New findings on the orientation of the mouse anterior-posterior (A-P) axis before and during the initiation of gastrulation using a more refined embryo staging

A clinically significant event of early mammalian embryogenesis is the generation and early development of the anterior-posterior (A-P) axis, the imaginary line along which the structures from head to tail will form. This axis not only appears before gastrulation but is also oriented in a specific way in relation to the long and short diameters of the bilaterally symmetric epiblast. In mice, the most widely used mammalian in vivo model of early embryogenesis, the A-P axis is normally aligned with the long epiblast diameter by the early streak (ES) stage, a time during early gastrulation around embryonic day 6.5 (E6.5). Incorrect orientation of the A-P axis by the ES stage, that is, being aligned with the short epiblast diameter, leads to failure in completing gastrulation and results in embryo death soon after. Knowing the orientation of this axis from when it forms before gastrulation (around E5.5) until just before the ES stage is crucial for: (a) understanding the ill-defined factors involved in its formation and early development since they must be spatially related to it, and (b) providing explanations for the underlying mechanism when it is incorrectly orientated. However, the orientation of the A-P axis in pre-ES embryos of the E5.5-E6.5 period remains unclear. Specifically, although it is thought that this axis initially aligns with the short epiblast diameter and subsequently changes its orientation to become aligned with the long diameter by an unidentified pre-gastrulation stage before the ES stage, this proposition remains unresolved. This is largely due to the lack of clearly defined morphological criteria for staging certain periods of pre-ES mouse embryos (especially when the A-P axis initiates and when gastrulation begins prior to the ES stage), which are a prerequisite for identifying A-P axis orientation at specific pre-ES stages. Furthermore, although the orientation of an extraembryonic trophoblast asymmetry, specifically the tilt of the ectoplacental cone (EPC), coincides with that of the A-P axis by the ES stage, it is unknown whether such an association also exists at pre-gastrulation stages during A-P axis formation. Knowing this would exclude or implicate this trophoblast asymmetry as an upstream factor in orientating the A-P axis when it forms. To address these issues, we established a more refined embryo staging for the E5.5-E6.5 period using a novel combination of live morphological criteria and used it to examine the orientation of the A-P axis and that of the EPC tilt at specific stages. First, contrary to current thinking, we show that when the A-P axis first appears at our newly described anterior visceral endoderm-1 (AVE-1) and AVE-2 stages, it aligns with the long epiblast diameter in all embryos. This orientation is maintained in most embryos at all subsequent pre-gastrulation stages, specifically at our AVE-3 and pre-streak stages (the remaining embryos of these stages had this axis aligned with the short epiblast diameter). Second, we identified for the first time the pre-ES stage when gastrulation initiates, which we named the nascent streak (NS) stage, and further subdivided it into NS-1 and NS-2. At variance with current belief, we provide evidence that the earliest stage just before the ES stage when all embryos align their A-P axis with the long epiblast diameter is not a pre-gastrulation stage, but the NS-2 stage (at NS-1, most but not all embryos had this A-P axis orientation). Third, we implicate the EPC tilt as a possible extraembryonic factor in promoting correct A-P axis orientation, as this tilt exists before the AVE-1 stage and its orientation coincided with that of the A-P axis in all embryos at AVE-1, AVE-2 and ES stages and almost all embryos at AVE-3, pre-streak and NS stages. Overall, our work: (a) identified the previously unresolved orientation of the mouse A-P axis within the epiblast before the ES stage during the E5.5-E6.5 period; (b) provides an alternative explanation for when this axis is incorrectly oriented by the ES stage, namely, its defective alignment with the short epiblast diameter by this stage could be due to its failure to align with the long epiblast diameter from the time of its formation; and (c) implicates the pre-existing orientation of the EPC tilt as a possible factor in orientating the newly formed A-P axis.