Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly
Many common structural birth defects appear to arise from a complex and ill-defined combination of genetic and environmental factors ( Krauss and Hong , 2016 ) . The notion that environmental agents affect penetrance and expressivity of predisposing mutations is speculated to underlie many human congenital anomalies , but has been difficult to demonstrate . Animal models are therefore valuable , but these have been slow to emerge . Some recent successful examples are a zebrafish model of craniofacial defects ( Pdgfra mutations and fetal alcohol ) , and mouse models of scoliosis ( Notch pathway mutations plus hypoxia ) , facial clefting ( Pax3 mutations plus 2 , 3 , 7 , 8-tetrachlorodibenzodioxin ( TCDD ) ) , and holoprosencephaly ( Hedgehog pathway mutations and fetal alcohol ) ( Hong and Krauss , 2012; Kietzman et al . , 2014; McCarthy et al . , 2013; Sparrow et al . , 2012; Zalc et al . , 2015 ) . Holoprosencephaly ( HPE ) is a common developmental defect in midline patterning of the forebrain and/or midface ( Muenke and Beachy , 2001 ) . HPE occurs approximately once per 250 human conceptions , with an associated in utero lethality of ~97% ( Leoncini et al . , 2008; Shiota and Yamada , 2010 ) . HPE is characterized by an unbroken continuum of rostroventral midline anomalies that range from complete failure to partition the forebrain into hemispheres plus cyclopia , through progressively less severe defects including partially partitioned forebrain , single nostril , and midface hypoplasia ( Cohen , 2006; Krauss , 2007; Muenke and Beachy , 2001 ) . Development of the rostroventral midline is initiated by signals derived from the prechordal plate ( PCP ) ( Kiecker and Niehrs , 2001; Marcucio et al . , 2011 ) . The PCP produces Sonic hedgehog ( SHH ) , initiating a sequence of events that progressively patterns the forebrain and midface ( Aoto et al . , 2009; Cordero et al . , 2004; Geng and Oliver , 2009; Kiecker and Niehrs , 2001; Rubenstein and Beachy , 1998; Zhang et al . , 2006 ) . HH ligands trigger signaling by binding the primary receptor Patched1 ( PTCH1 ) . In the absence of ligand , PTCH1 constrains the activity of a second membrane protein , Smoothened ( SMO ) . HH binding inhibits PTCH1 function , allowing SMO-dependent signals to activate GLI transcription factors and pathway-specific gene expression . HH ligand availability and signal reception are also regulated by a series of co-receptors and additional HH-binding proteins , including CDON , BOC , GAS1 , and LRP2 ( Beachy et al . , 2010; Kong et al . , 2019; Lee et al . , 2016; Petrov et al . , 2017; Willnow and Christ , 2017 ) . The PCP develops from the anterior primitive streak ( APS ) under the influence of Nodal pathway signaling ( Robertson , 2014; Schier , 2009; Shen , 2007 ) . Therefore , the Nodal pathway is developmentally upstream of the HH pathway in rostroventral midline patterning . Consistent with this conclusion , defective Nodal signaling at the primitive streak stage of development leads to defects in HH signaling and HPE ( Krauss , 2007 ) . Nodal is a TGFβ superfamily member and signals through a receptor complex including the type I and type II receptors , ALK4 and Activin receptor IIA/B ( ActRIIA/B ) , and a GPI-linked co-receptor , TDGF1 ( also called Cripto ) ( Robertson , 2014; Schier , 2009; Shen , 2007 ) . Receptor activation results in phosphorylation of the transcription factors SMAD2 and SMAD3 , and activation of genes required for PCP induction ( Robertson , 2014; Schier , 2009; Shen , 2007 ) . Among these genes are Foxa2 and Gsc ( Ang and Rossant , 1994; Belo et al . , 1998; Filosa et al . , 1997 ) . Nodal signaling also induces expression of Lefty1 and Lefty2 , which encode secreted inhibitors of the pathway that bind to TDGF1 and Nodal itself ( Robertson , 2014; Schier , 2009; Shen , 2007 ) . The etiology of HPE is complex , involving both genetic and environmental risk factors ( Addissie et al . , 2020; Dubourg et al . , 2018; Hong and Krauss , 2018; Johnson and Rasmussen , 2010; Krauss , 2007; National Birth Defects Prevention Study et al . , 2010; Muenke and Beachy , 2001; NISC Comparative Sequencing Program et al . , 2018; Roessler et al . , 2018; Summers et al . , 2018 ) . Heterozygous , loss-of-function mutations in components or regulators of the HH , Nodal , and FGF signaling pathways are associated with HPE ( NISC Comparative Sequencing Program et al . , 2018; Roessler et al . , 2018 ) . Epidemiology of HPE is less advanced than genetic analyses , but among the environmental risk factors implicated is fetal alcohol exposure ( Abe et al . , 2018; Cohen and Shiota , 2002; Croen et al . , 2000 ) , though this is not always observed ( Addissie et al . , 2020 ) . A full range of clinical phenotypes is seen in both sporadic and familial HPE ( Muenke and Beachy , 2001; Solomon et al . , 2010 ) . Many mutation carriers in pedigrees lack clinical manifestation , and many apparently sporadic cases have inherited a mutation from a minimally affected parent ( Lacbawan et al . , 2009; Solomon et al . , 2012 ) . It is likely , therefore , that HPE-associated mutations are not sufficient to produce midline defects , but are the substrate on which a complex landscape of genetic and/or environmental modifiers act . Statistical analysis is consistent with a multifactorial , ‘autosomal dominant with modifier’ model , wherein the penetrance and expressivity of a heterozygous driver mutation is graded by the modifier landscape ( Dubourg et al . , 2018; Hong and Krauss , 2018; Roessler et al . , 2012 ) . We have modeled this phenomenon in mice with high fidelity . CDON encodes a multi-functional co-receptor , including for the HH pathway , ( Lu and Krauss , 2010; Sanchez-Arrones et al . , 2012 ) . CDON loss-of-function mutations have been identified in human HPE patients ( Bae et al . , 2011; NISC Comparative Sequencing Program et al . , 2018 ) , but such variants are relatively common in the human population . Additionally , a patient with a rare homozygous CDON mutation displayed retinal coloboma , a mild HPE-associated eye phenotype also seen in Cdon-/- mice ( Berkun et al . , 2019; Pineda-Alvarez et al . , 2011; Zhang et al . , 2009 ) . Therefore , CDON mutations likely require additional insults to contribute to HPE . Studies with mice are consistent with this conclusion . Cdon-/- mice on a 129S6 genetic background have a subthreshold defect in HH signaling and are subject to HPE induced by genetic and environmental modifiers; among the latter is ethanol ( EtOH ) ( Hong and Krauss , 2018 ) . Cdon mutation or in utero EtOH exposure individually yielded little effect on 129S6 mice . The combination , however , synergized to inhibit HH signaling in the developing forebrain and produced a complete spectrum of HPE phenotypes with high penetrance ( Hong and Krauss , 2012 ) . Furthermore , fetal EtOH induced a low penetrance of HPE in C57BL/6J mice , and this was exacerbated by heterozygosity for Shh or Gli2 ( Kietzman et al . , 2014 ) . Consistent with the notion that a threshold of HH signaling activity is rate-limiting in midline patterning , genetic removal of one copy of the negative pathway regulator , Ptch1 , rescued 129S6 Cdon-/- mice from EtOH-induced HPE ( Hong and Krauss , 2013 ) . Defective HH pathway function may be a final common mechanism for all classical forms of HPE , so these results do not prove that HH signaling is the direct synergistic target of Cdon mutation and in utero EtOH exposure . Several lines of evidence argue that EtOH itself , rather than a consequence of its metabolism , is the HPE-inducing teratogen ( Hong and Krauss , 2017 ) . In this study , we demonstrate that the window of sensitivity to EtOH-induced HPE in 129S6 Cdon-/- mice is closed by embryonic day ( E ) 7 . 5 , with the sensitivity period overlapping Nodal-mediated specification of the PCP from the APS . Furthermore , we find that CDON interacts genetically and physically with regulators of Nodal signaling . Finally , EtOH inhibits Activin/Nodal pathway signaling in vitro in mouse epiblast stem cells ( mEpiSCs ) , which have transcriptional and functional properties that resemble APS cells ( Kojima et al . , 2014; Tsakiridis et al . , 2014 ) . Together these results argue that , unexpectedly , Cdon mutation and fetal alcohol synergize to induce HPE by interfering with Nodal signaling . These results illuminate mechanisms of gene-environment interaction in a high fidelity , multifactorial model of a common birth defect .
All studies were performed with mice on a 129S6 background and are referred to only by genotype unless otherwise noted . Our model uses one-hour timed mating of Cdon+/- mice , allowing rigorous identification of a window of sensitivity for EtOH’s teratogenic effects . The standard EtOH treatment regimen involves one dose at E7 . 0 and a second dose four hours later . This results in a full range of HPE phenotypes in Cdon-/- mice with overall penetrance of ~75% ( Hong and Krauss , 2012 ) . The protocol is analogous to the original regimen developed to model fetal alcohol spectrum disorders in C57BL/6J mice , wherein ~ 19% developed HPE phenotypes ( Aoto et al . , 2008; Sulik et al . , 1981 ) . HPE phenotypes were not induced in wild type C57BL/6J mice when EtOH was administered at E6 . 5 or E7 . 5 ( Aoto et al . , 2008 ) . We have shown that EtOH is ineffective in Cdon-/- mice when administered at E8 . 0 ( Hong and Krauss , 2012 ) . To further refine the temporal window of sensitivity , we assessed the ability of EtOH to induce HPE in Cdon-/- animals when administered at E7 . 25 and E7 . 5 , with embryos examined for external HPE phenotypes at E14 . 0 . EtOH was effective at E7 . 25 , but not at E7 . 5 ( Table 1 and Figure 1 ) . EtOH itself is the HPE-inducing teratogen in this system ( Hong and Krauss , 2017 ) . The developmental processes directly perturbed by EtOH must therefore occur: 1 ) during the sensitive time window; and 2 ) prior to EtOH clearance , even if defects in midline development occur later . When administered at E7 . 0 , maternal blood EtOH levels peaked at E7 . 25 and were no longer detectable by ~E7 . 75 ( Hong and Krauss , 2012 ) . Critically , EtOH is no longer effective at E7 . 5 , a time reported to be prior to SHH expression and function in prechordal plate-mediated rostroventral midline patterning ( Echelard et al . , 1993; McMahon et al . , 2003 ) . These results argue that EtOH targets processes other than , or in addition to , HH signaling in Cdon-/- mice . Consistent with this conclusion , the time of peak sensitivity to HPE induced by the direct SMO inhibitor , Vismodegib , was E7 . 5 , 6–12 hr later than for EtOH ( Heyne et al . , 2015 ) . Both the window of sensitivity to EtOH and peak EtOH levels overlap with induction of the PCP by Nodal signaling . We previously observed that expression of Foxa2 and Gsc , two Nodal target genes that mark the APS ( from which the PCP is derived ) , was diminished at E7 . 25 in EtOH-treated Cdon-/- embryos ( Hong and Krauss , 2012 ) . Both Cdon mutation and EtOH exposure were required for this effect . Expression of Lefty2 , a direct Nodal pathway target gene , was also reduced at E7 . 25 specifically in EtOH-treated Cdon-/- embryos ( Figure 2 ) . Taken together , these results showed that the synergy between Cdon mutation and in utero EtOH exposure occurred during Nodal-dependent specification of the PCP and that reduction of Nodal target gene expression required a combination of mutation and teratogen . The observation that loss of CDON was required for the effect suggests that , in addition to its role as a SHH co-receptor , CDON functions earlier in development to promote midline patterning , potentially via the Nodal pathway . In fact , Cdon expression initiates during gastrulation and is seen in embryonic mesoderm and ectoderm ( Hong and Krauss , 2012; Mulieri et al . , 2000 ) . Mice with mutations resulting in complete loss of Nodal signaling gastrulate abnormally and die early in development , but hypomorphic pathway mutants display a range of HPE phenotypes ( Andersson et al . , 2006; Chu et al . , 2005; Lowe et al . , 2001; Nomura and Li , 1998; Schier , 2009; Shen , 2007; Song et al . , 1999 ) . To further probe the linkage between CDON and regulation of Nodal signaling , we assessed whether Cdon interacted genetically with Tdgf1 and Lefty2 , direct positive and negative regulators of the Nodal pathway , respectively . When analyzed at E10 , ~14% of Cdon-/- embryos treated with EtOH had alobar HPE and cyclopia , dying in utero by E11 ( Hong and Krauss , 2012; Hong and Krauss , 2013 ) . When studied at E14 , ~70% of such embryos displayed lobar HPE and a range of craniofacial midline phenotypes , including fused upper lip and single nostril ( Hong and Krauss , 2012; Hong and Krauss , 2013 ) . To address genetic interactions between Cdon and Nodal pathway regulators , we took advantage of the high penetrance of phenotypes at E14 and analyzed embryos at this stage for these unambiguous , easily scored phenotypes . Targeted mutations in Tdgf1 and Lefty2 ( Ding et al . , 1998; Meno et al . , 1999 ) were placed onto the 129S6 genetic background and then crossed to Cdon mutants to generate double heterozygotes . Offspring of intercrosses of these mice were further crossed and pregnant females were treated with EtOH or saline as a control . Tdgf1 is essential for Nodal signaling . Lefty2 is induced in response to Nodal signaling to provide critical negative feedback as an inhibitor of the pathway . Since CDON and EtOH may act together at the level of Nodal signaling in HPE , we predicted that removal of one copy of Tdgf1 would sensitize Cdon-/- embryos to EtOH-induced HPE . In contrast , removal of one copy of Lefty2 would be predicted to rescue Cdon-/- embryos from EtOH-induced HPE . ( Null mutations in Tdgf1 and Lefty2 result in early lethality due to strong gastrulation defects ( Ding et al . , 1998; Meno et al . , 1999 ) , so studying homozygous double mutants with Cdon is impossible . ) To permit the detection of sensitization by Tdgf1 heterozygosity , we used a dose of EtOH which we previously reported gives ~30% penetrance of HPE at E14 ( 2 . 9 g/kg ) ( Hong and Krauss , 2012 ) . With this regimen , 28% of EtOH-treated Cdon-/-;Tdgf1+/+ embryos had HPE , whereas 55% of Cdon-/-;Tdgf1+/- embryos did ( p<0 . 014 ) ( Table 2 and Figure 3 ) . With the exception of a single EtOH-treated Cdon+/-;Tdgf1+/- embryo , EtOH-treated mice of other genotypes and saline-treated control mice did not display HPE . Removal of one copy of Tdgf1 therefore sensitized Cdon-/- mice to EtOH-induced HPE . Nodal-/- and Lefty2-/- embryos have opposite defects in gastrulation and display dosage-dependent genetic interactions , with Lefty2 functioning as a negative regulator of the pathway ( Meno et al . , 1999 ) . To test for phenotypic suppression in EtOH-induced HPE , we used the standard dose of EtOH ( 3 . 48 g/kg ) for these analyses , as it results in high penetrance . All embryos with HPE at E14 display a fused upper lip , while only some show the more severe single nostril phenotype . Approximately 70% of EtOH-treated Cdon-/-;Lefty2+/+ embryos had a fused upper lip , similar to our previous findings ( Hong and Krauss , 2012; Hong and Krauss , 2013 ) , whereas 54% of Cdon-/-;Lefty2+/- embryos did , revealing a trend toward overall reduction of HPE frequency ( p=0 . 14 ) . However , while 21% of EtOH-treated Cdon-/-;Lefty2+/+ embryos had a single nostril , only 6% of Cdon-/-;Lefty2+/- embryos did ( p=0 . 04 ) ( Table 3 and Figure 4 ) . Again , EtOH-treated mice of other genotypes and saline-treated control mice did not display HPE . Therefore , removal of one copy of Lefty2 rescued a more severe form of HPE associated with EtOH treatment . Taken together , these studies revealed that the Nodal signaling components Tdgf1 and Lefty2 act as heterozygous enhancer and suppressor genes , respectively , of EtOH-induced HPE in Cdon-/- embryos . The APS is a small , transiently existing cell population , making it hard to study directly . Cultured mEpiSCs have transcriptional and functional properties resembling APS cells ( Kojima et al . , 2014; Tsakiridis et al . , 2014 ) , making them a tractable in vitro surrogate model . mEpiSCs rely on FGF2 and Activin/Nodal signaling for maintenance as self-renewing , pluripotent stem cells ( Brons et al . , 2007; Vallier et al . , 2009 ) . Activin A and Nodal use the same receptor and signaling mechanism , except that Nodal , but not Activin A , requires the co-receptor TDGF1 . Therefore , we evaluated the effects of EtOH on Activin/Nodal signaling in mEpiSCs . A direct readout of Nodal signaling activity is receptor-mediated phosphorylation of SMAD2 at its C-terminus ( Robertson , 2014; Schier , 2009; Shen , 2007 ) . We assessed C-terminally phosphorylated SMAD2 ( p-SMAD2C ) and total SMAD2 levels in the mEpiSC line , EpiSC9 ( Najm et al . , 2011 ) , after treatment with EtOH for 6 hr . A relatively short treatment duration was chosen because: 1 ) the in vivo model is an acute exposure regimen; 2 ) the window of sensitivity to EtOH-induced HPE is <12 hr; and 3 ) high EtOH levels last only about 10 hr in pregnant females ( Hong and Krauss , 2012 ) . EtOH dose-dependently diminished p-SMAD2C levels in mEpiSCs , without altering total SMAD2 levels ( Figure 5A , B ) . EtOH stimulates activation of the MAP kinase , JNK , in specific cell types ( McAlhany et al . , 2000; Ren et al . , 2017 ) . SMAD2 is phosphorylated in its linker region by MAP kinases ( Massague , 2003; Rezaei et al . , 2012 ) , usually leading to inhibition of SMAD2 function ( Grimm and Gurdon , 2002; Kretzschmar et al . , 1999; Lessard et al . , 2018 ) . We therefore assessed total and phosphorylated ( activated ) forms of JNK1 MAP kinase in EtOH-treated mEpiSCs . EtOH dose-dependently increased phospho-JNK1 ( T183/Y185 ) levels ( p-JNK1; Figure 5A , B ) . Consistent with EtOH-induced p-JNK1 activation , phosphorylation of the SMAD2 linker region ( p-SMAD2L ) also occurred in an EtOH dose-dependent manner , with production of p-JNK1 and p-SMAD2L correlating well ( Figure 5A , B ) . Human embryonic stem cells ( hESCs ) resemble mEpiSCs ( Pauklin and Vallier , 2015 ) . Treatment of hESC or mEpiSC cultures with the ALK4 inhibitor , SB43152 , led to reduction of Activin/Nodal target gene expression and , eventually , induction of early markers of neuroectoderm differentiation ( Chng et al . , 2010; Li et al . , 2015; Liu et al . , 2018; Vallier et al . , 2009 ) . These studies generally used time courses of days to weeks , but Vallier et al . assessed expression of five Activin/Nodal target and/or pluripotency-related genes in hESCs treated with SB43152 for 6 hr , the same amount of time we treated mEpiSCs with EtOH . Levels of Nanog , Nodal , and LeftyA mRNA were reduced by SB43152 , whereas levels of Pou5f1 ( also called Oct4 ) and Sox2 underwent little or no change ( Vallier et al . , 2009 ) . We assessed expression of these five genes in mEpiSCs treated with EtOH for 6 hr ( Figure 5C ) . Similar to SB43152-treated hESCs , EtOH-treated mEpiSCs displayed significantly reduced levels of Nanog and Nodal mRNAs , while Pou5f1 and Sox2 mRNA levels were largely unchanged . Expression of the additional SB43152-inhibitable mEpiSC markers Fgf5 and T ( Liu et al . , 2018 ) were also reduced by EtOH treatment ( Figure 5C ) . In contrast , EtOH did not alter levels of Lefty1 or Lefty2 in mEpiSC cultures ( Figure 5—figure supplement 1 ) . As these are known Activin/Nodal target genes , this result might be related to differences in SB43152 vs . EtOH treatment , or differences in half-lives of these mRNAs in human vs . mouse cultures . Therefore , acute EtOH treatment of mEpiSCs resulted in an inhibitory SMAD2 phosphorylation pattern and changes in gene expression similar to those induced by a direct Activin/Nodal pathway inhibitor . Induction of neuroectoderm-specific genes ( e . g . , Hoxa1 , Six1 , and Gbx2 ) was observed in hESCs treated with SB43152 for 2–6 days ( Chng et al . , 2010; Vallier et al . , 2009 ) . After 6 hr of EtOH treatment , we found a varied response , with Hoxa1 levels strongly induced , Six1 expression displaying a biphasic dose-response , and Gbx2 levels reduced ( Figure 5—figure supplement 1 ) . Our findings argue that CDON plays an earlier role in development than its known function as a SHH co-receptor . The need for EtOH exposure to reveal this role suggests that CDON’s effects in primitive streak embryos may be redundant with other factors . The HH co-receptors BOC and GAS1 are not likely to be such factors , as Cdon;Boc;Gas1 triple knockout mice had a phenotype that is close to a complete loss of embryonic HH pathway function , but not Nodal pathway-like phenotypes ( Allen et al . , 2011 ) . LRP2 is an endocytic and auxiliary receptor for multiple morphogenetic ligands and receptors ( Willnow and Christ , 2017 ) . Lrp2-/- mice have HPE of variable penetrance and severity ( Christ et al . , 2012; Spoelgen et al . , 2005; Willnow and Christ , 2017; Willnow et al . , 2012 ) . We therefore constructed mice with mutations in Cdon and Lrp2 . Some Cdon;Lrp2 double mutants at E11 . 5 had a severe truncation of anterior head and face structures , a phenotype neither single mutant displayed ( Figure 6A ) . Double-mutant embryos studied at several stages displayed a range of phenotypes , including milder craniofacial truncation and strong HPE ( Figure 6B , C ) . Of 30 Cdon;Lrp2 double mutants scored between 20 and 37 somites , 24 ( 80% ) had HPE and 6 ( 20% ) displayed anterior truncations . Loss of anterior head structure is a more severe phenotype than that seen even in Smo mutants , which lack all HH signaling ( Zhang et al . , 2001 ) . These phenotypes – anterior truncations and HPE – are similar to those observed in mice with partial loss of Nodal pathway function ( e . g . , hypomorphic Nodal and Tdgf1 mutants , and Nodal+/-;Smad2+/- mutants , each have phenotypes that include truncation of anterior head structures and severe HPE ) ( Andersson et al . , 2006; Chu et al . , 2005; Lowe et al . , 2001; Nomura and Li , 1998; Song et al . , 1999 ) . The Cdon/Lrp2 genetic interaction is specific and selective: the Cdon paralog Boc is not expressed in primitive streak-stage embryos ( Mulieri et al . , 2002; Zhang et al . , 2001 ) , and Boc mutant mice do not synergize with Lrp2 mutants ( unpublished results ) , nor are they sensitive to EtOH-induced HPE ( Hong and Krauss , 2012 ) . CDON is a multifunctional co-receptor and promotes HH-independent signaling when associated with various other receptors and cell adhesion molecules . In addition to binding HH ligands and PTCH1 , CDON interacts with and influences signaling by classical cadherins; the Netrin receptor , Neogenin; and the WNT co-receptor , LRP6 ( Bae et al . , 2011; Bae et al . , 2009; Izzi et al . , 2011; Jeong et al . , 2014; Kang et al . , 2003; Kang et al . , 2004; Lu and Krauss , 2010; Tenzen et al . , 2006 ) . We hypothesized that CDON might work similarly in Nodal signaling . To address this possibility , we assessed whether CDON binds to Nodal receptor components . A secreted CDON ectodomain fused in-frame at its carboxy terminus with the IgG Fc region ( CDON-Fc ) was expressed in HEK293T cells with individual ectodomains of the Nodal receptor ( ActRIIA , ActRIIB , ALK4 , and TDGF1 ) fused in-frame at their carboxy termini with alkaline phosphatase ( AP ) . As positive and negative controls , SHH-N-AP and CD164-AP were used , respectively ( SHH-N is the active portion of SHH , CD164 is a cell surface sialomucin ) ( Kang et al . , 2002; Tenzen et al . , 2006 ) . The use of secreted ectodomains reduces the possibility that unknown , cell surface-bound factors promote binding or contribute directly to the complex . Conditioned medium was collected , and equivalent amounts of AP fusion proteins from conditioned media were pulled down and blotted for captured CDON-Fc . Conversely , equivalent levels of Fc were pulled down , and associated AP activity was quantified . Similar results were obtained in each pull-down: CDON-Fc interacted efficiently with TDGF1-AP , similar to the interaction between CDON-Fc and SHH-N-AP ( Figure 7A , B ) . CDON-Fc did not bind to the negative control CD164-AP , and it interacted only weakly with the AP-tagged versions of ALK4 , ActRIIA , and ActRIIB ( Figure 7A , B ) . As CDON and LRP2 interacted genetically to produce Nodal hypomorph-like phenotypes , we asked whether LRP2 , like CDON , binds to TDGF1 . The extracellular region of LRP2 harbors four repetitive modules , each comprising a cluster of complement-type repeats followed by EGF-type repeats and β-propellers . Each module is likely capable of independently binding ligands , as shown for the related receptor , LRP1 ( Croy et al . , 2003 ) . LRP2 is a huge polypeptide of 4660 amino acids , making it difficult to express in vitro . Therefore , we constructed a series of soluble ‘mini-receptors’ ( designated sR1 , sR2 , sR3 , and sR4 ) spanning virtually the entire ectodomain in four non-overlapping pieces , each fused in-frame at their carboxy termini to the IgG Fc region ( see Figure 7C ) . Each soluble ectodomain segment and TDGF1-AP were expressed in HEK293T cells and assessed for their ability to bind to each other . LRP2 binds SHH-N ( Christ et al . , 2012; McCarthy et al . , 2002 ) , so this ligand was used again as a positive control , with CD164-AP serving as a presumptive negative control . Each soluble LRP2 mini-receptor ectodomain ( i . e . , sR1 to sR4 ) pulled down SHH-N-AP , revealing that LRP2 harbors multiple SHH-N binding sites ( Figure 7D ) . These results were confirmed by expressing each ectodomain segment joined with the native LRP2 transmembrane and cytoplasmic domains ( mini-receptors R1 , R2 , R3 , and R4; Figure 7C ) in NIH3T3 cells . These cultures were incubated with recombinant SHH-N-GST fusion proteins for 2 hr , followed by immunoprecipitation with anti-LRP2 antibody recognizing all mini-receptors and western blotting with antibodies to LRP2 or GST . Again , all four mini-receptors pulled down SHH-N , whereas no pulldown of SHH-N was seen in control transfectants lacking LRP2 mini-receptors ( Figure 7—figure supplement 1 ) . Finally , we assessed LRP2 binding to TDGF1 with the soluble ectodomain binding assay . Similar to its multivalent interaction with SHH-N , the soluble LRP2 ectodomain segments sR3 and sR4 pulled down TDGF1-AP ( Figure 7D ) . Soluble LRP2 ectodomain segments sR1 and sR2 also pulled down TDGF1-AP , but greater variability in the interaction was observed , leading to p>0 . 05 ( p=0 . 14 and p=0 . 06 , respectively ) . The efficiency of LRP2 mini-receptor ectodomains to bind SHH-N-AP and TDGF1-AP was similar to that of CDON-Fc ( Figure 7D ) .
HPE is a common developmental disorder , but its etiology remains poorly understood . Mutations in the Nodal , HH , and FGF pathways are associated with HPE ( NISC Comparative Sequencing Program et al . , 2018 ) , but these alone are insufficient to drive aberrant development . Identifying and understanding the critical phenotypic modifiers of such mutations is important but still in its infancy . Many birth defects are thought to be caused by poorly defined interactions between genetic and environmental risk factors , but the mechanistic bases of these interactions are largely unknown ( Krauss and Hong , 2016 ) . Cdon mutation and fetal alcohol are each implicated in human HPE ( Abe et al . , 2018; Bae et al . , 2011; Cohen and Shiota , 2002; Croen et al . , 2000; NISC Comparative Sequencing Program et al . , 2018 ) , though neither may be sufficient . The Cdon mutation plus fetal alcohol model is noteworthy for its specificity and fidelity to many aspects of human HPE ( Hong and Krauss , 2012 ) . Therefore , illumination of how loss of Cdon interacts with in utero EtOH exposure provides insight into mechanisms of HPE and how fetal alcohol functions as a teratogen . The Nodal pathway is developmentally upstream from the HH pathway in patterning the rostroventral midline . While CDON is clearly a component of the HH pathway , it regulates signaling within several additional pathways as well ( Bae et al . , 2011; Bae et al . , 2009; Izzi et al . , 2011; Jeong et al . , 2014; Kang et al . , 2003; Kang et al . , 2004; Lu and Krauss , 2010; Tenzen et al . , 2006 ) . Multiple lines of evidence presented here argue that CDON also regulates Nodal signaling and that the Nodal pathway is a major point of synergistic interaction between mutation of Cdon and fetal EtOH exposure . The evidence includes: ( 1 ) EtOH-treated Cdon-/- embryos display defects in expression of Nodal target genes in the APS ( including Gsc , Foxa2 , and Lefty2 ) , whereas EtOH treatment or Cdon mutation alone do not ( Hong and Krauss , 2012; this study ) ; ( 2 ) the window of sensitivity to EtOH-induced HPE is very narrow and closed by E7 . 5 , a time reported to be prior to SHH expression and function in prechordal plate-mediated rostroventral midline patterning; ( 3 ) EtOH itself is the likely HPE-inducing teratogen and peak circulating EtOH levels coincide with Nodal signaling in the APS; ( 4 ) Cdon interacts genetically with the critical Nodal pathway factors Tdgf1 and Lefty2 in EtOH-induced HPE; ( 5 ) acute EtOH treatment dose-dependently diminishes p-SMAD2C and elevates p-SMAD2L levels , and inhibits Activin/Nodal-dependent gene expression , in mEpiSCs; ( 6 ) Cdon;Lrp2 double mutants display phenotypes similar to mice with hypomorphic Nodal pathway mutations , including anterior truncations , a phenotype not seen in mice lacking all HH signaling; and ( 7 ) CDON and LRP2 both bind efficiently to TDGF1 , an essential component of the Nodal receptor . We note that although EtOH inhibited p-SMAD2C production in mEpiSCs , and diminished expression of Nodal target genes in both mEpiSCs and Cdon-/- embryos , we have not yet demonstrated that EtOH inhibits p-SMAD2C production in embryos; additional work is therefore required to show the mechanisms in vitro and in vivo operate at the same level . Similarly , analysis of Nodal signaling in Cdon;Lrp2 double mutant embryos requires study . Together our findings are consistent with the following model: CDON and LRP2 function with overlapping or compensatory roles to regulate Nodal pathway signaling in the APS during induction of the PCP . Brief exposure of Cdon-/- embryos to EtOH during this period transiently and partially inhibits Nodal pathway activity . While all the mice successfully gastrulate , they emerge from this period predisposed towards development of HPE in a stochastic manner . If HH signaling strength fails to reach a required threshold early during rostroventral midline patterning , at the PCP stage , the outcome is alobar HPE and cyclopia; if it occurs at later stages of development , the outcomes are progressively less severe . Aspects of the model will require additional experimentation , but it is logically consistent with the synergistic effects of fetal alcohol and Cdon mutation within a narrow window of sensitivity , and also with the ‘mutation plus modifier’ view of human HPE . Our findings do not exclude that EtOH may also target HH signaling in Cdon-/- mice , but they are consistent with Nodal signaling being a major target of EtOH in HPE . The specific mechanisms whereby acute EtOH treatment reduces expression of Nodal target genes in the APS of Cdon-/- mice and diminishes Activin/Nodal signaling in mEpiSCs are not known . EtOH itself , rather than consequences of its oxidative metabolism , is very likely the HPE-inducing teratogen ( Hong and Krauss , 2017 ) . An alternative mechanism to metabolism-based toxicity is that EtOH itself functions to perturb cell membranes via its hydrophobicity ( Lyon et al . , 1981; McKarns et al . , 1997 ) . One possibility is that EtOH’s hydrophobic nature perturbs trafficking of Nodal receptor components or their stable assembly at the cell surface . TDGF1 is a GPI-linked protein , so might be especially vulnerable to hydrophobic membrane perturbation . However , exogenously provided Activin A is the major activator of SMAD2 in cultured mEpiSCs , and Activin does not require TDGF1 to signal through ActRII/ALK4 . EtOH reduces p-SMAD2C levels in these cultures , so TDGF1 may not be a direct critical target of EtOH . Nevertheless , mEpiSCs endogenously express Nodal ( Kojima et al . , 2014 ) , which does require TDGF1 , and its contribution to SMAD2 signaling in these cultures is not fully clear . Additionally , EtOH activates JNK1 in mEpiSCs , with a corresponding increase in p-SMAD2L levels . Phosphorylation of SMAD2 by MAP kinases is generally inhibitory to SMAD2 activity , independent of p-SMAD2C phosphorylation status ( Massague , 2003; Rezaei et al . , 2012 ) , so EtOH appears to promote or inhibit multiple signals that converge on SMAD2 phosphorylation . CDON regulates several signaling pathways , but it has not previously been implicated in Nodal signaling . How CDON functions to regulate Nodal signaling is of obvious interest . Cdon;Lrp2 double mutant mice display phenotypes that resemble Nodal pathway hypomorphs , and CDON and LRP2 each bind to TDGF1 . Therefore , as with the effects of EtOH , TDGF1 is a possible point of regulation . One potential mechanism is that CDON and LRP2 might function as components of a larger Nodal receptor complex , contributing via interaction with TDGF1 . Alternatively , they may work with TDGF1 in processing and trafficking of Nodal or other receptor components , or play a role in processing and trafficking of TDGF1 itself ( Constam , 2009 ) . There is evidence in other systems that CDON and LRP2 can act in such a manner . LRP2 is best understood as an endocytic receptor that controls internalization and intracellular trafficking of both soluble and membrane-bound proteins , including PTCH1 , the Na+/H+ exchanger NHE3 , and the intrinsic factor receptor , Cubilin ( Willnow and Christ , 2017 ) . Furthermore , CDON regulates subcellular localization of N-cadherin during neural crest migration in zebrafish ( Powell et al . , 2015 ) . Similarly , the Drosophila orthologs of CDON act not only as co-receptors for HH , but in trafficking of the primary HH receptor , PTC ( Zheng et al . , 2010 ) . Another possibility is in regulation of cell-cell contact and adhesion . CDON is localized to sites of cell-cell contact and functions with N-cadherin in cell adhesion-dependent signaling ( Kang et al . , 2003; Lu and Krauss , 2010 ) . In zebrafish , a positive feedback loop exists between Nodal signaling , E-cadherin expression , and duration of cell-cell contact , that directs PCP specification at the expense of endoderm specification ( Barone et al . , 2017 ) . It is conceivable that CDON could play a role in adhesion-mediated , higher order regulatory events like this as well . These varied potential mechanisms are not mutually exclusive . Our findings offer insight into how Cdon mutation synergizes with fetal alcohol in mice to produce a wide spectrum of HPE phenotypes , closely resembling the complex etiology and variable outcomes seen in humans at the population level . A combination of approaches , including window of sensitivity studies , genetic interactions in mice , and in vitro analyses revealed that , unexpectedly , a major point of synergy is at the level of Nodal signaling . These findings illuminate gene-environment interactions in the causation of a common birth defect , expand understanding of alcohol teratogenesis , and serve as a conceptual framework for additional developmental anomalies .
All animal procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committee ( IACUC ) of the Icahn School of Medicine at Mount Sinai and according to Max-Delbruck-Center guidelines following approval by local authorities ( X9007/17 ) . B6 . 129P2-Lefty2tm1Hmd mice were provided by Hiroshi Hamada . Tdgf1 mutant mice ( Cripto-LacZ mice ) were provided by Michael Shen . Both strains were transferred onto the 129S6/SvEvTac ( 129S6 ) background with the Taconic Speed Congenic Program , by backcrossing and mapping with the 1450 SNP array . Mice used for these experiments were estimated to be over 98% 129S6/SvEvTac background . These lines were crossed with Cdon+/tm1Rsk ( Cdon+/- ) mice on the 129S6 background ( Cole and Krauss , 2003; Hong and Krauss , 2012 ) to generate 129S6 . Cdon+/-;Lefty2+/- and 129S6 . Cdon+/-Tdgf1+/- double mutant mice . Because Cdon and Tdgf1 are both located on chromosome 9 , Cdon+/-;Tdgf1+/- mice were crossed with wild type mice and offspring screened for those that carried a recombinant chromosome 9 carrying both Cdon and Tdgf1 mutant alleles . Offspring of intercrosses of these mice were further crossed ( see Tables 2 and 3 for details ) and pregnant females treated with EtOH or saline as a control ( Hong and Krauss , 2012 ) . Briefly , two- to three-month old mice were mated for one hour in the dark and plugged females were collected . 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 solution of 30% EtOH in saline ( 3 . 48 g/kg ) at E7 . 0 and again 4 hr later . For window-of-sensitivity experiments , pregnant females were treated similarly , except the initial dose was given at E7 . 25 or E7 . 5 . For studies involving Cdon;Tdgf1 mutants , a lower dose of ethanol ( 25% EtOH in saline , 2 . 9 g/kg ) was used . Saline injections were used as controls throughout . Mice with targeted gene disruption of Lrp2 ( Lrp2tm1Her ) have been described ( Willnow et al . , 1996 ) . Lrp2+/tm1Her ( Lrp2+/- ) mice were kept on the C57BL/6N genetic background and crossed with Cdon+/- mice on the C57BL/6N genetic background . Offspring were further intercrossed , and Lrp2+/-;Cdon+/- mice were subjected to timed mating to collect embryos at the depicted embryonic day . Neither Cdon-/- mice nor Lrp2-/- mice on the C57BL/6N display anterior truncation phenotypes . Whole mount RNA in situ hybridization was performed according to standard protocols ( Hong and Krauss , 2012 ) . Briefly , embryos were dissected out and fixed in 4% paraformaldehyde in phosphate-buffered saline ( PBS ) , dehydrated through a graded methanol series , and stored at −20°C . Rehydrated embryos were treated with 10 μg/ml proteinase K ( Qiagen ) in PBS , 0 . 1% Tween-20 ( PBST ) according to stage . Embryos were rinsed with PBST , post-fixed and hybridized with digoxygenin ( DIG ) -labeled probe in hybridization mix ( 50% formamide , 1 . 3x SSC pH 5 . 0 , 5 mM EDTA , 50 μg/ml yeast tRNA , 100 μg/ml heparin , 0 . 2% Tween-20 , and 0 . 5% CHAPS ) overnight at 65°C . They were washed , blocked with 2% Roche blocking reagent , 20% heat-inactivated lamb serum in Tris-buffered saline with 0 . 1% Tween-20 ( TBST ) and incubated with alkaline phosphate-conjugated anti-DIG antibody ( 1:2000 , Roche ) in blocking buffer overnight at 4°C . After washing with TBST and NTMT ( 100 mM NaCl , 100 mM Tris pH9 . 5 , 50 mM MgCl2 , and 0 . 1% Tween-20 ) , embryos were stained with BM Purple AP substrate ( Roche ) in the dark . Stained embryos were cleared in 80% glycerol and photographed with a Jenoptik ProgRes C3 camera attached to Nikon SMZ 1500 stereomicroscope . Captured images were assembled by Helicon Focus software ( Helicon Soft ) . Mouse epiblast stem cells ( EpiSC9 cells ) were obtained from Jianlong Wang , authenticated by robust expression of pluripotency markers , and confirmed to be negative for mycoplasma infection . EpiSC9 cells were cultured as described ( Huang et al . , 2017 ) . Briefly , cells were cultured on 0 . 1% fibronectin-coated plates ( MilliporeSigma , 10 μg/ml/cm2 ) in N2B27 media supplemented with 20 ng/ml Activin A ( R and D Systems ) and 12 ng/ml Fgf2 ( R and D Systems ) . Media were changed daily . EpiScs were treated with EtOH for six hours in fresh EpiSC media with supplements . For protein analysis , cells were harvested with RIPA buffer plus protease inhibitor ( Sigma ) and phosphatase inhibitor ( Sigma ) . Western blotting was as described ( Bae et al . , 2009 ) . Images were scanned and quantified using ImageJ software . Statistical significance was calculated using Student’s t-test and a cutoff of p<0 . 05 . Primary antibodies used for western blot: SMAD2 ( Cell Signaling #5339 ) , p-SMAD2C ( Cell Signaling #3108 ) , p-SMAD2L ( Cell Signaling #3104 ) , JNK ( Cell Signaling #9252 ) , p-JNK ( Cell Signaling #9251 ) , and GAPDH ( Cell Signaling #97166 ) . For mRNA analysis , total RNA was extracted from EpiSCs using the RNAeasy kit ( Quiagen ) . Reverse transcription and cDNA production were performed with Superscript III first strand synthesis system ( Invitrogen ) . qPCR was performed using iQ SyBR green supermix ( BioRad ) on an iCycler iQ5 ( BioRad ) . Gene expression levels were normalized to Gapdh . Expression vectors encoding soluble , tagged forms of CDON , SHH-N , CD164 , ActRIIA , ActRIIB , ALK4 , TDGF1 , and LRP2 , and LRP2 mini-receptors that encoded the LRP2 transmembrane and intracellular domains , were constructed by standard methods; details are available on request . HEK293T cells were obtained from ATCC , confirmed to be negative for mycoplasma infection , and cultured in DMEM with 10% FBS and 1x Penicillin/Streptomycin . HEK293T cells in 10 cm dishes were transfected with a total of either 2 or 3 μg of plasmid vectors using Effectene reagent ( Qiagen ) . Media were changed to 2% FBS two days after transfection . Conditioned media were harvested five days afterwards . Soluble fusion proteins in conditioned media were quantified by dot blotting with human IgG Fc protein ( Jackson ImmunoResearch Laboratories ) as a standard , and by alkaline phosphatase ( AP ) enzyme activity using AP yellow liquid substrate ( Sigma ) . For analysis of CDON-Fc interactions with soluble AP-tagged proteins , the factors were coexpressed in HEK293T cells . For Fc pull down and AP activity assays with CDON-Fc , soluble fusion proteins were incubated with protein G agarose beads ( Roche ) for 2 hr at 4°C . For analysis of soluble LRP2-Fc mini-receptors interactions with soluble AP-tagged proteins , LRP2 sR1-sR4 and the AP-tagged counterparts were generated separately in individual transfections and CM mixed together . Pull down complexes were washed five times with ice-cold HNTG buffer ( 20 mM HEPES pH 7 . 5 , 150 mM NaCl , 0 . 1% Triton X-100 , 10% glycerol ) . Endogenous AP was inactivated by heat inactivation . AP activity in pull-down complexes was measured using AP yellow liquid substrate ( Sigma ) . For AP pull down followed by western blot analysis , soluble fusion proteins were incubated with anti-AP conjugated agarose beads ( Sigma ) overnight at 4°C . Pull-down complexes were washed five times with HNTG buffer , eluted from the beads by boiling , and separated by SDS-PAGE . Antibodies used for western blot were HRP conjugated with goat anti-human Fc . To test SHH-N interaction with membrane-bound LRP2 mini-receptors , NIH3T3 cells were transfected with the four individual LRP2 mini-receptors encoding transmembrane and intracellular domains and incubated with 20 μg/ml GST-SHH-N in the medium for two hours . Cell lysates were immunoprecipitated with polyclonal LRP2 antibody directed against full-length LRP2 , and subjected to western blot analysis with anti-LRP2 and anti-GST antibodies .
Holoprosencephaly ( HPE ) , a defect in midline patterning of the forebrain and midface , arises ~1 in 250 conceptions . It is associated with predisposing mutations in the Nodal and Hedgehog ( HH ) pathways , with penetrance and expressivity graded by genetic and environmental modifiers , via poorly understood mechanisms . CDON is a multifunctional co-receptor , including for the HH pathway . In mice , Cdon mutation synergizes with fetal alcohol exposure , producing HPE phenotypes closely resembling those seen in humans . We report here that , unexpectedly , Nodal signaling is a major point of synergistic interaction between Cdon mutation and fetal alcohol . Window-of-sensitivity , genetic , and in vitro findings are consistent with a model whereby brief exposure of Cdon mutant embryos to ethanol during gastrulation transiently and partially inhibits Nodal pathway activity , with consequent effects on midline patterning . These results illuminate mechanisms of gene-environment interaction in a multifactorial model of a common birth defect .
A common birth defect known as holoprosencephaly affects how the brain and face of a fetus develop in the womb . In many cases , the condition is so severe that the fetus dies before , or shortly after , birth . Mutations in certain genes that control how the fetus develops are associated with holoprosencephaly . For example , mutations in components of the Hedgehog and Nodal signaling pathways , which transmit information that help cells to become specialized , increase the risk that a fetus will develop holoprosencephaly . Environmental factors , such as exposure to alcohol in the womb , are also thought to contribute to this condition . A gene known as Cdon is a component of the Hedgehog signaling pathway . In 2012 , a team of researchers reported that mice with a mutation in the Cdon gene exposed to alcohol in the womb develop symptoms similar to holoprosencephaly in humans . Here , Hong et al . – including some of the researchers involved in the previous work – set out to understand how Cdon and alcohol work together to cause holoprosencephaly in the mutant mice . First , the team exposed pregnant mice to alcohol at different times during gestation to find out when their young were sensitive to developing holoprosencephaly . This showed that the young mice were most sensitive in early pregnancy when the Nodal pathway was active in their growing bodies . Further experiments found that alcohol and mutations in Cdon change Nodal signaling in cells . Together , these findings demonstrate that exposure to alcohol in the womb works together with the mutant form of Cdon via the Nodal signaling pathway , rather than the Hedgehog pathway , to cause holoprosencephaly in mice . The causes of many common birth defects are complex and difficult to distinguish at the level of individual cases . The work of Hong et al . illuminates how multiple risk factors during pregnancy , which may not create any problems on their own , may work together to produce birth defects in the fetus . The findings also offer new ways to understand how exposure to alcohol in the womb affects the fetus . Ultimately , understanding how birth defects form could lead to new strategies to prevent them in the future .