A major aim of this overview is to illustrate the morphogenesis of a wide range of teratogen-induced brain and facial abnormalities that comprise the HPE spectrum. As described in a recent review by Cohen and Shiota 
, HPE has been experimentally produced in a wide variety of non-mammalian and mammalian models by a number of different teratogenic agents. Dysmorphogenesis resulting from exposure of mouse embryos to a selected group of these teratogens during the critical period for HPE induction (GD 7-8.5) is described.
Among the earliest reports of experimentally-induced HPE were those in which ethanol was administered to chicks [Féré, 1899
], frogs [LePlat, 1913
] and fish [Stockard, 1910
]. More recently, the genesis of a HPE spectrum resulting from exposure of mice to this teratogen has also been described [Sulik and Johnston, 1982
; Sulik and Johnston, 1983
; Webster et al., 1983
; Sulik, 1984
; Schambra et al., 1990
; Godin et al., 2010
]. In the mouse, treatment of dams with high doses of ethanol administered as 2 doses given 4 hours apart early on the 7th
gestational day (when embryos are at the stages shown in ) results in the HPE phenotypes shown in and . In normal embryos having 29 somite pairs (late on GD 9), the olfactory placodes/developing nasal pits are evident and widely spaced on the frontonasal prominence (the tissue surrounding the telencephalon) (). In ethanol-exposed embryos at this same developmental stage, the olfactory placodes are too closely spaced, accompanied by narrowing of the frontonasal prominence (). On GD 11, a time when the nasal pits should be surrounded by well developed lateral and medial nasal prominences (the progenitors of the nasal alae, and the nasal tip along with the intermaxillary segment, respectively), affected embryos present with medial nasal prominences that are abnormally small and closely approximated to the point of appearing as a single band of median tissue between nostrils that are too closely spaced (). Deficiency in the median aspect of the forebrain accompanies that of the upper midface and is evidenced by abnormally close proximity (and in severe cases, median confluence) of the ganglionic eminences (). illustrates a spectrum of the ethanol-induced facial phenotypes as they appear on GD 14 in mice. Mildly affected animals have facial features consistent with those in fetal alcohol syndrome, i.e. a small nose, deficient philtrum, and a long upper lip, all of which are indicative of median tissue deficiencies (). More severely affected mice may present with a single nostril, as in human cebocepahly (), or with a median cleft lip associated with an absent intermaxillary segment (). Notably, in teratogen-exposed mice, even in inbred animals, widely ranging degrees of effect commonly occur within single litters. This is attributable, at least in part, to the fact that there is significant intralitter variation in developmental stages [see Sulik and Johnston [1982
] for details regarding the interrelationship of the developing face and brain in ethanol-induced HPE in the mouse].
Figure 5 Ethanol-induced dysmorphology in GD 9.5 and 11 mouse embryos following acute ethanol exposure on GD 7. Frontal views of the face of control (a, b) and ethanol-exposed embryos (d, e; g, h), along with views of the forebrain interior of the embryos in b, (more ...)
Figure 6 Ethanol-induced dysmorphology in GD 14 mice following acute ethanol exposure on GD 7, and corresponding human phenotypes. Illustrated are children with Fetal Alcohol Syndrome (b), cebocephaly (d), and median cleft lip (f), whose features are represented (more ...)
While cyclopia/synophthalmia have not been noted to result from ethanol treatment on GD 7 in mice, mild to severe microphthalmia or anophthamia occurs. While the eyes and forebrain remain sensitive to ethanol's teratogenicity when exposure occurs at later stages (extending through GD 9) [Kotch and Sulik, 1992
; Parnell et al., 2009
], HPE is an endpoint that appears to be specific to ethanol exposures that occur when gastrulation is just being initiated (GD 7 in the mouse). This temporal specificity for the induction of HPE with ethanol in mice is consistent with that for other species. Studies employing zebrafish as conducted by Blader and Strahle 
are particularly informative with respect to identification of a narrow window of vulnerability. These investigators showed that exposure of zebrafish embryos to ethanol over a time period as brief as 3 hours, encompassing late blastula and early gastrula stages, caused severe HPE. In the fish embryos, synophthalmia was a common endpoint.
Blader and Strahl's work has also shed light on the mechanisms involved in HPE induction. Using gooscoid
as a molecular marker for the prechordal plate (defined as that tissue that is derived from axial hypoblast cells that involute/ingress at the onset of gastrulation and migrate anteriorly ahead of the chordal mesoderm), they illustrated an abnormal caudal placement of this tissue and suggested that ethanol arrests its migration. Thus, absence of inducing tissue for the forebrain was considered to be the primary cause for the ethanol-induced HPE. Others have shown that in zebrafish, shh
mRNA injection can prevent ethanol-induced cyclopia, suggesting that abrogation of Hedgehog (Hh) signaling is one of the major effects of ethanol exposure [Loucks and Ahlgren, 2009
]. The specific cellular mechanism(s) by which ethanol perturbs this signaling remains to be definitively established as does the initial cell population and developmental event involved.
Another teratogenic agent that, when acutely administered on GD 7 in mice causes HPE is retinoic acid [Sulik et al., 1995
]. As with ethanol, the result of exposure to this agent is a wide range of severity within the HPE spectrum. As shown in , in addition to median upper face deficiencies, some of which are severe enough to yield proboscis formation (union of the lateral nasal prominences with absence of intermediate tissues), the developing lower jaw may be hypoplastic. Lower jaw deficiencies can also result from ethanol exposure on GD 7 [Godin et al., 2010
], but are not illustrated herein. Typically, the maxillary component of the developing upper jaw appears to be much less affected than the mandibular region. That the mesenchyme of the maxillary prominences is almost entirely derived from neural crest cells, while that of the mandibular prominences has a large mesodermal contribution may account for this differential effect. The neural crest cells that contribute to the maxillary and mandibular prominences begin to migrate into those regions from the mid and upper hindbrain levels of the neural folds on GD 8 in the mouse, while the mandibular mesoderm is laid down at very early gastrulation stages; i.e. at or very near the GD 7 time of insult. illustrates an extreme example of loss of prechordal tissues. In this retinoic acid-exposed mouse fetus, the entire forebrain and associated facial tissues, along with the mandibular prominences are absent. However, the mid and hindbrain, along with the maxillary prominences are preserved.
Figure 7 Retinoic acid-induced dysmorphology in mouse embryos. As compared to control embryos (a, d), those whose mothers are treated with retinoic acid on GD 7 present with defects consistent with those shown in & . In addition, retinoic acid (more ...)
Acute exposure to other teratogens on GD 8-8.5, when mice are at very early somite stages, also can yield defects in the HPE spectrum [Wei and Sulik, 1993
; Lipinski et al., 2008
]. illustrates defects caused by acute maternal treatment on GD 8 with Ochratoxin A (OA), a food-born mycotoxin. Some of the affected animals present with severe microphthalmia, while in others the eyes are less severely diminished in size, but very closely approximated, to the point of union (synophthalmia). In each of the specimens shown, virtually all of the medial and lateral nasal prominence tissues are absent. This is evidenced by median union of the maxillary prominences, producing a snout (not a proboscis) that is surrounded by rows of hair follicles. In some specimens with this facial morphology there was also failure of anterior neural tube closure, resulting in anencephaly (not shown). Study of the OA-mediated pathogenesis illustrated that within 6 hours of maternal drug treatment, a remarkable amount of cell death occurred in the presumptive telencephalon and forebrain floor [Wei and Sulik, 1993
]. Excessive cell death in selected cell populations/regions was also notable at 24 hours after maternal treatment. The frontonasal prominence was particularly affected.
Figure 8 Ochratoxin A (OA)-induced dysmorphology in mouse fetuses. Illustrated are the holospheric forebrain () in a cebocephalic fetus (a, b), extreme hypotelorism (c), and synophthamia (d), all of which followed acute maternal OA treatment on the 8th (more ...)
OA has also been employed is gene/environment interaction studies. Ohta et al 
have shown that in mice carrying a mutation in Gli3 (a downstream gene in the Hh signaling cascade) which results in HPE, polysyndactyly, and neural tube defects, OA treatment on GD 7.5 increases the incidence of abnormalities. Additionally this group showed that in these mice, maternal folinic acid administration at times surrounding the OA treatment reduces the incidence of teratogen-induced defects.
Also studying gene/environment interactions, Lanoue et al 
showed that in mutant mice in which cholesterol levels are low due to mutation of the apolioprotein b
gene, further reduction resulting from treatment with a cholesterol biosynthesis inhibitor induces HPE. In some individuals the forebrain and facial defects are accompanied by hindbrain and limb reduction abnormalities () and in others the cranial neural tube fails to close (). In this model, the drug-induced hypocholesterolemia was accomplished by treating dams on their 4th
day of pregnancy with the biosynthesis inhibitor. That the hindbrain and limbs (whose critical periods are after GD 7) were affected indicates that cholesterol remained reduced to teratogenic levels for some time beyond GD 7. Recognizing the requirement of cholesterol for normal Hedgehog signaling [Incardona and Roelink, 2000
] Lanoue et al 
pointed out the similarities between the defects observed in their model and in Shh
knockouts [Chiang et al., 1996
]. More recently, Li et al 
, have shown that in zebrafish, supplementing ethanol-exposed embryos with cholesterol rescues a loss of Shh signal transduction, and prevents embryos from developing HPE.
Figure 9 Cholesterol-deficiency-induced dysmorphology in mouse embryos. Comparison of a sagittal cut through the head of a control (a-c; b & c are reciprocal halves), and an affected embryo (d-f; f & g are reciprocal halves) illustrates deficiency (more ...)
Figure 10 Anencephaly/HPE in the mouse and human. As shown in a mouse fetus with cholesterol deficiency-induced dysmorphia (a), and in a child with a similar presentation (b), facial features typical of HPE may be accompanied by anencephaly/ rostral neural tube (more ...)
More directly examining teratogen-mediated insult to the Hh signaling pathway in mice, Lipinski et al [2008
and submitted] have employed cyclopamine and a potent synthetic analogue. These compounds inhibit the morphogenetic activity of the Hh pathway by binding to and preventing activation of the transmembrane protein, Smoothened (Smo) [Chen et al., 2002
]. In the absence of Hh ligand, its receptor, Patched (Ptc1) inhibits Smo activity, presumptively through a small molecule mediator [Taipale et al., 2002
; Bijlsma et al., 2006
]. Upon Hh binding to Ptc1, inhibition of Smo is relieved, triggering a complex downstream signaling cascade that culminates in target gene activation via the Gli family of transcription factors [Ingham and McMahon, 2001
]. As in the classic studies in which Binns and Keeler exposed sheep to cyclopamine [reviewed in Keeler, 1975
] this agent and the analogue also cause the HPE spectrum in mice. In the Lipinski et al studies, maternal drug treatment was initiated on GD 8.25 - 8.5; i.e. close to the end of the known critical period for HPE induction. In addition to the range of defects described herein for other teratogens (e.g. cebocephaly; ), unilateral and bilateral clefts of the lip as well as clefts of the secondary palate were induced (). In some of the mice with cleft lip, the intermaxillary segment is notably reduced in size and in others it appears normal. These phenotypes are also seen in clinical populations. In individuals in which intermaxillary segment reduction is recognized, median forebrain deficiencies are expected. However, in those that have what appears to be typical unilateral or bilateral cleft lip and palate, this is not the case. Noteworthy in this regard, is that histological sections of the cyclopamine-exposed mouse fetus featuring cleft lip and palate and shown in revealed agenesis of the anterior pituitary ().
Figure 11 Cyclopamine-induced dysmorphology in mouse fetuses and corresponding human phenotypes. A wide spectrum of dysmorphology including cebocephaly (a) and cleft lip (b, c) result from drug exposure initiated on GD 8.5. Notably, while the fetuses in b & (more ...)
Figure 12 Cyclopamine-induced CNS dysmorphology in fetal mice. As compared to frontal (coronal) histological sections made at the level of the eyes and pituitary in a control GD 16.5 fetus (a, b), abnormalities including union of the frontal lobes of the brain (more ...)