features, physiological processes, and lesion patterns that are the norm in embryos and neonates. Most investigators approach histopathological evaluation of late‐stage embryos (gestational day [GD] 15.5 or older), neonates, and juveniles (up to postnatal day [PND] 42) with confidence due to anatomic similarities between these developmental stages and adult mice. This assurance fades when the rapidly evolving morphology of earlier embryos and their extra‐embryonic membranes needs to be characterized.
This chapter offers a brief introduction to key anatomical and pathophysiological concepts in prenatal and early postnatal mice. More detailed coverage of specific mouse developmental anatomy and pathology topics is available in other books [1–5], book chapters [6–9], anatomic atlases [10–15], major review articles [16–27], and websites [15,28–30].
Four‐Dimensional Anatomy of the Developing Mouse
Stages of mouse development are defined in terms of major events in four dimensions: length, width, breadth, and time (Figure 5.1). Specific stages typically are assigned using the Theiler staging (TS) system based on visible anatomic landmarks [11, 15, 29, 31, 32]. Timing for each event varies by up to 12 hours among strains with different genetic backgrounds [33, 34]. Furthermore, the developmental stages in any litter differ by between 10 to 24 hours for the oldest and youngest embryos (Figure 5.2) [2, 11, 33]. Accordingly, developmental stage is not equal to developmental age for prenatal mice. In utero age is assigned as either “days post coitum” (dpc) or as “gestational day” (GD, often referred to as “embryonic day” [E]). When using the GD (or E) timing convention, it is imperative that investigators note whether the day of conception is counted as GD0 or GD1 since this choice will dramatically impact the times assigned to developmental landmarks. The convention used in this chapteris that the presence of a vaginal plug is designated as 0 dpc (or GD0).
Developmental Events in Embryos and Neonates
In mice, conception for all members of a litter occurs on average at GD0.5. At about GD1.0, the free‐floating zygotes (i.e. one‐celled embryos) begin the first of multiple rounds of cell division as they travel down the oviduct. Embryos enter the uterus at about GD2.5 as morulae (solid multi‐celled masses) and evolve into blastocysts with an off‐center, fluid‐filled cavity at about GD3.0. Dilation of this cavity is accompanied by differentiation of the first embryonic tissues: the inner cell mass (ICM), a crescentic group of pluripotent stem cells at one pole that will become the embryo proper, and the trophectoderm, which forms the outer wall of the blastocyst and will differentiate into the extra‐embryonic membranes (i.e. placenta). Evolution from morula to blastocyst is accompanied by increased activity of the embryonic genome and substantial strain‐specific metabolic changes.
Embryos implant in the uterine wall at about GD4.5. Prior to implantation, cross‐talk between the uterine wall and blastocyst results in production of a highly permeable, well‐vascularized endometrial layer, termed “decidua,” that promotes embryo attachment and survival [35, 36]. The decidual reaction provides the primary means for sustaining the embryos until they form their own placenta. Upon implantation, the trophectoderm proliferates, invades the decidua, and then differentiates into syncytiotrophoblasts (which border the maternal tissue) and cytotrophoblasts (which envelop the ICM). Reciprocal interactions between the embryos and decidua are essential in maintaining pregnancy [37, 38].
By GD5.0, the implanted round blastocyst morphs into an elongated “egg cylinder,” and distinct embryonic and placental features begin to form. At this stage, the ectoplacental cone appears as a triangle of extra‐embryonic tissue that invades the mesometrial decidua at one pole of the egg cylinder. Simultaneously, the ICM differentiates into the epiblast, an outer layer of columnar cells that will give rise to three embryonic germ layers (ectoderm, mesoderm, and endoderm) as well as some extra‐embryonic ectoderm and mesoderm, and the hypoblast, a thin inner layer of cuboidal cells that will produce the yolk sac (YS) endoderm and some extra‐embryonic mesoderm. Generation of germ layers by the epiblast begins on GD6.0 with the ectoderm and endoderm, with formation of the mesoderm (a process called “gastrulation”) beginning at about GD6.5 and lasting until about GD7.5.
Figure 5.1 Composite image of embryonic mouse developmental stages from just after conception (at embryonic day [E] 0.5, in upper left) throughout preimplantation (E1.5 to E3.5, remainder of upper row) and during early postimplantation (E6.5) to mid‐gestation (E13.5).
Source: Dr. Yi Zhang, Harvard Medical School and the Howard Hughes Medical Institute.
Figure 5.2 Mouse embryo littermates with a shared chronological age at gestational day (GD) 13 but having anatomic features demonstrating different developmental stages: GD13 (Theiler stage [TS] 21) on the left as shown by discrete linear gray rays denoting the sites of future digit separation on the paddle‐shaped forepaw (F) and hind paw (H) limb buds, but GD12 (TS20) on the right as shown by the absence of digital rays on the forelimb buds. Digital rays usually form on the forelimb bud at about GD12.3 and on the hind limb bud at about GD12.8.
Source: Bolon and La Perle [89] with permission of CRC Press.
Organogenesis begins at about GD7.5 and lasts until GD15.0. During this portion of embryonic development, all major organ precursors (termed “anlagen”) undergo initial specification and generation. Organ structures and the external appearance of the embryo only approximate the adult conformation at the end of organogenesis. Different organs, and parts of organs, are initiated at different “critical periods” throughout this time range so that the developmental path for most organs extends across several days [39]. These critical periods represent times of intense cell proliferation, migration, and differentiation, and thus define organ‐, region‐, and cell type‐specific times of heightened vulnerability to endogenous and exogenous insults [40, 41]. An oft forgotten fact is that for some organs, such as brain and lymph nodes, critical periods occur not only during gestation but also after birth [42, 43].
The last few days of gestation (GD15.0 to term) and the first few days after birth (up to PND7 or so) correspond to the fetal period in primate embryos. This phase represents a period of organ growth. With some exceptions, organs acquire their adult appearance at this time, although function often is decreased in neonatal and juvenile animals relative to adult systems.
Developmental Events in Placentation
The placenta is the first organ created by a newly implanted embryo (GD5.0), although its form varies considerably until the definitive placenta (DP) becomes active (GD12.5). The placenta acts as an interface for exchanging gases and organic molecules between the embryonic and maternal circulations, a hormone source that regulates maternal processes need to sustain pregnancy, and a protective environment for embryonic survival and growth. The decidua also serves as a barrier to reduce immunogenicity of embryonic proteins. The structure, function, and molecular signature of the placenta is revised greatly over time in response to the growing embryo's evolving metabolic demands [4, 16, 17, 44, 45].
