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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
[email protected]@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
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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
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2
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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
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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