetylated for lOmin in 0.1M triethanolamine with 1/400volume acetic anhydride. After rinsing with PBS, sectionswere dehydrated and dried. The probe was diluted toSxKf'ctsmin"1 fA~l in a buffer containing 50% formamide,2 x SSC, 10 mM Tris, 10 mM NaPO4 pH 6.8, 5 mM EDTA, 10 %dextran sulfate, 150 /̂g tRNAml"1, 150/igml"1 denaturedsalmon sperm DNA, 0.1 mM UTP, 10 mM S-ATP, lmMADP/3S, 50 mM DTT and 10 mM 0-mercaptoethanol. Thehybridization mix was boiled for 2min, directly applied ontothe sections and covered with siliconized coverslips. Hybridiz-

Pax-6, isolation and expression 1437

ation was done overnight in a humid chamber at 50°C,followed by a wash in washing buffer (50% formamide,2xSSC, 10 nun /S-mercaptoethanol) at 37 °C for 3h and aRNAase digestion at 37°C for 15min. A second wash inwashing buffer was done overnight followed by dehydrationin graded ethanol. Slides were dipped in Kodak NTB-2emulsion (diluted 1:1 with water) and exposed for 6-7 days at4°C. Developing was done at room temperature in Kodak D-19 for 3 min, followed by 1 min in 1 % acetic acid and 3 min in30% sodium thiosulfate. After extensive rinses with water,sections were stained with Giemsa, dried and coverslips wereapplied. Photomicrographs were taken with a Leitz Labovertbright-field/dark-field microscope.

Results

Isolation and structure of the Pax-6 cDNAsInitially Pax-6 was isolated by screening a genomiclibrary under low-stringency conditions with paired boxprobes of Pax-1, Pax-2, Pax-3 and gsb-d (Walther et al.1991). Subsequently four overlapping cDNA clonescp64, cp61, cp611 and cp6111 (Fig. 1) were isolatedfrom a 8.5 and a 11.5 (cp6111) day embryonic cDNAlibrary using part of the Pax-6 paired box as a probe.

Of these clones, cp61 may represent a partiallyunspliced Pax-6 mRNA. Intron sequences are presentat the 5' untranslated end of this clone, as indicated by asplice site consensus sequence and colinearity with thegenomic sequence that is spliced out in the other clones(not shown). At present, however, it cannot beexcluded that clone cp61 could also represent part of analternative splice product. Clone cp611 contains aninsertion of 42bp in the paired box, after nucleotideposition 303. This insertion represents an additionalexon as confirmed by analysis of the genomic sequence(not shown). Clone cp611 therefore represents analternative splice product of Pax-6. Otherwise, the fourcDNA clones are completely colinear. The longestcDNA clone, cp611 spans 2481bp of the Pax-6 mRNA.The complete nucleotide sequence of cp611 is shown inFig. 2. No putative polyadenylation signal is present atthe 3' end of this clone, which also lacks a poly(A) tract.Thus, clone cp611 does not contain the 3' end of the

q>«4

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Fig. 1. The structure of the overlapping Pax-6 cDNAclones is outlined. The coding region is represented by theopen box, the stippled area marks the paired domain andthe hatched area the paired-type homeodomain. Thedashed line represents the region of clone cp61 5' fromnucleotide position 31 in Fig. 2 that is not colinear with theother cDNA clones. The insertion in the paired box ofclone cp611 is indicated by a triangle. Restriction sites are:B, flg/II; E, EcoRl; N, Nhel.

Pax-6 mRNA. It is also possible that this clone lackspart of the 5' untranslated region, since a transcript ofabout 3kb is detected in northern blot analysis ofembryonic mouse RNA (Fig. 4).

The deduced Pax-6 proteinThe two longest cDNA clones, cp64 and cp611, containan open reading frame encoding a protein of 422 aminoacids or 436 amino acids, with the insertion, respect-ively. The predicted protein contains a paired domainand a paired-type homeodomain (Fig. 2). The openreading frame starts at nucleotide position 46 andcontinues until a stop codon is encountered at position1470 in Fig. 2. Only one in-frame translational start site,at position 163, is present between a stop codon at the 5'end of this reading frame and the paired box. Thesequence surrounding this AUG (CCAGCAUGC)agTees well with the consensus sequence derived fromeukaryotic translational initiation sites (CCPuC-CAUGG; (Kozak, 1986, 1987). Since this 5' region iscolinear in three independent cDNA clones (cp61,cp611, cp64) and was also analysed in genomic clones(not shown) a cDNA artefact can be excluded and thisAUG can be considered to represent the translationalstart site of the Pax-6 protein. This places the paireddomain very close to the amino terminus of the Pax-6protein, as seen with all other Pax proteins.

Comparison of the sequence of Pax-6 with otherpublished paired domains shows that the paired domainof Pax-6 represents a rather divergent new class ofpaired domain (Walther et al. 1991). The amino acididentity ranges from 66 % with Pax-3 to 75 % with Pax-2, which is significantly lower than between members ofthe same paired domain class (Burri et al. 1989;Goulding et al. 1991; Walther et al. 1991). Moreover,the sequence shows nonconservative amino acidchanges at a number of positions that are conserved inall other paired domains (Walther et al. 1991).Remarkably, the additional 14 amino acids present inclone cp611 are inserted into the most conserved regionof the paired domain.

The Pax-6 protein contains in addition to the paireddomain, 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 thehomeodomain of Pax-6 is rather divergent (Fig. 3).Only 54-61 % of the amino acid sequence of Pax-6 isidentical to the Drosophila and the other murinepaired-type homeodomains. In comparison, the aminoacid identity between all other paired-type homeo-domains ranges from 72 % (Pax-7/gsb-p) to 95 % (Pax-3/Pax-7). The sequence of the predicted second helix ofthe helix-turn-helix motif (HTH), the recognition helix,is highly conserved in all paired-type homeodomains,including Pax-6. Pax-6, however, shows a number ofnonconservative amino acid changes in the first helix ofthe HTH motif at positions that are conserved in allother paired-type homeodomains (Fig. 3).

The sequence homology between the Drosophilaproteins and Pax-3 and Pax-7, is extended by 18aminoacids at the amino terminal end of the homeo-


1 ACAACGACGAAAGAGAS^TGCCTCTTAAAGGCAGAAGACTTTAACC^ 90

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157 TyrAspLysLeuArgMetLeuAsnGlyGlnThrGlySerTrpGlyThrArgProGlyTrpTyrProGlyThrSerValProGlyGlnPro 186

187 ThrGlnAspGlyCysGlnGlnGlnGluGlyGlyGlyGluAsnThrAsnSerlleSerSerAsnGlyGluAspSerAspGluAlaGlnMet 216

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307 ThrSerValTyrGlnProIleProGlnProThrThrProValSerSerPheThrSerGlySerMetLeuGlyArgThrAspThrAlaLeu 336

337 ThrAsnThrTyrSerAlaLeuProProMetProSerPheThrMetAlaAsnAsnLeuProMetGlnProProValProSerGlnThrSer 366

367 SerTyrSerCysMetLeuProThrSerProSerValAsnGlyArgSerTyrAspThrTyrThrProProHlsMetGlnThrHlsMetAsn 396

397 SerGlnProMetGlyThrSerGlyThrThrSerThrGlyLeuIleSerProGlyValSerValProValGlnValProGlySerGluPro 426

427 AspMetSerGlnTyrTrpProArgLeuGln 436

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2251 CAAAAGTTAATTGTTCTTAGATATAGCTGTATTACTXnTCACAGTCCAATCATTTTGTGC^ 2340

24 31 GCCAAAGCCTTTGTATTTTCTTTATTACCCTTGACCGTAAGACATGAATTC 2481


Fig. 2. The DNA sequence and the deduced amino acidsequence of the longest cDNA clone of Pax-6, cp611, aredepicted. Paired domain and paired-type homeodomain areboxed. Stop codons and translation start site areunderlined. The 42 nucleotide insertion, present only inthis clone, is double underlined.

domain. In Pax-6, this extended homology is reduced tosix amino acids situated immediately adjacent to theamino terminus of the homeodomain. Sequence com-parison to Pax gene products that do not contain apaired-type homeodomain revealed that Pax-2 and Pax-8 contain downstream of the paired domain a regionthat is homologous to sequences in the first part of thePax-6 homeodomain. This region includes the first hehxof the homeodomain. Within a region of 24 amino acids(delineated by arrows in Fig. 3), 15 amino acids areidentical between Pax-6 and Pax-2 and 13 amino acidsare identical between Pax-6 and Pax-8 (Fig. 3). Ifconservative amino acid exchanges are included thehomology increases to 19/24 and 17/24 amino acids,respectively.

A third conserved sequence motif, the octapeptide, isusually present downstream of the paired domain(Burn et al. 1989; Goulding et al. 1991; Jostes et al.1991). Pax-6, like prd, does not contain this motif.

As with all murine paired domain proteins analyzedto date, the carboxy terminal region of the predictedPax-6 protein is very rich in proline, serine andthreonine (45 %) (Chalepakis et al. 1991; Dressier et al.1990; Goulding et al. 1991; Plachov et al. 1990). Asearch of the NBRF protein sequence bank with theregions outside of the two conserved domains revealedno significant homology to any known protein.

Pax-6 is expressed during embryogenesisIn order to determine the expression pattern of Pax-6,Poly(A)+ RNA from various adult tissues and fromembryos of day 10.5 to day 15.5 of gestation wasexamined by northern blot analysis, using a probe

encompassing the more divergent 3' part of the pairedbox.

A single Pax-6 transcript of about 3kb is detectedfrom day 10.5 to day 15.5 of mouse embryogenesis(Fig. 4). Using the amount of Hras transcript (Leon etal. 1987) for standardization, Pax-6 mRNA is mostabundant at day 10.5 p.c., with decreasing levelsthereafter (Fig. 4). In adult brain, a faint signal,corresponding to a mRNA of about the same size as inthe embryo, could be detected whereas adult kidney,heart, intestine, liver, lung, spleen, testis and uterusshow no Pax-6 expression even after prolongedexposure (not shown).

The precise spatial and temporal expression patternduring embryogenesis was analyzed by in situ hybridiz-ation to sections from day 6.5 p.c. to day 13.5 p.c. aswell as day 15.5 p.c, 16.5 p.c. and 18.5 p.c. embryos.Hybridization was performed with both sense andantisense RNA probes derived from the 3' part of thepaired box. These probes had already been shown to bespecific for Pax-6 in northern blot analysis. Moreover, ithas been shown that paired box probes even fromhighly homologous Pax genes do not cross hybridizeunder the conditions employed (Plachov et al. 1990;Jostes etal. 1991).

No Pax-6 transcripts were detected in day 6.5 p.c.and day 7.5 p.c. embryos (not shown). Expression wasfirst seen at day 8.0 of gestation and was confined toectodermal-derived structures throughout develop-ment, the only exception being pancreas, where Pax-6transcripts are seen in a small subset of cells (notshown).

Expression in the central nervous systemPax-6 expression was first detected in the presumptiveforebrain and hindbrain at day 8.0 of gestation;however, transcripts were absent from the presumptivemet- and mesencephalon (Fig. 5B,D,F). This overalldistribution of Pax-6 transcripts is maintained duringdevelopment with the exception of the metencephalonwhere expression is detected from day 15.5 of gestation

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onwards. In the developing spinal cord, expression wasfirst seen coincident with neural tube closure (seebelow). No transcripts were detected in the neuralgroove (Fig. 5C,D). Pax-6 expression in the neural tubeextends along the entire anteroposterior axis up to therhombencephalic isthmus (Fig. 5G,H and Fig. 7A,B)and is mainly restricted to mitotically active cells in theventral ventricular zone. These expression patterns willbe described in detail in the following sections.

Expression in the developing neural tubeThe distribution of Pax-6 transcripts in the developingspinal cord was analyzed by examining cross sections ofday 8.5 p.c. to day 15.5 p.c. embryos. Pax-6 expressionwas first observed in the closed neural tube at day 8.5 ofgestation (Fig. 6A,B)- In more caudal regions of theembryo, no signal could be detected in the neuralgroove. Since the developing spinal cord shows arostral-caudal gradient of maturation (Nornes andDas, 1974; Nornes and Carry, 1978), serial cross

sections of day 9.0 p.c. embryos were analyzed todetermine the onset of Pax-6 expression in the neuralepithelium. As seen in day 8.5 p.c. embryos, Pax-6 isexpressed in the closed neural tube but not in theadjacent mesoderm and the neural plate(Fig. 6A,B,C,D). Initially, at day 8.5 p.c, Pax-6transcripts are present in a broad band of cells in thetransverse plane of the neural tube, being absent onlyfrom the most dorsal and ventral cells (Fig. 6A,B).Beginning with day 9.0 of gestation cells in the dorsalneural tube appear to down-regulate Pax-6 sinceexpression is confined to the ventral part of the neuraltube, the basal plate (Fig. 6C,D). While high levels ofPax-6 transcripts are found in the basal plate, no sharpdorsal boundary of expression can be defined thatwould coincide with the dorsal border of the basal plate.Rather the signal extends dorsally into the alar platewhere it is however weaker than in the basal plate(Fig. 6E-N). Floorplate and the cells directly adjacentto the floorplate do not express Pax-6 at any of thedevelopmental stages examined (Fig. 6A-P). Fig. 6E,Falso illustrate that Pax-6 expression in the basal platestarts to regress dorsally at day 10.5 p.c. when theventral horns begin to form and before the ventricularzone (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, whichis radially arranged around the lumen (Sidman et al.1959; Sauer, 1959). Between day 11 and 12 a second celllayer, the intermediate zone, has formed lateral to theventricular zone. It consists of differentiating postmi-totic cells which migrate radially from the ventricularzone. In sections through thoracic and lumbar levels ofthe neural tube at day 11.5 p.c, Pax-6 transcripts arerestricted to the ventricular zone and the expressionpattern within this zone is identical to the previous day(Fig. 6G,H). In cross sections from the cervical level ofday 11.5 p.c. and in sections from day 12.5 p.c.embryos, a second signal appears in the ventralintermediate zone, spreading radially from the ven-tricular zone (Fig. 6I,J, arrows). The signal correspondsto the spotted band of expression seen in the close-up ofa sagittal section of a day 12.5 p.c. embryo in Fig. 6K,L(marked by arrows). Pax-6 expression in the basal platehas further regressed concordant with the reduction ofthe ventricular zone. The same pattern is seen at day13.5 p.c. (Fig. 6M,N). Pax-6 expressing cells are stillfound at day 15.5 of gestation (Fig. 6O,P). By this time,virtually all neurons have been born (Nornes and Carry,1978) and regression of the ventricular zone is virtuallycomplete. Transcripts of Pax-6 are present in a subset ofependymal cells around the wider part of the residuallumen of the neural tube, the neural canal, but not inthe ependymal layer lining the rest of the lumen.Another low signal is observed ventrally in a subset ofpostmitotic cells (Fig. 60,P).

Expression in the developing brainThe earliest expression of Pax-6 was observed at day 8.0of gestation in the neuroepithelium of forebrain and


Fig. 5. In situ expressionanalysis of Pax-6 at day 8.0, day8.5 and day 10.5 of gestation.Pictures at the left (A,C,E, G)represent bright-field images ofthe embryos, pictures at theright (B,D,F,H) represent thecorresponding dark-field images.(A) Transverse section of a day8.0 p. c. embryo showingforebrain, midbrain and allantois(see Fig. 51). (B) Dark-fieldimage of A. (C) Transversesection of the same embryo at alower level, showing hindbrainand neural groove (see Fig. 51).(D) Dark-field image of C.(E) Parasagittal section of a day8.5 p.c. embryo, anterior is tothe left, posterior is to the right.(F) Dark-field image of E. Theapparent gap of thehybridization signal betweenhindbrain and neural tube is dueto the plane of the section andnot to a gap of expression.(G) Parasagittal section of a day10.5 p.c. embryo. (H) Dark-fieldimage of G. The large arrowmarks the limit of Pax-6expression between thediencephalon and themesencephalon. The small arrowindicates the expression of Pax-6in the nasal pit. (I) Schematicrepresentation of the levels ofthe sections in Fig. 5A-D.Abbreviations: al, allantois; f,forebrain; d, diencephalon; h,hb, hindbrain; m, midbrain; ms,mesencephalon, mt,metencephalon; my,myelencephalon; ng, neuralgroove; np, nasal pit; nt, neuraltube; pr, preoptic recess; t,telencephalon.

5A.B

5C,D

hindbrain (Fig. 5A-D). This overall spatial expressionpattern is maintained during brain development fromday 8.0 p.c. to day 18.5 p.c; Pax-6 transcripts arepresent in the forebrain, the tel- and diencephalon, andin the myelencephalon, but not in the roof of themesencephalon (Fig. 5A-D and Fig. 7A-J). Ex-

pression in the metencephalon is first observed at day15.5 p.c. of development (Fig. 7E,F).

In the telencephalon, from day 10.5 p.c. to 18.5 p.c.,high levels of transcript are detected in the lateral anddorsal neural epithelium but not in the basal telen-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,Oshow transverse sections of the neural tube at consecutively later stages of neural tube development, photographsB,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) day10.5, (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 thesignal in postmitotic cells corresponding to the signal marked by arrows in the transverse section of a day 12.5 p.c. embryoin I,J. (M,N) Day 13.5, (O,P) day 15.5. Abbreviations: ap, alar plate; bp, basal plate; e, ependymal layer; dh, dorsalhorns; fp, floorplate; i, intermediate zone; nt, neural tube; rf, roof plate; s, somite; v, ventricular zone; vh, ventral horns.


lar zone throughout development (Fig. 5G,H andFig. 7A-J). The olfactory bulbs develop as protrusionsfrom the rostro-ventral telencephalon and also expresshigh levels of Pax-6 transcript (Fig. 7E-J).

The expression domain extends from the telencepha-lon into the diencephalon up to a sharp caudal border atthe posterior commissure, which delineates the bound-ary between diencephalon and mesencephalon. Thiscaudal limit of expression persists through all develop-mental stages analyzed (Fig. 5G,H; Fig. 7C-J; largefilled arrows). A second boundary lies within thediencephalon and coincides with the sulcus diencephali-cus medius (Niimi et al. 1962) which separates ventraland dorsal thalamus. Beginning with day 10.5 p.c. (notshown) before the intermediate and marginal zoneshave formed, a strong hybridization signal is seen in theventral thalamus (Fig. 7A-D). By day 15.5 p.c, thesize of the ventral thalamus and its germinal zone havebecome reduced and nucleus reticularis, zona incertaand ventral lateral geniculate are differentiating in theintermediate zone. Pax-6 is now expressed in a smallerarea probably corresponding to the presumptive zonaincerta (Fig. 7E,F), where it is still detected at day 18.5p.c. (Fig. 7G-J), when neurogenesis has essentiallybeen completed. A sharp drop in signal intensity isobserved dorsal to the sulcus diencephalicus medius,i.e., the dorsal thalamus shows a distinctly lower levelof expression (Fig. 7C,D; open arrow). This weakerexpression seen in earlier stages (day 10.5 to day 12.5p.c.) is no longer detected at day 15.5 p.c. (Fig. 7E,F).Ventrally, a signal extending into the anterior hypo-thalamus in the region of the preoptic recess of the thirdventricle is observed at day 10.5 p.c. to day 12.5 p.c.(Fig. 7A-D). Pax-6 mRNA is also present in theepithalamus before the intermediate zone is formed atday 13 (Fig. 7A-D) and at later stages in differentiatingstructures such as the nuclei habenulae (Fig. 7E,F,I,J).The pineal gland, an evagination of the epithalamus,expresses Pax-6 at all stages examined (Fig. 7E,F,I,J).

The metencephalon does not express Pax-6 at earlystages (Fig. 7A-D). Expression is however seen at day15.5 p.c. to 18.5 p.c. in the external granular layer(EGL) and in cells distributed in the dorsal cerebellum(Fig. 7E,F). The EGL represents a second germinalzone unique for the cerebellum, where mitotic activityincreases while it is decreasing in the ventricular zonebetween day 13 and 15 of gestation (Miale and Sidman,1961). It is formed by cells that migrated from therostral part of the rhombic lip of the myelencephalon tothe surface of the cerebellum. Cells of the EGL will giverise to the granular layer of the adult cerebellum, wherePax-6 is expressed at low levels (not shown). Pontinenuclei, originating from cells from the more caudal partof the rhombic lip also show expression of Pax-6(Fig. 7E-J), as well as inferior olivary nuclei (notshown). Both structures relay information to thecerebellum.

The expression pattern of Pax-6 in the myelencepha-lon is essentially the same as in the spinal cord.Transcripts are most abundant in the ventricular zone

and the expression decreases together with the re-gression of the ventricular zone (Fig. 7A-J).

Pax-6 is expressed in the developing eyePax-6 expression in the developing eye was firstdetected at day 8.5 p.c. in the optic sulcus, a lateralevagination at the base of the forebrain (not shown). Byday 9.5 p.c, this evagination has formed the opticvesicle which approaches the surface ectoderm. Tran-scripts of Pax-6 are present in the epithelial layer of thevesicle, the optic stalk and the overlying surfaceectoderm, which will give rise to the lens (Fig. 8A-D).Between day 10 and day 12, the optic vesicleinvaginates, forming the optic cup, while the lens formsby indentation of the thickened surface ectoderm,which eventually detaches from the surface. Theinvaginated neuroectoderm (inner layer and presump-tive neural retina), a mitotic highly active layer iscomparable in organization to the ventricular zone ofthe neural tube (Miale and Sidman, 1961; Pei andRhodin, 1970). Pax-6 is strongly expressed in the innerlayer of the cup and in the lens, whereas the outer layer(presumptive pigmental retina), a zone of low mitoticactivity, shows only a low level of Pax-6 expression(Fig. 8E-L). In addition, Pax-6 transcripts are detectedin the overlying surface ectoderm which is part of thefuture cornea (Fig. 8G-J, arrows). At day 12.5 p.c. thehybridization pattern is essentially the same as on theprevious day. The now differentiating lens fiber cells inthe posterior wall of the lens are still expressing Pax-6and transcripts are still detected in a narrow ventricularzone extending along the optic stalk (Fig. 8K,L). Byday 15.5 p.c., most of the structures of the adult eye arepresent, the lids have developed as has the cornea, thelens and the retina continue to differentiate. By thistime two layers have developed in the neural retina andcan be clearly distinguished, an outer layer of high celldensity and mitotic active cells and an inner layer oflower cell density containing mainly postmitoticganglion cells. Both layers express Pax-6 at about equalamounts (Fig. 8M,N). Expression is also observed inthe anterior epithelium of the cornea and in the lens(Fig. 8M,N).

Expression in the developing pituitary and theolfactory epitheliumThe anterior pituitary develops from an outpocketing ofthe roof ectoderm of the stomodeum (Rathke's pouch)in close association with the infundibulum. Theinfundibulum is formed from an evagination of the floorof the diencephalon which will give rise to the posteriorlobe (pars neuralis) of the mature pituitary. Beginningwith day 11.5 of gestation, Pax-6 transcripts are clearlydetectable in the developing adenohypophysis (anteriorpituitary). Expression of Pax-6 is restricted to Rathke'spouch and its derivatives, the pars intermedia anddistalis. No transcripts could be detected in theinfundibulum and the differentiating pars neuralis in thedevelopmental stages examined (day 10.5 p.c. to day18.5 p.c; Fig. 7C-L). Peak levels of Pax-6 expressionoccur at day 12.5 p.c. with decreasing signal intensity


Fig. 7. Expression of Pax-6 in the developing brain. The left panel of photographs shows the bright-field image, the rightpanel 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,shortly before all three parts begin differentiating, weaklabelling remains in the epithelium lining the remaininglumen of Rathke's pouch and in the anterior lobe(Fig. 7E,F). Expression just above background level is

still observed in these structures at day 18.5/?.c.(Fig. 71 J).

As evident from Fig. 5G,H and Fig. 7A-J, Pax-6 isalso expressed in the developing olfactory epithelium ofthe nose from the relative primitive olfactory pit stage


at day 10.5 to the adult-like nasal structure at day 18.5(Fig. 5G,H; Fig. 71,J).

Discussion

The Pax-6 gene is a divergent member of the murinepaired box gene family (Walther et al. 1991). It encodesa paired-type homeodomain in addition to the paireddomain. Hence the Pax-6 protein contains two DNA-binding domains as seen with Pax-3, Pax-7, prd, gsb-pand gsb-d (Goulding et al. 1991; Jostese/a/. 1991; Boppet al. 1986). The presence of these DNA-binding motifs

suggests that Pax-6 acts as a transcriptional regulator bybinding to DNA. Sequence comparison of the Pax-6paired domain and paired-type homeodomain withother known domains of these types reveals that bothare rather divergent. They show nonconservative aminoacid exchanges at a number of positions which areconserved in all other paired domains or paired-typehomeodomains. Pax-6, however, contains a glycine atposition 15 of the paired domain, like all other paireddomains described to date. Replacement of this glycineto a serine as seen with Pax-1 in the mouse mutant un,results in a dramatic decrease in DNA-binding affinity,concommitant with a reduced transcriptional activation


Fig. 8. Expression of fta-<5 in the developing eye. Close-ups of sections through embryos at: (A,B) day 9.0 p.c., arrowpoints 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 tosignal 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 layerof 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 thecornea; f, forebrain; il, inner layer of the neural retina; le, lens; li, lid; nr, neural retina; oc, optic cup; ol, outer layer ofthe 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 9of the recognition helix of the homeodomain, which hasbeen shown to be important for the binding specificityof the paired protein (Treisman et al. 1989), is alsoconserved in Pax-6. Experiments using deletion mu-tants or mutated paired domains have demonstratedthat the first part of the paired domain is required forDNA binding and is sufficient for conferring DNA-binding specificity (Treisman et al. 1991; Chalepakis etal. 1991). The sequence of Pax-6 in this region differs at

many positions from the other paired domain proteins(Walther et al. 1991), suggesting that Pax-6 might have adifferent DNA-binding specificity to the other paireddomain proteins. This is supported by our preliminaryexperiments (C.W., unpublished).

Pax-6 is also expressed in a variant form whichcontains an insertion of fourteen amino acids in themost highly conserved region of the paired domain. Thepotential importance of this alternative form of the Pax-6 protein is highlighted by its conservation during


evolution, being also found as a product of the zebrafishpax-6 gene (A. Piischel, pers. communication). Atpresent the function of this insertion is unclear. In thecontext of the DNA-binding ability of the paireddomain, it is, however, tempting to speculate that thisalternative form might change the DNA-bindingproperties of Pax-6.

To date no other murine gene has been isolated thatshows such extensive sequence homology to Pax-6, asseen between the paralogous genes Pax-3/Pax-7 andPax-21Pax-5/Pax-8 which are likely to have been arisenby duplication. Nevertheless, Pax-4 shows the samegenomic structure as Pax-6 (as analyzed so far) andcontains a number of amino acids that are unique forPax-6 and Pax-4 (Walther etal. 1991). In addition, Pax-4 encodes a homeodomain that at least in part is highlyhomologous to the homeodomain of Pax-6 (M. Asanoand P. Gruss, unpublished results). Pax-6 and Pax-4therefore, might have been derived from the sameancestral gene by duplication early in evolution whichcould account for the high degTee of sequencedivergence of their paired boxes.

Sequence comparison of the entire Pax-6 protein withall other Pax proteins revealed that sequences in Pax-2and Pax-8 downstream of the paired domain arehomologous to the first part of the Pax-6 homeodomainincluding the first helix. This raises the intriguingpossibility that Pax-2 and Pax-8 have a rudimentaryhomeodomain which has lost the helix-turn-helix motif.This part of the homeodomain might have been deletedin the duplication or exon shuffling events whichgenerated the Pax genes or alternatively was randomlymutated during evolution. In any case, it will beinteresting to see which function this truncated homeo-domain has aquired or retained.

The expression pattern of Pax-6 during embryogen-esis was examined by northern and in situ analysis. Pax-6 is expressed in distinct regions of the brain beginningat day 8 of gestation, in the neural tube, in thedeveloping eye, in the pituitary, the olfactory bulbs andin the olfactory epithelium.

In the developing neural tube, Pax-6 transcripts aredetected in the ventral ventricular zone but not in floorplate cells and cells immediately adjacent to the floorplate. Expression is detected prior to neural differen-tiation and declines with the regression of the ventricu-lar zone. At early stages in development, the spinalcord represents a pseudostratified epithelium of mitoti-cally active cells which are radially arranged around thelumen (Sidman etal. 1959; Sauer, 1959). Cells that haveundergone their final mitosis and start to differentiatemigrate radially out of the ventricular zone and settle inthe adjacent intermediate zone. A dorsal-ventral(D-V) polarity within the neural tube becomes evidentduring this process since functionally different neuronsoriginate from distinct regions (Jacobson, 1985). Motorneurons for example arise from the ventral partwhereas interneurons of the dorsal horns and neuronsof the sensory pathway originate primarily from thedorsal part of the neural tube (Altman and Bayer,1984). Notochord and floorplate were shown to have a

polarizing effect on the neural tube, by controlling atleast in part the neuronal differentiation pattern alongthe D-V axis (van Straaten et al. 1989; Placzek et al.1990; Hirano et al. 1991; Yamada et al. 1991). Themechanisms by which floorplate and notochord exerttheir polarizing activity is yet unclear. Possible candi-dates for morphogenetic signals emanating from thesestructures are retinoids, which are possibly present innotochord (Hornbruch and Wolpert, 1986) and floor-plate (Wagner et al. 1990) or growth factors like stemcell growth factor (SCF) shown to be expressed in thefloorplate (Matsui etal. 1990; Keshet etal. 1991). SincePax-6 is expressed early in neural tube developmentbefore neural differentiation begins, Pax-6 could beinvolved in interpreting such a signal into positionalinformation within the neural tube, thereby establishingthe ventral character of the neural tube and beinginvolved in the generation of motorneurons. Ex-pression could be regulated in a position- and/orconcentration-dependant manner, the threshold levelof morphogen required for transcription of Pax-6roughly coinciding with the position of the borderbetween basal and alar plate. This is at least partlysupported by recent experiments which have demon-strated that the notochord influences the expression ofPax-6. In the chicken, transplantation of an additionalnotochord lateral to the neural tube changes theexpression domain of the chicken homolog of Pax-6 (M.Goulding, A. Lumbsden and P. Gruss, unpublishedresults). Another murine Pax gene, Pax-3, is expressedin the dorsal part of the neural tube beginning with day8.5 of gestation and expression of the chicken homologof Pax-3 is also influenced by the notochord. (Gouldingetal. 1991) (M. Goulding, A. Lumbsden and P. Gruss,unpublished results). The expression domain of Pax-6extends into the alar plate, which generates a zone atthe border of alar and basal plate where the expressionof Pax-6 and Pax-3 overlaps. This raises the possibilitythat the Pax-3 gene product could act directly orindirectly on Pax-6 and/or the Pax-6 gene product onPax-3, thereby probably defining ventral and dorsal ofthe neural tube. A third Pax gene, Pax-7, shows asimilar expression pattern in the developing nervoussystem as Pax-3 (Jostes et al. 1991) and might thereforebe also involved in the generation of the ventral-dorsalpolarity of the neural tube. In contrast, Pax-2 and Pax-8begin to be expressed at later stages of neural tubedevelopment in discrete subsets of postmitotic cells andthus are not involved in these early steps of neural tubedevelopment.

In the brain, Pax-6 expression extends from the mostrostral part of the CNS, the telencephalon, to themyelencephalon, sparing the roof of the mesencepha-lon and the early metencephalon. This overall spatialpattern of expression of Pax-6 remains essentially thesame during brain development with the exception ofthe metencephalon where expression is detected fromday 15.5 p.c. onwards. Pax-6 is expressed in theforebrain and the hindbrain at day 8.0 p.c. andcontinues to be expressed in the telencephalon and thediencephalon which develop from the forebrain, and in


the myelencephalon which develops from the hind-brain. Especially striking is the sharp limit of expressionat the border between diencephalon and mesencepha-lon corresponding to a well-studied boundary betweenthese two regions of the brain (Vaage, 1969; Keyser,1972; Puelles et al. 1987). Within the diencephalon, theexpression domain of Pax-6 matches another regionalboundary, the zona limitans intrathalamica whichdelineates the border between ventral and dorsalthalamus (Niimi et al. 1962) and which corresponds tothe interparencephalic neuromeric boundary of Puelleset al. (1987) and the reticular protuberance of Altaianand Bayer (1986). Interestingly, the murine Distal-lessrelated gene, Dlx (Price et al. 1991), and two membersof the Wnt family, Wnt-3 and Wnt-3A (Roelink andNusse, 1991), show also a restricted expression patternwith respect to this boundary. A number of other genesis also expressed in distinct regions of the brain duringdevelopment, e.g. the Hox genes in the hindbrain (forreview see Kessel and Gruss (1990)), En-1 and En-2 inthe midbrain-hindbrain region (Davis and Joyner,1988; Davis et al. 1988; Davidson et al. 1988) and Wnt-3A also in the telencephalon (Roelink and Nusse,1991). Two other Pax genes, Pax-3 and Pax-7, showexpression in all brain regions at early stages indevelopment (day 8.5 p.c), the expression, however,regresses posteriorly during development. Thus, Pax-6in combination with other genes generates overlappingor complementary expression domains in the brain in asimilar way as in the transverse plane of the neural tubeor as the Hox genes along the anteroposterior axis. Thecombination of the gene products may define subdiv-isions in the brain at early stages and might later berequired for refinement and differentiation of thesesubdivisions. As in the spinal cord Pax-6 expression isnot exclusively restricted to mitotic active cells in theventricular zone. Expression is still detected at day 18.5in the ventral thalamus, epithalamus and myelencepha-lon when mitosis has ceased (Gardette et al. 1982). Thuscertain cell populations retain or reinitiate expression ofPax-6 after their final mitosis.

Pax-6 expression in the eye was examined from day8.5 to day 15.5 of gestation. During this developmentalperiod, the main structures of the eye are formed.Expression of Pax-6 is first detected at day 8.5 in theoptic sulcus. At later stages, the eye vesicle, the lens,the differentiating retina and the cornea show ex-pression of Pax-6. Interestingly, it is expressed in theectoderm overlying the optic vesicle before the lens hasformed. After lens formation, Pax-6 continues to beexpressed in the surface epithelium from which thecornea will develop. The eye has been studiedextensively with respect to the inductive processes thatgovern its development. Initially viewed as a rathersimple process e.g. the optic vesicle was thought to besufficient and necessary to induce formation of the lensfrom ectoderm (Spemann, 1901; Lewis, 1904), it turnedout to be a complex multistep process. This processstarts already at gastrulation and the major inductiveevents that determine the ectoderm to become lensoccur during neurulation (McAvoy, 1980; Saha et al.

1989). Thus the instructive inductive events (Gurdon,1987) act before the optic vesicle is formed, which theninduces the determined lens ectoderm to become lens.In addition, the retina seems to be important forgrowth, differentiation and maintenance of the lens. Asimilar multistep process seems to be required forcornea formation where in addition the lens is necessaryduring a certain period in development (Zak andLinsenmayer, 1985) and at later stages for maintenanceof the cornea (Hay and Revel, 1969). Pax-6 is firstexpressed at day 8.0 of gestation, i.e. not during theearly stages of gastrulation and neurulation, making itunlikely that Pax-6 is involved in the initial steps ofinduction. The expression pattern rather suggests thatPax-6 is involved in the regulation of the followinginductive events. In this context, it is noteworthy thatPax-6 is expressed in the inducing as well as in theresponding tissue. In addition, Pax-6 might be requiredfor growth, differentiation and maintenance of the lensand the cornea.

In summary, Pax-6 represents a rather divergentmember of the paired box gene family, encoding apotential transcription factor. Pax-6 is the earliestexpressed Pax gene described so far and is in contrast toall other Pax genes not expressed in segmentedstructures during murine embryogenesis. The ex-pression pattern suggests a regulatory role for Pax-6 inthe development of the CNS and the eye.

We thank K. Fahrner and B. Hogan for their generous giftof the cDNA library and R. AltschSffel for photograghicwork. We are grateful to Dr Riekmann for his help with theanatomy of the brain and we would also like to thank M.Goulding and particularly R. Fritsch for helpful discussionsand comments on the manuscript. We thank A. Ptischel forcommunication of unpublished results and continous dis-cussion. C.W. is supported by a Boehringer Ingelheim FondFellowship. This project was supported by the Max PlanckSociety and Deutsche Forschungsgemeinschaft grant No. En84/18-4.

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{Accepted 5 September 1991)

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Pax-6, a murine paired box gene, is expressed in the ... › content › develop › 113 › 4 › 1435...Pax-6, isolation and expression 1437 ation was done overnight in a humid chamber