Role of Folbp1 in the regional regulation of apoptosis and cell proliferation in the developing neural tube and craniofacies.

Folic acid is essential for many cellular reactions, including synthesis of nucleotides and regulation of cell cycle. Folic acid-binding protein one (Folbp1), a membrane-bounded protein, is the primary mediator of folic acid transport. Mice deficient in Folbp1 gene die in utero with multiple malformations, including severe exencephaly and craniofacial defects. Fusion of the neural tube and craniofacies require precisely regulated interactions of apoptosis, cell proliferation, and differentiation. To understand the role of Folbp1 in regulating the fusions of these primordia, levels of dead and proliferating precursor cells from Folbp1 embryos were quantified before the fusion processes. Massive apoptosis was detected in the Folbp1-/- defective tissues, with Bax and activated caspase-3 distributed evenly across the apico-basal axis of the lateral neural plate. 5-Bromodeoxyuridine (BrdU) and PCNA labeling assays revealed a reduced cell proliferation as well. However, telomerase activity was unaltered, arguing against telomere shortening and consequently, chromosomal instability, as the cause of the apoptosis. Notably, Islet-1 and 2H3 immunohistochemistry demonstrated the presence of differentiating neuronal cells, albeit in decreased numbers. Interestingly, Folbp1-/- embryos also elaborated novel neural structures that sprouted orthogonally from the embryonic neuraxis. Assays on the defective craniofacies exhibited similar phenomena, suggesting the neural crest precursor population that gives rise to both these structures is selectively vulnerable to Folbp1 inactivation. The results demonstrate a prominent role of Folbp1 in the regional regulation of apoptosis and cell proliferation that underlies the aberrant neural tube and craniofacial defects.

The extracellular matrix, p53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors, and reciprocally, Fas-ligand modifies estrogen control of cell-cycle proteins.

BACKGROUND: Apoptosis is important for normal cerebral cortical development. We previously showed that the Fas suicide receptor was expressed within the developing cerebral cortex, and that in vitro Fas activation resulted in caspase-dependent death. Alterations in cell-surface Fas expression may significantly influence cortical development. Therefore, in the following studies, we sought to identify developmentally relevant cell biological processes that regulate cell-surface Fas expression and reciprocal consequences of Fas receptor activation. RESULTS: Flow-cytometric analyses identified two distinct neural sub-populations that expressed Fas on their cell surface at high (FasHi) or moderate (FasMod) levels. The anti-apoptotic protein FLIP further delineated a subset of Fas-expressing cells with potential apoptosis-resistance. FasMod precursors were mainly in G0, while FasHi precursors were largely apoptotic. However, birth-date analysis indicated that neuroblasts express the highest levels of cell-surface Fas at the end of S-phase, or after their final round of mitosis, suggesting that Fas expression is induced at cell cycle checkpoints or during interkinetic nuclear movements. FasHi expression was associated with loss of cell-matrix adhesion and anoikis. Activation of the transcription factor p53 was associated with induction of Fas expression, while the gonadal hormone estrogen antagonistically suppressed cell-surface Fas expression. Estrogen also induced entry into S-phase and decreased the number of Fas-expressing neuroblasts that were apoptotic. Concurrent exposure to estrogen and to soluble Fas-ligand (sFasL) suppressed p21/waf-1 and PCNA. In contrast, estrogen and sFasL, individually and together, induced cyclin-A expression, suggesting activation of compensatory survival mechanisms. CONCLUSIONS: Embryonic cortical neuronal precursors are intrinsically heterogeneous with respect to Fas suicide-sensitivity. Competing intrinsic (p53, cell cycle, FLIP expression), proximal (extra-cellular matrix) and extrinsic factors (gonadal hormones) collectively regulate Fas suicide-sensitivity either during neurogenesis, or possibly during neuronal migration, and may ultimately determine which neuroblasts successfully contribute neurons to the differentiating cortical plate.