Neural and orofacial defects in Folp1 knockout mice [corrected

BACKGROUND

Folic acid is essential for the development of the nervous system and other associated structures. Mice deficient in the folic acid‐binding protein one (Folbp1) gene display multiple developmental abnormalities, including neural and craniofacial defects. To better understand potential interactions between Folbp1 gene and selected genes involved in neural and craniofacial morphogenesis, we evaluated the expression patterns of a panel of crucial differentiation markers (Pax‐3, En‐2, Hox‐a1, Shh, Bmp‐4, Wnt‐1, and Pax‐1).

METHODS

Folbp1 mice were supplemented with low dosages of folinic acid to rescue nullizygotes from dying in utero before gestational day 10. The gene marker analyses were carried out by in situ hybridization.

RESULTS

In nullizygote embryos with open cranial neural tube defects, the downregulation of Pax‐3 and En‐2 in the impaired midbrain, along with an observed upregulation of the ventralizing marker Shh in the expanded floor plate, suggested an important regulatory interaction among these three genes. Moreover, the nullizygotes also exhibit craniofacial abnormalities, such as cleft lip and palate. Pax‐3 signals in the impaired medial nasal primordia were significantly increased, whereas Pax‐1 showed no expression in the undeveloped lateral nasal processes. Although Shh was downregulated, Bmp‐4 was strongly expressed in the medial and lateral nasal processes, highlighting the antagonistic activities of these molecules.

CONCLUSIONS

Impairment of Folbp1 gene function adversely impacts the expression of several critical signaling molecules. Mis‐expression of these molecules, perhaps mediated by Shh, may potentially contribute to the observed failure of neural tube closure and the development of craniofacial defects in the mutant mice. Birth Defects Research (Part A) 67:209–218, 2003. © 2003 Wiley‐Liss, Inc.

     

Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of␣alveolar RMS by influencing the commitment␣and differentiation of the myogenic cell lineage.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Folic acid supplementation dysregulates gene expression in lymphoblastoid cells--implications in nutrition

For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ?four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics.