Role of Differential Cell Proliferation in the Tail Budin Aberrant Mouse NeurulationMARIAN C.E. PEETERS,1* BERT SCHUTTE,2 MARIE-HELENE J.N. LENDERS,1 JOHAN W.M. HEKKING,1JAN DRUKKER,1 AND HENNY W.M. VAN STRAATEN1

1Department of Anatomy and Embryology, University of Maastricht, Netherlands2Department of Molecular Cell Biology & Genetics, University of Maastricht, Netherlands

ABSTRACT In the mouse mutant curly tail,the phenotypes spina bifida and curled tail resultfrom a delay in closure of the posterior neuro-pore (PNP). At the developmental stage when thisdelay can first be recognized, the caudal region ofthe embryo demonstrates a transiently enhancedcurvature of the body axis which likely inhibitselevation, convergence, and fusion of the neuralfolds. The enhanced curvature is thought to bethe result of a decreased proliferation in theventrally located gut endoderm and notochord,together with a normal proliferation of the over-lying neuroepithelium of the PNP. However, theproliferation defect and the enhanced curvaturewere originally demonstrated at the same devel-opmental stage, while it is expected that reducedproliferation should precede enhanced curva-ture and delayed PNP closure. The caudal regionoriginates from the tail bud and we thereforepropose that the enhanced curvature is inducedby a disturbed dorso-ventral proliferation pat-tern in the tail bud. Using flow cytometry, prolif-eration patterns were determined separately forthe dorsal and ventral halves of the tail bud ofcurly tail and of control embryos as well as ofrecombinant embryos having the curly tail pheno-type with a genetic background which is matchedto the BALB/c control strain. In general, it ap-peared that about half of the cell cycle durationin tail bud cells was occupied by S phase, about40% by G0/G1 and the rest by G2/M. For the controlembryos, no dorso-ventral differences in relativephase duration were demonstrated. However,curly tail and recombinant embryos at the 21–25somite stage, prior to the onset of enhancedcurvature, exhibited ventrally a higher propor-tion of G0/G1 phase cells than dorsally, and acomplementary relationship for S phase cells. Weinterpret these observations as indicating a pro-longed G1 phase at the ventral side of the tail bud,resulting in a prolongation of the cell cycle andthus a decreased proliferation. In 26–30 somitestage embryos, prior to the normalization of cur-vature in curly tail embryos, the dorso-ventralproliferation balance was re-established. We con-clude that a reduced proliferation in the ventralpart of the tail bud of the curly tail embryo

precedes both the onset of enhanced curvatureand the previously observed reduction in prolif-eration of the hindgut and notochord, and is alikely candidate for an early event in the pathoge-netic sequence leading to the curly tail pheno-type. Dev. Dyn. 1998;211:382-389.r 1998 Wiley-Liss, Inc.

Key words: tail bud; curly tail; neurulation;mouse embryo; posterior neuropore;flow cytometry

INTRODUCTION

Neural tube defects (NTD), including spina bifida,are the result of multifactorial disturbances of thedevelopmental process of neurulation. The aetiologicaland pathogenetic factors underlying NTD can be eluci-dated by studying mutant mouse strains in which oneor more factors interfere directly or indirectly with theformation of the neural tube. One such mouse mutant isthe curly tail which was first described by Gruneberg(1954). Among curly tail mice, in our stock the frequen-cies of the phenotypes are 52% curled tail, 8% spinabifida along with curled tail, 0.07% spina bifida alongwith straight tail, and 40% straight tail; anencephalyhas not been observed. Both the curled tail and spinabifida are the likely result of a delay in closure of theposterior neuropore (PNP). While in nonmutant mouseembryos the PNP decreases rapidly in size and closes atthe 30 somite stage, the delayed closure in curly tailembryos can be recognized from the 25 somite stageonwards by an abnormal sized PNP and which remainsopen beyond the 30 somite stage (Copp, 1985; VanStraaten et al., 1992). It has been shown that ‘affected’curly tail embryos, with such a delay in PNP closure,exhibit an enhanced axial curvature of the caudalregion at the 27–29 somite stage compared to theirunaffected littermates (Brook et al., 1991). This en-hanced curvature is proposed to cause the delay inclosure by counteracting elevation, convergence, and

Grant sponsor: Netherlands Organisation for Scientific Research;Grant number: 431241 (M.C.E.P.).

*Correspondence to: M.C.E. Peeters, Department of Anatomy andEmbryology, Faculty of Medicine, University of Maastricht, P.O. Box616, NL-6200 MD Maastricht, Netherlands.E-mail: M.Peeters@ae.unimaas.nl

Received 3 October 1997; Accepted 29 December 1997

DEVELOPMENTAL DYNAMICS 211:382–389 (1998)

r 1998 WILEY-LISS, INC.

fusion of the neural folds. This hypothesis was con-firmed by studies in which experimentally alteredcurvature was shown to modulate PNP closure rate(Brook et al., 1991; Van Straaten et al., 1993; Peeters etal., 1996). In a more detailed study, in which curvature wasdescribed along the craniocaudal axis at several develop-mental stages, we found that the enhanced curvature ofthe PNP region in affected curly tail embryos is a tempo-rary phenomenon: affected curly tail embryos with28–31 somites exhibit an enhanced curvature, from thelevel of somite 26 onwards, whereas curvature normal-izes at the 32–34 somite stage (Peeters et al., 1997).

Axial curvature may be determined by differences ingrowth rates at the ventral and dorsal side of the body axis.Growth is the resultant of variations in the components cellproliferation, cell volume, cell death, cell migration, andvolume of the extracellular matrix. A cell proliferationdefect has been demonstrated in affected curly tail em-bryos: proliferation of hindgut endoderm and noto-chordal mesoderm appeared to be reduced, while prolif-eration of the neuroepithelium appeared normal, whencompared to unaffected curly tail embryos (Copp et al.,1988). It was therefore hypothesized that the reducedproliferation of the ventrally located tissues results inan imbalance of growth which causes an enhanced axialcurvature of the caudal region (Brook et al., 1991).However, proliferation was determined in that studyonly at the stage at which the aberrant curvaturebecomes manifest (i.e., 27–29 somites), while it mightbe predicted that aberrant curvature should be pre-ceded by reduced ventral proliferation.

The caudal region of the embryo originates from thetail bud, which is the continuation of the node andprimitive streak. The tail bud is a dense mass of cellslocated at the posterior end of the embryo and its cellshave the potential to give rise to the variety of tissues inthe caudal region (Schoenwolf, 1977; Tam, 1984; Grif-fith et al., 1992; Catala et al., 1995; Kanki and Ho,1997). We therefore propose that enhanced axial curva-ture of the caudal region in curly tail embryos arises asa consequence of earlier dorso-ventral differentialgrowth in the tail bud. Moreover, the subsequentnormalization of curvature likewise may be preceded bya rebalancing of growth in the tail bud.

In order to determine whether differential growth in thetail bud is indeed present and is correlated with axialcurvature of the PNP region, proliferation patterns (i.e.,relative cell cycle phase durations) were determined for thedorsal and ventral halves of the tail bud at the developmen-tal stage immediately preceding onset of enhanced curva-ture and at the subsequent stage that precedes re-establishment of normal curvature. The proliferationpatterns were determined in curly tail and nonmutant(BALB/c and CBA/J) embryos. Because the curly tailstrain differs in genetic background from all existinginbred strains, a BALB/c-curly tail recombinant strain,which possesses the curly tail characteristics on aBALB/c genetic background, was included. By this,

differences between the recombinant strain and thecontrol can be ascribed to curly tail specifically.

RESULTSProliferation Pattern in the Tail Budat the Developmental Stage Prior to Onsetof Enhanced Curvature

Proliferation pattern in the tail bud was determinedseparately for dorsal and ventral halves, which wereisolated as shown in Figure 1. In general, for youngembryos (21–25 somites) of all strains of mice it ap-peared that approximately half of the nuclei in thedorsal tail bud were in S phase, one-third of the nucleiwere in G0/G1 phase and less than 15% were in G2/Mphase (Table 1).

When the proliferation patterns were comparedwithin embryos an interesting strain difference emerged(Fig. 2). In the nonmutant strains, there was an approxi-mately even distribution of embryos with dorsal-dominance and those with ventral-dominance for eachcell cycle phase. Thus, five out of 14 BALB/c embryoshad a higher proportion of nuclei in S phase dorsallythan ventrally. For the G0/G1 six out of 14 exhibiteddorsal-dominance, and for G2/M 10 out of 14 embryosshowed dorsal-dominance. These dorso-ventral distribu-tions are not significantly different from a 50/50 distri-bution, indicating that the proliferation patterns of thedorsal and ventral halves evenly balanced in the BALB/cembryos. A similar finding was obtained from theCBA/J embryos. In contrast, proliferation patternsdiffered markedly between the dorsal and ventral halvesin the mutant (curly tail and recombinant-2) embryos.All 15 curly tail embryos exhibited a higher proportionof nuclei in S phase dorsally than ventrally (P ,0.0001), and, conversely, only one out of 15 had a higherproportion in G0/G1 dorsally than ventrally (P 5 0.001).An identical tendency was seen in recombinant-2 em-bryos; 15 out of 16 had a higher S proportion dorsallythan ventrally (P 5 0.0005), and all 16 exhibited a

Fig. 1. A 27 somite stage curly tail embryo of which the tail bud is splitinto a dorsal (d) and ventral (v) half. Dotted lines indicate the position ofthe transverse cut for isolating the tail bud halves. This example showing asimilar amount of tissue in the dorsal and ventral halves is representativefor the isolations made. n, Right lateral fold of the neuropore.

383DIFFERENTIAL PROLIFERATION IN THE MOUSE TAIL BUD

lower G0/G1 proportion dorsally than ventrally (P ,0.0001). In both mutant strains, the G2/M proportionswere similar in the dorsal and ventral halves of the tailbud. Thus, whereas the proliferation patterns in controlembryos were balanced between the dorsal and ventralhalves of the tail bud, a dorsal preference for S togetherwith a ventral preference for G0/G1 was demonstratedin the mutant embryos.

Proliferation Pattern in the Tail Budat the Developmental Stage Priorto Re-Establishment of Normal Curvature

As with embryos at the earlier developmental stage,embryos at the 26–30 somite stage also had about half

of their nuclei in S phase, about one-third in G0/G1phase and less than 15% in G2/M phase (Table 2).

The dorso-ventral distribution of cell cycle phases innonmutant embryos at this more advanced stage didnot differ from a 50/50 distribution, except for the G0/G1phase in the CBA/J embryos (P 5 0.03; Fig. 2). Incontrast to the findings at the 21–25 somite stage, curlytail and recombinant-2 embryos with 26–30 somitesalso demonstrated an equal proliferation pattern be-tween the dorsal and ventral halves of the tail bud. Theshift from S phase dominance dorsally at the 21–25somite stage to an equal dorso-ventral distribution atthe 26–30 somite stage was statistically significant forboth the curly tail and the recombinant-2 (P , 0.001)

Fig. 2. The proportion of embryos with a dorsal dominance (white) vs.the proportion with a ventral dominance (black) indicated for each cellcycle phase. Length of bar represents all embryos (100%) of a mousestrain at that developmental stage; number of embryos per group are inparentheses. Two mutant (curly tail and recombinant-2) and two nonmu-

tant (BALB/c and CBA/J) mouse strains are used at the developmentalstages prior to onset of enhanced (21–25 somites) and prior to re-establishment of normal (26–30 somites) curvature. Statistically signifi-cant differences are indicated (*).

TABLE 1. Proliferation Patterns in Tail Bud of Embryos at the Stage of 21–25 Somitesa

Mousestrain

Number ofembryos

Dorsal half Ventral half% G0/G1 % S % G2/M % G0/G1 % S % G2/M

curly tail 15 35 (1.5) 51 (1.8) 14 (0.9) 41 (0.6) 45 (0.7) 14 (0.7)recomb.-2 16 36 (1.2) 51 (1.8) 13 (1.1) 41 (1.1) 46 (1.5) 13 (0.7)BALB/c 14 34 (0.8) 55 (1.4) 11 (0.9) 34 (1.3) 56 (1.8) 9 (1.0)CBA/J 16 36 (1.2) 49 (1.2) 15 (0.7) 38 (1.3) 47 (1.8) 15 (0.8)

aFigures are the number of embryos used, the mean (and SEM) of the proportions for each cell cyclephase.

384 PEETERS ET AL.

embryos. A complementary shift occurred for the G0/G1phase dominance in the ventral tail bud for both (P ,0.005) mutant strains.

When comparing the proliferation patterns of thenonmutant strains of mice at the two developmentalstages, several consistent differences could be noted,although the magnitude of the differences were small(Tables 1 and 2). The proportion of nuclei in G0/G1 wasincreased at the older stages, although not significantlyfor the ventral tail bud of BALB/c embryos. The de-crease in proportion of nuclei in S was consistent,although not statistically significant.

DISCUSSION

We have shown that cells of the tail bud of mouseembryos, at the developmental stages during which thePNP closes, have a cell cycle of which half is occupied byS phase, about one-third by G0/G1, and the rest by G2/M.For nonmutant embryos no dorso-ventral difference inproliferation patterns could be demonstrated. However,both mutant mouse strains (curly tail and recombi-nant-2) showed, at the stage prior to the onset ofenhanced curvature, a ventral dominance for G0/G1phase together with a dorsal dominance for S phase.These proliferation patterns were no longer evident atthe later developmental stage studied, just prior tore-establishment of normal curvature. In the nonmu-tant mouse strains, the contribution of the G0/G1 phaseshowed a tendency to increase during development.

Changes in the Proliferation Pattern of the TailBud During Development

The proliferation pattern in the tail bud of the mouseembryo was determined using flow cytometry on cellsisolated from separated dorsal and ventral halves of thetail bud. The percentages of nuclei in G0/G1, S and G2/Mwere determined as a reflection of their contribution tothe total cell cycle duration. The method used in thisstudy has been described by Vindeløv (1983b) andproduces histograms of sufficient resolution for cellcycle analysis (mean coefficients of variation of 5–6%for the G0/G1 peak; Vindeløv et al., 1983a).

Proliferation characteristics of embryonic cells, par-ticularly in the rodent neural tube, have previouslybeen determined by the measurement of incorporationof 3H-thymidine or BrdU. These studies describe an Sphase duration of about 5 hours in neuroepithelial cells,which corresponds to approximately 50% of total cell

cycle duration, in good agreement with our findings.Data from the previous studies on the length of G0/G1and G2/M phases show more variation (Kauffman,1968; Wilson and Center, 1974; Peters and De Geus,1981; Wilson, 1982). Flow cytometry has previouslybeen used to determine proliferation patterns of com-plete rat embryos at the stage of about 20 somites(Rogers et al., 1995). The pool of various cell typesshowed an overall proliferation pattern of 38% G0/G1,49% S, and 13% G2/M phase, which is almost identicalto the proliferation pattern of the mouse tail bud asobserved in the present study.

During development, the contribution of cells inG0/G1 phase in nonmutant mouse embryos increased,while the contribution of S phase tended to decrease.This is in agreement with the results of incorporationstudies in neural tissue, which showed that the contri-bution of G0/G1 increased from about 2 hr on day 10(22% of total cell cycle duration) to 9.3 hr on day 14(62%), while S phase decreased from 6.4 hr on day 7(.80%) to about 5 hr on day 10 (55%), and subsequentlyto 3.8 hr on day 14 (25%) (Kauffman, 1968; Wilson andCenter, 1974; Poelmann, 1980; Peters and De Geus,1981; Wilson, 1982; Mirkes et al., 1989; Takahashi etal., 1993). The largest change was an increase in G1and, as a consequence, total cell cycle duration in-creased. The lengthening of G1, and of the total cellcycle, are likely related to the initiation of cell differen-tiation. This increase of G0/G1 during differentiationhas also been corroborated in studies using flow cytom-etry: the proportion of cells in G0/G1 phase was low(about 25%) in gastrulating embryos (MacAuley et al.,1993) and over 50% in more differentiated tissue of thefore limb bud (Francis et al., 1990). We have nowdemonstrated that such a shift in the proliferationpattern also occurs in tail buds of two closely relatedstages.

Incorporation studies have the disadvantage of therelatively extended time period necessary for incorpora-tion of thymidine or its analog BrdU into DNA. As theembryo continues to develop during the labelling pe-riod, it is difficult to determine proliferation differencesbetween closely-spaced developmental stages. Flow cy-tometry does not suffer from this disadvantage andproliferation patterns can be established at any develop-mental stage. Our study of proliferation in the tail budagrees closely with previously reported proliferationpatterns of mouse and rat embryos at comparable

TABLE 2. Proliferation Patterns in Tail Bud of Embryos at the Stage of 26–30 Somitesa

Mousestrain

Number ofembryos

Dorsal half Ventral half% G0/G1 % S % G2/M % G0/G1 % S % G2/M

curly tail 21 38 (0.8) 48 (1.0) 14 (0.7) 37 (0.9) 49 (1.1) 14 (0.7)recomb.-2 16 39 (1.3) 50 (1.5) 12 (0.7) 38 (1.2) 52 (1.9) 10 (0.9)BALB/c 16 38 (1.3) 51 (1.7) 12 (0.7) 38 (1.3) 51 (1.9) 11 (1.0)CBA/J 18 39 (0.8) 47 (1.2) 14 (0.7) 41 (0.8) 46 (1.0) 13 (0.6)

aFigures are the number of embryos used, the mean (and SEM) of the proportions for each cell cyclephase.

385DIFFERENTIAL PROLIFERATION IN THE MOUSE TAIL BUD

developmental stages and suggests that flow cytometryis a valuable method for cell cycle analysis in the earlymouse embryo.

Proliferation is Reduced in the Ventral Tail Bud

The flow cytometry technique necessitates that allnuclei pass individually through the laser beam. So,nuclei have to be extracted and the position of the cellswithin the tail bud is lost. Therefore, the tail bud wassplit mechanically before analysis, and the prolifera-tion pattern was established for each half separately. Asa consequence, the pool of cells from each half of the tailbud comprised mesoderm as well as ectoderm cells, andlikewise, cells from more caudal and cranial positionswithin the tail bud. This could be important if heteroge-neity of proliferation pattern exists within the tail bud,as has been shown for the tail bud of the zebrafish(Kanki and Ho, 1997). Because in a tail bud the lengthsof the dorsal and ventral halves were identical, compar-ing the proliferation pattern of the dorsal and ventralhalves within each embryo, therefore, tends to rule outpotential complications caused by a possible cranio-caudal gradient in proliferation.

Dorso-ventral comparisons in the mutant embryos atthe stage prior to onset of enhanced curvature revealeda ventral dominance for G0/G1 phase together with adorsal dominance for S phase, while in the nonmutantembryos no dorso-ventral differences were detected.The ventral dominance for G0/G1 and the dorsal domi-nance for S could be the result of several dorso-ventraldifferences, as follows.

(a) Cell loss at a particular phase may occur in one ofthe halves of the tail bud, due to apoptosis. In aprevious proliferation study of curly tail embryos it wasreported that the incidence of cell death was very low inthe caudal region (Copp et al., 1988). For the developingtail of the chick embryo, an incidence of cell death of1–10% has been demonstrated, with dead cells concen-trated mainly at the site of the ventral ectodermal ridgeand at the tip of the tail (Mills and Bellairs, 1989). In apilot study of cell death in curly tail and nonmutantmouse embryos, we only found few scattered dead cellsin the tail bud. Therefore, apoptosis is probably not animportant factor in producing the dorso-ventral differ-ences, although it remains to be determined moreprecisely whether the incidence of cell death is differentfor the dorsal and ventral halves of the tail bud as wellas how cell death in the tail bud is related to phases ofthe cell cycle.

(b) With flow cytometry, no distinction between G0and G1 cells can be made, due to their equal amount ofDNA. However, the growth fraction in the caudal regionof curly tail embryos is nearly 100% (Copp et al., 1988)and this is likely to be the case in nonmutant embryosas well. Therefore, any change in G0/G1 distributionmust be due to changes in G1, and not in G0.

(c) The ventral dominance for G1 phase together withthe dorsal dominance for S phase seems likely to resultfrom significant variation in one of these phases only.

Thus, the G1 phase is longer or the S phase is shorter atthe ventral side of the tail bud when compared to thedorsal side. There are three reasons to think thatprolongation of the G1 phase at the ventral side of thetail bud is the principal alteration in the cell cycle. (1)Previous proliferation data of affected curly tail em-bryos at the stage of 27–29 somites demonstrated aslightly prolonged S phase in the ventrally locatedtissues (gut and notochord) when compared to dorsally(neural plate; Copp et al., 1988), making it less likelythat the S phase in the ventral tail bud half is short-ened.

(2) Neuroepithelial cells of rodent embryos at stagessimilar to the present study have been reported to havea cell cycle duration of about 10 hr, with an S phase ofabout 5 hr (Kauffman, 1968; Wilson and Center, 1974;Peters and De Geus, 1981; Wilson, 1982). Moreover, thevarious cell types in the caudal region of curly tailembryos also exhibit an S phase of about 5 hr (Copp etal., 1988). Accordingly, we suppose that cell cycle dura-tion in the dorsal half of the tail bud is about 10 hr withan S phase of 5.1 hr, and consequently a G0/G1 of 3.5 hrand a G2/M of 1.4 hr. If S and G2/M in the ventral half ofthe tail bud would also take 5.1 resp. 1.4 hr, accordingto Table 1 G0/G1 would take 4.7 hr. As a result, cell cycleduration would be increased to about 11.2 hr, which isin agreement with the cell cycle duration observed inhindgut endoderm of affected curly tail embryos (Coppet al., 1988). Thus, the prolonged cell cycle duration inthe ventral half of the tail bud compared to the dorsalhalf indicates that proliferation is decreased ventrally.

(3) A reduced rate of cell proliferation would result ina reduced DNA content of the caudal region. This hasindeed been demonstrated: at the stage prior to theonset of enhanced curvature, the DNA content of caudalregions in curly tail embryos is 0.93 µg instead of the1.25 µg for BALB/c (Peeters et al., submitted).

Taken together, our findings suggest a reduction inproliferation due to G1 prolongation and increase intotal cell cycle duration in the ventral part of the tailbud in the curly tail mouse embryo in the periodpreceding onset of enhanced axial curvature.

Decreased Ventral Proliferation and EnhancedAxial Curvature

Previous studies of curly tail embryos have demon-strated a dorso-ventral discrepancy in growth betweenhindgut/notochord and the neural plate, which corre-lates spatially and temporally with enhanced curvatureof the body axis (Copp et al., 1988; Brook et al., 1991;Peeters et al., 1997). The present study extends thoseobservations by showing that the tail bud at the 21–25somite stage also exhibits a dorso-ventral proliferationimbalance. The tail bud at this early stage containsprogenitor cells for the hindgut and caudal notochordand for the neural plate at the later 26–30 somite stage,suggesting that a dorso-ventral proliferation discrep-ancy in the tail bud leads, via enhancement of axialcurvature, to failure of neural tube closure. By the

386 PEETERS ET AL.

26–30 somite stage, the balance of growth has beenrestored in the tail bud and, subsequently, normal axialcurvature is re-established. The disturbed proliferationin the tail bud occurs at a stage before affected curly tailembryos can be distinguished from their unaffectedlittermates, indicating that the disturbed proliferationin the tail bud is an early marker of the curly tailpathogenesis.

The recombinant-2 embryos showed an identicalreduction of proliferation in the ventral part of the tailbud. Although the genetic background contains 75%BALB/c, its proliferation pattern resembles that ofcurly tail rather than that of BALB/c. This emphasizesthat it is the curly tail genetic defect specifically whichinfluences the proliferation in the tail bud.

Regulation of Proliferation in the Tail Bud

In order to investigate further the origin of thedorso-ventral discrepancy in proliferation in the tailbud, genes must be identified that exhibit a dorso-ventral gradient of expression in the tail bud. Manygenes which appear to play key roles in caudal develop-ment have been studied recently in curly tail embryosat the developmental stage prior to re-establishment ofnormal curvature. The expression of HNF-3a, HNF-3b,Shh, T, Hox-b1, Fgf-8, Evx-1, and Wnt-5b are allreported to be normal in curly tail embryos, whileexpression of Wnt-5a is reduced in the caudal meso-derm at both stages investigated in the present study(Gofflot et al., 1996, 1997). Moreover, the expression ofthe retinoid acid receptors RAR-b and RAR-g has alsobeen found to be reduced in the curly tail embryo (Chenet al., 1995). Upregulation of RAR-b either by RA orinositol administration results in a normalization ofPNP closure and a reduction of the incidence of spinalNTD in curly tail mice (Seller et al., 1979; Chen et al.,1994, 1995; Greene and Copp, 1997). This indicatesthat RAR-b, RAR-g, and Wnt-5a are among the geneslikely involved in the pathogenesis of the curly tail.However, the relationship between their expressionpattern and the dorso-ventral discrepancy in prolifera-tion in the tail bud still has to be clarified.

In conclusion, the present study has demonstrated acell proliferation imbalance possibly involving lengthen-ing of the cell cycle owing to prolongation of G1 in theventral part of the tail bud of the curly tail mouseembryo at the stage prior to the development of en-hanced axial curvature, which precedes development ofneural tube defects. Like the enhanced curvature, theproliferation imbalance in the tail bud is a transientfeature, with rebalancing at the stage prior to re-establishment of normal axial curvature. The prolifera-tion disturbance of the tail bud is an early feature inmutant embryos of the pathogenetic sequence thatleads to the curly tail phenotype. Genes which deter-mine the dorso-ventral proliferation pattern may in-clude RAR-b, RAR-g, and Wnt-5a.

EXPERIMENTAL PROCEDURESMouse Strains and Embryo Preparation

The curly tail mutation arose spontaneously in afemale of the GFF inbred strain; this female wassubsequently mated with a CBA/Gr male (Gruneberg,1954). The curly tail stock has been derived from thismating and is kept as a closed, random-bred colony. Thenonmutant inbred BALB/c and CBA/J mice were ob-tained commercially (Charles River, Wiga GmbH, Ger-many). The BALB/c-curly tail recombinant strain wasconstructed according to a cross-intercross design.Briefly, curly tail mice were mated with BALB/c mice,followed by an intercross of the heterozygous offspring.The descendants of the intercross bearing a curly tailphenotype (curled or kinked tail, with or without spinabifida) were selected and mated for the second cross-intercross cycle with BALB/c. The curly tail phenotypesof the offspring of the intercross, having a geneticbackground of 75% BALB/c and 25% curly tail, werekept as a separate colony (recombinant-2). Mice re-ceived water and food ad libitum and were maintainedon a light-dark cycle with the dark period 8.00 p.m. to6.00 a.m..

Mice (curly tail, recombinant-2, BALB/c and CBA/J)were paired for mating overnight, after which femaleswere checked for copulation plugs (5 day 0). On day 9,9.5, or 10, females were sacrificed by cervical disloca-tion and the uterine horns were explanted into pre-warmed (37°C) Hanks’ buffer containing 1% penicillin(Gibco, Life Technologies, Breda, The Netherlands), 1%streptomycin (Gibco), and 10% newborn calf serum(Gibco). After removing myometrium, endometrium,Reichert’s membrane, yolk sac, and amnion, appropri-ate embryos were selected for analysis based on theirsomite numbers.

Measurement of Proliferation

Embryos of the developmental stages of 21–25 somites(prior to onset of enhanced curvature) and 26–30 somites(prior to re-establishment of normal curvature) werecollected for determination of the proliferation pattern.Each embryo was washed in PBS. The tail bud was splitinto a dorsal and ventral half (Fig. 1) and, subsequently,both halves were isolated by a transverse cut across thebody axis. Each half was placed in a Falcon vial (F2058,Becton/Dickinson, San Jose, CA) in approximately 50 µlPBS using a glass pipet. Cell cycle parameters weredetermined according to the method of Vindeløv (1983b).Briefly, nuclei were extracted by adding 450 µl 0.003%trypsin (Worthington Biochemical Corporation, Free-hold, NJ) in citrate buffer. During an incubation periodof 15 min at room temperature, the suspension wasgently mixed every 3 min. Next, 450 µl trypsin inhibitorsolution (0.05% trypsin inhibitor [type II-O, Sigma, St.Louis, MO], 0.01% ribonuclease [RNAse, Serva, Heidel-berg, Germany] in citrate buffer, pH 5 7.6) was addedand mixed gently for 15 min. DNA was labeled by theaddition of 375 µl propidium iodide (PI) solution

387DIFFERENTIAL PROLIFERATION IN THE MOUSE TAIL BUD

(0.0415% PI [Calbiochem 537059], 0.116% spermine[Sigma] in citrate buffer, pH 5 7.6). After at least 15min in the dark, the suspension was sieved through a50 µm nylon mesh (Stokvis & Smits, IJmuiden, theNetherlands). Using the eluate, the DNA content pernucleus was established using a FACSORT (BectonDickinson): PI was excited with a single 488 nm Argonlaser and PI fluorescence was detected through a 600nm LP filter. Pulse processing was used to excludedoublets and larger cellular aggregates. A DNA histo-gram was constructed for each half of the tail bud.Based on this histogram, cell cycle analysis (determina-tion of the proportions of nuclei in cell cycle phasesG0/G1, S and G2/M) was performed using the CellFITsoftware (Becton Dickinson). Tail buds with a coeffi-cient of variation (CV) for G0/G1 above 10% wereexcluded; the mean CV for G0/G1 was between 5 and 6%in all groups studied.

The procedure of embryo preparation and tail budisolation required maximal 20 min, which is very shortcompared to the total cell cycle duration and thereforewill hardly affect the proliferation pattern. Cell cyclechanges do not occur after adding the trypsin solutionto the isolated halves of the tail bud and fluorescence ofthe nuclei is reported to be stable for at least 3 hr afteradding the PI (Vindeløv et al., 1983b).

Proliferation Pattern and Statistical Analysis

Data were grouped by mouse strain and developmen-tal stage. Mean proportions for G0/G1, S, and G2/M werecalculated for all groups for the dorsal and ventralhalves of the tail bud. In order to compare the prolifera-tion patterns of the dorsal and ventral tail bud halves,embryos were classified according to whether the propor-tion G0/G1 was highest in the dorsal or in the ventralhalf. A similar analysis was done separately for S phaseand for G2/M. The percentage of embryos with dorsaldominance versus the percentage of embryos withventral dominance, within each group, was analysedusing a sign test. A possible shift in dorsal or ventraldominance from the 21–25 somite stage to the 26–30somite stage was tested using a chi-square test. Thepercentages of nuclei in the various cell cycle phaseswere compared between the two developmental stagesusing MANOVA with G0/G1, S, and G2/M as dependentvariables and developmental stage as factor (Wilk’stest). Differences were considered statistically signifi-cant when P , 0.05.

ACKNOWLEDGMENTS

We acknowledge Andrew J. Copp for critically read-ing the manuscript.

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389DIFFERENTIAL PROLIFERATION IN THE MOUSE TAIL BUD