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I Int..I. Dc\'. BioI. ~O: 369-377 (1996) 369 I

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Pax and vertebrate development I

ROLAND WEHR" and PETER GRUSS I Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany

I ABSTRACT Pax encode transcription factors sharing a highly conserved sequence, the

paired box. Their temporally and spatially restricted expression patterns during development indi- I cate that Pax genes are involved in important steps of nervous system formation. Mutations in Pax genes have been correlated with three mouse mutants (undulated, splotch, small eye) and two human diseases IWaardenburg syndrome, aniridia). Recent data demonstrated that deregulation I of Pax genes contributes to tumor formation. I KEYWORDS:Pax, Small f)'e, 5;plotch,~raardnibwg, aniridia

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Introduction Because of similarities in structure, genomic organization I and expression during development, the murine Pax genes can Based on the to sequence motifs in the genome of be classified into subgroups. designated as paralogous genes I Drosophila melanogaster different families have been iden- (Fig. 1). tified in the mouse: the homeodomain (McGinnis el al.. 1984a,b; I Kessel and Gruss, 1990), the paired domain (Bopp el al., 1986; Expression of Pax genes in the developing CNS Deulsch el al.. 1988), the heiix-ioop-helix domain (Murre el al., I 1989), the domain (Rosenberg el al., 1986), the fork The developmenl of the nervous system starts with the inter- head domain (Jurgens and Weigel, 1988), the POU domain action of the chordamesoderm and the overlying neuroectoderm (Herr el al.. 1988; Rosenleld 1991) and the resulting in the induction of the neural plate and subsequently in I (Land schultz el al., 1988). neural tube formation. Shortly after closure of the neural tube, it The family of paired box (Pax) genes share a highly con- differentiates regionally inlo Ihe spinal cord and three primary I served DNA sequence of 384 bp and was first described in brain vesicles: the prosencephalon, the mesencephalon and the

Drosophila genes (Bopp el al., 1986; Baumgartner rhombencephalon. Later, the prosencephalon is further subdi- I ef al., 1987). Besides Drosophila, genes containing the paired vided into diencephalon and lei encephalon, while the rhomben- box have been found in many vertebrate species such as cephalon gives rise to the metencephalon and myelencephalon. I zebrafish, frog, chick, mouse and human (Dressler el al., 1988; Burri el al., 1989; Krauss el al., 1991b; Puschel el al., 1992; Pax genes and brain development Goulding el al., 1993a) suggesting, as in the case of Hox genes, Along the anlerior-posterior axis from the rhombencephalon I that fundamental events in body patterning are conserved to the tail, segment identity appears to be specified by the differ- between insects and vertebrates. ential expression of Hox genes (Boncinelli el al., 1991; Kessel, I The murine Pax gene family consists of nine members (Pax1 1991; McGinnis and Krumlauf, 1992). Little is known about the molecular basis establishing regional properties more anterior in to Pax9) (Walther el al.. 1991; Wallin el al., 1993). In contrast to I Hox genes (Kessel and Gruss, 1990), Pax genes are not clus- mes- and prosencephalon, especially in the region overlying the tered and map on different chromosomal loci (Walther el al., prechordal mesoderm. On the basis of expression patterns it is 1991; Stapleton el al., 1993). Pax contain addilional likely that members of the Emx (Simeone ef al., 1992b), Dlx I conserved motifs, a conserved octapeptide (present in all except (porteus ef al., 1991), Nkx (Price, 1993), Olx (Simeone el al., Pax6 and Pax4) and/or a paired-type homeodomain (Pax3, 1992a), Wnt (Nusse and Varmus, 1992) gene families are impor- I Pax4, Pax? and Pax6). Pax2. Pax5 and Pax8 harbor a rudimen- tant in early prosencephalon patterning. tary homeodomain encoding only the first a-helix (Fig. 1). Pax All Pax genes, except Pax1 and Pax9 (Deulsch el al., 1988; I proteins act as transcription factors. This is shown by their in vit- Stapleton el al.. 1993; Wallin el al., 1993), show spatially and ro DNA.binding activity (Treisman el al.. 1989, 1991; Chalepakis temporally restricted expression during development of the cen- I elal., 1991; Goulding elal., 1991; Dressler and Douglass. 1992; tral nervous system. The first group of early expressed Pax Czerny el al., 1993; Epstein el al.. 1994), their transcriptionai genes, namely Pax3, Pax6 and Pax7, starts to be active around activation (Chalepakis el al.. 1991) and their localization in the I nucleus (Bopp el al., 1989; Dressler and Douglass, 1992). .1b{n.r1.liat;rms IIst'd il/illis papt'Y: p.c., po~t coinnn.

I "Address for reprints: Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, 37077 Goningen, Germany. FAX: 551.2011504. e-mail: [email protected] I 0214-6282/96/503.00 OUB(Pre.., Prinle,lmSpain I

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I 370 R. Wehr alld P. Gmss

I day 8.0-8.5 p.c. With the exception of Pax6, which excludes the these transcription factors can play an important role during the mesencephalic roof from its expression domains, these genes establishment of the dorso-ventral polarity in the spinal cord. are present in the entire developing neural tube (Fig. 2A) Regionalization 01 the early spinal cord appears to be con- I (Goulding et a/., 1991; Jostes et al., 1991; Walther and Gruss, trolled by signals originating from ventral midline cell groups, the 1991). Different Irom Pax6which is expressed in lorebrain in lat- axial mesodermal cells of the notochord and the Iloor plate cells er stages and even after birth (Stoykova and Gruss, 1994), in (Placzek et al., 1991). A signal coming Irom the notochord is I midgestation embryos, Pax3 and Pax? are retracted from the responsible for floor plate induction and subsequently the noto- telencephalon to a rostral limit in the diencephalon (Fig. 2B,C). chord and the floor plate will induce the differentiation of motor A second group 01 late expressed Pax genes (Pax2, Pax5, neurons (Placzek el al., 1991; Ruiz i Altaba and Jessell, 1993). I and PaxB) is confined to the epichordal part 01 the CNS (spinal In chick embryo, experiments of surgical ablation or ectopic graft- cord and hindbrain) and starts to be expressed at day 10.5 p.c. ing of notochord show that these early expressed Pax genes I in postmitotic cells of the intermediate zone. The midbrain-hind- modify rapidly their region of expression in response to signals brain boundary is the rostral limit of Pax2, Pax5 and PaxB coming from the notochord and Iloor plate centers (Goulding el expression, whereas Pax5 is also detected in the posterior mes- al., 1993a). These data indicate that the region specific expres- I encephalic tegmentum (Fig. 2B) (Nornes et a/., 1990; Plachov et sion of Pax genes is governed by the induction cascade originat- al., 1990; Asano and Gruss, 1992). ing from the notochord. This observation is consistent with the

The analysis of Pax gene activity in the developing brain idea that Pax genes might play an important role in the early I reveals that the spatial expression is delineated by anatomical establishment of the dorso-ventral polarity in the spinal cord. landmarks. First, Pax2, Pax5 and PaxB are detected around the The second subgroup of Pax genes without homeodomain midbrain-hindbrain boundary (Fig. 2B) (Names et al., 1990; (Pax2, Pax5 and Pax8) starts to be expressed around day 10.5 I Plachov el al., 1990; Asano and Gruss, 1992). In zebrafish, p.c. in a subset of post-mitotic neuroblasts located as two longi- injection 01 antibodies raised against the pax(zl-b] protein (most tudinalcolumns in the intermediate grey on both sides of the sul- similar to Pax2) show default development of the Isthmus cus limitans (Fig. 3B) (Nornes el al., 1990; Plachov et al., 1990; I (Krauss et al., 1992). suggesting that members 01 the Pax fami- Asano and Gruss, 1992). The expression analysis of Pax2 com- ly might playa critical role establishing the midbrain-hindbrain pared to the zebralish homologue pax [zl-b] identified these cells boundary. as interneurons 01 the intermediate grey (Krauss et al., 1991 a; I Two recently reported models described the neuromeric orga- Mikkola et al" 1992) supporting the idea that Pax genes may be nization of the forebrain in lour (01-04, Figdor and Stern, 1993) involved in the cell fate determination to specific neuronal phe- I or six (P1-P6 prosomeres, Puelles and Rubenstein, 1993) neu- notypes. romeres, which are consistent with the restricted expression domains of a number 01genes (Emx, Simeone el a/., 1992b; Olx, Pax, mouse mutants and human diseases I Porteus et al., 1991, Nkx, Price, 1993; Olx, Simeone el al., 1992a; Wnt, Nusse and Varmus, 1992). In the developing dien- It has been shown that three Pax genes are altered in classi-

cephalon Pax6 shows segment like expression confined to the cal mouse mutants (Hastie, 1991; Gruss and Walther, 1992) and I ventral thalamus (01, or the alar plate 01 P3) while Pax3, Pax6 two are associated with human diseases (Hill and van and Pax7 transcripts are detected in the epithalamus (region of Heyningen, 1992; Strachan and Read, 1994). Recently, an addi- 02 or P3) and pretectum (03-04 or PI). Additional experiments tional transgene-induced mutation (Krd) including a Pax2 dele- I are necessary to relate their expression domains to the pro- tion has been reported (Keller el al., 1994). posed two neuromeric subdivisions (03 and 04, Figdor and Stern, 1993) of the pretectum. Pax1 and undulated (un) I Pax gene expression was also detected in the adult brain The paralogous Pax! and Pax9 are the only Pax genes that (Fig. 2C) with a similar transcript distribution along the AP-axis are not expressed in the central nervous system (Deutsch et al., compared to midgestation embryos (Stoykova and Gruss, 1994). 1988; Wallin et al., 1993, 1994). At day 8.25 p.c. Pax! expres- I In many cases, a correlation exists between the expression of sion can be first detected in the ventral part of the somites (the particular Pax genes in adult brain structures and the embryonic prospective sclerotome) and subsequently in the developing ver- I region of their origin suggesting that Pax genes are not only tebral column, the facial skeleton, the shoulder girdle, the seg- involved in brain regionalization but also in the maintenance of mented sternum, the pharyngeal pouches and the thymus anlage (Deutsch el al., 1988; Timmons et al., 1994). With excep- the regional identity. I tion of the thymus anlage and the facial skeleton, the expression Pax genes in the developing spinal cord domains are confined to structures undergoing chondrogenesis

Pax genes harboring a complete paired-type homeodomain suggesting that Pax! plays a crucial role in that process includ- I (Pax6, and the paralogous Pax3 and Pax7) (Goulding et al., ing sclerotome specification. 1991; Jostes el al" 1991; Walther and Gruss, 1991) are the lirst All Pax1 expression domains are corresponding to malforma- Pax genes expressed in the spinal cord. They are detected tions affected in undulated mutant mice (Balling et al., 1988, I around day 8.5 p.c. in specific regions of the ventricular zone. 1992; Dietrich el al., 1993; Wallin el al., 1994). The Pax! gene While Pax6 is present in the basal and intermediate plate, Pax3 has been mapped on mouse chromosome 2 close to the reces- and Pax? are expressed in the alar plate. The expression sive mutation undulated (Fig. 1) (Balling et al., 1988). The analy- I domain of Pax6 is partially overlapping with Pax3 and Pax7 (Fig. sis of undulated mice revealed that there are three different 3A). This region specific pattern of expression suggests that undulated alleles harboring mutations within the Pax1 gene. I ""-..---...

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Pax alld \'ertebrate del'el0l'lIlellt 371 I

I The first one identified was undulated (un) showing a single developing eye, expression has been observed around day 9.0 base pair mutation in a highly conserved position of the paired p.c. in the optic vesicle, the optic cup and the optic stalk, in I box (Balling et al., 1988). This causes an exchange midgestation embryos in the optic disk and optic nerve. from glycine to serine reducing the DNA-binding activity of the Recently, a transgene-induced mutation (Krd, and reti- I protein dramatically (Chalepakis et a/., 1991). The undulated nal effects) on chromosome 19 has been described, showing a is characterized by a kinky tail, vertebral, shoulder gir- large deletion including the Pax2 locus (Keller et at., 1994). I dle and sternum malformations (Wright, 1947; GrOneberg, 1963). Although the deletion encompasses possibly around 400 genes, The undulated extensive (un"') allele (Wallace, 1979, 1980) some characteristic features of Krd are likely due to the deletion I presents a similar but more severe phenotype than undulated. of Pax2. Krd shows in heterozygous state kidney defects includ- The deletion of the 3' part of the Pax1 gene including the last ing aplastic, hypoplastic and cystic kidneys. These data are also I and the poly (A) signal leads to a decreased Pax! mRNA supported by observations in Pax2 transgenic mice revealing level (Dietrich and Gruss, 1995). severe functional and histological defects in the developing kid- I Finally, Undulated short tail (Uri') (Blandova and Egorov, ney (Dressler et al., 1993). Furthermore, Krd exhibits retinal 1975) presents the most severe phenotype, caused by the dele- defects including abnormal electroretinograms and a reduced I tion of the whole Pax1 gene (Balling et al., 1992). In contrast to number of cells in the inner and ganglion layers of the retina. the other recessive Pax1 alleles. Urf is a semi.dominant muta- Heterozygous mice show growth retardation whereas homozy- I tion resulting in late embryonal or neonatal death in homozygous gote Krd mice are early embryonic lethal. Another characteristic mice and similar vertebral malformations as the un allele in het- of the Krd mutation is the semidominance, that has been also I erozygous mice (Balling et al" 1992; Wallin et ai" 1993), described in the undulated, small eye and splotch mutations The comparison of of all three undulated alleles (Mansouri et al., 1994). I supports the idea that Pax! is an important regulator during ver- tebral column formation. Pax3 and splotch (Sp) I The Pax3 expression pattern (Goulding et al., 1991) and the Pax2 and kidney and retinal defects (Krd) correlation with defects in the mouse mutant splotch (Sp) I Pax2 expression during embryonic development is confined (Auerbach, 1954) provides evidence that the Pax3 gene is an to the neural tube, the kidney and to the eye. Pax2 starts to be important regulatory factor during neural tube development and I expressed in the developing kidney at day 9.0 p.c. in the pro- neural crest migration (Moase and Trasler, 1990). Initially, Pax3 and mesonephric tubules, the Wolffian duct and subsequently in has been mapped close to the sptotch locus on chromosome 1 I the ureter and the condensed metanephric mesenchyme. In the (Fig. 1) (Evans etal., 1988; Goulding etal" 1993b), Six different I Chromosomal Gene localisation Protein structure Mutants I Mouse Human N PD Oct HD C Mouse Human I Paxt 2 20p11 ---.- undulated (un) I Pax9 12 14q12 ------q13 I

I Pax2 19 10q25 (Krd) I Pax5 4 9p13 ------2q12 I Pax8 2 -q14 ---.... Fig. 1. Protein structure of mem- I bers of the murine and human Pax 2q35 splotch Waarden- gene family. Chromosomal localisa- I (Sp) burg tion of Pax genes and their corre- Pax7 4 1p36 sponding mouse mutants and I human diseases are indicated. The homeodomain and the paired domain are shown in c%ured box- I Pax4 6 7q32 es. the octapeptide (eire/e) and the putarive a-he/ices (ovals) in red. The I paralogous genes are grouped together. C, carboxy terminus; HO, I Pax6 2 I1p13 small eye Aniridia homeodomain; Krd, kidney and reti- (Sey) nal defects; N, amino terminus; Oct, I octapeptide; PO, paired domain. I

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372 R. Wehr and P. Gruss

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semi-dominant splotch alleles have been described in mouse: (Baldwin et al., 1992) and a small deletion in WS.OS (Tassabehji

two spontaneously mutations, splotch (Sp) (Russel 1947, et al., 1992) completely abolishes the DNA-binding ability of I Auerbach 1954) and splotch delayed (Sp") (Dickie, 1964; Vogan PAX3 (Chalepakis et al., 1994). Phenotypic features of patients et al., 1993), and tour induced, splotch retarded (Spr) , SplH, with WS 1 include a lateral displacement of the inner corner ot I Sp2H and Sp4H (Beechey and Searle, 1986). The molecular alter- the eye (dystropia canthorum), a prominent nasal root, deafness ations have been identified in all splotch alleles correspondingto and pigmentation disturbances indicated by Le. white forelock

deletions and point mutations in the Pax3 gene (Epstein st 81.. and eye lashes and heterochromia irides (Waardenburg, 1951; I 1991a,b, 1993; Goulding et al., 1993b). DNA-binding studies of McKusick, 1992). Patients with WS 2 show a very similar phe- different splotch alleles revealed that these alterations affect the notype as WS 1 except no dystropia canthorum (Tassabehji st DNA-binding property of Pax3 (Chalepakis et al., 1994). al., 1993). These phenotypes appear in a heterozygous state. I In a heterozygous state, all splotch alleles exhibit a phenotype Individuals homozygous for the most showing characteristic pigmentation abnormalities (white spot- likely do not survive. Furthermore, WS 3 patients can be distin- I ting of the belly, limbs and tail) originating from neural crest defi- guished from WS 1 by additional limb abnormalities (Hoth et aI" ciencies (Auerbach, 1954). in addition, heterozygous splotch 1993), This observed limb abnormality can be correlated with the

retarded (Spr) mice are characterized by retarded growth (Evans underdeveiopment of the muscle anlagen and a reduced size of I et al., 1988). the axial musculature in splotch mice (Bober et al., 1994; Hoth st Homozygous splotch mice are embryonic lethal around day al., 1993). Finally, also the observation of spina bifida and 13-14 p.c. The most prominent phenotype is observed during meningomyelocele in WS 1 patients are of particular interest I development of the neural tube. All splotch alleles cause severe (Arnvig, 19S9; Lavergne, 19S9; Pantke and Cohen, 1971; abnormalities during the proper closure of the neural tube Carezani-Gavin et al., 1992; Chatkupt et 1993), reminiscent al" I exhibiting spina bifida, cranial exencephaly, meningocele and of splotch mice. overgrowthof nervous tissue (Auerbach, 1954). Consistent with the Pax3 expression pattern in the dorsal part of the neural tube, Pax6 and small eye (Sey) I an important role during the proper closure of the neural tube Another developmental mutant could be associated with the can be suggested. Pax6 gene (Walther and Gruss, 1991). Small eye (Sey) Neural crest defects result in various severe deficiencies mutants are identified in mouse (Roberts, 1967; Hill et al., I affecting melanocytes, Schwann cells, truncus arteriosus, , 1991) and rat (Matsuo et al., 1993). Pax6 maps on chromo-

parathyroid, thymus, dorsal root ganglia and the cardiovascular some 2 close to the Sey locus (Fig. 1) (Walther et al., 1991). I system (Auerbach, 1954; Franz, 1989; Moase and Trasler, 1989, Early Pax6 expression is confined to the optic sulcus, the optic Grim et al., 1992). Therefore, the splotch mutants provide a good stalk and the optic vesicle, subsequentiy in the retina and the model for neural crest defects perfectiy matching with the Pax3 surface ectoderm later developing into the lens (Walther and I expression (Auerbach, 1954; Goulding et al., 1991). Thus, Pax3 Gruss, 1991), Furthermore, Pax6 is expressed during nasal might be a crucial regulator in emigration of neural crest cells. development. I Pax3 is also expressed in the dermomyotome of the devel- Heterozygous smalf eye mutants have a characteristic eye oping somite and in the limb buds. Recent analysis of the limb malformation phenotype in tissues where Pax6 is expressed dur-

phenotype suggests that the lack of limb musculature is corre- ing development. In particular, a reduced diameter of the eye, I lated with the absence ot Pax3 expressing cells that fail to cataracts, vacuolated and dislocated lenses, iris hypoplasia etc. migrate into the limb bud (Franz et al., 1993; Bober et al., 1994). are observed (Hogan et al., 1986, 1988). In comparison to wild- Also the dermomyotome and myotome was shown to be disor- type litter mates, small eye mice are about 10% smaller I ganized in splotch mutants (Yang and Trasler, 1991). Both, (Roberts, 1967). In a homozygous state, small eye embryos fail defects in neural crest and myoblast precursors suggest a prob- to develop eyes, nose, the optic nerve and the olfactory bulbs, I able function of Pax3 during cell migration and/or proliferation finally dying after birth because they cannot breath (Hogan et al., and specification. 1986, 1988). Also the defects in the nose coincides with Pax6 expression domains (Walther and Gruss, 1991). However, none I PAX3 mutations and the Waardenburg syndrome of the analysed Sey alleles showed defects in the spinal cord. Mutations in the human PAX3 gene have been described in This is possibly due to compensating effects of other Pax tamily I the genome of patients with Waardenburg syndromes (WS type members, as has been proposed for other families of transcrip- 1 and 3) (Fig. 1) (Waardenburg, 19S1). Waardenburg syndrome tion factors (Joyner et a/., 1989; Chisaka and Capecchi, 1991;

is an autosomal dominant hereditary disease with a frequency of McMahon et a/., 1992) or possibly subtie alterations exist which I approximately 1 in 100,000 (Tassabehji et al., 1993). The WS 1 have not been detected yet. has been mapped to a chromosomal region syntetic to the Four different semidominant smalf eye alleles have been region of the murine splotch locus (Ishikiriyama et al., 1989; Foy detected and analyzed yet: Seyand SeY'e" (Hill et a/., 1991) are I st al" 1990). A variety of PAX3 mutations have been identified in phenotypic similar, whereas SeyH and SeyDeyexhibit a more WS1 patients. Different mutations like splice site mutations drastic phenotype showing additionally Coloboma of the eyes I (Tassabehji et al., 1992, 1993), insertions and deletions (Morell (heterozygous) and prenatal death around implantation et al., 1992; Tassabehji et al., 1993), missense mutations (homozygous) (Theiler st al" 1978; Hogan et al., 1986; 1988; (Baldwin et al" 1992; Hoth et al., 1993; Tassabehji et al., 1993) Glaser et a/., 1990). The molecular analysis of these four alleles I etc. have been assigned to the paired box of PAX3, Previously it revealed that a total deletion of Pax6 in ssyH and SeyDey might was shown, that a point mutation in Waardenburg Brazil be responsible tor the stronger phenotype (Glaser et al., 1990), I I I Pax and vertehrate development 373 I

Meer de Jong et a/., 1990) and Peters' anomaly (Hanson et a/., I A 1994) (Fig. 1). Aniridiais an autosomal dominant disorder with a frequency of around 1 in 64,000 to 1 in 96,000 characterized by I defects affecting the iris, the lens, retina and optic nerve (Shaw ela/., 1960; Nelson ela/., 1984; Hittner, 1989). Aniridiamaps on I chromosome 11p13, which is the loci where PAX6 has been located (Fig. 1) (Davis el a/., 1989; Ton et a/., 1991). Molecular I analysis revealed that the PAX6 gene was mutated in several aniridia patients, having point mutations or various deletions I (Tonel a/., 1991; Glaser el a/., 1992; Jordan el a/., 1992).Acom- pound heterozygous child has been reported with very drastic I craniofacial abnormalities, absence of eyes, nose and adrenal gland (Glaser el a/., 1994). Recently, PAX6was also found to be I mutated in patients with Peters' anomaly characterized by defects of the anterior chamber of the eye and corneal malfor- I 8.5 dpc 13.5 dpc mations (Hanson el a/., 1994). Thus far, the malformations observed in aniridia and Peters' anomaly show striking similari- I ty to those described for Sey. Therefore, Sey provides a mouse c model for both human diseases. I Pax genes and oncogenesis I

Pax-] I _Pax.3 Many transcription factors have been classified as proto- _ Pax-6 oncogenes controlling cellular processes, Recent data show that _Pax-2 _ Pax.S Pax genes are involved in oncogenesis. Overexpression of I _ Pax-S Paxl, 2, 3, 6 and 8 in fibroblasts (NIH3T3) resulted in foci of transformed cells that are able to develop tumors when injected I into nude mice (Maul becker and Gruss, 1993). This oncogenic potential seems to depend on a functional paired domain since I the point mutation in the Pax1unprotein inhibits the transforming activity of Pax1. I These in vitro studies are supported by in vivo observations in diverse human cancers. PAX2 and PAXB are expressed in I Wilms' tumor, a kidney carcinoma, whereas both genes are nor- adultbrain mally expressed only during kidney development and not after I Fig. 2. Schematic representation of Pax gene expression in early terminal differentiation (Dressler and Douglass, 1992; Poleev et and midgestation embryos and adult brain. fA) Saggitaf section of a/., 1992). Data showing that Pax2 is required for conversion of I day 8.5 p.C. embryo. (B) Saggital section of day 13.5 p.c. embryo. IC) mesenchyme to epithelial aggregates is also consistent with the Overview of Pax gene expression in rhe adult mouse brain. AP. afar characteristically mesenchymal origin of Wilms' tumors I plate: BP. basal plate; Cb. cerebellum: 01, diencephalon; dpc, day post (Dressler el a/., 1993). Moreover, it has been shown that the co/rum; OT, dorsal thalamus; E, eye; ET. epithalamus; FP. floor plate; IZ, human PAX3 in alveolar rhabdomyosarcomas is linked with can- I Intermediate zone; Ms, mesencephalon Mt, metencephalon; My, mye- cer (Barr el a/., 1993). The rearrangement and translocation of lencephalon; NE. nasal epithelium; PM, pons; Pro, prosencephafon: PT, to the carboxy terminus of a fork head gene (FKHR) results I pretectum; Rh. rhombencephalon; Rp, roof plate: Sc, spinal cord, Sf, sul- PAX3 in the expression of a PAX-Forkhead fusion protein. Finally, cus limitans: TCX, telencephalic cortex; n. telencephalon; \IT. ventral I thalamus; VZ, ventricular zone; 4\1, 4th ventricle. rearrangements in highly malignant astrocytomas in the chro- mosomal region including the PAX5 locus suggests the involve- while Seyand Seyveu are characterized by a point mutation and ment of PAX5 in the progression of astrocytomas. Studies show- I a 116 bp insertion respectively (Hill el a/., 1991). ing altered PAX5 expression seem to support this idea (Stuart el The phenotypic features of all four Sey alleles correspond a/., 1994). I with the expression of Pax6 during the development of the eye In summary, inappropriate Pax gene expression seems to be and nose. Recently, ablation experiments in chick revealed that tightly linked to uncontrolled cell growth although the function of I Pax6 expression in the optic vesicle is independent from the Pax genes in this process is not yet understood. overlying head ectoderm (Li el a/., 1994), suggesting Pax6 to be I involved in the early establishment of competent regions in the Perspectives and possible functions of Pax genes head ectoderm. I Pax genes have turned out to be important transcriptional reg. PAX6 and human diseases ulators in murine development. The functional analysis is still in I Mutations in the human PAX6 gene have been identified in its infancy but there is clearly available evidence that Pax genes I patients with aniridia syndrome (Glaser et a/., 1990; van der can serve as a powerful tool to elucidate the molecular mecha- I

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I nisms underlying embryonic development, in particular that of A the nervous system. I First of all, the comparison of the detailed expression analy- sis and the loss-ol-function mutations in mouse and man provid- I ed us with information about their tissue specific roles. For two Pax mutants, splotch and small eye, human diseases have been described correlating with Waardenburg syndrome I (Waardenburg, 1951) and aniridia (Glaser et al., 1990; van der Meer de Jong et al., 1990). These mouse models present a good BP

system to analyse the molecular basis responsible for these I human diseases. So far, it is still nof clear whether the phenotypic alterations of I the mouse mutants can be correlated with a complete or partial 11.5dpc loss of function. On one hand, the analysis of all mutant alleles 8.5 dpc

suggests that Pax gene mutations are loss-of-function mutations I since different disruptions (point mutations or extensive dele- Pax-3 Pax-2 tions) of the Pax proteins result in similar phenotypes. This _ _ Pax-5 I would suggest that every functional subdomain (paired domain Pax-? _ and homeodomain for DNA-binding, C- and N-terminus for tran- _ Pax-6 _ Pax-8 scriptional activation or inhibition) is essential for the gene tunc. I tion (Chalepakis et 1991; Chalepakis and Gruss, unpub- Fig. 3. Schematic representation of Pax gene expression in the al" developing spinal cord. Restricted expression as observed in cross lished data). On the other hand in the undulated alleles residual sections along the dors()-ventrafaxis. {AI Embryo day 8.5 p.c. (S) gene activity seems to argue against this hypothesis. Whereas I Embryo day 17.5 p.c. Ap, alar plate: BP, basal plate: dpc, day post coitum: the classical undulated allele, characterized by a point mutation, IZ; intermediate zone; Fp, floor plate; SL. sulcus limitans; RP. roof plate; shows the least severe defect. the most severe phenotype is VZ; ventricular zone. exhibited by the undulated short tail (uns) mutant, missing the I whole Pax! gene (Blandova and Egorov, 1975; Balling et al., underlying pathogenesis. Therefore, differential screening meth-

1988). uns can be considered as a null allele although the dele- ods (subtracted eDNA-libraries (Wang and Brown, 1991) or dif- I tion encompasses more than the Paxl locus. Theretore, it can ferential display (Liang and Pardee, 1992) comparing wild-type not be excluded that contiguous genes contribute to the uns phe- mice with mutant mice or induced and non-induced cell culture notype. systems would be of advantage. The approach to identify target I Gain-of-function experiments expressing ectopically Pax pro- genes by immunoprecipitation at chromosomal protein.DNA tein in fibroblast cell lines (Maulbecker and Gruss, 1993) or the complexes might be another useful tool (Gouid et al., 1990). I overexpression of Pax2 in transgenic mice (Dressler et al., 1993) indicate that also positive deregulation ot Pax proteins causes Acknowledgments We would like to thank F. Pituel/o, A. Stoykova, L St-Dnge for critical abnormal cellular programs. I Another feature of Pax genes seems to be reflected by the reading of the manuscript and in particular R. Fritsch for comments and helpful suggestions. semidominant nature of the mutant alleles in mouse and man. The phenotypic variations of e.g. the splotch mutant ranges from I References minor pigmentation detects in a heterozygous state up to severe neural crest, neural tube and limb muscle defects in a homozy- ARNVIG. J. (1959). The syndrome of Waardenburg. Acta Genet 9:41-46. I gous state (Auerbach, 1954; Bober et al., 1994). This observa- ASANO, M. and GRUSS, P. (1992). Pax-5 is expressed at the midbrain-hindbrain tion could be explained by a dosage effect, meaning that vari- boundary during mouse development. Mech. Dev. 33: 27-38. able thresholds of Pax proteins are responsible for pathogenesis AUERBACH, R. (1954). Analysis of the developmental effects of a lethal mutation I in different organs. in the house mouse. J. Exp. Zool. 127: 305-329. To further elucidate the Pax gene function, two different BALDWIN. CT, HOTH, C.F., AMOS. J.A., DA-SILVA, E.O. and MILUNSKI, A. I approaches might be useful. Firstly, there is need tor more Pax (1992). An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome. Nature 355: 637-638. gene mutants that could be generated as gain-oj-function BALLING, R., DEUTSCH, U. and GRUSS, P. (1988). Undulated, a mutation affect- mutants in transgenic mice or as loss-of-function mutants by ing the development of the mouse skeleton, has a point mutation in the paired I homologous recombination, In particular the foss-ot-function box of Pax1. Gel/55: 531.535. approach could be performed by different methods taking into BALLING, R., LAU, C.F.. DIETRICH S., WALLIN. J. and GRUSS. P. (1992). account that the Pax genes show temporal and spatial restricted Development of the skelelal system. In Postimplantation Development In the I expression. Therefore, the establishment of systems for tempo- Mouse 165. Wiley, Chichester, pp. 132-143. ral induced and tissue specific knock-outs are in progress. BARR, F.G., GALIU. N., HOUCK. J., BIEGEL, J.A., ROVERA, G. and EMANUEL, I Secondly, since in vitro studies revealed that Pax proteins act B.S. (1993). Rearrangement of the PAX3pafred box gene in the pediatric solid tumor alveolar rhabdomyosarcoma. Nature Genet. 3: 113-117. as DNA-binding transcription factors the question for down- BAUMGARTNER, S., BOPP, D., BURAI, M. and NOLL. M. (1987). Structure of I stream target genes arises. The identification of targets would two genes at the gooseberry locus related to the paired gene and their spa- certainly help to understand the biological functions of Pax tial expression during Drosophila embryogenesis. Genes Dev. 1: 1247- genes in development as well as the molecular mechanisms 1267. I

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