Most of the writing I do nowadays is for university purposes, so I’m often more concerned with meeting a deadline than I am blogging information. To change things up a bit, I’ve decided to post a review paper I wrote recently about Fetal Alcohol Syndrome (FAS) for a developmental biology course. At first, I was less than enthusiastic about the topic, but FAS turns out to be really bloody fascinating, developmentally.

Due to these aforementioned deadlines, the ending trails off awkwardly, but the science within is damned cool.

Embryonic Development of Craniofacial Malformations in FAS
by Liz Shaw   |   Last modified February 21, 2011

A relative newcomer to the scientific world, the prenatal development involved in Fetal Alcohol Syndrome (FAS) was formally called to question in 1973, when Jones, Smith, Ulleland, and Streissguth noticed strikingly similar malformations in the children of alcoholic mothers. Five years later, the ramifications of maternal alcohol ingestion became unmistakable; craniofacial malformations in children such as a smooth philtrum, low nose bridge, thin upper vermilion border, short palpebral fissures, and retrognathism were statistically linked to maternal alcohol consumption (FIGURE 1; Sulik, 2005). These children also had significantly smaller heads and, accordingly, smaller brains (Gilbert, 2010). Alcohol was identified as a teratogen. As time progressed, more features associated with maternal alcohol ingestion were documented, and the phenomenon became known as FAS. Several diagnostic guidelines currently exist to determine the presence and severity of FAS, including the 4-Digit Diagnostic Code, guidelines by the Institute of Medicine, Hoyme FAS guidelines, and guidelines published by the Centers for Disease Control and Prevention (Anthony et al., 2010). Although each set of guidelines differs, there is one commonality throughout: an emphasis on the noticeable craniofacial malformations (Sulik, 2005).

Some craniofacial malformations associated with FAS.
FIGURE 1: Craniofacial malformations associated with FAS. Original image source.

Developmental studies indicate that maternally-ingested ethanol, after diffusing through the placenta, interferes with signaling transduction pathways, contributes to impromptu apoptosis, and is detrimental to specific populations of cells involved in cytoskeletal organization, particularly in the presumptive craniofacial region of the embryo. Rout, Krawetz, and Armant suggested that ethanol causes a rapid change in intracellular calcium ion concentration, thereby reestablishing body axes and altering signaling cascades in any developmental stage, which is expected to cause dysmorphogenesis (1997; Sulik, 2005). One of the pathways surmised to be influenced by this change is the hedgehog signaling pathway, which is required at early developmental stages for normal forebrain development, since the Sonic hedgehog ligand is critical for patterning the anterior-posterior axis in the central nervous system and establishing dorsal-ventral polarity in the neural tube (FIGURE 2; Sulik, 2005; Gilbert, 2010). One recent study found that Sonic hedgehog also prevents the ethanol-induced degradation of cranial neural crest cells, which ordinarily differentiate into structures such as cranial neurons, craniofacial cartilage, connective facial tissues, and the bones of the middle ear and jaw (Ahlgren, 2010; Gilbert, 2010). In a similar manner, the pathway for FGF8, a protein perhaps best known for establishing a proximal-distal gradient in limbs, is expected to be influenced by ethanol (Gilbert, 2010; Sulik, 2005). During neural tube differentiation, FGF8 is expressed in the organizing centers of the forebrain, most notably by a population of cells called the anterior neural ridge (ANR), located at the telencephalic midline (Sulik, 2005).

Dorsal-ventral polarity in the neural tube is established by BMPs and Sonic hedgehog (Shh)
FIGURE 2: Dorsal-ventral polarity in the neural tube is established by TGF-ß family proteins (BMPs) and Sonic hedgehog (Shh). The notochord secretes Shh, which induces the formation of the ventral neural tube to form the floor plate and a concentration gradient is set up from the ventral region. Ethanol is believed to interrupt signal transduction pathways that release Shh. Figure from Gilbert, 2010.

Through fate-mapping with dyes, the ANR has been identified as not only critical for proper craniofacial development and organization, but is seemingly very vulnerable to ethanol-induced apoptosis, more so than other exposed cell populations, indicating a genetic basis for sensitivity to ethanol (Sulik, 2005). As testimony to this observation, already within eight to 12 hours of maternal alcohol ingestion, significant apoptosis occurs at the ANR regardless of developmental stage (FIGURE 3; Kotch and Sulik, 1992). Apoptosis of the ANR essentially impairs the telencephalon organizer, which would ordinarily coordinate the proper development of structures involved in the ability to smell, make mental associations, and store memories (Gilbert, 2010). These results are phenotypically demonstrated in the significantly diminished olfactory bulbs and abnormally fused cerebral hemispheres of most organisms with FAS (FIGURE 4; Gilbert, 2010).

The anterior neural ridge (ANR) forms most nasal structures affected by FAS. It is highly susceptible to ethanol.
FIGURE 3: The anterior neural ridge (ANR) forms most nasal structures affected by FAS. It is highly susceptible to ethanol. Within 8–12 hrs of maternal ethanol treatment, embryos illustrate excessive cell death in the ANR (arrows in d, e, and f) as evidenced by cell staining (fate-mapping) with Nile blue sulfate (d and e) and Lysotracker red (c and f). This is apparent in embryos at presomite (a and d), early somite (b and e) and neural tube closure (c and f) stages of development. Figure and description from Sulik, 2005.


Normal mouse brain (D) compared with FAS brain (E).
FIGURE 4: MRI-based 3-D reconstruction of a normal 17-day embryonic mouse brain (D) compared with 17-day embryonic FAS mouse brain (E). The olfactory bulbs (pink) are completely absent from view and the cerebral hemispheres (red) are abnormally united in the midline (i.e. postclosure telencephalic midline).
Figure from Gilbert, 2010.

There are several important ANR derivatives that contribute to the development of important craniofacial structures, the most important of which are cells making up the epithelial lining of the nasal cavities and the band of nerve tissue between the hemispheres of the presumptive cerebrum (Sulik, 2005). Many of FAS’s facial malformations — in particular, the smooth philtrum, low nose bridge, and thin upper vermilion border — can be, in part, explained by the apoptosis of AER-derived epithelial lining, as these cells shape the nasal prominences of the developing face (Sulik, 2005). On the other hand, when the defect impairs the telencephalonic nerve tissue (i.e. the telencephalonic commissural plate), there is often an increase in deficiencies or total absence of the corpus callossum, which results in smaller brain size and is indicative of mental retardation in many subjects with FAS (Riley et al., 1995; Sulik, 2005).

Fate-mapping during development has clarified that neural crest cells are quite sensitive to ethanol exposure. The use of various dyes during development has revealed that neural crest cell migration patterns are clearly altered due to ethanol exposure, which has a number of repercussions for the final craniofacial product of the organism (Anthony et al., 2010). Cranial neural crest cells that would ordinarily undergo endomembranous ossification to form jaw bones — such as the mandible and maxilla — differentiate prematurely or not at all, contributing to retrognathism (Oyedele and Kramer, 2008; Gilbert, 2010). Some cranial neural crest cells, too, develop into cartilage prematurely when proper cytoskeletal organization is not established, leading to observed facial malformations (Gilbert, 2010). The severe impact on cranial neural crest cells indicates a lack of properly-functioning hedgehog signaling pathways, as the presence of Sonic hedgehog has been shown to ward off ethanol-induced apoptosis, although the specific reasons for this are not yet fully understood (Ahlgren et al., 2002).

One likely cause of ethanol-induced apoptosis is alcohol’s ability to generate superoxide radicals by dint of its chemical structure and interactions (Gilbert, 2010). These toxic radicals degrade cell membranes, thereby causing cells to lyse and die. In the presence of an enzyme known as superoxide dismutase, however, ethanol-induced apoptosis can be rendered practically nonexistent; the enzyme dismantles superoxide radicals into the less biologically harsh oxygen and hydrogen peroxide (FIGURE 5; Gilbert, 2010). Photographs from studies carried out in the head region of mice, appear to confirm that the ANR region would be less degraded in the presence of this enzyme. This would imply that craniofacial abnormalities — and other FAS-related abnormalities which arise following neural tube differentiation — could be lessened or prevented with superoxide dismutase. Two peptides known as PAL and NAP also appear to prevent alcohol-induced fetal anomalies, but these anomalies are isolated more to presumptive cerebellum development in the telencephalon vesicle — which determines qualities such as intelligence and the ability to make mental associations — not craniofacial phenotypic changes (Incerti et al., 2009; Gilbert, 2010; Sulik, 2005).

Control (A), Degradation of cell membranes due to superoxide radicals shown in black (B), and superoxide radicals plus superoxide dismutase (C)FIGURE 5: Superoxide dismutase prevents cellular degradation by ethanol-generated superoxide radicals. These mouse head images display a normal control (A), degradation of cell membranes due to superoxide radicals shown in black (B), and superoxide radicals plus superoxide dismutase (C). Figure from Gilbert, 2010.

There is currently no definitive cure for FAS, aside from refraining from consuming alcohol while pregnant; presently, the Centers for Disease Control and Prevention recommends complete abstinence from drinking alcoholic beverages while pregnant (Centers for Disease Control and Prevention, 2010). The variability of responses in developing children to ethanol exposure, while once elusive, is steadily becoming more predictable as embryogenesis and developmental processes are the subject of increasing study (Sulik, 2005). Variation in craniofacial effects was once posited to be due to differences in maternal metabolism, yet recent studies have indicated that metabolism plays a barely noticeable effect in phenotypic development, if any (de Licona, 2008; Sulik, 2005). Yet genetic variation among mothers does appear to play a part, as a 2010 study indicated that certain cell populations like the ANR and cranial neural crest are more resistant to apoptosis in different strains of mice under the same experimental circumstances (Anthony et al., 2010). At the root of the issue is ethanol-induced apoptosis, the most likely cause for the major visible abnormalities associated with FAS. Cellular death can be held responsible for the most obvious facial malformations (Sulik, 2005) and its occurrence can impair cytoskeletal organization, which influences cranial neural crest cells involved in the formation of specific craniofacial features (Oyedele and Kramer, 2008). The pattern of apoptosis seems to target the ANR, which initiates the development of some of the most well-documented facial regions subject to abnormalities in FAS (Sulik, 2005). The specificity of this apoptosis hints that certain cell populations are more sensitive to degradation during a given developmental time than others, possibly due to this genetic variation (Anthony et al., 2010). Yet what determines this variation and subsequent vulnerability is a topic still awaiting an answer.

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Ahlgren S.C., Thakur, V., Bronner-Fraser, M. (2002). Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure. Proceedings of the National Academy of Science U.S.A. 99:10476–10481.

Anthony, B., Vinci-Booher, S., Wetherill, L., Ward, R., Goodlett, C., and Zhou F.C. (2010). Alcohol-induced facial dysmorphology in C57BL/6 mouse models of fetal alcohol spectrum disorder. Alcohol 44, 659-671.

Centers for Disease Control and Prevention. “CDC – Fetal Alcohol Spectrum Disorders.” Centers for Disease Control and Prevention. 21 Oct. 2010. Web. 20 Feb. 2011.

de Licona, H.K., Karacay, B., Mahoney, J., McDonald, E., Luang, T., and Bonthius, D.J. (2008). A single exposure to alcohol during brain development induces microencephaly and neuronal losses in genetically susceptible mice, but not in wild type mice. Neurotoxicology 30, 459-470.

Gilbert, S.F. (2010). Developmental Biology. Sunderland, MA: Sinauer Associates, Inc.

Incerti, M., Vink, J., Benassou, I., Roberson, R., Abebe, D., and Spong, C. (2009). Prevention of the alcohol-induced changes in brain-derived neurotrophic factor expression using neuroprotective peptides in a model of fetal alcohol syndrome. [abstract] American Journal Obstetrics and 
Gynecology 201, 54.

Jones, K.L., Smith, D.W., Ulleland, C.N., and Streissguth, P. (1973). Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1(7815), 1267-1271.

Kotch, L.E., Sulik, K.K. (1992). Experimental fetal alcohol syndrome: proposed pathogenic basis for a variety of associated facial and brain anomalies. American Journal of Medical Genetics 44, 168–176.

Oyedele, O.O. and Kramer, B. (2008). Acute ethanol administration causes malformations but does not affect cranial morphometry in neonatal mice. Alcohol 42, 21-27.

Riley, E.P., Mattson, S.N., Sowell, E.R., Jernigan, T.L., Sobel, D.F., and Jones, K.L. (1995). Abnormalities of the corpus callosum in children prenatally exposed to alcohol. Alcohol: Clinical and Experimental Research 19, 1198–1202.

Rout, U.K., Krawetz, S.A., and Armant, D.R. (1997). Ethanol-induced intracellular mobilization rapidly alters gene expression in the mouse blastocyst. Cell Calcium 22(6), 463-474.

Sulik, K.K. (2005). Genesis of Alcohol-Induced Craniofacial Dysmorphism. Experimental Biology and Medicine 230(6), 366-375.

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