Development 104 Supplement, 181-186 (1988)Printed in Great Britain @ The Company of Biologists Limited 1988

Murine genes with homology to Drosophila segmentation genes

GREGORY R. DRESSLER" URBAN DEUTSCH1 , RUDI BALLING" DOMINIQUE SIMON2,

JEAN-LOUIS GUENET2 and PETER GRUSS1

rDepartment of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry,3400 Goettingen, FRG2(Jnitre de Genetique de Mammiferes, Institut Pasteur,25 rue de Dr Roux, Paris 75724 Cedex 15, France

Summary

As a potential method to define genes that controlvertebrate segmentation processes during embryogen-esis, murine genes containing a protein domain hom-ologous to the Drosophila paired box sequence wereisolated. The mouse genome contains at least threegenes with highly conserved paired box like sequences,

termed Pax genes, that are also conserved in othervertebrates. During embryogenesis, the PaxI gene isexpressed in ventral sclerotome cells and in the inter-vertebral disks of the vertebral column. Thus, PaxIclearly shows a segmented pattern of expression. A

181

second gene, Pax2, also is expressed in segmentedstructures of the intermediate mesoderm and in cer-tain regions of ectoderm derived tissues. The PaxI andPax2 genes map to chromosomes 2 and 7, respectively.Interestingly, several known mutations that affectvertebral column development map near the PaxIlocus.

Key words: paired box genes, segmentation, vertebratedevelopment, intervertebral disks.

Introduction

The genetic mechanisms controlling segmentationduring mammalian embryogenesis still remain poorlyunderstood. In contrast, many developmental genes

and mutations have been characterized in Dros-ophila, which has led to a more thorough understand-ing of segmentation , pattern formation, and theinteractions among these processes (for reviews see

Akam , 1987; Scott & Carroll , 1987). The hypothesisthat certain genetic mechanisms employed duringdevelopment are highly conserved even betweendivergent species has been substantiated by the iso-lation of many vertebrate genes based on homologyto conserved protein domains found among Dros-ophila homeotic and segmentation genes (McGinniset al. L984; Hart et al. 1985; Colberg-Poley et al. 1985;

Joyner et al. 1985). Recently, mouse homologues tosegmentation genes have been isolated based on a

conserved sequence found among several Drosophilagenes that specify inter- and intrasegmental bound-aries (Deutsch et Al. 1988). As in Drosophila, thereappear to be at least three genes sharing this particu-lar protein domain. Whether these murine segmen-

tation homologues are also involved in the subdiv-ision of the rostrocaudal axis during embryogenesisremains an intriguing possibility that, in light of theinterspecies divergence between Drosophila andmouse, would be a tribute to the design and adapta-bility of such a genetic regulatory system.

The Drosophila paired box

It has been proposed that segmentation during Dros-ophila embryogenesis entails a progressive subdiv-ision of the embryo into increasingly smaller units(Ni.isslein-Volhard & Wieschaus, 1980). This subdiv-ision is controlled by the gap, pair-rule and segmentpolarity genes whose respective mutations result inthe deletion of adjacent segments, the deletion ofalternate or parts of alternate segments and thedeletion of segment parts with the mirror-imageduplication of the remainder (Niisslein-Volhard &Wieschaus, 1980). Recently, a I28 amino acid con-served protein domain, termed the paired box, wasidentified among the segmentation genes paired(prd), gooseberry-proximal (gtb-p), and gooseberry-distal (gsb-d) (Bopp et al. 1986; Cote et al. 1987). As

I82 G. R. Dressler and others

the prd gene is a member of the pair-rule class and thegsb genes are segment polarity genes, it was sugges-ted that the conserved domains among these genesmay provide a functional link between the generationof individual segments and the polarization of seg-ments into anterior and posterior halves.

The spatial expression of the prd gene duringDrosophila development undergoes a shift from a 7 -

band striped pattern with double segment periodicityto a I4-band striped pattern with single segmentperiodicity at the blastoderm stage (Kilchherr et al.1986). The spatial expression of the gsb-d is similar toprd, initially showing 7 bands and dividing into 15

bands after germ band elongation (Bopp et al. 1986;Cote et al. L987). The gsb-p gene, however, onlyexhibits the l,S-band pattern with an additional stripeanterior to gsb-d (Bopp et al. 1986; Cote et al. 1987).Thus, the patterns of gene expression correlate di-rectly with the nature of the mutant phenotypes.

The murine Pax gene family

Using the Drosophila paired box sequence, Deutschet al. (1988) have isolated a mut'ine sequence from agenomic library that shows a high degree of aminoacid conservation with all three Drosophila pairedbox sequences. This mouse sequence, termed Paxlfor paired box containing gene, was used to investi-gate the presence of paired box sequences in othervertebrates. Fig. 1" shows the results obtained when3zP-labelled Pax DNAs were hybridized, under lowstringency conditions, to restriction-enzyme-digestedDNA from frog, turtle, chicken, hamster, rabbit,mouse and human. The multiple bands observed withboth PstI- and BamHl-digested DNA is evidence formultiple Pax sequences present in the genomes of allthe vertebrates examined.

With the isolation of several independent cDNAclones from mouse embryonic libraries, the presence

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Fig. 2. A comparison of amino acid conservation between mouse and Drosophila paired box sequences. The numbers

of identical amino acids between two sequences are shown in the corresponding squares. The intensity of shading

reflects the degree of homology between the sequences. The deduced protein sequences f.or prd (Frigerto et al. L986),

gsb-p and gsb-d (Bopp et al. 1986), and PaxI (Deutsch et al. 1988) are published. The protein sequence for Pax2 is

unpublished.

of multiple Pax genes in the mouse was firmlyestablished (G. Dressler & U. Deutsch, unpublisheddata). A comparison of amino acid conservation forthe paired box domains, as deduced from DNAsequences, is shown in Fig .2. The Drosophila pairedbox was originally described as bein g I28 amino acids

in length, with the gsb-p box having a three aminoacid internal deletion. The homologous mouse se-

quences, PaxI and Pax2, appear to be approximatelysix amino acids shorter. Thus, only I20 amino acids

were used for the comparison. As might be expected,the three Drosophila genes show more homology toeach other than to the mouse genes. However, PaxIand Pax2 are not significantly more homologous toeach other than to the Drososphila sequences. In fact,Pax2 shows a slightly higher level of conservation(87 lI20) to the gsb-d gene when compared to thePaxI gene (55 lI20).

In addition to the paired box, the Drosophila genes

prd, gsb-d and gsb-p also share a paired homeoboxdomain (Bopp et al. 1986). It remains to be deter-mined whether the murine PaxI and Pax2 genes also

contain homeoboxes, although a third murine gene,

Pax3, appears to contain both a paired box and ahomeobox (U. Deutsch, unpublished data).

Segmentation in the mouse and the expressionof Paxl

It is not altogether obvious that a mouse can beconsidered a segmented organiSffi, as compared to an

adult fruit fly for example. However, during thedevelopment of the mouse it is quite clear thatsegmentation plays an important role in the organiz-ation of the body plan and in the generation of

Murine Pax genes 183

5

individual tissues. Somitogenesis, the formation ofmetameric units along the rostrocaudal axis lateral tothe neural tube, is the most obvious example ofsegmentation in the mouse (reviewed by Hogan et al.1985). The somites subsequently differentiate intodermatome, ffiyotome and sclerotome, which ulti-mately generate the skin, skeletal muscles and axialskeleton, respectively. Ectoderm-derived tissues also

show segmented characteristics, particularly theneural plate which is divided into neuromeres (Tuck-ett et al. 1985; Sakai, 1987) and the spinal gangha, a

neural crest cell derivative. Segmentation in tissuesderived from the intermediate mesoderm is evident inthe pro- and mesonephric tubules, the embryonicexcretory organs.

The extent of PaxI expression during murineembryogenesis has been described in detail (Deutschet Al. 1988). Two representative micrographs and a

summ ary of the PaxI transcription pattern are pre-sented in Fig. 3. PaxI transcripts can first be detectedat 9 days post coitum (p.r.) and are restricted to thesclerotome cells of the differentiated somite. There-fore , PaxT expression is initiated after the primarysegmentation of the presomitic mesoderm. At 10 daysp.c., PaxI transcripts can be detected as a continuousband of hybridization in ventral sclerotome cellsalong the entire rostrocaudal axis beginnittg at ap-proximately the fourth occipital somite . PaxT-

expressing cells appear to migrate ventromedially tosurround the notochord. By 12 days p.c., the ex-pression has undergone a shift and now exhibits a

striped pattern, restricted to the anlagen of theintervertebral disks (Fig. 3A). By 14 days p.c., it isclear that the intervertebral disk cells are expressingPaxI (Fig.3B). Expression is also detected in thesternum and the thymus. At later times, expression in

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determined by Deutsch et al.(1988). (A) In situhybridizatron of PaxI tomidsagtttal section from aI2-day p.c. embryo showingdense labelling between theprevertebrae. (B) In situhybridization af PaxI tomidsagittal section from a14-day p.c. embryo showingdense labelling to theintervertebral disks. Thedorsal side is at the top androstral end is to the left.(C) Schematic representationof Paxl-expressing cells in afrontal view of the developingvertebral column.

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the developing vertebral column decreases until it canno longer be detected in newborn animals.

If the somite is considered the primitive segment,then subsequent somite differentiation and sclero-tome division into vertebrae and disk cells might beconsidered segment polaization events. Since PaxIexpression is initiated after primary segmentation has

occurred, Paxl may function, by analogy to Dros-ophila, os a segment polarity gene specific for one halfof the vertebral segment, the disk.

The Pax2 gene also exhibits a spatial and temporalexpression pattern in segmented structures duringembryogenesis. Pax2 can first be detected in theextending mesonephric duct and mesonephrictubules, as well as the developing metanephros(Dressler, unpublished). In addition, Pax2 tran-scripts can be detected in the neural tube at theborder of the ependymal and mantle layers and in theventral horns. It is of interest to note that Pax2-expressing cells in the intermediate mesoderm areadjacent to the mesoderm-derived ventral sclerotomecells which express Paxl.

The Pax gene expression patterns in the vertebralcolumn and the intermediate mesoderm extend alongthe rostrocaudal axis. In contrast, expression patternsof some mouse homeobox genes (Dony & Gruss,L987; Gaunt, I9B7; Holland & Hogan, L988; LeMouellic et al. 1988; Breier et al. 1988) are regionspecific in mesoderm-derived tissues. This importantdistinction could reflect the functional differencesbetween the Pax genes, required for segmentationalong the entire rostrocaudal axis, and homeoboxgenes, required for positional specification along theaxis.

The chromosomal location of murine Pax genes

In order to determine the chromosomal location ofthe Pax genes, the method of mouse interspeciescrosses was used (reviewed by Guenet, 1986). Re-striction fragment length polymorphisms (RFLPs),for PaxI and Pax2, between C57BLf 6 mice and aninbred Mus spretus line SPE/Pas (maintained in thelaboratory of J.-L. Guenet) were used to probeDNAs from a panel of backcross progeny of (C57BL|6 x SPE/Pas) F1 females x C57BL| 6 males. Thechromosomal assignment of the Pax genes was thendetermined by matching the RFLPs to known geneticmarkers. The PaxI gene is closely linked to the agoutilocus on chromosome 2. As 4163 recombinants werescored, the linkage distance between agouti and Paxlis approximately 6 + 3 cM. Similarly, the Pax2 gene islinked to the cHa-ras gene on chromosome 7

(23cM+ 6cM).Within 7 cM of the agouti locus on chromosome 2

Murine Pax genes 185

are two known mutations that, in light of the ex-pression pattern of PaxI , are worth mentioning. Therecessive mutation diminutive (d*) results in macro-cytic anaemia and consistent smaller body size. Inaddition, dm homozygotes have an additional rib atboth ends of the thorax, additional presacral ver-tebrae, malformed vertebrae, and fused ribs (Stevens& Mackensen, 1958). Potentially more relevant is therecessive mutation undulated (un). Mice homozygousfor un have malformed vertebrae along the entirebody axis because anterior sclerotome cells fail to jointhe posterior cells from the adjacent sclerotomeduring vertebrae formation. The result is a smallervertebra and alarger intervertebral disk (Gruneberg,1954). Although the anomalities described, particu-larly for uft, correlate with the time and position ofPaxI gene expression, &ny definitive correlation willrequire more precise linkage distance determi-nations.

Conclusions

The unusually high degree of amino acid conservationamong Drosophila paired box domains and murinePax domains suggests a conservation of function thatmay reflect similar genetic mechanisms. Thus, theisolation and characterization of murine paired boxcontaining genes is a potential method for unravellingthe morphogenetic basis of segmentation during em-bryogenesis. During the course of evolution, how-ever, the paired box domain may have been adaptedsuch that it retains its molecular function but inanother context. The possibility that the Pax genesare involved in cellular migration, intercellular com-munication, or other physiological processes associ-ated with the formation of structures such as thevertebral column cannot be ruled out. Although thePax gene expression patterns during mouse develop-ment are revealing, conclusive evidence regarding thedevelopmental nature of these genes will requiremutational analysis, perhaps through the applicationof gene-targetting techniques currently under devel-opment.

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