Journal: bioRxiv

Article Title: A Novel Mouse Model Reveals a Role for Mitochondria in Early Lineage Specification and Gastrulation

doi: 10.1101/2025.07.14.664670

Figure Lengend Snippet: A. E3.5 blastocysts from control ( R26R-mitoAtDarT , upper panel) and R26-mitoAtDarT (lower panel) embryos. Immunofluorescence staining shows SOX2 (inner cell mass marker, green), CDX2 (trophectoderm marker, magenta), and nuclei (DAPI, gray). Images were acquired by light-sheet microscopy and analyzed with Imaris software; the Spots module was used for cell quantification. Scale bars, 20 μm. N = 15 embryos per genotype. B. Quantification of total cell number (DAPI+) and lineage-specific cells (SOX2+ and CDX2+) per blastocyst. R26-mitoAtDarT embryos show significantly fewer total cells and reduced numbers of SOX2+ and CDX2+ cells. Notably, the proportion of cells co-expressing SOX2 and CDX2 is increased in R26-mitoAtDarT embryos. C. A stacked bar chart represents the average of the percentage of CDX2+, SOX2+ and CDX2 and SOX2 double positive cells in R26R-mitoAtDarT and R26-mitoAtDarT. The proportion of double-positive cells, relative to the total cell number, was significantly higher for the different markers, P < 0.002.

Article Snippet: Cryosections of post-implantation embryos were washed with 0.3% PBST three times for 20 minutes each to ensure permeabilization and complete removal of the OCT. Tissues were then blocked in 10% heat-inactivated goat serum (Himedia) and 1% BSA (Sigma) in 0.3 % PBST for 1 hour, followed by overnight incubation with primary antibodies: CdX2 (Biogenix-MU392A), SOX2 (Invitrogen-14-9811-82), SOX17 (R&D systems-af1924), Brachyury (Abcam-EPR18113), TOMM20 (Thermofisher-MA532148), FLAG-tag (Sigma-F1804), phospho-Histone H3 (pHH3) (Cell Signalling Technology - 9706S), and Cleaved caspase (Cell Signalling Technology - 9661S) at a 1:200 concentration diluted in the blocking buffer, overnight at 4°C.

Techniques: Control, Immunofluorescence, Staining, Marker, Microscopy, Software, Expressing

Journal: Nature

Article Title: Primate gastrulation and early organogenesis at single-cell resolution.

doi: 10.1038/s41586-022-05526-y

Figure Lengend Snippet: Fig. 2 | Developmental trajectories of PS and its derivatives from monkey embryos. a, UMAP showing PS and its derivatives from CS8–11 monkey embryos with trajectories inferred from RNA velocity analysis. Black arrows represent calculated velocity trajectories. b, Schematic diagram showing a CS8 embryo. Left, dorsal view; right, lateral view showing potential trajectories from PS to APS, DE, node and Nas.Meso. c, Joint hierarchical clustering (HC) showing the circular heatmap with representative TFs upregulated in PS (red), APS (green), node (purple), DE (orange) and Nas.Meso (grey). Genes marked with the same colour by HC show similar expression patterns in different cell types. d, Left, diagrams summarizing the distribution of indicated cell types in the region of interest, based on IF images on the right. Right, IF results showing localization of T+, CDX2+, OTX2+, FOXA2+, TBX6+ and SOX2+ cells in a monkey

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging 3 nature portfolio | reporting sum m ary M arch 2021 Antibodies Antibodies used Primary antibodies used for IF staining of embryos and embryonic stem cells: OCT4 (mouse, 1:100, Santa Cruz Biotechnology, sc5279, clone C-10) SOX2 (mouse, 1:100, Santa Cruz Biotechnology, sc365823, clone E-4) E-Cadherin (goat, 1:200, R&D Systems, AF748) OTX2 (goat, 1:100, R&D Systems, AF1979) Slug (rabbit, 1:100, Cell Signaling Technology, 9585S, clone C19G7) FOXA2 (rabbit, 1:200, Abcam, ab108422, clone EPR4466) T (rabbit, 1:100, Cell Signaling Technology, 81694, clone D2Z3J) AF9, MLLT3 (rabbit, 1:200, Abcam, ab154492) Brachyury, T (goat, 1:200, R&D Systems, AF2085) FOSB (goat, 1:200, R&D Systems, AF2214) TBX6 (goat, 1:200, R&D Systems, AF4744) YAP1 (mouse, 1:200, Abnova, H00010413-M01, clone 2F12) EOMES (rabbit, 1:100, Abcam, ab23345) CDX2 (mouse, 1:100, BioGenex, MU392A-5UC, clone CDX2-88) GATA6 (goat, 1:100, R&D Systems, AF1700) PDGF Receptor α (rabbit, 1:1, Cell Signaling Technology, 3174T, clone D1E1E) Secondary antibodies used for IF staining of embryos and embryonic stem cells: Alexa Fluor 488 Donkey anti-mouse (1:200, Thermo Fisher, A21202) Alexa Fluor 647 Goat anti-mouse (1:200, Thermo Fisher, A31571) Alexa Fluor 568 Donkey anti-rabbit (1:200, Thermo Fisher, A10042) Alexa Fluor 647 Donkey anti-rabbit (1:200, Thermo Fisher, A31573) Alexa Fluor 488 Donkey anti-goat (1:200, Thermo Fisher, A11055) Alexa Fluor 488 Donkey anti-rat (1:200, Thermo Fisher, A21208) Alexa Fluor 568 Donkey anti-sheep (1:200, Thermo Fisher, A21099) Validation All the above primary antibodies were validated in this work, the subcellular localization of the analyzed proteins has been previously reported.

Techniques: Expressing

Journal: Advanced Science

Article Title: Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1

doi: 10.1002/advs.202408852

Figure Lengend Snippet: Extended potency of Pdzk1 ‐KO ESCs. A) Sense (red) and antisense (green) insertions of PB in the Pdzk1 gene body from a previous screen of iTSCs via a haploid system. The rectangles indicate the exons. B) Schematic diagram of the strategy for deleting Pdzk1 in WT‐ESCs via the CRISPR/Cas9 system. C) Genotype PCR results of Pdzk1 ‐KO subclones. D) Genotyping of Pdzk1 ‐KO ESCs via DNA sequencing. E) Morphological images of the induced cell cultures of Pdzk1 ‐KO ESCs and WT‐ESCs before and after cell sorting with a TSC‐specific antibody (CDCP1). Scale bar, 100 µm. F) Percentages of CDCP1+ cells in induced cultures of Pdzk1 ‐KO ESCs and WT‐ESCs. G) Morphological images of Pdzk1 ‐KO iTSCs and WT‐TSCs. Scale bar, 100 µm. H) Immunofluorescence of TSC‐specific markers (CDX2 and EOMES) in Pdzk1 ‐KO iTSCs and WT‐TSCs. DNA was stained with Hoechst 33342. Scale bar, 50 µm. I) Morphological images of Pdzk1 ‐KO iXENs and WT‐ESC‐derived iXENs. Scale bar, 100 µm. J) Immunofluorescence of the XEN‐specific marker (GATA6) in Pdzk1 ‐KO iXENs and WT‐ESC iXENs. DNA was stained with Hoechst 33342. Scale bar, 50 µm. K) Summary of the potencies of Pdzk1 ‐KO ESCs and WT‐ESCs.

Article Snippet: The primary antibodies used were CDX2 (BioGenes, MU392A), OCT4 (Abcam, ab181557), GATA6 (CST, 5851), EOMES (Abcam, ab23345), TFAP2C (Santa Cruz, sc‐12762), and YAP1 (ABclonal, A1002).

Techniques: CRISPR, DNA Sequencing, FACS, Immunofluorescence, Staining, Derivative Assay, Marker

Journal: Advanced Science

Article Title: Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1

doi: 10.1002/advs.202408852

Figure Lengend Snippet: Advanced developmental potential of Pdzk1 ‐KO ESCs in chimeras. A) Schematic overview of the developmental contribution of microinjection of Pdzk1 ‐KO ESCs and WT‐ESCs into chimeric embryos. B) Immunofluorescence of OCT4 (red) in chimeric blastocysts derived from GFP‐labeled (green) Pdzk1 ‐KO ESCs and WT‐ESCs. DNA was stained with Hoechst 33342 (blue). Scale bar, 50 µm. C) Immunofluorescence of CDX2 (red) in chimeric blastocysts derived from GFP‐labeled (green) Pdzk1 ‐KO ESCs and WT‐ESCs. DNA was stained with Hoechst 33342 (blue). Scale bar, 50 µm. D) Immunofluorescence of GATA6 (red) in chimeric blastocysts derived from GFP‐labeled (green) Pdzk1 ‐KO ESCs and WT‐ESCs. DNA was stained with Hoechst 33342 (blue). Scale bar, 50 µm. E) Images of E12.5 chimeras (including fetus, placenta and yolk sac) derived from GFP‐labeled Pdzk1 ‐KO ESCs, with GFP‐labeled WT‐ESC‐derived chimeras used as a control. The white arrowheads indicate the fetus, placenta and yolk sac from the chimeric embryo. Scale bar, 5 mm. F) Heatmap representation of genes predominantly expressed in GFP+ cells from the chimeric placenta and chimeric yolk sac. The WT‐placenta and WT‐yolk sac data were downloaded from a previous study (Andergassen et al., 2017, Elife). Representative lineage markers are indicated.

Article Snippet: The primary antibodies used were CDX2 (BioGenes, MU392A), OCT4 (Abcam, ab181557), GATA6 (CST, 5851), EOMES (Abcam, ab23345), TFAP2C (Santa Cruz, sc‐12762), and YAP1 (ABclonal, A1002).

Techniques: Microinjection, Immunofluorescence, Derivative Assay, Labeling, Staining, Control

Journal: Advanced Science

Article Title: Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1

doi: 10.1002/advs.202408852

Figure Lengend Snippet: Generation and development of Pdzk1 ‐KO ESC‐derived blastoids. A) Blastoid formation efficiencies of Pdzk1 ‐KO ESCs and Dyrk1a ‐KO ESCs, with WT‐ESCs serving as a negative control. B) Images of Pdzk1 ‐KO blastoids, Dyrk1a ‐KO blastoids and E4.5 mouse blastocysts. Scale bar, 100 µm. C) Total number of cells in each E4.5 mouse blastocyst (n = 5) and each blastoid ( Pdzk1 ‐KO, n = 15; Dyrk1a ‐KO, n = 19). D) The diameter of each E4.5 mouse blastocyst (n = 19) and each blastoid ( Pdzk1 ‐KO, n = 19; Dyrk1a ‐KO, n = 19) separately. E) Immunofluorescence of OCT4 (red) and CDX2 (green) in Pdzk1 ‐KO blastoids. WT blastocysts were used as controls. DNA was stained with Hoechst 33342 (blue). Scale bar, 50 µm. F) Immunofluorescence of GATA6 (red) and CDX2 (green) in Pdzk1 ‐KO blastoids. WT blastocysts were used as controls. DNA was stained with Hoechst 33342 (blue). Scale bar, 50 µm. G) Schematic overview of the establishment of BESCs, BTSCs, and BXENs derived from GFP‐labeled blastoids. H) GFP and immunofluorescence images of BESCs, BTSCs, and BXENs derived from Pdzk1 ‐KO blastoids with specific markers. Scale bar, GFP images, 100 µm; Immunofluorescence images, 50 µm. I) Images of in vitro cultured (IVC) blastoids on Days 1–4. Scale bars, 100 µm. J) Immunofluorescence of OCT4 and CDX2 in IVC blastoids on Day 4. Scale bar, 50 µm. K) Immunofluorescence of CDX2 and GATA6 in IVC blastoids on Day 4. Scale bar, 50 µm.

Article Snippet: The primary antibodies used were CDX2 (BioGenes, MU392A), OCT4 (Abcam, ab181557), GATA6 (CST, 5851), EOMES (Abcam, ab23345), TFAP2C (Santa Cruz, sc‐12762), and YAP1 (ABclonal, A1002).

Techniques: Derivative Assay, Negative Control, Immunofluorescence, Staining, Labeling, In Vitro, Cell Culture

Journal: Advanced Science

Article Title: Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1

doi: 10.1002/advs.202408852

Figure Lengend Snippet: Single‐cell transcriptome analysis of Pdzk1 ‐KO blastoids and Pdzk1 ‐KO ESCs. A) UMAP plots of Pdzk1 ‐KO blastoids and WT blastocysts. B) Comparison of cells derived from Pdzk1 ‐KO blastoids and WT blastocysts. This panel shows lineage assignments: epiblast (purple), primitive endoderm (green) and trophectoderm (blue). C) The expression of representative marker genes for epiblast ( Oct4 ), primitive endoderm ( Gata6 ) and trophectoderm ( Cdx2 ) in Pdzk1 ‐KO blastoids at the single‐cell level. D) Principal component analysis (PCA) of Pdzk1 ‐KO blastoids compared with published datasets of mouse embryos. The embryonic stages are indicated. E) PCA of Pdzk1 ‐KO ESCs, WT‐ESCs, ciTotiSCs, TBLCs, L. EPSCs, D. EPSCs, TBLCs and mouse embryos at different stages at the single‐cell level. F) Hierarchical clustering analysis of Pdzk1 ‐KO ESCs, WT‐ESCs, ciTotiSCs, TBLCs, L. EPSCs, D. EPSCs, TBLCs and mouse embryos at different stages at the single‐cell level.

Article Snippet: The primary antibodies used were CDX2 (BioGenes, MU392A), OCT4 (Abcam, ab181557), GATA6 (CST, 5851), EOMES (Abcam, ab23345), TFAP2C (Santa Cruz, sc‐12762), and YAP1 (ABclonal, A1002).

Techniques: Comparison, Derivative Assay, Expressing, Marker

Journal: Advanced Science

Article Title: Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1

doi: 10.1002/advs.202408852

Figure Lengend Snippet: Validation of lineage inducers in the totipotent state. A) Expression levels of OE genes ( C1qa , C1qb , Fgf5 , and Cdx2 ) in OE‐ESCs, with the empty vector group and WT‐ESCs used as controls. The data are presented as the means ± SDs. *** p < 0.001. B) Images of E12.5 chimeras (including fetuses, placentas and yolk sacs) derived from GFP‐labeled C1qa ‐OE, C1qb ‐OE, Fgf5 ‐OE, and Cdx2 ‐OE ESCs, with the GFP‐labeled empty vector group used as a control. Scale bar, 5 mm. C) Summary of C1qa ‐OE, C1qb ‐OE, Fgf5 ‐OE, and Cdx2 ‐OE ESCs contributing to three lineages according to chimeric assays. D) Volcano plot of DEGs between Pdzk1 ‐KO ESCs and WT‐ESCs. C1qa , C1qb , Fgf5 , and Cdx2 are highlighted.

Article Snippet: The primary antibodies used were CDX2 (BioGenes, MU392A), OCT4 (Abcam, ab181557), GATA6 (CST, 5851), EOMES (Abcam, ab23345), TFAP2C (Santa Cruz, sc‐12762), and YAP1 (ABclonal, A1002).

Techniques: Biomarker Discovery, Expressing, Plasmid Preparation, Derivative Assay, Labeling, Control

Journal: Advanced Science

Article Title: Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1

doi: 10.1002/advs.202408852

Figure Lengend Snippet: Single‐cell transcriptome profiles of Pdzk1 ‐KO ESCs and WT‐ESCs. A) UMAP plots of Pdzk1 ‐KO ESCs and WT‐ESCs. B) UMAP plots of Pdzk1 ‐KO ESCs, WT‐ESCs, WT‐TSCs, and WT‐XENs. C) Normalized expression of C1qa , C1qb , Fgf5 , and Cdx2 in Pdzk1 ‐KO ESCs and WT‐ESCs according to the UMAP at the single‐cell level. D) Dot plots indicating the expression of C1qa , C1qb , Fgf5 , and Cdx2 in Pdzk1 ‐KO ESCs and WT‐ESCs.

Article Snippet: The primary antibodies used were CDX2 (BioGenes, MU392A), OCT4 (Abcam, ab181557), GATA6 (CST, 5851), EOMES (Abcam, ab23345), TFAP2C (Santa Cruz, sc‐12762), and YAP1 (ABclonal, A1002).

Techniques: Expressing