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Developmental Pathology of Engineered Mice Course Objectives • Why worry about wee wodents? wodents? • A (very) brief review of mouse development • Mellow methods for mincing minute mice Dr. Brad Bolon • Arranging the analysis to avoid annoyance GEMpath Inc. Cedar City, UT Phone: (435) 867867-4734 • Mining menageries of monster mice [email protected] Why Examine Developing Mice? • To discover biological roles for novel genes • To investigate mechanisms of disease A Brief Overview of Mouse Development • To evaluate potential new therapeutic targets • To screen agents for efficacy and/or toxicity PrePre-Implantation Development Gastrulation and Neurulation 1 Cell (E0.5) Gastrulation (E6.5) – initial formation of mesoderm 2 Cell (E1.5) Decidua Neurulation (E8) – initial generation of the nervous system (plate, folds, closure) 4 Cell (E2) 8 Cell (E2.5) Morula (E3) Blastocyst (E3.5) Implantation (E4.5) In situ cross section of trilaminar embryo (E8) in early neurulation Photographs by Joe Anderson (Amgen) 1 Evolution of the Embryonic Profile During Mouse Organogenesis E10.5 E11.5 E12.5 Placentation E13.5 Discoid, Hemochorial Placenta (human, primate, rodent) Epitheliochorial (horse, pig, ruminants) Hemochorial endothelium connective tissue Fetus epithelium epithelium Dam E16.5 E15.5 endothelium E14.5 Mouse Placental Anatomy % Exencephaly Near Term Chorionic Plate The Critical Period Concept of Developmental Susceptibility Reichert’s Membrane Large Maternal Vessels Decidua (Maternal) connective tissue Labyrinth Parietal & Visceral Layers of Yolk Sac Fetus 60 Litter 40 20 0 7-9 77-8 8-9 7 8 9 99-11 Gestational Day(s) of Maternal Methanol Inhalation Spongiotrophoblast Allantois Amnion Fundam Appl Toxicol 21: 508, 1993 Different Critical Periods Exist for Each Component of a System End of Critical Period for Gross Defects Birth Septum Amygdala Hippocampus Midbrain Cerebral Cortex Thalamus Corpus Striatum Hypothalamus Cerebellum 9 10 Methods for Rapid Phenotypic Evaluation of Developing Mice Olfactory Bulb 11 12 13 14 15 16 17 18 19 20 - - - 7 - - - 14 Time (days) Develop Med Child Neurol 22: 525, 1980 2 Removal of Early Embryos Fetal Removal Genotyping Conceptuses External Examination of Fetuses Placenta Limb N X E A X X H Tail Use material from embryo or extraembryonic membranes Skeletal Examination of Fetuses • • • • • Blocking of the Fetal Torso Euthanize NearNear-term Fetus Eviscerate Fore Limb Place in Hot Water (~75° (~75°C for 1 min) Skin Fetus Double Stain for 96 hrs in • • • • 1 70% ethanol containing Alcian Blue, 0.001% − cartilage Alizarin Red, 0.002% − bone Glacial Acetic Acid, 14% • Clear sequentially (12 hr each) in • 2% potassium hydroxide (KOH) • 1% KOH (repeat if needed) • 1:1 mix of 1% KOH and glycerin • Store in glycerin 2 * * Hind Limb * * * * Umbilicus Skull, NearNear-term (E18.5) Fetus Evaluation of two sections per near-term fetus allows for the consistent evaluation of 25 to 30 organs of all major systems Teratology 49: 497, 1994 3 Blocking of the Fetal Torso for Detailed Neurohistologic Analysis 1 2 3 4 Fore Limb * * * * Blocking of the Fetal Head * * 5 * * * Hind Limb * * * * * * Histologic Assessment of NearNear-term Mouse Fetuses Histologic Assessment of Early Mouse Embryos Abdominal cross section, E18.5 Fetus Untreated Both kidneys exhibit marked hydronephrosis and hydroureter. In this case, the change reflects maternal exposure to methanol during organogenesis (E7 to E9). Milder lesions of this type are a common background finding. Stage-matched neurulating (E8.5) embryos positioned to evaluate craniofacial and visceral anatomy Teratology 49: 497, 1994 Fundam Appl Toxicol 21: 508, 1993 Special Anatomic Methods for Assessing System Development Whole Mount, E15 Methanol-Exposed Clinical Pathology in Mouse Conceptuses • Endpoints – Hematology: cell counts, morphology, cell size, lineage differentiation – Sample types: whole blood, blood smears, tissue smears – Example: Genes Dev. 10: 154154-164, 1996 • Techniques – Harvest conceptus – Wash in PBS and blot dry to remove maternal blood cells – Collect blood for hematology by capillary tube from E15 mouse embryo with targeted insertion of bacterial lacZ at expression sites for the type II collagen promoter • Umbilical cord (E10.5 or older) • Heart (E9.5 to E10.5) J Clin Invest 107: 35, 2001 4 An EventEvent-Oriented Decision Tree for Developmental Pathology Work Experimental Design Features for Analysis of Developing Mice Are engineered neonates produced? No Are fetuses produced? No Yes Detailed morphologic analysis Is there evidence of early embryonic death? No Yes Are anomalies evident? No Functional assays Selection Criteria for Choosing a Gestational Age for Further Analysis • Characteristics of the Implantation Site Define affected stage, then assess an earlier embryo Yes Detailed morphologic analysis An EventEvent-Oriented Decision Tree for Developmental Pathology Work Are engineered neonates produced? – Early loss (reflecting resorption soon after implantation) Yes • A small, dark blotch embedded in the endometrium • No embryo to be found Are neonates viable? – Intermediate loss (associated with midmid-term embryolethality) Are anomalies evident? • Characteristics of the Affected Embryo No – Loss prior to organogenesis – flat or tubular embryo – Loss in early organogenesis – bulbous embryo with limb buds and branchial arches – Loss in late organogenesis – small and disproportionate but overtly “normal” normal” embryo A Standard Experimental Design for Mouse Development Studies Yes No • A midmid-sized, tan or green mass filling the uterine lumen • Autolyzed embryo present Functional and molecular assays Done Yes Detailed morphologic analysis Selection of Appropriate Controls • Developmental Age • Tier I: Screening – Purpose: Purpose: Basic assessment of the anatomic phenotype(s) elicited in a novel engineered construct – Subjects: Subjects: NearNear-term fetuses (E17 or E18) and placentae – Endpoints: Endpoints: Clinical observations (maternal), gross and microscopic anatomy • Tier II: Mechanistic Studies – Purpose: Purpose: Detailed characterization of the molecular events that produce a given anatomic phenotype – Subjects: Subjects: Depends on the phenotype (likely will include both early and late embryos, with associated placentae) placentae) – Endpoints: Endpoints: Gross and microscopic anatomy, in situ molecular assays, functional tests in vitro (cells, isolated organs, whole mounts) and in vivo (heart rate, blood flow) – Early (E0 to E12): choose stagestage-matched embryos using a combination of anatomic features (e.g., brain conformation, presence of limb buds, somite numbers) – Late (E13 and later): choose ageage-matched conceptuses • Treatment – Genetic studies: include wild type and engineered embryos (transgenic, or heterozygous and knockout) – Toxicity bioassays: include exposed and unexposed litters • Other variables to consider – Sex: select males and females (anogenital (anogenital distance) – Strain 5 Consequences of In Utero Damage Depend on the Gestational Age Interpretation of Lesions in the Developing Mouse • Pre Differentiation – Conceptus consists of pluripotent stem cells – Severe damage: diffuse cell death → embryonic death – Mild injury: partial cell survival → “normal” normal” embryo Consequences of In Utero Damage Depend on the Gestational Age Consequences of In Utero Damage Depend on the Gestational Age • • Pre Differentiation – – – • • Embryonic Stage – Organogenesis phase − with different critical periods for each organ – Conceptus consists of partially differentiated stem cells – Damage: focal to diffuse cell death → malformation – Pattern of anomalies depends upon timing of insult Consequences of Developmental Damage Depend on the Age • Fetal Stage – Growth phase – Conceptus consists of oligopotent and differentiated cells – Damage: cell death → functional deficit >> malformation • Postnatal Stage – Growth phase – Conceptus consists of oligopotent and differentiated cells – Damage: cell death → functional deficit, no malformations Pre Differentiation – – – Conceptus consists of pluripotent stem cells Severe damage: diffuse cell death → embryonic death Mild injury: partial cell survival → normal embryo Conceptus consists of pluripotent stem cells Severe damage: diffuse cell death → embryonic death Mild injury: partial cell survival → normal embryo Embryonic Stage – – – – Organogenesis phase − with different critical periods for each organ Conceptus consists of partially differentiated stem cells Damage: focal to diffuse cell death → malformation Pattern of anomalies depends upon timing of insult • Fetal Stage – Growth phase – Conceptus consists of oligopotent and differentiated cells – Damage: cell death → functional deficit >> malformation Disrupted Circulation is the Major Cause of Embryolethality • Placental malformations • Embryonic malfunction – – – – – Anemia Cardiac anomalies Cardiac arrhythmias Hypoxia (via altered neuroendocrine regulation of heart) Vascular dysgenesis (with hemorrhage) • Maternal sources – Anemia – Hemorrhage 6 Gestational Age ≠ Stage Embryonic Death The apparent age of these littermates was defined using digital rays, which appear at E12.3 on the fore limb and at about E12.8 on the hind Apparent Age: E13 Actual Age: E13 Heterozygote Urinary Tract Aplasia Knockout Wild Type * * * Transgenic E13.5 embryos, one of which expired at approximately E9.5 due to over-expression of a stem cell inhibitor throughout development Apparent Age: E12 Actual Age: E13 Renal Aplasia Wild Type Wild Type * Neonates (P1), the right one of which bears a lethal targeted null mutation of the Gfrα1 gene * Knockout * E11 embryos, the middle and right bearing a lethal targeted null mutation of the Gfrα1 gene Limb Aplasia Limb Aplasia Wild Type E18 fetuses, the right one of which bears a lethal targeted null mutation of the Fgf10 gene Knockout Heterozygote Knockout E9.5 embryos, the right one of which bears a lethal targeted null mutation of the Fgf10 gene 7 Dysplasia of the Cranial (Superior) Cervical Ganglion Major Causes of Perinatal Lethality • Airway malfunction Wild Type Embryo Transgenic Embryo – Agenesis or dysgenesis of pulmonary system – Decreased thoracic volume – Skeletal defects (reduced thoracic expansion) • Cardiac malfunction – Arrhythmias – Heart and/or vascular malformations • Other major anomalies E14 lesion resulting from over-expression of a trophic factor for sympathetic neurons throughout development – Functional: Immunodeficiency – Structural: Agenesis (kidney), ectopia (neural tube defect) Toxicol Pathol 32: 275, 2004 Spontaneous Malformations in Developing Mice • Common Variants = 0 to 35% – Examples: Renal pelvic cavitation, cavitation, supernumerary ribs, wavy ribs – Outcome: Incidental • Major Malformations = < 1% – Examples: Exencephaly, ventricular septal defect – Outcome: Lethal • Minor Visceral Malformations = 1 to 3% – Examples: Cranial displacement of gonads, hemorrhages – Outcome: Usually incidental • Minor Skeletal Anomalies = 1 to 5% Summary • The need for phenotypic evaluation of developing mice will increase in all fields of biomedical research • The examiner will have to have broad anatomic and physiologic knowledge of all developmental stages to perform a competent phenotypic examination • The skills required for such proficiency are mere extensions of the understanding acquired during a wellwell-rounded education in biology and medicine – Examples: Curly tail, sternebral asymmetry, unossified phalanges – Outcome: Incidental 8