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Histological and ultrastructural observations of tail bud formation in the chick embryo.

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Histological and Ultrastructural Observations of Tail Bud
Formation in the Chick Embryo
GARY C . SCHOENWOLF
Department of Anatomy, The Uniuersity of New Mexico School
Albuquerque, New Mexico 87131
of
Medicine,
ABSTRACT
Tail bud formation was studied in chick embryos by light and
electron microscopy. The caudal part of t h e neural groove at stage 11 is flanked
by widely separated neural folds and merges posteriorly with t h e shallow primitive groove. The neural groove and primitive streak partially overlap. The
depth of t h e neural groove gradually decreases antero-posteriorly within this
overlap zone while the dorso-ventral thickness of t h e streak progressively increases. The anterior end of t h e streak begins to form a spherical accumulation
of mesenchymal cells, t h e incipient tail bud, concomitant with closure of t h e
posterior neuropore. Formation of t h e posterior body fold results in consolidation of t h e remainder of t h e streak into t h e definitive tail bud. The overlap zone
between neural groove and primitive streak is retained as the tail bud forms.
Thus t h e posterior end of t h e neural tube and anterior end of t h e tail bud overlap. The latter undergoes cavitation to form t h e ventral part of t h e spinal cord
within this overlap region.
The tail bud is initially continuous with a n overlying, flattened layer of ectoderm and a n underlying, columnar layer of endoderm. A bilaminar ectodermal
epithelium forms directly above t h e developing neural tube as t h e dorsal portion of the tail bud undergoes cavitation. Most of t h e endodermal cells a r e displaced from t h e ventral surface of t h e tail bud by t h e posterior body fold and
condensed into a disk-shaped region which ultimately gives rise to tail gut.
The tail bud first forms in chick embryos a t
about Hamburger-Hamilton ('51) stages 13-14
and consists of a dense mass of mesenchymal
cells positioned a t t h e posterior end of the developing body. It gives rise to a considerable
portion of t h e embryo, contributing cells to
most of t h e major tail structures Ke., neural
tube, somites, and caudal arteries, but not notochord or tail gut) as well as some lumbosacral structures (Criley, '69; Schoenwolf,
'77, '78a). Mechanisms involved in tail bud
formation are incompletely understood. Light
microscopic studies indicate t h a t t h e tail bud
gradually derives from persisting remnants
of t h e primitive streak (Holmdahl, '25); specifically from only the anterior portion of
t h e streak since t h e posterior part becomes
widely dispersed during extraembryonic mesoderm formation a t early gastrulation stages
(Spratt, '47). However, t h e precise mechanisms involved in transformation of these
remnants into the tail bud have not been anaANAT. REC.
(1979) 193: 131-148.
lyzed. Holmdahl merely noted over 50 years
ago t h a t three major events appeared to be associated with tail bud formation: dorso-ventral thickening of a midline portion of t h e posterior blastoderm, formation of t h e posterior
body fold, and separation of t h e developing
tail bud from t h e overlying surface ectoderm.
The purpose of this investigation is to describe t h e specific morphological events involved in tail bud formation in the chick embryo, utilizing light microscopy of 1 - p m
plastic sections and scanning and transmission electron microscopy. The resolution provided by these techniques is far superior to
t h a t previously obtained by light microscopy
of whole embryos and paraffin sections. Furthermore, t h e resolution and depth of field
provided by scanning electron microscopy is
Received Mar. 7, '78. Accepted Aug. 11, '78.
' This paper is dedicated to the memory of the late Dr. Robert M.
Sweeoey, my friend, teacher, advisor, and colleague, who introduced
me to the excitement of embryogenesis.
131
132
GARY C. SCHOENWOLF
almost ideal for such a study since i t allows
detailed observations of changes in cellular topography and arrangement over a relatively
large field of view. The observations reported
here are a n advance over our previous knowledge and provide a morphological foundation
for future experimental analyses of tail bud
formation.
MATERIALS AND METHODS
Several dozen fertile White Leghorn eggs
were incubated in a forced-draft incubator at
38°C until embryos attained stages 11-20.
Eggs were opened into finger bowls containing
0.9%saline, and blastoderms were quickly cut
away from t h e yolk and washed with saline.
Processing for scanning electron
microscopy
Blastoderms were fixed for three hours at
room temperature with either 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2
(Sabatini et al., '63) or with a mixture containing a final concentration of 2% formaldehyde-2.5% glutaraldehyde (with or without
0.01% trinitrophenol) in 0.1 M cacodylate
buffer at pH 7.4 (Karnovsky, '65; Ito and Karnovsky, '68). No significant differences in cellular morphology were observed as a result of
these variations in fixation procedure. Embryos were dissected free from surrounding
tissues following primary fixation, pooled
according to stage, washed in several changes
of buffer, postfixed for one hour with buffered
1%osmium tetroxide, and dehydrated with
ethanol.
Intact embryos were critical point dried
from liquid C 0 2following dehydration in order
to examine external surface morphology. Surfaces of deeper cells were exposed prior to critical point drying either by transection with
razor blades during dehydration or by ethanolic cryofracture as described by Humphreys
et al. ('74). Fracture planes produced by transection with razor blades tended to pass between cells exposing their surfaces (plate 2).
Fracture planes obtained by cryofracture
usually passed through cells creating relatively flat fracture faces (fig. 14). All dried
embryos were affixed to aluminum stubs with
double-stick Scotch tape and silver paint,
coated with gold-palladium in a Hummer I
sputter coater, and examined with a n ETEC
Autoscan scanning electron microscope operated a t 10-20 kV.
Processing for light microscopy and
transmission electron
microscopy
Following brief washes with 0.9% saline,
blastoderms were fixed with either 2.0% glutaraldehyde in 0.1 M cacodylate buffer (pH
7.2) a t room temperature for two hours
(Sabatini et al., '63) or with a mixture containing a final concentration of 2%glutaraldehyde-1%osmium tetroxide in 0.1 M cacodylate
buffer (pH 7.2) on ice for one hour (Hasty and
Hay, '77). Slightly better overall preservation
of ultrastructure was obtained with t h e latter
fixative. Embryos were then dissected away
from surrounding tissues, pooled, washed in
buffer containing 0.1 M sucrose, postfixed with
buffered 1%osmium tetroxide (for one hour if
not previously exposed to osmium; for one-half
hour if previously fixed with osmium), dehydrated with ethanol, transferred t o propylene
oxide, and embedded in Epon 812 (Luft, '61).
Thick sections (1 pm) cut with glass knives on
a Sorvall Porter-Blum ultramicrotome were
mounted on slides and stained with methylene
blue-azure I1 (Richardson e t al., '60).Thin sections were cut with diamond knives, collected
on copper grids, stained with uranyl acetate
followed by lead citrate (Reynolds, '63), and
examined with a Hitachi HS-7S transmission
electron microscope a t 50 kV.
RESULTS
Changes i n the surface o f the blastoderm
during tail bud formation
A neural tube is present throughout t h e
length of the anterior two-thirds of the embryo at stage 11, and a neural groove occupies
the posterior one-third (fig. A). The posterior
end of t h e neural groove is flanked by widely
separated neural folds and merges indistinctly
with the shallow primitive groove (fig. 1). The
primitive groove and ridges are t h e only components of t h e primitive streak grossly visible
at this stage. The neural folds progressively
contact one another in t h e dorsal midline to
close the posterior neuropore at about stages
12-13 (fig. 2). The primitive groove begins to
disappear as t h e posterior neuropore closes
and t h e anterior end of the primitive streak
enlarges to form a spherical accumulation of
cells continuous with t h e posterior end of t h e
neural tube. This mass of mesenchymal cells
is the rudiment of t h e tail bud (fig. 2). Although the tail bud is well formed by stage 13
MORPHOLOGY OF CHICK TAIL BUD FORMATION
or 14, i t is still continuous posteriorly with a
persisting portion of t h e primitive streak
(fig. 3).
The posterior body fold usually first appears
during stages 13-14 as bilateral depressions,
one on either side of t h e short primitive streak
(fig. 3). Cells of t h e primitive streak are progressively displaced antero-ventrad as this
fold forms and deepens in the midline, and are
eventually incorporated into t h e developing
tail bud. The posterior end of t h e embryo is
clearly demarcated from t h e blastoderm as
early as stages 15-16 and the entire primitive
streak is consolidated into the tail bud (fig. 4).
During subsequent development the leg
buds become well defined and t h e tail undergoes considerable elongation (figs. 5, 6). The
tail bud occupies t h e tip of the lengthening
tail and continues to contribute cells to tail
structures throughout all stages examined.
Structure of the posterior blastoderm
j u s t prior to tail bud formation
Mesodermal structures are well developed
near t h e anterior end of the neural groove a t
stages 11-12 and clearly separated from one
another and from t h e ectoderm and endoderm
by spaces containing a fibrous material (fig.
7). Mesodermal structures are not well defined more posteriorly, however (fig. 8 ) . The
most posterior levels of the notochord are represented by a poorly defined, midline accumulation of mesenchymal cells intermingled
with the cells lining the neural groove. Paraxial mesoderm is not yet organized into discrete
segmental plates a t this level and,boundaries
are not obvious between paraxial mesoderm
and prospective notochord.
The neural groove and primitive streak partially overlap at more posterior levels (figs. 911) and the depth of t h e neural groove progressively decreases antero-posteriorly within
this overlap zone while t h e thickness of t h e
primitive streak progressively increases (cf.
figs. 9, 11). The cells lining t h e neural groove
are elongated and their apices are interconnected around their entire circumference (fig.
10). Their basal ends are intercalated with
cells of t h e primitive streak and exhibit n u merous thin cellular protrusions. Cells of t h e
primitive streak, by contrast, have a stellate
configuration within t h e overlap zone (fig.
11).
The primitive streak extends through t h e
entire thickness of the blastoderm at levels
133
posterior to t h e neural groove and cells of t h e
streak merge with cells of a well defined epiblast dorso-laterally (fig. 12). The uppermost
cells of t h e primitive streak are elongated dorso-ventrally while t h e deeper cells have a
characteristic mesenchymal shape and a r rangement. Prospective mesodermal cells are
still involuting through the streak at stages
11-12.
Structure of the definitive tail bud
The tail bud is clearly demarcated from the
blastoderm posteriorly as early as stage 15
(fig. C). Its anterior end tapers beneath t h e
posterior part of the developing neural tube
forming a compact wedge of cells. A short
overlap zone is thus present between the posterior portion of t h e neural tube and t h e anterior part of the tail bud (fig. 13). The dorsoventral thickness of the tail bud increases and
t h e diameter of the neural tube decreases
antero-posteriorly within this overlap zone.
No distinct boundaries are present between
neural tube and tail bud within this area;
rather, the cells of these two structures freely
interdigitate. The short overlap zone present
between developing neural tube and tail bud
at stage 15 is derived from t h e similar region
of overlap between neural groove and primitive streak present a t stages 11-12 (fig. 9).
The definitive tail bud occupies the entire
thickness of t h e blastoderm a t levels posterior
to t h e developing neural tube (fig. 14). Its cells
a r e contiguous with cells of the surface ectoderm dorsally, paraxial mesoderm laterally,
endoderm ventrally, and primitive streak posteriorly. The surface ectoderm covering the
paraxial mesoderm is organized into a bilaminar columnar epithelium (fig. 15) which
is underlain by a distinct basal lamina. A fibrous material, primarily extracellular as determined by transmission electron microscopy, is frequently observed in the cleft between
ectoderm and paraxial mesoderm. This material is particularly well illustrated in scanning
electron micrographs (fig. 16). Cells of the surface ectoderm and tail bud merge medially,
and as these two areas become confluent t h e
intervening basal lamina on each side fragments and disappears (fig. 151, and t h e ectoderm forms a squamous, single-layered sheet
covering t h e tail bud. Cells of t h e endoderm
and tail bud are likewise confluent (fig. 17). A
distinct double-layered endodermal epithelium is present beneath the paraxial meso-
134
GARY C . SCHOENWOLF
derm, however, and a well defined basal lamina lies adjacent to t h e deep cells of this epithelium (fig. 18).
Separation o f ectoderm and endoderm
f r o m tail bud
Examination of sections cut exactly through
the midsagittal plane reveals the progressive
separation of ectoderm and tail bud. The posterior neuropore is closed by stage 15 and t h e
dorsal portion of the tail bud is undergoing
cavitation to form posterior neural tube levels
(fig. 19). Separation of ectoderm from tail bud
is spatially correlated with t h e process of cavitation. The ectoderm overlying the area of
cavitation forms a distinct bilaminar epithelium (figs. 19,20) with a subjacent basal lamina. The outer squamous layer of this epithelium is present more posteriorly where it constitutes the dorsal covering of the tail bud
(figs. 20, 21). Hence separation of ectoderm
from tail bud primarily involves formation of
a deep (inner) layer of ectodermal cells which
are clearly demarcated from the prospective
neural ectodermal cells of the tail bud.
The endoderm underlying t h e area of the developing notochord likewise forms a distinct
epithelium which tends to be double layered.
Formation of this epithelium is spatially correlated with differentiation G f the ventral surface of the notochord (figs. 19, 22). A single
layer of endodermal cells extends posteriorly
covering the ventral surface of the tail bud
(figs. 22, 23); these cells tend to be columnar
rather than squamous (fig. 23).
Several changes occur in t h e region of the
tail bud due to formation and deepening of t h e
posterior body fold. Most of t h e cells of the
primitive streak are consolidated into the tail
bud by this fold as early as stage 15, and several of the last streak cells to become incorporated contain darkly stained particles (fig. 19).
The number of cells containing such particles
varies considerably from one embryo to
another. By following t h e relative positions of
cells containing these particles during stages
15-20i t can be determined t h a t t h e last streak
cells to become incorporated into t h e tail bud
are displaced antero-ventrad by t h e posterior
body fold (figs. 19, 24, 25, 27). Therefore,
prospective tail bud cells located most posteriorly (i.e., within t h e postero-most remnants
of t h e primitive s t r e a k ) a r e ultimately
brought forward to form t h e antero-ventral
portion of the tail bud.
The surface ectoderm initially covering the
dorsal surface of the tail bud (fig. 21) is progressively wrapped around the latter by the
posterior body fold, such t h a t the postero-ventral aspects of t h e tail bud become clearly
defined (figs. 19, 24, 2 5 ) . Formation of a
bilaminar ectodermal epithelium has progressed only as far posteriorly as the process of
cavitation, however (fig. 26). Separation of ectoderm and tail bud is not yet completed even
as late as stage 20 (fig. 27).
The endoderm initially covering the ventral
surface of t h e tail bud (fig. 23) is condensed by
action of the posterior body fold into a concave
disk continuous with the ventral surface of
t h e developing notochord and with the anteroventral portion of the tail bud (fig. 25). The
tail gut grows out from this disk into the elongating tail during subsequent development
(fig. 27).
One further action of t h e posterior body fold
is notable. As i t deepens, t h e orientation of the
cloaca1 membrane anlage and allantois rudiment change (figs. 19, 24, 2 5 ) and, concomitantly, t h e hindgut is established.
DISCUSSION
This study describes t h e events involved in
tail bud formation in t h e chick embryo by high
resolution light and electron microscopy. The
observations made complement and enrich
previous data obtained by light microscopy of
whole embryos and paraffin sections, and t h e
three-dimensional information provided by
scanning electron microscopy greatly i n creases our comprehension of the series of
complex events involved in formation of t h e
tail bud.
The transition from primitive streak to tail
bud is gradual and t h e exact stage a t which
t h e latter forms is thus impossible to determine. This fact has resulted in a problem with
terminology since a variety of names have
been applied to identical cellular areas. For
example, t h e more ventral cells in t h e area of
overlap illustrated in figure 9 can be designated by many terms including primitive
streak, incipient tail bud, tail bud, or end bud.
It seems pointless to enter a semantic argument over the designation of these cells since
as readily observed in this study they merely
represent a slightly different morphological
manifestation of t h e primitive streak of earlier stages. By stages 13-14, however, a large
percentage of these cells have consolidated
MORPHOLOGY OF CHICK TAIL BUD FORMATION
into a discrete, bud-like mass which is probably best called a tail (or end) bud.
Role o f posterior body fold in
tail bud formation
The depth of field afforded by scanning electron microscopy is particularly useful for
studying the three-dimensional events involved in formation and expansion of t h e posterior body fold. I t should be emphasized, however, t h a t mechanisms by which this fold
forms can not be determined by a purely descriptive study. Experimentation is necessary
to determine whether formation of this fold is
due to posterior growth of t h e tail bud as suggested by Gruenwald ('41: his figs. 2-6) or due
to a n active undercutting of t h e tail bud by
forward growth of t h e posterior blastoderm.
This investigation has demonstrated t h a t
t h e posterior body fold is involved in tail bud
formation as previously suggested by Holmdahl ('25). The postero-most remnants of t h e
primitive streak are displaced antero-ventrad
by this fold and thus consolidated into t h e developing tail bud. The postero-ventral aspects
of t h e tail bud a r e likewise established at t h e
same time a s t h e surface ectoderm is progressively wrapped around t h e tail bud. The posterior body fold is therefore involved in repackaging t h e length of t h e slender primitive
streak into a somewhat spherical mass of
cells. Other factors, such as regression of Hensen's node, a r e probably also involved in consolidation of t h e linear streak into a discrete
tail bud, since t h e incipient tail bud forms a
distinct swelling even prior to formation of
t h e posterior body fold (fig. 2). It is possible,
however, t h a t increased mitotic activity and
not node regression is t h e primary factor involved in formation of this initial swelling.
Hence, experimentation is necessary to determine exactly what roles node regression and
cell division may play in formation of t h e tail
bud.
Formation o f the lumbosacral
overlap zone
An area of overlap between t h e posterior
end of the neural groove and t h e anterior end
of t h e primitive streak is described in t h e present study a t stages 11-12 (fig. 9). Although
this overlap region was previously illustrated
(Klika and Jelinek, '69) its significance in
spinal cord formation was not understood. The
present investigation has shown t h a t t h e over-
135
lap zone between these two structures is
retained as t h e tail bud forms resulting in a n
overlap zone between a portion of t h e neural
tube formed by closure of t h e neural groove
(i.e., by primary neurulation) and t h e anterior
end of t h e tail bud (fig. 13). The latter cavitates (i.e., undergoes secondary neurulation)
to form t h e ventral portion of t h e spinal cord
within t h e overlap region (Criley, '69; Jelinek
e t al., '69; Schoenwolf, '78b). This overlap zone
of primary and secondary neurulation is
located within the future lumbosacral region
as shown by extirpation experiments (Criley,
'69). The high incidence of lumbosacral
myeloschisis observed in chick embryos following various experimental procedures
(Criley, '67) may be related to the fact t h a t
this portion of t h e spinal cord forms by two
mechanisms, but t h e etiology of this defect is
unknown.
Separation of ectoderm and endoderm
from tail bud
The cells of t h e tail bud are initially confluent with a n overlying flattened layer of ectodermal cells (fig. 21). Formation of a distinct ectodermal epithelium in this region is
spatially correlated with tail bud cavitation.
The ectoderm overlying t h e area of cavitation
becomes bilaminar and a distinct basal lamina
appears subjacent to t h e deep cells of this developing epithelium. The spatial relationship
between formation of a n ectodermal epithelium and tail bud cavitation can be observed
only in sections cut exactly in t h e midsagittal
plane. In a preliminary report based on slightly parasagittal cryofractures i t was erroneously reported t h a t ectoderm completely separates from tail bud concomitant with formation of t h e posterior body fold (Schoenwolf and
Waterman, '78).
The inner layer of surface ectodermal cells
overlying t h e developing neural tube appears
in histological sections to be derived from cells
of t h e tail bud. The origin of these cells from
tail bud was not confirmed by previous radioautographic mapping studies (Schoenwolf,
'771, but this may be due to t h e fact t h a t so few
cells a r e actually contained within this portion of t h e epithelium and thus these cells
may go undetected.
Cells of t h e tail bud are also initially confluent with a n underlying layer of endodermal
cells (fig. 23). Most of t h e endodermal cells are
displaced from t h e ventral surface of t h e tail
136
GARY C. SCHOENWOLF
bud as t h e posterior body fold deepens (figs.
19, 24, 251, and condensed into a plaque-like
structure continuous with t h e antero-ventral
surface of t h e tail bud (fig. 25). During subsequent development this disk of endodermal
cells gives rise to tail gut. Mechanisms
involved in formation of tail gut and related
structures are currently under study.
ACKNOWLEDGMENTS
I wish to express my appreciation to Doctor
Robert E. Waterman for advice during t h e
course of this investigation and in preparation
of the manuscript, to Ms. Helen Eason for
superb technical assistance, and to Ms. Anita
Kimbrell and to my wife, Patricia, for assistance with typing.
LITERATURE CITED
Criley, B. B. 1967 Analysis of the Embryonic Sources
and Mechanisms of Development of Posterior Levels of
Chick Neural Tubes. Ph.D. thesis, University of Illinois,
ChampaigdUrbana, Illinois.
1969 Analysis of the embryonic sources and
mechanisms of development of posterior levels of chick
neural tubes. J. Morph., 128: 465-501.
Gruenwald, P. 1941 Normal and abnormal detachment
of body and gut from the blastoderm in the chick embryo,
with remarks on the early development of the allantois. J.
Morph., 69: 83.125.
Hamburger, V., and H. L. Hamilton 1951 A series of normal
stages in the development of the chick embryo. J. Morph.,
88: 49-92.
Hasty, D. L., and E. D. Hay 1977 Freeze-fracture studies of
the developing cell surface. Formation of particle-free
membrane blebs during glutaraldehyde fixation. J. Cell
Biol., 75: 234a.
Holmdahl, D. E. 1925 Die erste Entwicklung des Korpers bei den Vogeln und Saugetieren, inkl. dem
Menschen, besonders mit Rucksicht auf die Bildung des
Ruckenmarks, des Zoloms und der entodermalen Kloake
nebst einem Exkurs uber die Entstehung der Spina bifida
in der Lumbosakralregion. I. Gegenbaurs Morphol.
Jahrb., 54: 333-384.
Humphreys, W. J., B. 0. Spurlock and J. S. Johnson 1974
Critical point drying of ethanol-infiltrated, cryofractured
biological specimens for scanning electron microscopy.
SEMl1974: 275-282.
Ito, S., and M. J. Karnovsky 1968 Formaldehyde-glutaral.
dehyde fixatives containing trinitro compounds. J. Cell
Biol., 39: 168a-169a.
Jelinek, R., V. Seichert and E. Klika 1969 Mechanism of
morphogenesis of caudal neural tube in the chick embryo.
Folia Morphol. (Praha), 17: 355-367.
Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde
fixative of high osmolality for use in electron microscopy.
J. Cell Biol., 27: 137a-138a.
Klika, E., and R. Jelinek 1969 The structure of the end and
tail bud of the chick embryo. Folia Morphol. (Praha), 17:
29-40.
Luft, J. H. 1961 Improvements in epoxy resin embedding
methods. J. Biophys. Biochem. Cytol., 9: 409-415.
Reynolds, E. S. 1963 The use of lead citrate a t high pH as
an electron-opaque stain in electron microscopy. J. Cell
Biol., 17: 208-212.
Richardson, K. C., L. J a r e t t and E. H. Finke 1960 Embedding in epoxy resins for ultrathin sectioning in electron
microscopy. Stain Technol., 35: 313-323.
Sahatini, D. D., K. Bensch and R. J. Barrnett 1963 Cytochemistry and electron microscopy. The preservation of
cellular ultrastructure and enzymatic activity by
aldehyde fixation. J. Cell Biol., 17: 19-58.
Schoenwolf, G. C. 1977 Tail (end) bud contributions to
t h e posterior region of the chick embryo. J. Exp. Zool.,
201: 227-246.
1978a Effects of complete tail bud extirpation
on early development of the posterior region of the chick
embryo. Anat. Rec., 192: 289-296.
1978b An SEM study of posterior spinal cord development in t h e chick embryo. SEM/1978/Vol. ZI:
739-746.
Schoenwolf, G. C., and R. E. Waterman 1978 Tail bud formation in the chick embryo: A scanning electron microscopic study. Anat. Rec., 190: 533-534.
Spratt, N. T., J r . 1947 Regression and shortening of the
primitive streak in the explanted chick blastcderm. J .
Exp. Zool., 104: 69-100.
PLATES
A b breuiations
A, Allantois
AR, Allantois rudiment
C, Cavitating region
CM, Cloaca1 membrane anlage
D, Debris marking site of
posterior neuropore closure
E, Endoderm
EP, Epiblast
ITB, Incipient tail bud
LB, Leg bud
M, Mesoderm
N, Notochord
NF,
NG,
NT,
PG,
Neural fold
Neural groove
Neural tube
Prlmltive groove
PM, Paraxial mesoderm
P N , Prospective notochord
PR, Primitive ridge
PS. Primitive streak
SE, Surface ectoderm
SP, Segmental plate
TB, Tail bud
TG, Tail gut
PLATE 1
EXPLANATION OF FlGIlRES
1 Dorsal view of t h e posterior end of a stage 11 embryo (see fig. A for orientation).
The neural and primitive grooves a r e directly continuous. x 120.
2
Stage 12. The anterior end of t h e primitive streak begins t o form a spherical accumulation of cells a s the posterior neuropore closes. x 120.
3 Stage 14. The posterior body fold first appears as bilateral depressions (arrow:;), one
on each side of t h e primitive streak. X 120
4
Stage 15. The posterior end of t h e body including the tail bud is delineated from t h e
blastoderm by t h e posterior body fold (arrows). x 120.
5
Stage 17 The tail is elongating as t h e leg buds become well defined. x 120.
6 Stage 20. Ventral view of t h e posterior end of a stage 20 embryo. The tail bud is positioned a t t h e tip of t h e tail which is flexed ventrad toward t h e allantois. X 30.
Fig. A Light micrograph of a. dorsal view of a stage 11 chick embryo. Photograph
made from a commercially prepared “33-hour” chick embryo whole mount (Macmillan
Science Co., Inc., Chicago, Illinois). Arrowheads delineate approximate extent of a similar area shown in figure 1. X 20.
MORPHOLOGY OF CHICK TAIL BUD FORMATION
Gary C Schoenwolf
PLATE 1
139
PLATE 2
EXPLANATION OF FIGURES
All figures illustrate cross sections through stage 11-12 embryos (see fig. B for orientation). Sections obtained by transection of blastoderms with razor blades during dehydration.
7
At the anterior end of the neural groove the mesoderm is organized into distinct
structures which are separated from one another and from the ectoderm and endoderm. A fibrous material occupies spaces between adjacent structures. X 700.
8
Mesodermal structures are not yet well organized a t this more posterior level. The
cells of the prospective notochord are intermingled with the cells lining the neural
groove and with those of the paraxial mesoderm. x 700.
9 At more posterior levels the neural groove and the anterior end of the primitive
streak partially overlap. X 320.
10 Enlargement of the central portion of figure 9. The cells lining the neural groove
are intermingled with those of the primitive streak. X 1,100.
11 A shallow neural groove lies above a dorso-ventrally thickened primitive streak a t
the posterior end of the overlap zone. X 700.
12 The primitive streak spans the entire thickness of the blastoderm posterior t o the
overlap zone. X 700.
Fig. B Light micrograph identical to that shown in figure A. Numbered arrowheads
indicate approximate section levels shown in figures 7-12.
MORPHOLOGY O F CHICK TAIL BUD FORMATION
Gary C. Schaenwolf
141
PLATE 3
EXPLANATION OF FIGURES
13 Stage 15. Cross section, obtained by transection of t h e blastoderm with a razor
blade during dehydration, shows region of overlap between posterior end of neural
tube and anterior end of tail bud (see fig. C for orientation). X 420.
14 Stage 15. Transverse cryofracture through tail bud (see fig. C for orientation). Regions similar t o those indicated by numbered arrowheads a r e shown in figures 1518. X 300
15 Transmission electron micrograph of an area similar to t h a t indicated by
arrowhead (15) in figure 14. The basal lamina beneath surface ectoderm becomes
fragmented a s cells of t h e ectoderm and tail bud merge. Arrows indicate positions
of fragmented portions of basal lamina which a r e difficult to discern a t t h e low
magnification shown. X 4,200.
16
Cryofracture of an area similar to t h a t indicated by arrowhead (16) in figure 14.
The surface ectoderm and paraxial mesoderm a r e separated by a space containing a
fibrous material. x 3,000.
Fig. C Light micrograph of a dorsal view of t h e posterior end of a stage 15 chick
embryo. Photograph made from a commercially prepared “48-hour” chick embryo whole
mount (Macmillan Science Co., Inc.). Numbered arrowheads indicate approximate section levels shown in figures 13 and 14. x 70.
MORPHOLOGY ot. CHI(‘K TAIL Burl FORMATION
Gary C Schoenwolf
t’l.ATE 3
143
PLATE 4
EXPLANATION OF FIGURES
17 Transmission electron micrograph of an area similar to that indicated by
arrowhead (17)in figure 14. The ventral surface of the tail bud lacks a distinct epithelial covering of endoderm. X 5,800.
18 Transmission electron micrograph of an area similar to
arrowhead (18) in figure 14. An enddermal epithelium and
lamina (arrow) are present on each side beneath the paraxial
Inset: Enlargement of basal lamina subjacent to endoderm. x
144
that indicated by
corresponding basal
mesoderm. >: 7,500.
45.000.
MOKPHOLOGY OF CHICK T A I L B1.D FOKMATION
Gary C Schoenwoll
145
PLATE 5
EXPLANATION OF FIGURES
All figures illustrate 1 - p m sections cut either precisely through the midsagittal plane (figs. 19-26) or
what obliquely to it (fig. 27).
some^
19 Stage 15. The cells of the primitive streak are largely consolidated into t h e tail bud. Many of the last
streak cells to become incorporated contain darkly stained particles (unlabeled arrowhead). Arrow indicates posterior body fold. Numbered arrowheads indicate areas enlarged in figures 20-23. X 90.
20 Enlargement of area indicated by numbered arrowhead (20) in figure 19. Surface ectoderm overlying the
roof of the neural tube is bilaminar. X 350.
21 Enlargement of area indicated by numbered arrowhead (21) in figure 19. Surface ectoderm overlying the
tail bud consists of a single layer of flattened cells. X 350.
22
Enlargement of area indicated by numbered arrowhead (22) in figure 19. Differentiation of the ventral
surface of the notochord and formation of a distinct layer of endoderm beneath it are spatially correlated. X 350.
23 Enlargement of area indicated by numbered arrowhead (23) in figure 19. The endoderrnal cells underlying the tail bud are elongated dorso-ventrally. X 350.
24 Stage 1 5 + . The posterior body fold is well formed in the midline. Arrowhead indicates position of cells
containing darkly stained particles. X 90.
25 Stage 16. The postero-ventral aspects of the tail bud are clearly demarcated. Continuity of endoderm and
tail bud is indicated by arrow. Numbered arrowhead indicates area enlarged in figure 26. Unlabeled
arrowheads indicate expanse of cells containing darkly stained particles. X 90.
26 Enlargement of area indicated by numbered arrowhead in figure 25. Formation of a bilaminar ectoderma1 epithelium (arrow) is spatially correlated with tail bud cavitation. X 350.
27 Stage 20. Separation of ectoderm from tail bud is still spatially correlated with neural tube formation,
but this fact can not be discerned in the slightly oblique sagittal section shown. Continuity between surface ectoderm and tail bud (arrows) can be observed more ventrally, however, where the section is exactly midsagittal. Arrowheads indicate expanse of cells containing darkly stained particles. X 180.
146
MORPHOLOGY OF CHICK TAIL
Gary C Schoenwolf
BUD FORMATION
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