DISTRIBUTION OF PRIMITIVE ENDODERM AND EPIBLAST LINEAGE SPECIFIC FACTORS IN LATE STAGE BLASTOCYSTS
Abstract
Mouse preimplantation development leads to the formation of three distinct cell types by the blastocyst, namely, the trophectoderm (TE) which is an apolarized epihthelial layer, the inner cell mass which forms primitive endoderm, and pluripotent lineage epiblast. Segregation of the lineages is related to the expression of some key factors such as Cdx2, Oct4, Nanog, Gata4 and Sox17. We aimed to understand the role and distribution of these factors in mouse late blastocyst stage embryos. Two cell stage embryos were flushed from mated female B6D2F1 mice 44 hours after human chorionic gonadotropin injection. They were cultured in KSOM-AA medium at 37 °C with 5 % CO2 in humidified air up to the late blastocyst stage. We performed immunofluorescent staining with antibodies specific to these factors. After that blastocysts were imaged and assessed using an fluorescence and confocal laser scanning microscope. We show that lineage specific factors, Gata4 and Sox17, were restricted to primitive endoderm while Nanog was restricted to epiderm lineage at late blastocyst stage. Moreover, the Oct4 protein is present in the nuclei in all cells but is strongly expressed in inner cell mass cells, whereas Cdx2 is localized only in TE cells. We conclude that lineage specific factors, Gata4, Sox17 and Nanog are essential for segregation of distinct lineages and blastocyst development.
Key words: blastocyst; epiblast; preimplantation; primitive endoderm; transcription factors
RAZPOREDITEV PRIMITIVNEGA ENDODERMA IN EPIBLASTNIH RODOVNO SPECIFIČNIH DEJAVNIKOV V POZNI FAZI BLASTOCIST
Mišji predimplantacijski razvoj vodi do tvorbe treh različnih tipov celic blastociste - trofektoderma (TE), ki je nepolarizirana epitelijska plast, notranje celične mase, ki tvori primitivni endoderm in pluripotentni epiblast. Ločevanje teh celičnih linij je povezano z izražanjem nekaterih ključnih genov oziroma prepisovalnih dejavnikov, kot so Cdx2, Oct4, Nanog, Gata4 in Sox17. Naš namen je bil raziskati vlogo in porazdelitev izraženosti teh genov v mišjih zarodkih v pozni fazi blastociste. Zarodki so bili izprani iz brejih samic miši B6D2F1 44 ur po injiciranju humanega horionskega gonadotropina. Do faze pozne blastociste so bili zarodki gojeni v mediju KSOM-AA pri 37 °C s 5 % CO2 v navlaženi atmosferi. Izvedli smo imunofluorescenčno barvanje s protitelesi, specifičnimi za prej naštete gene oziroma beljakovine. Blastociste so bile opazovane in ocenjene s pomočjo fluorescenčnega in konfokalnega mikroskopa. Pokazali smo, da je bila izraženost rodovno specifičnih genov Gata4 in Sox17 omejena na primitiven endoderm, medtem ko je bil gen Nanog omejeno izražen v epidermu v poznem stadiju blastociste. Poleg tega je bila beljakovina Oct4 prisotna v jedrih v vseh celicah, vendar najmočneje v notranji masi, medtem ko je bil Cdx2 lokaliziran samo v celicah TE. Iz rezultatov sklepamo, da je rodovno specifična izraženost genov Gata4, Sox17 in Nanog bistvenega pomena za ločevanje različnih linij in razvoja blastociste.
Ključne besede: blastocista; epiblast; predimplantacija; primitivni endoderm; prepisovalni dejavniki
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