Wydział Biologii

This dataset consists:

File 1 - with results of the level of activin A protein from the culture medium of Inhba^KO/KO and wild-type mouse embryonic fibroblasts.

Files 2A, B, C, D - Four .xlsx files with data of morphokinetic parameters from time-lapse imaging of activin A knockout (Inhba^KO/KO), heterozygotes (Inhba^KO/WT), and wild-type (Inhba^WT/WT) embryos:

• File 2A - in C57BL/6/Tar x CBA/Tar mixed genetic background that reached the blastocyst stage;
• File 2B - in C57BL/6/Tar x CBA/Tar mixed genetic background that were arrested in development;
• File 2C - in an inbred C57BL/6 genetic background that reached the blastocyst stage;
• File 2D - in an inbred C57BL/6 genetic background that were arrested in development.

Files 3A, B, C- Three .xlsx files with the number and percentage of cell lineages of Inhba^KO/KO, Inhba^KO/WT and Inhba^WT/WT:

• File 3A - 120 hours blastocysts in C57BL/6/Tar x CBA/Tar mixed genetic background;
• File 3B - 135 hours blastocysts in C57BL/6/Tar x CBA/Tar mixed genetic background;
• File 3C - in an inbred C57BL/6 genetic background.

Files 4A, B - Two .xlsx file with Inhba^KO/KO, Inhba^KO/WT and Inhba^WT/WT blastocyst outgrowths data:

• File 4A - blastocyst outgrowths area (cultured with or without activin A).
• File 4B - blastocyst that did not form outgrowths (culture without activin A)

File 5 - with the results of RT-qPCR analysis of mesoderm, endoderm, and ectoderm markers in Inhba^KO/KO, Inhba^KO/WT and Inhba^WT/WT embryoid bodies.

Briefly about the project and data acquisition methods:

In our project, we aimed to determine the role of activin A during crucial events of mouse embryogenesis and distinguish the function of the protein of zygotic origin and the one secreted by the maternal reproductive tract. To this end, we obtained a mouse line with a knockout of the zygotic activin A gene (Inhba-KO). The lack of activin A protein in knockout (Inhba^KO/KO) mouse was endorsed in the enzyme linked immunosorbent assay (ELISA) from the culture medium of mouse embryonic fibroblasts (MEFs) derived from Inhba^KO/KO and wild-type (Inhba^WT/WT) fetuses (File 1).

To compare the phenotype of Inhba^KO/KO embryos with stage matched Inhba^KO/WT and Inhba^WT/WT embryos, we analyzed the morphokinetic parameters by recording the progress of their development from the zygote to the blastocyst stage using time-lapse microscopy. The same analysis was performed on embryos in two genetic backgrounds (C57BL/6/Tar x CBA/Tar mixed background and on an inbred C57BL/6 genetic background). Morphokinetic data for embryos that reached the blastocyst stage and those arrested in development are summarized in separate files (respectively File 2A and 2B for C57BL/6/Tar x CBA/Tar, and File 2C and 2D for C57BL/6 embryos).

In order to determine the effect of depletion of zygotic activin A expression on cell lineage specification, we detected the presence of Nanog/SOX2, SOX17/GATA4, and CDX2, markers of epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE), respectively. Then, using Imaris software, we counted the number and percentage of cells contributing to the cell lineages (EPI, PE, and TE) of Inhba^KO/KO, Inhba^KO/WT and Inhba^WT/WT blastocysts in two genetic backgrounds (C57BL/6/Tar x CBA/Tar and on an inbred C57BL/6) and time points (120 h and 135 h after hCG injection)(Files 3A,B,C).

Furthermore, we used the outgrowth assay to assess the implantation competence of activin A–deficient embryos (File 4A and B). Therefore, C57BL/6/Tar x CBA/Tar blastocysts were cultured individually in ESC medium without LIF on gelatin-coated plates. We also attempted to rescue the Inhba^KO/KO outgrowths phenotype by supplementing the culture medium with recombinant activin A. The rate of trophoblast cell migration was determined by measuring the mean area of outgrowing embryos (the only ICM outgrowth and total area including trophoblast giant cells) using an image analysis system. Once the perimeter of the trophoblast or ICM cells was respectively selected using a computer mouse, the total outgrowth area and ICM area were calculated in px2 using ImageJ software, and converted into square micrometers using the conversion factor px2 = 0.255 μm2, determined using a calibration slide (File 4A).

Next, we compared the ability of Inhba^KO/KO and control (heterozygous and wild-type) ESCs to differentiate into tissues originating from three germ layers: ectoderm, mesoderm, and endoderm. To this end, we tested whether they could differentiate in vitro in embryoid bodies (EBs). We used RT-qPCR analyses to study the relative mRNA level of the mesodermal markers: Mesogenin 1 (Msgn1), Mesoderm posterior BHLH transcription factor 2 (Mesp2), Nodal, Eomesodermin (Eomes), Mix Paired-like Homeobox (Mixl1), the ectodermal markers: Zic Family Member 1 (Zic1), Paired Box 1 (Pax1), and Paired Box 6 (Pax6), and the endodermal marker Forkhead Box A2 (Foxa2)(File 5).