Analysis of neural tube defects in a mouse mutant using whole embryo culture

Toxic. in Vitro Vol. 7, No. 6, pp. 679~84, 1993 Printed in Great Britain.All rights reserved

0887-2333/93$6.00+ 0.00 Copyright © 1993PergamonPress Ltd

Session 1" Developmental Aspects ANALYSIS OF N E U R A L T U B E D E F E C T S IN A M O U S E MUTANT USING WHOLE EMBRYO CULTURE A. J. COPP*, A. S. W . SHUM t and F. A. BROOK:~ *Division of Cell and Molecular Biology, Instituteof Child Health, University of London, 30 Guildford Street,London W C I N IEH, tWellcome/CRC Institute,Univcrsity of Cambridge, Tennis Court Road, Cambridge CB2 IQR and :~ImperialCanccr Rcscarch Fund, Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OXI 3PS, U K

Abstract--The method of whole embryo culture permits a variety of experimental manipulations to be performed on the mammalian embryo. When used in conjunction with mouse mutants, this technique can provide information on the pathogenetic mechanisms underlying the development of birth defects. To illustrate this approach, we review in vitro studies on the development of embryos homozygous for the mutation curly tail (ct). These studies have involved making repeated observations on individual embryos, performing surgical manipulations, applying environmental influencesand metabolic labelling. As a result of this work, we have now partially elucidated the developmental sequence of events that precedes the appearance of spina bifida in the ct mutant.

Introduction

of teratogen-induced defects may be extremely complex and difficult to unravel. A large number of mouse mutations, which disrupt a wide variety of developmental systems, have been described (Green, 1989). In this review, we concentrate on mutations that produce NTD (Copp et al., 1990); a list of these mutant genes is shown in Table 1. It is noticeable that the mutations vary in the level of the lesion: some cause exencephaly (the developmental precursor of anencephaly), others produce spina bifida and one mutant gene ( L p ) causes the severe condition cranio-rachischisis, in which almost the entire neural tube remains open. This spectrum of conditions resembles the range of pathological types of NTD seen in humans (Seller, 1987), providing further support for the idea that the human population may harbour mutant genes that are homologous to a number of the mouse mutations, as already demonstrated for Sp.

Birth defects continue to be a major cause of spontaneous abortion, death in the perinatal period and handicap in childhood. Most major birth defects develop during the first few weeks of gestation, when the human embryo is relatively inaccessible for study. It is to animal model systems, therefore, that we must turn for information on the mechanisms of birth defect development. Mouse mutants as models of birth defects

Birth defects can arise in animals either as a result of the action of mutant genes or environmental teratogens, or both. We are studying the embryonic development of birth defects in mouse genetic mutants for the following reasons: first, cloning of the mutant genes will offer the possibility of relating the animal model directly to the human situation, through the identification of human genetic homologues. This has recently been achieved, for instance, in the case of splotch (Sp), a mouse mutation that causes neural tube defects (NTD) and other neural crest-related defects (Epstein et al., 1991). Sp is the mouse homologue of human Waardenburg type I syndrome (Baldwin et al., 1992; Tassabehji et al., 1992). The second reason for choosing genetic models is that it may ultimately be possible to relate the developmental defect to an abnormality in a single species of protein, the product of the mutant gene. Since environmental teratogens frequently have multiple cellular effects, the molecular pathogenesis AbbrevhTtion: NTD = neural tube defect.

Embryo culture: an opportunity to test developmental hypotheses

Most studies of developmental phenomena begin with a description of the system, for instance in terms of its morphology, biochemistry or gene expression pattern. Descriptive studies can lead to the formulation of hypotheses about the mechanisms that underlie the developmental event. To test these hypotheses, however, it is necessary to manipulate the developing embryo. Although this is possible with embryos developing in utero (Papaioannou, 1990), or in the 'exo utero' system (Muneoka et al., 1990), manipulations are much more readily performed in vitro, using the method of whole embryo culture.

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A.J. COPP et al. Table I. Mouse mutations that produce neural tube defects Mutation Gene symbol* Chromosomet Cranialdefect Spinaldefect Loop-tail Lp 1 Cranio-rachischisis Cranioschisis crn ND Exencephaly -Exencephaly xn ND Exencephaly -Extra toes Xt, Xt ~ph 13 Exencephaly -Rib fusions Rf ND Exencephaly -Bent tail Bn X Exencephaly Spina bifida Curly tail ct ND Exencephaly Spina bifida Splotch Sp, Sp a 1 Exencephaly Spina bifida Curtailed Tc 17 -Spina bifida Vacuolated lens vl 1 -Spina bifida *In the case of extra toes and splotch, more than one allele has been described. tND indicates genes that have not yet been mapped to a chromosome.

Table 2 shows some types of descriptive and experimental study that can be performed with cultured embryos. In the remainder of this paper, we will describe the ways in which we have used embryo culture to answer a number of questions regarding the development of spina bifida and other spinal N T D in one mouse mutant, curly tail ( c t ) . These experimental studies have led to the formulation of a 'chain of events' leading from a cellular abnormality, namely reduced proliferation of the notochord and gut endoderm, through an abnormality of curvature of the body axis, to development of spinal N T D .

Delayed closure of the posterior neuropore leads to spina bifida The curly tail mutant was first described in 1954 by Grfineberg. Homozygotes develop a tail defect (40% of cases), or spina bifida with a tail defect (20% of cases), or are morphologically normal (40% of cases). Grfineberg (1954) noted that the incidence of spinal N T D during late gestation in ct litters was similar to the incidence of delayed neuropore closure at mid-gestation. We confirmed this observation (Copp e t al., 1982) and set out to determine whether delayed neuropore closure is causally related to development of spinal N T D . Embryos were explanted from ct litters at 10.5 days of gestation, keeping the yolk sac and amnion intact. Neuropore size was measured through a small window created in a non-vascular region of the yolk sac and the embryos were then cultured for 32-36 hr, until the presence of spina bifida or a tail defect could be discerned. There was a strong correlation between the size of the neuropore at the start of culture and the subsequent development of spinal N T D (Copp, 1985). Mutant embryos with normal sized neuropores (Fig. la) developed normally in 63% of cases and developed tail defects

in 37% of cases; these embryos did not develop spina bifida. In contrast, mutant embryos with the largest posterior neuropores (Fig. lb) developed normally only in 7% of cases; 35% developed spina bifida with tail defects and 58% developed tail defects alone. Thus, the two types of N T D , spina bifida and tail defects, can be seen to correlate with the same embryonic abnormality, delay in neuropore closure, and differ only in the severity of this abnormality. To test whether the delay in neuropore closure is causally related to development of spinal N T D , we enlarged the neuropore experimentally by incising the roof plate of the most recently formed neural tube. Again, embryos were cultured for 36 hr and then examined for spinal N T D . Enlargement of the neuropore of non-mutant embryos led to the development of tail defects in 38% of cases, compared with 0% for unoperated controls (Fig. lc, d). The tail defects in these non-mutant embryos in which the neuropore was experimentally enlarged closely resembled those seen in c t / c t embryos (Copp, 1985). Enlargement of the neuropore in c t / c t mutant embryos (from the size seen in Fig. la to that in Fig lb) produced a marked increase in the incidence of spina bifida (Fig. le). This experiment demonstrates that delayed neuropore closure is the immediate developmental precursor of spinal N T D in the c t mouse mutant.

Curvature of the body axis leads to delayed neuropore closure Disturbance of curvature of the body axis has been suggested to affect the rate of neurulation in the cranial region of the mouse embryo (Jacobson and Tam, 1982). To determine whether such a mechanism operates in the caudal region of the c t mutant, we measured the angle of curvature of

Table 2. Experimental analysis of embryonic mechanisms in whole embryo culture Types of study using cultured embryos Examplesin this review Repeated observations Prospective study of neuropore size Surgical interventions Enlargement of posterior neuropore Insertion of eyelash tip into hindgut lumen Environmental effects Effect of hyperthermia on neuropore size Metabolic labelling Incorporation of [3H]glucosamine

Embryo culture and mouse mutant analysis the body axis at the level of the posterior neuropore. This study revealed a correlation between the angle of curvature and size of the posterior neuropore: embryos with large neuropores (destined to develop a high incidence of spina bifida, see Fig. 1) had a markedly greater angle of curvature than embryos with normal sized neuropores (Brook et al., 1991). To test whether this correlation represents a cause-and-effect relationship, we surgically inserted the tip of a human eyelash into the lumen of the hindgut of ct/ct embryos, in order to keep the posterior neuropore region straight during subsequent development. Embryos were explanted at 9.5 days of gestation, the eyelash tips were inserted, and they were then cultured for approximately 20 hr, until they reached the 27-29 somite stage, at which time neuropore length was measured (Brook et al., 1991). Embryos that received eyelash implants were found to develop significantly less curvature of the neuropore region than embryos that did not receive an eyelash (Fig. 2a). Moreover, neuropore closure was enhanced in embryos with eyelash implants: the mean length of the neuropore was significantly reduced in comparison with control embryos that were not operated on, or that had mock insertions of an

681

eyelash (Fig. 2b). This experiment demonstrates that delayed closure of the posterior neuropore in the ct mutant is the direct result of enhanced curvature of the body axis. A defect of cell proliferation leads to delayed neuropore closure The neurulation stage mouse embryo grows at a rapid rate, with a neuroepithelial cell cycle length of 8.5 hr at 9.5 days of gestation (Kauffman, 1968). To determine whether a disturbance of cell proliferation rate may underlie the development of NTD in the ct mutant, we compared the length of the cell cycle in the neuroepithelium, surface ectoderm, mesoderm, notochord and hindgut endoderm in ct/ct embryos with large and small neuropores. This analysis revealed that the notochord and hindgut endoderm proliferate significantly more slowly, with an approximate 25% increase in cell cycle length, in embryos with large neuropores than in those with small neuropores ( C o p p e t al., 1988a). In contrast, there was no difference in the rate of proliferation of the neuroepithelium, surface ectoderm or mesoderm between the two types of embryo. %

%

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Fig. I. Delay in closure of the posterior neuropore leads to the development of spinal neural tube defects. Each part of the Figure (a-e) depicts the developmental outcome in terms of the percentage of embryos developing spina bifida or tail flexion defects, following whole embryo culture for 32-36 hr. Numbers above the arrows indicate the number of embryos studied. (a) ct/ctembryos with normal sized neuropores at the start of culture. (b) ct/ctembryos with abnormally large neuropores. (c) Non-mutant not operated upon. (d) Non-mutant embryos in which the posterior neuropore was enlarged surgically before culture. (e) ct/ctembryos with normal sized neuropores, in which the neuropore was enlarged surgically before culture. Data are taken from Copp, JournalofEmbryologyandExperimentalMorphology1985,88, 39-54.

682

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Mock-operated(n=23) Eyelashinserted(n=21)/

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gestation and continued for approximately 18 hr, the embryos cultured under hyperthermic conditions contain 13% less DNA than controls maintained at the normal temperature. No structural abnormalities were noted in the growth retarded embryos. Cell cycle analysis in these embryos showed that the rate of cell proliferation is reduced in all tissues by hyperthermia, but that the greatest reduction is in the neuroepithelium (Copp et al., 1988b). Thus, the effect of growth retardation in vitro is to re-balance tissue growth rates within affected ct/ct embryos. The effect of growth retardation on posterior neuropore closure was to decrease the proportion of embryos with enlarged neuropores and increase the proportion with normal sized neuropores (Fig. 3). Indeed, some embryos in the growth retarded group underwent premature closure of the neuropore (Copp et al., 1988b). We conclude that the cell proliferation imbalance is a causal factor in the development of spinal NTD in the ct mutant. Since the affected tissues, the notechord and hindgut endoderm, are situated ventrally within the posterior neuropore region, and are firmly attached to the overlying neuroepithelium by extracellular matrix, it seems likely that the growth imbalance is responsible for generating enhanced ventral curvature of the body axis, which in turn causes delay in neuropore closure (Fig. 4). An extracellular matrix defect in

0 Closed Small MediumLarge Posterior neuropore size

Fig. 2. Experimental reduction in the angle of curvature of the body axis prevents the development of spinal NTD in ct/ct mouse embryos. (a) Diagrams showing the appearance of the caudal region of ct/ct embryos following 20 hr of culture either with (top) or without (bottom) the insertion of an eyelash tip into the lumen of the hindgut prior to culture. The eyelash tip (dotted line) acts as a splint to maintain the caudal region straight. Embryos subjected to mock operations (bottom), in which the eyelash was inserted and then immediately withdrawn, develop marked ventral curvature. Embryos with eyelash inserts have smaller neuropores after culture than those without. Abbreviations: hg, hindgut; nt, neural tube; pn, posterior neuropore. (b) Distribution of posterior neuropore lengths following culture in embryos that received an eyelash tip ( I ) and in embryos on which mock operations were performed ([]). There is a shift in the distribution towards normal sized neuropores in the embryos with eyelash inserts. Data are taken from Brook et al., Development 1991, 113, 671-678.

We hypothesized that an imbalance of cell proliferation rates may be responsible for causing the delay in neuropore closure observed in ct/ct embryos. To test this idea, we attempted to re-balance the growth rates within the neuropore region by slowing the growth of the whole embryo. It had been shown previously that mild hyperthermia (40.5°C v. 38°C) produces growth retardation in cultured rat embryos (Cockroft and New, 1975). We found that when ct/ct mouse embryo cultures are initiated at 9.5 days of

ct/ct

embryos

In an attempt to define the molecular basis of the ct defect, we examined the molecular composition of the extracellular matrix i n embryos at the stage of

60

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Fig. 3. Effect of hyperthermia on the size of the posterior neuropore in cultured ct/ct embryos. Embryos were cultured either at 38 or 40.5°C, for approximately 18 hr, until they had 27-29 somites. Embryos cultured at 38°C had predominantly medium sized neuropores after culture, whereas embryos cultured at 40.5°C, although containing 13% less DNA, had predominantly small sized neuropores, indicating a normalizing effect of hyperthermia. Data are taken from Coppet al., Development 1988, 104, 297 303.

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Embryo culture and mouse mutant analysis ct gene

ia unknown molecular mechanism =b )

reduced cell proliferation in notochord and hindgut endoderm

ic i

ventral curvature of the body axis

delayed closure of posterior neuropore ('-

,L,

spina bifida or tail flexion defect (

Fig. 4. The authors' current view of the sequence of pathogenetic events leading to the development of spinal NTD in ct/ct mouse embryos. Solid arrows indicate cause-and-effect relationships for which experimental evidence has been obtained, as detailed in the text. Dashed arrows indicate hypothetical relationships. Our experiments have provided evidence to support steps d, e and f in the sequence. The relationship between the cell proliferation defect and ventral curvature of the body axis (step c) seems likely but is, as yet, unproven. The molecular basis of the relationship between the ct gene and the cell proliferation defect (steps a and b) are unknown although, as described in the text, a possible role for the extracellular matrix molecule hyaluronan has been suggested. posterior neuropore closure. Previous studies had suggested that glycoconjugates in the extracellular matrix may play an important role in neurulation (Morriss and Solursh, 1978). Curly tail embryos were labelled in vitro with [3H]glucosamine, a metabolic precursor of a wide range of proteoglycans and glycoproteins. Labelled glycoconjugates were separated by ion-exchange chromatography. The only abnormality revealed by this analysis was a reduced accumulation of [3H]hyaluronan in ct/ct embryos with large neuropores (Copp and Bernfield, 1988a). This molecular abnormality was confined to the posterior neuropore region, where [3H]hyaluronan is the predominant glycoconjugate (Copp and Bernfield, 1988b), and was not seen in more cranially situated regions, where sulfated glycoconjugates are most abundant. To ascertain the spatial distribution of the [3H]hyaluronan, we prepared histological sections of labelled ct/ct embryos with large and small neuropores and subjected the sections to autoradiography, with or without pre-digestion with the hyaluronan-specific degrading enzyme Streptomyces spp. hyaluronidase. This analysis showed that, in embryos with small neuropores, [3H]hyaluronan accumulates in large quantities at the basal surface of the neuroepithelium and around the notochord. In contrast, embryos with large neuropores exhibited a marked reduction in the accumulation of [3H]hyaluronan at this site. We conclude that there is a temporal and spatial correlation between reduced

accumulation of [3H]hyaluronan and the cell proliferation defect in affected ct/ct embryos.

Conclusion

We have used the method of whole embryo culture to investigate the pathogenetic mechanism that underlies the development of spinal NTD in the ct mouse mutant. This series of studies illustrates the many applications of whole embryo culture for the analysis of developmental mechanisms. Repeated observations can be made on individual embryos, surgical interventions can be performed, environmental influences can be applied and radio-isotopic labelling can be carried out. To this list could be added the administration of other molecules such as antibodies or antisense oligonucleotides for further molecular analysis of developmental events. Clearly, as a result of embryo culture, the mammalian embryo is now available for the types of analysis that have previously been possible, among the vertebrates, only with avian and amphibian embryos. An additional advantage of the mouse embryo is its wealth of developmental mutants. The application of embryo culture analysis to mutant systems promises considerable advances in future in our understanding of the mechanisms of origin of birth defects, and may also indicate ways in which birth defects can be prevented.

Acknowledgement--The authors gratefully acknowledgethe financial support of the Imperial Cancer Research Fund for the original research described in this review. REFERENCES

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