Development ID, 1435-1449 (1991) 1435 Printed in Great Britain © The Company of Biologists Limited 1991 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 (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 protein 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, homeobox, 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 (Pax 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 pattern formation and morphogenesis (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 conserved sequence motifs axis of the neural tube and the hindbrain where it is such as homeobox, zinc-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 segmentation 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 homology 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 body plan. 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 mutation 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 thiocyanate (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)- phenotype (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.