aprosencephaly, cystic hygroma, hypoplastic mandible and small cranium. The investigators suggested that hypoxia and ischemia in the acardiac twin created the HPE phenotype owing to the increased vulnerability of the head to lower levels of oxygen

Craniofacial malformations, ranging from cleft lip and palate to complex disorders including holoprosencephaly (HPE), affect 84 in 10,000 people worldwide (WHO, 2003). These defects, which can be caused by genetic mutations, environmental factors or combinations of the two, have a mechanistic basis in the alteration of cellular processes during development. For example, mutations in Treacle (TCOF1) induce apoptosis of neural crest progenitor cells, leading to facial hypoplasia (Dixon et al., 2006; Jones et al., 2008), whereas defects in fibroblast growth factor (Fgf) signaling can lead to reduced cell proliferation, causing facial clefting (AbuIssa et al., 2002; Riley et al., 2007). Teratogenic agents, including cyclopamine, ethanol, nicotine and other drugs, cause similar malformations by altering the survival and proliferation of cells (Webster et al., 2006; Loucks et al., 2007; Perez-Aytes et al., 2008). A recent clinical report suggests that hypoxia might also be a cause of craniofacial anomalies in humans. Siebert (Siebert, 2007) describes a case of monochorionic, diamniotic twinning in which one twin was normal and the other twin had no heart, which created hypoxia and ischemia due to reversed arterial perfusion. Structural malformations in the acardiac twin included a spectrum of anomalies that resembled HPE (Cohen, 2006): cyclopia, aprosencephaly, cystic hygroma, hypoplastic mandible and small cranium. The investigators suggested that hypoxia and ischemia in the acardiac twin created the HPE phenotype owing to the increased vulnerability of the head to lower levels of oxygen (Siebert, 2007). Thus, hypoxia during pregnancy might affect facial development by increasing apoptosis or reducing cell proliferation. However, the relationship between hypoxia, cellular processes and craniofacial morphogenesis was not tested directly. The mechanistic link between hypoxia and craniofacial malformations is not well established because the effects of hypoxia on development are poorly understood. In the 1950s and 1960s, a number of researchers (Grabowski and Paar, 1958; Ancel, 1959; Nelsen, 1960; Grabowski, 1961; Grabowski, 1964; Yamamoto and Noguchi, 1964; Grabowski and Schroeder, 1968; Noguchi, 1969; Smith et al., 1969) undertook descriptive studies on the effects of hypoxia on the morphology of chick embryos. Chick embryos were made hypoxic by incubation in an atmospheric mix of air and nitrogen atmosphere, by incubation at high altitude (Smith, 1969), or by shellacking the shells to prevent gaseous diffusion (Ancel, 1959). In all, hypoxia led to high mortality, a variety of craniofacial malformations and, in a small number of embryos, brain defects including exencephaly and