Download A Brief Overview of Mouse Development

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