Pathology of Genetically Engineered and Other Mutant Mice. Группа авторов

basal cranial structures. Panel (b): Methanol‐exposed exencephalic embryo (following maternal inhalation throughout neurulation [GD7–9]) showing malformed basal cranium bones exhibiting less cartilage to bone conversion. Abbreviations: bs = basisphenoid, bo = basioccipital, e = ethmoid, n = nasal, p = petrous temporal bone.

      Source: Bolon et al. [97] with permission of John Wiley & Sons.

Figure 5.13 Developmental anatomy of a wild‐type GD16.0 mouse embryo demonstrating the anatomic relationship of key structures when viewed in mid‐axial section. Major organs are specified during organogenesis (the period from approximately GD8.0 to GD15.0), and near‐term are fairly easy to recognize with the aid of well‐annotated developmental anatomy atlases and their similarity to adult positions and contours. Major exceptions to this rule are the cerebellum (arrow) and lymph nodes, for which anlagen (organ primordia) form prenatally while the bulk of differentiation occurs after birth. Stain: H&E.

Figure 5.14 Missing structures represent a special situation in developmental pathology. In some cases, their absence is obvious – as is seen here by comparing the normal appearance of expanded air spaces in the lung parenchyma of a wild‐type (WT) mouse neonate (Panel (a)) to the thoracic cavity of an NK2 homeobox 1 (Nkx2‐1^tm1Shk) null mutant (KO) mouse neonate, which lacks all lung tissue except some cystic remnants (asterisks) representing aborted lobar development (Panel (b)). The absence of smaller organs or individual cell populations may be more subtle, and the tendency of the mind to “fill in” missing features may lead to a missed phenotype if the phenotypic evaluation is undertaken in a cursory manner. Stain: H&E.

Readouts for mouse developmental pathology endpoints may be confined to qualitative scores (i.e. “normal” or “abnormal”) and detailed descriptions of lesion, but in many cases, potential developmental phenotypes are better judged by assigning semi‐quantitative lesion scores [76]. A common scoring scheme typically includes multiple tiers: within normal limits (score = 0), or minimal (1+), mild (2+), moderate (3+), or marked (4+) lesions. Each tier must be founded on well‐defined criteria, which generally should be stated explicitly in publications to ensure their reproducibility by investigators seeking to interpret or replicate the results.

      Clinical Pathology Evaluation of Developing Mice

      Many mouse developmental pathology assessments are limited to anatomic pathology evaluation of macroscopic and microscopic defects. Clinical pathology analysis for mouse developmental pathology experiments usually is confined to hematology (in whole blood), clinical chemistry (in serum), or organ cytology. These samples are collected due to the common role of cardiovascular dysfunction and hematopoietic defects in developmental lethal phenotypes [8, 77]. Common tests performed for mouse developmental pathology projects, since they need only a small blood sample, are an automated complete blood count (CBC) and a limited chemistry battery (albumin, blood urea nitrogen, calcium, glucose, phosphorus, total protein).

      Blood collection for developing mice typically is undertaken as a terminal procedure since embryos and neonates have a very small blood volume. Common sampling options are decapitation and cardiac puncture [58, 77]. Blood is harvested into standard collection tubes (e.g. Microtainer^®). Greater blood volumes may be obtained from outbred mouse stocks relative to smaller inbred strains. In our experience, enough blood may be harvested from a single neonatal mouse (1.5 g or larger in weight) to permit either a CBC or a serum chemistry battery, but not both. Alternatively, collection of blood in a tube containing potassium or sodium ethylenediaminetetraacetic acid (EDTA) as an anti‐coagulant may allow measurement of both a CBC (from whole blood) and a very few chemistry values (from plasma obtained by centrifugation of the sample). For small subjects (embryos from GD15 to term), sample volumes typically need to be extended either by mixing whole blood with an equal volume of physiologic saline (pH 7.4) or by pooling blood or plasma samples from mice that have an identical genotype and/or that received the same treatment. In our experience, pooling blood from two (at GD18.5) to four (at GD15.0) embryos will provide enough sample for either a CBC or a chemistry battery, but not both.

      When investigating some lethal phenotypes, cytological assessment of isolated cells may be necessary to define the pathogenesis [58]. Cytological evaluations permit assessment of cellular and subcellular features at high resolution. Techniques such as smears for blood and bone marrow and either squash or touch preparations for organs with dense parenchyma like liver, spleen, and thymus are particularly useful in this regard. In general, results from cytological methods should be interpreted along with findings from conventional histopathological evaluation so that both tissue organization and individual cell characteristics can be assessed. For most embryo phenotyping projects, microscopic examination of tissue sections is used for the initial screen for both cell and tissue lesions, while cytological methods are employed as a secondary procedure when warranted to better understand the nature of the changes first identified by histopathologic analysis of cells within the context of their usual tissue organization.

Pathologic Patterns in Mouse Embryos and Neonates

      Gene targeted (null mutant or “knockout” [KO]) mouse embryos, and less often transgenic mouse embryos, develop abnormal organ‐ or tissue‐specific lesions due to the functional abnormalities arising from altering the role of the genetically engineered gene at different stages of normal development. In large‐scale knockout mouse programs, about 30% of all inactivated genes lead to developmental embryonic defects at various stages of gestation [78, 79]. In one study of 242 developmental lethal knockout mouse lines, 44% of lethal events occurred before GD9.5, 17% from GD9.5–12.5, 4% from GD12.5–18.5, and 36% from GD15.5 to weaning [78]. The specific developmental defect(s) in the 242 lines were not determined.

The stage at which injury (lack of normal development of the entire embryo, or specific tissues or organs) occurs will determine the outcome of developing mice (Table 5.1). For harm prior to implantation (i.e. before GD4.5), significant interference to normal development usually will lead to death of the embryo, while modest effects may engender developmental delays but minor consequences or no overt abnormalities. Such delays may undergo complete or partial compensatory repair as the remaining pluripotent stem cells restore lost tissue. Gross abnormalities (“birth defects” seen during gestation) most commonly result from damage incurred after implantation (GD5.0) but before the end of organogenesis (GD15.0), with the character of the malformation determined by the timing of the insult relative to various organ‐specific “critical periods.” For injury after organogenesis is finished (i.e. GD15.0 through the neonatal period), the embryo may exhibit slow growth or develop cell type‐ or tissue‐specific histopathological changes and/or functional abnormalities rather than gross defects; the exceptions to this rule are structures like the cerebellum and hippocampus that undergo major peaks of cell proliferation after birth. Embryonic lethality may result from many different causes, but common aberrations that reliably lead to death are decreased circulation (e.g. heart and/or vascular defects any time during gestation) and reduced oxygen exchange (e.g. erythrocyte deficiencies or hemorrhagic diatheses or placental insufficiency, especially occurring late in gestation).

Gross developmental defects that occur as spontaneous background lesions vary in frequency and severity among wild‐type (WT) mouse strains and lines. Minor incidental anomalies like renal pelvic cavitation (physiological hydronephrosis) and extra or wavy ribs are common (from 5% to 35% of animals) since they do not impact embryonic viability [80]. Major malformations like