We show that the occurrence of birth defects associated with a LOF mutation in Lrp6
is reduced among all conceptions by moderate supplementation of FA in the prenatal diet. However, in contrast to our studies in the Cd
mouse line that bears a GOF mutation, the same level of FA supplementation that rescued Lrp6Cd/Cd
embryos did not rescue LRP6-deficient embryos. Instead, FA supplementation of Lrp6−/−
embryos shifted an already severe phenotype to earlier embryonic lethality, so that LRP6-deficient embryos responded, but with an adverse reaction, to elevated FA levels. The fact that FA levels influence WNT signaling was evident in the effect of FA supplementation on gene expression profiles in Lrp6+/−
embryos and adults. In fact, of the hundreds of pathways and networks that are traceable with IPA algorithms, it is highly significant that cell cycle regulatory and WNT pathways displayed a strong gene–diet interaction. The relevance of WNT signaling was validated using two in vitro
assays of the WNT canonical pathway. In these assays, FA deficiency blunted pathway responses to WNT signaling, reflecting the importance of FA metabolism for early development (3
). However, these assays also indicate that FA supplementation, even to a modestly elevated level, attenuates WNT signaling. These data suggest that added dietary FA likely rescued the Lrp6Cd
defect by countering this net hyperactive Cd
allele, whereas FA supplementation further suppressed WNT signaling to exacerbate the LOF in the Lrp6
knockout line. This indicates a direct relationship between FA supplementation and the LRP6/WNT signaling pathway. Such a direct relationship would be consistent with our previous studies indicating that the Lrp6Cd
allele also impacts intracellular FA utilization (9
The mitotic index of PH3 labeling in neuroepithelial cells around the time of cranial neural tube closure (E9.5–10) showed that raising dietary levels of FA promotes proliferation in wild-type mice. However, Lrp6 LOF mutant embryos displayed an impaired proliferative response to FA supplementation. While these results could explain the failure of added FA to rescue NTDs in Lrp6−/− embryos, they could not explain the increased loss of heterozygous mice, since there was no statistical difference in mitoses of heterozygotes compared with wild-type on the control diet or between diets in the heterozygous cranial neural folds. Moreover, Lrp6 null embryos of equivalent stage did not display a statistically significant difference in mitoses on the two diets, undermining the possibility that a proliferation defect was the sole reason for earlier embryonic loss in Lrp6 nulls receiving the 10 ppm FA diet. Similarly, TUNEL labeling ruled out an increase in apoptosis upon FA supplementation to account for the observed increase in NTD in nulls and fetal loss of heterozygous LOF mutants.
Involvement of LRP6 in canonical WNT signaling suggested that FA effects on neurulation could entail transcriptional influences on embryogenesis. Indeed canonical WNT signaling in the cranial neural folds and midbrain of E9.5 embryos was diminished as assessed by the comparison of in situ β-galactosidase activity in the Lrp6+/+::TCF-LacZ versus Lrp6−/−::TCF-LacZ double mutants, when sibling embryos were processed in parallel. Therefore, the loss of LRP6 in null embryo cranial folds was not fully compensated by continued LRP5 expression.
assessment of WNT canonical pathway stimulation in the presence of varied FA levels revealed a bimodal relationship between WNT signaling and FA concentration, in which FA excess was as deleterious to WNT signaling as FA deficiency. Particularly striking was that relatively modest FA increases of 2.5-fold or more above the optimal level can attenuate WNT signaling in vitro
. There was no effect of FA alone on the basal levels of cytosolic β-catenin or TopFlash transcriptional reporter activity in NIH3T3 fibroblast cultures. Interestingly, the level of FA that provided optimal WNT activation in vitro
, 4 μg/ml, is the same concentration used in the complete Dulbecco's modified Eagle's medium that was previously optimized for growth and maintenance of cell lines (26
). FA concentrations of 10 μg/ml or higher blunted the cellular response to recombinant WNT3a in both β-catenin stabilization and TopFlash reporter assays. Therefore, FA supplementation has an impact on transcriptional activation by the canonical WNT signaling pathway.
We examined gene–diet interactions because of our interest in the impact of dietary folate supplementation on NTDs and expression profiles in wild-type and Lrp6
heterozygous mutants in two key tissues at different life stages. Our expression studies were focused on E9.5 crania because NTDs arise in embryonic cranial folds where key aspects of folate metabolism in mitochondria take place (27
) and on adult liver where most of the methylation reactions and 50% of all methionine metabolism occurs (28
). In addition, liver function in both embryos and adults depends on WNT signaling (29
), and methionine metabolism in liver can be adversely affected in mice that have mutations in WNT signaling pathway genes (9
). The statistical analysis used here is a new application of Bayesian hierarchical selection and interaction testing that provides a robust and highly reproducible result. Our interrogation of gene–diet interactions revealed several metabolic and signaling pathways that are significantly affected by Lrp6
mutation and FA supplementation in cranial development during embryogenesis, in liver function in adults or in both. Understanding the gene- and diet-dependent interplay among these physiological pathways may lead to general strategies for preventing NTDs, regardless of their responsiveness to folate. We propose that mutations in WNT signaling genes such as Lrp6
not only adversely affect methionine metabolism, but also secondarily affect methylation reactions, DNA synthesis (purine and pyrimidine metabolism), translation control and, through polyamine metabolism, the balance of cell proliferation, mitochondrial functions and perhaps metabolism of alternative methyl donors (Table ). This proposal nicely integrates our molecular and pathway studies with known functions of these pathways.
Pathway analysis of gene–diet interactions suggests a central role for methionine and polyamine metabolism in these developmental and physiological processes. In particular, S-adenosylmethionine (AdoMet, also known as SAM) is used for methylating a wide variety of molecules such as DNA, RNA, lipids and proteins, for polyamine synthesis, a source of AdoMet radicals and a component of riboswitches that control transcription and translation (31
). Availability of AdoMet for these reactions depends heavily on dietary folate. Polyamines (putrescine, cadaverine, spermidine and spermine), which are DNA-binding organic molecules with at least two amino groups, are ubiquitous in rapidly dividing cells (32
). AdoMet decarboxylase converts AdoMet to dcAdoMet, which then is used to make spermine and spermidine. Although cell growth is retarded or blocked when polyamine pools are depleted, cells rigorously maintain polyamine levels at the expense of methionine metabolism, which in turn leads to imbalanced AdoMet and nucleotide pools, thereby adversely affecting methylation as well as purine and pyrimidine metabolism (33
). The interdependency between methionine metabolism and polyamine biosynthesis exacerbates the functional consequences of folate deficiency, with ramifications for DNA synthesis, cell proliferation and mitochondrial functions.
Degradation of polyamines links methionine metabolism with mitochondrial function and alternative methyl donors. Degradation of spermine and spermidine uses acetyl-CoA and, under the action of succinate dehydrogenase (SDH1), leads to production of succinate, which is essential for complex II functions in mitochondria. Thus, demands of polyamine metabolism impact methionine metabolism in two ways, first by preferentially using AdoMet for polyamine synthesis rather than for methylation reactions, and second by using acetyl-CoA for polyamine degradation rather than for choline metabolism, which could affect availability of betaine, an alternative methyl donor. Together, these results and observations suggest that disruption of methionine metabolism in WNT mutant mice leads to diverse metabolic consequences that are remediated or exacerbated, depending on genetic conditions, with supplementary folate.
These studies provide a novel perspective to the gene–environment interactions that contribute to NTD formation. With respect to developmental signaling pathways, not only FA deficit but also elevated FA levels can impair WNT pathway function. Depending on individual genotype, FA supplementation can be deleterious to neurulation and embryogenesis. In the Lrp6 LOF mutants, the effects of supplementation on proliferation did not explain the negative impact of added FA on the heterozygous and homozygous mutants. Similarly, we did not detect an enhancement of apoptotic cell death in mutant embryos compared with wild-type. Thus, the impact of the detected pathway changes related to genotype and diet likely rests not in cell proliferation or cell death but in cell function and possibly cell morphology (oxidative stress and cytoskeletal regulation). Finally, these data in Lrp6 mutant mice also demonstrate that the mechanism of birth defect prevention by FA supplementation (reduction of NTD occurrence by amelioration versus increased embryonic loss) can vary not only with the genes associated with increased risk, but also with the type of mutation within those genes (i.e. LOF versus GOF). Clearly, a detailed understanding of genetic risk and interactions between folate metabolism and other developmentally important pathways will be required to optimize birth defect prevention strategies for individual families.