Gli2 gene dosage and gene-environment interaction illuminate the

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DMM Advance Online Articles. Posted 1 September 2016 as doi: 10.1242/dmm.026328
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Gli2 gene dosage and gene-environment interaction illuminate
the etiological complexity of holoprosencephaly
Galen W. Heyne1,*, Joshua L. Everson1, 2,*, Lydia J. Ansen-Wilson1, Cal G. Melberg1, Dustin
M. Fink1, Kia F. Parins1, Padydeh Doroodchi1, Caden M. Ulschmid1, and Robert J. Lipinski1, 2,‡
1Department
of Comparative Biosciences, School of Veterinary Medicine, University of
Wisconsin-Madison, Madison, WI, United States of America
2Molecular
and Environmental Toxicology Center, School of Medicine and Public Health,
University of Wisconsin-Madison, Madison, WI, United States of America
* These authors contributed equally to this work
‡Corresponding
author:
E-mail: rjlipinski@wisc.edu
Keywords: Holoprosencephaly, birth defects, gene-environment, Hedgehog signaling
Summary Statement: This work illustrates how a specific genetic predisposition in
combination with an environmental exposure can result in a severe birth defect, providing a
Disease Models & Mechanisms • DMM • Advance article
novel opportunity to develop prevention strategies.
Abstract
Holoprosencephaly (HPE) is a common and severe human developmental abnormality
marked by malformations of the forebrain and face. While several genetic mutations have
been linked to HPE, phenotypic outcomes range dramatically and most cases cannot be
attributed to a specific cause. Gene-environment interaction has been invoked as a premise
to explain the etiological complexity of HPE but identification of interacting factors has been
extremely limited. Here, we demonstrate that mutations in Gli2, which encodes a Hedgehog
pathway transcription factor, can cause or predispose to HPE depending upon gene dosage.
On the C57BL/6J background, homozygous GLI2 loss of function results in the characteristic
brain and facial features of severe human HPE, including midfacial hypoplasia,
hypotelorism, and medial forebrain deficiency with loss of ventral neurospecification. While
normally indistinguishable from wildtype littermates, we demonstrate that mice with singleallele Gli2 mutations exhibit increased penetrance and severity of HPE in response to lowdose teratogen exposure. This genetic predisposition is associated with a Gli2 dosagedependent attenuation of Hedgehog ligand responsiveness at the cellular level. In addition
to revealing a causative role for GLI2 in HPE genesis, these studies demonstrate a
mechanism by which normally silent genetic and environmental factors can interact to
produce severe outcomes. Together, these findings provide a framework for understanding
the extreme phenotypic variability observed in human mutation carriers, and a paradigm for
reducing the incidence of this morbid birth defect.
Introduction
Holoprosencephaly (HPE) is the most prevalent human forebrain malformation, and one of
the most common of all developmental abnormalities, with an estimated prevalence of 1 in
250 conceptuses (Leoncini et al., 2008, Orioli and Castilla, 2010, Matsunaga and Shiota,
1977). HPE is defined by incomplete midline division of the embryonic forebrain and
frequently co-occurs with facial abnormalities, including hypotelorism, ophthalmologic
anomalies, midfacial hypoplasia, and orofacial clefts (Richieri-Costa and Ribeiro, 2010,
Cohen, 2006, Pineda-Alvarez et al., 2011). In surviving patients, HPE causes severe
intellectual disability and learning, behavior, and motor impairment (Cohen, 2006). More
often, the severity of effects results in prenatal or perinatal mortality (Solomon et al., 2010).
Clinical presentation of HPE is extremely variable. True HPE ranges from alobar forms,
marked by a single ventricle and no separation of the cerebral hemispheres, to lobar forms
with minor midline deficiencies (Solomon et al., 2010). However, obligate carriers of HPEassociated mutations frequently exhibit facial dysmorphology in the absence of detectable
brain abnormalities or no apparent phenotype (Solomon et al., 2012). The dramatic
variability in phenotypic expression is believed to stem from a complex, heterogeneous
etiology involving the interaction of genetic and environmental factors (Krauss and Hong,
2016). While this premise has become widely accepted, supportive experimental evidence
demonstrating specific interacting factors is limited. This knowledge gap hinders clinical
development of prevention strategies (Mercier et al., 2010).
In humans and animal models, HPE has been linked to chemical and genetic disruption of
the Hedgehog (Hh) signaling pathway (Roessler et al., 1996, Chiang et al., 1996, Heyne et al.,
2015a). Initiated by the Sonic Hedgehog (SHH) ligand, Hh signaling is required for ventral
patterning and expansion of the medial forebrain, as well as outgrowth of the processes that
form the midface. Hh signal transduction culminates in regulation of tissue-specific target
genes by the Gli family of zinc finger transcription factors, with GLI2 acting as the dominant
transcriptional activator (Lipinski et al., 2006). Serving as a poignant example of the
complexity that has frustrated basic and translational research efforts, the role of GLI2 in
HPE has remained unclear for two reasons. First, while single-allele GLI2 mutations have
been detected in individuals with HPE-like phenotypes, the majority of mutation carriers do
not exhibit the full manifestation of HPE or are clinically unaffected (Bear et al., 2014,
Franca et al., 2010, Roessler et al., 2003, Bertolacini et al., 2012, Rahimov et al., 2006).
Second, Gli2 knockout mice generated on an outbred CD-1 background do not recapitulate
management of HPE by limiting the accuracy of genetic counseling and stymying
the forebrain and facial abnormalities that characterize human HPE (Matise et al., 1998,
Park et al., 2000).
We examined the effect of GLI2 loss of function by backcrossing a null allele to the C57BL/6J
(B6) background, which has been shown to exacerbate craniofacial phenotypes, including
HPE. We found that a homozygous Gli2 mutation on the B6 background causes the salient
features of severe human HPE, including abnormalities of the face and forebrain. B6 Gli2+/mice and cells were then used to test whether normally silent single-allele mutations increase
sensitivity to a class of teratogens that includes environmental compounds. These functional
in vivo and mechanistic in vitro assays demonstrated a gene-environment interaction that
provides a basis to potentially reduce the incidence of this etiologically-complex disease in
susceptible human populations.
Results
GLI2 loss of function causes HPE
Mating of B6 Gli2+/- mice generated Gli2-/- fetuses at the expected Mendelian ratio (n=13/49)
at gestational day (GD)15. Midfacial hypoplasia and hypotelorism were observed in all Gli2-/animals on the B6 background (Fig. 1). These abnormalities co-occurred with absence of the
phenotypes observed in Gli2-/- fetuses is shown in Figure S1. Significant reductions in both
snout width and interocular distance were identified by linear measurement. Gli2-/- animals
also displayed microphthalmia and decreased head width suggestive of microcephaly. Facial
morphology in B6 Gli2+/- fetuses was indistinguishable from that of wildtype littermates.
Crown-rump length and limb morphology were not different between B6 Gli2+/+, Gli2+/-, and
Gli2-/- fetuses (Fig. S2).
Gli2-/- animals were also compared to wildtype B6 fetuses exposed to a single 40mg kg-1 dose
of the potent Hh signaling pathway antagonist vismodegib at GD7.75. This teratogenic
exposure regimen was recently reported to result in severe HPE phenotypes (Heyne et al.,
2015a). A remarkable degree of overlap in facial dysmorphology was observed among Gli2-/fetuses and those acutely exposed to vismodegib (Fig. 1). Importantly, these facial
phenotypes in mice closely mimic those observed in humans with severe forms of true HPE.
This is illustrated in the neonate shown in Figure 1G, who exhibits severe midfacial
hypoplasia, hypotelorism, and a single central nostril. MRI conducted prior to birth showed
upper lip notch and a single central or two closely opposed nostrils. The range of facial
that these facial features co-occurred with HPE, illustrated by an undivided cerebral cortex
and single ventricle.
We next investigated whether facial dysmorphology resulting from GLI2 loss of function cooccurs with forebrain abnormalities. Gli2-/- fetuses exhibited deficiency of the midbrain and
forebrain (Fig. 2). Narrowing of the anterior aspect of the cerebral cortices extended to the
olfactory bulbs, which were hypoplastic and abnormally closely spaced. A ventral view of the
forebrain further illustrates the medial deficiency observed in Gli2-/- fetuses (Fig. S3).
Vismodegib exposure in wildtype animals resulted in more pronounced deficiency of the
cerebral cortices and olfactory bulb aplasia. Gross brain morphology was indistinguishable
between Gli2+/- fetuses and their wildtype littermates.
The relationship between face and forebrain abnormalities resulting from GLI2 loss of
function was investigated in more detail by histologic examination. This revealed specific
features of midfacial hypoplasia including absence of the nasal septal cartilage, closely
spaced or fused nasal passages, and vomeronasal organ deficiency observed in each
examined Gli2-/- fetus (n=6/6) (Fig. 3C). One Gli2-/- fetus exhibited olfactory bulb agenesis,
while in each of the others (n=5/6), the olfactory bulbs were present but spaced abnormally
close (Fig. 3G). Medial forebrain deficiency, evidenced by absence of the septal region, was
observed in each B6 Gli2-/- fetus that was examined. The majority (n=4/6) exhibited
incomplete division of the cerebral cortices with a single communicating ventricle, the
defining feature of HPE (Fig. 3K). Severe abnormalities in the diencephalic region of the
included attenuation of the third ventricle, along with absence of both the anterior and
posterior lobes of the pituitary (Fig. 3O). The brains and faces of Gli2+/- fetuses were
histologically similar to those of their wildtype littermates.
The face and brain phenotypes observed in Gli2-/- fetuses were largely recapitulated by
vismodegib exposure at GD7.75. Teratogen-exposed B6 wildtype mice exhibited absence of
the nasal septal cartilage and forebrain septal region, and incomplete division of the cerebral
cortices (Fig. 3D,L). Notable differences detected in vismodegib-exposed fetuses included
higher penetrance of olfactory bulb agenesis, subtler diencephalic dysmorphology, and a
hypoplastic anterior pituitary (Fig. 3H,P).
Both GLI2 and the protein target of vismodegib, Smoothened, are expressed in SHH ligandresponding cells, where they are required for transduction of the downstream signaling
cascade (Lipinski et al., 2006, Robarge et al., 2009). Given the overlap in face and brain
forebrain were also observed. In all Gli2-/- fetuses, dysmorphology of the hypothalamus
phenotypes between Gli2-/- and vismodegib-exposed wildtype embryos, we hypothesized that
tissue deficiency in these animals involves populations of cells that are responding to SHH
signaling at or shortly after GD7.75. To test this hypothesis, we used a Tamoxifen-inducible
lineage reporter model to trace temporally-specific Hh-responsive cells (Ahn and Joyner,
2005). Tamoxifen administration at GD7.75 revealed Hh-responsive cell lineages populating
tissues that are deficient in Gli2-/- mice (Fig. 3, Q-T). Regions with positive staining included
the vomeronasal organs, connective tissue between the olfactory bulbs, cerebral cortices,
hypothalamus, and anterior pituitary gland, indicating that these areas contain the progeny
of cells that are responsive to Hh signaling at or shortly after GD7.75. This premise is
further supported by the observation that expression of the conserved Hh pathway target
gene Gli1 is reduced in the anterior neural plate/folds as early as GD8.0 in both Gli2-/- and
vismodegib exposed embryos (Fig. S4).
Hh signaling plays two distinct roles in early forebrain development. It acts as a mitogen,
promoting expansion of medial forebrain tissue and separation of the initially singular eye
field. At the same time, SHH ligand secretion from the notochord and floor plate of the
neural plate/tube specifies ventral neuroprogenitor cells. We tested whether attenuated
forebrain expansion caused by GLI2 loss of function coincides with disruptions in early
dorsal-ventral forebrain specification. At GD11.0, the morphogenesis of HPE was clearly
evident in Gli2-/- embryos (Fig. 4C). This was highlighted by incomplete separation and
hypoplasia of the telencephalic vesicles, along with absence of the medial nasal processes
that form the median aspect of the upper lip and nose. As shown in a wildtype control that
eminences and the ventral aspect of the diencephalon, while Pax6 is present in dorsal
aspects of the telencephalon and diencephalon (Corbin et al., 2003). Gli2-/- embryos
exhibited agenesis of the medial ganglionic eminences and absence of Nkx2.1 expression in
the telencephalon (Fig. 4G). Concurrently, the expression domain of Pax6 was expanded
into the ventral telencephalon (Fig. 4K). The altered forebrain patterning observed in Gli2-/embryos was largely recapitulated by vismodegib exposure. However, the diencephalic
domain of Nkx2.1 expression was drastically reduced in Gli2-/- embryos compared to those
exposed to vismodegib. The expression domains of Nkx2.1 and Pax6 were indistinguishable
between Gli2+/- and Gli2+/+ embryos.
We also examined expression of Shh and Gli1, with the latter serving as a reliable indicator of
Hh pathway activity. At GD11, Shh is normally expressed in the mantle region of the medial
ganglionic eminences, along the ventral aspect of the diencephalon, and in the zona limitans
intrathalamica (Fig. 4M). Gli1 expression reflects its paracrine responsiveness to stimulation
was hemisected in the sagittal plane, Nkx2.1 is normally expressed in the medial ganglionic
by secreted SHH. In Gli2-/- embryos, expression of Shh and Gli1 was nearly absent (Fig.
4O,S). In vismodegib-exposed wildtype embryos, Shh and Gli1 expression was diminished
but detectable in the ventral diencephalon and zona limitans intrathalamica (Fig. 4P, T).
Gli2 heterozygosity increases sensitivity to teratogen-induced HPE
In surviving human cohorts with HPE-associated phenotypes, variants in the GLI2 gene have
been identified as heterozygous loss of function mutations (Roessler et al., 2003). While
severe phenotypes have been observed in some GLI2 mutation carriers, most exhibit
incomplete HPE expressivity or are clinically normal. The data described above show that
mice with single-allele Gli2 mutations on the C57BL/6J background are phenotypically
indistinguishable from their wildtype littermates. These observations argue that GLI2 is
haplosufficient in the absence of additional genetic or environmental influences. To test
whether normally silent single-allele Gli2 mutations increase teratogenic sensitivity, direct
comparison was made between wildtype and heterozygous littermates by mating Gli2+/+
female and Gli2+/- male mice. As opposed to a 40mg/kg dose utilized in the experiments
described above, pregnant mice were exposed to a single dose of 2.5 or 10mg kg-1 vismodegib
at GD7.75. In the 2.5mg kg-1 vismodegib exposure group, HPE-associated facial
dysmorphology was detected in 15% (n=2/13) of Gli2+/- fetuses but in none (n=0/16) of the
wildtype littermates (Fig. 5). In the 10mg/kg vismodegib exposure group, dysmorphology
was detected in 67% (n=8/12) of Gli2+/- fetuses versus 17% (n=3/18) of wildtype fetuses. The
heterozygous fetuses compared to their wildtype littermates. Histologic examination
confirmed that the degree of observed facial dysmorphology closely corresponds with the
severity of forebrain abnormalities.
We next investigated the effect of Gli2 gene dosage on cellular responsiveness to SHH ligand
using embryonic fibroblasts from B6 Gli2+/+, Gli2+/-, and Gli2-/- mice. As expected, Gli2
mRNA abundance was dependent upon the number of functional Gli2 alleles (Fig. 6A). In
Gli2+/+ cells, stimulation with SHH peptide resulted in significant upregulation of Hh target
genes Gli1 and Ptc1. Comparison of expression levels in Gli2+/+, Gli2+/- and Gli2-/- cells
revealed a significant gene dosage effect. Specifically, the SHH-induced expression of these
genes incrementally decreased with loss of each functional Gli2 allele (Fig. 6B-C). We then
examined the effect of Gli2 haploinsufficiency on pathway sensitivity to vismodegib. Gli2+/+
and Gli2+/- cells appeared equally responsive to pathway inhibition by vismodegib. However,
severity of vismodegib-induced facial dysmorphology was also dramatically increased in Gli2
because of diminished capacity to respond to SHH stimulation, target gene expression was
lower in Gli2+/- cells at all vismodegib concentrations (Fig. 6D). Gli2+/- cells reached the level
of gene expression observed in Gli2-/- cells at a lower concentration of vismodegib than
Gli2+/+ cells. This in vitro result is consistent with the increased teratogenic sensitivity
observed in Gli2 heterozygous mice.
Discussion
Considerable effort has been made to elucidate the complex etiology of HPE, with recent
studies focusing on underlying genetic factors. However, most identified mutations are
heterozygous and demonstrate incomplete penetrance/expressivity. The tenuous connection
between GLI2 and HPE had typified this paradigm. While the defining medial forebrain
deficiency of true HPE has been identified in several individuals with single allele GLI2
mutations, most cases involve more subtle abnormalities, such as pituitary deficiency and/or
facial dysmorphology (Bear et al., 2014, Roessler et al., 2003, Bertolacini et al., 2012, Franca
et al., 2010, Rahimov et al., 2006). One explanation, proposed by Roessler et al., is that HPE
results from additional environmental or genetic influences superimposed on
the GLI2 haploinsufficent state (Roessler et al., 2003). This has become a widely accepted
overarching premise of HPE etiology, but while some interacting factors have been
identified, the majority of cases cannot be explained (Kietzman et al., 2014, Hong and
The studies described herein directly address the complex etiology of HPE, and provide a
framework to understand the extreme phenotypic variability observed in human GLI2
mutation carriers. We provide the first evidence definitively linking GLI2 to HPE, by
demonstrating that a homozygous mutation causes the defining brain and face
malformations. We also show that a single-allele mutation is normally silent but
dramatically increases sensitivity to HPE induced by low-dose teratogen exposure. These
findings establish a novel model to further elucidate the intricate gene-environment
interactions that have frustrated clinical management of this common human birth defect.
In addition to variable clinical findings, the uncertain role of GLI2 in HPE pathogenesis was
obfuscated by the initial characterization of Gli2 knockout mice. These mice were generated
and maintained on an outbred CD-1/129 background and described to have incompletely
penetrant abnormalities of the vertebrae and limbs, as well as secondary palate clefts,
pituitary hypoplasia, and midbrain deficiency (Mo et al., 1997, Matise et al., 1998, Park et al.,
Krauss, 2012, Roessler and Muenke, 2010).
2000). However, the forebrain and midfacial deficiency phenotypes characteristic of HPE
were not observed (Blaess et al., 2006). This discordance extended to cellular and molecular
findings. Gli2-/- fetuses on an outbred CD-1 background were found to have intact
expression of the ventral neurospecification marker Nkx2.1 and grossly normal medial
ganglionic eminences (Park et al., 2000). These transient developmental structures give rise
to inhibitory cortical interneurons, cell populations that are depleted in individuals with true
HPE (Fertuzinhos et al., 2009). Here, we demonstrate that when backcrossed to the
C57BL/6J background, homozygous Gli2 mutations cause the characteristic brain and face
abnormalities of severe HPE, including severe diminishment of the medial ganglionic
eminences, medial forebrain deficiency, hypotelorism, and midfacial hypoplasia. These
structural malformations followed diminished Hh signaling pathway activity and severely
disrupted forebrain patterning.
Backcrossing gene mutations to the C57BL/6 background has previously been shown to
reveal or exacerbate HPE penetrance and/or expressivity. Specific examples include
homozygous mutations in the Hh ligand co-receptor Cdo, a predicted truncating mutation in
Tgif, and single- allele mutations in Six3, a transcription factor upstream of Shh (Kuang et
al., 2006, Zhang et al., 2006, Geng et al., 2008). These reports, along with the findings
presented in this study, suggest that the C57BL/6 background includes one or more yet-tobe-identified HPE modifier genes. Demonstration that GLI2 loss of function on the B6
inbred strain results in severe HPE phenotypes with complete phenotypic penetrance
provides a new opportunity to uncover background-specific interacting genetic variations
While this study focused on gene-environment interaction, gene-gene interactions have been
postulated to play a role in the complex etiology of HPE. As a basic test of this concept, we
generated mice heterozygous for Gli2 and Shh, the latter being the most commonly mutated
gene identified in in non-chromosomal HPE. When backcrossed to the C57BL/6J
background, Shh-/- embryos exhibited severe HPE phenotypes, including severe midfacial
hypoplasia and a proboscis (Fig. S5). However, Shh+/-Gli2+/- fetuses were apparently normal
and indistinguishable from single heterozygotes and wildtype littermates. This suggests that
Shh heterozygosity does not interact with Gli2 haploinsufficiency. A functional interaction
with a downstream gene is perhaps more likely given the role of GLI2 as a direct activator of
Hh target genes. However, the tissue specific Hh target genes that are regulated by GLI2 and
mediate the pathogenesis of HPE are not known. Moving forward, use of the
complementary genetic and teratogenic models of HPE characterized here provides an ideal
that may represent novel and clinically-relevant predisposing factors.
platform to identify Hh target genes involved in HPE pathogenesis and to test novel genegene interactions.
While GLI2 loss of function causes chronic Hh pathway disruption, the effect of acute
pathway disruption can be examined by targeted exposure of the potent and specific
inhibitor vismodegib. We found that the anatomical and molecular phenotypes caused by
GLI2 loss of function were largely recapitulated by acute exposure to vismodegib at GD7.75.
This observation suggests that the abnormalities we observed in GD15 fetuses are caused in
part by changes to cell populations that respond to SHH signaling during the early
neurulation stage of embryogenesis. This period of development (GD8.0 and shortly after)
corresponds to the fourth week of human gestation and has been shown to be sensitive to
other chemicals that cause HPE (Lipinski et al., 2010). This premise was further supported
by genetic fate mapping using an inducible system that labels Hh-responsive cells and their
progeny at discrete periods of development. Tamoxifen exposure at GD7.75 demonstrated
Hh-responsive cell lineages in specific medial facial and forebrain compartments
corresponding with tissues that were deficient in Gli2-/- and vismodegib-exposed mice.
However, several differences were also observed between the genetic and teratogenic models
of HPE that we investigated. Gli2-/- animals exhibited more severe pituitary and
diencephalic abnormalities but subtler olfactory bulb deficiency than those exposed to
vismodegib. Loss of GLI2 function also resulted in a more extensive disruption of Hh
pathway activity and ventral specification in the diencephalic region of the forebrain. These
differences likely reflect, at least in part, the pathogenic effects of chronic versus acute
these differences, like differential effects on Gli3 repression, are also possible and should be
considered in future investigation.
Directly addressing the premise of gene-environment interaction, our study demonstrates
that normally silent single-allele Gli2 mutations dramatically increase teratogenic sensitivity.
This result is congruous with our previous study examining the effect of prenatal alcohol
exposure in the context of Gli2 heterozygosity (Kietzman et al., 2014). These findings
illustrate that a functional predisposition can exacerbate the teratogenic effects of two
unique classes of environmental influences, resulting in severe birth defects with largely
overlapping phenotypes. Together these findings provide a construct for understanding the
extremely variable phenotypes exhibited in clinical populations, while highlighting the
emerging consensus that HPE and other birth defects likely result from complex interactions
of genetic and environmental factors.
attenuation of the Hh pathway inherent in these models. Additional factors contributing to
We utilized the potent Hh pathway antagonist vismodegib to test the concept of geneenvironment interaction in the context of Gli2 heterozygosity. Importantly, vismodegib is
part of a larger class of compounds that inhibit Hh signaling by binding the transmembrane
protein Smoothened (Robarge et al., 2009, Chen et al., 2002, Keeler, 1975, Chen, 2016).
With both synthetic and natural small molecules, this group includes more than 20
compounds and continues to grow with increasing attention focused on the biological effects
of Hh pathway inhibition. With respect to HPE etiology, perhaps the most intriguing Hh
pathway inhibitors are identified environmental small molecules, including dietary alkaloids,
an antifungal agent, a human dietary supplement, and a pesticide synergist present in
hundreds of insecticide formulations (Lipinski et al., 2007, Lipinski and Bushman, 2010,
Wang et al., 2012). These compounds are less potent than vismodegib and appear unlikely
to act independently to cause birth defects at typical environmental concentrations.
However, the findings presented here illustrate the “multiple hit” hypothesis of complex
disease and should prompt careful investigation of the etiological role of environmental Hh
pathway inhibitors in the context of predisposing mutations in genes like Gli2. Building
upon the study presented here, continued elucidation of the intricate gene-environment
interactions that cause HPE provides a direct path to improving clinical management and
developing effective prevention strategies.
Animals models. Mouse (Mus musculus) studies were carried out in strict accordance with
the recommendations in the Guide for the Care and Use of Laboratory Animals of the
National Institutes of Health. The protocol was approved by the University of Wisconsin
School of Veterinary Medicine Institutional Animal Care and Use Committee (protocol
number 13-081.0). Gli2+/- mice (Matise et al., 1998) were backcrossed to the C57BL/6J
background for more than 15 generations. C57BL/6J wildtype mice were purchased from
The Jackson Laboratory (Bar Harbor, ME, USA). Gli1CreERT2 (stock no: 007913) and
Rosa26lacZ (stock no: 003474) mice were purchased from The Jackson Laboratory. All mice
were housed under specific pathogen-free conditions in disposable, ventilated cages
(Innovive, San Diego, CA) in rooms maintained at 22 ±2 degrees Celsius and 30-70%
humidity on a 12 hr light, 12 hr dark cycle. Mice were fed 1919x Irradiated Harlan Teklad
Global Soy Protein-Free Extruded Rodent Diet. For timed matings, 1-3 female mice between
8 and 20 weeks of age were placed with a single male for 1-2 hrs and subsequently examined
for the presence of copulation plugs. The beginning of the mating period was designated as
Materials and Methods
GD0. True pregnancy was confirmed by assessing weight gain between GD7 and 10 before
dam treatment or embryo harvest as previously described (Heyne et al., 2015b).
Hedgehog signaling antagonist exposure. Vismodegib was purchased from LC Laboratories
(Woburn, MA, USA) was suspended as 3 mg ml-1 in 0.5% methyl cellulose with 0.2% tween
as previously described (Heyne et al., 2015a). Individual suspensions were prepared within
30 min of administration. Pregnant mice were administered 2.5, 10, or 40 mg kg-1
vismodegib (aka GDC-0449) by oral gavage at GD7.75.
Dissection, imaging, and fetal phenotyping. Pregnant dams were euthanized at indicated
stages of development by CO2 asphyxiation and subsequent cervical dislocation. Fetal
specimens were fixed in 10% formalin or Bouin’s solution for at least 1 week prior to imaging.
Images were captured using a micropublisher 5.0 camera connected to a Nikon SZX-10
stereomicroscope. Linear facial measurements of formalin-fixed GD15 fetuses, were
produced in Photoshop v14.1.2 as previously described (Lipinski et al., 2014). For
comparison of face and brain morphology, images of Bouin’s-fixed tissue were captured and
converted to grey-scale. The severity of HPE phenotypes in GD17 fetuses was assessed by a
single rater blinded to treatment based upon a semi quantitative scale as previously
described (Heyne et al., 2015a, Kietzman et al., 2014).
Histology. GD15 fetuses were fixed in Bouin’s solution for at least one week, and then
transferred to 70% ethanol. GD17 fetuses were fixed in 10% formalin. Following paraffin
Fate mapping. Gli1CreERT2+/- male mice were mated with Rosa26lacZflox/flox females. 50mg
ml-1 Tamoxifen dissolved in corn oil was administered to pregnant dams by IP injection at
GD7.75. GD15 fetuses were fixed overnight in 2% paraformaldehyde with 0.2%
glutaraldehyde. Embryonic tissues were then embedded in 4% agarose and 150μm coronal
sections were produced by vibrating microtome and x-gal stained as described previously
(Mehta et al., 2011).
In situ hybridization. ISH was performed using an established high-throughput technique
that allows multiple treatment groups to be processed identically and as a single unit (Abler
et al., 2011). For GD11 embryos, mid-sagittal hemisection using a scalpel was performed
before staining. Hemisected embryos were incubated in proteinase K (2.5µg ml1)/collagenase
(250µg ml-1) for 2 minutes prior to initial washes. Younger embryos were not
subjected to proteinase K/collagenase treatment. ISH probe primers were resuspended as
embedding, 10μm sections were produced and stained with H&E by standard protocols.
stock solutions of 100mM in TE buffer pH7.0 (Ambion). Working stocks were made as
10mM solutions containing both forward and reverse primers. Primer sequence for probe
generation are listed in supplemental table S1.
Mouse embryonic fibroblast isolation and treatment. Mouse embryonic fibroblasts were
harvested from GD15 embryos as previously described (Lipinski et al., 2006). Cells were
grown to confluence in DMEM [with l-glutamine, 4.5g L-1 glucose, without sodium pyruvate],
with 10% FCS and 1% Pen/Strep. They were then trypsinized and plated in 24 or 48 well
tissue culture plates (Falcon, Franklin Lakes, NJ, USA) at 4.0 × 105 cells ml-1 media. Cells
were allowed to attach for 24 hr and media were replaced with DMEM containing 1% FBS ±
SHH-N peptide (R&D Systems, Minneapolis, MN, USA) at 0.4 μg ml-1, ± vismodegib (LC
Laboratories) at indicated concentrations. For cell culture experiments, vismodegib was
dissolved in DMSO.
RNA isolation and Real Time RT-PCR. RNA was isolated using GE Illustra RNAspin
kits. 100 ng of RNA was reverse transcribed using the Promega GoScript Reverse
Transcription System. Both kits were used according to manufacturer's instructions. Realtime PCR was performed using a BioRad CFX96 Touch real-time PCR detection system.
Reaction mixtures contained 6 µl SSoFast EvaGreen Supermix (BioRad Laboratories,
Hercules, CA, USA), 4.75 µl ddH2O, 0.75 µl cDNA, and 0.5 µl 10 mM combined forward and
reverse gene-specific primers. Primers were resuspended as stock solutions of 100mM in TE
buffer pH7.0. Working stocks were made as 10mM solutions containing both forward and
follows: 1 cycle at 95°C for 3 min, then 40 cycles of 95°C for 10 s followed by 30 s at 60°C
(annealing temperature). To confirm specificity, primer sequences were analyzed with
BLAST and melt curves were examined for a single peak in the expected temperature range.
Gapdh was used as the housekeeping gene and analysis was conducted with the 2^-ddCt
method.
Statistics. Analysis of linear measurements was made using one-way ANOVA followed by
Tukey's HSD test using Graphpad Prism software (v6.04). Differences in the frequency of
facial dysmorphology between experimental groups were assessed by one-tailed Fisher’s
exact test using Graphpad Prism software. T-tests were used to determine whether gene
expression was changed by SHH stimulation in mouse embryonic fibroblasts. To test
whether basal and Shh-induced target gene expression preserved the ordering implicitly
suggested by the genotypes, a Jonckheere–Terpstra test was performed using Mstat version
reverse primers. Primer sequences are listed in Table S2. Reaction conditions were as
6.1.4 (http:/mcardle.oncology.wisc.edu/mstat/download/index.html). An alpha value of
0.05 was maintained for all analyses.
Human images. Parental written informed consent was received for inclusion and
publication of patient images in Figure 1.
Acknowledgements
The authors thank Dr. Ruth Sullivan for consultation of study design, discussion of results,
and review of the manuscript. We also thank Dr. Paul Kruszka and the patient’s family for
use of images depicting the clinical manifestation of HPE.
Competing Interests
No competing interests declared.
Author Contributions
RJL, GWH, LAW designed studies; JLE, CGM, LAW, GWH, DMF, KFP, PD, CMU, and RJL
conducted experiments and acquired data; JLE, KFP, DMF, and RJL analyzed data; RJL
wrote the manuscript.
Funding
This work was supported by grants R00DE022101 to RJL from the National Institute of
Dental and Craniofacial Research/National Institutes of Health, T32ES007015-37 to JLE
from the National Institute of Environmental Health Sciences/National Institutes of Health,
pilot funding from the University of Wisconsin Molecular and Environmental Toxicology
Center.
T35OD011078 to LAW from the National Institutes of Health/Office of the Director, and
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Figures
Fig. 1. GLI2 loss of function causes HPE-associated facial dysmorphology. (A-D)
GD15 B6 Gli2+/+, Gli2+/-, Gli2-/- fetuses are shown along with a Gli2+/+ fetus exposed to 40mg
kg-1 vismodegib (Vis) at GD7.75. Snout width (SW), interocular distance (IOD), and head
width (HW) was measured as illustrated by dashed lines shown in E. (F) Linear
plot. Values represent the mean ± s.e.m. (Gli2+/+ n=6; Gli2+/- n=9; Gli2-/- n=6; Gli2+/++ Vis,
n=12). * p≤0.05 by one-way ANOVA followed by Tukey's HSD. Scale bar = 1 mm.
measurements were normalized to Gli2+/+ control group values and shown on a semi-log
Fig. 2. Facial dysmorphology in Gli2-/- fetuses co-occurs with brain
abnormalities. (A-D) Shown are animals from the same experimental groups described in
Fig. 1. To visualize correlative phenotypes, images of the dorsal surface of the brain along
with facial images of the same animal. Images were captured following Bouin’s fixation and
converted to grayscale. In the Gli2-/- brain, hypoplasia of the cerebral cortices (cc) and
midbrain (mb) is apparent. The olfactory bulbs (ofb) are hypoplastic and abnormally
approximated. In the Gli2+/+ fetus exposed to vismodegib, more severe deficiency of the
cerebral cortices and absence of the olfactory bulbs is observed. Scale bar = 1 mm.
Fig. 3. GLI2 loss of function results in deficiency of medial forebrain and facial
tissue. (A-P) Serial histological images are shown from the same experimental groups
described in Figs. 1 and 2. Dashed boxes in low magnification images on the left provide
context for the coronal sections shown in each row. Gli2-/- and Gli2+/+ vismodegib-exposed
fetuses exhibit absence of nasal septal (ns) cartilage and diminished vomeronasal organs
deficient in the Gi2-/- fetus, while the olfactory bulbs are absent in the Gli2+/+ vismodegibexposed fetus. These fetuses also have severe medial forebrain deficiencies, including
absence of the septal region (s) and communicating lateral ventricles (lv). In the Gli2-/- fetus,
attenuation of the third ventricle (tv) and absence of the anterior pituitary (ap) lobe are also
apparent. The anterior pituitary is present but hypoplastic in the Gli2+/+ vismodegibexposed fetus, which also exhibits subtle attenuation of the third ventricle. (Q-T) A genetic
fate mapping system (23) was used to identify Hh-responsive cell lineages. Embryos
exposed to Tamoxifen at GD7.75 were sectioned and stained with X-gal to visualize Hhresponsive cells and their progeny. 100 uM sections were produced at planes comparable to
those stained by H&E. Tongue (t). Scale bar = 0.25 mm.
(vo). The midline connective tissue separating the olfactory bulbs (ofb) marked by the (*) is
patterning. Shown are GD11 embryos of the same experimental groups described in Figs 13. (A-D) Frontal images show forebrain and facial morphology. Gli2-/- and Gli2+/+ fetuses
exposed to vismodegib exhibit hypoplasia and abnormal approximation of the telencephalic
vesicles (t), absence of the medial nasal processes (mnp), and a single central nostril (n).
Embryos were hemisected near the midline and subjected to ISH staining for the indicated
genes. In the Gli2+/+ control, Nkx2.1 is expressed in the medial ganglionic eminences (mge)
and the ventral aspect of the diencephalon (d), while Pax6 is present in the dorsal aspect of
Fig. 4. GLI2 loss of function results in altered dorsal-ventral forebrain
the telencephalon and diencephalon. Shh is expressed in the mantle region of the medial
ganglionic eminence, along the ventral aspect of the diencephalon, and in the zona limitans
intrathalamica (zli). Gli1 expression reflects its paracrine responsiveness to stimulation by
secreted SHH. Eye (e). Scale bar = 1 mm.
Fig. 5. Gli2 heterozygosity increases teratogenic sensitivity. Wildtype dams
carrying Gli2+/+ and Gli2+/- embryos were exposed to 2.5 or 10mg kg-1 vismodegib at GD7.75.
The graph at the left shows percentage of GD17 fetuses classified as having mild, moderate,
or severe HPE-associated facial dysmorphology. * p<0.05 by Fisher’s exact test. n=5
litters/treatment group. (A-D) Categories of facial morphology are illustrated by GD17
fetuses of indicated genotype and dose group. Animals that were indistinguishable from
controls were assigned “normal” (A). Those with a diminished area of pigmentation between
the nostrils were assigned as “mild” (B). Animals with a gross deficiency of the median lip
notch but some remaining pigment remaining at the tip of the nose were assigned as
“moderate” (C). Those with an absent lip notch and single central nostril were assigned
degree of facial dysmorphology corresponds to forebrain abnormalities. Note deficiency of
the septal region (s) in (B’) and absent septal region and single communicating lateral
ventricle in (C’) and (D’). Scale bar = 1 mm.
“severe” (D). (A’-D’) Histologic sections through the cerebral cortices illustrate that the
Gli2+/-, Gli2-/- mouse embryonic fibroblasts (MEFs) were treated ± SHH ligand for 48 hrs.
Expression of Gli2 and the conserved Hh target genes Gli1 and Ptc1 were measured by Real
Time RT-PCR and normalized to Gapdh. *p<0.05 as determined by t-test for genotypespecific gene expression. Arrows indicate a significant gene dosage effect determined by
Jonckheere-Terpstra test. (D) Gli2+/+ and Gli2+/- fibroblasts were treated ± SHH ligand and
graded concentrations of vismodegib for 48 hrs. Induction of Ptc1 (SHH/Vehicle) relative to
Gli2+/+ cells is shown in the semi-log plot. The dashed line shows relative Ptc1 induction in
Gli2-/- fibroblasts to illustrate a theoretical threshold of teratogenicity. For each graph,
values represent the mean ± s.e.m. of 6 replicate experiments.
Fig. 6. Gli2 heterozygosity diminishes SHH-ligand responsiveness. (A-C) Gli2+/+,

Gli2 gene dosage and gene-environment interaction illuminate the

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