Staging of ovine embryos and expression of the T-box genes Brachyury and Eomesodermin around gastrulation

Abstract

The high rates of embryonic mortalities which follow in vitro production of ruminant embryos have emphasized the need for increased knowledge of early development. It is likely that early failures in embryonic development and placenta formation involve abnormal differentiation of mesoderm. The aim of this study was to investigate the pattern of expression of two T-box genes known to control the gastrulation process, Brachyury and Eomesodermin, by whole-mount in situ hybridization. To allow a more precise comparison of both expression patterns between embryos, we describe a new staging of pre-implanted ovine embryos by gross morphology and histology from pre-gastrulation stages to the beginning of neurulation. In pre-streak embryos primitive mesoderm cells delaminated in between the primitive endoderm and the epiblast. At that stage, no expression of Brachyury or Eomesodermin could be detected in the embryos. Early expression of both T-genes was observed by the early-streak stages in epiblast cells located close to the presumptive posterior pole of the embryos. Later on, during gastrulation both genes followed a pattern of expression similar to the ones described in other mammals. These observations suggest that other genes, which remain to be identified, are responsible for extra-embryonic mesoderm differentiation in ruminant embryos.

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

“…The lack of pluripotency in the pig ICM may explain the diffi culty in establishing ESC from blastocysts. Previous reports have demonstrated that pig ICM derived from Day 8 blastocysts transit to an epiblast phenotype after 2-3 days in culture [ 34 ] and they rapidly differentiate [ 27 ]. Here, we cultured isolated epiblasts using conditions that support hESC and mEpiSC growth.…”

“…The epiblast at this stage of development transitions from a round oblong disc to a developmentally advanced pear-shaped embryonic disc with a narrow posterior end [ 26 ]. Early round oblong epiblasts identifi ed under a stereomicroscope can be easily separated from the remaining trophoblast cells on the basis of their contrasting appearance [ 27 ]. The underlying PE cells, which are transparent cells and have a globular phenotype, can be distinguished from the epithelial cells of the epiblast.…”

“…In mouse blastocysts, NANOG is expressed in conjunction with OCT4 and SOX2 conferring pluripotent properties to the ICM [ 32 , 33 ]. In the pig the concerted expression of these factors is delayed until the epiblast stage [ 27 ] suggesting that the ICM is a transitional stage that has not yet acquired pluripotency. The lack of pluripotency in the pig ICM may explain the diffi culty in establishing ESC from blastocysts.…”

Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formation

“…The expansion of the trophoblast provides an increased placental surface area to enable maternal:conceptus cross-talk and nutrient exchange that are essential for the survival of the conceptus (Stroband & Van der Lende 1990). Accompanying elongation is the degradation of the sheath of trophoblasts cells covering the embryonic disc (Rauber's layer) exposing the cells of the embryonic disc to the maternal milieu (Marrable 1971, Guillomot et al 2004). In the ewe and cow blastocyst, trophoblast elongation is initiated around gestational day 11 (gd11) and gd12 respectively, and transition from the ovoid to filamentous stage is complete after several days (ewe, gd16;cow, gd18;Bazer et al 1993, Guillomot et al 2004.…”

“…Accompanying elongation is the degradation of the sheath of trophoblasts cells covering the embryonic disc (Rauber's layer) exposing the cells of the embryonic disc to the maternal milieu (Marrable 1971, Guillomot et al 2004). In the ewe and cow blastocyst, trophoblast elongation is initiated around gestational day 11 (gd11) and gd12 respectively, and transition from the ovoid to filamentous stage is complete after several days (ewe, gd16;cow, gd18;Bazer et al 1993, Guillomot et al 2004. In cattle, trophoblast cells start to elongate at gd14 and the embryonic membrane can extend the entire length of both uterine horns by gd24.…”