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Effects of complete tail bud extirpation on early development of the posterior region of the chick embryo.

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Effects of Complete Tail Bud Extirpation on Early
Development of the Posterior Region
of the Chick Embryo
GARY C. SCHOENWOLF '
Department of Genetics and Deuelopment, University of Illinois, Urbana, Illinois 61801
ABSTRACT
The tail bud was completely extirpated down t o the yolk from
65 embryos a t stages 13-17 to determine whether the posterior part of the notochord originates from the tail bud or from a more anterior region (i.e., prospective notochordal region). About 40% of the 44 surviving embryos developed
near-normal tails, containing a localized defective region beginning near the
base of the tail and extending a short distance posteriorly, about 15%developed
truncated, cone-shaped tails, containing a defective region beginning near the
base of the tail and extending towards the tip, and about 45% developed short,
ventral tail remnants, containing a localized defective region beginning near
the base of the tail and extending a short distance posteriorly. The tail was absent in only one embryo. These differences were probably due primarily to variation in the amount of healing and regeneration that occurred, and were independent of the stage a t which the operation took place. The tail region has a
tremendous capacity for regeneration since a near-normal tail frequently developed. The location of the beginning of the defective region near the base of
the tail suggests that the tail bud primarily gives rise to tail structures. All
embryos had neural tube defects, about 30% developed large, midline somites
within the defective region, and about 25% developed an ourenteric outgrowth.
The notochord was always normal within the defective region. These results are
consistent with the view that the tail bud contributes cells to the posterior part
of the neural tube, but not to the notochord.
The tail bud first appears in chick embryos
a t about Hamburger-Hamilton ('51) stages
13-14, and consists of a dense mass of cells
located a t the posterior end of the embryo. The
origin of the posterior part of the neural tube
from the tail bud has been documented by histological (Braun, 1882; Holmdahl, '25a,b;
Schumacher, '27; Criley, '69; Jelinek e t al.,
'69; Klika and Jelinek, '691, microsurgical
(Criley, '691, and radioautographic (Schoenwolf, '77) studies. The origin of the posterior
part of the notochord has been disputed, however. Histological studies (Holmdahl, '25a,b;
Schumacher, '27; Criley, '69; Jelinek et al.,
'69; Klika and Jelinek, '69) suggested that it
originates from the tail bud, while more recently i t has been suggested t h a t i t originates
from a region which merges with the anterior
end of the tail bud, the so-called prospective
notochordal region (Schoenwolf, '77). Criley
ANAT. REC.
(1978)192: 289-296.
('69) reported that defects, including apparent
gaps, were often observed within the posterior
part of the neural tube after extirpation of the
anterior portion of the tail bud. This result
suggests that the posterior part of the neural
tube originates from the tail bud. In addition,
Criley implied t h a t gaps were also occasionally present within the posterior part of the notochord. This operation should never result in
notochordal discontinuities if the posterior
part of the notochord originates from a region
anterior to the tail bud. Complete extirpation
of the tail bud should resolve the question of
whether the posterior part of the notochord
originates from the tail bud or from a more anterior region and has been done (Graper, '33;
Zwilling, '42a), but development of the posteReceived Feb. 15, '78. Accepted Apr. 11. '78.
I Present address: Department of Anatomy, University of New
Mexico, School of Medicine, Albuquerque, New Mexico 87131.
289
290
GARY C. SCHOENWOLF
rior part of t h e notochord was not examined.
The purpose of this investigation is to determine experimentally if t h e posterior part of
the notochord originates from t h e tail bud or
from a more anterior region by examining notochord development in embryos following
complete extirpation of t h e tail bud. The results obtained suggest t h a t t h e posterior part
of t h e notochord is derived from a region anterior to the tail bud and not from t h e tail bud.
MATERIALS AND METHODS
Cross-bred, fertile eggs (Columbian hens X
New Hampshire Red roosters) were incubated
a t 38°C for 50 t o 59 hours and windowed by
standard techniques. After vital staining, t h e
entire thickness of t h e tail bud was extirpated
down to t h e yolk from 65 embryos at stages 1317 with finely sharpened tungsten needles,
exactly as previously described (Schoenwolf,
'77). An attempt was always made to cut just
inside of t h e tail bud boundaries t o prevent
injury to or removal of surrounding areas. Incisions were made in t h e tail bud as follows:
(1)anteriorly, just posterior t o t h e region of
continuity between tail bud and neural tube;
t h e posterior end of t h e notochord and t h e socalled prospective notochordal region would
be left intact by this incision since these structures terminate beneath the posterior end of
t h e neural tube (Schoenwolf, '77; figs. 3-51; (2)
laterally, just medial to t h e regions where intensity of vital staining, and therefore cellular density, began t o decrease; (3) posteriorly,
just anterior to either t h e cloaca1 membrane
anlage, in earlier stages, or the posterior
boundary of t h e body, in later stages. Windows
were then sealed with tape and t h e eggs were
reincubated to a total incubation time of 88 t o
96 hours, after which embryos were fixed with
Bouin's fluid. Posterior regions of selected embryos were traced in toto with a camera lucida
after light staining with nile blue sulfate. The
posterior third of all embryos was then prepared for paraffin serial sectioning. Embryos
were sectioned transversely (relative to t h e
trunk) a t 8 wm and sections were stained with
Harris hematoxylin (Humason, '72).
RESULTS
Gross observations
Three types of tails developed following tail
bud extirpation: (1)about 40% of t h e 44 surviving embryos developed near-normal tails
(figs. 2, 3), (2) about 15%developed truncated,
cone-shaped tails (fig. 41, and (3) about 45%
developed short, ventral tail remnants (fig. 6).
The tail was absent in only one embryo (fig. 5 ) .
The length and shape of t h e near-normal tails
closely resembled t h a t of t h e tails of unoperated (control) embryos (cf. fig. 1 with figs. 2,
3). The length and shape of t h e tails of t h e remaining embryos (figs. 4-61, however, were
drastically altered. In embryos which developed only a short, ventral tail remnant (fig.
61, one or both leg buds were frequently abnormal or absent.
All embryos exhibited a grossly discernable
defective region which began near the base of
t h e tail. Within this region, t h e neural tube
was either obviously abnormal or appeared t o
be lacking (figs. 2-51. Paired somites flanked
t h e entire defective region in some embryos
(fig. 21, while in others, large, midline somites
developed within t h e defective region (figs.
3-51.
The stage at which t h e operation was done
had no affect on t h e antero-posterior position
of t h e beginning of t h e defective region, on t h e
structures t h a t were defective, or on the types
of tails which developed, as reported in similar
studies (Graper, '33; Zwilling, '42a; Criley,
'69; Schoenwolf, '77).
Histological observations
All embryos were examined histologically
t o determine what specific structures were
located within t h e defective region, posterior
to this region, and in t h e short, ventral tail
remnants. All embryos had defects of t h e neural tube, about 30% developed large, midline
somites within t h e defective region, and about
25% developed a n ourenteric outgrowth (an
abnormality in which a portion of the embryo
containing t h e neural tube andlor notochord
grows into t h e hindgut or allantois). The notochord was always normal within the defective
region, however.
Sections through t h e defective region of t h e
embryo illustrated by figure 2 are shown in
figures 7a,b. The notochord is normal and
spans t h e entire defective region (fig. 7a). In
contrast, t h e neural tube probably contains a
short gap, flanked by paired somites, since
only isolated fragments of neural ectoderm
can be observed within t h e defective region
(fig. 7b). It is possible, however, t h a t fine
strands of neural ectoderm, thinner than t h e
section thickness, interconnect these fragments but cannot be resolved. Similar neural
tube discontinuities were observed in three
other embryos (one with a near-normal tail,
291
TAIL BUD EXTIRPATION IN THE CHICK EMBRYO
2
3
Fig. 1 Camera lucida tracing of the dorsal surface of t h e posterior region of an unoperated (control) 4-day embryo
showing t h e normal shapes and positions of the neural tube (NT), somites (S),and leg buds (L). The base of each leg bud
is located a t approximately the level of somites 26-32.Ca. x 15.
Figs. 2-5 Camera lucida tracings of the dorsal surfaces of t h e posterior regions of 4-day embryos showing neural tube
and somite defects after tail bud extirpation. Sections of the embryos illustrated are shown in figures 7a-lob. D, defective region; MS, somites within defective region; TT,truncated tail. Ca. x 15.
Fig. 6 Camera lucida tracing of the ventral surface of the posterior region of a 4-day embryo t h a t formed only a
short, ventral tail remnant after tail bud extirpation. Sections of the embryo illustrated are shown in figures Ila,b. VT,
ventral tail remnant; VV, vitelline vessel. Ca. X 15.
one with a truncated, cone-shaped tail, and
one with a n ourenteric outgrowth [see belowl).
However, notochordal discontinuities were
never observed; thus the notochord always
spanned the defective region.
Figures 8a-c show sections through the
defective region of the embryo illustrated by
figure 3. The notochord is normal (fig. 8a), but
the neural tube is grossly abnormal (fig. 8b).
Large, unpaired somites are present in the
defective region dorsal to the notochord and
defective neural tube (fig. 8c).
Figures 9a-d show sections through the
truncated, cone-shaped tail of the embryo illustrated by figure 4. The tip of the tail contains only loosely-packed mesenchyme (fig.
9a). More proximally, the tail contains a noto-
chord (fig. 9b) which is continuous with the
trunk portion of the notochord (fig. 9c). The
neural tube ends abruptly a t the base of the
tail (fig. 9d).
The embryo illustrated by figure 5 is the
only one which lacks a tail. Sections show,
however, that it contains an ourenteric outgrowth. The distal end of this outgrowth projects freely into the hindgut (fig. 10a); more
proximally, i t is continuous with the trunk.
This outgrowth contains a notochord, which is
continuous with the trunk portion of the notochord (fig. lob), and a neural tube, which
fades out before connecting with the trunk
portion of the neural tube.
Sections through the ventral tail remnant
of the embryo illustrated by figure 6 are
292
GARY C. SCHOENWOLF
shown in figures lla,b. The trunk and tail portions of the notochord are directly continuous
(fig. l l a ) . The trunk portion of the neural tube
exhibits severe myeloschisis within the defective region, but is still continuous with the
tail portion of the neural tube (fig. l l b ) . The
tail does not contain somites. About 95%of the
tails of this type contain a notochord, about
85%contain a neural tube, and only about 25%
contain somites.
DISCUSSION
Three types of tails developed following tail
bud extirpation: (1) near-normal tails, (2)
truncated tails, and (3) short, ventral tail
remnants. The tail was absent in only one embryo. Other investigators have reported a
much higher frequency of tailless embryos following total or partial extirpation of the tail
bud (Graper, '33; Zwilling, '42a; Criley, '691,
but in some cases the tail was designated a s
absent when actually a small tail remnant
could be identified in their published photographs. The differences in the types of tails
that developed are probably due primarily to
variation in the amount of healing and regeneration that occurred. The tail region has a
tremendous capacity for regeneration since
near-normal tails developed in about 40% of
the embryos. However, since all embryos contained a defective region, complete tail restitution never occurred. The location of the
beginning of this defective region a t the base
of the tail is consistent with the results of
Schoenwolf ('77) that the tail bud primarily
gives rise to tail structures.
In many of the embryos which formed only a
short, ventral tail remnant, one or both leg
buds were abnormal or absent. These embryos
are bent sharply ventrad, and the regions of
the lateral plates which form leg buds are
compressed. This compression probably prevented normal leg bud development in most of
these embryos. Alternatively, the leg buds
may have been injured since they form near
the level of the incipient tail bud (Graper, '33).
The notochord was always normal within
the defective region. The neural tube was always abnormal within this region, however,
and may have contained a short gap in four
embryos. These results strongly suggest that
the cells which form the posterior part of the
neural tube and those which form the posterior part of the notochord originate from two
different areas. The fact that the neural tube
was always abnormal confirms that the posterior part of the neural tube originates from
the tail bud. This result is in agreement with
numerous previous investigations (Braun,
1882; Holmdahl, '25a,b; Schumacher, '27;
Criley, '69; Jelinek et al., '69; Klika and
Jelinek, '69; Schoenwolf, '77). The fact that
development of the notochord is relatively
unaffected by complete extirpation of the tail
bud strongly suggests that the posterior part
of the notochord originates from a region anterior to the tail bud and grows into the developing tail rather than forming in situ from
cells of the tail bud. However, the specific
mechanisms involved in this posterior growth
are unknown. It is likely that the posterior
part of t h e notochord originates by rearrangement of the cells of the so-called prospective
notochordal region since this area is sandwiched between the tail bud and notochord,
and has the same distinctive pattern of tritiated thymidine labeling as the notochord, a
pattern which is quite different from that of
the tail bud (Schoenwolf, '77).
Ourenteric outgrowths were observed in
about 25% of the embryos. Ourentery has also
been observed by others in dominant rumpless
embryos (Zwilling, '42b), in insulin-induced
rumpless embryos (Moseley, '471, in embryos
following extirpation of the anterior portion of
the tail bud (Criley, '691, and in embryos following homotopic transplantation of the tail
bud (Schoenwolf, '77). Ourenteric outgrowths
apparently form primarily by growth of the
tail bud and/or notochord antero-ventrad into
the developing hindgut or allantois (Moseley,
'47: figs. 5a-f).
ACKNOWLEDGMENTS
I wish to thank Mrs. Alice Prickett, who
prepared figures 1-6, Doctor Ray L. Watterson, for his advice during this investigation,
and Doctor Robert E. Waterman, for his critical review of the manuscript.
LITERATURE CITED
Braun, M. 1882 Entwickelungsvorgange am Schwanzende bei einigen Saugethieren mit Berucksichtigungder
Verhatnisse beim Menschen. Arch. Anat. Physiol., Anat.
Abt., 6: 207-241.
Criley, B. B. 1969 Analysis of the embryonic sources and
mechanisms of development of posterior levels of chick
neural tubes. J. Morph., 128: 465-501.
Graper, L. 1933 Beitrag zur Frage der sekundaren Korperentwicklung und der Entwicklung der hinteren Extremitaten beim Huhnchen. Arch. Entwmech. Org., 128:
766-794.
-
TAIL BUD EXTIRPATION I N THE CHICK EMBRYO
Hamburger, V., and H. L. Hamilton 1951 A series of normal
stages in t h e development of t h e chick embryo. J . Morph.,
88: 49-92.
Holmdahl, D. E. 1925a 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 entcdermalen Kloake
nebst einem Exkurs uber die Entstehung der Spina bifida
in der Lumbosakralregion. I. Gegenbaurs Morphol.
Jahrb., 54: 333-384.
1925b Die erste Entwicklung des Korpers bei
den Vogeln und Saugetieren, inkl. dem Menschen, besonders mit Rucksicht auf die Bildungdes Ruckenmarks, des
Zoloms und der entodermalen Kloake nebst einem Exkurs
uber die Entstehung der Spina bifida in der Lumbosakralregion. 11-V. Gegenbaurs Morphol. Jahrb., 55:
112-208.
Humason, G. L. 1972 Animal Tissue Techniques. Third
ed. W. H. Freeman and Company, San Francisco.
293
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.
Klika, E., and R. Jelinek 1969 The structure of the end and
tail bud of t h e chick embryo. Folia Morphol. (Praha), 17:
29-40.
Moseley, H. R. 1947 Insulin-induced rumplessness of
chickens. IV. Early embryology. J. Exp. Zool., 105:
279-316.
Schoenwolf, G. C. 1977 Tail (end) bud contributions to
the posterior region of the chick embryo. J . Exp. Zool..
201: 227-246.
Schumacher, S. 1927 Uber die sogenannte Vervielfachung
des Medullarrohres (bzw. des Canalis centralis) bei Embryonen. Z. Mikrosk.-Anat. Forsch., 10: 75-109.
Zwilling, E. 1942a Restitution of the tail in the early
chick embryo. J. Exp. Zool., 91: 453-463.
1942b The development of dominant rumplessness in chick embryos. Genetics, 27: 641-656.
Abbreviations
H, Hindgut
M, Neural tube with myeloschisis
MS, Somites within defective region
N, Notochord
NE, Neural ectoderm
NT, Neural tube
0, Ourenteric outgrowth
TT, Truncated tail
PLATE 1
EXPLANATION OF FIGURES
Some structures are cut frontally as well as transversely because of the caudal flexure. Micrographs of sections through single embryos are lettered in antero-posterior
sequence.
7a.b Selected sections of the embryo drawn in figure 2.
X
35
8a-c Selected sections of the embryo drawn in figure 3. Figures 8a,b
x 80.
X
35; figure 8c
9a-d Selected sections of the embryo drawn in figure 4. Arrow in figure 9d indicates
the posterior end of the neural tube. x 35.
10a,b Selected sections of the embryo drawn in figure 5. x 35
l l a , b Selected sections of the embryo drawn in figure 6. Some structures are cut frontally as well as transversely because the tail projects ventrad. X 35.
TAIL BUD EXTIRPATION IN THE CHICK EMBRYO
Gary C. Schoenwolf
PLATE 1
295
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