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Dr. Brad BolonDr. Brad Bolon

GEMpath Inc.GEMpath Inc.Cedar City, UTCedar City, UT

Phone: (435) 867Phone: (435) 867--4734 4734 brad@gempath.netbrad@gempath.net

Developmental Pathology Developmental Pathology of Engineered Miceof Engineered Mice

Course ObjectivesCourse Objectives

•• Why worry about wee Why worry about wee wodentswodents??

•• A (very) brief review of mouse developmentA (very) brief review of mouse development

•• Mellow methods for mincing minute miceMellow methods for mincing minute mice

•• Arranging the analysis to avoid annoyanceArranging the analysis to avoid annoyance

•• Mining menageries of monster miceMining menageries of monster mice

Why Examine Developing Mice?Why Examine Developing Mice?

•• To discover biological roles for novel genesTo discover biological roles for novel genes

•• To investigate mechanisms of diseaseTo investigate mechanisms of disease

•• To evaluate potential new therapeutic targetsTo evaluate potential new therapeutic targets

•• To screen agents for efficacy and/or toxicityTo screen agents for efficacy and/or toxicity

A Brief Overview of A Brief Overview of Mouse DevelopmentMouse Development

PrePre--Implantation DevelopmentImplantation Development

1 Cell (E0.5)

2 Cell (E1.5)

4 Cell (E2)

8 Cell (E2.5)

Blastocyst (E3.5)

Morula (E3)

Implantation (E4.5) Photographs by Joe Anderson (Amgen)

GastrulationGastrulation and Neurulationand Neurulation

Gastrulation (E6.5) – initial formation of mesoderm

Neurulation (E8) – initial generation of the nervous system (plate, folds, closure)

In situ cross section of trilaminar embryo (E8) in early neurulation

Decidua

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Evolution of the Embryonic Profile Evolution of the Embryonic Profile During Mouse OrganogenesisDuring Mouse Organogenesis

E10.5 E11.5 E12.5 E13.5

E16.5 E15.5 E14.5

PlacentationPlacentation

Discoid, Hemochorial Placenta(human, primate, rodent)

Hemochorial

epithelium

connective tissue

endothelium

endothelium

connective tissue

epithelium

Epitheliochorial(horse, pig, ruminants)

Fetus

Dam

Mouse Placental AnatomyMouse Placental Anatomy

Decidua (Maternal) Large Maternal Vessels

Spongiotrophoblast Allantois

Parietal & Visceral Layers of Yolk Sac

ChorionicPlate

Amnion

Labyrinth

Reichert’s Membrane

The Critical Period Concept of The Critical Period Concept of Developmental SusceptibilityDevelopmental Susceptibility

%

% E

xenc

epha

lyEx

ence

phal

yN

ear T

erm

Nea

r Ter

m

Gestational Day(s) of Maternal Methanol InhalationGestational Day(s) of Maternal Methanol Inhalation

77--9 79 7--88 88--99 77 8 8 9 99 9--1111

FetusFetusLitterLitter

6060

4040

2020

00

Fundam Appl Toxicol 21: 508, 1993

Different Critical Periods Exist Different Critical Periods Exist for Each Component of a Systemfor Each Component of a System

End of Critical PeriodEnd of Critical Periodfor Gross Defectsfor Gross Defects BirthBirth

SeptumAmygdala

HippocampusMidbrain

Cerebral Cortex

Corpus StriatumHypothalamus

Cerebellum Olfactory Bulb

Time (days)Time (days)9 10 11 12 13 14 15 16 17 18 19 20 - - - 7 - - - 14

Thalamus

Develop Med Child Neurol 22: 525, 1980

Methods for Rapid Methods for Rapid Phenotypic Evaluation Phenotypic Evaluation

of Developing Miceof Developing Mice

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Removal of Early EmbryosRemoval of Early Embryos Fetal RemovalFetal Removal

Genotyping ConceptusesGenotyping Conceptuses

Use material from embryo or extraembryonic membranesUse material from embryo or extraembryonic membranes

PlacentaPlacenta

TailTail

LimbLimb

External Examination of FetusesExternal Examination of Fetuses

N E X

X XA H

Skeletal Examination of FetusesSkeletal Examination of Fetuses•• Euthanize NearEuthanize Near--term Fetusterm Fetus•• EviscerateEviscerate•• Place in Hot Water (~75Place in Hot Water (~75°°C for 1 min)C for 1 min)•• Skin FetusSkin Fetus•• Double Stain for 96 hrs inDouble Stain for 96 hrs in

•• 70% ethanol containing70% ethanol containing•• AlcianAlcian Blue, 0.001% Blue, 0.001% −− cartilagecartilage•• Alizarin Red, 0.002% Alizarin Red, 0.002% −− bonebone•• Glacial Acetic Acid, 14%Glacial Acetic Acid, 14%

•• Clear sequentially (12 hr each) inClear sequentially (12 hr each) in•• 2% potassium hydroxide (KOH)2% potassium hydroxide (KOH)•• 1% KOH (repeat if needed)1% KOH (repeat if needed)•• 1:1 mix of 1% KOH and glycerin1:1 mix of 1% KOH and glycerin

•• Store in glycerinStore in glycerin

Skull, NearSkull, Near--term (E18.5) Fetusterm (E18.5) Fetus

Teratology 49: 497, 1994

Blocking of the Fetal Torso Blocking of the Fetal Torso

**

Fore Limb 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

**

**1

2

4

Blocking of the Fetal Torso forBlocking of the Fetal Torso forDetailed Neurohistologic Analysis Detailed Neurohistologic Analysis

**

Fore Limb Hind Limb

1 2

**

3

**

4

**

5

**

Blocking of the Fetal Head Blocking of the Fetal Head

* ** * *

Histologic Assessment of Histologic Assessment of NearNear--term Mouse Fetusesterm Mouse Fetuses

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.

Abdominal cross section, E18.5 Fetus

Fundam Appl Toxicol 21: 508, 1993

Histologic Assessment of Histologic Assessment of Early Mouse EmbryosEarly Mouse Embryos

Stage-matched neurulating (E8.5) embryos positioned to evaluate craniofacial and visceral anatomy

Untreated Methanol-Exposed

Teratology 49: 497, 1994

Special Anatomic Methods for Special Anatomic Methods for Assessing System DevelopmentAssessing System Development

E15 mouse embryo with targeted insertion of bacterial lacZ at expression sites for the type II collagen promoter

Whole Mount, E15

J Clin Invest 107: 35, 2001

Clinical Pathology Clinical Pathology in Mouse Conceptusesin Mouse Conceptuses

•• EndpointsEndpoints–– Hematology: cell counts, morphology, cell size, lineage Hematology: cell counts, morphology, cell size, lineage

differentiationdifferentiation–– Sample types: whole blood, blood smears, tissue smearsSample types: whole blood, blood smears, tissue smears–– Example: Example: Genes Dev.Genes Dev. 10: 15410: 154--164, 1996164, 1996

•• TechniquesTechniques–– Harvest conceptusHarvest conceptus–– Wash in PBS and blot dry to remove maternal blood cellsWash in PBS and blot dry to remove maternal blood cells–– Collect blood for hematology by capillary tube fromCollect blood for hematology by capillary tube from

•• Umbilical cord (E10.5 or older)Umbilical cord (E10.5 or older)•• Heart (E9.5 to E10.5)Heart (E9.5 to E10.5)

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Experimental Design Experimental Design Features for Analysis Features for Analysis of Developing Miceof Developing Mice

An EventAn Event--Oriented Decision Tree Oriented Decision Tree for Developmental Pathology Workfor Developmental Pathology Work

Are engineered neonates produced?

Detailed morphologic analysis

No

Are fetuses produced?

YesNo

Is there evidence of early embryonic death?

YesNo

Are anomalies evident? Define affected stage, then assess an earlier embryo

Detailed morphologic analysis

YesNo

Functional assays

Selection Criteria for Choosing a Selection Criteria for Choosing a Gestational Age for Further AnalysisGestational Age for Further Analysis

•• Characteristics of the Implantation SiteCharacteristics of the Implantation Site–– Early loss (reflecting resorption soon after implantation)Early loss (reflecting resorption soon after implantation)

•• A small, dark blotch embedded in the A small, dark blotch embedded in the endometriumendometrium•• No embryo to be foundNo embryo to be found

–– Intermediate loss (associated with midIntermediate loss (associated with mid--term embryolethality)term embryolethality)•• A midA mid--sized, tan or green mass filling the uterine lumensized, tan or green mass filling the uterine lumen•• AutolyzedAutolyzed embryo presentembryo present

•• Characteristics of the Affected EmbryoCharacteristics of the Affected Embryo–– Loss prior to organogenesis Loss prior to organogenesis –– flat or tubular embryoflat or tubular embryo–– Loss in early organogenesis Loss in early organogenesis –– bulbous embryo with limb buds bulbous embryo with limb buds

and and branchialbranchial archesarches–– Loss in late organogenesis Loss in late organogenesis –– small and disproportionate but small and disproportionate but

overtly overtly ““normalnormal”” embryoembryo

An EventAn Event--Oriented Decision Tree Oriented Decision Tree for Developmental Pathology Workfor Developmental Pathology Work

Are engineered neonates produced?Yes

Are neonates viable?

Yes

Done

No

Are anomalies evident?

YesNo

Detailed morphologic

analysis

Functional and molecular

assays

A Standard Experimental DesignA Standard Experimental Designfor Mouse Development Studiesfor Mouse Development Studies

•• Tier I: ScreeningTier I: Screening–– PurposePurpose: Basic assessment of the anatomic phenotype(s) elicited : Basic assessment of the anatomic phenotype(s) elicited

in a novel engineered construct in a novel engineered construct –– SubjectsSubjects: Near: Near--term fetuses (E17 or E18) and term fetuses (E17 or E18) and placentaeplacentae–– EndpointsEndpoints: Clinical observations (maternal), gross and : Clinical observations (maternal), gross and

microscopic anatomymicroscopic anatomy

•• Tier II: Mechanistic StudiesTier II: Mechanistic Studies–– PurposePurpose: Detailed characterization of the molecular events that : Detailed characterization of the molecular events that

produce a given anatomic phenotype produce a given anatomic phenotype –– SubjectsSubjects: Depends on the phenotype (likely will include both early : Depends on the phenotype (likely will include both early

and late embryos, with associated and late embryos, with associated placentaeplacentae))–– EndpointsEndpoints: Gross and microscopic anatomy, : Gross and microscopic anatomy, in situin situ molecular molecular

assays, functional tests assays, functional tests in vitroin vitro (cells, isolated organs, whole (cells, isolated organs, whole mounts) and mounts) and in vivoin vivo (heart rate, blood flow)(heart rate, blood flow)

Selection of Appropriate ControlsSelection of Appropriate Controls

•• Developmental AgeDevelopmental Age–– Early (E0 to E12): choose stageEarly (E0 to E12): choose stage--matched embryos using a matched embryos using a

combination of anatomic features (e.g., brain conformation, combination of anatomic features (e.g., brain conformation, presence of limb buds, presence of limb buds, somitesomite numbers)numbers)

–– Late (E13 and later): choose ageLate (E13 and later): choose age--matched conceptusesmatched conceptuses

•• TreatmentTreatment–– Genetic studies: include wild type and engineered embryos Genetic studies: include wild type and engineered embryos

(transgenic, or heterozygous and knockout)(transgenic, or heterozygous and knockout)–– Toxicity bioassays: include exposed and unexposed littersToxicity bioassays: include exposed and unexposed litters

•• Other variables to considerOther variables to consider–– Sex: select males and females (Sex: select males and females (anogenitalanogenital distance)distance)–– StrainStrain

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Interpretation of Interpretation of Lesions in the Lesions in the

Developing MouseDeveloping Mouse

Consequences of Consequences of In In UteroUtero Damage Damage Depend on the Gestational AgeDepend on the Gestational Age

•• Pre DifferentiationPre Differentiation–– Conceptus consists of pluripotent stem cellsConceptus consists of pluripotent stem cells–– Severe damage: diffuse cell death Severe damage: diffuse cell death →→ embryonic death embryonic death –– Mild injury: partial cell survival Mild injury: partial cell survival →→ ““normalnormal”” embryoembryo

Consequences of Consequences of In In UteroUtero Damage Damage Depend on the Gestational AgeDepend on the Gestational Age

•• Pre DifferentiationPre Differentiation–– Conceptus consists of pluripotent stem cellsConceptus consists of pluripotent stem cells–– Severe damage: diffuse cell death Severe damage: diffuse cell death →→ embryonic death embryonic death –– Mild injury: partial cell survival Mild injury: partial cell survival →→ normal embryonormal embryo

•• Embryonic StageEmbryonic Stage–– Organogenesis phase Organogenesis phase −− with different critical periods for with different critical periods for

each organeach organ–– Conceptus consists of partially differentiated stem cellsConceptus consists of partially differentiated stem cells–– Damage: focal to diffuse cell death Damage: focal to diffuse cell death →→ malformationmalformation–– Pattern of anomalies depends upon timing of insultPattern of anomalies depends upon timing of insult

Consequences of Consequences of In In UteroUtero Damage Damage Depend on the Gestational AgeDepend on the Gestational Age

•• Pre DifferentiationPre Differentiation–– Conceptus consists of pluripotent stem cellsConceptus consists of pluripotent stem cells–– Severe damage: diffuse cell death Severe damage: diffuse cell death →→ embryonic death embryonic death –– Mild injury: partial cell survival Mild injury: partial cell survival →→ normal embryonormal embryo

•• Embryonic StageEmbryonic Stage–– Organogenesis phase Organogenesis phase −− with different critical periods for each organwith different critical periods for each organ–– Conceptus consists of partially differentiated stem cellsConceptus consists of partially differentiated stem cells–– Damage: focal to diffuse cell death Damage: focal to diffuse cell death →→ malformationmalformation–– Pattern of anomalies depends upon timing of insult Pattern of anomalies depends upon timing of insult

•• Fetal StageFetal Stage–– Growth phaseGrowth phase–– Conceptus consists of Conceptus consists of oligopotentoligopotent and differentiated cellsand differentiated cells–– Damage: cell death Damage: cell death →→ functional deficit >> malformationfunctional deficit >> malformation

Consequences of Developmental Consequences of Developmental Damage Depend on the AgeDamage Depend on the Age

•• Fetal StageFetal Stage–– Growth phaseGrowth phase–– Conceptus consists of Conceptus consists of oligopotentoligopotent and differentiated cellsand differentiated cells–– Damage: cell death Damage: cell death →→ functional deficit >> malformationfunctional deficit >> malformation

•• Postnatal StagePostnatal Stage–– Growth phaseGrowth phase–– Conceptus consists of Conceptus consists of oligopotentoligopotent and differentiated cellsand differentiated cells–– Damage: cell death Damage: cell death →→ functional deficit, no malformationsfunctional deficit, no malformations

Disrupted Circulation is Disrupted Circulation is the Major Cause of Embryolethalitythe Major Cause of Embryolethality

•• Placental malformationsPlacental malformations

•• Embryonic malfunctionEmbryonic malfunction–– Anemia Anemia –– Cardiac anomaliesCardiac anomalies–– Cardiac arrhythmiasCardiac arrhythmias–– Hypoxia (via altered neuroendocrine regulation of heart)Hypoxia (via altered neuroendocrine regulation of heart)–– Vascular Vascular dysgenesisdysgenesis (with hemorrhage)(with hemorrhage)

•• Maternal sourcesMaternal sources–– AnemiaAnemia–– HemorrhageHemorrhage

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Gestational Age Gestational Age ≠≠ StageStage

Apparent Age: E13Actual Age: E13

Apparent Age: E12Actual Age: E13

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

Embryonic DeathEmbryonic DeathWild Type Transgenic

E13.5 embryos, one of which expired at approximately E9.5 due toover-expression of a stem cell inhibitor throughout development

Renal AplasiaRenal Aplasia

Neonates (P1), the right one of which bears a lethal targeted null mutation of the Gfrα1 gene

Wild Type Heterozygote Knockout

* *****

Urinary Tract AplasiaUrinary Tract Aplasia

E11 embryos, the middle and right bearing a lethal targeted null mutation of the Gfrα1 gene

Wild Type Knockout Knockout

Limb AplasiaLimb Aplasia

E18 fetuses, the right one of which bears a lethal targeted null mutation of the Fgf10 gene

Limb AplasiaLimb Aplasia

E9.5 embryos, the right one of which bears a lethal targeted null mutation of the Fgf10 gene

Wild Type Heterozygote Knockout

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Dysplasia of the Cranial Dysplasia of the Cranial (Superior) Cervical Ganglion(Superior) Cervical Ganglion

Wild Type Embryo Transgenic Embryo

E14 lesion resulting from over-expression of a trophic factor for sympathetic neurons throughout development

Toxicol Pathol 32: 275, 2004

Major Causes of Perinatal LethalityMajor Causes of Perinatal Lethality

•• Airway malfunctionAirway malfunction–– Agenesis or Agenesis or dysgenesisdysgenesis of pulmonary systemof pulmonary system–– Decreased thoracic volumeDecreased thoracic volume–– Skeletal defects (reduced thoracic expansion)Skeletal defects (reduced thoracic expansion)

•• Cardiac malfunctionCardiac malfunction–– ArrhythmiasArrhythmias–– Heart and/or vascular malformationsHeart and/or vascular malformations

•• Other major anomalies Other major anomalies –– Functional: ImmunodeficiencyFunctional: Immunodeficiency–– Structural: Agenesis (kidney), Structural: Agenesis (kidney), ectopiaectopia (neural tube defect)(neural tube defect)

Spontaneous Malformations Spontaneous Malformations in Developing Micein Developing Mice

•• Common VariantsCommon Variants = 0 to 35%= 0 to 35%–– Examples: Renal pelvic Examples: Renal pelvic cavitationcavitation, supernumerary ribs, wavy ribs, supernumerary ribs, wavy ribs–– Outcome: IncidentalOutcome: Incidental

•• Major MalformationsMajor Malformations = < 1%= < 1%–– Examples: Exencephaly, ventricular Examples: Exencephaly, ventricular septalseptal defectdefect–– Outcome: LethalOutcome: Lethal

•• Minor Visceral MalformationsMinor Visceral Malformations = 1 to 3%= 1 to 3%–– Examples: Cranial displacement of gonads, hemorrhagesExamples: Cranial displacement of gonads, hemorrhages–– Outcome: Usually incidentalOutcome: Usually incidental

•• Minor Skeletal AnomaliesMinor Skeletal Anomalies = 1 to 5%= 1 to 5%–– Examples: Curly tail, Examples: Curly tail, sternebralsternebral asymmetry, asymmetry, unossifiedunossified phalangesphalanges–– Outcome: IncidentalOutcome: Incidental

SummarySummary

•• The need for phenotypic evaluation of developing The need for phenotypic evaluation of developing mice will increase in all fields of biomedical researchmice will increase in all fields of biomedical research

•• The examiner will have to have broad anatomic and The examiner will have to have broad anatomic and physiologic knowledge of all developmental stages physiologic knowledge of all developmental stages to perform a competent phenotypic examination to perform a competent phenotypic examination

•• The skills required for such proficiency are mere The skills required for such proficiency are mere extensions of the understanding acquired during a extensions of the understanding acquired during a wellwell--rounded education in biology and medicinerounded education in biology and medicine