Transcription factor ASCL2 is required for development of the glycogen trophoblast cell lineage

Abstract

The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2.

Fig 1. Loss of imprinting at Ascl2 in +/Del^7AI placentae.

(A) Simplified map of the imprinted region on distal mouse Chr7. Maternally expressed genes are shown in red, paternally expressed genes in blue. The gametic differentially methylated regions acting as imprinting centres (IC) in the H19 and Kcnq1ot1 sub-domains are labelled as IC1 and IC2, respectively. Location of the Del^7AI deletion is shown on the left, transcriptional orientation of each gene (arrows), on the right. (B) Allele-specific expression analysis for Ascl2, Tssc4, Cdkn1c, and Phlda2, on E13.5 placental cDNA. M and P show the position of the maternal and paternal bands, respectively. The 2M+P band contains two maternal bands and 1 paternal band; M+P, co-migrating maternal and paternal bands. Informative SNPs are boxed. For the Cdkn1c SNP, CAST = C and 129 = T; and for Phlda2, CAST = C and the 129 = A. C: WT CAST allele; +: WT 129 allele. (C) Maternal to paternal allele expression ratio (M to P ratio) for wild-type (C/+) and paternal deletion mutants (C/Del^7AI). (D) Total expression levels in E15.5 placentae determined by RT-qPCR for three biological replicates per genotype. Error bars: SD for biological replicates. Expression is relative to Ppia levels. *p<0.005.

Fig 2. Phenotypic rescue of the mid-gestational lethality of Ascl2 mutants by paternal inheritance of Del^7AI.

(A) Number of rescued embryos at E12.5 to E18.5 from crosses between Ascl2^+/lacZ or Ascl2^+/KO females and +/+ or +/Del^7AI males. ^¶: includes 3 small and necrotic embryos. The data for individual litters are presented in S1 Table. (B) Number of live Ascl2 mutant pups at birth obtained from similar crosses. (C) Bar charts showing the percentage observed over expected values from crosses analyzed in utero (left) and at birth (right). X-axis: male genotypes. Values from crosses involving Ascl2^+/lacZ and Ascl2^+/KO females are shown in grey and black, respectively. The probability values were obtained by the chi-square test of the null hypothesis of Del^7AI = WT, where the rescue frequency of the wild-type allele, 0.901%, is based on all crosses involving the Ascl2^lacZ allele (3/333 Ascl2^lacZ/+ progeny). * p<1×10⁻¹⁰.

Fig 3. Growth retardation of rescued Ascl2^lacZ/Del^7AI heterozygotes.

(A) Scatterplots showing weights of rescued live Ascl2^lacZ/Del^7AI pups (–/Del^7AI, n = 7) and their wild-type littermates (n = 14) at postnatal days 0 (P0, birth), 7, and 21. Scatterplots of E15.5 placental (B) and embryonic (C) weights of Ascl2^lacZ/Del^7AI conceptuses (–/Del^7AI, n = 10) and wild type littermates (n = 25). In all graphs, the bars show the average weight ± SD.

Fig 4. Hyperplastic parietal trophoblast giant cell layer in Ascl2^lacZ/Del^7AI placentae at E15.5.

Heamatoxylin and eosin (H&E, top) and DAPI (bottom) staining of wild-type, +/Del^7AI and rescued Ascl2^lacZ/Del^7AI placental sections at E15.5. DAPI staining readily identifies the large polyploid nuclei of the cells stacked at the giant cell layer in the mutants. dec, decidua; P-TGC, parietal trophoblast giant cells; SpT, spongiotrophoblast cells; GlyT, glycogen trophoblast cells; lab, labyrinthine layer. Dashed lines indicate the P-TGC layer boundary. Scale bar: 0.5 mm.

Fig 5. Abnormal spongiotrophoblast and glycogen trophoblast cell development in Ascl2^lacZ/Del^7AI placentae at E15.5.

Frozen sections of E15.5 placentae were analyzed for the expression of Ascl2, Tpbpa, Pcdh12, and Cdkn1c by in situ hybridization. Periodic acid-Schiff (PAS) staining of glycogen was performed on paraffin sections of E15.5 placentae. Multiple sections from two placentae of each genotype, labeled at the top, were assessed and representative pictures are shown. SpT, spongiotrophoblast cells; lab, labyrinthine layer; GlyT, glycogen trophoblast cells; P-TGC, parietal trophoblast giant cells; dec, maternal decidua. Scale bars: 0.5 mm.

Fig 6. Absence of glycogen cell precursors in Ascl2 mutant placentae.

(A) Histological sections of wild-type and rescued Ascl2^KO/Del^7AI placentae at E16.5 were stained with heamatoxylin and eosin (H&E, top) or PAS with heamatoxylin nuclear counter-stain (bottom). SpT, spongiotrophoblast cells; lab, labyrinthine layer; GlyT, glycogen trophoblast cells; P-TGC, parietal trophoblast giant cells. Scale bar: 0.5 mm. (B) Immunofluorescence staining with PCDH12 polyclonal antibodies (red) and phalloidin (green) on cryosections of E8.5 wild-type and Ascl2^lacZ/+ conceptuses, counterstained with DAPI (blue). Scale bar: 0.1 mm.

Fig 7. RNA-seq analysis of wild-type and Ascl2-deficient E7.5 ectoplacental cones.

(A) Scatter plot showing average RPKM values for 25,071 autosomal RefSeq genes, from the RNA-seq analysis of dissected E7.5 EPCs from three wild-type (Ascl2^+/+) and three mutant (Ascl2^lacZ/+) littermates. Significantly upregulated genes (72 genes, Z > 1) and downregulated genes (217 genes, Z < −1) are shown in blue and red, respectively. r: Pearson correlation coefficient. (B) Average RPKM values for selected genes known to be expressed in spongiotrophoblast and glycogen trophoblast derivatives of EPC progenitors, from the RNA-seq analysis of E7.5 EPCs of given genotypes. Ppia is included as a housekeeping gene control. * p<0.05; ** p<0.01; *** p<0.005. (C) Fold change in RPKM values (mutant/wild-type) for the genes analyzed in B. Graphs show average and SD for three biological replicates per genotype.

Fig 8. Differentiation defects of Ascl2^lacZ/+ trophoblast stem cells.

(A) Wild-type (Ascl2^+/+) and mutant (Ascl2^lacZ/+) TSCs were differentiated by FGF4 and conditioned medium withdrawal for 8 days and RNA samples were collected at the time points shown. Expression levels for Ascl2, Cdx2, Pcdh12, Tpbpa, Car2, and Prl3d1 were analyzed by RT-qPCR and expressed as levels relative to those of the house-keeping gene Ppia. The graphs show the average ± SD for technical triplicates. (B) Flow cytometric analysis of ploidy during TSC differentiation, performed by DNA staining with propidium iodide. Cells of higher ploidy (8n and 16n) are seen in differentiated cells of both genotypes starting at day 6 of differentiation. FACS profiles are shown in S7 Fig.