Rescue of Holoprosencephaly in Fetal Alcohol-Exposed Cdon Mutant Mice by Reduced Gene Dosage of Ptch1

Mingi Hong; Robert S. Krauss

doi:10.1371/journal.pone.0079269

Abstract

Holoprosencephaly (HPE) is a commonly occurring developmental defect in which midline patterning of the forebrain and midface is disrupted. Sonic hedgehog (SHH) signaling is required during multiple stages of rostroventral midline development, and heterozygous mutations in SHH pathway components are associated with HPE. However, clinical presentation of HPE is highly variable, and carriers of heterozygous mutations often lack apparent defects. It is therefore thought that such mutations must interact with more common modifiers, genetic and/or environmental. We have modeled this scenario in mice. Cdon mutant mice have a largely subthreshold defect in SHH signaling, rendering them sensitive to a wide spectrum of HPE phenotypes by additional hits that are themselves insufficient to produce HPE, including transient in utero exposure to ethanol. These variable HPE phenotypes may arise in embryos that fail to reach a threshold level of SHH signaling at a specific developmental stage. To provide evidence for this possibility, here we tested the effect of removing one copy of the negative regulator Ptch1 from Cdon^−/− embryos and compared their response to ethanol with that of Cdon^−/−;Ptch1^+/+ embryos. Ptch1 heterozygosity decreased the penetrance of HPE in this system by >75%. The major effect of reduced Ptch1 gene dosage was on penetrance, as those Cdon^−/−;Ptch1^+/− embryos that displayed HPE did not show major differences in phenotype from Cdon^−/−;Ptch1^+/+ embryos with ethanol-induced HPE. Our findings are consistent with the notion that even in an etiologically complex model of HPE, the level of SHH pathway activity is rate-limiting. Furthermore, the clinical outcome of an individual carrying a SHH pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors like fetal alcohol exposure.

Citation: Hong M, Krauss RS (2013) Rescue of Holoprosencephaly in Fetal Alcohol-Exposed Cdon Mutant Mice by Reduced Gene Dosage of Ptch1. PLoS ONE 8(11): e79269. https://doi.org/10.1371/journal.pone.0079269

Editor: Xin Zhang, Columbia University, United States of America

Received: June 13, 2013; Accepted: September 25, 2013; Published: November 11, 2013

Copyright: © 2013 Hong and Krauss. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by NIH grant R01AA018825 to RSK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, results from disruption in patterning of the rostroventral midline [1]. HPE occurs with high frequency, ∼1∶250 human conceptions, the great majority of which are lost during pregnancy [2], [3]. Both genetic and environmental factors are implicated in the etiology of HPE [4]–[6]. Heterozygous mutations in components of the Sonic hedgehog (SHH) signaling pathway are found in both inherited and sporadic forms of HPE [5]. These include the SHH ligand itself and the receptors, PTCH1, CDON, and GAS1 [5], [7], [8]. Among the non-genetic factors suggested to elevate the risk of HPE are preexisting maternal diabetes and fetal alcohol exposure [9], [10].

Experiments with mice and other vertebrate model organisms indicate that development of the rostroventral midline occurs by a progressive patterning mechanism whereby SHH produced by one midline structure induces Shh expression in a successive midline structure. SHH produced by the prechordal mesendoderm (PCM) is a key early step in development of the midline of the forebrain and midface [1], [11]–[13]. SHH produced by the PCM induces expression of pathway target genes (including Shh itself) in the rostral diencephalon ventral midline of the developing forebrain, which in turn leads to Shh expression in the ventral telencephalon [13]–[18]. As development proceeds, SHH produced by the forebrain induces expression of Shh in the ectoderm of the frontonasal and maxillary processes of the developing face, helping to pattern the craniofacial midline [17], [19]–[21].

Clinical presentation of HPE is extremely variable, and the range of defects falls within a continuum known as the HPE spectrum [4], [22]. The most severe form, alobar HPE, is characterized by complete failure to partition the forebrain into left and right hemispheres; this is usually associated with the most severe midfacial phenotypes, including cyclopia. The spectrum encompasses progressively less severe forebrain defects (semilobar and lobar HPE) and also mild facial midline abnormalities (so-called HPE microforms) that can occur in the absence of brain malformations.

A full spectrum of HPE phenotypes is seen both in sporadic and familial cases [23]. Furthermore, many mutation carriers in pedigrees are without apparent clinical manifestation, and mutations in many sporadic HPE patients are inherited from an apparently unaffected parent [23], [24]. Statistical analysis of these observations and gene mutation frequencies have led to an “autosomal dominant with modifier” model, in which the phenotypic outcome associated with a heterozygous mutation is influenced by more common genetic variants and/or environmental exposures [25]. It is important, therefore, to identify interactions between bona fide mutations and additional factors that grade penetrance and expressivity, resulting in the wide range of defects encompassed by the HPE spectrum. Animal models are well placed to do so. We have established Cdon mutant mice as such a model. Mice lacking CDON on a 129S6 genetic background have a largely subthreshold defect in SHH signaling that renders them sensitive to induction of HPE by second hits that are, themselves, insufficient to produce HPE (e.g., removal of one copy of Shh; gene dosage-sensitive removal of the Cdon paralog, Boc) [26]–[28].

We recently reported use of these mice to develop a model of gene-environment interaction in HPE. In utero ethanol exposure at E7.0 had little or no effect on wild type 129S6 embryos, but produced a wide range of HPE phenotypes with >75% penetrance in Cdon^−/− embryos [29]. The spectrum of HPE phenotypes produced in ethanol-treated Cdon^−/− mice ranged from alobar HPE with an undivided eye field, to lobar HPE with single nostril, to microform HPE and defects in palatogenesis [29]. A model consistent with production of phenotypes at both the severe and mild ends of the spectrum in ethanol-treated 129S6.Cdon^−/− mice is as follows: ethanol initiates defects in midline patterning specifically in genetically sensitized embryos; the variable severity of the HPE phenotype may arise as a consequence of individual embryos failing to reach a threshold level of SHH signaling at specific developmental stages, the stage of the deficit occurring stochastically and dictating phenotypic severity [29]. This hypothesis argues that pathway signaling strength is an important determinant of phenotypic outcome. PTCH1, the primary SHH receptor, functions as a negative regulator of pathway signaling [30]. To test the hypothesis, we removed one copy of Ptch1 from 129S6.Cdon^−/− embryos and assessed ethanol-induced production of HPE. As predicted by the model, Ptch1 heterozygosity effectively rescued such embryos.

Results

Rescue of HPE in fetal alcohol-exposed Cdon mutant mice by reduced Ptch1 gene dosage

Loss of Cdon or in utero ethanol exposure in 129S6 mice gave little or no phenotype individually, but together produced defects in midline patterning, inhibition of SHH signaling in the developing forebrain, and a broad spectrum of HPE phenotypes [29]. When analyzed at E10.0, ∼13.5% of ethanol-treated 129S6.Cdon^−/− embryos displayed severe phenotypes that included alobar HPE and failure to divide the eye field. These embryos died in utero and were resorbed by E11.0. When analyzed at E14.0, ∼70% of 129S6.Cdon^−/− embryos displayed phenotypes ranging from lobar through microform HPE, and variously included single nostril, defective palatogenesis, diminished nasal septal cartilage, and rudimentary vomeronasal organs. In asking whether removal of one copy of Ptch1 could rescue HPE in these animals, we analyzed E14.0 embryos to take advantage of the high penetrance of the phenotypes observed at this stage.

Mouse strain-dependent modifier genes regulate HPE phenotypes associated with Cdon mutation [27], [28]. Because our ethanol studies have been performed on a congenic 129S6 background, we first generated a congenic 129S6.Ptch1^+/− line by back-crossing. These mice were bred with 129S6.Cdon^+/− mice to generate double heterozygotes. Various offspring of intercrosses of these animals were further crossed, and pregnant females received IP injections of ethanol or saline control at E7.0 and four hours later ([29] and see Materials and Methods). Embryos of four genotypes were studied: Cdon^+/+;Ptch1^+/+, Cdon^+/+;Ptch1^+/−, Cdon^−/−;Ptch1^+/+, and Cdon^−/−;Ptch1^+/−.

Whole embryos were initially analyzed at E14.0 for external HPE features, including single nostril, deficient philtrum, and hypotelorism. Cdon^+/+;Ptch1^+/+ and Cdon^+/+;Ptch1^+/− embryos with or without ethanol did not have HPE, nor did saline-treated Cdon^−/−;Ptch1^+/+ and Cdon^−/−;Ptch1^+/− embryos (Figures 1 and S1, Table 1). Approximately 65% of ethanol-treated Cdon^−/−;Ptch1^+/+ embryos displayed external HPE phenotypes, similar to our previous results. In contrast, only ∼15% of ethanol-treated Cdon^−/−;Ptch1^+/− embryos displayed HPE, significantly lower than for Cdon^−/−;Ptch1^+/+ embryos (Figure 1, Table 1). Therefore, reduced gene dosage of Ptch1 substantially, though partially, rescued HPE associated with the synergistic interaction of Cdon mutation and fetal alcohol exposure.

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Figure 1. Effect of reduced Ptch1 gene dosage on ethanol-induced HPE in Cdon^−/− embryos: external features and lobar HPE.

(A–E) Frontal views of E14.0 embryos. About 65% of ethanol (EtOH)-treated Cdon^−/−;Ptch1^+/+ embryos displayed strong facial features of HPE, including a single nostril (arrowhead in C) and smooth, pointed philtrum (arrow in C). In contrast, only ∼15% of EtOH-treated Cdon^−/−;Ptch1^+/− embryos had external features of HPE. Two ethanol-treated Cdon^−/−;Ptch1^+/− embryos are shown, one that did not have external HPE features (D) and one that did (E; note the single nostril (arrowhead) and smooth, pointed philtrum (arrow)). (F-J) H&E-stained coronal sections of E14.0 embryos. Note that the ethanol-treated Cdon^−/−;Ptch1^+/+ embryo (H) displays continuity across the ventral midline of the forebrain (arrow), indicative of lobar HPE. Two ethanol-treated Cdon^−/−;Ptch1^+/− embryos are shown, one that did not have external HPE features (I) and one that did (J). Note that only the embryo that had external HPE (J) also had continuity across the ventral midline of the forebrain (arrow). See Figure S1 for saline-treated control embryos. See Table 1 for quantification.

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Table 1. Frequency of HPE defects in ethanol-treated mice.

https://doi.org/10.1371/journal.pone.0079269.t001

Hematoxylin and eosin (H&E)-stained sections of E14.0 embryos were examined to identify defects in forebrain and midline craniofacial structures. We compared four ethanol-treated Cdon^−/−;Ptch1^+/+ embryos that had external HPE features with six ethanol-treated Cdon^−/−;Ptch1^+/− embryos, 50% of which had external HPE. Saline-treated embryos of these genotypes, as well as saline- and ethanol-treated Cdon^+/+;Ptch1^+/+ and Cdon^+/+;Ptch1^+/− embryos, all lacked external HPE features and were used as controls (n = 4 for each). As expected [29], these controls showed normal forebrain and craniofacial patterning (Figures 1, 2, S1, and S2, Table 1). Two of the four ethanol-treated Cdon^−/−;Ptch1^+/+ embryos displayed lobar HPE, whereas one of six ethanol-treated Cdon^−/−;Ptch1^+/− embryos did so (Figure 1F-J, Table 1). All four of the ethanol-treated Cdon^−/−;Ptch1^+/+ embryos had defective palatogenesis, whereas two of six ethanol-treated Cdon^−/−;Ptch1^+/− embryos did so (Figure 2A-E, Table 1). All four of the ethanol-treated Cdon^−/−;Ptch1^+/+ embryos had diminished nasal septal cartilage and vomeronasal organs; three of the six ethanol-treated Cdon^−/−;Ptch1^+/− embryos had deficits in these structures (Figure 2F-J, Table 1). Finally, all four ethanol-treated Cdon^−/−;Ptch1^+/+ embryos had narrow midfacial regions that also contained additional presumptive mesenchymal tissue (Figure 2K-O, Table 1), a phenotype seen in Cdon mutants of a mixed genetic background [31]. Three of six ethanol-treated Cdon^−/−;Ptch1^+/− also showed this phenotype (Figure 2K-O, Table 1). A critical point here is that the ethanol-treated Cdon^−/−;Ptch1^+/− embryos that displayed defects in forebrain and midline craniofacial structures in sections were always among the three that showed external features of HPE; none of the ethanol-treated Cdon^−/−;Ptch1^+/− embryos that lacked external HPE features showed abnormalities upon sectioning (Table 1).

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Figure 2. Effect of reduced Ptch1 gene dosage on ethanol-induced HPE in Cdon^−/− embryos: palatogenesis and midfacial features.

(A-O) H&E-stained coronal sections of E14.0 embryos. (A–E) Palatogenesis. Note that the ethanol-treated Cdon^−/−;Ptch1^+/+ embryo (C) displays defective development of the palatal shelves (asterisks). Two ethanol-treated Cdon^−/−;Ptch1^+/− embryos are shown, one that did not have external HPE features (D) and one that did (E). Note that only the embryo that had external HPE (E) also had defective palatogenesis (asterisks). (F–J) The nasal septal cartilage and vomeronasal organ are denoted in (F) by an arrowhead and arrow, respectively. Note that the ethanol-treated Cdon^−/−;Ptch1^+/+ embryo (H) displays diminished nasal septal cartilage and vomeronasal organ (arrow). Two ethanol-treated Cdon^−/−;Ptch1^+/− embryos are shown, one that did not have external HPE features (I) and one that did (J). Note that only the embryo that had external HPE (J) also had reduced nasal septal cartilage and vomeronasal organ (arrow). (K–O) Note that the ethanol-treated Cdon^−/−;Ptch1^+/+ embryo (M) displays a narrow midfacial region with additional presumptive mesenchyme (asterisk). Two ethanol-treated Cdon^−/−;Ptch1^+/− embryos are shown, one that did not have external HPE features (N) and one that did (O). Note that only the embryo that had external HPE (O) also had a narrow midfacial region with additional presumptive mesenchyme (asterisk). See Figure S2 for saline-treated control embryos. See Table 1 for quantification.

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Taken together, there may be a small diminution in HPE phenotypic expressivity in ethanol-treated Cdon^−/−;Ptch1^+/− embryos, relative to similarly treated Cdon^−/−;Ptch1^+/+ embryos. However, in most cases, embryos of either genotype that displayed external HPE features showed roughly similar defects in forebrain and midfacial regions. These results suggest that the major effect of removing one copy of Ptch1 is to decrease the penetrance of HPE in ethanol-treated Cdon^−/− mice, rather than to selectively alter the severity of some HPE phenotypes and not others.

Effect of Ptch1 gene dosage on Shh and Nkx2.1 expression in the ventral forebrain of ethanol-treated Cdon^−/− mice

We next tested the expression of Shh and a direct SHH pathway target gene, Nkx2.1, in the forebrain by whole-mount in situ hybridization at E10.25. We previously found that about half of the ethanol-treated Cdon^−/− embryos at this stage had significantly reduced expression of these genes in the developing ventral telencephalon [29]. In this set, we observed that three of eight ethanol-treated Cdon^−/−;Ptch1^+/+ embryos had an obvious diminution in Shh expression and five of nine embryos of this genotype had an obvious reduction in Nkx2.1 expression (Figure 3). In contrast, one of eight ethanol-treated Cdon^−/−;Ptch1^+/− embryos showed a noticeable change in the Shh expression pattern and no Cdon^−/−;Ptch1^+/− embryos had alterations in the expression pattern of Nkx2.1, as compared to controls (n = 7) (Figures 3 and S3).

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Figure 3. Reduced Ptch1 gene dosage rescued defective expression of Shh and Nkx2.1 in ethanol-induced HPE in Cdon^−/− embryos.

Whole mount in situ hybridization analyses of Shh (A–D) and Nkx2.1 (E–H) expression in E10.25 (31–35 somites) ethanol-treated embryos of the indicated genotype. Three out of eight and five out of nine ethanol-treated Cdon^−/−;Ptch1^+/+ embryos showed defective Shh and Nkx2.1 expression in the forebrain, respectively (arrows in C and G). None of the ethanol-treated Cdon^−/−;Ptch1^+/− embryos displayed alterations in Nkx2.1 expression (n = 7), and only one of eight such embryos showed reduced Shh expression. See Figure S3 for saline-treated control embryos.

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Discussion

HPE is etiologically complex, involving interactions between the timing and strength of developmental signaling pathways, genetic variation, and exposure to environmental agents [4], [22]. The preponderance of the evidence is consistent with an “autosomal dominant plus modifier” model, wherein inherited or de novo heterozygous mutations in one of at least 12 genes interact with more common modifier loci and/or environmental exposures to generate a wide spectrum of rostroventral midline patterning defects [25]. The mutations are found in genes encoding components of the SHH pathway or in genes that, directly or indirectly, regulate SHH expression or pathway activity in the early midline or forebrain [4], [5], [16], [30], [32], [33].

Mouse models of HPE provide support for this model. We have shown that mice lacking the SHH co-receptor CDON display HPE with strain-dependent penetrance and severity, consistent with an important role for silent modifier genes in grading the spectrum of phenotypes [27], [28]. CDON is partially redundant with other co-receptors [27], [34], [35], and 129S6.Cdon^−/− animals have only a substhreshold defect in SHH signaling that renders them sensitive to induction of HPE by additional insults, genetic or environmental, that are themselves insufficient to produce HPE [26]–[29], [34], [35].

Epidemiological evidence suggests that fetal alcohol exposure raises the likelihood of HPE [9]. We recently showed that in utero exposure to ethanol produces a nearly complete spectrum of HPE phenotypes in 129S6.Cdon^−/− embryos, but is not teratogenic to 129S6.Cdon^+/+ embryos [29]. We hypothesized that a brief exposure to ethanol during gastrulation in a genetically sensitized embryo sets up a situation whereby the variable severity of HPE arises as individual embryos fail to reach a threshold level of SHH signaling at a specific developmental stage, with the stage of the deficit occurring stochastically and determining the severity of the outcome. This is consistent with observations in model systems demonstrating that SHH pathway activity is required during multiple stages of rostroventral midline patterning, including forebrain partitioning, bilateral division of the eye field and non-neural facial structures, and development of the primary and secondary palates [19], [36], [37].

This hypothesis argues that the level of SHH pathway signaling activity is an important determinant of phenotype. To provide experimental evidence for this possibility we tested the effect of removing one copy of the negative regulator Ptch1 from Cdon^−/− embryos and compared their response to ethanol-induced HPE with that of Cdon^−/−;Ptch1^+/+ embryos. As predicted, reduced gene dosage of Ptch1 substantially reduced the penetrance of HPE in this system, from ∼65% in Cdon^−/−;Ptch1^+/+ embryos to ∼15% in Cdon^−/−;Ptch1^+/− embryos, a reduction of >75%. The major effect of Ptch1 heterozygosity in this system was on penetrance, as those Cdon^−/−;Ptch1^+/− embryos that displayed HPE did not show major differences in phenotypic spectrum or selectivity from Cdon^−/−;Ptch1^+/+ embryos with ethanol-induced HPE.

At what developmental stage(s) might the rescue of HPE by Ptch1 haploinsufficiency occur? The PCM is required for induction of the ventral forebrain and subsequent partitioning of the forebrain and eye field, as well as patterning of the midfacial midline [1], [11]–[13]. The PCM serves as a source of SHH early during this multistep process, but SHH is also required for normal development of the PCM itself [1], [13], [14]. Cdon is expressed in the PCM but not in the ventral forebrain, even though the latter structure is affected in Cdon^−/− mice; this observation and additional results argue that loss of Cdon function in the PCM is the most likely cause of HPE in such mice [26]-[28], [38]. CDON and PTCH1 bind to one another as components of the SHH receptor complex [7], [35] and, like Cdon, Ptch1 is expressed in the PCM [14]. Therefore, it seems likely that rescue of HPE phenotypes in ethanol-treated Cdon^−/−;Ptch1^+/− embryos may occur as a consequence of enhanced SHH signaling function in the PCM itself. Alternatively, enhanced responsiveness to PCM-derived SHH may occur in the Ptch1^+/− ventral forebrain, exclusive of co-expression of Cdon and Ptch1. Finally, Cdon and Ptch1 are also co-expressed in the developing facial mesenchyme, and restoration of SHH signaling there may contribute to rescue of facial midline phenotypes. These possibilities are not mutually exclusive, and conditional mutagenesis will be required to address this issue. The mechanism whereby ethanol synergizes with Cdon mutation to inhibit SHH signaling is also not fully resolved but must involve early patterning events between E7.0 – E8.0, as ethanol is no longer detectable by E8.0 [29].

Studies on individuals with HPE and animal models conclusively demonstrate that HPE is associated with loss of SHH pathway function. The fact that haploinsufficiency for Ptch1 rescues HPE phenotypes in ethanol-treated Cdon^−/− embryos is consistent with PTCH1 functioning as a negative regulator of SHH signaling. However, heterozygous, germline missense mutations in PTCH1 have been identified in HPE patients [39], [40]. PTCH1 functions in the absence of Hedgehog ligand to inhibit the activity of a second membrane protein, Smoothened (SMO). Binding of SHH to PTCH1 relieves this inhibition, and SMO signals to activate pathway target genes [41]. Because HPE is caused by loss, not gain, of SHH pathway function, it is highly unlikely that the PTCH1 mutations found in HPE are complete loss-of-function mutations. In fact, heterozygous, germline loss-of-function mutations in PTCH1 result in a different disorder, Basal Cell Nevus Syndrome (also called Gorlin syndrome) [42]. Rather, such PTCH1 variants would be predicted to maintain the ability to inhibit SMO activity but to be insensitive to ligand-induced pathway activation. A synthetically constructed deletion mutant with such properties is the widely used PTCH1^Δloop2 variant [43].

Our findings are consistent with the notion that even in an etiologically complex animal model of HPE (one that models the complexities of human HPE), the level of SHH pathway activity is rate-limiting. These findings are highly relevant in the emerging era of human genomics and soon-to-be widespread personal access to genome sequences. Systematic analyses of common variants in SHH pathway components may reveal not only alleles that predispose offspring of heterozygous HPE mutation carriers to severe phenotypes but also alleles with protective effects. With a core database of variants – which will need to be functionally validated – it may be possible to predict the sum effect of deleterious and beneficial alleles on penetrance and expressivity of bona fide mutations and, conceivably, their interaction with non-genetic risk factors like fetal alcohol exposure.

Materials and Methods

Ethics Statement

All animal work was approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC). Our animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Mice

STOCK.Ptch1^tm1Mps/J mice [44] were purchased from the Jackson Laboratory and transferred onto the 129S6/SvEvTac (129S6) background with the Speed Congenics Program at Taconic, employing back-crossing and SNP arrays. Mice used for these experiments were estimated to be >98% 129S6. 129S6.Ptch1^+/− mice were crossed with 129S6.Cdon^+/− animals to generate 129S6.Cdon^+/−;Ptch1^+/− double mutant mice. These animals were then used to generate Cdon^−/−;Ptch1^+/−, Cdon^+/−;Ptch1^+/−, and Cdon^+/−;Ptch1^+/+ mice, which were then variously crossed for ethanol treatment as previously described [29]. Briefly, two to three month-old mice were mated for one hour in the dark. The time of the plug was designated as embryonic day (E) 0.0. Pregnant female mice were injected intraperitoneally twice with 15 µl per g body weight of a solution of 30% ethanol in saline (3.48 g/kg), first at E7.0 and again 4 hr later. Saline injections were used as a control.

Histology and Whole Mount In Situ Hybridization

Embryos were processed and analyzed as previously described [29]. Briefly, embryos were dissected out, fixed in 4% paraformaldehyde/PBS, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned at 8 µm. H&E staining was performed as described [27]. Sections were dehydrated through graded ethanol and xylene and mounted with Permount (Fisher Scientific). For whole mount in situ hybridization, embryos were prepared as described previously [45], except that they were treated with 10 µg/ml proteinase K (QIAGEN) in phosphate-buffered saline, 0.1% Tween-20 (PBT) according to stage. Embryos were rinsed, postfixed and hybridized with digoxygenin-labeled probes in hybridization mix [50% formamide, 1.3x SSC, 5 mM EDTA, 50 µg/ml yeast RNA, 0.2% Tween 20, 0.5% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate, and 100 µg/ml heparin] overnight at 65°C. They were then washed, blocked and incubated overnight with alkaline phosphatase-conjugated anti-digoxigenin antibody (1∶2000; Roche) in blocking buffer (2% blocking reagent [Roche]), 20% heat-inactivated lamb serum in 100 mM maleic acid, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 [MABT]). After washing in TBST (Tris-buffered saline with 0.1% Tween-20) and NTMT (100 mm NaCl, 100 mm Tris-HCl, pH 9.5, 50 mm MgCl2, and 0.1% Tween -20), signals were developed with BM Purple AP Substrate (Roche). Embryos were photographed with a Jenoptik ProgRes C3 camera attached to a Nikon SMZ 1500 stereomicroscope. Captured images were assembled by Helicon Focus software (Helicon Soft).

Supporting Information

Figure S1.

In utero exposure to saline does not induce external or forebrain features of HPE. (A–D) Frontal views of E14.0 embryos. (E–H) H&E-stained coronal sections of E14.0 embryos. This figure serves as a control for Figure 1.

https://doi.org/10.1371/journal.pone.0079269.s001

(TIF)

Figure S2.

In utero exposure to saline does not induce defects in palatogenesis or development of the midfacial midline (A–L) H&E-stained coronal sections of E14.0 embryos. This figure serves as a control for Figure 2.

https://doi.org/10.1371/journal.pone.0079269.s002

(TIF)

Figure S3.

In utero exposure to saline does not alter expression of Shh or Nkx2.1. Whole mount in situ hybridization analyses of Shh (A–D) and Nkx2.1 (E–H) expression in E10.25 (31–35 somites) saline-treated embryos of the indicated genotype. This figure serves as a control for Figure 3.

https://doi.org/10.1371/journal.pone.0079269.s003

(TIF)

Acknowledgments

We are grateful to Kuen Cheng for technical support and Katie Fantuozzo and Daisuke Chihara for critiquing the manuscript.

Author Contributions

Conceived and designed the experiments: MH RSK. Performed the experiments: MH. Analyzed the data: MH RSK. Wrote the paper: RSK.

References

1. Muenke M, Beachy PA (2001) Holoprosencephaly. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic & Molecular Bases of Inherited Disease. Eighth Edition ed. New York: McGraw-Hill. pp. 6203–6230.
2. Orioli IM, Castilla EE (2010) Epidemiology of holoprosencephaly: Prevalence and risk factors. Am J Med Genet C Semin Med Genet 154C: 13–21.
- View Article
- Google Scholar
3. Shiota K, Yamada S (2010) Early pathogenesis of holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 22–28.
- View Article
- Google Scholar
4. Krauss RS (2007) Holoprosencephaly: new models, new insights. Expert Rev Mol Med 9: 1–17.
- View Article
- Google Scholar
5. Roessler E, Muenke M (2010) The molecular genetics of holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 52–61.
- View Article
- Google Scholar
6. Solomon BD, Mercier S, Vélez JI, Pineda-Alvarez DE, Wyllie A, et al. (2010) Analysis of genotype-phenotype correlations in human holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 133–141.
- View Article
- Google Scholar
7. Bae GU, Domené S, Roessler E, Schachter K, Kang JS, et al. (2011) Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors. Am J Hum Genet 89: 231–240.
- View Article
- Google Scholar
8. Ribeiro LA, Quiezi RG, Nascimento A, Bertolacini CP, Richieri-Costa A (2010) Holoprosencephaly and holoprosencephaly-like phenotype and GAS1 DNA sequence changes: Report of four Brazilian patients. Am J Med Genet A 152A: 1688–1694.
- View Article
- Google Scholar
9. Johnson CY, Rasmussen SA (2010) Non-genetic risk factors for holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 73–85.
- View Article
- Google Scholar
10. Miller EA, Rasmussen SA, Siega-Riz AM, Frías JL, Honein MA, et al. (2010) Risk factors for non-syndromic holoprosencephaly in the National Birth Defects Prevention Study. Am J Med Genet C Semin Med Genet 154C: 62–72.
- View Article
- Google Scholar
11. Kiecker C, Niehrs C (2001) The role of prechordal mesendoderm in neural patterning. Curr Opin Neurobiol 11: 27–33.
- View Article
- Google Scholar
12. Muenke M, Beachy PA (2000) Genetics of ventral forebrain development and holoprosencephaly. Curr Opin Genet Dev 10: 262–269.
- View Article
- Google Scholar
13. Rubenstein JL, Beachy PA (1998) Patterning of the embryonic forebrain. Curr Opin Neurobiol 8: 18–26.
- View Article
- Google Scholar
14. Aoto K, Shikata Y, Imai H, Matsumaru D, Tokunaga T, et al. (2009) Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis. Dev Biol 327: 106–120.
- View Article
- Google Scholar
15. Cordero D, Marcucio R, Hu D, Gaffield W, Tapadia M, et al. (2004) Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes. J Clin Invest 114: 485–494.
- View Article
- Google Scholar
16. Geng X, Speirs C, Lagutin O, Inbal A, Liu W, et al. (2008) Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly. Dev Cell 15: 236–247.
- View Article
- Google Scholar
17. Marcucio RS, Cordero DR, Hu D, Helms JA (2005) Molecular interactions coordinating the development of the forebrain and face. Dev Biol 284: 48–61.
- View Article
- Google Scholar
18. McMahon AP, Ingham PW, Tabin CJ (2003) Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol 53: 1–114.
- View Article
- Google Scholar
19. Helms JA, Cordero D, Tapadia MD (2005) New insights into craniofacial morphogenesis. Development 132: 851–861.
- View Article
- Google Scholar
20. Hu D, Helms JA (1999) The role of Sonic hedgehog in normal and abnormal craniofacial morphogenesis. Development 126: 4873–4884.
- View Article
- Google Scholar
21. Hu D, Marcucio RS (2009) A SHH-responsive signaling center in the forebrain regulates craniofacial morphogenesis via the facial ectoderm. Development 136: 107–116.
- View Article
- Google Scholar
22. Cohen MM Jr (2006) Holoprosencephaly: clinical, anatomic, and molecular dimensions. Birth Defects Res Part A Clin Mol Teratol 76: 658–673.
- View Article
- Google Scholar
23. Ming JE, Muenke M (2002) Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am J Hum Genet 71: 1017–1032.
- View Article
- Google Scholar
24. Mercier S, Dubourg C, Garcelon N, Campillo-Gimenez B, Gicquel I, et al. (2011) New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases. J Med Genet 48: 752–760.
- View Article
- Google Scholar
25. Roessler E, Vélez JI, Zhou N, Muenke M (2012) Utilizing prospective sequence analysis of SHH, ZIC2, SIX3 and TGIF in holoprosencephaly probands to describe the parameters limiting the observed frequency of mutant gene×gene interactions. Mol Genet Metab 105: 658–664.
- View Article
- Google Scholar
26. Tenzen T, Allen BL, Cole F, Kang J-S, Krauss RS, et al. (2006) The cell surface membrane proteins Cdo and Boc are components and targets of the hedgehog signaling pathway and feedback network in mice. Dev Cell 10: 647–656.
- View Article
- Google Scholar
27. Zhang W, Hong M, Bae G-U, Kang J-S, Krauss RS (2011) Boc modifies the holoprosencephaly spectrum of Cdo mutant mice. Dis Model Mech 4: 368–380.
- View Article
- Google Scholar
28. Zhang W, Kang J-S, Cole F, Yi MJ, Krauss RS (2006) Cdo functions at multiple points in the Sonic Hedgehog pathway, and Cdo-deficient mice accurately model human holoprosencephaly. Dev Cell 10: 657–665.
- View Article
- Google Scholar
29. Hong M, Krauss RS (2012) Cdon mutation and fetal ethanol exposure synergize to produce midline signaling defects and holoprosencephaly spectrum disorders in mice PLoS Genet. 8: e1002999.
- View Article
- Google Scholar
30. Ingham PW, Nakano Y, Seger C (2011) Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 12: 393–406.
- View Article
- Google Scholar
31. Cole F, Krauss RS (2003) Microform holoprosencephaly in mice that lack the Ig superfamily member Cdon. Curr Biol 13: 411–415.
- View Article
- Google Scholar
32. Taniguchi K, Anderson AE, Sutherland AE, Wotton D (2012) Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway. PLoS Genet 8: e1002524.
- View Article
- Google Scholar
33. Warr N, Powles-Glover N, Chappell A, Robson J, Norris D, et al. (2008) Zic2-associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation. Hum Mol Genet 17: 2986–2996.
- View Article
- Google Scholar
34. Allen BL, Song JY, Izzi L, Althaus IW, Kang J-S, et al. (2011) Overlapping roles and collective requirement for the co-receptors Gas1, Cdo and Boc in Shh pathway function. Dev Cell 20: 775–787.
- View Article
- Google Scholar
35. Izzi L, Lévesque M, Morin S, Laniel D, fWilkes BC, et al. (2011) Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev Cell 20: 788–801.
- View Article
- Google Scholar
36. Marcucio RS, Young NM, Hu D, Hallgrimsson B (2011) Mechanisms that underlie co-variation of the brain and face. Genesis 49: 177–189.
- View Article
- Google Scholar
37. Schachter KA, Krauss RS (2008) Murine models of holoprosencephaly. Curr Top Dev Biol 84: 139–170.
- View Article
- Google Scholar
38. Mulieri PJ, Okada A, Sassoon DA, McConnell SK, Krauss RS (2000) Developmental expression pattern of the cdo gene. Dev Dyn 219: 40–49.
- View Article
- Google Scholar
39. Ming JE, Kaupas ME, Roessler E, Brunner HG, Golabi M, et al. (2002) Mutations in PATCHED-1, the receptor for SONIC HEDGEHOG, are associated with holoprosencephaly. Hum Genet 110: 297–301.
- View Article
- Google Scholar
40. Ribeiro LA, Murray JC, Richieri-Costa A (2006) PTCH mutations in four Brazilian patients with holoprosencephaly and in one with holoprosencephaly-like features and normal MRI. Am J Med Genet A 140: 2584–2586.
- View Article
- Google Scholar
41. Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15: 801–812.
- View Article
- Google Scholar
42. Lindström E, Shimokawa T, Toftgård R, Zaphiropoulos PG (2006) PTCH mutations: distribution and analyses. Human Mutation 27: 215–219.
- View Article
- Google Scholar
43. Briscoe J, Chen Y, Jessell TM, Struhl G (2001) A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol Cell 7: 1279–1291.
- View Article
- Google Scholar
44. Goodrich LV, Milenković L, Higgins KM, Scott MP (1997) Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277: 1109–1113.
- View Article
- Google Scholar
45. Mulieri PM, Kang J-S, Sassoon DA, Krauss RS (2002) Expression of the boc gene during murine embryogenesis. Dev Dyn 223: 379–388.
- View Article
- Google Scholar

[ref1] 1. Muenke M, Beachy PA (2001) Holoprosencephaly. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic & Molecular Bases of Inherited Disease. Eighth Edition ed. New York: McGraw-Hill. pp. 6203–6230.

[ref2] 2. Orioli IM, Castilla EE (2010) Epidemiology of holoprosencephaly: Prevalence and risk factors. Am J Med Genet C Semin Med Genet 154C: 13–21.
View Article
Google Scholar

[3] View Article

[4] Google Scholar

[ref3] 3. Shiota K, Yamada S (2010) Early pathogenesis of holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 22–28.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref4] 4. Krauss RS (2007) Holoprosencephaly: new models, new insights. Expert Rev Mol Med 9: 1–17.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref5] 5. Roessler E, Muenke M (2010) The molecular genetics of holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 52–61.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref6] 6. Solomon BD, Mercier S, Vélez JI, Pineda-Alvarez DE, Wyllie A, et al. (2010) Analysis of genotype-phenotype correlations in human holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 133–141.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref7] 7. Bae GU, Domené S, Roessler E, Schachter K, Kang JS, et al. (2011) Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors. Am J Hum Genet 89: 231–240.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref8] 8. Ribeiro LA, Quiezi RG, Nascimento A, Bertolacini CP, Richieri-Costa A (2010) Holoprosencephaly and holoprosencephaly-like phenotype and GAS1 DNA sequence changes: Report of four Brazilian patients. Am J Med Genet A 152A: 1688–1694.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref9] 9. Johnson CY, Rasmussen SA (2010) Non-genetic risk factors for holoprosencephaly. Am J Med Genet C Semin Med Genet 154C: 73–85.
View Article
Google Scholar

[24] View Article

[25] Google Scholar

[ref10] 10. Miller EA, Rasmussen SA, Siega-Riz AM, Frías JL, Honein MA, et al. (2010) Risk factors for non-syndromic holoprosencephaly in the National Birth Defects Prevention Study. Am J Med Genet C Semin Med Genet 154C: 62–72.
View Article
Google Scholar

[27] View Article

[28] Google Scholar

[ref11] 11. Kiecker C, Niehrs C (2001) The role of prechordal mesendoderm in neural patterning. Curr Opin Neurobiol 11: 27–33.
View Article
Google Scholar

[30] View Article

[31] Google Scholar

[ref12] 12. Muenke M, Beachy PA (2000) Genetics of ventral forebrain development and holoprosencephaly. Curr Opin Genet Dev 10: 262–269.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref13] 13. Rubenstein JL, Beachy PA (1998) Patterning of the embryonic forebrain. Curr Opin Neurobiol 8: 18–26.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref14] 14. Aoto K, Shikata Y, Imai H, Matsumaru D, Tokunaga T, et al. (2009) Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis. Dev Biol 327: 106–120.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref15] 15. Cordero D, Marcucio R, Hu D, Gaffield W, Tapadia M, et al. (2004) Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes. J Clin Invest 114: 485–494.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref16] 16. Geng X, Speirs C, Lagutin O, Inbal A, Liu W, et al. (2008) Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly. Dev Cell 15: 236–247.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref17] 17. Marcucio RS, Cordero DR, Hu D, Helms JA (2005) Molecular interactions coordinating the development of the forebrain and face. Dev Biol 284: 48–61.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref18] 18. McMahon AP, Ingham PW, Tabin CJ (2003) Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol 53: 1–114.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref19] 19. Helms JA, Cordero D, Tapadia MD (2005) New insights into craniofacial morphogenesis. Development 132: 851–861.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref20] 20. Hu D, Helms JA (1999) The role of Sonic hedgehog in normal and abnormal craniofacial morphogenesis. Development 126: 4873–4884.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref21] 21. Hu D, Marcucio RS (2009) A SHH-responsive signaling center in the forebrain regulates craniofacial morphogenesis via the facial ectoderm. Development 136: 107–116.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref22] 22. Cohen MM Jr (2006) Holoprosencephaly: clinical, anatomic, and molecular dimensions. Birth Defects Res Part A Clin Mol Teratol 76: 658–673.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref23] 23. Ming JE, Muenke M (2002) Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am J Hum Genet 71: 1017–1032.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref24] 24. Mercier S, Dubourg C, Garcelon N, Campillo-Gimenez B, Gicquel I, et al. (2011) New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases. J Med Genet 48: 752–760.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref25] 25. Roessler E, Vélez JI, Zhou N, Muenke M (2012) Utilizing prospective sequence analysis of SHH, ZIC2, SIX3 and TGIF in holoprosencephaly probands to describe the parameters limiting the observed frequency of mutant gene×gene interactions. Mol Genet Metab 105: 658–664.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref26] 26. Tenzen T, Allen BL, Cole F, Kang J-S, Krauss RS, et al. (2006) The cell surface membrane proteins Cdo and Boc are components and targets of the hedgehog signaling pathway and feedback network in mice. Dev Cell 10: 647–656.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref27] 27. Zhang W, Hong M, Bae G-U, Kang J-S, Krauss RS (2011) Boc modifies the holoprosencephaly spectrum of Cdo mutant mice. Dis Model Mech 4: 368–380.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref28] 28. Zhang W, Kang J-S, Cole F, Yi MJ, Krauss RS (2006) Cdo functions at multiple points in the Sonic Hedgehog pathway, and Cdo-deficient mice accurately model human holoprosencephaly. Dev Cell 10: 657–665.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref29] 29. Hong M, Krauss RS (2012) Cdon mutation and fetal ethanol exposure synergize to produce midline signaling defects and holoprosencephaly spectrum disorders in mice PLoS Genet. 8: e1002999.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref30] 30. Ingham PW, Nakano Y, Seger C (2011) Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 12: 393–406.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref31] 31. Cole F, Krauss RS (2003) Microform holoprosencephaly in mice that lack the Ig superfamily member Cdon. Curr Biol 13: 411–415.
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref32] 32. Taniguchi K, Anderson AE, Sutherland AE, Wotton D (2012) Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway. PLoS Genet 8: e1002524.
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref33] 33. Warr N, Powles-Glover N, Chappell A, Robson J, Norris D, et al. (2008) Zic2-associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation. Hum Mol Genet 17: 2986–2996.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref34] 34. Allen BL, Song JY, Izzi L, Althaus IW, Kang J-S, et al. (2011) Overlapping roles and collective requirement for the co-receptors Gas1, Cdo and Boc in Shh pathway function. Dev Cell 20: 775–787.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref35] 35. Izzi L, Lévesque M, Morin S, Laniel D, fWilkes BC, et al. (2011) Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev Cell 20: 788–801.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref36] 36. Marcucio RS, Young NM, Hu D, Hallgrimsson B (2011) Mechanisms that underlie co-variation of the brain and face. Genesis 49: 177–189.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref37] 37. Schachter KA, Krauss RS (2008) Murine models of holoprosencephaly. Curr Top Dev Biol 84: 139–170.
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref38] 38. Mulieri PJ, Okada A, Sassoon DA, McConnell SK, Krauss RS (2000) Developmental expression pattern of the cdo gene. Dev Dyn 219: 40–49.
View Article
Google Scholar

[111] View Article

[112] Google Scholar

[ref39] 39. Ming JE, Kaupas ME, Roessler E, Brunner HG, Golabi M, et al. (2002) Mutations in PATCHED-1, the receptor for SONIC HEDGEHOG, are associated with holoprosencephaly. Hum Genet 110: 297–301.
View Article
Google Scholar

[114] View Article

[115] Google Scholar

[ref40] 40. Ribeiro LA, Murray JC, Richieri-Costa A (2006) PTCH mutations in four Brazilian patients with holoprosencephaly and in one with holoprosencephaly-like features and normal MRI. Am J Med Genet A 140: 2584–2586.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref41] 41. Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15: 801–812.
View Article
Google Scholar

[120] View Article

[121] Google Scholar

[ref42] 42. Lindström E, Shimokawa T, Toftgård R, Zaphiropoulos PG (2006) PTCH mutations: distribution and analyses. Human Mutation 27: 215–219.
View Article
Google Scholar

[123] View Article

[124] Google Scholar

[ref43] 43. Briscoe J, Chen Y, Jessell TM, Struhl G (2001) A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol Cell 7: 1279–1291.
View Article
Google Scholar

[126] View Article

[127] Google Scholar

[ref44] 44. Goodrich LV, Milenković L, Higgins KM, Scott MP (1997) Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277: 1109–1113.
View Article
Google Scholar

[129] View Article

[130] Google Scholar

[ref45] 45. Mulieri PM, Kang J-S, Sassoon DA, Krauss RS (2002) Expression of the boc gene during murine embryogenesis. Dev Dyn 223: 379–388.
View Article
Google Scholar

[132] View Article

[133] Google Scholar

Figures

Abstract

Introduction

Results

Rescue of HPE in fetal alcohol-exposed Cdon mutant mice by reduced Ptch1 gene dosage

Effect of Ptch1 gene dosage on Shh and Nkx2.1 expression in the ventral forebrain of ethanol-treated Cdon−/− mice

Discussion

Materials and Methods

Ethics Statement

Mice

Histology and Whole Mount In Situ Hybridization

Supporting Information

Figure S1.

Figure S2.

Figure S3.

Acknowledgments

Author Contributions

References

Effect of Ptch1 gene dosage on Shh and Nkx2.1 expression in the ventral forebrain of ethanol-treated Cdon^−/− mice