molecular and phenotypic characterization of a new mouse … · 2002. 7. 5. · phenocal...

Copyright 8 1995 by the Genetics Society of America

Molecular and Phenotypic Characterization of a New Mouse Insertional Mutation That Causes a Defect in the Distal Vertebrae of the Spine

Jeffrey J. Schrick,* M. E. Dickinson,t” B. L. M. Hogan,t P. B. Selb9 and R. P. Woychikf

*University of Tennessee, Graduate School for Biomedical Sciences, Oak Ridge, Tennessee 37831-8080, tHoward Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232

and $Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8080 Manuscript received November 26, 1994 Accepted for publication April 5, 1995

ABSTRACT We have identified and characterized the phenotype of a new insertional mutation in one line of

transgenic mice. Mice carrying this mutation, which we have designated TgN(Imusd)37ORpw, display undulations of the vertebrae giving rise to a novel kinky-tail phenotype. Molecular characterization of the insertion site indicates that the transgene integration has occurred without any substantial alterations in the structure of the host sequences. Using probes that flank the insertion site, we have mapped the mutation to chromosome 5 near the semidominant mutation, thick tail ( Tht) . Thick tail does not comple- ment the TgN(Imusd)37ORpmutation; compound mutants containing one copy of each mutation display a more severe phenotype than either mutation individually.

T HE recent analyses of several mouse mutants that exhibit defects in tail development have led to

the identification of genes that play important roles in establishing the pattern of the body axis. In one of these mutations, Brachyu?y (ZJ, heterozygotes have malformed sacral vertebrae and show varying reduc- tions in tail length. Homozygous embryos die at around embryonic day 10 and exhibit abnormalities in mesodermal structures, such as the notochord and somites. These characteristics can be traced back to late gastrulation when precursors for these cells arise in the primitive streak (reviewed in WILSON 1990 and references therein). The Tgene encodes a novel DNA- binding protein (HERRMANN et al. 1990; KISPERT and HERRMANN 1993; STOTT et al. 1993) that may control mesoderm formation by regulating gene expression in the early embryo. Two other genes that have been disrupted using homologous recombination show abnormalities in tail formation that are caused by defects in the primitive streak. The first of these, Wnt-3a, causes shortened tails in heterozygotes, and homozygotes show severe disruptions in the formation of somi- tic mesoderm and the extension of the embryonic axis (TAKADA et al. 1994). Wnt-3a is expressed in the primitive streak in a pattern that overlaps that of T gene expression during late gastrulation (TAKADA et al. 1994). Additionally, mice homozygous for mutations in the proto-oncogene int2 (Fd3) have shortened tails and abnormal caudal vertebrae ANSOU SOUR et al. 1993);

Corresponding author: Richard P. Woychik, Biology Division, Oak Ridge National LaboratoIy, P.O. Box 2009, MS 8080, Bear Creek Rd., Y-12, Oak Ridge, TN 37831-8080. E-mail [email protected]

‘Present address: Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138.

Genetics 140 1061-1067 (July, 1995)

their phenotype is less severe than that observed for mutations in Tor Wnt-3a. Tail abnormalities observed in int2 mutants may result from the loss of activity of this factor in the posterior primitive streak, a region where int2 is expressed during early development (WILKINSON et al. 1988; MANSOUR et al. 1993).

Tail abnormalities can also result from defects in patterning mesodermal derivatives during later stages of development. For instance, undulated (un) mutants have a kinked-tail phenotype that appears to result from a defect in somite patterning such that the vertebral bodies do not form properly (DEUTSCH et al. 1988; KO- SEKI et al. 1993; WALLIN et al. 1994). In all un alleles there is a disruption in expression or function of the Pax-1 gene, a member of the paired-box gene family (BALLING et al. 1988; WALTHER et al. 1991; WALLIN et al. 1994). Consistent with un phenotypes, Pax-1 is normally expressed in the ventral portion of the somite, in the sclerotome cells that migrate ventromedially and con- dense around the notochord to form the vertebral bodies (WALLIN et al. 1994).

Of the 126 skeletal mutants in the Genetic Variants and Strains of the Laboratory Mouse (GREEN 1989), 76 include defects of the tail and appendages. Numerous mutations exhibit strictly a tail-length phenotype, while others affect structures of the entire vertebral column (GRUNEBERG 1963; GREEN 1989). The phenotypic display of tail mutations can range from the absence or severe shortening of the tail to a slight decrease in length and size. In many cases, fusions, undulations and tail kinks can be observed (GREEN 1989). As with the mutations described above, the characterization of other mouse mutants with tail defects could provide the means to identify additional genes that function in axial development. For this reason, we have initiated

1062 J. J. Schrick et al.

an effort to characterize a new insertional mutation that A. causes a defect in the vertebrae. Here we describe the +I+ nature of the skeletal phenotype in these animals and the molecular cloning and mapping of the mutant lo- 1" cus. We also show that a dominant mutation called thick tail (BEECHEY and SEARLE 1980) maps to the same gen- eral region on chromosome 5 and does not comple- ment this new mutation.

MATERIALS AND METHODS

Animals: The transgenic line TgN(Zmusd)37ORpw was generated as part of a large-scale insertional mutagenesis program in the Biology Division, Oak Ridge National Laboratory. Transgenic mice were generated by the microinjection of a linearized 10-kb DNA fragment containing ,&actin chloram- phenocal acetyltransferase (PAC) (GUNNING et al. 1987) into fertilized mouse embryos (HOGAN et al. 1986) from the FVB/ N mouse line (TAKETO et al. 1991). The TgN(Zmusd)370Rpw line was maintained by intercrossing mutants and backcross- ing to wild-type FVB/N mice. The thick tail mutation was ob tained as a kind g& from A. SEARLE (Harwell) and was maintained in the Mammalian Genetics Section by intercrossing the Tht allele to the FVB/N strain.

Identification of transgenic mice: Genomic DNA from tail biopsies was isolated, digested with restriction endonucleases, electrophoresed on 0.8% agarose gels (GIBCO-BRL) and Southern blotted to GeneScreen nylon membranes (AUSUBEL et al. 1988). Mutant mice were initially identified by hybridization to sequences from within the transgene. Once the mutant locus was cloned, unique copy sequences from within the region flanking the transgene insertion, probes A-C Figure 2 A , were used to distinguish heterozygotes from homozygotes (WOYCHIK et al. 1985; AUSUBEL et al. 1988; SAMBROOK et al. 1989).

Skeletal analpis: Skeletons of TgN(Zmusd)37ORpw, thick tail, and control mice were visualized by staining with Alizarin Red (SELBY 1987). Skeletons were visualized and photographed under a dissecting microscope.

Genomic cloning and sequencing: Genomic liver DNA from a TgN(Zmusd)37ORpu homozygote was partially digested with Sau3A and size-fractionated on a 10-40% sucrose gradi- ent. Fragments of 18-20 kb were ligated into AEMBL3 arms to generate a genomic library (SAMBROOK et al. 1989). The library was screened using a radiolabeled probe corresponding to the transgene sequences (SAMBROOK et al. 1989). Transgene-positive A-clones that also contained the host- flanking sequences were initially identified by hybridization with a probe comprised of total mouse genomic DNA (WOY- CHIK et al. 1985). To clone the wild-type region corresponding to the mutant locus, FVB/N genomic liver DNA was used to prepare a genomic library that was screened with a probe (probe A, Figure 2) from the genomic region flanking the transgene integration site. All wild-type and mutant fragments were subcloned into the pGEM plasmid vectors (Promega) and sequenced with T7 DNA polymerase (Sequenase, USB) utilizing either the T7 or SP6 universal primers or oligonucle- otide primers prepared from a specific region within the insert (SAMBROOK et al. 1989). BXD recombinant inbred mapping: Genomic DNA was iso-

lated from frozen kidneys of C57BL/6J, DBA/2J mice and 25 mice representing the BXD recombinant inbred lines (JEN- KINS et al. 1982). (RI and parental mice were purchased from the Jackson Laboratory.) To differentiate alleles from the different genetic backgrounds, genomic DNA was digested with HinJ to distinguish the C57BL/6J and DBA/2J alleles frag-

Rostral Caudal ""_ I I I z z

TgN37ORpw/rgnr37ORpw

L Rostral Caudal

TgN37ORpw.gN37ORpw d9 neonate

.

I 1

FIGURE 1. -The adult and neonatal kinky-tail phenotype in the TgN370Rpuinsertional mutant. Arrows show the defective caudal region of the oss@ng vertebrae. (A) Alizarin clearance preparations of the wild-type adult mouse (top) and the homozygous TgN370Rpzu adult kinky-tail mouse (bottom). Arrows indicate the defective region of the vertebra. Ex- panded regions show one specific vdrtebrae, Ca25, from normal and mutant tails. The diagram outlines the Ca vertebrae for the defective region. (B) The kinlq-tail phenotype in a day 9 neonatal mouse. Alizarin clearance preparations of the TgN37ORpu homozygote show a distinct kink at the distal end of the tail that is only slightly visible on gross examination (data not shown). White arrows indicate the defective region of the vertebrae.

ment sizes of 1.0 and 0.43 kb or 0.3,0.55 and 0.43 kb, respectively. The genomic DNAs were then Southern blotted and hybridized with probe C, which corresponds to the region flanking the transgene insertion site (Figure 2). Strain distribution patterns from these mice were analyzed with computer software supporting statistical estimates based on SILVER (1985). Strain distribution patterns from these mice were analyzed with RI Map Manager computer software that utilizes statistical estimates based on SILVER (1985).

Crosses between thick tail and T g N ( Z m d ) 3 7 0 ~ Reci- procal crosses involving Tht/+ and TgN(Zmusd)37ORpw homozygotes were used to generate F1 compound heterozygotes (Tht/+;TgN(Zmusd)37ORp/+). The TgN(Zmusd)37ORpw allele was followed by genotyping the animals utilizing genomic DNA from the tail and a probe corresponding to the transgene sequences. The Tht dominant allele was followed by scoring the animals for the thick tail phenotype (BEECHEY

T$V(Imusd)370RjIro Tail Mutation 1063

E Wild type I E B H H H X E E E S BE H

6 Earn HI Hind 111

-. .~ ~. , -22kb

-20kb 1Bkb - 16kb -

- 2kb

Bkb -

C WILD TYPE

S...AATTACATGTAAAGTATlT'llAllTGAAGC...S

...g caggttcctctag ... ... aaggaattctagcc ... 5 IWactln Transgene 3'swo

and SEARLE 1980), which we determined was fully penetrant in the F1 animals.

RESULTS

Generation and phenotypic characterization of the TgN(Imusd)37ORpw mutant lime: As part of a large-scale insertional mutagenesis program at the Oak Ridge Na- tional Laboratory, we identified a new insertional mutation that causes an undulation of the spine and a kinky- tail phenotype. This mutation was named TgN(Imusd)- 370Rp-u (Im, insertional mutation; w d , undulated spine distal) in accordance with the new nomenclature rules for transgenic mice (GORDON 1992) and will be abbrevi- ated TgN37ORpw. The transgene in the TgN37ORpw line segregates in a simple Mendelian fashion. Mice heterozygous for the transgene in this line are phenotypically normal. Approximately 25% of the offspring from heterozygous transgenic parents develop a specific kinky- tail phenotype, and all of these animals are homozygous for the transgene integration site (data not shown). A

FIGURE 2.-Molecular structure of the TgN37ORfiu mutant and corresponding wild-type locus. (A) The TgN3iOR~nu mutant locus was isolated from a homozygous mutant genomic DNA library using p-actin CAT, the transgene, as a probe. One genomic clone (X20-12) from the mutant library contained a 1.9-kb Xl,nI/EcoRI fragment immediately downstream from the transgene integration site (probe A) that was used as a probe to isolate the genomic clone, X19-1, from a wild- type FVB/N genomic library. X19-1 spans the transgene insertion site and was used to identify a 0.7-kb Hind11 fragment (probe B) that was determined to map immediately upstream of the transgene integration site. Probe B was used to isolate a genomic clone (h20B-2) from the mutant library that contains the junction between the transgene and the host sequences upstream of the insertion site. Probe C was used for mapping experiments. R, transgene; R, BnmHI; E, EroRI; H, HindII; X, XbnI; S, SnQ. (B) Southern blots with 10 pg of genomic DNA from a wild-type (+/+). heterozygous (TgN37ORpw/+) and homozygous (TgN370Rfiu/T~~370I~~u) animal digested with BnmHI, Hind11 or EcoRI and hybridized with probe A. Numbers refer to the size of the detected fragments in kb. (C) Sequence of the transgene insertion site. A 3.0-kb Hind11 fragment from X20B-2, a 2.6kb XbnI/SnlI fragment from X20- 12, containing the left and rightjunction sites, respectively, and a 1.2-kb XbnI fragment from h19-1, which spans the insertion site, were sequenced and compared. Host genomic sequence is in uppercase and the transgene sequence is in lowercase. One end of the transgene corresponds to the promoter region of P-actin (left) and the other end contains the SV40 sequence on the transgene construct (right). The TA and T nucleotides in open boxes represent duplicated nucleotides at the integration site.

total of 563 homozygous offspring analyzed thus far all express the mutant trait, which indicates that the phenotype is essentially 100% penetrant. Based on the recessive genetics of the mutant trait coupled with the fact that none of the numerous other lines that have been prepared with the same transgene DNA construct showed the same kinky-tail defect, we conclude that the phenotype in the TgN370RW line arose from a mutation caused by the integration of the transgene sequences into the host genomic DNA.

The mutant animals show only the kinky-tail defect and do not contain any obvious soft tissue abnormalities in the mice examined thus far (J. J. SCHRICK and R. P. WOYCHIK, unpublished results). However, since we have not yet conducted an indepth analysis for any other defects, we cannot exclude the possibility that subtle soft tissue defects may become apparent upon further investi- gation. To characterize the kinky-tail defect in more de- tail, we prepared Alizarin-Red-stained skeletons from adult mice (day 65+) using the protocol from SELBY (1987) and systematically analyzed them. All of the skeletal structures in the TgN37ORpU mutant mice appear normal except for the distal region of the tail (Figure 1A). In all cases, tails from mutant animals have a disrup tion in the alignment of the last seven caudal vertebrae (Ca 24-30 in the FVB/N background) caused primarily by misshapen development of the posterior portion of

1064 J. J. Schrick et al.

individual vertebrae (Figure 1A). Consequently, the vertebrae are positioned at right angles to each other rather than in a normal linear arrangement, and it is this defect that may be causing the visible kink in the tail. In mutant neonates, the kinky-tail phenotype first becomes apparent at -9 days after birth. At this time only a few vertebrae in the tail are affected (Figure 1B).

Cloning the mutant locus: To characterize the mutation in the TgN37ORpw line at the molecular level, we have cloned and characterized both the mutant locus and the corresponding wild-type region (Figure 2A). Flanking sequences were isolated from a genomic library prepared from a TgN37ORpw homozygote using the transgene as a probe (see MATERIALS AND METH- ODS). Utilizing the cloned sequences, we were able to deduce the structure of the mutant and corresponding wild-type regions (Figure 2A). We confirmed that the cloned region corresponds to the mutant locus by testing genomic DNA from wild-type, heterozygous and homozygous animals within the TgN37ORpw mutant line (Figure 2B). With probe A from one side of the insert, wild-type 6.0- and 12-kb mutant allele-specific EcoRI fragments were detected. Similarly, with probe B (Fig- ure 2A) cloned from the other side of the insertion, 6.0-kb wild-type and 12-kb mutant allele-specific EcoIU fragments were observed (data not shown). With both probes, only the wild-type fragment was detected in wild-type animals, only the mutant allele-specific fragment is observed in the mutant animals, and, as ex- pected, both fragments were detected in animals heterozygous for the mutation. The fact that both probe A and probe B from opposite sides of the transgene insertion detected the same size wild-type fragment indicates that there are no major structural alterations in the host DNA at the transgene integration site. Se- quence analysis of the mutant and corresponding wild- type regions indicates that a single T nucleotide is duplicated on one side of the transgene, and a duplication of two nucleotides, TA, has occurred on the other end of the transgene (Figure 2C). Moreover, standard Southern analysis coupled with pulsed-field gel electro- phoresis failed to reveal any structural alterations in the mutant region apart from the integration of the transgene (data not shown).

Mapping the TgN37ORpw wild-type locus: To deter- mine if the TgN37ORpw locus is linked to any known mutant locus, we mapped the transgene integration site utilizing recombinant inbred strains (TAYLOR 1978). A single-copy 1.6-kb BumHI/SuZI genomic fragment from a region adjacent to the transgene integration site (probe C in Figure 2A) was used to identify a restriction fragment length polymorphism (RFLP) in C57BL/6J and DBA/2J mice. Probe C was hybridized to HznjI-digested genomic DNA from BXD recombinant lines to generate the strain distribution pattern (SDP) shown in Figure 3A. The lowest recombination fraction (ratio of recombinants to informative strains)

A BXD recombinant inbred strains

MARKER i ~ s 8 8 ~ i 2 3 4 s 6 8 s o 1 2 3 4 s 7 8 9 0 1 2 DNA# 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3

D4S43h B D D B B B D B B B B D B D D B D B D B B D B D D D X

QmkQG B D D B B B D B B B B D B D D D D B D B f D B D D D Pmv-5 B D D B B B D B B B B D B D D D D B D B D D B D D D

B

Mouse Chromosome 5

Hm, Hx

TgN370Rpw probe C I I:: 4 1 D4S43h

26.8 P mv-5, Tg(A RSmu) 30.2 D5Ndsl

Tht

y. a FIGURE 3.-Mapping the TgN37ORpo insertional mutant

to mouse chromosome 5. (A) Genomic DNA samples from recombinant inbred strains (BXD) were analyzed with probe C (see Figure 2A), with the greatest linkage to the proximal end of chromosome 5, as shown in the strain distribution pattern. (B) Mapping of the TgN37ORpw locus to chromosome 5 near thick-tail (Tht), a dominant mutation affecting the tail (GREEN 1989; KOZAK and STEPHENSON 1993).

was found between TgN37ORpw and those for known loci on mouse chromosome 5. TgN37ORpw is most closely linked to Pmu-5 and D5H4S43, in which only one discordant sample was observed with each of these markers. Therefore, we have positioned TgN37ORpw between these two loci with a recombination distance -+ SE of 1.02 2 1.06 cM between each marker (Figure 3B). Interestingly, close to the TgN37ORpw transgene integration site is another tail phenotype mutant called thick tail (Tht).

Complementation testing TgN37ORpw and Tht: On the basis of the close proximity of Tht and TgN37ORpw, we performed an allelism test by generating individual animals that were heterozygous for both mutations (see MATERIALS AND METHODS). From heterozygous Tht and homozygous TgN37ORpw parent animals, a total of 52 progeny were generated, 25 of which were compound heterozygotes ( Tht/+; TgN37ORpw/+) and 27 were TgN37ORpw heterozygotes (+/+; TgN37ORpw/+). Tht heterozygotes alone have shortened thickened tails that are -'/3 normal length and develop undulations as the tail grows (BEECHEY and SEARLE 1980; J. J. SCHRICK, P. B. SELBY, and R. P. WOYCHIK, unpublished data). Compound heterozygotes resemble Tht/+ mice, except that these mice have even shorter tails (half the average length of wild type) and also show a kink at the end of their tails (Figure 4D). This combined phenotype is never observed in either one of the mutant lines by itself

T~N(Irnusd)37ORpw Tail Mutation 106

FIGURE 4.-Complementation test of the Tht and TgN37ORpw mutations. Alizarin Red preparations of the distal tails from a wild-type FVB/N (+/+) (A), a homozygous TgN37ORpw (B), a heterozygous Tht (Tht/+) (C), and a compound heterozygous (Tht/+;TgN37ORpw/+) animal (D). Tails in A and B also shown in Figure 1. In the compound mutant shown in D, the distal tail vertebrae are severely disrupted and appear as small bone fragments near the end, which is unlike that which occurs with either of the mutants individually. Differences in the size of vertebrae between samples is primarily due to variability in magnification of the skeletons.

(Figure 4, B and C). While TgN37ORpw homozygotes possess kinks in the last seven caudal vertebrae as seen in Figure 4B (see also Figure lA), Tht/+;TgN370- Rpw/+ compound mutants (Figure 4D) show a more severe phenotype in which bone fragments, rather than distinct vertebrae, form in the most distal part of the tail. By itself the Thtmutant phenotype does not express any overt skeletal phenotype in the distal vertebrae (see Figure 4C).

DISCUSSION

A novel mouse insertional mutation with a tail defect: We have undertaken the phenotypic and molecular characterization of a new recessive mutation, TgN37ORpw, that causes a kink at the end of the tail. The skeletons of these animals contain an abnormality that is confined to the distal spine and involves a structural abnormality in the individual vertebrae that ultimately gives rise to an undulation of the distal tail. As a first step in beginning to characterize TgN37ORpw at the molecular level, we were able to clone the mutant locus utilizing the transgene as a molecular tag.

Noncomplementation suggests close linkage with f i t : Of the existing mutations that have a defect in the tail, only the pintail mutation has a phenotype that is similar to the TgN37ORpw mutant (BERRY 1960). Since pintail maps to chromosome 4 and TgN37ORpw maps to chromosome 5, the two cannot be allelic. On the

other hand, our mapping experiments did reveal that TgN37ORpw maps to a region of mouse chromosome 5 near another mutation, Tht, that causes a defect in the tail. Thick tail (Tht) causes the tail to be short and thickened at birth and to become undulated later in development. This anomaly may be caused by insufficient carti- lage formation in the vertebral bodies (GREEN 1989; J. J. SCHRICK, P. B. SELBY, and R. P. WOYCHIK, unpublished results). Compound heterozygotes containing both the Tht and the TgN37ORpw mutant alleles express a unique phenotype that overall is more severe than either mutation by itself. This result could indicate that Tht repre- sents a dominant mutation of the same gene associated with TgN37ORpw. To explore this possibility, we analyzed the Tht allele utilizing DNA probes derived from the region flanking the transgene integration site of TgN37ORpw. Even though Tht may have been induced by radiation, we have been unable to detect any obvious DNA structural alterations with the available probes (J. J. SCHRICK and R. P. WOYCHIK, unpublished results). This result does not rule out the possibility that the two mutations are allelic since Tht may be a point mutation or some other change in the structure of the DNA that cannot be readily detected with our molecular probes. Once we have identified the gene directly associated with the TgN37ORpw mutation, further experiments will be performed to help to resolve this issue.

Similar to the situation described in this report, POHL et al. (1990) characterized a recessive insertional muta-

1066 J. J. Schrick et at.

tion called anterior digzt defmity ( a d d ) , which was initially mapped to a region near the dominant mutation extra-toes ( X t ) . The recessive add insertional mutation causes subtle defects in the anterior digits of only the forelimbs in the mutant animals, and the existing X t dominant mutation is characterized by polydactyly on all four limbs (GREEN 1989). The production of compound heterozygotes between add and Xt generated an enhanced phenotype on both the fore- and hind limbs that was more severe than either allele alone (POHL et al. 1990). Ultimately, it was determined that the Gli3 gene, which was mapped relative to Xt, was affected by intragenic deletions in the X t and X t J alleles (SCHIM- MANG et al. 1992; VORTKAMP et al. 1992; HUI andJOmER 1993). The transgene insertion in the add mutation integrated within the 5' region of the Gli3 gene, and as a result, negatively influences Gli3 expression and causes a more subtle phenotype than the dominant Xt (VAN DER HOEVEN et ai. 1993). Based on the similarity of these data to those described in this report, it may be that the transgene insertion in the TgN37ORpw line has also occurred within a regulatory region of a gene associated with the mutant locus, and that the Tht mutation may be a deletion of the coding sequences of the same gene.

A simple insertional mutation: Previous reports have indicated that some insertional mutations in transgenic mice generated by pronuclear microinjection contain rearrangements and deletions of the host genomic sequences adjacent to the transgene insertion site, thereby complicating the molecular analysis of these mutations (SINGH et al. 1991). Our data clearly indicate that i t is possible for a transgene to integrate essentially without any rearrangements of the host DNA. Therefore, based on previous reports where it has been shown that a large deletion can occur in the host sequences at the transgene integration site, we are led to suggest that a whole range of different kinds of mutations can probably occur in the host sequences at the transgene integration site, including essentially simple insertions of the exogenous DNA. Although it is presently unknown how often insertional mutations more similar to simple insertions occur as compared to those like large deletions or complex structural alterations, it is becoming increasingly clear that many insertional mutations generated with pronuclear microinjection are like the TgN37ORpw mutation in that they can be readily accessed at the molecular level (reviewed in MEISLER 1992; HODGUNSON et al. 1993; MOVER et al. 1994).

Nature of the developmental defect in the mutant animals: Based on our limited characterization of the phenotype in the TgN37ORpw line, we are speculating that the defect occurs during late somite and tail bud formation. Since the defect is apparent only in the caudal vertebrae in the TgN37ORpw and the Tht/ Tg37ORpw compound mutant animals, we suggest that the defect

occurs within the late stage somite or in the sclerotome that gives rise to these vertebrae. Specifically how the defect occurs is presently unclear, although we expect that in situ hybridization experiments with the gene associated with the TgN37ORpw, once it is identified, will be useful in this regard.

We thank Drs. LISA STUBBS, DABNEYJOHNSON, EDWARD J. MICHAUD and WILLIAM G. RICHARDS for their critical reading of this manuscript, BARFWRA B w m for the embryo microinjections and GENE BARKER for other support activities involving the maintenance of transgenic mice. Dr. B. L. M. HOGAN is an investigator of the Howard Hughes Medical Institute. This work was supported by a grant from the Na- tional Institutes of Health (R01 HD-25323) (R.W.) and from the Office of Health and Environmental Research, US. Department of Energy, under subcontract DE-AC05-840R21400 with Martin Mari- etta Energy Systems, Inc.(R.W.).

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