J.C. Craig’s research while affiliated with Texas A&M University and other places

Neural tube defects (NTDs), although prevalent and easily diagnosed, are etiologically heterogeneous, rendering mechanistic interpretation problematic. To date, there is evidence that mammalian neural tube closure (NTC) initiates and fuses intermittently at four discrete locations. Disruption of this process at any of these four sites may lead to a region-specific NTDs, possibly arising through closure site-specific genetic mechanisms. Although recent efforts have focused on elucidating the genetic components of NTDs, a void persists regarding gene identification in closure site-specific neural tissue. To this end, experiments were conducted to identify neural tube closure site-specific genes that might confer regional sensitivity to teratogen-induced NTDs. Using an inbred mouse strain (SWV/Fnn) with a high susceptibility to VPA- induced NTDs that specifically targets and disrupts NTC between the prosencephalon and mesencephalon region (future fore/midbrain; neural tube closure site II), we identified a VPA-sensitive closure site II-specific clone. Sequencing of this clone from an SWV neural tube cDNA library confirmed that it encodes the r1 subunit of the cell cycle enzyme ribonucleotide reductase (RNR). The abundance of rnr-r1 mRNA was significantly increased in response to VPA drug treatment. This upregulated expression was accompanied by a significant decrease in cellular proliferation in the closure site II neural tube region of the embryos, as determined by ELISA cellular proliferation assays performed on BrdU-pulsed neuroepithelial cells in vivo. We hypothesize that rnr-r1 plays a critical role in the development of VPA-induced exencephaly.

Although the teratogenicity of valproic acid (VPA) has been well established, the mechanism(s) by which this anticonvulsant drug induces malformations remains controversial. Using the combined molecular techniques of in situ-transcription (IST) and antisense RNA (aRNA) amplification we analyzed VPA-induced alterations in the gene expression for 10 genes within the neural tubes of embryos from two murine strains that have been shown to differ in their susceptibility to VPA-induce neural tube defects (NTD). Pregnant dams from both SWV (susceptible) and LM/Bc (resistant) strains were either treated with saline (control) or VPA (600 mg/kg) on gestational day (GD) 8:12 (day:hour). Neural tubes were isolated from control or VPA exposed embryos at three gestational time points, which represented the beginning (GD 8:18), middle (GD 9:00), and end (GD 9:12) of neural tube closure (NTC) in both of these murine strains. Using univariant statistics we demonstrated that in LM/Bc embryos with NTDs, the expression of bdnf, ngf, and trk, ngf-R were significantly elevated at all three time points, and the cytokine, cntf was significantly decreased at GD 9:00. In contrast, the major gene alterations observed in SWV embryos were a significant increase in tfgα and tgfβ1–3 at GD 9:00. In an effort to better define the more intricate interactions between VPA exposure and the expression of these genes, we analyzed our data using Principal Component Analysis. The results from this analysis demonstrated that embryos from these two stains behaved differently, not only in response to a VPA exposure, but also under control conditions, which may explain the multifactorial nature of NTDs in these mice.

Although the teratogenicity of valproic acid (VPA) has been well established, the mechanism(s) by which this anticonvulsant drug induces malformations remains controversial. Using the combined molecular techniques of in situ-transcription (IST) and antisense RNA (aRNA) amplification we analyzed VPA-induced alterations in the gene expression for 10 genes within the neural tubes of embryos from two murine strains that have been shown to differ in their susceptibility to VPA-induce neural tube defects (NTD). Pregnant dams from both SWV (susceptible) and LM/Bc (resistant) strains were either treated with saline (control) or VPA (600 mg/kg) on gestational day (GD) 8:12 (day:hour). Neural tubes were isolated from control or VPA exposed embryos at three gestational time points, which represented the beginning (GD 8:18), middle (GD 9:00), and end (GD 9:12) of neural tube closure (NTC) in both of these murine strains. Using univariant statistics we demonstrated that in LM/Bc embryos with NTDs, the expression of bdnf, ngf, and trk, ngf-R were significantly elevated at all three time points, and the cytokine, cntf was significantly decreased at GD 9:00. In contrast, the major gene alterations observed in SWV embryos were a significant increase in tfgalpha and tgfbeta1-3 at GD 9:00. In an effort to better define the more intricate interactions between VPA exposure and the expression of these genes, we analyzed our data using Principal Component Analysis. The results from this analysis demonstrated that embryos from these two stains behaved differently, not only in response to a VPA exposure, but also under control conditions, which may explain the multifactorial nature of NTDs in these mice.

The molecular basis for the well-established hierarchy of susceptibility to valproic acid-induced neural tube defects in inbred mouse strains was examined using in situ transcription and anti-sense RNA amplification methodologies with both univariate and multivariate analyses of the resulting gene expression data. The highly sensitive SWV strain demonstrated a significant reduction in the expression of the folate binding protein (FBP-1) following the teratogenic insult at gestational day 8:18, while the more resistant LM/Bc embryos were up-regulating this gene in response to valproic acid treatment. More importantly, at all 3 gestational timepoints spanning the period of murine neural tube closure examined in this study, the LM/Bc embryos had significantly higher MTHFR (5,10-methylenetetra-hydrofolate reductase) gene expression levels compared to the SWV embryos. As this folate pathway enzyme is important in homocysteine and methionine metabolism, it suggests that the SWV embryos may be hypomethylated, and essential gene expression during critical periods of neural tube closure is compromised by the teratogenic exposure to valproic acid. This study represents the first evidence of a strain difference in transcriptional activity in response to a teratogenic exposure that might be causally related to the development of the teratogen-induced congenital malformations.

The molecular techniques of in situ transcription and antisense RNA amplification (IST/aRNA) have allowed for the monitoring of coordinate changes in the expression of multiple genes simultaneously. However, the analysis of their concurrent behavior during murine embryogenesis has been problematic. Studies involving the investigation of temporal and spatial gene expression during embryogenesis have focused solely on the analysis of isolated, single gene events. Such an approach has failed to provide an integrative picture of genetic control over the varied and complicated cellular processes governing embryogenesis. In order to interpret the enormous amount of gene expression data generated by these procedures, we have attempted to develop an analytical framework by employing the statistical concepts of principal components analysis (PCA). For the current study, we performed IST/aRNA on neural tubes dissected from the highly inbred LM/Bc murine strain collected during four gestational time periods. A subset of these genes, representing a partial signaling pathway in the developing neuroepithelium, was then subjected to PCA. Here, we report that PCA highlighted the transcriptional interplay among the genes p53, wee-1, Tgf beta-2, and bcl-2 such that the combined reciprocal regulation of their gene products is suggestive of a predominant proliferative state for the developing neuroepithelium. The application of PCA to the gene expression data has elucidated previously unknown interrelationships among cell cycle genes, growth, and transcription factors on a transcriptional level during critical stages of neurulation. The information gleaned from this analysis, while not definitive, suggests distinct hypotheses to guide future research.

The teratogenic potential of valproic acid has been well established both in experimental models and in human clinical studies. As with all human teratogens, there are genetically determined differences in individual susceptibility to the induction of congenital defects. Using a mouse model of valproate-induced neural tube defects, a study was undertaken to examine differential changes in gene expression for selected transcription factor (Pax-3, Emx-1, Emx-2, c-fos, c-jun, creb) and cell cycle checkpoint genes (bcl-2, p53, wee-1) during neural tube closure. In general, exposure to teratogenic concentrations of valproic acid elicited GD 9:12 control levels of transcription factor mRNA expression in GD 9:0 embryos of both strains. This accelerated developmental profile is marked by significant elevation of Emx-1, Emx-2, c-fos, c-jun, and creb expression. There was also a significant over expression of the cell cycle genes p53 and bcl-2 in the LM/Bc embryos in response to the teratogenic insult. Examination of the ratio of expression of these genes clearly favored bcl-2, which supports the hypothesis that altered neuroepithelial cell proliferation rates, rather than increased apoptosis, is the underlying mechanism by which valproic acid alters normal neural tube morphogenesis. An investigation into interactive effects of these genes on the molecular profile of GD 9:0 embryos further validated this observation. That is, the overall proliferative state among the control embryos was prematurely modified into a more differentiated state following teratogenic insult. These results suggest that alterations in the expression of multiple genes are most likely responsible for valproic acid-induced neural tube defects.

Abstract Potentially teratogenic agents enter the environment at a rate that greatly exceeds current capabilities to effectively evaluate their reproductive toxicities. This is due, in part, to costly, labour-intensive methodologies involving mammalian embryonic screening assays that are currently in use worldwide. Therefore, we sought to develop a rapid, less expensive screening system with which to identify molecular biomarkers of teratogenicity using a non-mammalian system. Embryos of the topminnow, Fundulus heteroclitus, offer several advantages in terms of reproductive toxicity screening efficiency as compared with mammalian embryonic systems. These embryos are easily manipulated and develop normally at ambient temperature in air, water, or air-saturated mineral oils, making them readily adapted for field studies. In the present study, developing f. heteroclhs embryos were exposed to teratogenic concentrations of sodium valproate (VPA) or arsenic acid (arsenate), and the frequency and types of induced malformations were evaluated. Using in situ transcription and antisense RNA (aRNA) amplification procedures (IST/aRNA), we attempted to correlate the teratogenic outcomes to specific alterations in the expression of a panel of developmentally regulated genes. Preliminary studies identified treatment concentrations of arsenate and VPA that induced abnormal development in 95 % of the surviving embryos. Among the F. heteroclitus embryos, the structural defects most commonly induced by these compounds were cardiac and neural tube malformations. The genetic expression profiles revealed a number of genes whose expression levels were significantly altered by exposure to the test compounds. Molecular analysis of f. heteroclitus embryonic development represents a novel, inexpensive approach to screen for potential teratogens, and identify genes whose expression patterns may be used as biomarkers, or indicators, of teratogenicity.