neurulation abnormalities secondary to altered gene expression in neural tube defect susceptible...

Neurulation Abnormalities Secondary toAltered Gene Expression in Neural TubeDefect Susceptible Splotch EmbryosGREGORY D. BENNETT,1 JIE AN,1 JOHANNA C. CRAIG,1 LISA A. GEFRIDES,1JAMES A. CALVIN,2 AND RICHARD H. FINNELL1*1Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 778432Department of Statistics, Texas A&M University, College Station, Texas 77843

ABSTRACT The murine mutant Splotch (Sp) isa well-established model for studying neural tube clo-sure defects. In the current investigation, the progres-sion through neural tube closure (NTC) as well as theexpression patterns of 12 developmentally regulatedgenes were examined in the neural tissue of wildtype(1/1), Splotch heterozygous (Sp/1), and Splotchhomozygous (Sp/Sp) embryos during neurulation. Theoverall growth of the embryos, as measured by thenumber of somite pairs, did not differ significantlybetween the three genotypes at any of the collectiontime-points. There was, however, a significant delay inthe progression through NTC for both the Sp/1 andSp/Sp embryos. A univariate analysis on the expressionof the 12 candidate genes (bcl-2, FBP-2, Hmx-2, Msx-3,N-cam, N-cad, noggin, p53, Pax-3, Shh, Wee-1, wnt-1)revealed that although 11 were statistically altered,across time or by genotype, there were no significantinteractions between gestation age and genotype forany of these genes during NTC. However, a multivariatestatistical analysis on the simultaneous expression ofthese genes revealed interactions at both gestation day(GD) 8:12 (day:hour) and 9:00 among Pax-3, N-cam,N-cad, bcl-2, p53, and Wee-1 that could potentiallyexplain the aberrant NTC. The data from these studiessuggest that a disruption in the genes that govern thecell cycle or extracellular matrices of the developingneural tube might play a critical role in the occurrenceof the NTDs observed in Splotch embryos. Teratology57:17–29, 1998. r 1998 Wiley-Liss, Inc.

The genetic regulation of mammalian neurulation ishighly complex, involving a multitude of events at thecellular, biochemical, and molecular levels, most ofwhich are not yet fully understood. Disturbance of thetightly regulated morphogenetic processes by eithergenetic or environmental factors could potentially re-sult in abnormal neural tube closure (NTC), leading tothe development of a neural tube defect (NTD) (Camp-bell et al., ’86; Hall et al., ’88; Copp et al., ’90). Theetiology of most isolated NTDs is thought to be multifac-torial in nature, possessing both genetic and environ-mental components. Several genes have been identifiedin recent years that are thought to play significant roles

in regulating mammalian neurulation. These genes canbe categorized into several classes, including thoseinvolved in cell proliferation/apoptosis (bcl-2, p53, Wee1,and FBP-2), cellular adhesion (N-cad and N-cam),neural induction, (Shh, wnt-1, and noggin), and neuralpattern formation (Pax-3, Hmx-2, and Msx-3). Whiledefinitive studies have not directly linked any of theaforementioned genes to abnormalities of the neuraltube, based strictly upon their temporal and spatialpattern of expression, they are considered to be impor-tant for the regulation of events involved in NTC.

An effective strategy for studying the normal regula-tion of gene expression during neural development hasbeen to exploit systems in which this regulation hasbeen altered, and work backwards from the abnormalto try to understand the molecular events that governnormal NTC. One such approach has been the use ofanimal models that have a high spontaneous rate ofNTDs (Campbell et al., ’86). Of the many murinemutants that fit this description, the Splotch (Sp)mouse, whose phenotype includes both spina bifidaand/or exencephaly, has become one of the most impor-tant animal models for studying the development ofNTDs. The Splotch mutation was initially identified asa spontaneous semi-dominant allele that occurred in amouse colony (Russell, ’47). It has since been mapped tomurine chromosome 1 (Skow et al., ’88). There aremultiple alleles at this locus that have arisen spontane-ously like Splotch-delayed (Spd; Dickie, ’64), or wereradiation-induced (Spr, Sp1H, and Sp2H; Beechey andSearle, ’86). Among the X-ray-induced Sp alleles, bothdeletions and single base substitutions were later iden-tified in the transcription factor Pax-3 gene (Epstein etal., ’91, ’93; Goulding et al., ’93). Although the role ofPax-3 during NTC is unknown, the pattern of expres-

Contract grant sponsor: National Institute of Environmental HealthSciences; Contract grant numbers: ES01365, DE11303; Contractgrant sponsor: NIH; Contract grant sponsor: National Institute ofDental Research.

*Correspondence to: Dr. Richard H. Finnell, Department of VeterinaryAnatomy and Public Health, Texas A&M University, College Station,TX 77843-4458. E-mail: [email protected]

Received 11 August 1997; Accepted 18 November 1997

TERATOLOGY 57:17–29 (1998)

r 1998 WILEY-LISS, INC.

sion suggests functional importance in these processes,as Pax-3 is expressed prior to NTC at the dorsal aspectof the prosencephalon, in the region associated withclosure site II, with expression extending to the poste-rior neuropore. The gene is also expressed in spinalganglia, dorsal root ganglia, somitic mesoderm, andcraniofacial regions that are derived from neural crest(Goulding et al., ’91).

Splotch heterozygotes (Sp/1) are characterized bythe appearance of white patches on the belly andextremities, which result from failure of neural crestcell derived melanocytes to migrate into these regions(Auerbach, ’54). Significant, life-threatening defects ofneural crest cell derivatives, such as agenesis of spinalganglia or persistent truncus arteriosus, have also beenreported in the Splotch embryos (Auerbach, ’54; Eff-mann and Whitman, ’86). Splotch homozygous (Sp/Sp)embryos display abnormalities of neural crest migra-tion, as well as exhibiting spina bifida, with andwithout exencephaly. The cephalic neuroepithelium ofthe Sp/Sp embryos relative to comparably staged 1/1or Sp/1 embryos, display an increased cell-cycle length.Additionally, these same cells in Sp/Sp embryos haveabnormal cellular processes, an increase in extracellu-lar spaces and in the number of gap junctional vesiclesin the posterior neural tube, when compared to compa-rably staged wildtype or heterozygous Splotch (Sp/1)embryos (Wilson, ’74; Wilson and Finta, ’79; Morris andO’Shea, ’83). Given the severity of the developmentaldefects observed in homozygous Splotch embryos, theygenerally die at approximately gestation day (GD) 14(Kapron-Bras and Trasler, ’88).

In the present study, we sought to determine theinteractions of the Splotch gene with important develop-mentally regulated genes that may be involved in theregulation of murine neurulation. Since Pax-3 is atranscription factor, we hypothesized that the Sp geneproduct would alter the pattern of expression of avariety of developmentally regulated genes. To this end,we dissected the neural tubes of embryos from all threeSplotch genotypes (Sp/Sp, Sp/1, and 1/1), and usingin situ transcription/anti-sense RNA (RT/aRNA) ampli-fication procedures (Eberwine et al., ’92a,b; Wlodarczyket al., ’96; Finnell et al., ’97), we examined the coordi-nate regulation of twelve genes during NTC, using twostatistical approaches. The goal, therefore, was to iden-tify candidate genes whose expression pattern in Splotchembryos with NTDs differs from those embryos whosuccessfully complete NTC, suggesting a possible caus-ative role in the pathogenesis of this malformation.

MATERIALS AND METHODS

Experimental animals

LM.B6 (F1LM/Bc.C57BL/6J) mice were maintainedon a 12-hour light cycle in the Laboratory AnimalResources and Research Facility at the College ofVeterinary Medicine, Texas A&M University. Up to fivehealthy females were housed per polycarbonate cage,and were allowed free access to Purina rodent breeder

chow and tap water. Virgin LM.B6 Sp/1 females,50–70 days of age, were bred overnight to experiencedLM.B6 Sp/1 males. Females were examined the nextmorning for the presence of vaginal plugs. The begin-ning of gestation (day 0, hour 0) was set at 10 P.M. of theprevious evening, the midpoint of the dark cycle (Snellet al., ’48). At the assigned hour (GD 8:12, 9:00, 9:12,and 10:00), pregnant dams were killed by cervicaldislocation, the abdomen opened, and the uterine con-tents removed. The location of all viable embryos andresorption sites were recorded. Using watchmaker’sforceps, the embryos were dissected free of the decidualcapsule and its chorion and amnion while in coldphosphate buffered saline, under a Wild M8 dissectingmicroscope (Heerbrugg, Switzerland). The embryos weregrossly examined morphologically and were classifiedon the basis of their stage of NTC, using previouslydescribed standardized staging criteria (Cole andTrasler, ’80; Macdonald et al., ’89; Golden and Chernoff,’93). For all fetuses and embryos, the amniotic mem-branes and internal viscera were isolated and used forthe PCR-based genotyping.

Removal of neural tube from NTC stage embryos

Using fine-tipped watchmaker’s forceps, the neuraltube proper was dissected away from the supportingparamesodermal tissue of the NTC stage embryosunder a dissecting microscope (Stemple and Anderson,’92; Taylor et al., ’95; Wlodarczyk et al., ’96). Oncedissected free, it was examined to ensure that only theintact neural tube tissue had been collected, free fromextraneous tissues. In preparation for the in situ stud-ies, this tissue was placed in ice-cold hybridizationbuffer consisting of 5 mM DTT, 100 U RNasin (Pro-mega, Madison, WI) and 0.1% digitonin. Following abrief sonication pulse at 4°C, which completely dis-rupted the cells of the dissected neural tube, 50 U ofRNasin was added to the hybridization buffer and thetissue was frozen at 280°C until further processing.

Genotyping the splotch gene using PCR

DNA isolation. Extraembryonic membranes andassociated viscera that were separated from each em-bryo during neural tube isolation were placed in a1.5-ml microcentrifuge tube containing 0.5 ml of diges-tion buffer (100 mM NaCl, 10 mM Tris Cl, pH 8.0, 25mM EDTA, pH 8.0, 0.1 mg/ml proteinase K). The DNAisolation procedure was essentially that of Gross-Bellard and coworkers (’72). Briefly, the tissue samplewas incubated with the digestion buffer at 50°C over-night. Three separate phenol/chloroform/isoamyl alco-hol (25/24/1) extractions were performed on the di-gested sample. Following the last extraction, theaqueous layer was transferred to a new eppendorf tube,ethanol precipitated, and resuspended in TE buffer (10mM Tris Cl, pH 7.5, 1 mM EDTA, pH 8.0).

Genotyping tissue from embryonic membranesby PCR. Two primers (spall 58TAGGGAGAGGGTT-GAGTACG38 and common 58CTCGCTCACTCAGGAT-

18 G.D. BENNETT ET AL.

GCC38) were used to amplify a region of interest in thePax 3 gene, approximately 1.1 kb, from the extractedgenomic DNA using a Stratagene Robocycler (Strata-gene, La Jolla, CA). Each PCR reaction consisted of 35cycles of denaturation (94°C for 1 min), annealing (65°Cfor 2 min), and elongation (72°C for 1 min), with thefinal concentration of the following reagents: 50 ngDNA, 1 3 PCR Buffer (Promega, Madison, WI), 1.5 mMMgCl2, 4% DMSO, 1 mg/ml BSA, 0.2 mM each primer,200 mM each dNTP, and 1 U/ml Taq polymerase(Promega). The amplified products were visualized byethidium bromide staining on a 1% agarose gel. ThePCR product was purified using the QIAquick-spinPCR Purification Kit (QIAGEN Inc., Chatsworth, CA)and the protocol and reagents described therein. Thesepurified products were used as template in the dideoxyfingerprinting (ddF) reaction.

Dideoxy fingerprinting of known mutations(ddF). Using ddF, which is analogous to running asingle DNA sequencing lane (dTTP was used in thepresent study), it was possible to compare bandingpatterns between samples to determine the genotype ofindividual embryos. Cycle sequencing was performedusing the Thermo Sequenase radiolabeled terminatorcycle sequencing kit (Amersham, Arlington Heights,IL) with final concentrations of the reagents as follows:500 ng DNA template (purified PCR product), 1 3Reaction Buffer, 0.25 µM 38 common primer, 2 µMdGTP termination master mix, 0.2 µM a-33P ddTTP,and 1 U of Thermo Sequenase polymerase. The reactionconditions were 40 cycles of denaturation (95°C for 1min), annealing (60°C for 1 min), and elongation (72°Cfor 1 min). After the reactions were amplified, one-halfvolume of stop solution (Amersham) was added to eachreaction, and the reactions stored at 220°C until theywere loaded onto a gel.

Gel electrophoresis. The samples were heated to95°C for 10 minutes and immediately placed on iceuntil loaded onto a 6% denaturing gel (Genomyx, FosterCity, CA). The reactions were run for 2 hours using ashort sequencing program of the GenomyxLR Program-mable DNA Sequencer (Genomyx). The gels werewashed, dried, and exposed to radiographic film forvisualization.

In situ transcription and aRNA amplification

In situ transcription and anti-sense RNA amplifica-tion procedures were performed according to the meth-ods described by Eberwine and coworkers (’92a,b; Wlo-darczyk et al., ’96; Finnell et al., ’97). The initial stepsinvolved making an RNA/DNA hybrid molecule byadding to the population of mRNAs present in each ofthe sonicated neural tube samples an oligo-dT-T7 oligo-nucleotide primer that hybridizes to the poly A tail ofthe mRNAs along with avian myeloblast reverse trans-criptase (Seikagaku America Inc., Bethesda, MD). Thesingle-stranded DNA produced in this reaction wasisolated and allowed to form double-stranded compli-mentary DNA (cDNA) by hairpin loop formation, which

was then used to prime the second strand synthesis.The hairpin loop was digested with S1 nuclease. Thisdouble-stranded cDNA was used to produce radiola-beled, amplified, anti-sense RNA (aRNA) by the addi-tion of T7 RNA polymerase (Epicentre Technologies,Madison, WI) in the presence of [32P]-CTP. The aRNA,representing the entire population of mRNAs from theneural tube tissue, was then hybridized to ‘‘reverse’’Northern blots in order to quantitatively determine thepattern of gene expression for the candidate genesselected for investigation.

Genetic expression profiling

Equimolar concentrations of the cDNA clones of the12 candidate genes were immobilized on a nylon mem-brane (Zetaprobe, Bio-Rad, Richmond, CA) along withan internal control cDNA (cyclophilin), using a Bio-Radslotting apparatus, following the manufacturer’s proto-col. The resulting slot blots were then hybridized withan aRNA probe using conditions detailed previously byWlodarczyk and colleagues (’96). Briefly, each blot wasprehybridized for 30 minutes in hybridization buffer(7% SDS [w/v], 0.12 M Na2HPO4 [pH 7.2], 0.25 M NaCl,and 50% formamide) at 42°C. The heat denaturedaRNA probes were then added to the buffer and allowedto hybridize for 24 hours. Subsequently, the slot blotswere washed at medium stringency with 0.1 3 SSCcontaining 0.1% SDS at 42°C, dried, wrapped in plasticwrap, and placed in an Ambis 101 two-dimensionalradioanalytics imaging detector (Scanalytics, Billerica,MA), which directly measured the radioactivity (CPMs)of each slot on the ‘‘reverse Northern blots.’’ The indi-vidual signals were normalized to cyclophilin geneexpression. The selection of cyclophilin as the normaliz-ing cDNA simply enables us to make comparisonsbetween different blots, as the individual hybridizationintensities of each cDNA on each blot can be expressedas a ratio of its expression to cyclophilin. There is noparticular significance to the selection of this gene,other than the fact that it is a constituitively expressedgene found in high abundance in the mouse brain,thymus, and embryo (Danielson et al., 1988), and maybe the best internal standard for use in multiprobeassays when examining low abundance messages.

Statistical analysis

Univariate (simple) and multivariate (complex coordi-nate) statistical procedures were employed. The meannumber of somite pairs per fetus, as well as thefrequency distribution of the stages of NTC, weresubjected to simple statistical analysis using a two-wayanalysis of variance and a Chi-square goodness of fittest, in order to determine if any of the Splotch geno-types differed significantly (P , 0.05) at a given time-point with respect to embryonic growth and develop-ment (Sokal and Rohlf, ’81). Follow-up analyses ofsignificant effects observed in the analysis of variancetesting were performed using a Student-Newman-Keuls multiple comparison test. For statistical signifi-

GENE EXPRESSION IN SPLOTCH EMBRYOS 19

cance in the gene expression profiling studies, leastsquares means describing the means of subpopulationswithin the experiment were analyzed, with a P value of,0.05 set for significance (Sokal and Rohlf, ’81). Theuse of least square means has specific advantages overarithmetic means when there are unequal sizes of theexperimental groups. The least square means are com-puted from the estimates of the parameters in themodel used to describe the data. Data were analyzedwith the SAS (Statistical Analysis System, 1990) pro-gram on an IBM computer.

A complex multivariate statistical procedure knownas principal components analysis (PCA) was used toassess the coordinate expression of a set of genessimultaneously. For a detailed discussion of PCA seeRao (’73) or Rohlf and Bookstein (’90). In the currentstudy, two separate PCAs were performed on expres-sion data from six genes representing the GD 8:12 and9:0 1/1 embryos. The selected genes encode cell cycleproteins (p53, Wee-1, and bcl-2), cell adhesion molecules(N-cad, N-Cam), and the Pax-3 transcription factor.This approach was utilized in an effort to identifypatterns among these genes whose interplay is thoughtto be important in regulating normal neural crest cellmigration, and has been previously described by Craigand co-workers (’97). Briefly, the PCA constructs newvariables that represent novel combinations of all theoriginal genes. The number of new variables, or princi-pal components (PCs), generated by the PCA is equal tothe number of original variables used as input to thePCA. Each PC describes a percentage of the between-embryo variability for the gene combination, and issorted based on this variability. Thus, the first newvariable, called PC1, is the combination of the originalmeasurements (gene expression values) that are themost variably expressed among embryos, while PC6represents the least variability expressed among em-bryos. Therefore, the lower order PCs would best de-scribe differences among embryos, while the higherorder PCs would represent embryo similarities, or acohesive embryonic response. Furthermore, the PCscan be thought of as ratios, as their construction isbased upon log-transformed data (Craig et al., ’97;Rohlf and Bookstein, ’90). For this study, we wereinterested in exploring gene ratios that elicited aunified embryonic response among the 1/1 embryos toestablish a ‘‘molecular fingerprint’’ for comparisonsacross genotypes. Therefore, we focused on the higherorder PCs, in which the numerator and denominatorgenes were expressed in parallel to one another fromone 1/1 embryo to the next. For each GD, PC1 throughPC6 was computed, and the combination of genes goinginto each PC was assessed for biological viability. Oncea PC was selected, we were able to utilize these ratioscomprised of six genes each to discuss biological interac-tions among the set of genes, and develop new hypoth-eses with which to extrapolate coordinate gene expres-sion to the three Splotch genotypes. Statistics for thesefollow-up ratio analyses were performed utilizing the

Hartley’s F-Max test for equality of variance, as well asa test for equality of means using the LSMEANSprocedure in SAS.

RESULTS

To address whether or not Splotch embryos developat a slower rate than their wildtype or heterozygouslittermates, embryos from Sp/1 3 Sp/1 crosses werecollected at several timepoints during the period ofNTC, genotyped, and evaluated as to their stage of NTCaccording to the criteria of Cole and Trasler (’80). Theprogression of embryos through NTC from each of thethree genotypes is summarized in Table 1. With respectto the overall embryonic growth parameters, there wereno significant differences in the mean somite number ofembryos among the different genotypes at any of thegestational ages (P . 0.05). In general, at GD 8:12,approximately one third of the embryos examined hadinitiated NTC, with fusion just beginning in the area ofclosure sites I and II. However, the Sp/Sp and Sp/1embryos appeared to be slightly delayed in the initia-tion of fusion at closure site II, compared to 1/1embryos (Table 1). At GD 9:0, while half of the wildtypeembryos had completed NTC, only 43% of the Sp/1 and17% of the Sp/Sp embryos had completed this process(Table 1). Similarly, homozygous Splotch embryos col-lected at GD 9:12 demonstrated a statistically signifi-cant degree of developmental delay of the neural tube,as only 32% of these embryos had completed NTC,while approximately 67% of the Sp/1 and 90% of 1/1embryos had closed neural tubes (P , 0.05; Table 1). ByGD 10:0, nearly all of the wildtype embryos had com-pleted NTC, with the exception of two embryos exhibit-ing delayed development. On the other hand, only 31%of the Sp/Sp embryos had fully closed neural tubes,which was significantly lower than the percentages ofheterozygotes and wildtypes (P , 0.05; Table 1). Inter-estingly, almost half of the Sp/Sp embryos failed to fusethe neural fold at closure site II, and nearly one-fifth ofSp/Sp embryos still had openings at closure sites IIand/or IV (Table 1). The Sp/1 heterozygotes had anintermediate number of embryos that had completedNTC, while approximately 37% of the embryos failed toachieve NTC by this gestational timepoint (Table 1).

The pattern of gene expression was analyzed inneural tube tissue dissected free from embryos of allthree genotypes collected at four gestational stages(8:12, 9:0, 9:12, and 10:0) during the period of NTC. Forthese experiments, no fewer than ten embryos pergenotype per time period, which amounted to a total of160 embryos, were analyzed. Our initial statisticalapproach was to explore the univariate regulation ofthe 12 candidate genes that were believed to havesignificant roles in the morphogenesis of the mamma-lian neural tube. Of these 12 genes, only the cell cyclecheckpoint gene, Wee1, maintained a more or lessconstant level of expression during the selected timeframe of these experiments. Each of the remaining 11genes demonstrated significantly different levels of


expression depending upon the gestational age of theembryo experiments (P , 0.05; Fig. 1A and B), whileonly three genes showed significant genotypic differ-ences (P , 0.05; Fig. 2). Although rigorously investi-gated, there did not appear to be any significantinteractions between time (gestational age) and Spgenotype (Sp/Sp, Sp/1, 1/1) for any of the genes,during the gestational timepoints examined in thisstudy (P . 0.05; data not shown).

Eleven of the 12 genes were significantly alteredacross gestational time. There were six genes whosepattern of expression consistently peaked at the earli-est gestational timepoints (GD 8:12), and then droppedto their lowest levels at GD 9:12, and, for four of thesegenes, to rise slightly by GD 10:00 (Fig. 1A). Thesegenes included: bcl-2, p53, Msx-3, noggin, N-Cam, andHmx-2. The expression of bcl-2 and noggin was signifi-cantly higher in the early embryos (GD 8:12), than wasthe level of expression observed at either of the twolatter gestational timepoints (GD 9:12 and GD 10:00;Fig. 1A) (P , 0.008). The p53 and Hmx-2 genes hadhigher expression levels prior to GD 9:00, than thoseobserved in embryos collected after GD 9:12 (P , 0.008).For Msx-3, a significant difference was found betweenexpression levels of GD 8:12 and GD 9:12 embryos(P , 0.008; Fig. 1A). N-Cam expression was relativelyunchanged at GD 8:12 and GD 9:00; however, there wasa significant reduction in expression at GD 9:12(P , 0.008; Fig. 1A).

In addition to the aforementioned genes whose pat-tern of expression tended to decrease with gestationalage, there were five other developmentally regulatedgenes who had their peak expression on GD 9:0. Thesegenes included: Shh, Pax-3, FBP-2, wnt-1, and N-cad(Fig. 1B). With the sole exception being wnt-1, whichdemonstrated a significantly lower expression level atGD 10:00 compared to GD 9:00, the other four genes(Pax-3, FBP-2, Shh, and N-cad) all had significantlydecreased expression levels at both GD 9:12 and GD10:00, when compared with the expression observed atGD 9:0 (P , 0.008).

The level of gene expression of three candidate genes(Pax-3, N-cad, and Hmx-2) was found to be significantlydifferent among the three Splotch genotypes (P , 0.05;Fig. 2). Specifically, all three of these genes wereconsistently expressed at significantly higher levels inthe neural tube tissue obtained from Sp/Sp embryosthroughout NTC, when compared to 1/1 embryos(P , 0.017; Fig. 2). With respect to N-cad, the expres-sion levels in the Sp/1 embryos were significantlylower as compared to the Sp/Sp embryos (P , 0.017;Fig. 2). There were no significant differences in the levelof expression between the Sp/1 and the 1/1 embryosfor any of these genes (P . 0.05).

Multivariate statistical analysis

Two PCAs were performed on select cell cycle, cellu-lar adhesion molecule, and transcription factor geneexpression data generated from GD 8:12 and GD 9:0wildtype embryos. Three cell cycle genes (p53, bcl-2,wee 1) and the three genes implicated in affecting theSplotch phenotype in Sp/1 and Sp/Sp mice (Pax-3,N-Cam, N-cad) were utilized in an effort to identify anassociation between cell cycle length and neural crestcell migration, and to clarify their interactive roles inthe developing Splotch neuroepithelium. The goal wasto have the PCAs identify gene combinations thatmight better clarify the normal molecular events in-volved in the patterning and the cellular activities atthis stage of development. We could then examine howthe identified gene combinations change across Splotchgenotypes.

The percentage of variation in the selected PCs isgiven in Table 2. For each PCA, six genes were reducedto one PC, which accounted for .2.0% of the totalvariation (Table 2). The fifth and six PCs were selectedfor the two PCAs conducted in this study, respectively,as they described the smallest percentage of the totalvariability, and were deemed to be the most biologicallyinteresting. As stated earlier, the variability describedby each PC represents the combined gene expressiondifferences between each wildtype embryo. Since the

TABLE 1. Progression through neural tube closure in wildtype and splotch embryos

Gestationalage

Litters(no.)

Embryogenotype

Embryos(no.)

Somite/embryo(mean 6 SEM)

Closure sites (%)

Open I II III IV II and IV Closed

8:12 11 Sp/Sp 21 7.43 6 0.49 71 24 5 0 0 0 0Sp/1 28 7.54 6 0.44 68 18 14 0 0 0 01/1 27 7.41 6 0.45 71 7 22 0 0 0 0

9:00 7 Sp/Sp 12 19.00 6 1.17 0 0 25 0 0 58 17Sp/1 21 19.33 6 0.73 0 0 14 0 19 24 431/1 26 18.16 6 0.81 0 0 33 0 0 17 50

9:12 7 Sp/Sp 19 24.79 6 0.46 0 0 37 0 0 32 32*,**Sp/1 18 25.72 6 0.55 0 27 0 0 6 0 671/1 20 25.20 6 0.51 0 0 5 0 5 0 90

10:00 7 Sp/Sp 16 33.69 6 2.35 0 0 50 0 0 19 31*Sp/1 21 34.67 6 1.16 0 10 0 0 0 27 63*1/1 26 34.57 6 1.05 0 8 0 0 0 0 92

*Significantly different from wildtypes (1/1), P , 0.05.**Significantly different from heterozygotes (Sp/1), P , 0.05.


Fig. 1. Plots of the relative expression for the genes analyzed in thisstudy. A: The expression pattern for bcl-2, p53, Msx-3, noggin, N-cam,and Hmx-2. B: Illustrates a different expression pattern for Shh,Pax-3, Folate receptor, wnt-1, and N-cad, during neurulation. Data isplotted as the least squares mean (LSMEAN) of relative intensity bygestation time. a 5 GD 8:12 values were significantly different thanGD 9:12 values, P , 0.008. b 5 GD 8:12 values were significantly

different than values at GD 9:12 and 10:00, P , 0.008. c 5 GD 8:12 and9:00 values were significantly different than GD 9:12 values, P ,0.008. d 5 GD 8:12 and 9:00 values were significantly different thanvalues at GD 9:12 and 10:12, P , 0.008. e 5 GD 9:00 values weresignificantly different than GD 9:12 and 10:00 values, P , 0.008. f 5GD 9:00 values were significantly different than values at GD 10:00,P , 0.008.


lower order PCs describe the majority of this between-embryo variability, the rationale for selecting the higherorder PCs was to identify a gene pattern that wasvirtually identical from one wildtype embryo to thenext. In this manner we could assess unified (possiblycharacteristic) expression patterning, and how it maybe disrupted across the remaining Splotch genotypes.The PC that represents this cohesive embryonic re-sponse can then be viewed in terms of its coefficients asa ‘‘multivariate ratio.’’ The numeric sign of the valuesthat make up this ratio (positive in the numerator andnegative in the denominator) indicate an opposinginteraction between these genes (Table 2). This type ofcomparison allows us to examine the changes in thegene expression pattern that is observed across variousgenotypes.

The principal components plots in Figure 3 representthe genotypic group relationships determined by theinitial PCA analyses. In these two graphs (Fig. 3A andB), the embryos representing the three Splotch geno-types are plotted against the PC ratio determined fortheir gestational age, with the denominator genes

comprising the x-axis and the numerator genes compris-ing the y-axis. Since each point represents a singleembryo, these plots illustrate the embryonic distribu-tions of the expression of these genes within eachgenotype. In this manner, the means and variances ofthe ratio responses of the three genotypes are clearlyrevealed. At GD 8:12, the y-axis is comprised of threegenes with positive eigenvector scores (PC coefficients)(Pax-3, bcl-2, and p53) in the numerator, as well asthree genes with negative PC coefficients (N-cam, N-cad,and Wee-1) in the denominator. The ratio shifts slightlyat GD 9:0, with Pax-3 and bcl-2 remaining in thenumerator, and p53 joining the original three genes inthe denominator. The coefficients for the PCs at GD8:12 and GD 9:0 indicate high values (.0.2) for all sixgenes at GD 8:12, with p53 and bcl-2 having thegreatest impact, and N-Cad and Wee-1 having minimal,although some, influence (Table 2). At GD 9:0, thedominant genes were p53 and bcl-2. Figure 3A and Billustrates a tight clustering among the wildtype em-bryos along their lines that, along with the Hartley’sF-Max test for variance equality, serve to distinguishtheir gene expression patterns from that of the othergenotypes at both GD 8:12 and GD 9:0 (P , 0.05). TheLSMEAN analysis comparing PC ratios among geno-types within gestational days revealed no significantmean differences (P . 0.05). However, the mean com-parisons between GD 8:12 and GD 9:00 1/1 embryoswere statistical significance (P , 0.05; Fig. 3A and B).The Hartley’s F-max test of variance equality revealedthat the 1/1 embryo distribution was significantlydistinct (P , 0.05) from the Sp/1 and Sp/Sp distribu-tions for the GD 8:12 gene ratio (Fig. 3A). By GD 9:0,this ratio was altered slightly in the 1/1 embryos,with p53 falling into the denominator. At this timepoint, there was a significant distinction (P , 0.05)between the 1/1 and Sp/Sp embryo distributions, asrevealed by Hartley’s F-max, indicating a genotypicdeviation from the gene patterning observed for the1/1 group (Fig. 3B).

DISCUSSION

Several different mouse mutants have been utilizedas model systems in which to study the morphogeneticprocesses involved in both normal and abnormal NTC.The Splotch mouse is one of the most valuable of theavailable models, and has provided considerable in-sight at both the morphological and biochemical levelsconcerning potential mechanisms underlying the devel-opment of NTDs. What has been lacking thus far, is theidentification of the primary downstream affect of theSplotch gene on these mechanisms involved in thepathogenesis of NTDs. The present studies representour efforts to better understand the progression ofneurulation in these mutant embryos, as well as theexpression patterns of several genes that may interactwith the Sp gene product, and may be causally relatedto the development of the NTDs.

Fig. 2. Plots of the relative gene expression of Hmx-2, Pax-3, andN-Cad in Sp/Sp, Sp/1, and 1/1 embryos. Data is expressed as theleast squares mean (LSMEAN) of the relative intensity at gestationday 9.0. a 5 Expression was significantly different from wildtype(1/1) embryos, P , 0.017. b 5 Expression was significantly differentfrom hetrozygous (Sp/1) embryos, P , 0.017.

TABLE 2. PC coefficients for the PCAs performed onGD 8:12 and GD 9:00 wildtype embryos

GenesGD 8:12 (1/1)

PC5CD 9:00 (1/1)

PC6

N-Cam 20.637 20.189N-Cad 20.157 20.023Pax-3 0.454 0.091bcl-2 0.474 0.771p53 0.320 20.600wee-1 20.193 20.027

Variance 1.5% 0.1%


In terms of the timing and progression through NTCamong the three Splotch genotypes, developmentallystaged embryos from Sp/1 dams were examined overthe entire period of NTC. The Sp/Sp homozygotes, andto a lesser extent, the Sp/1 heterozygotes, exhibited asignificant delay in the rate at which they completeddifferent stages of NTC (Table 1). Not only were therates of NTC altered, but the Sp/Sp embryos tended todeviate from the normal pattern of NTC, attempting tocomplete NTC without closure II. These findings werenot unlike those previously reported in the SELH/Bcmouse stock, which also has a high spontaneous rate(10–20%) of NTDs (Macdonald et al., ’89; Juriloff et al.,’93). All SELH/Bc embryos lack the initiation of NTCsite II. Those embryos that did manage to completenormal NTC, did so by the extension of closure IIIcaudally beyond its normal limits (Harris et al., ’94).Although the SELH stock pups that were liveborn didnot exhibit an apparent NTD, between 5 and 10% ofthese animals developed ataxia later in life, suggestinga more subtle neurological deficit (Macdonald et al., ’89;Juriloff et al., ’93; Harris et al., ’94).

In the present study, the RT/aRNA technique wasused to reveal interactions among the expression pat-terns for a group of genes that are believed to playsignificant regulatory roles during the developmentand closure of the neural tube. In an initial set ofunivariate statistical analyses, the developmental ex-pression of 12 genes was examined in embryos of all

three Splotch genotypes. Although 11 of the 12 genesdisplayed significant developmental regulation at oneor more of the gestational stages examined, only threegenes (Pax-3, Hmx-2, and N-Cad) were found to differsignificantly (P , 0.05) in terms of their quantitativelevel of expression across Splotch genotypes.

Since it is not feasible to envision all possible relation-ships among the genes of interest by looking at eachgene individually, PCA was used to provide greaterinsight into the complicated interactions of these geneswithin the three Splotch genotypes. Thus, we examinedthe expression data using PCA, focusing on six of theoriginal 12 genes that potentially interact in such amanner as to have a direct effect on the facilitation ofNTC in the Splotch embryos. It is well established thatthe protein products for three of these genes, Pax-3,N-Cad, and N-cam, are required for normal neuralcrest cell migration (Edelman, ’84; Thiery et al., ’82;Neale and Trasler, ’94; Goulding et al., ’91; Bonner-Fraser et al., ’92), while the remaining three genes,Wee-1, bcl-2, and p53, directly and/or indirectly, regu-late important cell cycle events that may impact thesuccess of NTC and neural crest cell migration (Larsen,’94; Vogelstein and Kinzler, ’92; Sah et al., ’95; Tang etal., ’93).

From these PCAs, we determined that the 1/1embryonic gene expression patterns were consistentwith reports that the protein products may interactdirectly, or indirectly, to influence the emigration of

Fig. 3. Plots of the numerator and denominator genes comprising thePC ratios. The results were based on the PCAs performed on the GD8:12 and GD 9:00 wildtype (1/1) embryos. Each point represents asingle 1/1, Sp/1, or Sp/Sp embryo. A: GD 8:12 embryos plottedagainst the PC5 ratio. B: GD 9:00 embryos plotted against the PC6ratio. The numbers on the ordinate and abscissa for each plot reflectthe scale resulting from the construction of separate linear gene

combinations for the numerator and denominator representing eachPC. A tight clustering of the points indicates a unified embryonicresponse to the ratio. Dispersal from the central ‘‘line’’ representsdifferent embryonic responses. * 5 variance is statistically significantfrom 1/1 embryos within a gestational day. 1 5 mean is statisticallysignificant from GD 9:00 1/1 embryos.


neural crest cells from the developing neural tube. Thedata were also consistent with previously reportedobservations that bcl-2 mediates the apoptotic andgrowth arrest effects of the p53 protein in the neuroepi-thelium (Ryan et al., ’94). Furthermore, these six genesmay be interacting simultaneously, through different,albeit, complementary pathways, to aid in proper NTCand neural crest cell migration in the wildtype em-bryos. During NTC, the neuroepithelium is goingthrough a myriad of changes that are regulated bycellular events involving adhesion, migration, differen-tiation, proliferation, and, in all likelihood, these pro-cesses are initiated at a molecular level. As illustratedby the coefficients for the PCA on the GD 8:12 1/1embryos (Table 2), the contribution of each gene to PC5is fairly heavy, with the possible exception of N-Cad.However, the scores for the PCA performed on the GD9:0 1/1 embryos reveal a predilection for the cell cyclegenes, p53 and bcl-2. This may mean that these twogenes are the most influential of all the genes analyzedduring the early stages of NTC, including Wee-1, sug-gesting that the neuroepithelial cells are attempting toregulate their entry into mitosis.

The early interactive influence of Pax-3 and N-camon these embryos, detected by this multivariate analy-sis, is consistent with the documented biological activi-ties of these genes during NTC. Pax-3 is a member ofthe paired box family of transcription factors (Chalepa-kis et al., ’94b) located on the murine chromosome 1(Goulding et al., 1991). Pax-3 transcripts have beendetected in the dorsal neuroepithelium just prior toNTC, and are restricted to dorsally located mitoticneuroepithelial cells throughout early neurogenesis inregions of the neural tube from which neural crest cellsare known to arise and migrate. The second of thesegenes, Hmx-2, is also a transcription factor, and is amember of the highly conserved H6 homeobox genefamily (Stadler et al., ’95). Essentially, univariate analy-sis showed that the expression levels for both genespeaked during NTC, and gradually decreased oncefusion of the neural folds was completed.

The Pax-3 gene of the Sp/Sp mutant mouse has beenfound to have a mutation in the 38 splice acceptor site,resulting in the generation of four aberrant mRNAtranscripts (Epstein et al., ’93). Three of the mRNAshave frameshift mutations that lead to prematuretermination of translation of the protein (Epstein et al.,’93). The fourth transcript encodes a Pax-3 proteinlacking exon 4, which encodes the C-terminal portion ofthe DNA-binding paired domain, as well as the octapep-tide domain. These truncated proteins have been shownin vitro to have a lower binding affinity to the paireddomain cis element and to be missing part of theprotein required for homodimerization (Chalepakis etal., ’94a,b), which, in all likelihood, results in proteinsthat are not functional in Sp/Sp embryos. It is interest-ing to note that Pax-3 was found in our study to beoverexpressed in the Sp homozygous embryos. Onepossible explanation is an inability of Pax-3 to autoregu-

late its own expression, as is seen for other transcrip-tion factors.

Similar to Pax-3, Hmx-2 was observed to be overex-pressed in Sp homozygous embryos. Transcription fac-tors, such as Hmx-2, are members of the Pax and Hoxgene families, which are known to bind to specificsequences within the N-cam promoter and serve toregulate the expression of this gene (Akam, ’89; Chalepa-kis et al., ’94a). N-cam is considered to be one of themost important regulatory factors for NTC, as celladhesion plays a critical role in the control of cellmovements, aggregation, and migration, and may influ-ence cell proliferation and differentiation. A homeodo-main binding site in the N-cam promoter interacts withseveral different homeodomain proteins, while a differ-ent promoter region contains paired domain bindingsites for the Pax gene products (Chalepakis et al., ’94b;Holst et al., ’94; Edelman and Jones, ’95). The presenceof these specific binding domains, along with the co-localization of the Hox and Pax gene products withN-cam, provides a clear indication of the intricateregulatory relationship between these two families oftranscription factors and N-cam gene expression. Thisrelationship must be rigorously maintained in order toensure that high N-cam levels are maintained at theonset of neural tube formation within the neural plate,only to drop off at the time of neural crest cell migration(Edelman, ’84; Thiery et al., ’82).

Several critical events in neural development havebeen correlated with changes in N-cam expression(Edelman, ’84; Rutishauser and Jessell, ’88; Thiery,’89). N-cam has previously been shown to be tightlyregulated at both the transcriptional level, and viaalternative splicing of the hnRNA (Goridis and Brunet,’92). Furthermore, N-cam undergoes post-translationalglycosylation, which results in the addition of polysialicacids (PSA) into the molecule. Interestingly, Sp/Spembryos display an altered form of N-cam, in additionto the two isoforms found in wildtype embryos that maybe due to post-translational modifications such as glyco-sylation, phosphorylation, or sulphation (Moase andTrasler, ’91). This altered form of N-cam has thepotential to interfere with cell adhesion mediated notonly by N-cam itself but other types of cell adhesionmolecules as well (Rutishauser, ’92). N-cam has ahighly restricted pattern of expression throughout theneural plate prior to actual neural tube formation(Crossin et al., ’85; Kintner and Melton, ’87; Prieto etal., ’89).

In addition to its proposed role in neural tube forma-tion, N-cam appears to play a significant role in neuralcrest cell migration, which is dependent upon a reduc-tion in N-cam expression, presumably by reducing theadhesiveness between these cells (Thiery et al., ’82;Neale and Trasler, ’94). The Sp/Sp embryos have fewerneural crest cells leaving the neural tube, and thosethat do leave migrate later in development when com-pared to unaffected, wildtype embryos (Kapron-Brasand Trasler, ’88; Moase and Trasler, ’90). We found that


although the levels of N-cam expression decreasedslightly between the onset of NTC (GD 8:12) and thetime when closure site II presumably fuses (GD 9:00),the most precipitous fall in N-cam expression in theSp/Sp embryos did not occur until GD 9:12 (Fig. 1), atwhich point NTC had been completed in 90% of thewildtype embryos. This prolonged expression of N-Cammay be the reason for the delayed migration of theneural crest cells in Sp/Sp embryos.

Neural cadherin (N-Cad), another important mol-ecule for cellular migration, was similarly examinedand found to be differentially expressed among thedifferent Splotch genotypes, with the highest level ofexpression being in the Sp/Sp embryos. Like othercadherins, N-Cad belongs to a family of CAMs that arecalcium dependent. In the absence of calcium, cadher-ins undergo a conformational change that renders themsusceptible to breakdown by proteases (Takeichi, ’88,’91). N-Cad was found to be important for the normalintegrity of the cranial neural tube, and for facilitatingthe emigration of neural crest cells away from theclosing neural tube (Bonner-Fraser et al., ’92). In thepresent study, the expression of N-Cad changed mark-edly over the period of NTC, with peak levels occurringat GD 9:00 (Fig. 1), the critical period for closure II.These high levels of expression significantly decreasedover the latter portion of NTC. It is possible that theelevated N-Cad levels of expression in Splotch homozy-gous embryos inhibit normal cell migration due toincreased intercellular adhesiveness, much like thatwhich has been suggested for N-cam. This is furthersupported by the observation that neural tube develop-ment has been shown to be delayed in the Sp/Spembryos compared to the Sp/1 and 1/1 embryos.Thus, the aberrant cellular migration might be directlyrelated to the failure of NTC in these embryos.

The cell cycle is obviously important to many of theevents that lead to normal NTC, which was alsoreflected in both PCA analyses. It is believed that cellscomprising the ‘‘hinge points’’ of the folding neural plateexperience changes in their cell cycle length in order toalter the position of the cell’s nucleus so that the neuralplate can effectively bend and fold to accommodateneural fold fusion (Schoenwolf and Smith, ’90). Inaddition, the cell cycle regulates the transitions be-tween differentiation and proliferation that occur atvarious time periods of embryonic development. Whileboth growth and differentiation are likely to be occur-ring concurrently within the developing neural tube, itis probable that the former is the more favored stateduring NTC. This is simply due to the increasednumber of cells that are required to facilitate elevation,folding, and fusion of the neural folds, as opposed tothose necessary for the differentiation events that occurwithin the neural tube or supporting tissues.

The checkpoint genes Wee-1, p53, and bcl-2, areknown to operate at various phases of the cell cycle in

epithelial tissues, and may thereby influence the devel-opment of the murine neural tube via cell cycle regula-tion. The PCA ratio relationship of these genes at 8:12,as well as the opposing and dominant effects of p53 andbcl-2 at GD 9:0, may explain a biological relationshipwhich is meaningful to the regulation of the cell cycle inthe neuroepithelium. For example, p53 and bcl-2 are onthe same side of the equation at GD 8:12, but areopposed at GD 9:0. This opposition may indicate apossible shift in the regulatory behavior of these twogenes, as the neural tube is still undergoing growthwhile preparing for differentiation. The antagonisticeffects of the p53 and bcl-2 gene products have beenwell documented. p53 is a multi-functional protein thathas been implicated in modulating normal develop-ment, in part by regulating gene transcription andsafeguarding cell cycle checkpoints (Clarke et al., ’93).Its main function is to induce growth arrest at the G1

phase of the cell cycle, in order for the cell to assess itsDNA integrity (Clarke et al., ’93; Mercer et al., ’90;Diller et al., ’90). It has also been suggested that theprotein product of p53 may have a role in regulating thecellular transition from proliferation to differentiationduring NTC (Sah et al., ’95). In contrast, the bcl-2 geneproduct is a potent inhibitor of p53-induced apoptosisand cell cycle arrest that is believed to regulate cellproliferation and differentiation (Hockenberry, ’95; Reed,’94; Vaux et al., ’88, ’92). The growth arrest andapoptotic functions regulated by the p53 and bcl-2 geneproducts are thought to be activated when the equilib-rium between the expression of these genes favors p53(Miyashita et al., ’94; Wang et al., ’93). Although nostatistical comparison was performed, the univariateanalysis indicated a higher mean level of bcl-2 relativeto that of p53 at GD 8:12 and equivalent levels at GD9:0. The relationship between these two genes is sugges-tive of a shortened cell cycle. However, the overexpres-sion of Wee-1, a gene that negatively regulates entryinto mitosis and may antagonize any of the p53/bcl-2effects, was also observed. A high expression of Wee-1,which has been demonstrated to delay proliferation andultimately lead to apoptosis, could be a symptom of yetanother molecular pathway, such as the CAMs, that isdisrupted in the Splotch embryos and whose alteredfunction may induce aberrant neurulation. Taken to-gether, the shift of p53 from the numerator in the GD8:12 to the denominator in the GD 9:0 PCA ratio, as wellas the equivalent levels of these two genes at thisgrowth period may suggest cellular efforts to maintainthe growth phase until NTC is complete. The signifi-cant difference (P , 0.05) between the 1/1 and Sp/Spdistributions for this ratio at GD 9:0 implies that theSp/Sp embryos are not unified in terms of this expres-sion pattern, which agrees with the theory of a length-ened cell cycle leading to NTC delay in a subset of theseembryos.


The mitotic inhibitor Wee-1 encodes a protein kinasethat negatively regulates cell entry into mitosis. Theoverexpression of this gene has been shown to inhibitthe cellular transition from G2 to mitosis, which canultimately lead to cell death (Davey and Beach, ’95;Igarashi et al., ’91). Wee1 was found to be important inthe GD 8:12 PCA, and thus may represent a biologicalcounterbalance to the combined regulatory effects ofp53 and bcl-2 at G1. As stated above, the reciprocaleffects of p53 and bcl-2 dominated the GD 9:0 PCA,while the effect of Wee-1 diminished dramatically. Takentogether, these data suggest that the low level of Wee-1among the GD 9:0 1/1 embryos reflects the minimaloperation of its protein product at G2 to allow swiftprogression through the cell cycle, thus complementingthe growth-inducing counterbalance of the bcl-2 andp53 gene products at G1.

Simultaneous examination of this data via PCAargues against the induction of neuroepithelial growtharrest and/or apoptosis as a mechanism responsible forthe observed NTDs. The counteractive effects of thesesix genes suggest a cooperative effort to regulate rapidcycling of the neuroepithelial cells, to facilitate theproliferative status required during active NTC, as wellas to facilitate successful emigration of the neural crestcells in the wildtype embryos. Disruption of the afore-mentioned ratio associations, as seen across Splotchgenotypes, may lead to the observed phenotypic effects,including delay of NTC.

The results of the present study serve to illustratethe importance of the extracellular environment to cellmigration, and specifically, the role of the adhesionmolecules N-cam and N-Cad on NTC. Using RT/aRNAapproaches to examine coordinate gene expression inthe murine neural tube between gestational days 8:12and 10:00, it became clear that the observed alterationin Pax-3 expression in homozygous Splotch embryoswas consistent with the pattern of altered gene expres-sion that was observed among highly inbred SWVembryos that were exposed in utero to concentrations ofsodium arsenate that induced exencephaly (Wlodarc-zyk et al., ’96). This specific alteration in gene expres-sion leading to the pathogenesis of a NTD in bothspontaneous and teratogen-induced models of NTDs ishighly suggestive of a broad, common mechanism ofaction. It is clear that the precise regulation of bothN-cam and N-Cad during the period of NTC is criticalto the successful completion of this crucial morphoge-netic process and warrants further experimental consid-eration.

ACKNOWLEDGMENTS

This work is supported in part by research grantsES01365 and DE11303 from the National Institute ofEnvironmental Health Sciences, NIH, and the NationalInstitute of Dental Research. Its contents are solely theresponsibility of the authors and do not necessarily

represent the official views of the NIEHS, NIH. Dr. Anwas supported in part by the Jeane B. Kempner Schol-arship from the University of Texas Medical Branch atGalveston. The authors express their appreciation toDr. Bogdan Wlodarczyk and Ms. Laurie Cook, Ms.Michele Davda, and Ms. Amanda Lightfoot for theirtechnical assistance and for the well-being of the mice.We thank Dr. Daphne Trasler for providing primers forthe PCR determinations of the Splotch genotypes. Theexcellent clerical assistance of Ms. Jennifer Wormuth isalso sincerely appreciated. We thank the followingindividuals for providing cDNA clones: F. Ruddle, D.Epstein, R. Harland, T. Lufkin, A. Brown, S. Miyatani,M. Wrzos, A. McMahon, and J. Eberwine.

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neurulation abnormalities secondary to altered gene expression in neural tube defect susceptible...

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