Copyright 0 1993 by the Society of America

Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Within thepid-Hbb Region of Mouse Chromosome 7

Eugene M. Rinchik, Donald A. Carpenter and Carol L. Long Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8077 Manuscript received May 12, 1993 Accepted for publication August 21, 1993

ABSTRACT As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7,four loci [1(7)1Rn, 1(7)2Rn,1(7)3Rn, 1(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutationswere mapped by means of trans complementation crosses to mice carrying lethal deletionsof the mouse chromosome-7 albino(c) locus. Each locuswas assigned to a defined subregionof the deletionmap at the distal endof the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pad and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethalswas found to arrest development after uterine implantation; the specifictime of postimplantation death varied, and depended on both the itself and on whether it was hemizygous or homozygous. Based on their map positions outsideof and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not havebeen discovered by phenotypicanalyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals definedby deletion breakpoints suggestsa number of positional-cloning strategies for the molecular isolation of these . Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segmentof the human genome (perhaps human1 lql3.5).

HE overlapping deletion mutations that encom- Mice homozygous for the largest of the c deletions T pass the albino (c) locus within the Fes-Hbb die at preimplantation stages of development, presum- region of mouse chromosome7 are providing auseful ably due to the deficiency of a or genes at the series of reagents with which to develop correlated preimplantationdevelopment ($id) locus (LEWIS physical and functional maps of megabase regions of 1978; GLUECKSOHN-WAELSCH1979; RUSSELLand amammalian chromosome (RUSSELL,RUSSELL and RAYMER1979; RUSSELL,MONTGOMERY and RAYMER KELLY 1979; GLUECKSOHN-WAELSCH1979; RUSSELL, 1982; RINCHIKand RUSSELL1990; RINCHIKet al. MONTGOMERYand RAYMER1982; reviewed in RIN- 1993). The early lethal phenotype exhibited by these CHIK and RUSSELL1990). These deletions have been homozygotes makes it impossible to estimate the num- useful for both genetic and physical mapping of loci ber of transcription units that might map within these defined by DNA probes and/or by phenotypes (RIN- large deletions. It is likewise impossible to determine CHIK and RUSSELL1990). Moreover, the mutant phe- the phenotypic effects of genes mapping outside of notype exhibited by mice homozygous for a deletion the relatively shorter c deletions, but within the longer has, in several cases, provided insights into the genetic deletions, if these genes normally function at stages control of developmental processes involved in liver subsequent to this preimplantation block. and kidney function in thelate fetus and neonate To generate afine-structure functional/mutation (ERICKSON,GLUECKSOHN-WAELSCH and CORI 1968; map of the Fes-Hbb region in which individual genes GLUECKSOHN-WAELSCH1979), spermiogenesis and their corresponding effects on development can (LEWIS,TURCHIN and WOJTOWICZ1978), gastrulation be defined and characterized, we have been utilizing (LEWIS,TURCHIN and GLUECKSOHN-WAELSCH1976; the presumptive point-mutation inducer, N-ethyl-N- NISWANDERet al. 1988, 1989), and in cleavage of the nitrosourea (ENU),and a screening protocolbased on preimplantation embryo (LEWIS1978). Moreover, a deletion hemizygosity, toinduce and recover both particulardeletion homozygote (cz4c0s/c’4c0s) that is lethal and viable single-gene mutations that map deficient for fumarylacetoacetate hydrolase (Fah) has within a large c deletion that is lethal at preimplanta- been proposed as an animal model for human type-1 tion stages of development(RINCHIK, CARPENTER and tyrosinemia (KLEBIG,RUSSELL and RINCHIK1992; SELBY1990). This report describes the more refined RUPPERTet al. 1992). deletion mapping of four such mutations, which are

Genetics 135 1117-1 123 (December, 1993) 1118 E. M. Rinchik, D. A. Carpenter and C. L. Long

lsqm-qtrr g mlq14.31 ESP ??? gllp15.51 llq13.5

9FR60Hb IFR6OHb

2R145L 3R145L lOFRQL 146G, 39SAS 26DVT 12FR60Hb

FIGURE .-Deletion 1 mapping of four ENU-induced lethal mutations within the pid-Hbb region of mouse chromosome 7. The map of the Fes-Hbb region of chromosome 7 presented in RINCHIKet al. (1993) (accompanying report) has been modified by incorporatingthe complementation data from Table 1. The loci l(7)lRn through 1(7)4Rnare defined by ENU-induced prenatally lethal mutations described in the text and areshown in larger type along with D7Rn6 and Omp (RINCHIKet al. 1993). Below the map are shown the extents of a subset of c-locus mutations that are most relevant for the mapping these four loci. (Table 1 lists additional c deletions that are not pictured here.) The thicker lines represent deletions that genetically bracket pid-Hbb-region loci defined by the ENU-induced mutations. The bracket above 1(7)2Rnand 1(7)4Rn indicates that these two loci cannot yet be ordered with respect to each other; likewise, the relative order of the l(7)IRn and D7Orl loci with respect to Omp and sh-I is not known. Estimates of the genetic distance (in cM) between c (Tyr) and Hbb ranges from 3.46 k 0.64 to 6.19 k 0.65 (DAVISSON,RODERICK and DOOLITTLE1989), and the c~~~~~ deletion has been estimated to be 6-1 1 cM in length (RUSSELL,MONTGOMERY and RAYMER 1982;RINCHIK, CARPENTER and SELBY 1990).The boxes above the map denote known human homology regions; human 1 1 q homologies are based on data of EVANSet al. (1993). lethal shortly after implantation, to a subregion of the nonalbino progeny were assumed to carry an ENU-induced Fes-Hbb interval distal to the pid locus. Thus, these viable mutation closely linked to the c locus. mutations define loci within the c-deletion complex The mutant c 1 chromosomes, recovered from cCh+/c 1 (light-chinchilla) Gn siblings, were propagated by crossing cCh that could never have been identified by analysis of +/c 1 mice to mice of the inbred strain 47BS-cch/cch, which deletion homozygotes. Moreover, the deletion map- exhibit the full (darker) chinchilla color. The c mutation ping reported here,in conjunction with the molecular serves as a marker for all l's, and because it can potentially analysis of a number of preimplantation-lethal c dele- be separated from 1 by crossing over in cCh+/c 1 carriers, cch +/c 1 males in each generation were first crossed to cCh+/ tions (RINCHIK et al. 1993), suggests strategies for c26DVT identifying these loci on the physical map of the females to progeny-test for the presence of 1. Males pid- that produced zero or one albinos in at least 30 progeny of Hbb region. this testcross were considered to be proved cCh+/c 1 carriers (RINCHIK, CARPENTERand SELBY 1990). Lethal albino deletions [Dxc)] were maintained by cross- MATERIALS ANDMETHODS ing light-chinchilla heterozygotes [c"'/DfTC)] to mice of the non-inbred, chinchilla stock 2A-cCh/cch.New ENU-induced The origin of ENU-induced c-region mutations has been described in detail elsewhere (RINCHIK,CARPENTER and lethal mutations were mapped with respect to breakpoints SELBY1990). Briefly, each mutation was induced by treat- of lethal albino deletions by crossing proved carrier males (cCh+/c ment of BALB/cRl(c/c) Generation-0 (Go)males with a total I) with females heterozygous for lethal albino dele- (but fractionated) dose of 400 mg/kg of ENU to spermato- tions [cCh/Dj'c)].Specific ENU-induced lethal mutations used gonial stem cells. Newly induced mutations that are closely for these mapping analyses include: 1(7)IRn'8'SB;1(7)2Rn'75SB; linked to c were identified by crossin GI females (+/c) with 1(7)3Rn677Sn;and 1(7)4Rn"08Sn(RINCHIK, CARPENTER and males carrying the Dfl;Mod-2 sh-IpDVTdeletion (abbrevi- SELBY1990). ated c26DVT;see Figure 1) opposite the cch (chinchilla) marker. To determine the approximate time of death caused by GI females that yielded no GPalbino progeny were hemizygosity for each of the four new ENU-induced lethal considered to carry an ENU-induced lethal mutation, de- mutations, proved carrier males (cch +/c 1) were crossed to noted here as "1," mapping within the limits of the c~~~~~ cCh+/c~~~~ females. At 14.5 days post coitum (E14.5) (where deletion. Likewise, GI females that gave rise to GP albino the morning of finding a vaginal plug was considered to be progeny manifesting abnormal phenotypes (e.g., differential E0.5), the females were sacrificed, and the ovaries and size, abnormal balance, etc.) that were not observed in uterus were removed. The number of corpora lutea within Deletion Mapping in Mapping Deletion the Mouse 1119 the ovary was determined, and the contents of the uterus stages) caused by hemizygosity for each of the four were examined as described RUSSELL in and RAYMER(1 979). mutationsmapped in Figure1 was determined by Some of the experiments designed to estimate the time of death of embryos/fetuses homozygous for a particular 1 crossing cch +/c~~~~females to proved cch +/c 1 males. involved crossing proved carrier males (cch+/c I) to proved As a control, cch +/c~~~~females were crossed to cch carrier females; these proved females were obtained by a +/c ~h-1’~’~males, which carry an ENU-induced mu- pro en testcross of presumed cCh +/c 1 females to cCh +/ tation at theshaker-1 locus (RINCHIK,CARPENTER and c,’ c,’ :ales. However, most of these experiments involved SELBY1990). At E14.5, the females were sacrificed, crossing proved carriermales (+ cCh+ +/+ c 1 +) to females (pcCh +ti/+ c 1 +) obtained from crosses of p cCh+fr/+ c 1 the uterine contents were analyzed, and the corpora + mice to p cch +fr/p cch +fr mice @,pink-eyed dilution;fr, lutea within the ovaries were counted (Table 2). frizzy]. These p cch +fr/+ c 1 + females were considered to Because themutant chromosome in each case is be “pseudoproved” carriers of 1, because a rare double marked by the c mutation, the absence of fetuses with crossover would be necessary to lose 1 in the cch/c progeny albino eyes (that is, the absence of fetuses that lack of this cross. eye pigment) suggests that hemizygosity for the cor- RESULTS responding lethal arrests development before El 1.5, at which time eye pigment normally appears. Indeed, Deletion mapping of ENU-induced lethal muta- for three of the four mutantloci examined, no albino tions: Four loci [1(7)1Rn through 4Rn] defined by fetuses were found and only one such fetus (a pre- complementing ENU-induced lethal mutations (RIN- sumed recombinant that had lost I from the c 1 chro- CHIK, CARPENTERand SELBY1990) were mapped to mosome) was observed in the cross involving one intervals of the Fes-Hbb region of chromosome 7 by mutation, 1(7)2Rn375SB(Table 2). Furthermore,the determining if their respective lethal phenotypes frequency of resorption moles in each of the crosses could be complemented intrans by chromosomes (36%-43%) was significantly greaterthan the fre- carrying albino (c) deletions of varying length. Table quencyobserved in the control cross (20%).This 1 summarizes the results obtained when proved car- increased number of resorption moles suggests that rier males [e.g., cch +/c 1(7)lRn181SB]were crossed to hemizygosity for each of the mutations is killing the females heterozygousfor c deletions. Crosses that embryoafter implantation but before El 1.5. Fre- produced zero or one albino offspring [c 1(7)/Dxc)] in quency of preimplantation loss, as calculated from the at least 30 classified progeny were considered to be ratio of thenumber of totalimplantations tothe noncomplementing combinations (RINCHIK, CARPEN- number of eggs ovulated (i.e., the number of corpora TER and SELBY1990). Typically, more than 30 off- lutea), is similar among the four experimentalcrosses, spring were raised for each noncomplementing com- bination. and is actually lower than the unusually high preim- As shown in Table 1, several patterns of comple- plantation loss observed in the control cross (Table mentation were observed with the loci identified by 2). [Althoughthe numberof preimplantation embryos ENU-induced lethal mutations. The 1(7)1Rn181SBmu- lost can vary widely depending on the genotype and tation is complemented by all albino deletions except strain of the female, most studies show that this quan- CIZFR60Hb and tity averages around 10-15% (L. B. RUSSELLand W. 26DvT, the latterof which was the deletion originally used for its detection. The 1(7)2Rn375SBand M. GENEROSO,unpublished data).] Even in absolute 1(7)4Rn1108SBmutations are complemented by all terms, the amount of preimplantation loss observed deletionsexcept cQPB, c1zFR60”b and c2‘jDVT. The in each of the four experimental crosses cannot ac- 1(7)3Rn677SBmutation displays yet anotherpattern, count for the missing albino class. The lack of evi- being complemented by all deletions except cIFDFoHrc, dence for preimplantation loss and the increase in the c4PB , cIZFR60Hb and c26DvT. frequency of resorption moles in each experimental These results suggest that these four loci map within cross, make it evident that hemizygosity for each of the pid-Hbb interval (Figure 1). Furthermore, two of the complementing mutations kills the embryo after the Bp-group deletions,dFDFoHrc and cQPB(see RINCHIK implantation. et al. 1993, accompanying report), which both affect Table 2 alsoshows thatthere was a substantial thepid locus, must extend moredistally than the other increase (from -2%--12%) in the observed number six members of the Bp complementation group, with of “dead lates” (DL) in crosses involving 1(7)2Rn375sB. c*‘~ extending the most distal. Indeed, assuming that The DL category includes fetuses that had progressed the radiation-induced c deletions are continuous and beyond the typical resorption-mole stage, but could linear, the loci can be arranged in a proximal-distal be classified as clearly malformed,underdeveloped order of Pid-D7Rn6-1(7)3Rn-[1(7)2Rn, 1(7)4Rn]- fetuses that in no way resembled their normal litter- [1(7)IRn,sh-11-Hbb. mates. Thus, it is likely that a substantial number of Lethal period for the four ENU-induced muta- embryos hemizygous for 1(7)2Rn375SBdie later thando tions: The approximate time of death (preimplanta- embryos hemizygous for any of the other three mu- tion,postimplantation, late fetal stages or neonatal tations, and/orthat this mutationmight be more 1120 E. M. Rinchik, D. A. Carpenter and C. L. Long

TABLE 1 Deletion mapping of lethal ENU-induced Fes-Hbb-region mutations"

Albino deletion [Df(c)t AlbinosfNo. classifiedC Complementation-group Allele designation I( 7)IRn181SB I( 7)2Rn37'SB I(E(7)4Rn"08sB 7)3Rn6""

Ai 1 5/28 8/20 8/29 8/25 Ai2 9/37 5/42 NDd 6/15 Ai3 3/15 6/22 5/24 5/23 Bem 1 6/26 611 7 7/22 7/32 BemP 6/28 8/35 411 7 6/28 Bem3 4/25 7/20 5/40 612 1 Bex 1 7/25 4/45 7/23 5/25 BPI 6/23 12/47 0/52 5/35 BP2 412 1 10150 12/44 10130 BP3 5/32 6/38 10124 8/47 BP4 5/42 5/2 1 12/36 10/32 BP5 6/27 0/57 0/54 BP6 14/38 4/33 813 1 12/69 BP7 8/25 813 1 5/43 6/27 BPS 5/17 5/30 8/45 5/30 c1 8/46 9/40 8/25 12/29 Di 1 4/22 511 8 9/30 5/22 Di2 6/28 6/30 4/26 6/28 Di3 6/27 6/32 9/36 513 1 Di4 316 8/22 713 1 811 4 Dj 1 7/34 8/29 3/19 11/24 Dj 2 511 7 7/27 6/22 411 5 7/27 6/25 7/36 8/26 10134 6/26 7/17 13/49 9/48 10130 7/32 5/23 4/26 8/23 10132 7/16 1/81e 0/42 -1/51e 0/45 -0135

Proved carrier males (c"+/c I) were crossed to females heterozygous for albino deletions [c"/Df(c)]. RUSSELL, MONTGOMERYand RAYMER(1982); NISWANDERet al. (1989); see also RINCHIKet al. (1993) (accompanying report). Progeny were classified at 3 weeks of age. In complementing combinations, albinos should comprise 25% of the progeny. Absence of albinos, or albinos with an abnormal phenotype, indicated noncomplementation. Noncomplementing combinations are indicated by the un erline. i ND, not done. Normal albinos were presumed to be recombinants that lost 1 from the c 1 chromosome. These presumed recombinants were not progeny tested.

TABLE 2 Time of death of embryos hemizygousfor ENU-induced prenatally lethal mutations mapping to pid-Hbbthe region of mouse chromosome 7

No. of Uterine contents: No. of

Sire" Litters Sire" corpora lutea c/c ~/c P/db DL~ moles %Prec %postd %DL'

c"+/c 1(7)1Rn18'SB 17 148 0 47 33 3 47 12.2 38.5 2.3 c"+/c 1( 7)2Rn"'"222 28 1 2581 40 6.8 60 41.1 12.1 cCh+/c 1( 7)3Rn677SB 33 280 0 112 53 4 89 7.9 36.0 1.6 cch+/c 1( 7)4Rn1108SB 9 67 0 1 21 12 24 13.4 43.1 1.7 c"+/c sh-lZaB266 32 31 5 84 46 35 24.4 19.9 2.5

Males of the indicated genotype were crossed to cch+/Dfc sh-I)z6DWfemales, and uterine contents were examined 14 days after finding a vaginal plug. The eye color of fetuses provided a means to determine the genotype (c/c, no pigment; ccA/c, light pigment; cch/ccA,dark pigpent). DL, dead late: fetuses that were clearly abnormal and underdeveloped, but had progressed beyond the resorption mole stage. E Percent preimplantation death, calculated as: 1 - (no. total implants/no. corpora lutea) X 100. The number of total implants equals the su of all live and dead fetuses plus moles. 'Percent postimplantation death, calculated as: (no. moles + no. dead lates/no. total implants) X 100. Percent dead lates, calculated as: (no. dead lates/no. total implants) X 100. Deletion MappingDeletion in the Mouse 1121

TABLE 3

Time of death of embryos homozygousfor ENU-induced prenatally lethal mutations mapping to the pid-Hbb region of mouse chromosome 7

Cross" No. of contents: Uterine No. of Corpora Female Litters luteaMale Litters Female CIC PIC Pp DL* Moles

c"+/c 1( 7)1Rn'8'SB c"+/c 1(7)1Rn'8'SB323 32 68 (51)' 123 (14)' 51 (5)' 2 35 cch+/c 1( 7)2Rd7''* cCh+/c1(7)2Rn'75sB 10 84 5 (3)' 34 11 1 11 cCh+/c 1( 7)3Rn677SB cLh+/c 1(7)3Rn677sB 8 80 0 39 12 0 17 cCh+/c 1( 7)4Rn"08sB cCh+/c I( 7)4Rn"08SB 9 81 0 30 13 1 24

' In each of these crosses, some females also were heterozygous for both p (pink-eyed dilution) andfr (frizzy). That is, some females were p crh+ fr/+ c 1 +, and some were +cCh + +/+ c 1 + (see MATERIALS AND METHODS). Uterine contents were examined 14 days after finding a vaginal plug. The eye colorof fetuses provided a means to determine the genotype(c/c, no pigment; cCh/c,light pigment; cCh/cch,dark pigment). Note that males were always wild type (+/+) at the p locus (see MATERIALS AND METHODS), and that resultant fetuses could be either +/p or +/+; this had no effect on the ability to determine genotypesat the c locus by examining eye color. DL, dead late; fetuses that were clearly abnormal and underdeveloped, but had progressed beyond the resorption mole stage. The number in parentheses indicates the number of fetuses included in the total that were visibly smaller than littermates.

"leaky" in the hemizygous state, allowing development same (early)time, and we have yet to notice any to proceed to a slightly later, and morevariable, point leakiness in the lethal period in embryos/fetuses either after implantation. This hypothesis is consistent with hemizygous or homozygous for either of these muta- the data in Table 3, which show that fetuses homo- tions. zygous for 1(7)2Rn375SBcan actually be found at E14.5, although some reduction in size with respect to their DISCUSSION nonalbino littermates is observed. Thus, having two The panel of radiation-induced, overlapping albino doses of this somewhat leaky ENU-induced lethal (c) deletions provides a set of genetic reagents that mutation appears to allow the fetus to develop to a facilitates the development of refined,interrelated point further than would be observed with one dose. physical and functional maps of approximately 10% However, we have neverobserved any albino off- of mouse chromosome 7. One component of this spring at birth in 359 progeny of an intercross of cch physical/functional analysis is the use of these dele- +/c 1(7)2Rn375SBmice. Thus, although (size-reduced) tions in a long-term mutagenesis experiment, first to fetuses homozygous for canfound be at select new ENU-induced (presumed point) mutations E14.5, these fetuses are never born alive. that map to the region immediately surrounding the This "lethal leakiness" is also observed in a striking c locus (RINCHIK, CARPENTERand SELBY1990), and way in crosses involving 1(7)1Rn'8'SB.We had noticed second, to fine-map these mutations with respect to (E.M.R. and D.A.C., unpublisheddata) thatinter- the deletion breakpoints on which the evolving phys- crosses of 1(7)1Rn"lSB heterozygotes often produce ical map is based. Thisreport describes such fine- extremely runty albino progenythat always die before structure deletion mapping for four newly identified 7 days of age. The data in Table 2 demonstrate that loci identified by ENU-induced lethal mutations. The hemizygosity for 1(7)1Rn'x'SBleads to embryonic death ease of mapping these mutations to defined intervals shortly after implantation, with little variability in the of chromosome 7 underscores the power of coordi- time of death [note that no albinos are observed at nated physical and fine-structure functional analysis E14.5 and that the frequency of DLs is not elevated, of a region of the mouse genome covered by a series as it isin crosses involving 1(7)2Rn375SB].However, of overlapping germline deletions (RINCHIKand RUS- 1(7)1Rn'8'SBhomozygotes appear to be fully viable to SELL 1990). El 4.5 (Table3), although themajority of these homo- The placement of the new loci to distal regions of zygotes were already distinguishable (in terms of re- the albino deletion complex,between the pid and Hbb duced fetal size) from their nonalbino, normal litter- loci, immediately suggests positional-cloning strategies mates. Thus, two doses of the ENU-induced with which to aid in identifying DNA sequences cor- 1(7)1Rn'8'SBmutation allow development to progress responding to the loci defined by these ENU-induced to late fetal and even neonatal stages, whereas one mutations. In fact, the complementation pattern ob- dose always kills the embryo shortly after implanta- servedfor mutations atthe 1(7)3Rn, 1(7)2Rn and tion, even before any type of DL-type fetus can be 1(7)4Rn loci suggests that all three map between recognized. By contrast, we observe that hemizygosity D7Rn6 (RINCHIKet al. 1993) andOmp (RINCHIKet al. (Table 2) or homozygosity (Table 3) for 1(7)3Rn677SB 199 1; BROWNet al. 1992) (see Figure 1).For example, or 1(7)4Rn''"8SBkills the embryo at approximately the a series of overlapping YAC clones, spanningthe 1122 E. M. Rinchik, D. A. Carpenterand C. L. Long D7Rn6-0mp (or D70rI) interval, should include the deletion used to select the ENU-induced mutations loci defined by these three ENU-induced mutations (RINCHIK,CARPENTER and SELBY1990), as well as the c14FR60Hb C3R60L cIOFR60L [and possibly also the l(7)IRn locus]. The breakpoints 9 , , and c39sAs deletions which of c deletions that map to this region will be valuable complement the mutations, are all preimplantation physical landmarks for establishing intervals in which lethals (RUSSELLand RAYMER 1979; RINCHIKet al. the loci defined by thesemutations will be found. 1993). Thus,this mutagenesis scheme has refined the Identification of the most distally mapping breakpoint functional mapof the pid-Hbb region, over andabove from among the c14FR60Hb c3R60L cIOFR60L C146G , 9 , and what would be possible by analysis of the c deletions c39SAS deletions will define the proximal boundary of themselves, by identifying genes involved in develop- the 1(7)3Rn locus, whereas the distal breakpoint of the mental processes occurring after the period that re- CIFDFoHrc deletion will either map within the locus or quires the product(s) encodedby the pid locus. define its distal boundary. Similarly, the cIFDFoHrCdistal The observation of “leakiness” in the lethal pheno- breakpointdefines the proximalboundary of the type of mutations at two different loci demonstrate physical intervalcontaining both the 1(7)2Rn and another potentially useful aspect of a series of ENU- 1(7)4Rn loci, whereas the distal breakpoint of the c4pB induced mutations in a defined segment of the ge- deletion either maps within either of the loci or de- nome. It is known that ENU is useful for generating fines the distal boundary of their map interval.Finally, intermediate alleles at coat-color and isozymeloci the distal breakpoint of the c4pB deletion defines the (RUSSELLet al. 1979; RUSSELL1986; PETERSet al. proximal boundary of the intervalcontaining the 1990). These types of leaky mutations are likely to be l(7)IRn locus. quite useful in the analysisof thefunction of the Because the distance between D7Rn6 and Omp corresponding protein product over developmental could prove to be very large physically, it is worth time, ranging from peri-implantation stages to later noting that new nucleation points for the continued stages of embryonic, fetal and neonatal life. Likewise, development of the physical map of this region and leaky mutations could be useful in the analysis of the for the identification of the loci identified by the new mutantprotein product itself, because they might ENU-induced lethal mutations could be generatedby allow this product to be isolated from the fetus or identifying and cloning rearranged restriction frag- neonate in greater quantity than exists at peri-implan- ments associated with the c4pB and clFDFoHTCdeletions, tation stages (provided, of course, the gene continues or as well as with any other deletion that has been to be expressed at later stages). Also cogent in this found to break between D7Rn6 and 1(7)3Rn. Access context is the potential derivation of an allelic series to the proximal breakpoints of these deletions, from of ENU-induced mutations at a single locus, which loci mapping in the D7CwrI8-c(Tyr) interval, could would be comprised of mutations that differ in sever- result in the corresponding distal deletion breakpoint ity of effect. Indeed, we have evidence (to be pre- clone, in anmanner similar tothat used forthe sented elsewhere) for one allele of 1(7)3Rn (among derivation of a probe for theD7Rn6 locus itself, which five recovered to date) that, when hemizygous, causes was accomplished by cloning a restriction fragment only a very mild runting syndrome, and, when homo- containing the c9FR60Hb breakpoint starting from the zygous, has no apparent effect on the external phen- D7Rt336 locus (RINCHIKet al. 1993). toype of the mouse. It is these types of mutations, It is likely that each ENU-inducedmutation de- rather than the null mutations associated with dele- scribed here is intragenic, since intragenic lesions have tions, that will be useful forexamining structure- been demonstrated for a number of mutations in- function relationships of developmentally necessary duced by ENU in stem-cell spermatogonia (POPPet al. proteins within the context of the whole organism. 1983; RUSSELLand RINCHIK1987; RUSSELLet al. Continued mappingof any evolutionarily conserved 1990; PETERSet al. 1990; ZDARSKY,FAVOR and JACK- sequences derived from the pid-Hbb region to human SON 1990;BRANNAN et al. 1992).Thus, the lethal chromosomes will refine the regions of homology phenotypeexhibited by hemi- or homozygotes for within the mouse and human genomes. It is known each of the four mutations is likely to be the result of that the human HBB locus maps to the short arm of a single-gene lesion. Similarly, if any pleiotropy is chromosome I lp (1 1~15.5)(JUNIEN and VAN HEYNIN- observed in these lethal phenotypes, that pleiotropy, GEN 199 1). However,the recent mappingof olfactory too, will probably be attributable to the dysfunction markerprotein (OMP) humanto chromosome of a single gene. This is in contrast to the analysis of IIq13.5 (EVANSet al. 1993) suggests that the mouse a lethal phenotype(s) associated with homozygosity for chromosome-7 segmentimmediately surrounding the a deletion,in whichthe number andnature(s) of genes Omp and sh-I loci is homologous to this region in the contributing to thephenotype can be uncertain.That long arm of human chromosome 11. If, by further each mutation described here is lethal after implan- mapping analyses, this 1 lq13.5 homology region is tation is of particular interest, given that the PDVT extended to include the entire pid-sh-1 interval ana- Deletion Mapping in the Mouse 1123 lyzed here and in the accompanying report (RINCHIK postimplantation survival in the mouse. Development102: 45- et al. 1993), the phenotypes associated with the ENU- 53. NISWANDER,L., D. YEE, E. M. RINCHIK,L. B. RUSSELLand T. induced lethal mutations reported here may be useful MAGNUSON,1989 The albino-deletion complex in the mouse predictors of the occurrenceof similar loci within the defines genes necessary for development of embryonic and 1 lql3.5 segment, as well as serving as useful models extraembryonic ectoderm. Development 105: 175-182. for genes and developmental processes active shortly PETERS,J., J. JONES, S. T. BALLand J. B. 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