This report describes MRM-based discovery and documentation of structural abnormalities resulting from early gastrulation stage ethanol insult in mice. In addition to providing a 3D perspective of ethanol-induced HPE, with its range of median forebrain deficiencies, other CNS and craniofacial abnormalities were also identified. As discussed below, these findings extend our understanding of the spectrum of ethanol-induced birth defects and of the critical periods for their induction.
It is clear that, as with other teratogens, both dosage and timing (developmental stage) dictate the consequences of prenatal ethanol exposure. Regarding the former, with the objective of identifying even the most severe of ethanol’s dysmorphogenic effects, a previously-reported maternal ethanol dose high enough to yield abnormalities without substantially increasing resorption rates was selected. This dosage was somewhat higher (yielding peak maternal BECs of approx. 380 vs. 440 ± mg/dl) than utilized for an MRM-based GD 8 ethanol exposure study by Parnell et al, 2009a
publication in a series of which this is a part). On GD 8, exposure to the higher ethanol dose typically yields severe heart defects and substantial embryo lethality. As shown in previous studies that employed the same treatment paradigm as for the current investigation, peaking within 30 minutes of the last dose, the maternal BEC remains above 100 mg/dl for a total of approximately 9 hours and reaches 0 within a few more hours (Kotch et al., 1992
; Webster et al., 1983
). Thus, exposure to ethanol totals less than 12 hours, including a period for which the concentration is expected to be less than teratogenic. An intraperitoneal (ip) route of maternal ethanol administration was employed for both the GD 8 and the GD 7 studies. As compared to maternal dietary ethanol intake, ip administration provides interlitter outcomes that are more consistent. It is recognized that with the ip treatment, embryos may experience a higher peak ethanol concentration than occurs in the maternal blood (Clarke et al., 1985
). However, it is notable that abnormalities consistent with those described herein also occur following a dietary exposure paradigm that yields maternal BECs comparable to those in the current study (Webster et al, 1983
Regarding timing, it is clear that ethanol is teratogenic at virtually every post-implantation stage. Remarkable is that ethanol exposure occurring within a relatively narrow window in 2 hr. time-mated inbred animals can yield not only a range of defects within a single spectrum, but also defects that appear to be virtual opposites. This is exemplified by the occurrence of median forebrain and facial deficiencies typical of semilobar and alobar HPE (a narrow snout and forebrain) in some fetuses and median split face accompanied by widely spaced cerebral hemispheres and olfactory bulbs in others; all following acute insult on GD 7. Undoubtedly, the fact that in C57Bl/6J mice there is significant intra-litter variation, representing as much as 12 hours difference in developmental staging among littermates, plays an important role in this variability (Parnell et al., 2009b
). HPE has been the most commonly-reported dysmorphology following GD 7 ethanol exposure in mice (Higashiyama et al., 2007
, Myers et al., 2008
; Schambra et al., 1990
; Sulik and Johnston, 1982
; Sulik et al., 1984
; Webster et al., 1983
), and was also observed in the current study population. GD 8 has previously been identified as the time in mouse development when median facial clefts and excessive brain width are induced by ethanol (Kotch and Sulik 1992
; Parnell et al., 2009a
; Webster et al., 1983
), while HPE is not a typical result of GD 8 ethanol treatment. Thus, it appears that among the dysmorphic fetuses described herein, those without HPE were more developmentally advanced at the time of ethanol insult than those with HPE. While insult on each individual day of mouse development is expected to yield a specific pattern of dysmorphology, it is also expected that, due to inter- and intra- litter variability in developmental stages, there will be some overlap.
With respect to HPE, via individual scans and 3D reconstructions, MRM has made it possible to readily show the range and severity of median forebrain deficiency that occurs in the absence of overt hindbrain dysmorphology. In those cases with semilobar and alobar forms of HPE, the severity of brain effect is consistent with that of the upper midface as evidenced, to a large extent, by the proximity of the nostrils. In all of the mouse fetuses whose nostrils are too closely positioned the median portion of the upper lip is too long (from nose to oral cavity). Notable was one fetus in which an effect on nostril positioning was subtle (if present), and that still had an unmistakably long upper lip. In this fetus the cerebrum had a complete interhemispheric fissure. It is expected that this phenotype is consistent with lobar HPE. Ongoing studies employing diffusion tensor imaging (DTI) and 3D facial analyses based on MRM reconstructions (Hammond et al., 2005
) are designed to enable identification of subtle changes in facial morphology and to better define the brain fiber tracts in fetuses such as this.
The genesis of the HPE-related facial dysmorphology has previously been described as resulting from ethanol-induced loss of medial nasal prominence tissue (i.e. the progenitor of both the nasal tip and the intermaxillary segment, the latter of which becomes the philtrum of the lip and the primary palate) and subsequent overconvergence of the maxillary prominences, yielding the excessively long upper lip (Sulik and Johnston, 1983
). DeMyer (1975)
recognized hypoplasia of the intermaxillary segment as being pathognomonic of brain malformation; the greater the deficiency of intermaxillary tissue, the greater the likelihood of a malformed brain. In the HPE spectrum, the human face presents with an absent or indistinct philtrum accompanied by a thin (vermillion) upper lip border; a phenotype that undoubtedly results from medial nasal prominence deficiency. These facial features are also characteristic of FAS.
In addition to ethanol exposure, other environmental agents (e.g. retinoic acid, cyclopamine, cholesterol biosynthesis inhibitors) and mutations in a number of different genes including sonic hedgehog (SHH), ZIC2, SIX3, and TGIFβ can cause HPE and the associated facial abnormalities. (Cohen, 2006
; Monuki, 2007
; reviewed by Muenke and Cohen, 2000
). Of particular note is interference with sonic hedgehog signaling (Shh-s) as a basis for these defects. Shh-s is a primary event in neural plate induction. Studies by Ahlgren and her co-workers in chicken (2002) and fish embryos (Loucks and Ahlgren, 2009
) and also by Li et al. in the latter species (2007), have illustrated that ethanol interferes with this signaling. Strongly supporting this as a key mechanism underlying ethanol-induced defects is that enhancing Shh-s can diminish the teratogenesis (Loucks and Ahlgren, 2009
). The prevalence of alcohol (ethanol) use and abuse and the multiple genes involved in the genesis of HPE contribute to the likelihood that via gene-environment interactions ethanol significantly factors into the high (1/250) incidence of HPE among human conceptuses (Matsunaga and Shiota, 1974).
Along with the forebrain and upper midfacial defects that characterize HPE, other defects that are associated with this spectrum were noted in this study. Micrognathia commonly occurs both in human HPE and in FASD (Ades and Sillence, 1992
; Blaas et al., 2002
; Cohen, 1989
; Jones and Smith, 1975
; Lemoine et al., 1968
; Majewski, 1981
; Pauli et al., 1981
), and was clearly evident in a third of the 19 ethanol-exposed mouse fetuses. 3D facial analyses are expected to also identify more subtle mandibular deficiencies resulting from ethanol exposure on GD 7. Severe micrognathia was accompanied by narrowing of the cerebral aqueduct in some specimens. The latter abnormality is commonly and causally associated with hydrocephalus, a condition that co-occurs with human HPE (Barr and Cohen, 1999
; Dickinson et al., 2006
) and that was previously noted to result from GD 7 ethanol treatment in mice (Sulik and Johnston, 1983
). Micro/aglossia and cleft palate, as seen in this study, also co-occur with HPE (Cohen, 1989
; Pauli et al., 1981
; Porteous et al., 1993
). In part, due to the relatively long period of genesis of the secondary palate, clefting of this structure (a recognized feature of FASD) is expected to also result from ethanol insult at later developmental stages. Of these (“other”) defects, for the fetuses in this study, certainly aqueductal stenosis, and probably cleft palate would not have been readily recognized without MRM.
Also with MRM, tissue that appears to correspond to misplaced olfactory nerves was found in the overtly holoprosencephalic animals. Normally, the olfactory nerves should project from the nasal epithelium, through the cribriform plate, to synapse in the olfactory bulbs. In the absence of olfactory bulbs, these nerves still extend upward, but lacking a target, form an intracranial mass that remains unattached to the brain. Recent analyses of holoprosencephalic mouse fetuses whose defects resulted from Shh-inhibition via in utero exposure to a potent cyclopamine analog revealed comparable olfactory nerve masses (R.J. Lipinski, personal communication).
Two of the ethanol-exposed fetuses in this study have small, widely spaced olfactory bulbs. Of these, one is anophthalmic and has an enlarged third ventricle (indicating hypothalamic deficiency), no pituitary, apparent absence of the corpus callosum, and markedly small/stenotic nasal cavities. The other has a median facial cleft. The collection of defects in these mice is consistent with the following recognized human syndromes/associations: 1) median cleft face syndrome; a condition for which agenesis of the corpus callosum and anomalies of the pituitary gland have been reported (DeMyer, 1967
), 2) septo-optic dysplasia; a syndrome characterized by absence of the septum pellucidum, pituitary hormone deficiency, and optic nerve hypoplasia; features of which a clinical report by Coulter et al. (1993)
attributed to prenatal ethanol exposure, and 3) CHARGE association which includes nasal cavity narrowing, growth and mental retardation, along with a variety of structural brain abnormalities including absence/hypoplasia of the olfactory bulbs and tracts, dysgenesis /hypoplasia of the frontal lobes and optic nerves, and agenesis of the corpus callosum and septum pellucidum. CHARGE association was highlighted in the Parnell et al. (2009a)
report as resulting from GD 8 ethanol exposure in mice. Indeed, although each has its own key features, there is significant overlap between HPE and these 3 clinical conditions (Bomelburg et al., 1987
; de Toni et al., 1985
; Fitz, 1994
; Lin et al., 1990
; Polizzi et al., 2005
). This is also true for the dysmorphology resulting from GD 7 versus GD 8 ethanol exposure in mice.
The MRM-based discovery of cerebral cortical dysplasia/heterotopias resulting from acute GD 7 ethanol exposure is novel and is expected to be of significant clinical importance. Nearly 35 years ago the first autopsy report by Jones and Smith (1975)
of a newborn with FAS described a large heterotopia encompassing the left cerebral hemisphere. Under this mass of tissue, the cortex was thin and disorganized and the lateral ventricles were enlarged. In more recent studies of rodent FASD models, one of which was conducted utilizing cultured GD17 fetal rat cortical slices (Mooney et al., 2004
) and one which employed maternal dietary ethanol exposure on days 10 through 21 in the rat (Komatsu et al., 2001
; Sakata-Haga et al., 2004
), cortical heterotopias have also been found. The cortical defects noted in the current study ranged from extremely small and isolated, to involving the medial aspect of both cerebral hemispheres. In most cases, the morphology is consistent with leptomeningeal heterotopia, though a more accurate descriptor for the most extensive defects is probably cortical dysplasia. Cortical heterotopias are generally considered as resulting from neuronal migration errors (Verotti et al., 2009). It is remarkable that they can result from an acute teratogenic insult occurring as early as the time of neural plate induction.
The presence of cortical heterotopias is highly correlated with seizure activity. Indeed, Verroti et al. (2009) state that “neuronal migration disorders are considered to be one of the most significant causes of neurological and developmental disabilities and epileptic seizures in childhood”. Among individuals with FAS the prevalence of epilepsy is higher than in the general population (1%), with estimates varying from 3–21% (Dorris, 1989
; Ioffe and Chernick, 1990
; Jones et al., 1973
; Majewski, 1981
; Marcus, 1987
; Murray-Lyon, 1985
; Olegard et al., 1979
; O’Malley and Barr, 1998
; Streissguth et al., 1978
). Work directed toward identifying pathologic changes that may underlie alcohol-induced seizure threshold reduction has shown an association with hippocampal abnormalities induced during the human 3rd
trimester equivalent (Bonthius et al., 2001 a
). These studies employed a rat FASD model in which both behavioral and electrographic seizure thresholds were examined. Similar testing of postnatal animals following acute ethanol exposure during early gastrulation is needed.
Linear and volume measurements made in this study from MRM scans and 3D reconstructions are consistent with the visually-assessed dysmorphology. Notable in the ethanol-exposed animals are reduced frontothalamic and brain width measures and lateral ventricular enlargement; features that can be readily assessed in human fetal ultrasounds. Work by Kfir et al. (2009)
showing that both 2nd
trimester ultrasound can detect frontothalamic reductions in the fetuses of moderate to heavy alcohol users, is consistent with the mouse data (Sulik et al., 2009
). Together, the human and experimental studies illustrate the diagnostic potential of early (prenatal) forebrain measures.
In conclusion, this work contributes significantly to defining the CNS dysmorphology that results from ethanol insult at times corresponding to the middle through the end of the 3rd week of human development. Individual MRM scans and 3D reconstructions of fetal mouse brains have facilitated this effort, allowing documentation and discovery of ethanol-induced CNS defects and appreciation of their relationship to co-occurring facial abnormalities. These results promise to aid in clinical recognition, diagnosis, and prevention of FASD.