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Pathology of the Developing Mouse Course Objectives • A (very) brief review of mouse development • Mellow methods for mincing minute mice Brad Bolon GEMpath Inc. Longmont, CO Phone: (720) 209209-1105 • Arranging the analysis to avoid annoyance • Mining menageries of monster mice [email protected] Glossary: Terms for the Beast Conceptus – the embryo or fetus with the placenta A Brief Overview of Mouse Development Embryo – the animal until organs are first formed • Zygote – a oneone-celled embryo • Morula – a multimulti-celled, solid embryo • Blastocyst – a multimulti-celled, cavitated embryo Fetus – an unborn animal after organs are first formed Neonate – the newly born animal Perinatal – the time from just prior to just after birth Glossary: Terms for the Evil Twin Decidua – the epithelial part of the uterine endometrium Placenta – the conceptus’ conceptus’s organ for nutrient exchange • • • • Allantois – a tubular, vascular extension of the yolk sac Amnion – the inner extraembryonic membrane Chorion – the outer embryonic membrane Reichert’ Reichert’s membrane – a tough, protective extraembryonic membrane (unique to rodents) that overlies the yolk sac Trophoblast – the placental region derived from the blastocyst wall (inner = cytocyto-, outer = syncytiosyncytio-) Yolk sac – the extraembryonic membrane that is the main absorptive surface prior to placenta formation Glossary: Terms for the Process Anlage –undifferentiated cells from which an organ forms Embryogenesis – that portion of development from fertilization until major organs are first formed Gastrulation – that portion of embryogenesis during which the mesoderm is formed Neurulation – that portion of embryogenesis during which the central nervous system is delineated Organogenesis – that portion of embryogenesis during which major organs are first formed Vasculogenesis – a process by which blood vessels are first derived – not angiogenesis (i.e., new vessels from old) 1 Conventions for Timing Developmental Events Gestational Age ≠ Stage • Paradigms 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 – Arbitrary assignment derived from time of mating (dpc (dpc)) • Overnight mating schedule • Plug time assumed to be midnight – “Specific” Specific” calculation estimated from timed mating • Limited breeding - 1 to 2 hrs • Plug time assumed to be start of the mating period • Standards – Designation of plugplug-positive day = E0 or E1 – Fractionation of gestational days = 9.25 or 96 • Caveats Apparent Age: E13 Actual Age: E13 Apparent Age: E12 Actual Age: E13 – – Timing of Some Key Events in Mouse Development Chronological age varies by 12 to 24 hours within a litter Chronological age varies between mouse strains PrePre-Implantation Development 1 Cell (E0.5) Implantation ~ 5.0 Primitive Streak 8.0 First Somite Forms 8.0 Tenth Somite Forms 8.5 S-Shaped Heart 8.5 Cranial Neuropore Closes 9.0 Caudal Neuropore Closes 9.5 Cerebral Hemispheres Form 10.0 Fore Limb Buds 9.75 Hind Limb Buds 10.25 Palatal Folds Unite 15 Length of Gestation 19 Gastrulation and Neurulation Gastrulation (E6.5) – initial formation of mesoderm Decidua Neurulation (E8.0) – initial generation of the nervous system (plate, folds, closure) 2 Cell (E1.5) 4 Cell (E2) 8 Cell (E2.5) Morula (E3) Blastocyst (E3.5) Implantation (E4.5) Photographs by Joe Anderson Hit the Books Early and Often Exocoelom Ectoplacental Cone Amnion Mouse Embryo, E7.75 Chorion Yolk Sac Reichert’s Membrane Head Fold Allantois Heart Hindgut Primitive Streak In situ cross section of trilaminar embryo (E8) in early neurulation Yolk Cavity Foregut Amniotic Cavity 2 Evolution of the Embryonic Profile During Mouse Organogenesis E10.5 E11.5 E12.5 E13.5 Physiology of Developing Mice • Circulation and Hematopoiesis – The embryonic heart beat is initiated by ~E9.5 and increases over time (125 bpm at E10.5; 147 at E12.5; 195 at E14.5) – Embryonic hematopoiesis begins at E7.5 in the yolk sac – Definitive hematopoiesis is initiated in the aortoaorto-gonadogonadomesonephric ridge (AGM) at E8 and in liver at E10 before moving to bone marrow and lymphoid organs near term • Metabolism – Embryos first attain the capacity for xenobiotic metabolism at the blastocyst stage – Biotransformation systems in conceptuses (especially prior to implantation) are incomplete E16.5 E15.5 – The relative contributions of embryonic and maternal metabolic pathways are rarely known in detail E14.5 StrainStrain-Related Differences Placentation • Basic Physiology – Anatomic • C57BL/6 passes through each gestational stage faster than CBA/J • NZW have more spontaneous neural tube defects than do DBA/2 Discoid, Hemochorial Placenta (human, primate, rodent) – Functional • C57BL/6N blastocysts have more CYP450 activity than do DBA/2N Epitheliochorial (horse, pig ruminants) • Differential Sensitivity to Environmental Insults – C57BL/6 have more exencephaly than do SWV with teratogens – Early CF1 embryos more sensitive than BALB/C to radiation – Targeted deletion of epidermal growth factor receptor yields • Death near implantation in CF1 via disruption of inner cell mass • Death during organogenesis in 129/Sv via placental dysgenesis • Survival until weaning with multiple anomalies in CD1 Mouse Placental Anatomy – Early Decidua (Maternal) Large Maternal Vessels Chorionic Plate Hemochorial endothelium Fetus connective tissue epithelium epithelium Dam connective tissue endothelium Mouse Placental Anatomy - Late Reichert’s Membrane Labyrinth Parietal & Visceral Layers of Yolk Sac Spongiotrophoblast Giant Cells Allantois Amnion Image provided by Dr. Jerrold Ward 3 The Critical Period Concept of Developmental Susceptibility Different Critical Periods Exist for Each Component of a System % Exencephaly Near Term End of Critical Period for Gross Defects 60 Birth Fetus Septum Litter Amygdala Hippocampus 40 Midbrain Cerebral Cortex 20 Thalaus Corpus Striatum Hypothalamus 0 7-9 77-8 8-9 7 8 9 99-11 Cerebellum Gestational Day(s) of Maternal Methanol Inhalation Olfactory Bulb 9 10 11 12 13 14 15 16 17 18 19 20 - - - 7 - - - 14 Time (days) Fundam Appl Toxicol 21: 508, 1993 Develop Med Child Neurol 22: 525, 1980 Clinical Endpoints of Developmental Events Methods for Rapid Phenotypic Evaluation of Developing Mice • Maternal characteristics – behavior, body weight • Implantation sites per dam – total, viable • Offspring per litter – total, live • Number of affected litters • Deaths per litter – resorptions, resorptions, embryolethality • Malformations per litter – total, lethal, organorgan-specific • Malformations per conceptus – total, lethal, organorgan-specific • Offspring metrics – body weight, body length, profile Tools of the Trade, Original Opening Initial Entry Achieved 4 Tools of the Trade, Take Two Digging Deeper Fetal Removal Removal of Early Embryos The Devil is in the Details Critter Containers 5 Euthanasia of Conceptuses Possibilities for Processing • Acceptable Measures – Decapitation • Complete • Partial – through trachea and carotid arteries but not vertebrae – Rapid Freezing (liquid nitrogennitrogen-cooled isopentane) isopentane) • Unacceptable Measures – – – – Carbon dioxide (gas or dry ice) Evisceration Slow Freezing (refrigerator or regular ice) Suffocation Fixation of Developing Mice Genotyping Conceptuses • Bouin’ Bouin’s Solution Placenta Limb – 70% saturated picric acid, 5% glacial acetic acid, and 24% saturated formaldehyde in water – Advantages: available, hardens soft tissues, decalcifies bone – Disadvantages: low contrast, marked tissue shrinkage, poor blood cell preservation, corrosive, and potentially explosive • Formalin (10%) – 4% formaldeyhde (a 1:10 dilution of saturated solution) in water – Advantages: readily available – Disadvantages: often yields inadequate hardening of tissues, stabilizers may destroy antigens • Modified Davidson’ Davidson’s Solution Tail Use material from embryo or extraembryonic membranes Gross Examination of Developing Mice – 14% ethanol, 6.25% glacial acetic acid, and 37.5% saturated formaldehyde in water – Advantages: hardens tissue, better contrast, less shrinkage – Disadvantages: commercial sources are hard to find External Examination of Fetuses • Rationale for the Exam – To evaluate features for normal location, shape, and size (developmental landmarks) – To provide a rapid, qualitative assessment of anomalous events (conceptus death, malformation, and/or resorption) N E X • Components of the Exam – External evaluation – all conceptuses (including placentae) placentae) – Internal examination – 50% of conceptuses X A X H • FreeFree-Hand Sectioning (Wilson’ (Wilson’s technique) – fetus • Histopathology – embryo / fetus and placenta – Skeletal examination – 50% of fetuses 6 • • • • Internal Examination of Fetuses Internal Examination (Head/Brain) Skeletal Examination Rapid Histologic Assessment of the Developing Mouse Eviscerate Place in hot water (~75° (~75°C for 1 min) Skin fetus or neonate DoubleDouble-stain for 96 hrs in • • • • • Orientation – Longitudinal (torso) – Transverse (head) • Rationale for Selected Orientations 70% ethanol containing Alcian blue, 0.001% − cartilage Alizarin red, 0.002% − bone Glacial acetic acid, 14% – Torso • Clear sequentially (12 hr each) in • 2% potassium hydroxide (KOH) • 1% KOH (repeat if needed) • 1:1 mix of 1% KOH and glycerin Skull, NearNear-term (E18.5) Fetus • Store in glycerin – Head Teratology 49: 497, 1994 Blocking of the Fetal Torso Fore Limb 1 2 * * • Requires only two longitudinal cuts to trim nearnear-term fetus • Cuts are made using consistent external landmarks that are not altered by variations in muscle contraction • Decapitation is a preferred means of fetal euthanasia • Brain symmetry is better appreciated in coronal section • Blocks are acquired by Wilson’ Wilson’s method for gross exam Blocking of the Fetal Head * * Hind Limb * * * * * * * * Umbilicus Evaluation of two sections per near-term fetus allows for the consistent evaluation of 25 to 30 organs of all major systems 7 Histologic Procedures for Use in the Developing Mouse Histologic Assessment of NearNear-term Mouse Fetuses Abdominal cross section, E18.5 Fetus • Conventional methods – Hematoxylin and eosin (HE) – Special stains – Ultrastructure • Routine “functional” functional” procedures – Apoptotic cells: antianti-caspasecaspase-3, TUNEL – Dividing cells: BrdU, Ki67, PCNA • Gene expression – – – in situ hybridization (for mRNA) immunohistochemistry (for protein) enzyme histochemistry (for functional protein) Both kidneys exhibit marked hydronephrosis and hydroureter. In this case, the change reflects maternal exposure to methanol during organogenesis (E7 to E9). However, milder lesions commonly occur as an incidental background finding. Fundam Appl Toxicol 21: 508, 1993 Histologic Assessment of Early Mouse Embryos Quantitative Histology Untreated Untreated Methanol-Exposed Methanol-Exposed Skull O C I S N Lateral Ventricle Stage-matched neurulating (E8.5) embryos positioned to evaluate craniofacial and visceral anatomy Teratology 49: 497, 1994 Thickness of Frontal Cortex in Overtly Normal Mouse Fetuses MethanolMethanol-Exposed Fetuses Control Fetuses All Litters Lateral Ventricle Teratology 49: 497, 1994 Special Anatomic Methods for Assessing System Development Whole Mount, E15 Litters Lacking Dysraphism No. Litters 24 16 6 No. Fetuses 56 39 14 Neuroepithelium 98.5 ± 1.3 108.8 ± 2.1** 107.1 ± 2.5** Intermediate Cortex, Subventricular Plate 229.8 ± 3.3 190.9 ± 3.7** 193.6 ± 3.1** Cortical Plate 129.6 ± 1.4 127.3 ± 2.3 128.2 ± 4.0 Cortical Layer 1 30.2 ± 0.6 22.9 ± 0.6** 23.4 ± 1.2** Total Thickness 488.1 ± 3.9 449.9 ± 5.9** 452.3 ± 7.4** Teratology 49: 497, 1994 E15 mouse embryo with targeted insertion of bacterial lacZ at expression sites for the type II collagen promoter J Clin Invest 107: 35, 2001 8 Clinical Pathology in Mouse Conceptuses • Endpoints Imaging for Virtual Histology in Mouse Developmental Pathology Magnetic Resonance Ultrasound – 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 • Umbilical cord (E10.5 or older) • Heart (E9.5 to E10.5) Gross Examination of Mouse Placenta Trimming Mouse Placenta • Rationale for the Exam – To evaluate features for normal location, shape, and size (developmental landmarks) – To provide a rapid, qualitative assessment of abnormal events (conceptus death, malformation, and/or resorption) • Components of the Exam – External evaluation – entire conceptus (both embryo / fetus and placenta) – Internal examination – 25% to 50% of placentas • FreeFree-Hand Sectioning – one or two steps • Histopathology Histologic Assessment of the Mouse Placenta • Orientation – Transverse – Horizontal (if warranted) • Rationale for Selected Orientations – Transverse • Requires only one cut to trim placenta • Cuts are made using consistent external landmarks that are not altered by variations in postpost-fixation contracture Experimental Design Features for Analysis of Developing Mice – Horizontal • Provides a focused assessment of embryoembryo-derived tissue 9 An OutcomeOutcome-Oriented Decision Tree for Developmental Pathology Selection Criteria for Choosing a Gestational Age for Further Analysis Are neonates produced? Early Resorption No Are fetuses produced? No Yes Detailed morphologic analysis Is there evidence of early embryonic death? No Yes Are anatomic anomalies evident? No Functional assays Yes Define affected stage, then assess an earlier embryo • Loss prior to organogenesis – A small conceptus (dark red or green) – No embryo to be found OR – Small, flat or tubular embryo • Loss in early organogenesis – Bulbous embryo with limb buds and branchial arches – Indistinct external features Detailed morphologic analysis Selection Criteria for Choosing a Gestational Age for Further Analysis An OutcomeOutcome-Oriented Decision Tree for Developmental Pathology Are neonates produced? Later Resorption Yes Are neonates viable? • MidMid-term embryolethality – A midmid-sized conceptus (tan or white) – Autolyzed embryo present Yes No Are anatomic anomalies evident? • LateLate-stage fetal lethality No – Small and disproportionate but overtly “normal” normal” fetus OR – Structurally normal fetus A Standard Experimental Design for Mouse Development Studies Functional 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 developmental lethal phenotype – Subjects: Near-term fetuses (E17 or E18) and placentae Subjects: Near– 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 10 Animal Numbers • Qualitative study – Basic morphologic description using conventional anatomic tools and/or in situ molecular techniques – Groups include normal (unexposed or wild type) and affected (xenobiotic(xenobiotic-exposed and/or engineered) individuals – n = 2 to 4 per group • Quantitative study – Strict calculation of altered cell or organ structure using special anatomic methods (e.g., cell counts, morphometry) – Groups include normal (unexposed or wild type) and affected (xenobiotic(xenobiotic-exposed and/or engineered) individuals – n = 5 or more per group Impact of Embryo Transfer on Mouse Developmental Pathology • Embryo Transfer Protocol – – – Mass release of ova elicited by hormone priming of female Zygotes harvested, microinjected with DNA, and cultured Embryos (n = 25 to 30) transferred to pseudopregnant female • Consequences of Embryo Transfer Protocols – Developmental delay • All gestational stages – reduced length and weight • Early gestational stages – chronological age exceeds that suggested by developmental landmarks – Increased resorption rate (early especially) • Proposed Explanation is a modest nutritional deficit (via too many conceptuses) leading to a physiologic response to maintain a normal litter size (6 to 12) Categories of Pathology Changes in the Developing Mouse Interpretation of Lesions in the Developing Mouse • Alterations to the norm – – – – Color Consistency Shape Size • Aberrations from the norm – Altered Structures – Missing structures – New structures Wilson’ Wilson’s Principles of Teratogenesis Consequences of In Utero Damage Depend on the Gestational Age I. Susceptibility depends on the interaction between genotype and environment • Pre Differentiation II. Susceptibility varies with the developmental age at exposure III. Teratogens disrupt embryogenesis by specific mechanisms – Conceptus consists of pluripotent stem cells – Severe damage: diffuse cell death → embryonic death – Mild injury: partial cell survival → normal embryo IV. The final manifestations of aberrant development are death, malformation, growth retardation, and functional deficits V. Access of potential teratogens to the conceptus depends on the nature of the agent VI. Development deviations increase in degree as the dose rises 11 Consequences of In Utero Damage Depend on the Gestational Age Consequences of In Utero Damage Depend on the Gestational Age • • Pre Differentiation – – – Pre Differentiation – – – 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 Consequences of Developmental Damage Depend on the Age 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 • Fetal Stage – Growth phase – Conceptus consists of oligopotent and differentiated cells – Damage: cell death → functional deficit >> malformation • Embryonic malfunction • Postnatal Stage – Growth phase – Conceptus consists of oligopotent and differentiated cells – Damage: cell death → functional deficit, no malformations – – – – – Anemia Cardiac anomalies Cardiac arrhythmias Hypoxia (via altered neuroendocrine regulation of heart) Vascular dysgenesis (with hemorrhage) • Maternal sources – Anemia – Hemorrhage Renal Aplasia Wild Type * * Heterozygote * Urinary Tract Aplasia Knockout * Neonates (P1), the right one of which bears a lethal targeted null mutation of the Gfrα1 gene * Wild Type Knockout Knockout * E11 embryos, the middle and right bearing a lethal targeted null mutation of the Gfrα1 gene 12 Morphologic Changes are Predicted by Gene Expression Ureteral Aplasia Ret Wild Type Knockout Knockout E17 near-term fetuses, the middle and right bearing a lethal targeted null mutation of the Gfrα1 gene GFRα GFRα-1 GDNF Ganglion Aplasia Wild Type Limb Aplasia Knockout E17 near-term fetuses, the right one of which bears a targeted null mutation of the Gfrα1 gene Limb Aplasia Wild Type Heterozygote E18 fetuses, the right one of which bears a lethal targeted null mutation of the Fgf10 gene Adrenal Medulla Dysplasia Knockout Wild Type Transgenic Kidney Liver E9.5 embryos, the right one of which bears a lethal targeted null mutation of the Fgf10 gene Kidney Liver E14 lesion resulting from over-expression of a trophic factor for sympathetic neurons throughout development 13 Dysplasia of the Cranial (Superior) Cervical Ganglion Wild Type Transgenic Abnormal Vasculogenesis of the Placental Labyrinth Wild Type Knockout Spinal Cord E14 lesion resulting from over-expression of a trophic factor for sympathetic neurons throughout development Abnormal Vasculogenesis of the Placental Labyrinth Proc Natl Acad Sci USA 99, 9248, 2002 Lesions in Mouse Trophoblast Degeneration Wild Type Transgenic Dysplasia Eosinophilic Droplets Images provided by Dr. Jerrold Ward Image provided by Dr. Jerrold Ward Major Causes of Perinatal Lethality • Airway malfunction – 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 – Functional: Immunodeficiency – Structural: Agenesis (kidney), ectopia (neural tube defect) 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% – Examples: Curly tail, sternebral asymmetry, unossified phalanges – Outcome: Incidental 14 Maternal Causes of Aberrant Development • • • • • • • • • • References – General Aberrant maternal behavior (postnatal impact) Altered nutritional status Autoimmune disease Decreased uterine blood flow (including anemia) Diabetes Fever Hormonal Imbalance Inadequate milk production (postnatal impact) Placental toxicity Stress The Right Stuff References – Anatomic Peaceful Pathologist Good Design Proper Processing The Road to Ruin Poor Design Best Way to Save Your Ass(ets) Perturbed Pathologist Incorrect Processing Call the developmental pathologist before: Designing an embryology experiment X Harvesting embryos and placentas Processing specimens Delivering the slides He hunts you down and hurts you 15