ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development

Abstract

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast cell lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this study, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant up-regulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation, as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both down-regulation of EVT cell-associated transcripts and up-regulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis, resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a modulator of the syncytiotrophoblast lineage.

Keywords: differentiation; invasion; placenta; trophoblast.

Fig. 1.

ASCL2 expression in invasive trophoblast cells is conserved in hemochorial placentation. (A) Schematic depicting the location of invasive trophoblast progenitor cells in human and rat. (B) Human placental tissue specimen obtained at 12 wk of gestation probed for ASCL2 using in situ hybridization. ASCL2 (red) is expressed in extravillous trophoblast column cells but not in syncytiotrophoblasts or cytotrophoblasts. Rat placental tissue specimen obtained from a gd 12.5 rat probed for Ascl2 using in situ hybridization. Ascl2 (red) is robustly expressed in the junctional zone compared with the decidua and labyrinth zones. (Scale bar, 50 μm.) (C) First trimester human uteroplacental tissue specimen probed for HLA-G (green) and ASCL2 (red) using in situ hybridization. DAPI marks cell nuclei (blue). HLA-G is a known marker of EVT. The arrow indicates endovascular EVTs lining the decidual spiral arterioles (SpAs). (Scale bars, 50 μm.) (D) Rat placental tissue specimen obtained from a gd 12.5 rat probed for Prl7b1 and Ascl2 using in situ hybridization. (Scale bars, 200 μm.) Dotted lines depict the region shown below in higher magnification. DAPI marks cell nuclei (blue). Prl7b1 (green) is a known marker of rat invasive trophoblast cells. Arrows indicate endovascular invasive trophoblast which line the decidual SpA. (Scale bars, 100 μm.)

Fig. 2.

ASCL2 is required for EVT differentiation. (A) Transcript levels of six invasive trophoblast markers (ASCL2, HLA-G, MMP2, IGF2, PCSK6, and SNAI1) on day 8 of EVT differentiation following transduction with lentivirus containing a control shRNA (Ctrl) or one of two independent ASCL2-specific shRNAs (Sh1 or Sh2). ASCL2 knockdown decreased levels of transcripts characteristic of invasive trophoblast cells (n = 3 transductions). *P < 0.05; **P < 0.01. All graphs depict means ± SD. (B) Phase-contrast images depicting cell morphology of EVT differentiated cells transduced with control shRNA (Control) or ASCL2-specific shRNA (ASCL2 shRNA 1 or 2) lentivirus on day 8 of EVT cell differentiation. (Scale bars, 500 μm.) (C) HLA-G (green) expression was evaluated in EVT differentiated cells on day 8 following transduction with lentivirus containing control shRNA (Control) or ASCL2-specific shRNA (ASCL2 KD) using immunocytochemistry. Phase-contrast images depict cell morphology, and DAPI labels cell nuclei. Merged fluorescence images overlay HLA-G and DAPI images. (Scale bars, 500 μm.)

Fig. 3.

ASCL2 knockdown alters the human TS cell transcript profile. (A) Volcano plot and heat map depicting RNA-seq analysis results of control (blue) and ASCL2 shRNA-treated (red) EVT cells (n = 4 per group). Blue dots represent significantly down-regulated transcripts with P ≤ 0.05 and a logarithm to base two-fold change of less than or equal to −2. Red dots represent significantly up-regulated transcripts with P ≤ 0.05 and a logarithm to base two-fold change of ≥2. The heat map depicts difference in gene expression between four control and ASCL2 knockdown (KD) samples. The heat map color key represents z-scores of RPKM values. (B) Validation of RNA-seq results by qRT-PCR confirms that ASCL2 knockdown significantly decreases EVT-specific transcripts (HLA-G, CCR1, NOTUM, and PLOD2) and significantly increases syncytiotrophoblast (ST)-specific transcripts (SDC1, CYP19A1, CGB5, and ADM). n = 4 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C) Schematic depicting trophoblast cell differentiation from the stem/progenitor state toward either EVT cell or syncytiotrophoblast fates. (D) Relative expression of CYP19A1, ADM, CGB5, and SDC1 in human TS cells cultured under the following conditions: stem state (Stem), following 8 d of EVT cell differentiation without transduction (EVT) or with transduction using control shRNA (Control) or ASCL2-specific shRNA (ASCL2 KD), or 6 d of syncytiotrophoblast differentiation using the ST2D or ST3D protocol (34). n = 3 to 4 per group. **P < 0.01; ***P < 0.001; ****P < 0.0001. (E) Relative expression of ERVW-1, ERVFRD-1, SLC1A5, and MFSD2 in human TS cells following 8 d of EVT cell differentiation with transduction using control shRNA (Control) or ASCL2-specific shRNA (ASCL2 KD). n = 3 to 4 per group. *P < 0.05; **P < 0.01; ****P < 0.0001. All graphs depict mean ± SD values.

Fig. 4.

Ascl2 mutant placentation sites do not form an intact junctional zone. (A) Immunohistochemical analysis of vimentin in gd 12.5 WT and Ascl2 mutant rat placentation sites. (B) In situ hybridization for Prl3d1 (white) in gd 12.5 WT and Ascl2 mutant rat placentation sites. DAPI marks cell nuclei (blue). (C) Volcano plot and heat map depicting results of RNA-seq analysis from gd 12.5 placentas dissected from WT and Ascl2 mutant (Mutant) specimens (n = 4 per group). The x-axis of the volcano plot depicts logarithm to base 2 of the fold change (logFC). Blue dots represent significantly down-regulated transcripts with P ≤ 0.05 and a logarithm to base two-fold change of ≤−2. Red dots represent significantly up-regulated transcripts with P ≤ 0.05 and a logarithm to a base two-fold change of ≥2. The heat map depicts difference in gene expression between four WT and mutant samples. The heat map color key represents z-scores of RPKM values. (D) Validation of RNA-seq results by qRT-PCR confirms up-regulation of Adm, Cyp11a1, Prl3d1, and Hsd3b3 and down-regulation of Mmp15, Prl7b1, and Ncam1 in maternally inherited Ascl2 mutant specimens (Ascl2^+/−; gray) compared with WT specimens (Ascl2^+/+; black). n = 4 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All graphs depict mean ± SD values. (E) Immunohistochemical analyses of MKI67 (white) and cytokeratin (green) in gd 12.5 WT and Ascl2 mutant rat placentation sites. (F) Mki67 transcript levels are significantly decreased in gd 12.5 Ascl2 mutant placentas compared with WT. n = 4. P < 0.001. (Scale bars, 500 μm.)

Fig. 5.

Intrauterine trophoblast cell invasion is impaired in ASCL2-deficient placentation sites. (A) Schematic depicting the primary tissue zones comprising the rat placenta (BioRender). (B) Immunohistochemical analysis of cytokeratin (trophoblast marker) expression in gd 12.5 placentation sites from WT and Ascl2 mutant specimens. Arrows indicate invasive endovascular trophoblast cells. (C) Relative expression of invasive trophoblast cell-specific transcripts Prl7b1, Ncam1, and Mmp15 in dissected decidua tissue obtained from WT (^+/+) and maternally inherited Ascl2 mutant (^+/−) placentation sites (WT, n = 9; mutant, n = 10). *P < 0.05; **P < 0.01. All graphs depict mean ± SEM values. (D) WT and Ascl2 mutant rat placental tissue specimens obtained on gd 12.5 rats probed for Ascl2 using in situ hybridization. Ascl2 (red) is robustly expressed in the junctional zone and the invasive trophoblasts in the mesometrial decidua in WT, but not Ascl2 mutant, placentation sites. The arrow indicates the location of invasive endovascular trophoblast cells. (Scale bars, 200 μm.)