Pathology of Genetically Engineered and Other Mutant Mice. Группа авторов

dark band (asterisk) in the abdomen (due to chronic hepatic congestion). Relative to the WT animal (Panel (b)), the microscopic lesions in the KO animal (Panel (c)) stem from myocardial dysgenesis (shown by the larger size and random arrangement of ventricular myofibers). Stain: H&E.

      Source: Tullio et al [98] with permission of the U.S. National Academy of Sciences.

Several gross and microscopic patterns occur commonly in development phenotypes that arise during gestation (Figure 5.17). Cell degeneration (potentially reversible) and/or necrosis (irreversible) may be evident and often presents as organs with aberrant cellular features (for degeneration) or that are small (for either, but especially for necrosis). Heterotopiae are clusters of ectopic (displaced) cells that usually indicate prior abnormalities in migration. Kinetic defects appear as a surplus or dearth in either proliferation or programmed cell death (PCD) for a particular population. Any or all of these intrinsic mechanisms as well as other extrinsic tissue changes like hemorrhage may produce gross defects. In general, inflammation is not a prominent tissue response in mouse embryos since the immune system is functionally immature until fairly late in gestation or after birth. The gross appearance of the uterus and implantation sites (Figure 5.17a) as well as the microscopic lesion spectrum in a dying embryo varies with the tissue or organ involved (Table 5.1), but once viability is lost, the microscopic pattern invariably will include first focal or multifocal necrosis in various organs (Figure 5.17e) and then diffuse end‐stage necrosis of all organs (Figure 5.17d,f). Nonviable conceptuses grossly will appear as small implantation sites either lacking an embryo (a finding termed a “resorption,” and typically appearing as a pale, dark green, or purple tissue mass) or with cloudy intra‐amniotic fluid surrounding a white, friable embryo (Figure 5.17b,d).

Many different developmental disturbances may lead to neonatal lethality. Death within one to two hours of birth is a reliable indicator of functional or structural defects that prevent effective oxygen uptake and distribution. Commonly affected systems in this regard include the heart and vessels, lungs, hematopoietic tissue (red blood cells), and brain (respiratory centers). Mice can be born without an intact brain, endocrine glands, limbs, lungs, and skin because these organs are not needed for viability in utero, but only after birth. Acute but slower death (i.e. 6–36 hours after birth) typically results from impaired energy metabolism (especially glucose utilization) or suckling difficulties (Figure 5.18). The pathogenesis of these effects may be simple and obvious (e.g. small or no lung lobes), but often the cause is associated with subtle (e.g. reduced neurons in brainstem centers that control respiration) or no (e.g. altered neurotransmission in synapses of respiratory muscles) structural lesions. For this reason, investigations of developmental lethal phenotypes require a team approach.

For mutant mouse lines, the time of death may be probed further by evaluating the genotype of viable embryos and neonates. The first clue that a given genotype may be responsible for an embryonic or neonatal lethal phenotype often is an altered Mendelian ratio and/or a decrease in the expected litter size (Figure 5.17a) [87]. The developmental stage at which lethality occurs may be estimated by the gross appearance of abnormal individuals relative to the expected appearance of developmental stage‐matched embryos or neonates. If all implantation sites contain structurally normal embryos with a wild‐type (WT) or heterozygous (HET) genotype, then the null mutant (“knockout” [KO]) embryos likely died prior to implantation (i.e. before GD4.5). If some implantation sites contain no embryo or a small dead embryo, then the KO embryos likely expired during initial early organogenesis before generation of the DP (i.e. between GD8.5–9.5). If implantation sites at mid‐gestation (i.e. GD12.5) include apparently normal KO embryos, then the embryo lethal phenotype likely is expressed near or just after birth. Dead pups often are eaten immediately by the dam, so harvesting embryos right before birth (i.e. GD17.5–18.5) might be required to confirm that animals were viable throughout gestation. Given this spectrum of time‐dependent defects, most developmental pathologists tend to perform the initial evaluation to characterize a genetically engineered lethal phenotype by examining litters toward the end of gestation (between GD16.0–18.5; Figure 5.19) or at weaning (PND21; Figure 5.20) [22, 88, 89]. If necessary to pinpoint the time of death and/or characterize the phenotype, follow‐up examinations usually are conducted by working backward at two‐day intervals during gestation (GD14, GD12, GD10, etc.) or two‐ to three‐day intervals between birth and weaning (PND18, PND15, PND12, PND9, PND6, PND4, PND2) until viable but affected individuals are observed.

      For developmental toxicity studies, the developmental outcome also is predicted chiefly by the timing of exposure relative to critical periods of development. However, other factors also play a role in the vulnerability of developing mice. For example, some strains have an inherently higher sensitivity to some forms of birth defects because their background incidence is high (e.g. A/J mice and cleft palates [90, 91], SWV [Swiss Webster Vancouver] mice and neural tube closure defects [92]). This strain‐specific sensitivity usually is for a specific spectrum of birth defects and/or teratogenic agents, and not for all kinds of malformations and teratogens generally. Similarly, male and female embryos may respond differently to toxicants based on their proximity to littermates of the opposite sex during gestation and the nutritional status of the dam. Therefore, the pathologist will need to be familiar with the background strain sensitivity as well as the specific intrauterine environment experienced by the embryo prior to performing developmental pathology evaluations.

In the simplest scenario, a lethal developmental phenotype will reliably produce a common spectrum of lesions affecting the same organs in all individuals with a given genetic mutation or that were exposed to a particular dose of toxicant. However, in many cases (for mutant lines and infectious diseases more so than toxicant exposures), lethal phenotypes exhibit variable penetrance, with some embryos exhibiting substantial defects and/or death early in development while a few live well after birth. Typically, no explanation can be defined to account for the differing penetrance, although factors like the genetic background, gene dosage, and maternal age commonly are implicated. The pathologist's role in such investigations is to properly describe the lesions associated with lethality, and to record any shifts in the lesion constellation noted in the long‐term survivors.

Figure 5.17 Embryonic death is demonstrated by a spectrum of macroscopic and microscopic changes. Panel (a): Gravid uterus at GD10.0 in which all but three implantation sites exhibit various degrees of regression (termed “resorption”). Panel (b): An isolated implantation site at GD15.0 in which a dead (pale) embryo is obscured by opaque, red‐tinged amniotic fluid (i.e. containing increased protein and cells, including blood). A viable wild‐type GD14.5 embryo (Panel (c)) with tan skin and many branching blood vessels is contrasted to a white, avascular, wild‐type littermate (Panel (d)) that died at approximately GD13.5 (as shown by the obvious albeit short digits on the forepaw and digital rays (which arise about GD12.8) but very minimal digits on the hind paw). The grossly visible pallor corresponds to diffuse necrosis microscopically. Panel (e): Focal necrosis and acute hemorrhage in a GD13.5 embryo are the first harbingers of imminent embryonic death. Panel (f): Diffuse necrosis leading to death in a GD13.5 embryo (asterisk) is a consequence of widespread primary placental necrosis and acute hemorrhage at