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. 2024 May 14;19(5):689-709.
doi: 10.1016/j.stemcr.2024.04.004. Epub 2024 May 2.

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development

Shoma Nakagawa  1 , Davide Carnevali  1 , Xiangtian Tan  2 , Mariano J Alvarez  3 , David-Emlyn Parfitt  4 , Umberto Di Vicino  1 , Karthik Arumugam  1 , William Shin  2 , Sergi Aranda  1 , Davide Normanno  5 , Ruben Sebastian-Perez  1 , Chiara Cannatá  1 , Paola Cortes  1 , Maria Victoria Neguembor  1 , Michael M Shen  6 , Andrea Califano  7 , Maria Pia Cosma  8
Affiliations

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development

Shoma Nakagawa et al. Stem Cell Reports. .

Abstract

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

Keywords: NKX1-2; RNA polymerase I; Wnt signaling; embryonic stem cell; master regulator analysis; mouse embryo; nucleolus; ribosome biogenesis; systems biology.

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Conflict of interest statement

Declaration of interests A.C. is founder, equity holder, and consultant, and M.J.A. is Chief Scientific Officer at DarwinHealth Inc., a company that has licensed some of the algorithms used in this manuscript from Columbia University. Columbia University is also an equity holder in DarwinHealth Inc.

Figures

Figure 1
Figure 1
Differential gene expression analysis of β-catenin clones and Tcf3 −/− ESCs and MR analysis (A) PCA analysis of S33Y-β-catenin #1 (dark blue dots), S33Y-β-catenin #2 (yellow dots), S33Y-β-catenin isogenic E14 WT line (light blue dots), Tcf3 −/− KO (green dots), and Tcf3 isogenic E14tg2a WT line (red dots). n = 6 independent experiments per condition. (B) Differential mRNA expression levels of pluripotency and differentiation genes normalized to the WT. False discovery rate (FDR) < 0.05. (C) Top active (red) or inactive (blue) MRs in the S33Y-β-catenin #1 or #2 mESC lines or the Tcf3 KO mESC line, ranked according to the cumulative Stouffer score. n = 6 independent experiments per condition. (D) Heatmap showing MR activity across the different early mouse developmental stages (active, red; inactive, blue). (E) Violin plot showing the activity of the top 18 MRs from Figure 1C (FDR <0.005).
Figure 2
Figure 2
Nkx1-2 is essential for preimplantation development and regulates translation and ribosome biogenesis (A) Representative images of embryos injected with CTL-MO (top) or Nkx1-2-MO (for KD; bottom). Zoomed images of E3.5 CTL-MO embryos (black line) or of Nkx1-2 KD embryos with a cavity (red line) or shrunk (blue line) formation. Scale bars, 400 μm (non-zoomed) and 50 μm (zoomed). (B) Preimplantation development of CTL-MO (n = 152 embryos) or Nkx1-2 MO-mediated KD embryos (n = 136 embryos), injected at E0.5 zygotes; preimplantation development was evaluated at E4.5. n = 10 independent experiments. ∗∗∗p < 0.01. (C) Representative images of embryos injected with CTL-siRNA (top), Nkx1-2 siRNA#1 (middle), or Nkx1-2 siRNA#2 (bottom). Scale bars, 400 μm. (D) Preimplantation development of embryos injected with CTL-siRNA (n = 87 embryos), Nkx1-2 siRNA#1 (n = 87 embryos), or Nkx1-2 siRNA#2 (n = 84 embryos) at the E0.5 stage and evaluated at E4.5. n = 5 independent experiments. ∗∗∗∗p < 0.0001. (E) Single-cell clustering through UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction of morula embryos injected with CTL-MO (n = 96 blastomeres; circles) or Nkx1-2-MO (n = 95 blastomeres; triangles). Of the three separate clusters of single cells identified, cluster 0 only contained CTL embryos, and cluster 2, only Nkx1-2 KD embryos. (F) GO enrichment analysis by Metascape (https://metascape.org/gp/index.html#/citations/) of significantly (adj. p < 0.01) downregulated genes in the Nkx1-2 KD cluster 2 versus the CTL cluster 0 in the morula stage, or versus all other clusters in blastocyst embryos (p < 0.05). (G) Single-cell clustering through UMAP dimensionality reduction of CTL (n = 86 blastomeres) and Nkx1-2 MO (n = 91 blastomeres) injected blastocyst embryos. CTL-MO injected cells, circles; Nkx1-2 KD cells, triangles. Four separate clusters of single cells were identified. (H) Circos plot showing downregulated genes and overlapped genes between morula and blastocyst embryos after Nkx1-2-MO injection. Downregulated genes in Nkx1-2 KD embryos were identified by comparing the marker genes of MC0 vs. MC2 (for morula) and BC0 vs. BC1, -2, and -3 (for blastocyst). Each arc in the plot represents a gene list, whereby each gene has a spot on the arc. Dark orange represents genes expressed in both morula and blastocyst; light orange, genes expressed uniquely in one. Purple lines link the same genes that are expressed in both morula and blastocyst, and blue lines link different genes falling into the same ontology term. (I and J) Mean fluorescent intensity (normalized by area) of NKX1-2 in CTL-MO or Nkx1-2 KD (MO-mediated) embryos (I), or in CTL-siRNA or Nkx1-2 KD (siRNA-mediated) embryos (J). For (I), the following blastomeres were analyzed: CTL-MO E2.5 (gray dots, n = 85 blastomeres), Nkx1-2 KD E2.5 (red dots, n = 178 blastomeres), CTL-MO E3.5 (gray dots, n = 36 blastomeres), Nkx1-2 KD cavity E3.5 (pink dots, n = 48 blastomeres), and Nkx1-2 KD shrunk E3.5 (red dots, n = 17 blastomeres). n = 3 independent experiments. Representative images of NKX1-2 immunostaining are shown for E2.5 and E3.5 CTL-MO embryos and Nkx1-2 KD embryos (right). For (J), the following blastomeres were analyzed in E3.5 embryos: CTL-siRNA (gray dots, n = 152 blastomeres), Nkx1-2 siRNA#1 KD shrunk (red dots, n = 43 blastomeres), Nkx1-2 siRNA#1 KD cavity (orange dots, n = 29 blastomeres), Nkx1-2 siRNA#2 KD shrunk (red dots, n = 64 blastomeres), and Nkx1-2 siRNA#2 KD cavity (orange dots, n = 35 blastomeres). n = 3 independent experiments. Representative images of NKX1-2 immunostaining are shown for E3.5 CTL-siRNA or Nkx1-2 KD embryos (right). For both (I, J), green, NKX1-2 (anti-NKX1-2); magenta, DNA (DRAQ5). Scale bars, 5 μm.
Figure 3
Figure 3
NKX1-2 colocalizes with UBTF and regulates rRNA expression in mouse oocytes and preimplantation embryos (A) Representative images of NKX1-2 and UBTF showing co-localization in growing mouse germinal vesicle (GV) oocytes but not in fully developed oocytes. Green, NKX1-2 (anti-NKX1-2); red, UBTF (anti-UBTF); cyan, DNA (DRAQ5). Scale bars, 5 μm. (B) Representative images of NKX1-2 and UBTF in preimplantation embryos. UBTF is expressed starting from the 4-cell stage and then always colocalizes with NKX1-2. Green, NKX1-2 (anti-NKX1-2); yellow, UBTF (anti-UBTF); magenta, DNA (DRAQ5). Scale bars, 5 μm (zygotes), 2 μm (zoomed), or 10 μm (low magnification). (C) Representative images of EU incorporation and mean fluorescent intensity in Nkx1-2 KD or CTL-MO embryos. Green, EU; gray, DNA (DRAQ5). The EU signal was significantly reduced in Nkx1-2 KD embryos, both with shrunk (red dots, n = 29 blastomeres) or cavity (pink dots, n = 58 blastomeres) formation, as compared to CTL-MO embryos (gray dots, n = 70 blastomeres). n = 3 independent experiments. ∗∗∗p < 0.01. Scale bars, 20 μm. (D) RT-qPCR of 45S, mature 18S, and mature 28S in Nkx1-2 KD and CTL-MO embryos. Data are presented as mean ± SEM. n = 3 independent experiments. p values, t test. (E–H) Representative images of NKX1-2 and RNA POL I immunostaining of CTL-MO or Nkx1-2 KD (MO-mediated) embryos, at E1.5 (2-cell) (E), E2.0 (4-cell) (F), E2.5 (morula) (G), or E3.5 (blastocyst) (H). Green, NKX1-2 (anti-NKX1-2); red, RNA POL I (anti-RPA194); gray, DNA (DRAQ5). Scale bars, 3 μm. (I) Representative images of transmission electron microscopy images of Nkx1-2 KD or CTL-MO embryos at E2.5 (morula) or E3.5 (blastocyst). In the low-magnification images, red asterisks, hollows; yellow asterisks, condensed nucleoli; black arrows, disrupted nucleoli; and yellow arrows, tight junctions. Scale bars, 5 μm.
Figure 4
Figure 4
Nkx1-2 KD leads to severe chromosome missegregation and mitotic division errors (A) Time-lapse images of Nkx1-2 KD and CTL-MO embryos. Binucleation, white arrow; lagging chromosomes, red arrow; micronucleoli, white asterisk. Chromosomes and plasma membrane were visualized by H2B:EGFP and Gap43:EGFP, respectively. (B–D) Analyses of CTL-MO or Nkx1-2 KD embryos after nocodazole synchronization, cold shock, and heating (at E2.0) for 15 min (B, C) or 20 min (D). In (B), representative images show that CTL-MO embryos (top) had normal microtubules (MTs), while Nkx1-2 KD embryos frequently had multipolar spindles (middle) or disorganized spindle formation (bottom). In (C), spindle formation was categorized as bipolar (gray), multipolar (red), or disorganized (black). Nkx1-2 KD; n = 18 spindles, CTL-MO; n = 18 spindles, n = 3 independent experiments. ∗p = 0.0116, ∗∗∗∗p = 0.0002. In (D), representative images show that bipolar spindles had end-on kinetochore-MT (K-MT) attachment in CTL-MO embryos (zoomed) but frequently showed merotelic/lateral K-MT attachment in Nkx1-2 KD embryos (Nkx1-2 MO, zoomed). For (B) and (D): green, microtubules (anti-β-TUBULIN); red, kinetochores (anti-CREST); gray, DNA (DRAQ5). Scale bars, 5 μm (for non-zoomed) or 1 μm (for zoomed images). (E) K-MT attachment was classified into three categories: end-on, merotelic/lateral, or unattached. Green, MTs; red, kinetochore; gray, chromatid. (F) Quantification of K-MT attachment in CTL-MO or Nkx1-2 KD embryos. Gray, end-on; red, merotelic/lateral; black, unattached. Nkx1-2 KD; n = 36 embryos, CTL-MO; n = 18 embryos, n = 3 independent experiments. ∗∗∗∗p = 0.0002. (G) Representative images (at E3.5) of mitotic division in CTL-MO embryos (left) or Nkx1-2 KD embryos (middle and right). Images show different z stacks of a single embryo. Micronuclei were present in the Nkx1-2 KD shrunk (middle, yellow arrows) and cavity (right, yellow arrows) embryos. Gray, E-cadherin; magenta, DNA (DRAQ5). Scale bars, 10 μm. (H and I) Quantification of the total number of blastomeres (H) or micronuclei (I) in CTL-MO (n = 15 embryos) or Nkx1-2 KD (shrunk, n = 20 embryos; cavity, n = 7 embryos; total, n = 27 embryos) E3.5 embryos. n = 3 independent experiments. (J) Time-lapse images of blastocoel formation in CTL-MO or Nkx1-2 KD embryos. Cavity expansion was observed after each expansion/collapse cycle in CTL-MO embryos but not in Nkx1-2 KD embryos. (K) Timings of the first blastocoel formation in CTL-MO (n = 20 embryos) or Nkx1-2 KD (n = 7 embryos) embryos. Only embryos showing more than five expansion/collapse cycles were analyzed. n = 3 independent experiments. (L) Quantification of cavity diameter in CTL-MO (n = 20 embryos) or Nkx1-2 KD (n = 7 embryos) embryos. Cavity diameter was measured at each cycle of expansion (mean ± SEM). n = 3 independent experiments.
Figure 5
Figure 5
Severe impairment of TJ sealing, actin/myosin expression, and cell fate specification in Nkx1-2 KD embryos (A and B) Representative images of the TJ proteins ZO-1 and vinculin in (A) CTL-MO and Nkx1-2 KD (MO-mediated) embryos; and (B) CTL-siRNA or Nkx1-2 KD (siRNA-mediated) embryos. ZO-1 and vinculin were not detected in Nkx1-2 KD embryos in either KD condition. Red, ZO-1; green, vinculin. Scale bars, 15 μm. (C) Representative images of E3.5 embryos with Nkx1-2 KD (MO-mediated) (top) or CTL-MO (bottom), and incubated in medium supplemented with rhodamine-dextran (3,000 MW). Only cavity embryos were analyzed (CTL-MO, n = 26 embryos; Nkx1-2 KD, n = 12 embryos). n = 2 independent experiments. Scale bars, 20 μm. (D) Representative images and quantification of microlumen coarsening in CTL-MO (n = 16 embryos) or Nkx1-2 KD (shrunk, n = 20 embryos; cavity, n = 10 embryos) embryos at E3.5. Microlumens are indicated with black asterisks. Gray, E-CADHERIN. Scale bar, 20 μm. Red indicates embryos with numerous microlumens. n = 3 independent experiments. (E) Representative immunofluorescence images of E-CADHERIN, F-ACTIN, and MYOSIN in CTL-MO or Nkx1-2 KD embryos (at E3.5). Green, E-CADHERIN; red, MYOSIN (using anti-PHOSPHO-MYOSIN LIGHT CHAIN 2 [Thr18/Ser19]); gray, F-ACTIN (using anti-PHALLOIDIN). Scale bars, 20 μm. (F) Representative immunofluorescence images of E-CADHERIN and ACTIN in CTL-MO or Nkx1-2 KD embryos (at E3.5). Green, E-CADHERIN; gray, F-ACTIN (anti-PHALLOIDIN). Line scans were quantified in the middle areas of embryos (red line), and results are shown in the plots (CTL-MO, 25 embryos; Nkx1-2 KD shrunk, 16 embryos; Nkx1-2 KD cavity, 7 embryos). Scale bars, 20 μm; for zoomed images, 5 μm for CTL-MO and Nkx1-2 KD cavity, and 2 μm for Nkx1-2 KD shrunk. n = 3 independent experiments. (G) Representative immunofluorescence images of NANOG (epiblast cells) and GATA6 (primitive endoderm cells) in CTL-MO or Nkx1-2 KD embryos (E4.5). Green, NANOG (anti-NANOG); red, GATA6 (anti-GATA6). Scale bars, 20 μm. (H and I) Quantification of the number of NANOG-positive cells (H) or GATA6-positive cells (I) in CTL-MO (n = 21 embryos) or Nkx1-2 KD (shrunk, n = 12 embryos; cavity, n = 25 embryos) embryos (at E4.5). n = 3 independent experiments. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01. (J) Quantification of the number of NANOG and GATA6 double-positive cells in CTL-MO (n = 49 embryos) or Nkx1-2 KD (shrunk, n = 24 embryos; cavity, n = 20 embryos) embryos (at E4.5). n = 3 independent experiments. ∗∗p < 0.01. (K) Representative immunofluorescence images of NANOG (epiblast cells) and GATA6 (primitive endoderm cells) in CTL-siRNA and Nkx1-2 KD (siRNA-mediated) embryos (E4.5). Green, NANOG (anti-NANOG); red, GATA6 (anti-GATA6). Scale bars, 20 μm. (L) Representative immunofluorescence images of CDX2 (trophectoderm cells) and NANOG (epiblast cells) in CTL-MO and Nkx1-2 KD (MO-mediated) embryos (at E3.5). Green, CDX2 (anti-CDX2); red, NANOG (anti-NANOG). Scale bars, 20 μm. (M) Quantification of the number of CDX2 (trophectoderm cells) and NANOG (epiblast cells) double-positive cells in CTL-MO (n = 9 embryos) and Nkx1-2 KD (shrunk, n = 4 embryos; cavity, n = 5 embryos) embryos at E3.5. n = 2 independent experiments. ∗∗p < 0.01. (N) Representative immunofluorescence images of OCT4 (ICM marker) in CTL-MO or Nkx1-2 KD embryos (at E3.5). Gray, OCT4 (anti-OCT4); magenta, DNA (DRAQ5). Scale bars, 20 μm.
Figure 6
Figure 6
Preimplantation development and cell fate specification defects in Nkx1-2 KD embryos are rescued after Nkx1-2 mRNA injection (A) Preimplantation development in Nkx1-2 KD (MO-mediated) embryos was significantly improved by Nkx1-2 mRNA injection (at E4.5). ∗∗∗p < 0.001 (CTL-MO, n = 116 embryos; Nkx1-2 KD, n = 97 embryos; rescued, n = 63 embryos). n = 10 independent experiments. Representative images are shown. Scale bars, 400 μm. (B) Representative immunofluorescence images of NKX1-2 and POL I in CTL-MO, Nkx1-2 KD, and rescued embryos. Green, NKX1-2 (anti-NKX1-2); yellow, RNA POL I (anti-RPA-194); magenta, DNA (DRAQ5). Scale bars: 5 μm. (C) NKX1-2 mean fluorescent intensity in CTL-MO (gray; n = 23 blastomeres), Nkx1-2 KD (red; n = 54 blastomeres), and rescued (orange; n = 67 blastomeres) embryos. n = 5 independent experiments. ∗∗∗∗p < 0.0001. (D) Mean fluorescent intensity of RNA POL I in CTL-MO (gray dots, n = 37 blastomeres), Nkx1-2 KD (red dots, n = 52 blastomeres), and rescued (orange dots, n = 64 blastomeres) embryos. n = 5 independent experiments. ∗∗∗∗p < 0.0001. (E) Representative immunofluorescence images of NANOG (epiblast cells) and GATA6 (primitive endoderm cells) in CTL-MO, Nkx1-2 KD (MO-mediated), and Nkx1-2 mRNA rescued embryos (E4.5). Green, NANOG (anti-NANOG); red, GATA6 (anti-GATA6). Scale bars, 20 μm. (F) Quantification of the number of NANOG-positive cells in CTL-MO (n = 26 embryos), Nkx1-2 KD (n = 23 embryos), and Nkx1-2 mRNA rescued (n = 15 embryos) embryos (at E4.5). n = 4 independent experiments. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001. (G) Quantification of the total number of blastomeres in CTL-MO (n = 26 embryos), Nkx1-2 KD (n = 21 embryos), and Nkx1-2 mRNA rescued (n = 15 embryos) embryos (at E4.5). n = 4 independent experiments. ∗∗∗p < 0.001. (H) RT-qPCR of 45S, in CTL, Nkx1-2 MO, and Nkx1-2 mRNA rescued embryos. Data are presented as mean ± SEM (n = 3 independent experiments; the same CTL-MO and Nkx1-2 KD samples as used for Figure 3D were plotted). ∗p = 0.0189. (I) CTL-MO, Nkx1-2 KD, and rescued embryo transfer. All embryos were transferred into the uterus of a foster mother at the E3.5 stage, and pups were counted after birth. n = 3 independent experiments.
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