Gene Name Gene symbol Function C h ro m / NTD" Reference
Activating protein 2 Ap-2 Transcription factor 13 Exencephaly Schorle et a i, 1996, Zhang et al., 1996
Apolipoprotein B apo B Lipid transport 12 Exencephaly’' Deltour et a i, 1995, Homanics et al., 1993
Breast cancer 1 Brcal Zinc finger protein 11 Exencephaly and spina bifida Gov/en et a i, 1996
Cartilage homeoprotein 1 Cart-1 Transcription factor 10 Exencephaly Zhao et al., 1996
connexin 43’ cx43, Gjal Gap junction component 10 Exencephaly E w arteta/., 1997
csk csk Negative regulator or Src
family tyrosine kinases
15 Open neural tube at death Immamoto and Soriano, 1993
Nada et al., 1993 Fibroblast growth factor
receptor 1
Fgfr-1 Growth factor receptor 8 Spina bifida Deng et a i, 1997
Hox-al Hox-al Transcription factor 6 Exencephaly Lufkin et a/., 1991
Hairy and Enhancer of Split homologue-1
Hes-1 Transcription factor 16 Exencephaly Ishibashi et a/., 1995
iumonji^ m i Homology to RBP-2 13 Exencephaly Takeuchi et a i, 1995
MARCKS macs Protein kinase C
substrate 10 Exencephaly’' Stumpo et a l, 1995 MARCKS-related protein F52, Mrp, MacMARCKS Protein kinase C substrate
4 Exencephaly and spina bifida Chen et a l, 1996 Wu et a l, 1996 Mesoderm/mesenchyme
forkhead 3
Mf3 Transcription factor 9 Exencephaly Labosky et a l, 1997
myc c-myc Proto-oncogene 15 Failure or retardation of neural tube closure
Davis et a l, 1993
p53 Trp53 Tumour suppressor gene 11 Exencephaly" Armstrong et a l, 1995, S ah et a l, 1995
RBP-jx RBP-jK Transcription factor ND Persistent open ANP Oka et a l, 1995
Retinoid acid receptors RAR (double mutant of a and y)
Nuclear retinoid receptors
11 and 15 Exencephaly Lohnes et a l, 1994
Sonic hedgehog’ Shh Signalling peptide 5 Exencephaly Echelard et a l, 1993
Twist Twist Basic HLH transcription
factor
12 Exencephaly Chen and Behringer, 1995
Mouse chromosome to which gene is mapped Category of NTD
'Connexin 43 and Sonic hedgehog are overexpressed (not targeted null mutation)
Female excess among NTD-affected embryos
The study of single gene mutations has provided information on the role of particular genes in the neurulation process but does not explain the complex interactions known to occur during development. The study of such interactions has been attempted by intercrossing different genetic mutants and assessing the severity of the phenotypes in the offspring. This provides information on whether the individual mutations act in the same pathway, and hence which gene is epistatic to the other, or whether the genes act in distinct pathways for which a combination of mutations may exacerbate the
phenotypic defect (reviewed further in Section 1.4.1.3). It is widely believed that the inheritance of NTD in the human population is polygenic with multiple genes acting additively to determine the risk of defects (Copp et a l 1990), hence, the study of gene interactions is particularly important to aid understanding of the human condition.
1.4.1.1 Models of polygenic inheritance
The SELH mouse strain, in which 17% of offspring have exencephaly, demonstrates polygenic inheritance - it is only in the presence of a combination of the ‘mutant’ genes that NTD are seen (Juriloff et a l 1989). Various backcross studies have estimated that the SELH strain harbours 2 or 3 loci that contribute to NTD (Juriloff et a l 1989; Gunn et a l 1992). The study of the SELH strain is further reviewed in Section 4.1.1.
1.4.1.2 Major genes and modifiers
Other mouse mutations show partial penetrance, for example the curly tail mutation, in which only a proportion of homozygous embryos develop NTD (Embury et a l 1979). In contrast to the SELH mutation, in curly tail there appears to be a major ‘obligatory’ mutant gene which has been mapped to distal chromosome 4 (Letts et a l 1995). Several modifying genes, known to be polymorphic between different mouse strains, have been shown to determine the penetrance of the major gene (Letts et a l 1995). Axial defects (Axd) is another spontaneous mutation in which the penentrance of the
defect is affected by genetic background: the frequency of NTD increases when the mutation is backcrossed to BALB/c (Essien, 1992). This variation in the incidence of defects with respect to genetic background is explored further in Chapter 4 in the case of the splotch (Sp^^) mutant.
1.4.1.3 Major gene - major gene interactions
In addition to the interactions already described, the interaction between major genes in affecting the process of neural tube closure has been studied by the generation of double mutant mice. Mice doubly mutant for curly tail and splotch have an increased
incidence and severity of the spina bifida defect, suggesting that the two pathogenic mechanisms summate to cause a more severe defect than the action of either gene alone (Estibeiro etal 1993). The severity appears to come from the combination of the two defects as individual features of the defect in the single mutations (neural crest
migration in splotch and ventral curvature in curly tail) do not become any worse in the double mutant. This suggests that the interaction between the two genes operates on a relatively distal feature in the neurulation pathway causing a double insult which effectively prevents neural tube closure (Estibeiro et a i 1993).
1.4.1.4 The role of genetic background in the effect of mutant genes causing NTD
As discussed in the previous section, modifier genes play an important role in the penetrance of certain phenotypes. The effect of genetic background on the incidence of defects has become important recently in the generation of null-mutant mice. Since
1990, gene targeting has been used to inactivate specific genes. Homologous
recombination in ES cells followed by the transmission of mutations through the germ line of a chimera produces embryos with a null copy of the targeted gene. The
production of null mutations has become a valuable tool in studying the role of single genes in developmental processes (reviewed by Copp, 1994). The analysis of such
mutants has been complicated however, by the variable phenotype often seen when mutations are expressed on different genetic backgrounds. The Cart-1 knockout shows such variability with genetic background. The incidence of exencephaly is 65% on the C57B1/6 background and 100% on the 129 SvEv background (Zhao et a l 1996). Although this complicates the analysis of such null mutations, it provides valuable information on how genetic background can affect the severity or expression of a phenotype and suggests that on some backgrounds, a phenotype may be obscured. Such observations suggest a need for caution in the interpreting the role of a single gene in a given developmental process.
1.4.2 Environmental models of NTD
The study of teratogens on rodent embryonic development has provided a wealth of information on the environmental factors that influence neural tube closure (reviewed in Chapter 4). Of the teratogens which disrupt neural tube closure, the vast majority cause exencephaly whereas the development of spina bifida is relatively rare (reviewed in Copp era/., 1990).
The relative uniformity of defects seen in teratogen-induced NTD suggests that, unlike the genetic models, these agents may be acting in a similar fashion to produce a
relatively uniform set of cellular effects. It is possible that this wide range of teratogens acts through a small number of final common pathways to disturb the neurulation process. Such pathways may result in the inhibition of cell proliferation and induction of cell death, both common sequelae of teratogenicity (Copp et a l 1990). In summary, it is thought that the majority of teratogens act by inhibiting embryonic growth in some way and therefore produce a dramatic effect on cranial neurulation, leading to
1.4.2.1 Genetic background affects environmental models of NTD
Variation in genetic background is commonly cited as an explanation of the altered susceptibility to NTD between various inbred mouse strains when treated with the same teratogen. Work on hybrids of high and low incidence strains has shown an
intermediate response in the offspring, irrespective of the mother’s strain (Finnell et a l 1986). Furthermore, certain strains appear to be consistent in either being particularly susceptible (in the case of the SWV strain) or resistant (in the case of the DBA/2 strain) to exencephaly caused by a variety of teratogens. The loci responsible for these strain differences could be localised by recombinant inbred strain analysis (reviewed in Section 4.4). The identification of such modifier genes would not only be useful in understanding the genetic control of neurulation in murine development, but may also provide information on the analogous background genetic differences seen in human populations. The subject of genetic background and susceptibility to teratogens is dealt with further in Chapter 4.
1.4.3 Gene-teratogen interactions
The study of gene-teratogen interactions has proved valuable in understanding the developmental pathways that lead to NTD. As with the effect of double gene mutations, the combination of teratogen and mutant gene could produce a variety of effects. The interaction may cause an exacerbation of the defect, with the additive effect of the two factors working on independent pathways, or on the same pathway with the teratogen acting to increase the penetrance of the gene defect. Alternatively, the teratogen may produce no alteration of the defect seen with the gene defect alone. This would suggest that both gene and teratogen act through a single pathway and that the gene defect is fully penetrant. Finally, if the two influences act through antagonistic mechanisms, the effect of teratogen and mutant gene may cancel each other, producing
an ameliorating effect. Some known gene-teratogen interactions are summarised in Table 1.3. The majority of interactions produce an increase in defects, suggesting an additive effect. The exception to this is the effect of many teratogens on the curly tail (ct) gene. As discussed in Section 1.2.2.2, the ct defect is caused by a proliferation imbalance leading to increased ventral curvature of the caudal region. Culture of curly tail embryos in conditions known to reduce proliferation such as a mild hyperthermia, or cytotoxic drugs such as5- fluorouracil and mitomycin C, corrects the proliferation defect and again allows normal PNP closure (reviewed in Copp et a l, 1988).
These agents are known teratogens in normal mouse strains and are not models for human prevention as they are acting to correct a specific defect in curly tail. More recently however, myo-inositol has been shown to reduce the incidence of NTD in this defect and has been postulated to be a model for human folate-resistant NTD (Greene and Copp, 1997).
1.5 Mouse models of folate-sensitive NTD
Recent studies have attempted to provide a model for folate-sensitive NTD seen in the human population, as such a model could be used to study how this vitamin produces its ameliorating effect. Both teratogenic models, such as VPA (Trotz et a l 1987), and gene mutants, such as Axd (Essien, 1992) and Carr-7(Zhao et a l 1996), report a reduction in the incidence of NTD when treated with various folate-related compounds but have failed to establish how such compounds produce an ameliorating effect.