1. Developmental Biology
  2. Genetics and Genomics

Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly

  1. Mingi Hong
  2. Annabel Christ
  3. Anna Christa
  4. Thomas E Willnow
  5. Robert S Krauss  Is a corresponding author
  1. Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mount Sinai, United States
  2. Max-Delbruck-Center for Molecular Medicine, Germany
https://doi.org/10.7554/eLife.60351
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Cite this article
  1. Mingi Hong
  2. Annabel Christ
  3. Anna Christa
  4. Thomas E Willnow
  5. Robert S Krauss
(2020)
Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly
eLife 9:e60351.
https://doi.org/10.7554/eLife.60351
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7 figures, 4 tables and 1 additional file

Figures

Figure 1
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Time course of EtOH-Induced HPE in Cdon-/- Embryos.

Frontal views of E14.0 embryos. Treatment of Cdon-/- embryos with EtOH at E7.25, but not E7.5, results in HPE (see Table 1 for quantification). The Cdon-/-embryo treated with EtOH at E7.25 displays a fused upper lip and single nostril (black arrow), whereas the Cdon-/-embryo treated with EtOH at E7.5 does not and resembles the saline control (red arrow).

Figure 2
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Defective Expression of Lefty2 at the Primitive Streak Stage of EtOH-Treated Cdon-/- Embryos.

Whole mount in situ hybridization analyses of Lefty2 in embryos of the indicated genotype and treatment harvested at E7.25. Lefty2 expression was decreased specifically in EtOH-treated Cdon-/- embryos. Numbers of embryos with similar results: Cdon+/- (saline) = 6; Cdon+/- (EtOH) = 4; Cdon-/- (saline) = 9; Cdon-/- (EtOH) = 9. Scale Bar, 200 μm.

Figure 3
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Tdgf1 Heterozygosity Enhances EtOH-Induced HPE in Cdon-/- Embryos.

Frontal views of E14.0 embryos. Removal of one copy of Tdgf1 enhanced the response of Cdon-/- embryos to a dose of 2.9 g/kg EtOH (see Table 2 for quantification). The EtOH-treated Cdon-/-;Tdgf1+/- embryo displays a fused upper lip (arrow).

Figure 4
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Lefty2 Heterozygosity Suppresses EtOH-Induced HPE in Cdon-/- Embryos.

Frontal views of E14.0 embryos. Removal of one copy of Lefty2 suppressed formation of single nostril in Cdon-/- embryos in response to a dose of 3.48 g/kg EtOH (see Table 3 for quantification). The EtOH-treated Cdon-/- embryo displays a fused upper lip and single nostril (black arrow), whereas the EtOH-treated Cdon-/-;Lefty2+/- embryo has a fused upper lip and two nostrils (red arrow).

Figure 5 with 1 supplement
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Acute EtOH treatment inhibits activin/nodal signaling in mEpiSCs.

(A) Representative western blot of mEpiSCs treated with the indicated doses of EtOH for 6 hr. GAPDH was used as a loading control. (B) Densitometric quantification of p-SMAD2C levels, relative to total Smad2; p-JNK1 levels, relative to total JNK1; and p-SMAD2L levels, relative to total SMAD2, with the indicated doses of EtOH. N = 3 experiments with two biological replicates (as shown in (A)) in each experiment. (C) qRT-PCR analysis of Nanog, Nodal, Pou5f1, and Sox2 expression in mEpiSCs treated with the indicated doses of EtOH for 6 hr. Expression was normalized to Gapdh expression. N = 3 experiments with two biological replicates in each experiment. Values for (B) and (C) are means ± SEM, *p<0.05, **p<0.01, ***p,0.001 by Student’s t-test.

Figure 5—source data 1

Source data for quantification of western blot results shown in Figure 5A and B.

https://cdn.elifesciences.org/articles/60351/elife-60351-fig5-data1-v1.xlsx
Figure 5—source data 2

Source data for quantification of qRT-PCR results shown in Figure 5C.

https://cdn.elifesciences.org/articles/60351/elife-60351-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
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Analysis of gene expression in EtOH-treated mEpiSCs.

qRT-PCR analysis of Lefty1, Lefty2, Hoxa1, Six1, and Gbx2 expression in mEpiSCs treated with the indicated doses of EtOH for 6 hr. Expression was normalized to Gapdh expression. N = 3 experiments with two biological replicates in each experiment. Values are means ± SEM, *p<0.05, **p<0.01, ***p,0.001 by Student’s t-test.

Figure 5—figure supplement 1—source data 1

Source data for quantification of qRT-PCR results shown in Figure 5.

Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/60351/elife-60351-fig5-figsupp1-data1-v1.xlsx
Figure 6
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Cdon and Lrp2 Interact Genetically to Produce HPE and Anterior Truncations in Mice.

(A) Whole mount E11.5 embryos of the indicated genotype. Note the loss of anterior head structures in the Cdon-/-;Lrp2-/- embryo (arrow). (B) E18.5 embryos of the indicated genotype in whole mount and alizarin red/alcian blue-stained skeleton preparations. Note the craniofacial truncation in the Cdon-/-;Lrp2-/- embryos (arrows). (C) Whole mount P0 Cdon-/-;Lrp2-/- embryo showing severe HPE.

Figure 7 with 1 supplement
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CDON and LRP2 Bind to TDGF1.

(A) The indicated AP-tagged proteins were co-expressed with CDON-Fc, and equivalent amounts of AP proteins in conditioned medium (CM) were pulled down. Levels of CDON-Fc in CM and in the pull down were detected by western blot. (B) CDON-Fc was pulled down from CM derived from transfections as in (A), the amount of CDON-Fc from various CM normalized, and associated AP enzyme activity quantified, N = ≥4 for (A) and (B). (C) Schematic of full length LRP2 and recombinant mini-receptor variants used. Each mini-receptor spans one of the four repetitive modules of the extracellular receptor domain and was expressed either as soluble ectodomain fragments fused to IgG Fc (designated sR1- sR4) or as a membrane-bound mini-receptor fused to the authentic transmembrane and intracellular domains of LRP2 (designated R1- R4). Soluble ectodomain fragments sR1 – sR4 were studied in panel (D), whereas mini-receptors R1 – R4 were used in Figure 7—figure supplement 1 . Amino acid numbers next to the full-length receptor polypeptide indicate the extent of individual receptor domains. (D) The indicated Fc-tagged proteins were pulled down from CM derived from transfections as in (A), the amount of Fc-tagged protein from various CM normalized, and associated AP enzyme activity quantified, N = 3. Values for (B) and (D) are means ± SEM, *p<0.05, **p<0.01 by Student’s t-test.

Figure 7—source data 1

Source data for quantification of AP activity in CDON-Fc pulldown experiments shown in Figure 7B.

https://cdn.elifesciences.org/articles/60351/elife-60351-fig7-data1-v1.xlsx
Figure 7—source data 2

Source data for quantification of AP activity in CDON-Fc and soluble LRP2 minireceptor-Fc pulldown experiments shown in Figure 7D.

https://cdn.elifesciences.org/articles/60351/elife-60351-fig7-data2-v1.xlsx
Figure 7—figure supplement 1
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HEK293 cell transfectants expressing the indicated LRP2 mini-receptor were incubated with GST-SHH-N, and cell lysates immunoprecipitated with antibody to LRP2 and blotted with antibodies to LRP2 and GST.

Tables

Table 1
Time course of EtOH-Induced HPE in Cdon-/- Embryos
EtOH treatment:E7.25E7.5
SalineEtOHEtOH
Phenotype*Cdon+/-Cdon-/-Cdon+/-Cdon-/-Cdon+/-Cdon-/-
Fused upper lip0/172/140/2113/24**0/130/22
Single nostril0/170/140/215/240/130/22
Proboscis0/170/140/212/240/130/22

  1. * All embryos with HPE had fused upper lip, a fraction of these showed single nostril and proboscis.

    **p=0.0165 by Fisher’s two-tailed exact test, when compared to EtOH-treated Cdon+/- embryos.

Table 2
Tdgf1 Heterozygosity Enhances EtOH-Induced HPE in Cdon-/- Embryos.
TreatmentGenotype (# embryos with HPE/total (%))*
Cdon+/-;Tdgf1+/-Cdon-/-;Tdgf1+/+Cdon-/-;Tdgf1+/-
Saline0/23 (0%)0/11 (0%)2/27 (7.4%)
EtOH (2.9 g/kg)1/48 (2.1%)9/32 (28.1%)31/56 (55.4%)**

  1. * Crosses between the following genotypes were used to generate the genotypes scored above:

    Cdon+/-;Tdgf1+/- x Cdon+/-;Tdgf1+/-.

  2. Cdon+/-;Tdgf1+/- x Cdo-+/-;Tdgf1+/-.

    Cdon+/- x Cdon+/-;Tdgf1+/-.

  3. Cdon+/- x Cdon-/-;Tdgf1+/-.

    Cdon-/- x Cdon+/-;Tdgf1+/-.

  4. No HPE was found in offspring genotypes other than those shown.

    ** p=0.014 by Fisher's two-tailed exact test, when compared to EtOH-treated Cdon-/-; Tdgf1 +/+ embryos.

Table 3
Lefty2 Heterozygosity Supresses EtOH-Induced HPE in Cdon-/- Embryos.
TreatmentGenotype (# affected/total (%))*
Total HPESingle nostril
Cdon-/-;Lefty2+/+Cdon-/-;Lefty2+/-Cdon-/-;Lefty2+/+Cdon-/-;Lefty2+/-
Saline1/12 (8.3%)0/10 (0%)0/12 (0%)0/10 (0%)
EtOH (3.48 g/kg)33/47 (70.2%)26/48 (54.2%)10/47 (21.3%)3/48 (6.3%)**

  1. * Crosses between the following genotypes were used to generate the genotypes scored above:

    Cdon+/-;Lefty2+/- x Cdon+/-;Lefty2+/-.

  2. Cdon+/-;Lefty2+/- x Cdo-+/-;Lefty2+/-.

    Cdon+/- x Cdon+/-;Lefty2+/-.

  3. Cdon+/- x Cdo-/-;Lefty2+/-.

    Cdon-/- x Cdon+/-;Lefty2+/-.

  4. No HPE was found in offspring genotypes other than those shown.

    **p=0.04 by Fisher's two-tailed exact test, when compared to EtOH-treated Cdon-/-;Lefty2+/+ embryos with a single nostril.

Key resources table
Reagent type (species)
or resource		DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (Mus musculus)Cdon-MGIMGI:1926387 
Genetic reagent (Mus musculus)Cripto-MGIMGI:98658
Genetic reagent (Mus musculus)Lefty2-MGIMGI:2443573
Genetic reagent (Mus musculus)Lrp2-MGIMGI:95794
Cell line (Human)HEK293TATCC 
Cell line (Mus musculus)EpiSC9Najm et al., 2011 Huang et al., 2017
Kojima et al., 2014
Antibodyanti-Smad2 (Rabbit mAB)Cell Signaling#5339WB (1:1000)
Antibodyanti-phospho-Smad2C (Rabbit mAB)Cell Signaling#3108WB (1:1000)
Antibodyanti-phospho-Smad2L (Rabbit mAB)Cell Signaling#3104WB (1:1000)
Antibodyanti-JNK (Rabbit polyclonal)Cell Signaling#9252WB (1:1000)
Antibodyanti-phospho-JNK (Rabbit polyclonal)Cell Signaling#9251WB (1:1000)
Antibodyanti-Gapdh (Mouse mAB)Cell Signaling#97166WB (1:5000)
Antibodyanti-DIG AP conjugatedRoche11093274910WM in situ (1:2000)
Antibodyanti-LRP2 (Goat)Willnow et al., 1996WB (1:1000)
Recombinant DNA reagentSHH-N-AP (plasmid)This paper
Recombinant DNA reagentCD164-AP (plasmid)This paper
Recombinant DNA reagentActRIIA-AP (plasmid)This paper
Recombinant DNA reagentALK4-AP (plasmid)This paper
Recombinant DNA reagentCripto-AP (plasmid)This paper
Recombinant DNA reagentLRP2 sR1-Fc (plasmid)This paper
Recombinant DNA reagentLRP2 sR2-Fc (plasmid)This paper
Recombinant DNA reagentLRP2 sR3-Fc (plasmid)This paper
Recombinant DNA reagentLRP2 sR4-Fc (plasmid)This paper
Recombinant DNA reagentCdon-Fc (plasmid)Kang et al., 2003 
Peptide, recombinant proteinHuman Plasma Fibronectin purified proteinMilliporeSigmaFC01010 μg/ml/cm2
Peptide, recombinant proteinActivin AR and D Systems338-AC20 ng/ml
Peptide, recombinant proteinFGF2R and D Systems 234-FSE12 ng/ml
Peptide, recombinant proteinIgG Fc (human)Jackson Laboratories009-000-008 
Peptide, recombinant proteinProtein G-agarose beadsRoche11243233001
Peptide, recombinant proteinAnti-AP-conjugated agarose beadsSigmaA2080
Commercial assay or kitAP yellow liquid substrateSigmaP7998
Commercial assay or kitBM PurpleRoche11442074001
Commercial assay or kitDIG-labeling kitRoche11277073910
Commercial assay or kitEffectene transfection reagentQiagen 301425 
Commercial assay or kitRNA easy mini kitQiagen 74104 
Commercial assay or kitSuperscript III First strand synthesis systemInvitrogen18080051
Commercial assay or kitiQ SyBR Green SupermixBioRad1708882
Software, algorithmPrism 8GraphPadPrism 8 for MacOS ver 8.4.3
Sequence-based reagentGAPDH_FInvitrogen
Dong et al., 2008		PCR primersAACGACCCCTTCATTGAC
Sequence-based reagentGAPDH_RInvitrogen;
Dong et al., 2008		PCR primersTCCACGACATACTCAGCAC
Sequence-based reagentFgf5_FInvitrogen;
Liu et al., 2018		PCR primersGCTGTGTCTCAGGGGATTGT
Sequence-based reagentFgf5_RInvitrogen;
Liu et al., 2018		PCR primersCACTCTCGGCCTGTCTTTTC
Sequence-based reagentGbx2_FInvitrogen;
Harvard Primer Bank 133892275c2		PCR primersGCAACTTCGACAAAGCCGAG
Sequence-based reagentGbx2_RInvitrogen;
Harvard Primer Bank 133892275c2		PCR primersCCTTGCCCTTCGGGTCATC
Sequence-based reagentHoxa1_FInvitrogen;
Matt et al., 2005		PCR primersCGCACAATGTTCTGATGTCC
Sequence-based reagentHoxa1_RInvitrogen;
Matt et al., 2005		PCR primersTGCAAGCTTCATGACAGAGG
Sequence-based reagentLefty1_FInvitrogen;
Liu et al., 2018		PCR primersAACCGCACTGCCCTTAT
Sequence-based reagentLefty1_RInvitrogen;
Liu et al., 2018		PCR primersCGCGAAACGAACCAACTTGT
Sequence-based reagentLefty2_FInvitrogen;
Liu et al., 2018		PCR primersCAGCCAGAATTTTCGAGAGGT
Sequence-based reagentLefty2_RInvitrogen;
Liu et al., 2018		PCR primersCAGTGCGATTGGAGCCATC
Sequence-based reagentNanog_FInvitrogen;
Chng et al., 2010		PCR primersGGACTTTCTGCAGCCTTACG
Sequence-based reagentNanog_RInvitrogen;
Chng et al., 2010		PCR primersGCTTCCAAATTCACCTCCAA
Sequence-based reagentNodal_FInvitrogen;
Liu et al., 2018		PCR primersCCTGGAGCGCATTTGGATG
Sequence-based reagentNodal_RInvitrogen;
Liu et al., 2018		PCR primersACTTTCTGCTCGACTGGACA
Sequence-based reagentPou5f1_FInvitrogen;
Liu et al., 2018		PCR primersAGTTGGCGTGGAGACTTTGC
Sequence-based reagentPou5f1_RInvitrogen;
Liu et al., 2018		PCR primersCAGGGCTTTCATGTCCTGG
Sequence-based reagentSix1_FInvitrogen;
Chng et al., 2010		PCR primersTTAAGAACCGGAGGCAAAGA
Sequence-based reagentSix1_RInvitrogen;
Chng et al., 2010		PCR primersGGGGGTGAGAACTCCTCTTC
Sequence-based reagentSox2_FInvitrogen;
Liu et al., 2018		PCR primersGCGGAGTGGAAACTTTTGTCC
Sequence-based reagentSox2_RInvitrogen;
Liu et al., 2018		PCR primersCGGGAAGCGTGTACTTATCCTT
Sequence-based reagentT_FInvitrogen;
Liu et al., 2018		PCR primersCTCGGATTCACATCGTGAGAG
Sequence-based reagentT_RInvitrogen;
Liu et al., 2018		PCR primersAAGGCTTTAGCAAATGGGTTGTA

Additional files

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https://cdn.elifesciences.org/articles/60351/elife-60351-transrepform-v1.pdf

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  1. Mingi Hong
  2. Annabel Christ
  3. Anna Christa
  4. Thomas E Willnow
  5. Robert S Krauss
(2020)
Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly
eLife 9:e60351.
https://doi.org/10.7554/eLife.60351