The integration of advanced technologies for small animal imaging and shape analysis implemented here represents an innovative tool for defining concurrent abnormalities of the face and brain in 3D. The MRM imaging parameters utilized provided a high-throughput capability (35 min scan/animal), allowing examination of a large number of animals encompassing the phenotypic range resulting from each of two acute ethanol exposure times. Acquired 3D image sets allowed segmentation of regional brain structures as well as extraction of remarkably accurate and detailed facial surfaces from fetal mice measuring just 15 mm from the crown to rump. In addition to linear and volumetric measurements, this enabled application of DSM and assessment of 3D changes in facial surfaces and brain regions. While this technique has proven useful in modeling and delineating face shape differences across populations with various genetic syndromes, this study represents its first application in defining concurrent face-brain shape changes. These results will be readily comparable to the findings of ongoing clinical studies using DSM to define facial shape in populations with FAS, as well as those not meeting diagnostic criteria but having documented heavy prenatal ethanol exposure 
The results of this study have important conceptual and practical implications for diagnostic and interventional strategies in FASD. Conceptually, they demonstrate that early prenatal ethanol exposure can cause more than one pattern of facial dysmorphology. Practically, these results should encourage new avenues for clinical FASD research by highlighting abnormalities of specific facial features and brain regions that have previously received little attention. The pattern of craniofacial abnormalities resulting from insult during early gastrulation, including microcephaly, shortened palpebral fissures and an elongated upper lip with a smooth/deficient philtrum, is consistent with that currently recognized for FAS. Median forebrain deficiencies have been well described in populations with FAS 
, and recent MRI studies have demonstrated that palpebral fissure length is significantly correlated with abnormal cortical thickness and corpus callosum reduction 
. The correlation data presented here extend these findings by suggesting that the type and severity of brain abnormalities can be predicted, in part, by dysmorphology of the upper lip and hypoplasia of the midface.
Among the GD7 ethanol-exposed subjects, increased upper lip length was associated with hypoplasia of the olfactory bulbs and septal region. Remarkably, ethanol insult just 1.5 days later yielded a unique, and partially opposing pattern of dysmorphic features, including an abnormally shortened upper lip associated with increased septal region volume. As illustrated in and Movie S1
, and as previously suggested 
, the GD8.5 ethanol-induced facial dysmorphia appear more similar to those in individuals with DiGeorge syndrome (OMIM# 188400) 
. This malformation sequence commonly results from a 22q11.2 deletion but has also been reported in humans as a consequence of prenatal ethanol exposure 
. Individuals with DiGeorge syndrome and FASD also share a substantially increased incidence of depression and psychotic disorders 
. Particularly notable are findings relative to schizophrenia, which is common in DiGeorge syndrome and also occurs as part of FASD 
. Clinical imaging studies have shown an increased incidence of cavum septum pellucidum in adults with schizophrenia, as well as in those with DiGeorge syndrome 
. These investigations have not focused on the septal region, per se. However, its developmental and anatomical relationship to the septum pellucidum 
, along with the results of the current study, which provide a testable hypothesis to explore the underlying source of neurobehavioral phenotypic variation associated with FASD, indicate that such analyses are warranted.
Unique facial dysmorphology induced by stage-specific ethanol exposure in the mouse corresponds to distinct clinical phenotypes.
Cumulatively, the results of this study provide a proof of principle that early prenatal ethanol exposure can cause more than one temporally-dependent pattern of defects involving the face and brain and illustrate the predictive nature of the facial features for associated brain abnormalities. Numerous mechanisms for ethanol teratogenesis have been proposed. While outside the scope of the work presented here, the finding that ethanol elicits stage-specific teratogenic effects should provide new opportunities to examine these mechanisms within specific embryological contexts. Of clinical importance, these results suggest that an expansion of current diagnostic criteria to better represent the range of facial phenotypes induced by prenatal ethanol exposure could significantly advance early diagnosis and intervention strategies that are critical for the management of FASD.