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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