The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor

Cell, Vol. 79, 1025-1034,

December

16, 1994, Copyright

0 1994 by Ceil Press

he Mouse Segmentation Gene kr ncodes a Novel Basic Domain-Leucine Zippe ranscription Factor Gregory S. Barsh Departments of Pediatrics and Genetics and Howard Hughes Medical Institute Stanford University Stanford, California 94305

The mouse kreisier (kr) mutation causes segmentation abnormalities in the caudal hindbrain and defective inner ear development. Based on an inversion discovered in the original kr allele, we selected a candidate cDNA highly expressed in the developing caudal hindbrain. This cDNA encodes a basic domain-leucine zipper (bZlP) transcription factor and was confirmed to represent the kr gene by analysis of a second kr allele, generated by chemical mutagenesis, in which a serine is substituted for an asparagine residue conserved in the DNA-binding domain of all known bZlP family members. The identity, expression, and mutant phenotype of kr indicate an early role in axial patterning and provide insights into the molecular and embryologic mechisms that govern hindbrain and otic development.

Embryonic segmentation is a widely conserved developmental process whereby groups of cells along a particular body axis become organized into a multimeric structure in which individual multimeres exhibit related patterns of gene expression and developmental fates. One of the earliest and best-studied events in vertebrate segmentation occurs during hindbrain development, when cells of the rhombencephalon are transiently partitioned into seven or eight subunits known as rhombomeres (Vaage, 1969). All rhombomeres share certain features, such as the relative distribution of cell density, cell orientation, and mitotic activity (Guthrie et al., 1991; reviewed by Keynes and Lumsden, 1990). However, each rhombomere expresses a unique combination of Hox genes, in addition to genes encoding other transcription factors, receptors, and intercellular signaling molecules, which together are thought to be important determinants of developmental fate (reviewed by Wilkinson, 1993). Cell tracer studies show mixing within but not between rhombomeres (Fraser et al., 1990), and embryologic studies show that the developmental program of an individual rhombomere persists after transplantation to different anterior-posterior coordinates (Guthrie et al., 1992; Kuratani and Eichele, 1993). In all these respects, rhombomeres are functionally similar to parasegmentat compartments that are established during development of Drosophila embryos (Akam, 1987; LawAn important difference between rhombomeres and their invertebrate counterparts is likely to lie in the mechanisms that establish boundaries between adjacent seg-

ments, since the formation of Drosophiia parasegments is dependent on molecular interactions that occur in asyncitial blastoderm (reviewed by Pankratz and Jaeckle, 1993). One approach to identifying genes that control the formation of rhombomere boundaries is first to select candidates based on their predicted protein sequence, expression patterns, or both and then to determine mutant phenotypes for these genes. For example, inactivation of Krox20, a gene that encodes a zinc finger transcription factor expressed in rhombomere 3 (r3) and rhombomere 5 (r5), produces a shortened hindbrain with the loss of morphologic boundaries between r2 and r6 (Schneidermaunoury et al., 1993; Swiatek and Gridley, 1993). A second approach relies on the analysis of preexisting mouse mutations that affect hindbrain development, which may identify novel gene products of developmental significance without prerequisite k~owledg@ of their nature. Although many mouse mutations have been described that affect neural development in general, only one, kreisler (kr), is known specifically to affect rhombomere formation (Deol, 1964). The kr mutation was identified in an X-ray mutagenesis experiment because mutant animals exhibit a hyperactive pattern of behavior characterized by head-tossing and running in circles (Hertwig, 1942). Kreisler is the German word for circler. kr/kr animals are also deaf and cannot swim, a constellation of findings characteristic of mutations that affect inner ear function. In kr/kr embryos the otic vesicle, which normally lies adjacent to r5 and r6, is displaced laterally and develops into a cystic structure without an organized vestibular apparatus or cochlea (Hertwig, 1944; Deol, 1964). However, in contrast with most other mutations that result in deafness and circling, kr is unique in that it has been determined to affect both inner ear and rhombomere development. As described by Deol (1964), the area of the developing hindbrain caudal to rl-r3 in kr/kr embryos faiis to exhibit morphologic boundaries and appears instead as a large unsegmented bulge. Because transplantation studies in avians and amphibians had suggested that development of the otic vesicle was dependent on position-specific signals emanating from the neural tube, Deol suggested that defective otic vesicle devetopment in kr/kr animals was secondary to an underlying abnormality in hindbrain segmentation. To gain further insight into this possibility, we recently examined the expression patterns of several rhombomere-specific genes in kr/kr embryosandfoundthatKrox2O,~oxb~,~oxb3,and Hoxb4 each exhibited a qualitative disturbance in its domains of expression (Frohman et al., 19933, suggesting that kr affected a fundamental aspect of hindbrai~ segmentation. Mutations caused by X-rays are often due to chromosomal rearrangements and have helped to provide molecular access to several mouse genes of loam-standing interest. Here we report the positional cloning of the kr gene, based on the identification of a submicroscopic inversion detected by physical mapping with closely linked motecutar markers. Starting with reagents derived from the inver-

Cell 1026

yrnl-i’, Sac!, X/x+ + kr + kr + kr

Figure 1. Cloning a Rearrangement Sreakpoint Associated with the Original kr Ailele by a Chromosomal Walk from the Src Gene

(A) Southern blot autoradiograms of high molecular weight genomic DNA digested with the indicated enzymes, fractionated by contourclamped homogeneous electric field gel electrophoresis (three left panels) or by conventional gel electrophoresis (right panel), and hybridized with the indicated probes. The origin of each probe is described in Experimental * kb Procedures; the 1183 and Src probes ware hybridized to the same filter, and removal of resids1.0 ual probe between hybridizations was checked by autoradiography. The 1183 probe and the Src probe detect identical patterns of Mlul and Nrul fragments, but different patterns of Notl fragments, The differences detected by the Src Sacll Notl probe between (+/+) and kr/kr Not1 fragments Src are no longer detected with probe 1183, indiII cating that the kr-associated breakpoint lies Pi-151 cosll I cos8 closer to Src than it does to 1183. The Src and noncos8 mutant 10 kb the S2.6 probes detect identical fragments in Pi-4; l-----l / (+/+) DNA but different fragments in kr/kr DNA, indicating that the region of DNA between Src and S2.6 is interrupted by the kr-associated MIul Sacll Sacl I Nod Sacll breakpoint. (Very high molecular weight frag1 Src I II ments migrating at the limit of resolution in the autoradiograms depicted here were resolved kr on pulsed field gels run at longer switching times.) The Sl .O probe spans the kr-associated breakpoint and therefore detects two frag ments in (+I+) DNA digested with Sacl and Xbal; a second BamHl fragment of 5.0 kb is \ ,’ also visible on the original autoradiogram. WV 3.2 ? 1.2 0.6 k 0.4 0.4 F 0.4 , ’ ’ (B) Position of genomic clones and rare-cutting I -restriction sites relative to probes 1183, Src, Ada Emv-15 Src S2.6, and S1.0. The clones cos6, cos8, and t/I a kr cosl6 (data not shown) were obtained by 0.97 k 0.32 CM screening a C57BLI6J cosmid library with the Src probe; an end fragment from cos8 was then used to obtain cosli and cos5 (data not shown). Primers to isolate Pl-4 and Pi-3 (data not shown) were obtained from the 3’ end of the Src gene; an end fragment of Pl-4 was used to generate primers for the isolation of Pl-151, In the region of overlap, the BamHl restriction fragments obtained for all clones were identical. The Notl and Sacll sites shown in cosll, cos8, and cos6 were identified and mapped in cloned DNA and were also cleaved in genomic DNA as demonstrated with the 1183 probe and an end fragment probe from cos8 (data not shown). Another Sacll site and a Mlul site were identified in cloned DNA immediately 5’ to the Src probe but were not cleaved in genomic DNA. The positions of the Sacll, Notl, and Mlul sites in DNA that was not cloned were estimated from the sizes of fragments observed in (A). The coskr7.3 and five other overlapping cosmids that spanned the kr-associated breakpoint were obtained by screening a krfkr cosmid library with an end probe from PI-4. The arrows in coskr7.3 indicate the location of BamHl sites. (C) The consensus genetic map surrounding a, Emv75, and Src are according to Siracusa and Abbott (1992) and the distance between a and kr are according to The Jackson Laboratory data base.

“Fl

I

I

C

sion, we have isolated a cDNA expressed in the caudal hindbrain of (+I+) but not kr/kr embryos that encodes a basic domain-leucine zipper (bZIP) transcription factor closely related to the Maf subfamily (Fujiwara et al., 1993). Proof that thiscDNAencodes kreisler isevident from structural analysis of a kr allele we have generated by chemical mutagenesis. Our results demonstrate that this bZlP transcription factor plays an early role in the formation of rhombomere boundaries, suggest that additional members of the Maf subfamily may be involved in other aspects of mammalian segmentation, and provide insight into the relationship between development of the hindbrain and the inner ear.

Results The Original kr Mutation Is a Chromosomal Inversion Genetic crosses that segregate kr produce normal litter sizes, and there are no gross cytogenetic abnormalities in cells derived from mutant animals (data not shown), which suggests that kr is not a translocation or large inversion (Hertwig, 1942). Genetic mapping studies have previously localized kr 0.97 f 0.32 CM distal of the agouti (a) coat color locus on mouse chromosome 2 (Hertwig, 1942; Davisson and Roderick, 1989). The order and distances of molecular markers closely linked to A are

The Mouse Segmentation 1027

Gene kr

Cen-A-O.6 + 0.4 cM-Emv15-0.4 -I- 0.4 CM -Sk3.2 +: 1.2 CM -Ada (Figure 1C). We have described previously close genetic linkage between kr and the insertion site of EmvfS (0 of 93 recombinants) and the application of a polymerase chain reaction (PCRf-based polymorphism at the Emv75 locus to the genotypic identification of homozygous mutant embryos segregating in a kr/+ x kr/+ intercross (Frohman et al., 1993). Based on the hypothesis that the X-ray-induced lesion responsible for kr was a submicroscopic deletion or inversion, we determined whether hybridization probes from Emv75, Src, and Xmv70 (which lies between Emv75 and Src) would detect high molecular weight restriction fragments of abnormal size in kr/kr DNA. In DNA digested with Mlul, Notl, Nrul, or Sacll and fractionated by pulsed field electrophoresis, Emv75 and Xmv70 probes detect Notl, Nrul, and Sacll fragments of identical size in (+I+) compared with kr/kr DNA (data not shown), but a Src probe detects Mlul, Notl, and Nrul fragments of different sizes in (+/+) compared with kr/kr DNA (Figure 4 A). The Notl fragment in (+I+) DNA is 150 kb, suggesting that a kr-associated chromosomal breakpoint must lie within this distance from Src. To isolate this kr-associated breakpoint, we used theSrc probe as the starting point for a bidirectional chromosome walk. After the first step of the walk, it became apparent that the kr-associated breakpoint lay 3’ of Src, because a probe 5’of Src, 11 B3, was found to detect the same pattern of Mlul and Nrul fragments as the Src probe in (+/+) and kr/kr DNA, but did not detect the (+/+) 150 kb Notl fragment disrupted by the kr-associated breakpoint (Figures 1A and 16). Genomic clones that extended 3’ of Src could not be recovered in cosmid, h, or yeast artificial chromosome (YAC) vectors, but a bacteriophage Pl system allowed the region containing the breakpoint to be isolated in the clone Pl-151 (Figure 16). A single-copy probe from Pl151, S2.6, was shown to lie distal to the breakpoint because it detected several fragments identical to the Src probe in (i-l+) DNA, but detected a different pattern of fragments in kr/kr DNA (Figures 1A and 16). The breakpoint itself was isolated from kr/kr DNA on cosmid coskr7.3 and localized to a 1 kb Sac1 fragment, Sl .O, that contains no internal sites for BamHl or Xbal and yet, when used as a hybridization probe, detected two genomic fragments in (+/+) DNAdigested with BamHI, Sacl, or Xbal (Figure 1A). Further mapping indicated that S2.6 and S1.0 lie 42 kb and 40 kb, respectively, past the B’end of Src (Figure 1B). Thus, even though S2.6 detects a completely different pattern of high molecular weight restriction fragments in (++I+) compared with kr/kr DNA (Figure lA), sequences distal to the breakpoint are not deleted in kr/kr DNA. To characterize the nature of the kr-associated rearrangement further, we used the S2.6 probe, which lies distal to the region containing the breakpoint in (+/+) DNA, to clone coskrl6 from kr/kr DNA. We also used the fragment Dbp, which lies distal to the breakpoint in kr/krDNA, to clone P!-222 from (+I+) DNA (Figure 2A). Restriction map and DNA sequence analysis indicated that coskrl6 contained a second kr-associated breakpoint and that the

A

breakpoint

breakpoint

1

2

B

Figure 2. Identification, Associated Inversion

Cloning,

and Characterization

of the kr-

(A) Position and BamHl restriction maps (arrows) of genomic clones spanning the inversion breakpoints. Breakpoint 1 is identical to the one depicted in Figure 1. The clones PI -151 and coskr7.3 are described in the legend to Figure 1. The PI-222, Pl-221, and Pl-223 clones (only PI-222 is shown) were isolated using primers from an end fragment of coskr7.3, and all span the region of breakpoint 2. The coskrl6 and coskr21 (data not shown) clones were isolated using probe S2.6; both clones spanned breakpoint 2. The Mlul and Sactt sites indicated in PI-222 correspond to those mapped on the krchromosome in Figure I. The probes S2.6, S1.0, and Dbp are described in Experimental Procedures. (B) DNA sequence spanning the breakpoints was determined from subclones of the cosmid or Pl clones shown in (A). (6) The length of the inversion, 0.6 + 0.8 CM, was determined by genetic mapping of the Gbp probe, which detects a variant EcoRl site (indicated with an asterisk) that is present in C57EU6J, kr/kr, and Mus castaneus DNA, but absent from the strain of laboratory mice used to generate the intersubspecific backcross panel (Winkes et al., 1994). The location of the Dbp probe is indicated relative to adjacent BamHl and EcoRl sites in PI-222; other EcoRl sites present on the clone were not mapped. Segregation of the proximal inversion breakpoint was determined with a Src probe and Mspl.

molecular lesion responsible for krwas likely to have been a simple inversion with centromere-proximal and centromere-distal breakpoints in Pl-151 and Pl-222, respectively (Figure 2A). To establish the orientation and length of the inversion, we determined the genetic map positionof probes from the two breakpoints with respect to closely linked molecular

Cell 1028

markers that had been characterized previously in an intersubspecific backcross (Winkes et al., 1994). Analysis of 129animals typed forA, Emvl5, Src, Dbp, andAda placed Dbp between Src and Ada with a genetic distance and orderofcen-Src-0.8 + 0.8cM-Dbp-1.5 f 1.1 CMAda-tel (Figure 2C), indicating that the kr inversion is 0.8 f 0.8 CM in length with breakpoints that lie between EmvlSand Ada. The minimum physical length of the inversion is 1 Mb, since a Notl fragment of this size is detected by Src in kr/kr but not in (+I+) DNA (see Figure 1A; data not shown). There are no high molecular weight restriction fragments detected in common by probes from the kr inversion breakpoints, and therefore we are unable to place a maximum physical limit on the size of the inversion. However, given the95Vo confidence limit for the maximum genetic distance between Src and Dbp, 1.6 CM, the inversion is likely to be less than several megabases in length. Comparison of the sequences immediately flanking the inversion breakpoints from Pl-151, Pl-222, coskr7.3, and coskrl6 revealed that a small amount of DNA had been deleted from each breakpoint on the kr chromosome: 3 bpfrom the centromere-proximal inversion breakpoint and 16 bp from the centromere-distal inversion breakpoint (Figure 2B). Thus, the inversion is likely to have been caused by nonhomologous breakage and reunion and is consistent with the discovery of kr among the F2 offspring of an irradiated male. Taken together, these observations suggest that the inversion is causally related to the kr/kr phenotype and that the effects of kr on hindbrain and inner ear development are caused by disruption of a single gene located very close to one of the inversion breakpoints. Two Candidate Genes Close to the Distal kr Inversion Breakpoint Based on the genetic map position of a kr allele that we generated by chemical mutagenesis (see below), our search for the kr gene focused on the region disrupted by the distal inversion breakpoint. Previous phenotypic characterization of kr/kr embryos had established a relatively narrow spatiotemporal window within which the kr gene was likely to act (Deol, 1964; Frohman et al., 1993), and therefore our initial approach to isolate genes close to this breakpoint was based on cDNA selection rather than exon trapping (Lovett et al., 1991). Embryonic cDNA from 8.5 days postcoitum (dpc) was amplified by PCR and selected with 120 kb of genomic DNA spanning the distal inversion breakpoint as described in Experimental Procedures (Figure 3A). From approximately 150 clones that had inserts of 200-500 bp, approximately d5% did not hybridize to the genomic DNA used for selection. However, the remainder of the clones represented one of two cDNAs, described below as cDNA1 and &NAP, for which extended fragments 1.9 kb and 1.3 kb in length, respectively, were isolated from an embryonic cDNA library. Based on hybridization of the 1.9 kb and 1.3 kb fragments back to the initial pool of selected cDNA clones, we estimated that cDNA2 was approximately five times as abundant as cDNA1. Placement of the cDNAs on the genomic contig indicated that cDNA1 and cDNA2 are located 55 kb centro-

A

cDNA2 (1.3 kb)

distal kr breakpoint

Figure 3. Characterization kr Breakpoint

of Two cDNAs Associated

coNAl (1.9 kb)

with the Distal

(A) Orientation and position of genomic and cDNA clones spanning the distal breakpoint (referred to as breakpoint 2 in Figure 2), as determined by Southern blot hybridization. The PI-729 and PI-730 clones were obtained using primers from an end fragment of Pl-222. The 1.9 kb cDNA1 fragment lies on a genomic IO kb BamHl fragment from the centromere-distal end of Pl-730; the 1.3 kb cDNA.2 fragment lies on a genomic 4 kb BamHl fragment from the centromere-proximal end of Pl-222, which also contains Mlul and Sacll restriction sites mapped in genomic DNA by pulsed field electrophoresis (see Figures 1A, 18, and 2A). Orientation of the Pl clones relative to the centromere is based on the results depicted in Figure 2. The orientation of cDNA1 has not been determined; the orientation of cDNA2 is based on sequence comparison to genomic DNA. (B) The cDNA1 and cDNA2 clones were isolated as described in the text and Experimental Procedures. The sequence of cDNA2 is shown; the sequence of cDNA1 is available from GenEank. (C)Alignment of the cDNA2 predicted amino acid sequence with c-Maf according to the Lipman-Pearson algorithm. The sequence similarity is 49%, and the bZlP regions of both proteins are shown in bold.

The Mouse Segmentation 1029

Gene kr

C (cDNA2; +I+; IO s)

D (cDNA2; +I+; 14 s) 0 (cDNA2:

7 s)

E(Hoxb7;

+/+; 14s)

Whole-mount in situ hybridization was performed as described in Experimental Procedures, and representative embryos were photographed using dark-field microscopy. Magnjfication is 28.8 x except for (A) and the lower portion of (B), which is 57.6 x Two nonoverlapping probes for each cDNA are described in Experimental Procedures and were hybridized a minimum of three times separately to at least six kr/kr, frr/+, and (+/+) embryos. For cDNA2, multiple timepoints were examined between the 7-somite (s) stage at 8.5 dpc (B) and the I4-somite stage at 9.0 dpc (C and D). The expression domain of cDNA2 was determined by its position relative to the otic vesicle (arrows) and the first branchial arch (arrowheads). For comparison, expression of Noxbl in r4 is shown in (E). The curved arrow points to expression of cDNA2 in neural crest.

mere distal and 30 kb centromere proximal, respectively, of the kr inversion breakpoint, that neither clone contains sequences that cross the breakpoint, and that cDNA2 appears to be associated with a CpG island (Figure 3A). The sequence of cDNAl is not similar to previously cloned sequences in GenBank, but includes a 384 amino acid open reading frame apparently incomplete at its amino terminus, followed by approximately 694 bp of 3’ untransBated sequence and a poly(A) tail. The 1.3 kb cDNA2 fragment contains a 323 amino acid reading frame with approximately 120 bp of 5’ untranslated and 300 bp of 3’ untranslated sequence (Figure 3C). The protein predicted by cDNA2 is 49% similar to that encoded by the chicken v-maf oncogene (Figure 3D), a bZlP transcription factor originally identified by its ability to promote the formation of fibrosarcomas (Nishizawa et al., 1989). More recently, some members of the Maf family have been shown to be important for erythroid-specific transcription and development in mammals (Andrews et al., 1993).

Expression of cDNA1 and cDNA2 in (+/+I and kr/kr Embryos To help distinguish between cDNAf or A2 as candidates for the kr gene, we determined the pression patterns at 8.5 dpc, the time at which rhomobomere boundaries are beginning to form. For cDNA1, antisense probes detected diffuse expression throughout the headfold and in the tailbud of (+/+) and kr/krembryos with no discernible differences in the level or pattern between the two genotypes (Figure 4A). For cDNA2, antisense probes detected a restricted domain of high level expression in the caudal hindbrain of (++t) embryos with a sharp rostra1 border at the r4/r5 boundary and a diffuse caudal border that extends partway through r6 (Figures 4C-4E). In addition, cDNA2 is expressed at high levels in a thin stream of neural crest adjacent to the caudal hindbrain and at low levels throught the embryonic headfold and tailbud, but is notably sent from the developing otic vesicle. Expression of

Cell 1030

cDNA2 is detectable by the 7-somite stage at 8.5 dpc, continues through the 1Csomite stage at 9.0 dpc, fades by the 22-somite stage at 9.5 dpc (Figures 4B-4D), and is not expressed in adult tissues (data not shown). In kr/kr embryos, cDNA2 is still expressed at low levels in the headfold, but the domain of high level expression in the caudal hindbrain is completely extinguished (Figure 48). In experiments in which (+I+), kr/+, and kr/kr embryos were examined in parallel, heterozygotes exhibited reduced caudal hindbrain expression consistent with gene dosage (data not shown). The striking correlation between the expression domain of cDNA2 and the krmutant phenotype and the absence of this expression in kr/kr embryos are strong evidence that cDNA2 is the kr gene. Generation of a kr Allele by Chemical Mutagenesis The existence of multiple alleles can be an invaluable aid to positional cloning experiments, but most mouse genes cloned on the basis of their genetic map position have made use of preexisting alleles or have relied on complementation of the original mutation by transgenesis to confirm the identity of a particular candidate sequence. To aid in the cloning and analysis of kr, we designed a chemical mutagenesis experiment that would allow the identification of kr alleles. Male (BALBlcJ x SJL/J)Fl mice were treated with four weekly doses of N-ethyl-N-nitrosourea (ENU), a powerful mutagen that induces primarily point mutations in the male germline (Justice and Bode, 1986; Dove, 1987), and then were mated to a kr/A (+) females. The genotype of the nonmutant males, A (+)/A (+), was chosen to allow the use of a closely linked visible marker at the a locus to distinguish between a potential kr allele and theoriginal krallele. Females heterozygousforkrwere chosen because kr/kr females are often infertile and the few that do become pregnant make very poor mothers. Among 597 progeny screened for deafness and circling behavior, we identified one mouse that carried a kr allele, kP’. Subsequent breeding studies showed that kFNuI+ animals are fully viable and phenotypically normal, that kFNu/kr animals are phenotypically indistinguishable from kr/kr animals, and that krENu/kFNuanimals exhibit a hindbrain phenotype (to be described in more detail elsewhere). In addition, kFNu maps 1.8 + 1.2 CM centromere distal to Src, which is nearly coincident with the distal inversion breakpoint of the original kr allele. A cDNA2 Point Mutation in krENU As a complementary approach to identifying the kr gene, we examined the structure and expression of cDNA2 in kFNu. Whole-mount in situ hybridization experiments indicated that the level of expression of cDNA2 was not altered in kFNu/kFNu embryos compared with (+I+) embryos (data not shown). To determine whether kFNu was associated with a structural alteration in cDNA2, we used PCR to amplify the entire protein-coding sequence and flanking regions from genomic DNA of BALBlcJ, SJUJ, and kFNu/ kFNu animals, and we directly sequenced the amplified products with internal primers. We identified an A to G

transition at nucleotide 748, predicted to cause an asparagine to serine substitution at amino acid residue 248 (Figure 5) associated only with the kPNU allele. The mutation was observed in five kFNu/kFNu animals from three separate litters, was not observed in the two possible parental strains BALB/cJ and SJL/J (Figure 5) or in C57BU6J mice (data not shown), and was also not observed in three independent cDNA clones from C57BU6J embryos or the Pl222 genomic clone (data not shown). Alignment of the protein predicted by cDNA2 with other bZlP transcription factors reveals that the mutated asparagine residue lies in the putative DNA-binding domain and is conserved among all known family members (Figure 5). Structural and functional studies on GCN4 have demonstrated that this asparagine is in direct contact with the major groove of the DNA (Ellenbergeret al., 1992) and that, when changed into a serine, completely abolishes transcriptional activation (Tzamarias et al., 1992). These observations provide compelling evidence that cDNA2 is the kr gene.

acidic domain ----p77///f////f/flJ 1

l~uclne

basic domain ?&%I -IIIIct---235 265

110

323

amino acid residue

BALB/cJ

SJUJ

kr--“/krEfJ”

ACGTACGTACGT ~ non-mutant Asn 736 CTG AAG Am COG DGC TG AAD AGC CD0 GGC SW

kr

v-maf mafK mafF NRL P13 v-fOS fni-1 fosB

v-fun jU”B jU”D CREB GCN4 CIEBP

VIRLKQKRRT VIRLKQKRRT

L L

RGYAQSC RGYAQSC

EEEERRRVRR EEEEKRRVRR QERIKAERKR QERIKVERKR QERIKAERKR EAARKREVRL ESSDPAALKR KNSNEYRVRR

E E M L L i-4 A E

KLAAAKC KLAAAKC RIAASRS RLAATKC RIAASKC REAAREC TEAARRS NIAVRKS

RYKRVQQKHH RFKRVQQRIIV RIKRVTQKEE RVKRVCQKEE RSKRLQQRRG RIKRVTQKEE

265 L L L L L L

RNRRRELTDT RNRRKELTDF RNRRRELTDR RKRKLERIAR RKRKLERIAR RKRKLERISR RRKKKEYVKC RARKLQRMKQ RDKAKQRNVE

L L L L L L L L T

Figure 5. A Point Mutation in kr ENUAffects the Putative DNA-Binding Domain The upper panel shows a diagram of the predicted kreisler protein sequence with functional domains delineated according to comparison to other bZlP proteins. The middle panel shows a portion of a DNA sequencing gel for which genomic DNA from the indicated animals was PCR amplified and sequenced as described in Experimental Procedures. The A to G mutation was observed in PCR-amplified DNA from five kP”/krENu animals, but was not observed in two SALB/cJ animals, four SJLN animals, and three C57SUBJ animals (data not shown). No other differences in the predicted protein sequence were observed. The lower panel shows alignment of basic domains from different bZlP proteins, demonstrating conservation of the asparagine (N) mutated in kr?

The Mouse Segmentation

Gene kf

1031

iscussion

+I+ r7

or l-5

r5

+lkr r4

r3

krikr r2

r7

PF

r4

r3

i

The isolation and characterization of preexisting mouse mutations is a powerful approach to studying biological processes relevant to mammalian development and human disease. Here we show that two independent alleles of the mouse kr locus exhibit alterations in a gene that encodes a bZlP transcription factor related to the Maf subfamily. RNA for this transcription factor, which we have provisionally named kreisler, is expressed at greatly reduced levels in the original kr allele and, in the kFNu allele, contains a point mutation predicted to abolish transcriptional activity. Although it remains to be determined whether kr and kFNu are hypomorphic, amorphic, or antimorphic in nature, identification of the kr gene is an important step in defining both the molecular pathways of normal segmentation and the pathophysiologic defects that result in abnormal structure or function of the inner ear. Molecular Genetics of the Two kr Alleles The high level expression domain of kr RNA in the caudal hindbrain is eliminated in the original kr allele, but low levels of expression persist in the embryonic headfold. Cur preliminary experiments suggest the transcriptional initiation site for kr is located close to the protein-coding sequence, 30 kb away from the inversion breakpoint, and it seems likely that the effect of the inversion on kr expression is caused not by physical disruption of transcribed sequences, but instead by separation of the kr transcription unit from a positive regulatory element at the centromere-distal breakpoint or, alternatively, by juxtaposition of the transcription unit next to an inhibitory element at the centromere-proximal breakpoint. The region of DNA between the Src gene and the proximal inversion breakpoint could not be cloned in cosmid, h, or YAC vectors, and sequence elements from this region may well have an unusual structurecapable of exerting an inhibitory position effect Distinguishing between these alternatives should’ be possible as the kr 5’ flanking region is characterized in more detail. The asparagine to serine substitution in the basic domain of kFNu is likely to affect DNA binding but not the ability to dimerine. Although this might be expected to produce a dominant negative effect, kFNu behaves genetically as a recessive mutation. This does not exclude the potential for dominant negative effects at an altered dosage ratio, especially since we do not yet know whether the protein product of kFNu is present at normal levels. Because kreisler may interact not only with itself but with other bZlP proteins (see below), the phenotype of kFNu/ kFNu embryos may differ from those homozygous for a kr null allele. What Causes the krikr Phenotype? The pattern of kr gene expression in normal and kr/kr animals (Figure 6) provides insight into mechanisms of hindbrain segmentation and the pathogenesis of inner ear abnormalities that result in loss of vestibular and auditory function. Expression of kr is detectable at 8.0 dpc (one somite) as a band in the caudal hindbrain (unpublished

Figure 6. Schematic Representation of Gene Expression opment in Nonmutant and kr/kr Embryos

r2

I

I

and Devel-

The patterns of kr expression are based on the results described in this paper (Figure 4, cDNA2) using 8.5 dpc embryos; expression patterns of other genes are based on our earlier results using 9.6 dpc embryos (Frohman et al., 1993). In k&r embryos, there are no morphologic boundaries caudal to r3/r4; we suggest that cells in the region of r5 fail to acquire their proper identity, but at least some part of r6 is formed correctly since cartilaginous neural crest derivatives of this rhombomere are present in kr/kf mice (see text for discussion). The abnormally long rhombomere in kr/kr embryos exhibits antiparallel gradients of expression for Hoxb? and tfoxb3 that may be caused by mixing of cells in r4 and r6; lighter colors represent lower levels of expression. Boundaries of gene expression that do not coincide with morphologic boundaries are shown as broken lines. k RNA is not expressed in the region of the developing otic vesicle; its lateral displacement and failure to develop properly in Mkrembryos may be due to loss of diffusibie factors normally produced by the caudal hindbrain.

data), and by 8.5 dpc (six to eight somites), the high level domain exhibits a sharp rostra1 edge coincident with the r4/r5 boundary and a diffuse caudal edge located midway through r6 (Figure 4; Figure 6). Thus, expression of kr RNA precedes the establishment of rhombomere boundaries and coincides with the refinement of Hoxgene expression that occurs during hindbrain segmentation. Absence of the high level expression domain in kr/kr embryos results in the loss of visible boundaries from r4 to r6 and altered expression domains of Hoxb 7, Hoxb3, Hoxb4, Krox20, and Fgf3 (Frohman et al., 1993). Although these findings cannot be explained simply by deletion, addition, or homeotic transformation of one or more rhombomeres, certain components of the kr/kr phenotype may reflect a secondary response and not the underlying developmental abnormality. Based on the region in which kr is normally expressed, we favor the interpretation that the fundamental defect in kr/kr embryos is a failure of cells in the region of r5 to acquire their proper identity. Rhombomere transplantation studies predict that deletion of r5 should result in an unsegmented rhombomere of double length containing a mixture of cells from r4 with those from rCj’(Guthrie and Lumsden, 1991). Furthermore, cells in such adouble rhombomere would be expected to retain their original patterns of gene expression; therefore, failure to form r5 would explain both the caudal expansion of Hoxbl expression and the rostra1 expansion of Hoxb3 observed in kr/kr embryos (Figure 6). boss of r5 in kr/kr embryos would not account for the absence of Fgf3 expression in the region corresponding to r6. Some findings in kr/kr embryos that cannot be explained simply by the loss of r5 are the persistence of Krox20 ex-

Cell 1032

pression in r3 as well as the ectopic low level expression of Fgf3 in the rostra1 hindbrain (Figure 6). Because kr RNA is not expressed at high levels in the rostra1 hindbrain, these latter observations are likely to reflect secondary effects of loss of kr function. McKay et al. (1994) have recently confirmed our observations regarding expression of Hoxbl and Kiox20 in kr/krembryos and suggest a fundamental defect in which r5 and r6 are lost. It is possible that a portion of r6 fails to form normally in kr/kr embryos, especially since the expression of kr RNA in 8.5 dpc embryos extends partway through the region in which r6 will form. However, a complete deletion of r6 is unlikely since its cartilaginous neural crest derivatives are present in kr/ kr animals (Frohman et al., 1993). Potential Transcriptional Targets of Kreisler It is not yet clear whether the absence of kr causes cells in the region of r5 to die, to become incorporated into adjacent rhombomeres by default, or to acquire the identity of r4 or r6 owing to the expression of genes other than kr. Nonetheless, our findings indicate that kr acts either directly or indirectly to activate the expression of Fgf3 and Krox20 in the caudal hindbrain since, in kr/kr embryos, fgf3 is not expressed in the region corresponding to r5 and r6 and KroxPO is not expressed in the region corresponding to r5 (Figure 6). At 8.5 dpc, the expression domain of krextends partway through r6 and therefore overlaps that of Fgf3 (Wilkinson et al., 1988). More detailed studies will be required to determine whether there are some cells positive for Fgf3 and negative for kr, but existing data are compatible with the suggestion that expression of Fgf3 in the caudal hindbrain is a direct target for transcriptional activation by kreisler. The question of Krox20 activation by kreisler is more complex, since their expression patterns intersect but do not coincide (Figure 6). Nonetheless, gene products of both are required for the ultimate formation of r5, and, in principle, could act in parallel or in series. However, unlike the situation that pertains in kr/kr embryos, loss of Krox20 function does not prevent the expression of Krox20 RNA in r5 (Schneidermaunoury et al., 1993; Swiatek and Gridley, 1993). With regard to r5, then, kr must lie genetically upstream of Krox20, and even though kr is required for expression of Krox20, the converse may not be true. How Does Kreisler Affect Inner Ear Development? kr RNA is expressed in the hindbrain and adjacent neural crest, but is notably absent from the otic vesicle. Thus, as suggested by Deol(1964), faulty development of inner ear structures in kr/kr embryos must reflect abnormal inductive interactions rather than a requirement for kr in the otocyst itself. Certain pathologic features are common to kr/kr embryos (Hertwig, 1944; Deol, 1964) and those in which Fgf3 has been inactivated (Mansour et al., 1993), including failure to develop a normal endolymphatic duct, a tendency for cystic expansion later in development, and frequent asymmetry of the inner ear defects. However, many animals that lack Fgf3 function have a vestibular apparatus sufficient to prevent circling, and therefore the effects of kr on inner ear development cannot be mediated

solely by Fgf3. In particular, failure of the otic vesicle to move adjacent to the hindbrain is both an early and a prominent feature of kr/kr embryos but not those mutated for Fgf3. Transplantation studies have shown that early development of the otic vesicle is dependent on physical proximity to the caudal hindbrain (Detwiler and Van Dyke, 1950; Yntema, 1950), and biochemical approaches to identify targets of kreisler may reveal molecules that mediate this inductive effect. A Role for bZlP Transcription Factors in Segmentation Among bZlP transcription factors, kreisler is most similar to proteins of the Maf subfamily, named after an oncogene from an avian retrovirus that causes musculoaponeurotic fibrosarcomas (Nishizawa et al., 1989). Some members of this family, including c-Maf, are widely expressed (Fujiwara et al., 1993) but other members, including kreisler and NRL (the mammalian gene product for neural retina leucine zipper) are expressed in specific spatiotemporal domains (Swaroop et al., 1992). Although a mouse c-Maf has not yet been described, the low sequence similarity between kreisler and chicken c-Maf (49%) and the difference in their expression patterns indicate that kreisler is not the mouse homolog of c-Maf. All Maf-related proteins examined to date form heterodimers with Fos, Jun, or both that exhibit binding specificities distinct from either homodimer (Kataoka et al., 1994; Kerppolaand Curran, 1994). It will be interesting tocharacterize the binding specificities that result from heterodimerit interactions of kreisler with other bZlP proteins and to determine whether there are Maf subfamily members in addition to kreisler expressed in other regions of the hindbrain. Proteins such as Fos and Jun that are expressed widely during development may play a role in tissue-specific activation of target genes if their binding specificity is altered when paired with kreisler. A transcriptional regulatory network of heterodimeric bZlP proteins could also serve to establish and refine compartment boundaries during vertebrate segmentation by positionspecific activation of homeotic selector genes and would have implications for the acquisition of segment diversity during evolution. Concluding Remarks The presumptive loss of r5 in kr/kr embryos might be expected to cause more than circling and deafness. It is possible that kr is hypomorphic and that low level expression prevents a more severe defect or that the adult derivatives of the hindbrain segments affected by loss of krfunction are physiologically redundant. In either case, mutations of a human kr gene, which is likely to lie on chromosome 20q (Siracusa and Abbott, 1992), may be responsible for one of the many syndromes that include inherited deafness (reviewed by Deol, 1968). Although abnormal otic vesicle development often leads to circling in mice, primates rely more heavily on visual cues, and loss of vestibular function in humans affects balance but not locomotion. Determination of segment fate by the combinatorial action and autoregulation of Hox genes is a common feature

The Mouse Segmentation 1033

Gene kr

of distantly related organisms, but kr could not have been isolated on the basis of sequence similarity to Drosophila segmentation genes. Our results exemplify the potential of using phenotype as both a starting point and as an endpoint for studying segmentation in vertebrates, since the chromosomal inversion in kr was a crucial signpost in guiding a search for transcribed sequences and since the point mutation in kFNUprovided genetic proof of molecular identity. kr is one of many preexisting mouse mutations, ut the development of new techniques for mutagenesis and screening will allow the approach we have taken to be applied to biologic processes for which mutations do not yet exist. Experimental Procedures Mice and Breeding Studies Thekrmutationwasdiscovered among theF2offspringof an irradiated male prior to the derivation of current inbred strains and was obtained originallyfromThe Jackson Laboratory. Repeated cyclesof backcrossing a kr/a kr males to C3HIHeJ A (+)/A (+) females and intercrossing the progeny have been used as a source of mutant kr/kr and control (+/t) animals for the studies described here. Close linkage between a and kr allows a convenient screen for nonpenetrance; among several hundred progeny examined, the only nonagouti mice that have not exhibited the kreisler phenotype were found to be rare recombinants between a and kr, indicating that kr is fully penetrant. Genetic mapping of the kF” allele was based on analysis of 96 meioses from a kPY+ intercross and will be described fully elsewhere. DNA Probes DNA probes from Emvld, XmvlO, and Src have been described previously (Ollmann et al., 1992). For the chromosome walk, DNA frag ments from the ends of cosmids or Pi bacteriophage were identified by partial restriction mapping, subcloned, and then used to generate single-copy probes. Internal single-copy probes are identified by the BamH! fragment from which they originate as follows, The 52.6 probe is a2.6 kb Sac1 fragment located within a IO kb BamHl fragment whose position within R-151 is indicated in Figures 1 and 2. The Sl .O probe is a 1 kb Sac1 fragment located within an 8 kb BamHl fragment and contains 231 bp from the Src-proximal and approximately 800 bp from the Src-distal side of kr breakpoint 1 (Figures 1 and 2). The 1183 probe is a 500 bp Pstl fragment located within a 3 kb BamHl fragment at the end of cosli as indicated in Figure 1. The Dbp probe is a 1 kb Hindlll-Pstl fragment located within a 12 kb BamHl fragment whose position within PI-222 and coskr7.3 is indicated in Figure 2. Analysis of Genomic DNA Contour-clamped homogeneous electric field gel electrophoresis, conventional gel electrophoresis, limited acid hydrolysis, capillary transfer, and hybridization in the presence of 10% dextran sulfate to probes radiolabeled by random priming were all performed according to standard procedures. Cosmid libraries were prepared from genomic DNA of C57BU6J mice partially digested with SaulllA and from kr/kr genomic DNA partially digested with EcoRl using restriction-minus packaging extracts and hosts (Stratagene). Bacteriophage Pl clones were obtained from a commercial library constructed from a cell line of the mouse strain Rlll (Genome Systems) by PCR-based screening with the oligonucleotide primer pairs S’CAAAGCTTCTGGGCTCATC-3’ and YCTGCTGACAAGAGACTG-3’ (Pl-4 and Pi-3) 5”CAGAGGGACTCCACACAACACB’ and 5”CTCTGACATCACTCAGCACAG-3’ (PI-151), 5’-CACAGATCGCTAACAG-3’and 5”CTGACTGACAATGGCTC-3’ (Pl-222, Pl-221, and Pl-223), and Y-GGACTACTGGTCTTAG-3’ and 5’~CACACGGTGATTGGAA-3’ (Pl-730 and Pi-729). The PCR was performed for 35-40 cycles using denaturation and extension conditions of 94OC for 1 min and 72OC for I min. The respective annealing conditions were 65°C for 1 min, 58°C for 1 min, 52°C for f min, and 45’C for 1 min. Genomic clones were aligned by comparing their BamHI restriction maps, which were determined by indirect end labeling and partial digestion. In some regions, the order of several

BamHl fragments could not be determined, in Figures l-3 with parentheses,

and these are indicated

ENU Mutagenesis (SJUJ x 5alblcJ)Fl hybrid males were treated with four weekly intraperitoneal injections of 100 mglkg ENU as described by Justice and Bode (1986) and Rinchik et al. (1990). Of 15 males treated with ENU, 10 recovered fertility after 8-12 weeks and were bred to a kr/A (+) females. A total of 597 progeny were recovered at weaning; two males exhibited circling and deafness, and one female circled but was not deaf. Of the two animals that circled and were deaf, one carried the k?allele, and the other carried a dominant mutation unlinked to the a locus. The male that sired the kP allele had a total of 38 progeny, of which one exhibited growth retardation and did not reproduce. The k~““allele has been transmitted through four generations by breeding to C57BU6J ala animals; heterozygotes of presumptive genotype A kP”/a (+) are phenotypically normal but, when test bred to a kr/A {+) females, produce agouti circling (A kP”/a kr) and noncircling mice consistent with the expected Mendelian proportion of 1:3. Whole-Mount in Situ Hybridization Mutant embryos between 8.0 dpc and 9.0 dpc were obtained from naturally mated kr/kr males and kr/+ females, and their genotype was determinedaspreviouslydescribed (Frohmanetal., 1993). Nonmutant (+/+) embryos were obtained from naturally mated CD-I and FVB/N mice. Expression of cDNA1 and cDNA2 was examined with probes from the coding and the 3’ untranslated regions subcloned into Bluescrtpt KS II (Strategene). In cDNA1, probe l-la contains nucleotides i-299, and probe I-2a contains nucleotides 1126-1860. In cDNA2, probe X215 contains nucleotides 150365, and probe Nco4cks contains nucleotides 1183-1411. A Hoxbl probe, provided by M. Frohman, was used as a positive control. The images shown in Figure 4 were obtained with probe I-2a (cDNA1) and X215 (cDNA2); identical results were obtained with probe l-2b (cDNAl) and Nco4cks (cDNA2). Antisense and control sense probes were generated using T3 or T7 polymerase and digoxygenin-UTP (Boehringer-Mannheim), and hybridization was performed as described by Conlon and Rossant (1992). Embryos were photographed with Ektachrome 64T using a Nikon Diaphot equipped for dark-field photomicrography; representative slides chosen for presentation were scanned with a Nikon slide scanner and printed with a Kodak dye sublimation printer. Comparisons among embryos of different genotype were performed on embryos from the same experiment photographed under identical conditions. Other Procedures Biotinylated DNA from Pl-222 and PI-730 was used as a substrate to select cDNA from 30-40 8.5 dpc embryos. Modifications from the protocol described by Lovett et al. (1991) are available upon request. Small cDNA clones obtained from the selection were used to screen an 8.5 dpc cDNA library. A single cDNA1 cione was recovered from IO6 bacteriophage, and three cDNA2 clones were recovered from 2 x lo6 bacteriophage. Sequence of the kFNu allele was performed by first PCR amplifying the protein-coding region from genomic DNA of BALBlcJ, SJUJ, C57BL/6J, (+I+), krV+, and kP”/kF DNA with the primers 5’~CTGAGCTCGCTTTTAG-3’ and 5’-GCAGAATAGGGAGTCTG-3’ (94OC for 1 min, 45OC for 1 min, and 72OC for 1 min for 40 cycles). Amplified products were gel purified and sequenced directly using the original primers as well as internal primers corresponding to positions 369-347,349-363,506-489,566-582,745-609,729-745,814-831, 91 B-901, and 1153-l 137 in cDNA2. Sequence analysis was performed with the DNASTAR suite of programs. Acknowledgments We thank Ben Winkes for typing the backcrcss panel for Src, Nancy Ouintrell for the Src probe, Mike Frohman for the Hoxbl probe, and Brigid Hogan for the 8.5 dpc cDNA library. We are grateful to Monica Justice and Gene Rinchik for advice regarding ENU mutagenesis; Mike Frohman for advice regarding in situ hybridization; Nila Pate1 and Andy Peterson for advice regarding cDNA selection; Stan Nelson for advice regarding biotinylation; Paul Marker, Jennifer King, and Elaine Storm for advice with direct sequencing of PCR products; and

Cell 1034

Sarah Millar for comments on the manuscript. S. P. C. was supported by American Cancer Society grant PF-03790. G. S. B. 1s an Assistant investigator of the Howard Hughes Medical Institute. Received August 16, 1994; revised September

30, 1994.

expression of a homeodomain

Lawrence, P. A. (1990). Developmental tebrates? Nature 344, 382-383.

protein. Development

biology: compartments

in ver-

Lovett, M., Kere, J., and Hinton, L. M. (1991). Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc. Natl. Acad. Sci. USA 88, 9628-9632.

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Accession

Numbers

The accession numbers for the sequences L36434 and L36435.

reported in this paper are