An Atlas for Staging Mammalian and Chick Embryos 1st

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 ACKNOWLEDGMENTS

We wish to thank Irene Partridge and Brenda Peters for typing the manuscript and
Osman Kademoglu for the photographic work.
Embryos of different species pass through identical embryonic stages before acquiring their specific features
(modified from Haeckel, E., Anthropogenic ou Histoire de L'Evolution Humaine, C. Reinwald et Cie,
Paris, 1877.)
 TABLE OF CONTENTS

Introduction

I. Man 1

II. Baboon 31

III. Rhesus Monkey 53

IV. Common Marmoset 81

V. Lesser Galago 83

VI. Mouse and Rat 89

VII. Chinese Hamster and Golden Hamster 103

VIII. Guinea Pig 117

IX. Rabbit 131

X. Sheep 145

XI. Pig 155

XII. Tree Shrew 169

XIII. Chicken 171

Correlation Graphs 187

References 205

Index 207
 INTRODUCTION

During experimental or observational investigations of mammalian development it

is essential to be able to:

1. define a sequence of developmental stages for a given species which can be used
to identify the stage of development of individual specimens.
2. utilize these developmental stages when comparing embryos of different species,
since embryos of the same length or age are frequently at very different stages of
development.

Embryos have been placed in a sequential order of development by measuring their

length. Mall' arranged 266 human embryos, ranging from 2 to 25 mm in length into
14 stages according to their mean lengths. According to O'Rahilly2 the most useful
single measurement is " . . . the greatest length of the embryo as measured in a
straight line (that is, caliper length) without any attempt to straighten up the natural
curvature of the specimen" (Figure 1). Early embryos are straight but as development
proceeds they develop an increasing ventral curvature forming a flattened spiral since
the tail lies to one or other side of the head. Thus the greatest length, as defined by
O'Rahilly,2 becomes the crown rump length (C.R. length) which is shorter than the
true greatest length (Figure 1). A slight change in the ventral curvature of the embryos
will cause a considerable change in the C.R. length. The ventral curvature of the em-
bryo is greatest during the period of organogenesis, a period of critical importance in
teratological studies, and differences in ventral curvature are undoubtedly one reason
for the variation in the C.R. length at any given stage in this period (Figure 2). During
the fetal period the fetus begins to straighten up and the ventral curvature almost
completely disappears (Figure 1). The C.R. length is now referred to as the sitting
height and it becomes a more reliable indication of age. It has become customary to
measure the length of embryos after fixation since human embryos obtained in the
operating room or embryos of wild animals were fixed immediately before being trans-
ported to the appropriate laboratory for examination. For purposes of standardiza-
tion, Streeter' recommended measurements to be made after 2 weeks in 10% formalin.
It should be noted that the degree of shrinkage or swelling varies considerably from
one fixative to another. The length of embryos at equivalent stages of development
shows considerable species variation; e.g., the embryos of man, lesser galago, and
mouse at the Carnegie stage 22 of development have a C.R. length of 25.0, 12.0, and
11 to 12.0 mm, respectively. Thus it is clear that length alone is a misleading and
unreliable way of classifying embryos, and such expressions as " . . . at the 18.0 mm
stage" should never be used. However, the length of an embryo should be accurately
recorded after fixation, since it acts as a pointer to its appropriate developmental stage.
Theoretically, the age of an embryo dated from a known time of ovulation or an
estimated time of fertilization should be the most reliable method of staging embryos
but in practice there are many pitfalls. Firstly, embryos develop at variable rates after
fertilization, and the stated days of gestation must be regarded as modal and not inclu-
sive ranges.' In humans the timing of gestation is further complicated by the unrelia-
bility of data on the menstrual cycle and the time of coition and particularly by the
lack of obvious and overt signs indicating ovulation. O'Rahilly2 has drawn up what
may be regarded as the most accurate table of the age of human embryos. The aging
of embryos of laboratory animals is more accurate since the female usually shows clear
indications of impending ovulation. Animals may be, therefore, paired at the appro-
priate time for a specific length of time or until copulation is observed. Then a vaginal
ii An Atlas for Staging Mammalian and Chick Embryos

A B C

FIGURE 1. Sketches illustrating methods of measuring the length of

embryos. A = greatest length. B and C = crown rump length. Note how
the increasing ventral curvature of the embryo decreases the greatest
length as defined by O'Rahilly.2

smear can be examined for spermatozoa or in some species, e.g., rat and mouse, a
vaginal plug is observed. However, as Juurlink and Fedoroff showed,' there may be
considerable variation in the stage of development reached by embryos at any partic-
ular day of gestation (Figure 3). This variation was found to be an intralitter as well as
interlitter phenomenon. The reasons for these differences are not clear, but differences
in time of implantation and individual rates of development are probably involved.
However, it is clear that gestational age is not an entirely reliable indication of the
stage of development reached by an individual embryo. Another source of error is the
manner in which the time of mating is used to estimate the commencement of embry-
onic development. Some authors call the day on which sperm or vaginal plug are found
day zero of gestation, others, day one of gestation. In this work the day on which
sperm or a vaginal plug is found will be regarded as day one of gestation.
In mammals it is customary to divide gestation into three main periods:

1. The period culminating in implantation of the blastocyst and during which the
fetal membranes are established and the germ layers are laid down in the embry-
onic disc.
2. The embryonic period during which there is rapid growth and differentiation
resulting in the laying down of all the main systems and organs of the body, and
the major features of external body form are established.
3. The fetal period which terminates at birth. This is a period of rapid increase in
size associated with quite slow changes in body form. It is also a period of histo-
logical differentiation and the onset of function.

It is to be remembered that development is a continuous process and that these divi-

sions of gestation are arbitrary.

Embryonic Staging
It has long been realized that there is a need for defining standardized stages in
embryonic development of various organisms for the purpose of accurate description
 111

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ESTIMATED OVULATION AGE — DAYS

FIGURE 2. Graphic plot of specimens in the Carnegie Embryological

Collection which have been surveyed and assigned to horizons XI to
XXIII, i.e., Carnegie Stages 11 to 23 under the new terminology. Note
the considerable spread in length at all stages but particularly in the older
stages. (From Streeter, G. L., Contrib. Embryo]. Carneg. Inst., 34, 165,
1951. Carnegie Institution of Washington, Department of Embryology,
Davis Division. With permission.)

of normal development and to enable accurate comparisons to be made between de-

velopment in different species. Such stages are arbitrarily defined segments of the con-
tinuous process of development. O'Rahilly2 defines embryonic stages thus: "Stages are
based on the apparent morphological state of development, and hence are not directly
dependent on either chronological age or on size. Furthermore, comparison is made of
a number of features of each specimen, so that individual differences are rendered less
significant and a certain latitude of variation is taken into account."
iv An Atlas for Staging Mammalian and Chick Embryos

80

63%
60

10 11 12 13 14
Developmental Stage

FIGURE 3. Proportion of mouse embryos at various developmental

stages on day 8 of gestation. No. of litters = 13, no. of embryos = 135.
Note, in this graph the morning of the vaginal plug is considered day 0 of
gestation. (Modified from Juurlink, B. H. J. and Fedoroff, S., In Vitro,
15, 86, 1980.)

Although embryonic staging was introduced towards the end of the nineteenth cen-
tury, there is no doubt that G. L. Streeter's "Developmental Horizons in Human
Embryos"6 i° forms the basic guideline for staging mammalian embryos. He divided
human embryonic development into 23 stages or developmental horizons. He only
considered horizons XI to XXIII since there was, at that time, insufficient material
available for the first ten horizons. The onset of marrow formation in the humerus is
rapid and easily observed and was "arbitrarily adopted as the conclusion of the embry-
onic and the beginning of the fetal period of prenatal life: It occurs in specimens about
30 mm in length".9 The transition from embryo to fetus is also defined as the time of
closure of the secondary palate.' Horizon X was described by Heuser and Corner in
1957." The earlier horizons were described by O'Rahilly2 who introduced a new no-
menclature and re-assessed the age of the embryos in the light of more recent investi-
gations. He replaced the term "horizon" by "stage" because the latter is the simple
term employed for all other vertebrate embryos and had been applied to human em-
bryos as early as 1914 by Mall.' He concludes: "The term is simpler, clearer, of wide-
spread usage, and can be employed as a verb (to stage an embryo) as well as a partici-
pial adjective (a staging system)". Furthermore, it should be pointed out that such
expressions as "at the 3 mm stage", should be replaced by "at 3 mm". In other words,
the length of an embryo is a single criterion that is not in itself sufficient to establish a
"stage". Other changes include replacement of Roman by Arabic numbers and elimi-
nation of the scientifically meaningless term "ovum" which was so frequently applied
to pre-implantation stages. Thus, now the embryonic development of the human is
divided into Carnegie stages 1 to 23.
On the basis of somite number and internal characters, it is possible to arrange chick
embryos into Carnegie stages 9 to 23. These stages can be recognized on the basis of
their length, external characters, and incubation age. Thus it is now possible to com-
pare mammalian and chick embryos at equivalent stages of development.
This atlas of mammalian and chick embryos provides a means of staging them dur-
ing the period of organogenesis, i.e., from the appearance of the somites to the tran-
 V

Table 1
DIAGNOSTIC FEATURES OF MAMMALIAN EMBRYOS — FROM CARNEGIE
STAGES 9 TO 23

Carnegie Features
stage

9 1 to 3 pairs of somites; open neural plate

10 4 to 12 pairs of somites; neural folds begin to fuse; 2 branchial bars; otic placode invagination
11 13 to 20 pairs of somites; cranial neuropore closes
12 21 to 29 pairs of somites; caudal neuropore closes; C-shaped embryo; 3 branchial bars; begin-
ning cranial limb buds
13 4 limb buds present; maxillary and mandibular processes; closed otocyst; flat olfactory plac-
ode; tail bud
14 Cervical flexure; Nackengrube; open lens pit; definitive tail
15 Forelimb footplate; nasal pit; closed lens vesicle; early auricular hillocks; umbilical hernia
16 Retinal pigment; hindlimb footplate; nasal pits face ventrally
17 Finger rays; 6 distinct auricular hillocks; nasolacrimal groove
18 Notched handplate; elbow; toe rays; beginning eyelids; vibrissae; mammary buds
19 Early notched footplate; limbs extend nearly straight forward; head begins to be raised beyond
a right angle; continued fusion of auricular hillocks
20 Arms increased in length and bent at elbow; forelimb digits separating; hindlimb digits visible;
eyebrow follicles
21 Forelimb digits longer and have touch pads; feet approach each other; helix and antihelix
22 Thickened eyelids encroaching on eyeballs; tragus and antitragus
23 Fusion of secondary palate; limbs longer and more developed; all digits completely separated;
eyelids cover most of eye; definitive shape of auricle; hair follicles on body

sition stage from embryo to fetus. The standard used is Carnegie stages 9 to 23 of
human embryos, and all other embryos are, wherever possible, compared to these.
Ideally, it is necessary to analyze the following data for each embryo under considera-
tion:

1. Greatest length as defined by O'Rahilly2

2. Known or estimated postovulatory age
3. Description of external features
4. Serial sections of the embryo are examined to establish the stage of development
of the various diagnostic organs.

The diagnostic organs vary from stage to stage of development and are fully described
for human embryos by Streeter6 1° and O'Rahilly.2
This degree of examination is, however, only required when staging embryos of any
particular species for the first time. For the purpose of everyday laboratory identifi-
cation of embryonic stages, only greatest length, known or estimated age, and external
features will be used. Tables 1 and 2 list the diagnostic features of mammalian and
chick embryos.
vi An Atlas for Staging Mammalian and Chick Embryos

Table 2
DIAGNOSTIC FEATURES OF CHICK EMBRYOS — FROM CARNEGIE
STAGES 9 TO 23

Carnegie Features
stage

9 1 to 3 pairs of somites; open neural plate

10 4 to 12 pairs of somites; neural folds begin to fuse; otic placode; closure of caudal neuropore
11 13 to 20 pairs of somites; 2 branchial bars; otic pits; cranial neuropore closed
12 21 to 29 pairs of somites; closure of caudal neuropore; 3 branchial bars; olfactory placode;
distinct cranial limb buds
13 4 limb buds present; tail bud; otocyst closed
14 35 to 39 pairs of somites; lens vesicle closes; maxillary process; deep olfactory pit
15 Increased length of limb buds; very prominent maxillary process; retinal pigment appearing
16 Toe plate on leg bud; 6 distinct auricular hillocks; cervical sinus indistinct; umbilical hernia
17 Leg bud longer than wing bud; elbow and knee present; toe rays beginning in foot plate; most
ventral auricular hillock forms the "collar"
18 Distinct toe rays; the "collar" is more distinct, but remaining auricular hillocks are beginning
to fuse; distinct beak by end of stage
19 Leg still markedly longer than wing; both have distinct digital rays; beak more prominent and
egg tooth appearing; distinct eyelids; increased length of neck between "collar" and mandi-
ble; other auricular hillocks flattened and fusing; never more than 2 scleral papillae; feather
germs
20 All digits delineated and considerably lengthened; anterior tip of mandible has reached beak;
"collar" disappearing; up to 8 scleral papillae; more feather germs
21 Toes separating and first digit of wing distinct; webs between digits; 9 to 14 scleral papillae and
circle complete; nictitating membrane well developed; more feather germs
22 Fingers and toes more separated; webs disappearing; all feather germs more conspicuous; eye-
lids forming
23 Secondary palate fused; eyelids beginning to close; toes completely separated and claws ap-
pearing
 1

Table 3
CARNEGIE STAGES 1 TO 8 OF HUMAN EMBRYOS

Carnegie Age
stage (days) Features

1 1 Fertilization
2 2-3 From 2 to about 16 cells
3 4-5 Free blastocyst
4 5-6 Attaching blastocyst
5 7-12 Implanted though previllous
6 13-15 Chorionic villi; primitive streak may appear
7 15-17 Notochordal process
8 17-19 Primitive pit; notochordal and neurenteric canals

Based on O'Rahilly, R., Developmental stages in human embryos, including

a survey of the Carnegie collections. Part A. Embryos of the first three
weeks. (Stages 1 to 9), Carnegie Institute of Washington, Washington, D.C.,
1973.

I. MAN

Carnegie Stages of Human Embryos [Homo sapiens]

The 23 Carnegie stages of human embryos will be used as the reference base in this
atlas and will be presented in two ways:

1. The presomite stages (Carnegie stages 1 to 8) are listed (Table 3) but not illus-
trated because: (a) teratological research is rarely, if every carried out during this
period; (b) staging requires microscopic examination; and (c) staging is largely
dependent upon details of implantation which are not comparable with those of
other species.
2. Stages 9 to 23 cover the all-important period of embryogenesis. These stages will
be illustrated and a brief account of the internal features diagnostic of each stage
will be given. The criteria used to identify a particular stage are: (a) greatest
length, (b) known or estimated age, and (c) external features. In general, the
greatest length and external features will be the most reliable criteria.
 2 An Atlas for Staging Mammalian and Chick Embryos

A B

Tr ogeminal area

Prose/wet*

aural groove
1•t Pharyng.
POuch
Mesenceph
Hyoid area
01 ic disc Rhombenceph
Yolk sac

SOT teS

Plano
of
Amnion
(cut edg.)

Primitive node

Primitive streak

Umbdlcal as

Connecting
AI lantoic diver Woken
stalk

p1 0 Sew

FIGURE 4. (A) Left lateral and dorsal views of a reconstruction of Carnegie embryo No. 1878. (B) Left lateral
view of the same embryo showing ectodermal areas of head region (stippled) and the mesencephalic flexure.
(From O'Rahilly, R., Developmental stages in human embryos, including a survey of the Carnegie collections.
Part A. Embryos of the first three weeks (Stages 1 to 9), Carnegie Institute of Washington, D.C., 1973. With
permission.)

Man. Carnegie Stage 9

Embryos of stage 9 vary from approximately 1.5 to 2.5 mm in length and have a
postovulatory age of 20 days. The characteristic feature of this stage is the appearance
of from one to three pairs of somites. As seen from the dorsal aspect, the embryo is
frequently described as having the shape of the sole of a shoe. Many embryos have a
dorsal concavity or "lordosis". Very abrupt kinks are probably abnormal. The neural
groove is now quite deep but open along its complete length. The cranial half of the
neural groove represents the future brain. The elevated cranial ends of the neural folds
are separated by a terminal notch which leads to the buccopharyngeal membrane. In
more advanced specimens the cranial (or mesencephalic) flexure appears at the mid-
brain. The otic disc (or plate) appears during this phase, but the optic primordia are
not visible. A primitive groove may be found but a distinct node is not always recog-
nizable. The yolk sac has numerous blood islands and a vitelline plexus is visible.
 3

Optic area
Prosencephalon
Optic groove
Mesencephalon
Tngeminal prominence
Branchial arch 1
Branchial groove 1 Rhombencephalon
Region of
bronchial arch 2
Otic placode Preotic sulcus
and prominence
Head fold
Pericardial sac
Postotic sulcus
Rostra! neuropore Somite 1 loccipitall
under a thin layer of ectoderm
Amnion
(cut edgel
Neural tube under a
thin layer of ectoderm

Yolk sac

Lateral
body fold

Somite 10 IC 6)

Caudal Neural fold

neuropore

Connecting 0 5 mm
stalk

Tail fold

FIGURE 5. Dorsal view of a 10 somite embryo. (From Gasser, R. F., Atlas of Human Embryos,
Harper and Row, Maryland, 1975. With permission.)

Man. Carnegie Stage 10

External Features
Embryos of stage 10 vary from 2.0 to 3.5 mm in length and have a postovulatory
age of 22 days. They have from 4 to 12 pairs of somites. This stage is marked by the
commencement of the fusion of the neural folds. It begins in embryos with 6 to 7 pairs
of somites and by the end of the period extends from the otic region to the level of the
latest formed somite. The otic disc is beginning to invaginate. There is considerable
elongation of the embryo and expansion of the yolk sac. The mandibular and hyoid
bars begin to be visible externally. The head and tail folds appear. Towards the end of
this period the increasing size of the heart makes the pericardial region a prominent
feature of the external form.

Internal Features
The heart endothelium is enclosed in a jelly-like envelope which in turn is enclosed
in a layer of contractile tissue, the primordium of the myocardium. All the blood
vessels consist only of simple endothelium and contain very few blood cells. The optic
evaginations and the primordia of the corpus striatum, thalamus, and tegmentum are
present. The parts of the neural crest are emerging. Pronephric rudiments appear at 8
somites, and the first definitive mesonephric vesicle at 10 somites. There is a solid
mesonephric duct which extends for a varying distance caudal to somite ten.
4 An Atlas for Staging Mammalian and Chick Embryos

FIGURE 6. (A) Dorsal view of a 13-somite embryo (Carnegie embryo

No. 6344). (B) Left lateral view of the same embryo. (C) Left lateral view
of a 19-somite embryo (Carnegie embryo No. 6050) to show the dorsal
concavity or "lordosis" so frequently seen at this stage (arrow). A = cra-
nial neuro pore; B = yolk sac; C = body stalk. (From Streeter, G. L.,
Contrib. Embryo]. Carneg. Inst., 30, 211, 1942. Carnegie Institution of
Washington, Department of Embryology, Davis Division. With permis-
sion.)