Veterinary Pathology

49(1) 71-84
Pathology Methods for the Evaluation of ª The Author(s) 2012
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Embryonic and Perinatal Developmental DOI: 10.1177/0300985811429811
http://vet.sagepub.com
Defects and Lethality in Genetically
Engineered Mice

J. M. Ward1, S. A. Elmore2, and J. F. Foley2

Abstract
The normal embryonic development of organs and other tissues in mice and all species is preprogrammed by genes. Inactivation of
a gene involved in any stage of normal embryonic development can have severe consequences leading to embryonic or postnatal
developmental defects and lethality. Pathology methods are reviewed for evaluating normal and abnormal placenta and embryo,
especially after E12.5. These methods include pathology protocols for necropsy and histopathology in addition to references that
will provide additional knowledge for embryo assessment including histology atlases and advanced embryo imaging techniques.

Keywords
mouse embryo, embryonic lethality, placenta, histology atlas, imaging, phenotyping

Mice are often used in research to study functions of genes that
have been modified by various genetic engineering methods.
Complete or partial inactivation of a gene may lead to loss or
modification of gene function in cells and tissues during any
stage of gestation. The effects of gene modification can be ubi-
quitous in the embryo or targeted to specific tissues or cells.
Modification of gene function by either spontaneous mutations
or genetic engineering can result in an embryonic or postnatal
lethality phenotype.46 The aim of this article is to provide basic
pathology evaluation procedures that will assist investigators in
determining possible causes of embryonic and postnatal mor-
tality. It should be noted, however, that the use of pathology
and laboratory procedures described by us and other investiga-
tors may still not allow one to determine the cause of embryo-
nic or postnatal mortality.
Stages of Gestation
Mouse development occurs after fertilization of the egg. Devel-
opment in embryonic days (E) may be identified as going
through 4 basic periods: include pre- and peri-implantation
(E0-4.5), differentiation of the germ layers (E6-9), development
of the cardiovascular system and placenta (E9-12), and tissue and
organ differentiation (E12-19) (http://www.emouseatlas.org/
emap/ema/theiler_stages/house_mouse/book.html; http://www
.informatics.jax.org/greenbook).31,38,44,47 The morning that a
vaginal plug is found in mated females is generally defined as
E0.5 of gestation.23 Embryonic growth is at a specified rate and
in stages, as defined by Theiler stage, head to rump length, and
somite pairs.23
Gene Expression
Genes are expressed in the various cells and tissues during
gestation, and expression patterns have been described
(http://www.emouseatlas.org/emage/home.php). Many genes
function in normal development, growth, and differentiation
of the embryonic organs and tissues. To determine the cause
of embryonic death or to determine developmental defects
found in live or dead embryos or neonatal, perinatal, or postna-
tal mice, it is crucial to first determine the cells and tissues
expressing the gene in question. Several methods are available
to assess gene expression, including in situ hybridization,
immunohistochemistry, Lac-Z/b-gal expression,2 and real-
time qualitative PCR (q-PCR).30,31 The effect of gene inactiva-
tion may be evident in the tissues normally expressing the
gene.14 Genes normally expressed at high levels during in utero
development may have profound effects on organogenesis and
differentiation. Secondary effects of gene mutations may also
be found. A good example is the Nkx2-1 gene, the inactivation
of which causes lack of normal lung development (lung bud
branching is absent, which is the cause of death), absence of the
pituitary gland and thyroid, and an abnormal hypothalamus.
Mice live through gestation but die at birth or postnatally.25
1
Global VetPathology, Montgomery Village, Maryland
2
Cellular & Molecular Pathology Branch, National Toxicology Program,
NIEHS, NIH, Research Triangle Park, North Carolina
Corresponding Author:
Jerrold M. Ward, Global VetPathology, Montgomery Village, MD 20886
Email:globalvetpathology@gmail.com
72
Pituitary development is altered not because of gene inactiva-
tion in the tissue but rather is secondary to inactivation of the
Nkx2-1 transcription factor gene, which controls regulation of
other genes important for pituitary pouch formation and thus
pituitary development.45
Necropsy Methods
The dam is euthanatized by carbon dioxide, the abdominal cav-
ity is opened, and the uterus and ovaries are removed intact.5,40
For embryos over E15, euthanasia may be induced by the injec-
tion of chemical anesthetics, induction of hyperthermia, or
other methods specified by institutional policy, procedures,
or guidelines. The uterus is spread out, and the location and
sizes of placental sites (Fig. 1) are recorded on a uterus/
embryo necropsy sheet as previously described.49 Extraem-
bryonic membranes (amnion, yolk sac, chorion, allantois)
from each embryo are examined for viability (blood present
in the yolk sac vessels) and then removed and detached from
its placenta (Fig. 2).
A gross evaluation of the embryo and placenta is then per-
formed. The rump to head length is recorded, as are any other
gross findings. The placenta is examined to determine whether
it appears grossly normal or abnormal. Depending on gesta-
tional age, the yolk sac, an appendage, the tail, or the entire
embryo may be collected for genotyping. Any portion of the
embryo not needed for histological examination may be used.
Mouse embryonic death may occur spontaneously27 or may
be induced by genetic engineering. Thus, any grossly abnormal
embryo found may not only be an induced mutant. Examples of
abnormalities and progressive gross changes leading to
embryonic death are shown in Figs. 3–8. Growth retardation,
growth arrest, or delayed development are reported, but we see
embryonic lethality represented as early embryonic death
(focal or diffuse degenerative and necrotizing lesions.
Histopathology Methods
For evaluation of placenta and embryo histopathological char-
acteristics, the placenta should be separated from the embryo.
Embryos at most gestational stages are best fixed in Bouin’s
fixative.5,40 Embryo fixation is dependent upon gestational
age: embryos less than E12 are fixed for 4 hours, E12.5–
E16.5 for 24 hours, and greater than E16.5 for 24–48 hours.
If in situ hybridization, immunohistochemistry, or molecular
studies are to be done, 4% paraformaldehyde is the fixative
of choice. Optimal embryo fixation and embedding protocols
have been previously published.5,23,24,40 The entire uterus can
also be sectioned for overviews of uterus, placenta, and embryo
(Figs. 9, 10). Dissection of all components (placenta, extraem-
bryonic membranes, and embryo) usually results in the ability
to perform more extensive examinations (Figs. 11–15). At
embedding, embryos are oriented in the coronal (frontal), trans-
verse, or sagittal plane. One tissue screening method for
embryos older than E14 is as follows: at the time of necropsy,
open the abdomen to allow better penetration of the Bouin’s
Veterinary Pathology 49(1)
fixative, and after optimal fixation, trim the embryo sagittally,
making 1 cut down the middle (midsagittal) and trimming 1
remaining half sagittally into 2 pieces so that almost all tissues
and organs can be found in the 3 long sections (except for small
anatomical regions in the brain). Midsagittal sections alone
make for good tissue scanning sections for most, but obviously
not for all major organs (Fig. 15). The use of specific types of
sections (frontal, sagittal, or transverse) is crucial for compar-
ison, and the optimal type of sectioning can be organ specific.
For example, a sagittal section of the embryo is excellent for an
overall view of many organs, but the most useful section for the
evaluation of the brain is coronal (frontal). Transverse sections
are optimal for the evaluation of the spinal cord. With the use of
a stereomicroscope, individual tissues of the larger later gesta-
tion embryos can be carefully dissected out of the body,
trimmed, and embedded. The intact placenta is fixed in forma-
lin (48 hours), not Bouin’s, with or without the uterus attached.
Bouin’s is not used to fix the placenta because it alters the
appearance of erythrocytes (Figs. 16–21). After fixation, the
placenta is bisected adjacent to the umbilical vessels. Both
halves are embedded face down within 1 paraffin block.
Histological Characteristics of the Normal
Mouse Placenta
The mouse placenta can be described as discoid and hemotri-
chorial and has been compared to the discoid hemochorial pla-
centa of humans and monkeys (http://placentation.ucsd.edu/).16
The placenta, composed of both embryonic and maternal tis-
sues, plays an important role in embryonic development and
growth. The structure of the placenta allows nutrients to travel
to the embryo via maternal blood and the trophoblast–embryo-
nic blood interaction. The development of the mouse placenta
has been well described.6–8,21,28,34,39,47,49,50 Pregnancy dating
in the rat placenta is similar to that in mice.9 By maturity of
the placenta at E12, all structures are present (Figs. 11–13,
16–21) including the labyrinth with labyrinth trophoblasts and
embryonic blood vessels lined by embryonic endothelium
(Figs. 16–18). Vascular spaces are lined by embryonic labyr-
inth trophoblasts, contain mature maternal erythrocytes, and
are also present in the spongiotrophoblast and giant cell tro-
phoblast zones and decidua. The decidua, which is 100%
maternal, is composed of blood vessels and macrophage-
like cells expressing macrophage antigens. The metrial gland
can be seen in the outer portion of the decidua.47 If suboptimal
sections are prepared of the placenta, one may see mostly
decidua (Fig. 9). Normal age-related placental histological
characteristics in wild-type embryos should be compared with
those of the genetically engineered mouse when assessing
mechanisms of embryonic death and possible abnormal pla-
cental development.
Many genes are expressed in the placenta, including many
involved in adult carcinogenesis.11,12,14,17,36,43 Inactivation of
a number of genes that play a role in cancer development have
also have been reported to result in embryonic mortality, espe-
cially involving the placenta.48 Thus, how a gene functions in
Ward et al 73

Figure 1. Mouse uteri at E12, E10, and E10 showing 1, 2, and 7 abnormal implantation (placental) sites. Figure 2. E12 embryo showing normal
embryo, its umbilical vessels connected to the labyrinth of the placenta (red discoid area). Note pigmented retina. Figure 3. E13 showing nor-
mal sized embryo with massive hemorrhages due to platelet defects. Note no blood in the yolk sac blood vessels. The placenta is seen at the
bottom. Figure 4. E12–E13 Dead embryo due to lack of normal erythropoiesis in yolk sac. Note no blood in yolk sac. Figure 5. E12–E13.
Early lethality. Light from below; embryo on left is normal, embryo on right side is dying. Note smaller size of the embryo and loss of
normal color and tissue structures. Figure 6. E14 showing smaller embryo that had focal necrosis of various tissues histologically . Note
some small hemorrhages. Figure 7. Viable embryo, yolk sac, and placenta on right side and adjacent end-stage necrotic embryo on left.
Figure 8. Exencephaly in a newborn mouse.

the embryo and placenta may differ greatly than those in adult
mice. Immunohistochemistry studies and in situ hybridization
can reveal cells and tissues expressing such genes (http://
www.brain-map.org/; http://genex.hgu.mrc.ac.uk/). Inactiva-
tion of some genes can cause abnormal development and
growth of the placenta and its membranes, leading to placen-
tal failure as an organ providing nutrition and oxygen to the
embryo.39,49 Without vital nutrients, embryonic tissues
develop degenerative and necrotizing lesions, leading to
embryonic death.
The embryo can be aged, somewhat, by the appearance of
erythrocytes and their precursors in the embryonic vessels of
the placenta (Figs. 16–21) and yolk sac. These findings may
be related to the mouse strain. Immature erythroblast-like cells
74
(Figs. 16, 17) are seen in early placental development, whereas
erythrocyte maturation occurs over the next 3 days (E11–E14)
and a population of mostly metarubricytes is observed (Figs.
18, 19). Histologically, by day 15, most embryonic erythro-
cytes have extruded their nuclear material (Figs. 19, 20).
Degenerative changes commonly develop in the maternal
decidua and placental trophoblasts (Fig. 21) as the placenta
ages, including atrophy, vacuolation, hydropic change, and
necrosis. Degenerative changes in wild-type and mutant pla-
centas must be compared to determine whether there is an
increase of these changes or even appearance of degenerative
changes not normally seen, including hyperplasia, dysplasia,
and multinucleated giant cells.49
Causes and Evaluation of Embryonic
Mortality After E12.5
Genetic engineering has produced many mouse developmental
defects resulting in embryonic lethality and postnatal mortality.
Information for many of the embryonic lethal mutants can be
found at the Jackson Laboratory Mouse Genome Informatics
Web site (http://www.informatics.jax.org) under Phenotypes
& Disease Models. In an accompanying article, the authors dis-
cuss evaluation of embryonic lethality prior to E12.5.30 We will
review embryonic lethality as it applies to placental lesions
prior to E12.5, embryonic lesions after E12.5, and after birth
(due to abnormal in utero development).
Necropsy Strategy for Determining the
Causes of Embryonic Lethality
It has been estimated that perhaps 5%–15% of wild-type ferti-
lized embryos may be embryonic lethal.6,27,32 Thus, not all
dying or dead embryos are a result of induced genetic changes.
If null mice are absent after birth, it is most likely that the null
mutation is embryonic lethal. To determine whether embryo-
nic lethality begins post implantation and to assess the histo-
pathogenesis of lethality, a strategy would be to begin
collecting litters at E16. If null embryos are still absent and
abnormal placentation sites can be seen, work backward and
collect litters at 2-day gestation intervals (E14, E12, E10) to
pinpoint the initial developmental abnormality leading to
defects or lethality.
Gross Abnormalities of the Uterus, Placenta,
and Embryo
Initial evidence of embryonic mortality is a decrease in size and
change in color of the normal placental site in the uterus
(Fig. 1). As the embryo dies, the placenta also undergoes
degenerative and eventually necrotizing lesions. The number
of abnormal placental (implantation) sites that develop during
gestation depends on the cause and the nature of the defect dur-
ing normal development of the placenta or embryo. If vascular
abnormalities develop in the placenta, hemorrhagic lesions
may be seen (Fig. 14).18 Initial embryonic changes may include
Veterinary Pathology 49(1)
loss of a normal heartbeat or abnormal appearances or loss of
blood vessels or erythrocytes within the visible blood vessels.
Focal hemorrhages may appear, and large areas of hemorrhage
are often indicative of megakaryocyte or platelet defects
(Fig. 3), almost always after E13. As more severe gross
changes develop, there is loss of normal embryo structure as
it becomes more amorphous in nature (Figs. 4–6). Eventually,
the embryo dies. Over time, the embryo is gradually reduced in
size and becomes a discolored, amorphous structure, unrecog-
nizable as an embryo (Fig. 7).
Evaluation of the Placenta
The development of the placenta, which is complete by E12.5,
may fail because of a variety of developmental membrane
defects (eg, failure of allantois to fuse to the chorion, yolk sac
failure, mesodermal/endodermal defects, lack of normal ery-
thropoiesis in the yolk sac, placental defects in vasculogenesis,
trophoblast formation, and decidua formation).35,43,49 The dam
is often heterozygous or homozygous null for a gene, and thus
the expression of the gene in the decidua may be altered, pos-
sibly affecting decidua and placental development.15
If the placenta fails to develop normally, the consequences
are disruption of nutrition and oxygen to the developing
embryo. Embryonic growth is restricted. Lack of normal
growth and development leads to initiation of embryonic leth-
ality often by E10 and commonly before E13. Histologically
in the embryo, one can see focal, multifocal, or diffuse necro-
sis of various cells and tissues. After embryonic death, the
remaining tissue is resorbed but may leave residual lesions
at the placental site (Fig. 1). The structure of the labyrinth and
other anatomical regions may be compromised, causing dis-
ruption of normal placental anatomy and, therefore, causing
hemorrhage within the labyrinth.
Phenotypes From E12 to Birth
Gene inactivation causing initial developmental changes at
E9–E10 may also cause initial histological changes in the pla-
centa and embryo at that time. These progressive changes could
result ultimately in embryo death within a few days or over a
period time (several days or longer). Thus, at E12, embryos
dead or dying from earlier developmental alterations can be
observed as smaller placental sites in the uterus (Fig. 1).
Embryos with initial changes after E12 may survive the entire
gestation period or die after E12 but prior to birth. Common
causes of embryonic death after E12 include heart defects
involving failure of the cardiac conduction system and septa-
tion40 and hematopoietic defects involving erythroid and
megakaryocyte differentiation.20 The majority of dying or
dead embryos found after E12 have defects that have started
prior to E12. However, developmental defects initiated in any
stage of gestation for brain, kidney, liver, and lung may allow
the embryo to survive in utero but could result in neonatal or
postnatal mortality.
Ward et al 75

Figure 9. The pregnant mouse uterus at E11–E12 showing the various sections of placentas (P) and embryos. The decidua (D) is prominent in
some sections. Hematoxylin and eosin (HE) staining. Figure 10. The pregnant uterus at E14 showing larger placentas (P) and embryos. Note
the very thin uterine wall. HE. Figure 11. E14 embryo and placenta. Note the thin uterine wall and well-developed organs. HE. Figure 12.
E12.5 placenta showing the well-developed mature labyrinth and decidua. HE. Figure 13. The E12.5 placenta showing the various anatomical
zones. HE. Figure 14. E11–E12 placenta and dead embryo from a knockout mouse with abnormal placental vasculogenesis causing disrupted
normal growth of the labyrinth. HE.
76
Figure 15. Mid-sagittal sections of the mouse embryo at different stages of gestation showing general organ development. Not to actual embryo
size. Hematoxylin and eosin (HE) staining.
Phenotypes in the Perinatal Period and
Beyond
Mice may survive the gestation period with an organ or system
developmental defect, especially defects of the skin, skeleton,
lungs, kidneys, liver, and central nervous, immune, endocrine,
and reproductive systems. Defects involving the placenta, ery-
throid, or platelet development and the heart often result in
intrauterine mortality. Defects initiated in utero may be lethal
at some age postnatally, such as cystic kidneys,1,19 or mice may
be viable with nonlethal lesions such as loss of 1 kidney or
reproductive failure. Death, secondary to these defects, can
occur quickly at the time of birth, such as when lack of normal
lungs or brain dramatically impairs normal postnatal viability.
When postnatal mortality occurs, investigators can return to the
embryo and neonate to study the histopathogenesis of the post-
natal lethal lesions and determine the developmental defect that
led to death. Developmental defects, however, may also be
compatible with life, with or without clinical consequences.
In humans, these include cleft lip and palate, neural tube
defects, ciliopathies, polycystic kidney disease, laminopathies,
deafness, and the muscular dystrophies. Some mouse models
exist for these human conditions.
There are numerous reports of mouse mutants that breathe
briefly at birth and subsequently die. For some of these
mutants, the cause of respiratory insufficiency is unknown. In
our experience with these cases, lung histological status is usu-
ally normal or only slightly deviant from normal. Death may be
related to the breathing mechanism, rather than a defect in lung
anatomical, histological, or physiological characteristics.
Research of the breathing mechanism is a specialty involving
knowledge of respiratory physiological characteristics and ner-
vous system gene expression patterns. An excellent example of
neonatal respiratory insufficiency was reported in necdin (Ndn)
null mice.37 Absence of the gene normally expressed during
neuronal development resulted in disruption of pre-Bötzinger
complex, which is essential for the generation of respiratory
Veterinary Pathology 49(1)
rhythm. In this case, the investigators knew the anatomical
brain location of Ndn gene expression before they created their
mutant mice, which aided significantly in their ability to iden-
tify the cause of death.
Resources for the Evaluation of Mouse
Embryo Organ Development
With the rapidly expanding use of genetically engineered mice in
biomedical research and the widespread development of mouse
models to study the pathogenesis of human diseases, additional
and improved histology atlases are required to evaluate embryo-
nic malformations and lethality. Pathologists and biomedical
researchers have historically relied on atlases for illustrations and
descriptions of normal structures relevant to the most important
developmental events in the embryonic mouse.5,40,23,24,47 Other
useful resources include an online tutorial of normal mammalian
development using scanning electron micrographs (http://
www.med.unc.edu/embryo_images/), a high-resolution magnetic
resonance histology atlas of the embryonic and neonatal mouse,33
and the Edinburgh 3D mouse embryo anatomy atlas (http://
www.emouseatlas.org/emap/home.html).
The complexity of organ development underlies the need for
such resources. For example, the developing heart undergoes a
complicated 3-dimensional structural change when the heart
tube loops to the right during embryonic days 8.5–10.5 and the
venous pole subsequently moves cranially and dorsally.40
Concomitant with the rightward looping, lengthening heart
tube and further ballooning of the future chambers take place.
Additional structural changes occur until the final cardiac
architecture is achieved. Although the liver doesn’t undergo
such complicated architectural changes, there are changes
at the cellular level, which can best be illustrated with high-
magnification images.5 Embryonic liver venous drainage
and embryonic hematopoiesis are also important factors to
consider.
Ward et al 77

Figure 16. E9.5 mouse placental labyrinth. The embryonic endothelial cells (arrows) line embryonic blood vessels containing nucleated
erythrocyte precursors. Labyrinth trophoblasts (the cells with the largest nuclei) can be seen lining blood spaces containing maternal blood
(M) with mature erythrocytes. Hematoxylin and eosin (HE) staining. Figure 17. E10.5 labyrinth with some maturation of the erythrocyte pre-
cursors in embryonic blood vessels lined by embryonic endothelium (arrows). E, embryonic blood vessels; M, maternal blood spaces, LTB, labyr-
inth trophoblasts. HE. Figure 18. E12.5 labyrinth showing many metarubricytes in embryonic blood vessels (E). HE. Figure 19. E14.5 labyrinth
with few nucleated erythrocyte precursors evident. HE. Figure 20. E16.5 labyrinth showing that all embryonic erythrocytes are mature.
Trophoblasts are atrophic with little cytoplasm. HE. Figure 21. E17.5 labyrinth is atrophic, showing trophoblast atrophy and more prominent
vascular spaces containing mature erythrocytes. Embryonic endothelium (arrows) lining embryonic vessels can be seen. HE.
78 Veterinary Pathology 49(1)

Figure 22. Representative images of the E14.5 mouse heart. Upper left image: Schematic illustration of the E14.5 heart from a ventral view
demonstrating the estimated plane corresponding to the site of the hematoxylin and eosin (HE)–stained sagittal section (D). Lower right image:
Schematic illustration of the left lateral view demonstrates the heart of the E14.5 mouse, and it is used here to show the estimated planes cor-
responding to the sites of HE-stained E14.5 transverse (B) and frontal (C) sections. Figures 23–25. HE-stained sections of an E14.5 mouse
heart showing most major structures. Figure 23. Frontal section. Figure 24. Transverse section. Figure 25. Sagittal section. The asterisk in Figure
25 demonstrates the fibrous raphe at the site of fusion of the conal outflow tract ridges in the wall between the pulmonary and aortic roots.
Reprinted with permission from Sage Publications (Savolainen et al,40 Toxicologic Pathology, Vol. 37, No. 4, 2009). Ao, aortic trunk; AVL, aortic
valve leaflet; AVS, atrioventricular septum; CT, cushion tissue; DAo, descending aorta; E, esophagus; IVC, inferior vena cava; IVS, interventricular
septum; LA, left atrium; LV, left ventricle; LSV, left superior vena cava; MB, main bronchus; PR, pulmonary root (infundibulum); PVL, pulmonary
valve leaflet; RA, right atrium; RAVC, right anterior cardinal vein. RV, right ventricle; SP, septum primun; SS, septum secundum; SSp, septum
spurium; TVL, tricuspid valve leaflet; VS, vestibular spine; VV, venous valve.
Ward et al 79

Figure 26. A transverse section through the developing heart at E13.5 demonstrates the newly formed conal septum (arrow) separating the 2
ventricular outlets, pulmonary trunk, and aorta. Figure 27. A higher magnification of this region demonstrates foci of apoptotic cell debris
(arrows) in the cushion tissue surrounding the newly formed conal septum. A mitotic cell (arrowhead) is also present. Reprinted with permis-
sion from Sage Publications (Savolainen et al,40 Toxicologic Pathology, Vol. 37, No. 4, 2009). Ao, aortic trunk; CT, cushion tissue; DAo, descending
aorta; E, esophagus; LA, left atrium; LSV, left superior vena cava; MB, main bronchus; PA, pulmonary artery; PT, pulmonary trunk; RA, right
atrium; RSV, right superior vena cava. RV, right ventricle.

Knowledge of how the organ was sectioned (frontal,
sagittal, or transverse) is crucial for comparison and the optimal
type of sectioning can be organ specific. For the heart and hepa-
tobiliary system, frontal, transverse, and sagittal views at each
stage of development may be useful (Figs. 22–25). Providing
all 3 views can be helpful when there are structures that can
be seen in one view but not in another view. When appropriate,
high-magnification images can illustrate important cellular
detail such as apoptosis in the outflow tract cushions of the
heart (Figs. 26,27) and hematopoietic cells, hepatoblasts, and
erythrocyte morphological changes in the liver.5,40 Special
stains can be used to illustrate specific cellular and tissue
morphology such as macrophages within erythroblastic islands
and the presence of granulocyte progenitors in the developing
liver.5
The overall goal for histology atlases and online resources is
to provide tools that pathologists and biomedical scientists can
use for proper evaluation of developing mouse organs. Critical
to the phenotypical evaluation of any embryo organ system is
a thorough knowledge of early mouse development, main
developmental events, normal histological characteristics, all
features unique to that organ system, major abnormalities
seen at each embryonic stage, and important early postnatal
developmental events.
Modalities for Imaging the Mouse Embryo in
3D: High-Resolution Anatomical and Anato-
mical Pathological Methods, Low-Resolution
Histological and Histopathological Methods
Besides the availability of enhanced histology atlases for eval-
uating mice, one can also use advanced imaging techniques to
assist with the traditional histological methods of the embryo
assessment. Using conventional histological methods to iden-
tify and characterize possible developmental defects is labor
intensive and time consuming. High-throughput 3D imaging
methods improve screening embryos for possible developmental
phenotypes. Imaging is basically a high-resolution anatomical
and anatomical pathological method but a low-resolution histo-
logical method. Technological advances in the 2 primary meth-
ods of nondestructive, noninvasive imaging—micro–magnetic
resonance imaging (mMRI) and micro–computed tomography
(mCT)—allow the imaging of small specimens such as mouse
embryos (Fig. 28). Other optical modalities of high-throughput
imaging, such as episcopic fluorescence imaging capturing
(EFIC) and optical projection tomography (OPT), have been
developed to examine mouse embryos in 3D. These imaging
methods are discussed below as they apply to mouse embryo
phenotyping.
80 Veterinary Pathology 49(1)

Figure 28. Representative images for methods of imaging mouse embryos in 3 dimensions. (a-c) mMRI of an E17.5 mouse embryo at 19.5-mm
isotropic resolution. Isosurface, coronal, and transverse rendered images. (d) 3D rendering of a mCT scan (10-mm resolution) of the skeletal
system from an E18.5 mouse embryo. Dorsal view of a wild-type mouse versus a transgenic littermate overexpressing human COX-2. (e-l)
3D rendering from a mCT scan of an E11.5 mouse embryo. (E, F) Isosurface renderings of a mCT-scanned embryo. (g-l) Comparison of
hematoxylin and eosin (HE) and computed tomography sections of an E11.5 mouse embryo scanned at 8 mm. (m) High-resolution episcopic
Ward et al 81

Micro–Magnetic Resonance Imaging
mMRI is a nondestructive imaging modality that provides
accurate geometric representation of mouse anatomy and
can also be used when phenotyping the mouse embryo
(http://www.birncommunity.org/data-catalog/duke-center-for-
in-vivo-microscopy-civm-high-resolution-mri-images). Mag-
netic resonance imaging is based on the alignment of hydrogen
atoms of the specimen within a strong magnetic field, subject-
ing them to pulses of radio waves and then detecting the 3D
distribution of further radio waves that are emitted as a
response to the excitation.42 High-contrast images can be
obtained of soft tissue structures at about 25 mm resolution
in the mouse (Fig. 28a-c). Since the first studies using mMRI,
it has emerged as a technique to facilitate high-throughput
phenotyping of genetically engineered mice and ENU muta-
genesis screens and to investigate development effects second-
ary to teratogens and the environment.41 As a preferred
instrument for studying soft tissue structures, mMRI has been
most powerful in the rapid identification of cardiac malforma-
tions such as atrial and ventricular septal wall defects, outflow
tract malformations, and aortic arch remodeling defects.3,4,41
Advances in instrumentation, such as stronger magnets, and
replacement of complicated methods of sample preparation
by simple immersion staining techniques have allowed for
enhanced visualization of soft tissues and, more recently, miner-
alized tissue.13 Disadvantages of using mMRI include increased
costs attributable to complex instrumentation typically housed at
large medical/research centers and scan time. Low image resolu-
tion is another drawback. At 25-mm resolution, mMRI is not
capable of differentiating cells and some tissues.
Micro–Computed Tomography
Traditional methods for examining the skeletal system of the
mouse embryo involve a dual-stain procedure for the detection
of cartilage (alcian blue) and bone (alizarin red). Limitations of
this conventional embryo skeletal staining method consist of
time-consuming and laborious specimen preparation, tissue
integrity loss, and fragile samples, which are difficult to image
since they easily fragment when handled. mCT is a nondestruc-
tive tomographic imaging method that circumvents many of
these problematic issues. Mathematical models create a 3D
reconstruction of internal organs, vessels, and bone from a
large series of 2D X-ray data taken around a single axis of rota-
tion from a series of digital images.26
mCT is used extensively for imaging mineralized tissue,
since the radio opacity of mineralized bone makes it easy to
detect (Fig. 28d). Skeletal analysis can be accomplished at a
lower resolution of 38 mm, thus decreasing scan time, compu-
tational power, and expense.29 For bone metrics, mCT also
allows for the generation of accurate data such as bone volume
and average bone mineral density, which is not possible in
conventional-stained embryos.
Widespread use of mCT for nonmineralized tissues has been
restricted by the low X-ray contrast of soft tissues; however,
recent advances in contrast agents and instrumentation with
increased x-ray strength are changing this limitation.26 In com-
parison with mMRI, mCT offers higher resolution of soft tissue
scans, 6–10 mm versus 25 mm (Fig. 28e, f, j-l). Histology
equivalents of Fig. 28j-l are also shown (Fig. 28g-i). Scanning
time is rapid for mCT, 2 hours versus 9–14 hours.22 Since scan-
ning time is decreased, costs associated with mCT are also
reduced. As for mMRI, instruments for mCT are expensive,
typically are housed at large medical/research centers, and
require a skilled technician for instrument operation and soft-
ware rendering of 3D images.
Episcopic Fluorescence Image Capturing
EFIC is a histological method that provides accurately aligned,
high-resolution (2 mm) digital images that can be examined
through computer animations of stacked images, surface ren-
dering, or volume rendering procedures. Embryos E10.5 or
later are routinely processed according to gestational age and
blocked in paraffin. Since the tissue has the intrinsic property
of autofluorescence, episcopic fluorescence microscopy is used
during sectioning to capture digital images of the freshly cut
block surface.52 Because the block surface rather than individ-
ual cut sections is imaged, precise alignment of the specimen is
obtainable. In addition, tissue distortions produced by routine
histological sectioning and staining are avoided, since digital
images are acquired prior to sectioning and no special staining
is required of the specimen. Embryos less than E10.5 are not
optimal for EFIC given that there is little tissue to for autofluor-
escence. Stacked images can be looped with QuickTime or the
public domain software Image J. Although it is not possible to
discern specific cellular structure and only the largest intercel-
lular spaces can be identified, recent developments in this field
have allowed for the 3D visualization of molecular markers
such as GFP and LacZ in the embryo. In high-resolution epi-
scopic microscopy (HREM), embryos are embedded in a resin

Figure 28 (continued). microscopy (HREM) used to compile a series of 2-mm, 2D block surface images into a 3D image from an E15.5 mouse
embryo. (n-o) Fluorescence OPT imaging of multiple signals within an E10.5 mouse embryo with HNF3b antibody. (n) 3D rendering of an isosur-
face generated from the voxel reconstruction of the autofluorescent signal. (o) The isosurface has been rendered transparent, enabling the 3D
shapes of the antibody staining patterns to be observed within the embryo (blue, HNF3b expression; green, neurofilament). The high autofluor-
escence of blood in the heart has been rendered in red. Figures 28a–c courtesy of the Duke Center for In Vivo Microscopy, supported by NIH/
NCRR P41RR005959 and NCI U24 CA092656. Figures 28e–l reprinted according to the Creative Commons Attribution License; 2006. Johnson
et al. Virtual histology of transgenic mouse embryos for high-throughput phenotyping. PLoS Genet. 2006;2:e61. Figure 28m courtesy of Dr. T. J.
Mohun, supported by the Medical Research Council through the MRC National Institute for Medical Research. Figures 28n–o reprinted from
Sharpe J, Ahlgren U, Perry P, et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science.
2002;296:541-545. Reprinted with permission from AAAS.
82
with added fluorescein dyes, which enhance the captured digital
image (Fig. 28m).51,52
Optical Projection Tomography
With regard to 3D imaging of the mouse embryo, Sharpe,42 the
inventor of OPT, which is based on the ability of light to be
transmitted through the sample, describes the technique as fill-
ing the gap between confocal microscopy and mMRI. OPT is
the optical equivalent of mCT, which scans and records a quan-
titative shadow of the object, whereas OPT uses image-forming
optics to create a focused image on a CCD camera chip. Digital
images can be acquired from specimens up to 15 mm thick
(embryos <E10.5) with a resolution of 5–10 mm. Like EFIC,
OPT offers the advantage of visualizing RNA and protein
expression in an intact embryo; however, OPT allows for the
visualization of molecular markers in a 3D rendered image
(Fig. 28n-o).42
Although attempts have been made to render conventional
2D histological mouse embryo specimens into 3D structures
by aligning numerous digital images of serial cut, HE-stained
sections, the results cannot compare with more sophisticated,
3D imaging modalities. Whereas 2D histological analysis is the
only method to ascertain high-resolution images at the cellular
level, this level of detail is not necessarily required for the iden-
tification of anatomical development defects. Tables summar-
izing the advantages and limitations of the 3D modalities for
the detection of a morphological phenotype in a developing
mouse embryo are available for review.10,22
As described in this article, postimplantation evaluation of
the mouse embryo can be challenging. Using the described
basic pathology methods, along with provided histological
atlas references and possible 3D imaging techniques, the
pathologist can determine embryonic lethality with confidence
and assist other biomedical researchers using genetically engi-
neered mice.
Summary
We have provided information on pathology methods and pro-
cedures that may be used for evaluation of the causes of mouse
developmental defects and embryonic lethality due to induced
gene manipulation. Determination of gene expression in tissues
and cells and optimal necropsy, histopathological, and imaging
methods will make such evaluations much easier. Resources
such as Web sites, books, and other publications can also con-
tribute. Collaboration between pathologists, developmental
biologists, and other scientists is important to determine the
causes of embryonic developmental defects and lethality.
Acknowledgements
We are grateful to Dr Deborah Gillette and Beth Mahler for photoedit-
ing, Dr Piper Treuting for the whole pregnant uterus slides, and
Deborah Devor-Henneman for previous assistance with mouse embry-
ology. We would also like to thank the journal reviewers who contrib-
uted to the editing of this paper.
Veterinary Pathology 49(1)
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: This work
was supported, in part, by the Intramural Research Program of the
National Institute of Environmental Health Sciences, National Insti-
tutes of Health.
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