Glycosylphosphatidylinositol biosynthesis and remodeling are required for neural tube closure, heart development, and cranial neural crest cell survival

  1. Marshall Lukacs
  2. Tia Roberts
  3. Praneet Chatuverdi
  4. Rolf W Stottmann  Is a corresponding author
  1. Cincinnati Children’s Medical Center, United States
  2. University of Cincinnati, United States
10 figures, 4 tables and 4 additional files


The Clpex mutant phenotype is caused by a hypo-morphic mutation in Pgap2.

Whole mount E18.5 (A,E) and E15.5 (G) WT embryos. Whole mount E18.5 (B, F) and E15.5 (H) Clpex mutant embryos. H&E staining of WT E15.5 (C) and Clpex (D) coronal sections. Skeletal preparation of WT skull ventral view (I), dorsal view (K). Skeletal preparation of Clpex mutant skull ventral view (J) and dorsal view (L). Asterick indicates absent palatine bone in mutant (L). Skeletal preparation of WT limb (M), and Clpex mutant limb (N). Quantification of WT and mutant radial (O) and humeral (P) length normalized to the crown to rump ratio. Mapping data for Clpex mutation (Q). Sanger sequencing of Pgap2 exon three in WT and Clpex mutant with exon three highlighted starting at the initiating methionine (R). Scale bar indicates 500 μm in C,D and 1 mm in I-N. (**p<0.01).
Figure 1—source data 1

Length of radius and humerus in wildtype and Clpex mutants.
Figure 2 with 1 supplement
Pgap2null allele fails to complement Pgap2Clpex allele.

Whole mount image of E13.5 WT (A,C,E,G) and Pgap2Clpex/null mutant (B,D,F,H). Penetrance of some key phenotypes is compared in I. Cardiac histology of E14.5 WT (J) and Pgap2Clpex/null mutant (K,), scale bar indicates 1 mm. Higher power images of the ventricular septum (L, M), valve (N, O), and myocardial wall (P, Q). Scale bar indicates 100 μM in L-Q.
Figure 2—figure supplement 1
Pgap2 alternative transcripts.

UCSC Genome Browser view of Pgap2 with multiple alternative transcripts with Pgap2-225, Pgap2-203, and Pgap2-204 labeled with the start codon mutated in Clpex allele boxed in red and the start codon utilized by Pgap2-203 boxed in blue (A). Protein sequence alignment of canonical transcript Pgap2-225 and variant Pgap2-203 (B). The alignment shows a 89.9% identity in which most of the unaligned amino acids are in the C-terminal tail due to alternative start site usage.
Figure 3 with 1 supplement
Pgap2 is expressed in neural and craniofacial tissues during development.

Whole mount Pgap2 Xgal staining in E7.5 (A–B), E8.5 (C), E9.0 (D) E9.5 (E, G, H), E10.5 (I–L), and E11.5 (M,N). Pgap2 RNA in situ hybridization at E9.5 (F). Transverse section through the lip at E10.5 (L) at the future site of lip closure. Expression is seen in the ganglion cell layer of the retina at E11.5 (M). LNP = lateral nasal process, MNP = medial nasal process, NP = nasal pit.
Figure 3—figure supplement 1
Pgap2 expression at later embryonic and early postnatal stages.

Xgal section staining of Pgap2-LacZ in the eye (A), salivary gland (B), epidermis (C), stomach (D), nasal conchae (E), myocardium (F), lung parenchyma (G), kidney (H), ear (I), cerebral cortex (J, K), genital tubercle (L) and brain/choroid plexus (M). Scale bar indicates 200 um.
Figure 4 with 1 supplement
Pgap2 is required for proper anchoring of GPI-APs.

(A) FLAER staining of WT (orange) and Clpex (blue) MEFs, (unstained control in gray) with quantification of Mean Fluorescence Intensity (MFI) (B). FLAER staining of WT (orange) Clpex KI Clone 7 (purple), PGAP2-/- (green), PIGA-/- (blue) HEK293T cells, and unstained control (red) (C) with quantification of MFI (D). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 4—source data 1

FLAER Staining (MFI) of MEFs and HEK clones.
Figure 4—figure supplement 1
Sequencing of CRISPR/Cas9 generated PIGAnull/null, PGAP2 null/null, and Clpex KI 293T clones.

WT human sequence and Sanger Sequencing of PIGA null/null, clone showing a 29 bp deletion in PIGA exon 3 (A). PCR of exon 3 of PIGA showing a heterozygous clone with a small deletion and the KO with the 29 bp deletion (B). Sanger Sequencing of WT 293T PGAP2 exon three and PGAP2-/- clone showing a 121 bp deletion (C). PCR of PGAP2 exon three showing WT 293T, heterozygous clone with two deletions and the PGAP2-/- clone with a single large deletion (D). Sanger Sequencing of PGAP2 exon three in WT 293T and Clpex Knock-in (KI) clone with the highlighted A > G mutation (E). PCR of PGAP2 exon three with WT 293T and Clpex KI clone DNA (F).
Trafficking of FOLR1 to the cell membrane requires GPI biosynthesis and remodeling.

Wheat germ agglutinin (WGA) staining in WT (A), PIGAnull/null (D), PGAP2null/null (G) HEK293T cells. FOLR1-myc staining in WT (B), PIGAnull/null (E), PGAP2null/null (H) HEK293T cells. Merge of WGA and FOLR1 for WT (C), PIGAnull/null (F), and PGAP2null/null (I). Pearson Coefficient for co-localization of WGA and FOLR1-myc (J). Western blot for αmyc-FOLR1 (green) and αTubulin (red) loading control from cell lysates of WT, PIGAnull/null, and PGAP2 null/null cells overexpressing N-myc tagged FOLR1 (K) and Rabbit IgG control for the same cell lysate (L). ****p<0.0001, Scale bar indicates 100 μM.
Figure 5—source data 1

Quantification of co-localization of WGA and Folr1-myc staining.
Figure 6 with 1 supplement
Clpex cNCCs and neuroepithelium undergo apoptosis at E9.5.

Wnt1-Cre, R26R NCC lineage trace in WT (A, C, E) and Clpex mutant (B,D,F) at E9.5 (A,B) and E11.5 (C–F). WT E9.5 embryo stained for DAPI (G), AP2 (H) CC3 (I) and merged image in (J). Clpex E9.5 embryo stained for DAPI (K), AP2 (L) CC3 (M), and merged image in (N). Higher power image of WT (O) and Clpex mutant (P) neuroepithelium stained with CC3 and DAPI. Quantification of CC3+ cells over AP2+ cells in the first branchial arch Region of Interest (Q). **p<0.01. Scale bar indicates 100 μm.
Figure 6—source data 1

Quantification of CC3-positive and AP2-positive cells in wildtype and mutant.
Figure 6—figure supplement 1
Clpex mutants do not display a defect in barrier formation.

E18.5 WT (A), Clpex cleft palate mutant (B), Clpex cleft lip/cleft palate mutant (C), and Clpex neural tube defect mutant (D) stained with Toludine Blue.
Figure 7 with 1 supplement
Folinic Acid treatment in utero partially rescues the cNCC apoptosis and cleft lip in Clpex mutants.

Schema of diet regimen to evaluate apoptosis and phenotypic rescue in Clpex mutants treated with control, 25ppm folic acid, or 25ppm folinic acid diet in utero (A). Phenotypes observed in Clpex mutants from litters treated from E0-E16.5 with control diet (blue), 25ppm folic acid (orange), or 25ppm folinic acid (gray) (B). Summary of the phenotypes of Clpex mutants from litters treated with the indicated diets (C). (*p<0.05).
Figure 7—figure supplement 1
Folinic acid treatment of Clpex embyos does not rescue neural crest cell apoptosis.

WT E9.5 embryo (A) and E9.5 Clpex mutant embryo (B) from litters of pregnant dams fed a control diet from E0.5-E9.5. WT E9.5 embryo (C) and E9.5 Clpex mutant embryo (D) from litters of pregnant dams fed a 25ppm folinic acid diet from E0.5-E9.5. Coronal sections of the first arch stained for AP2 (red), Cleaved Caspase 3 (green), and counterstained with DAPI. Quantification of CC3 +spots/AP2 +spots in the first arch Region of Interest (E). n.s. = not significant.
Figure 8 with 1 supplement
Piga is expressed in the first branchial arch, medial nasal process, limb bud and deletion of Piga in the Wnt1-Cre lineage results in NCC cells that lack GPI biosynthesis.

WMISH of WT E11.5 embryo stained with αsense Piga probe (A, C, E) or sense Piga probe (B, D, E). FLAER flow cytometry staining of WT (orange, blue) and Piga hemizygous cKO MPNMCs (green). FLAER MFI quantified (H). Fb = Forebrain, Mb = Midbrain, BA1 = Branchial Arch 1, MNP = Medial Nasal Process, Lb = Limb bud. **p<0.01.
Figure 8—figure supplement 1
GPI biosynthesis genes show increased expression in the first branchial arch, limb bud, and forebrain.

WT E11.5 RNA in situ hybridization with α-sense (Pigp) (A,B) and sense control probe (G,H); Pigu α-sense probe (C,D) and sense control probe (I,J) and Pigx α-sense probe (E,F) and sense control probes (K,L).
Conditional knockout of Piga abolishes GPI biosynthesis in NCCs and leads to median cleft lip/palate and craniofacial hypoplasia.

Whole mount images of E15.5 WT (A), mosaic Piga cKO (D), E16.5 WT (G) and hemizygous Piga cKO (J). Ventral view of the secondary palate of E15.5 WT (B), Mosaic cKO (E), E16.5 WT (H) and hemizygous cKO (K). H and E staining of E15.5 WT (C), mosaic cKO (F), E16.5 WT (I), and hemizygous cKO (L), arrowhead indicates cleft palate. Alazarin red and alcian blue staining of E16.5 WT skull (M–O) and hemizygous Piga cKO skull (P–R). Asterick indicates cleft palate. Fr = Frontal bone, Pa = Parietal bone, iPa = interparietal bone, Zy = Zygomatic bone, Mn = Mandible, pMx = Premaxilla, Nas = Nasal bone. Scale bar indicates 500 μM in C, F, I, L and 1 mm in M-R.
Author response image 1
Commercial Pgap2 antibody does not detect overexpressed or endogenous PGAP2.

10μg lysate from overexpression of mouse Pgap2-myc tagged clone (Origene #MR203189) in WT (lane 2) or Pgap2 KO 293T (lane 3) or lysate from mock transfection of WT (lane 4) or Pgap2 KO 293T (lane 5) probed with 1:500 Rb αPGAP (Thermo #PA5-64091, Green). (A) 10μg lysate from overexpression of mouse Pgap2-myc tagged clone in WT (lane 2) or Pgap2 KO 293T (lane 3) or lysate from mock transfection of WT (lane 4) or Pgap2 KO 293T (lane 5) probed with 1:1000 Rb αMyc (Abcam #ab9106, Green) or Ms αTub (Red) (B).


Table 1
Exome analysis identifies variant in Pgap2.
Variant filtersNumber
Total variants145,956
Homozygous in all three mutants120,393
Chromosome 75854
81–125 Mb2196
‘High’ impact9
Not in dbSNP7
Single base pair change1: Pgap2
Table 2
RNA sequencing ToppGene pathway enrichment analysis.
GO Category
GO Category
Hit Count in
Query List
Hit Count in
Hit in
Query List
GO:0043565Sequence-specific DNA binding1.79E-071.53E-041.53E-041.12E-03391096CDX2, CDX4, CIART, MYT1, EVX1,
GO:0017127Cholesterol transporter activity1.10E-069.41E-044.71E-043.45E-03514ABCA1, APOA1, APOA2,
GO:0034185Apolipoprotein binding2.34E-062.01E-036.68E-044.90E-03516ABCA1, MAPT, PLG, PCSK9, LIPC
Table 3
Anterior/posterior transcription factors differentially expressed in Clpex mutants compared to controls.
Table 3A
Anterior TFsTPM
GeneLog2 FCp-valueWild-typeClpex
Table 3B
Posterior TFsTPM
GeneLog2 FCp-valueWild-typeClpex
Key resources table
Reagent type
(species) or
DesignationSource or
Genetic reagent (Mus Musculus)PigafloxRikenRiken:B6.129-Pigatm1
Genetic reagent (Mus Musculus)Wnt1-CreJackson LaboratoriesJAX:B6.Cg-H2afvTg(Wnt1-cre)11RthTg(Wnt1-GAL4)11Rth/J
Genetic reagent (Mus Musculus)R26R LacZ reporterJackson LaboratoriesJAX:B6.129S4 Gt(ROSA)26Sortm1Sor/J; R26RTg
Genetic reagent (Mus Musculus)Pgap2nullEUCOMMEUCCOM: Pgap2tm1a(EUCOMM)Wtsi
Genetic reagent (Mus Musculus)ClpexStottmann et al., 2011In house: Pgap2Clpex
Cell line (Homo sapiens, kidney)HEK 293 T cellATCCATCC: #CRL-11268
Antibodysheep polyclonal anti-DigoxigeninRocheRoche: #11093274910
Antibodyrabbit polyclonal anti-mycAbcamAbcam: #ab9106
Antibody488-congugated goat polyclonal anti-rabbitThermoThermo: #A11008
Antibodymouse monoclonal anti-AP2Developmental Studies Hybridoma BankDSHB: #3B5 supernatant
Antibodyrabbit polyclonal anti-Cleaved Caspase 3Cell Signaling TechnologyCST: #9661
AntibodyMouse monoclonal anti-mycSigmaSigma: #M4439-100UL
AntibodyMouse monoclonal anti-TubulinSigmaSigma: #T6199
Antibodygoat anti-rabbit IRDye 800CWLICORLICOR: # 926–32211
Antibodygoat anti-mouse IRDye 680RdLICORLICOR: #926–68070
Sequence-based reagentpaired-end RNA sequencingBeijing Genomics Institute-Americas
Peptide, recombinant proteinBBSI enzymeNew England BiolabsNEB: R0539S
Commercial assay or kitMEGAclear Transcription Clean-up kitThermoThermo: #AM1908
Chemical compound, drugAlexafluor-488 proaerolysin (FLAER)CedarLane Labs, Burlington, OntarioCedarlane Labs: #FL1-C, 25 µg
Chemical compound, drug25ppm folic acid dietEnvigoEnvigo: Custom diet TD.160472
Chemical compound, drug25ppm folinic acid dietEnvigoEnvigo: Custom diet TD.160746
Chemical compound, drugControl DietEnvigoEnvigo: Custom diet TD.160112
Chemical compound, drug5-Bromo-4-chloro-3-indolyl β-D-galactopyranosideSigmaSigma: #B4252
Chemical compound, drugAlcian BlueSigmaSigma: #A3157
Chemical compound, drugAlazarin RedSigmaSigma: #A5533
Chemical compound, drugToluidine BlueSigmaSigma: #89640
Chemical compound, drugwheat germ agglutinin Texas Red ConjugateThermoThermo: #W21405(5 µL WGA/1 mL PBS)
Chemical compound, drugFolinic acidSigmaSigma: #F7878-500MG
Software, algorithmGraphpad PrismGraphPad Software, San Diego, CARRID:SCR_002798
Software, algorithmImaris 9.2.1, colocalization functionOxford InstrumentsRRID:SCR_007370
Software, algorithmNikon Elements Software, birghtspot analysisNikon Instruments Inc.RRID:SCR_014329
Software, algorithmFASTQC
Software, algorithmRSEM-v1.3.0Li and Dewey, 2011RRID:SCR_013027
Software, algorithmToppgene
Software, algorithmComputational Suite for Bioinformaticians and Biologists
Software, algorithmBenchling sgRNA design softwareBenchling, San Fransisco CARRID:SCR_013955
Recombinant DNA reagentmouse Piga plasmidOrigeneOrigene: #MR222212
Recombinant DNA reagentmouse Pgap2 plasmidOrigeneOrigene: #MR2031890
Recombinant DNA reagentmouse Pigp plasmidOrigeneOrigene: #MR216742
Recombinant DNA reagentmouse Pigu plasmidOrigeneOrigene: #MR223670
Recombinant DNA reagentmouse Pigx plasmidOrigeneOrigene: #MR201059
Recombinant DNA reagentmouse Lhx8 plasmidOrigeneOrigene: #MR226908
Recombinant DNA reagentmouse Tbxt plasmidOrigeneOrigene: #MR223752
Recombinant DNA reagentmouse Alx3 plasmidDNASUDNASU: #MmCD00081160
Recombinant DNA reagentCRISPR/Cas9 PX459M2 puromycin-resistance vectorRan et al., 2013
Recombinant DNA reagentUltramer with 5' and 3' phosphorothiolate bondsIntegrated DNA Technologies
Transfected construct (Homo sapiens)Human N-myc tagged Folr1 plasmidSinobiologicalSinobiological: #HG11241-NM

Additional files

Supplementary file 1

Pgap2 alternatively spliced transcripts.
Supplementary file 2

Primers used in this study.
Supplementary file 3

Clpex mutants display defects in expression of anterior/posterior patterning genes.

E9.5 WT (A,C) and Clpex mutant (B, D) RNA in situ hybridization with α-sense Alx3 probe, an anterior pattering gene. E9.5 WT (E) and Clpex mutant (F) RNA in situ hybridization with α-sense Lhx8 probe, an anterior patterning gene. E9.5 WT (G) and Clpex mutant (H) RNA in situ hybridization with α-sense Tbxt (Brachyury) probe, a posterior patterning gene. E8.5 WT (I) and Clpex mutant (J) RNA in situ hybridization with α-sense Tbxt (Brachyury) probe, a posterior patterning gene.
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  1. Marshall Lukacs
  2. Tia Roberts
  3. Praneet Chatuverdi
  4. Rolf W Stottmann
Glycosylphosphatidylinositol biosynthesis and remodeling are required for neural tube closure, heart development, and cranial neural crest cell survival
eLife 8:e45248.