Download Axial Elongation in the Mouse and its Retardation in Homozygous

Axial Elongation in the Mouse and its Retardation
in Homozygous Looptail Mice
by L. JEAN SMITH1 and KATHRYN F. STEIN 2
From the Clapp Laboratory, Mount Holyoke College
WITH ONE PLATE
INTRODUCTION
M I C E carrying the gene Looptail were first described by Strong & Hollander
(1949), who named the mutation from the looped or kinked appearance of the
tail in heterozygotes. Animals homozygous for Lp, as the gene was designated,
have neural folds closed only as far posteriorly as the myelencephalon, and die
shortly before or at birth. Stein & Rudin (1953) traced the abnormality of the
nervous system back to the time of closure and found that it resulted from the
failure of the neural folds to fuse rather than from a secondary breakdown of
the roof of the neural tube, and that histological differentiation of the various
tissues of the embryo was comparatively normal. Various skeletal abnormalities
were reported by Stein & Mackensen (1957), who considered them secondary
effects of the abnormality in the neural tube.
This paper is concerned with the finding that, at the time closure of the neural
tube begins, the notochord, somites, and gut, as well as the neural tissue, show
abnormalities in growth possibly traceable to a failure of the primitive streak to
shorten normally. It presents a striking illustration of the manifold effects of
a disturbance in the delicately balanced relationships between the processes
involved in the elongation of the normal embryo.
MATERIALS AND METHODS
With the exception of a few older embryos, the mice used in this study resulted
from matings within lines inbred brother by sister for 20 to 25 generations.
Embryos from 14 to 19 days' gestation (by Griineberg's (1943) criteria) were
observed by gross inspection only, and are from six different lines all derived
from the same heterogeneous population. Embryos 9 to \2\ days of age came
from timed matings within only one of these, line 55. Line 55 females were
checked for vaginal plugs in the morning, and 1 a.m. of the day a plug was
1
Present address: Albert Einstein College of Medicine, Department of Anatomy, New York,
N.Y., U.S.A.
2
Address: Clapp Laboratory, Mount Holyoke College, South Hadley, Mass., U.S.A.
[J. Embryol. exp. Morph. Vol. 10, Part 1, pp. 73-87 March 1962]
74
L. J. SMITH AND K. F. STEIN
found was arbitrarily assigned as the time of fertilization. Since physiological
age may vary by as much as a day between different strains of mice, estimates
of somite numbers or stage of development also are given. Embryos to be sectioned were fixed in Carnoy's fluid, cut at either 7 or 10 /x and stained with
Azure B.
Two methods of estimating length were used in this study. For structures
confined to the posterior part of the body, the number of sections in which
these structures appeared was determined in embryos cut in true cross-sections.
This number was recorded relative to a fixed point, the tail-tip. Because of the
curvature of the trunk, however, it is impossible to get only cross-sections
through an entire embryo. Therefore, to compare the relative lengths of spinal
cords, for example, in two embryos, scale reconstructions were made on graph
paper. Relatively equal magnification in both dimensions of the reconstruction
was secured by the following method. Projection drawings at X 100 magnification were made of every 15th section of embryos cut at 7 [x. Dorsal-ventral
distances on the graph were plotted from direct measurement of structures in
the midline of each projected section. Points for successive sections were plotted
10 mm. apart on the graph and those for like structures were then connected by
lines. This gave, for structures in the midline, longitudinal outlines from which
direct measurements could be made (Text-fig. 1). It should be noted that placing
the points 10 rather than 10-5 mm. apart resulted in a magnification in length of
the embryo of only 95 rather than 100 times. As a result the ratio of length to
depth in the magnified outlines of embryos sectioned at 7 /x is slightly less
than 1.
RESULTS
1
Embryos at 141 to 19i days' gestation
The phenotype of the 69 looptailed homozygotes obtained from six different
lines (Table 1) differs little from that previously described by Strong & Hollander
TABLE 1
Numbers of embryos of different genotypes at 14$-19j days of gestation from
matings between heterozygous Looptails (Lp/+) of six inbred lines
Number of embryos
Line
8
16
44
55
66
71
TOTAL
ALL LINES
LpILp
17
21
3
10
12
6
Lpl+
+ /+
39
52
3
17
28
32
24
35
5
7
14
21
Total
80
108
11
34
54
59
69
171
106
346
Per cent
LpjLp
21
20
27
29
22
10
20
75
AXIAL ELONGATION IN LOOPTAIL MICE
(1949) and Stein & Rudin (1953). All but one or two were markedly smaller
than their normal littermates but the decrease in size was not proportional in
all parts of the body. The trunk seems relatively short for the size of the head
and the nervous system looks too large for the body. Superficially, the trunk of
the Lp/Lp embryo appears compressed along the anterior-posterior axis. The
picture of six sibs from line 8 (Plate, fig. A) illustrates this disproportionate
decrease in size. It also shows the frequently observed 'crooks' in the back. The
hernia described as characteristic for the homozygotes by Strong & Hollander
has disappeared from all our lines except line 8, in which not only intestine
but liver is found outside the body-wall. This does not show clearly in the figure
but was present in all Lp/Lp homozygotes of this line.
TABLE 2
Length in mm. of umbilical cords of embryos of different genotypes at 19-20 days'
gestation
(Each horizontal line contains embryos from one litter)
Length of individual umbilical cords in mm.
Line
8
$
Lp/ +
X
<J
Lp/ +
LpILp
2
6
7-5
6,4
16
44
Lp/+
+/+
Lp/+
Lp/ +
+ /+
Lp/ +
5,7
5,5
9-5,* 12, 10
9, 10, 9, 9, 9
10
12, 11, 11, 5*
6,* 6* 11, 10,11
9,9
8,* 9
55
Lp/+
Lp/ +
+ /+
Lpl +
9, 10, 10, 10
9,9, 11
9, 11, 12, 12
9-5
7,* 9, 10
8, 7, 10 12
9
14, 14
6,7, 8 10, 12, 12, 12
12
12
10, 11
10
12
11
7-5*
13, 15, 9*
12
11,9*
11
7,* 9, 11,9, 10
14
15, 15, 14, 16
12
Total number of embryos
16
44
26
Average cord length in mm.
6-4
10
11-4
Average cord length: cf.
pseudencephalic class
6-4
10-5
121
* Pseudencephalic.
The abnormal concave flexure in the back, seen especially well in the Lp/Lp
embryo on the upper left (Plate, fig. A), suggests that embryos in this line do not
complete the rotation around their long axis which normally produces the change
from the S-shape characteristic of rodent neural-plate embryos, to the C-shape
found in all mammalian embryos. Perhaps for this reason the somatopleure in
the posterior trunk region is unable to fuse ventrally and herniation results.
76
L. J. SMITH AND K. F. STEIN
Lp/Lp embryos from other lines show abnormal flexure of the back, but rotation
is more nearly complete and the most obvious result is therefore a shortening
of the right side relative to the left rather than a hernia. This was noted previously
by Stein & Rudin (1953).
The neural tissue in all these embryos, from the posterior border of the
metencephalon back into the extremely shortened tail, consists of a flat plate
with a deep median groove. In the brain region of some specimens, flaps of
more posterior levels of tissue are apparently pushed forward so that they
overlie the diencephalon and, in some cases, the cerebral hemispheres, which
are collapsed.
Skin is absent over all portions of the nervous system. The age at which
ectoderm over the closed brain region is lost was not determined, but presumably its loss is due to rupture by the expanding brain, since it is still present
at 10| days. The failure of closure of the neural folds of course accounts for the
absence of skin over the rest of the nervous system.
Another interesting difference characteristic of the older abnormal foetuses is
their shorter umbilical cords (Table 2). The average length for 16 individuals
at 19-20 days of gestation was 6-4 mm. (range 2-10). Comparatively, cords of
44 heterozygous Looptails averaged 10 mm. (range 5-14), those of 26 straighttailed 11-4 mm. (range 7-16). Included in the latter two groups were 11 pseudencephalic specimens which also had shorter cords. Elimination of these, on the
basis of evidence that this condition is genetically distinct from that produced
by the Looptail gene, gives 10-5 mm. (range 9-14) as the average cord length
for heterozygotes and 12*1 mm. (range 9-16) for normal straight-tailed foetuses.
Variation is too great within these two groups for us to conclude that any significant difference exists between heterozygotes and normals, but the abnormal
homozygotes would appear to have definitely shorter cords. This conclusion is
supported by the lack of any overlap within individual litters.
Embryos at 9 to 121 days of gestation
Twenty-four litters 9 to \2\ days of age from matings of line 55 Looptail
parents (Lp/+) were examined (Table 3). Of the 177 embryos obtained, 43 had
open hindbrains and spinal cords; 17 of the other 134 were abnormally small
and had failed to change from the S-shape characteristic of embryos of 13
somites or younger (Table 3—'S-shape'). All 134, however, had closed neural
tubes. The 43 abnormal embryos were considered to be Lp/Lp homozygotes on
the basis of the observed abnormality, and their number agrees very well with
the theoretically expected 44-4. Evidence that the 17 retarded embryos, all of
which were at less than 10| days' gestation, were not of the Lp/Lp genotype was
obtained when matings of L p / + X + / + parents of the same line produced
6 similarly small embryos out of a total of 32. Therefore they are excluded from
any further data. Of the 117 normals, only those 11 days or older could be
classified as heterozygotes (Lp/+) or true normals ( + / + ) . The genotypes of
77
AXIAL ELONGATION IN LOOPTAIL MICE
the 9-10|-day embryos called normal in the following descriptions may thus be
either Lp/+ or + / + .
TABLE 3
Numbers of embryos of different phenotypes at 9-12j days of gestation from
matings between heterozygous Looptails (Lp/-{-)from line 55
Numbers of embryos
Lpl+
Age
No. of
litters
or
LpjLp
+ 1+
8
18
12*
11*
10*
1
3
10i
1
9|
9*
2
9i
4
9
1
2
4
8
3
3
14
7
2
24
43
TOTALS
5
7
Closed neural
tube; embryo
S-shape
Total
10
22
21
2
—
—
1
2
9
3
2
117
17
177
19
5
18
26
27
9
23
49
31
6
Embryos from the one 12^-day-old litter were examined only grossly. The
abnormal homozygotes in this litter were phenotypically similar to the older
Lp/Lp embryos except that the neural tube was less obviously degenerate,
although a haemorrhagic area was present in the flap of metencephalon. Similar
haemorrhages were seen in three of the twelve 10^-11 ^-day-old Lp/Lp embryos
in the eight litters examined at these ages. No evidence of pycnosis could be
found in these areas, but earlier observations by Stein, Lievre, & Smoller (1960)
of an apparent reduction in cell density in portions of the mantle layer of the hindbrain and cord were confirmed. These areas of 'sparse, loosely arranged cells'
could be traced back to 10| days and may be the first indication of the subsequent degeneration which the open neural tube undergoes. However, Stein,
Lievre, & Smoller (1960) observed this abnormal appearance of the neural
tissue only at 10 to 13 days of gestation. Examination of comparable sections
of the brains of normal and abnormal 11 ^-day-old embryos suggested that,
in spite of these areas of decreased cell density, tract formation in the Lp/Lp
embryos is essentially normal, since almost identical patterns of darker and
lighter staining cells were observed in both.
The 10|-day-old abnormal embryos offered evidence that the open neural
canal, at least in the hindbrain region, may not be due to an inherent inability of
the neural fold to fuse but to mechanical difficulties which may cause them to
rip apart. Figures D and E of the Plate are of successive sections of an Lp/Lp
embryo in which the cells of the stretched roof of the metencephalon have pulled
apart, apparently under tension. In the normal embryos even the membranous
covering of the myelencephalon shows no such signs of stretching.
A ventral ectodermal ridge of the tail as described by Griineberg (1956) was
78
L. J. SMITH AND K. F. STEIN
observed in both the normal and the abnormal 10|-day-old embryos. Since outgrowth of the posterior portion of the body of the abnormals is disturbed prior
to the time of appearance of the ridge, it is believed that a defect in this structure
could not be a primary cause of the failure of tail-growth in these animals.
Even at 9£ days, or shortly after the mid-gut has closed, the Lp/Lp homozygotes show grossly all the anomalies which characterize them at later stages
except that their brain vesicles are not yet collapsed and folded. Neural folds
are open and lie spread out over their backs, producing an appearance of excess
neural tissue. Their bodies are relatively shorter than normal, at least from the
level of the otocysts backwards, and although anterior somites appear normal in
size, posterior somites are often very small and/or irregular in shape, making an
TABLE 4
Lengths of gut and nervous system in LpjLp and normal embryos
(Measurements in mm. from level of posterior limit of right otocyst to posterior limit of tissue
measured, made from reconstruction drawings magnified x 100)
Exp. no. of Somite number of
$ parent normal embryo
M15
C20
449
16
163
163
163
12-13
18-19
22
22-25
»>
>>
Nervous system
Gut
LpjLp
Normal
LpILp
Normal
140
200
22-5
27-8
30-5
340
350
260
300
300
360
38-5
440
47-0
130
198
18-3
24-5
240
280
25-5
23-5
26-5
260
29-8
313
38-8
380
accurate count impossible. For example, in a litter in which 18-25 pairs of
somites could be counted in normal animals, one Lp/Lp homozygote appeared
to have 22; two others 10. The heads of all three were about the same size and
stage of development as those of normal littermates. When the two with 10
somites were sectioned, they were found to have closed guts, a condition which
is usually not present before the 18-19-somite stage. Whether these somitic
irregularities are due to reduced growth rates, to originally smaller somites, or
to destruction of some of the cells, cannot be decided on the basis of present
evidence. Extensive areas of pycnosis were, however, found in somites of some
of the 9£-day-old Lp/Lp embryos. All the abnormal embryos of this age show
a typical bend in the region of the forelimbs which might produce the pycnosis
by compression or conversely might be the result of cell loss. Slightly younger
abnormal embryos appear to have smaller somites and little or no evidence of
pycnosis.
Although the total amount of neural tissue at 9\ days may be similar in normal
and abnormal embryos, the neural anlage in the region of the floor plate is
considerably shorter in the latter. This difference can be noted in the scaled
( x 100) reconstruction outlines of a typical pair of littermates (Text-fig. 1) and
also in measurements made on a series of such outlines (Table 4).
AXIAL ELONGATION IN LOOPTAIL MICE
79
The gut and notochord are also shorter in Lp/Lp homozygotes. This is obvious
from the reconstruction outlines (Text-fig. 1) and from measurements for the
gut (Table 4). Neither notochord nor gut in the abnormal embryo show any
curves or bends not found in the floor plate of the neural 'tube'. That both
TEXT-FIG. 1. Projection drawings (median sagittal) constructed to scale from cross-sections of embryo
9£ days of age. (Measurement in Table 4 obtained from similar reconstructions at x 100 magnification.)
these structures, and particularly the gut, are relatively more shortened than
the neural floor plate is indicated by their wider separation from it in the scale
reconstruction of the abnormal compared with the normal (Text-fig. 1). Further
evidence, but less convincing because of the paucity of data and greater chances
of error, is also furnished by the measurements. Averaged data for the five
sets of embryos with 22-25 somites (Table 4) show that while the neural floor
plate of the Lp/Lp embryo is 23 per cent, shorter than that of a normal embryo,
the gut is 27 per cent, shorter. Also worthy of note in Text-fig. 1 are the greater
diameter and extent of the thickened portion of the notochord in the abnormal
embryo relative to the normal.
Lp/Lp embryos seem to be more closely fastened to the yolk sac than their
normal littermates and this, together with the shortness of the embryos, may
account for their apparent difficulties in changing from the S to the C shape.
Whether there is an actual difference in length of the allantois which may later
5584.10
80
L. J. SMITH AND K. F. STEIN
be reflected in the difference (previously noted) in umbilical cord length was
not determined.
Many of the differences reported above were traced to even earlier stages of
development, i.e. in embryos of 9|-9 days' gestation (16-8 somites). Text-fig. 2
Primative streak -—=*-—y
Hind gut
- ^ ^ / \
E^r
fi\
Notochord
Foregut
Neural floor
plate
lOOu
10mm
TEXT-FIG. 2. Projection outlines (median sagittal) of an abnormal embryo Lp/Lp (A) and a normal
littermate (B) at beginning of 9th day of gestation. (Dotted lines represent region of separation between
numbered somites.)
shows projection outlines of embryos from the youngest of the litters examined.
The normal had 8 somites and a neural tube just beginning to close; the abnormal, 9 somites and no evidence of neural-tube closure. A comparison of these
projection outlines with photographs of embryos of similar stage (Plate,figs.B,
C) indicates that the outlines reliably reflect differences in form. Well shown in
these reconstructions is the difference in length, the 9-somite Lp/Lp embryo
being much shorter especially in the region of the somites than the 8-somite
normal. Even at this age, therefore, there is a difference in somite size between
normal and abnormal embryos. In addition, greater thickness and length of
the primitive streak and of the enlarged portion of the posterior notochord
characterize the abnormal. The primitive streak was considered as extending
from the posterior limit of the embryo to a point where neural tube and notochord seemed distinct structures by virtue of the smooth outline of the ventral
AXIAL ELONGATION IN LOOPTAIL MICE
81
surface of the neural plate and the regular arrangement of its cells. Photographs
of the 50th, 40th, and 30th sections from the tail-tip of two slightly older
embryos clearly show the differences in diameter of the notochord, and the
more anterior point of separation of neural tube and notochord from primitive
streak in the Lp/Lp (Plate; compare figs. G-I with J-L). They also show the
slightly larger tail gut of the abnormal embryos. The same differences characterized all the pairs examined.
Text-fig. 3 gives in graphic form, for all available embryos, the number of
sections from the tail-tip to (1) the point where the umbilical artery joins the
110
100
90
B0
70
60
50
40
30
20
10
0
Somite No. 9-10,8 10-11,9-11
Age (in days) |
|
g
Litter No.
503
862
Embryo No. 2,1
2,1,5,3
9-11,11-12
703
1,7,5,8
13,12-14
491
5,6,3,8,7
1314-16
705
6,5,7,1
13,15-17
561
5,8,7,4,2
11,16 18
489
2,1,3,7
14,17-19
405
4,1,5,2
12-13,19 22
2
163
R 3,2,4
TEXT-FIG. 3. Graph of primitive-streak lengths and of relative distances from tail-tip of indicated
structures in abnormal (Lp(diag.)Lp) embryos and normal littermates 9-9£ days of gestation. (Streak
length, abnormal H i , normal a ; area in which notochord forms part of gut roof in abnormal embryo
•^ — • , in normal < — > ; point of entrance of umbilical artery into dorsal aorta in abnormal • ,
in normal x.)
dorsal aorta, (2) the anterior end of the primitive streak, and (3) and (4) the
most posterior and anterior points between which the gut roof has not yet fused
under the notochord. These embryos are arranged in order of average physiological litter age of normal embryos, but this arrangement is somewhat arbitrary.
Relative position within the group timed as 9£ days of age was determined on
the basis of somite numbers of normal embryos. This order corresponds fairly
well (litter 705 excepted) with increasing distance between the tail-tip and point
of entrance of the umbilical artery into the dorsal aorta of the normal embryo
with increasing age. In spite of the variability in physiological age, even within
litters, and that resulting from differences in the angle of the cross-sectional cuts,
several striking differences between normals and abnormals are apparent. In
the normals the primitive streak becomes progressively shorter with increasing
82
L. J. SMITH AND K. F. STEIN
age. In the abnormals the pattern is less regular and the streak, in each of these
embryos, is longer than in any of their normal littermates. Average lengths are
compared in Table 5.
TABLE 5
Primitive-streak lengths in LpjLp and normal embryos
No. of embryos
Average no. of sections in P.S.
Average length in /x of P.S.
Age in days
LpjLp
Normal
LpjLp
Normal
LpjLp
9
2
8
3
4
14
3
60
44-5±5-3
32
42-5
24-8±l-8
15-3
450
334
240
Normal
319
186
114
The length of the abnormal embryo, measured from the point of exit of the
umbilical artery posteriorly, also fails to increase in the regular fashion of the
normal embryo. There seems to be an inverse relationship between the length
of the streak and the length of the embryo from this point, even though the
primitive streak of the Lp/Lp does shorten somewhat during the period of time
represented. This relationship seems to hold also for the normal embryo in
litter 491, which has a longer than average streak for its age and a shorter trunk.
The time of completion of the gut roof (see Plate, fig. F for incomplete gut roof)
by fusion of the gut endoderm under the notochord was also markedly different
in normal and abnormal embryos (Text-fig. 3). Not only was this fusion complete
in many of the older abnormal embryos at a time when it was still incomplete in
all but the oldest of the normal embryos: it was also farther advanced in the
younger abnormal embryos than in their normal littermates.
DISCUSSION
The early mesodermal cells of the mouse segregate out of, or are proliferated
from, the median ventral surface of the more posterior portion of the embryonic
epiblast; the head process comes from the anterior end of this same region. This
region of epiblast is, of course, the primitive streak. Normally, the primitive
streak shortens during the course of development. By the time the mouse embryo
has assumed a C-shape, at the 13-14-somite stage, it is only about three-fifths
as long as at the time of the beginning of neural-tube closure and is further
shortened by the time of mid-gut closure. According to Spratt (1947) and Vakaet
(1960), decrease in length of the primitive streak in the chick involves both
regression of the anterior portion and a 'shortening' at the posterior end. In
this study it was found that the substitution of two Lp genes for their normal
alleles in the mouse causes a delay in shortening and perhaps in the regression
of the primitive streak during and following the period of neural-tube closure.
Axial elongation of notochord and associated somites and neural tissue is
also less than normal in Lp/Lp embryos. This could be a direct consequence of
an abnormality of the Lp/Lp primitive streak which retarded conversion of its
AXIAL ELONGATION IN LOOPTAIL MICE
83
cells into definitive notochord cells. Increased bulk of the notochord and the
smallness and irregularity of the somites of Lp/Lp embryos might also result if
some presumptive paraxial mesoderm cells were organized to become notochord cells instead. This would be in line with the experiments of Spratt (1957)When he forced the 'notochord organizing center' in cultured pieces of chick
embryos to deviate so that it organized presumptive somite and lateral plate
cells into notochord, the diameter of the resulting notochord was approximately
twice as great as in the normal non-deviated part. Notochord cells, whether
derived from paraxial mesoderm or not, might also be abnormal and this
abnormality might then delay the reorganization which normally produces a
change in width of the differentiating notochord (10-12 cells wide at its juncture
with the primitive streak; 1-3 cells wide in the region of the somites). Since
it seems reasonable to suppose that it is this reorganization which normally
accounts for part of early notochord elongation, such a delay in reorganization
would explain the increased length of the bulky portion of the notochord of
Lp/Lp embryos and the decreased length of the total notochord.
The abnormal reaction of the gut-roof endoderm as expressed in its precocious
fusion under the notochord in the abnormal embryos would seem to support
the idea of abnormality in the notochord cells. Since the roof of the mid-gut is
itself believed to originate from the head process (Snell, 1941, p. 24), its precocious fusion may be another and direct expression of abnormality in Lp/Lp head
process or notochord cells. The failure of elongation of the notochord would
account for the shortness of the 'uncovered' portion.
The implied view of normal notochord and somite differentiation in the mouse
contained in the above attempt to account for some of the abnormalities in the
development of Lp/Lp embryos is based on Spratt's (1955) efforts to explain
normal axial formation in the chick from results of his carbon-marking, vitalstaining, and isolation experiments.
4
The results of many types of experiments seem to show that the upper (epiblastic)
portion of the node (prospective posterior spinal cord and tail bud epidermis) along
with at least a part of the surrounding posterior portion of the neural plate "slips
over" (i.e. moves posteriorly relative to) the ventral and originally more posterior
chorda and somite forming cells in the mesoblast. At least some of the chorda and
somite center cells remain in close association with the node epiblast and regress with
it, leaving behind (anteriorly) the differentiated notochord and somites' (Spratt, 1955,
p. 160).
It remains to be demonstrated by similar experiments how appropriate this
view will be in explaining axial organization in the mouse. It also remains to be
demonstrated, as Spratt has pointed out, what the relationship is between the
elongation and regression of neural plate and endoderm, and the regression of
the node. A discussion of causal relationships between the shortness of the neural
plate in the abnormal embryo and the shortness of the notochord becomes
therefore even more speculative. It is possible that regression of the ' organizing
84
L. J. SMITH AND K. F. STEIN
center' is responsible not only for the major and initial increase in length of the
notochord in early embryos but also for the elongation of the posterior neural
plate, perhaps by orientation of its cells. If this is the case, failure of notochord
elongation in Lp/Lp embryos would then account for the failure of the neural
plate to elongate normally.
On the other hand, reorganization and elongation of the differentiating neural
plate may move the node with its postulated notochord-organizing centre
posteriorly. It might then be postulated that 'intrinsic' failure of elongation of
the neural plate in Lp/Lp is the primary site of abnormality and that shortness
of notochord and irregularities of somites are secondary. The shortness of
neural tube and notochord might produce a mechanical pressure resulting in
shortening and fusion of the somites. Holtfreter's (1945) experiments, in which
growing Amblystoma larvae were prevented from elongating by encasement in
agar, did result in embryos with a bulkier than normal notochord, mesoderm
occupying less space relative to other tissues than normal, and tail somites which
lacked an orderly segmental pattern.
Many experiments in amphibians seem to indicate that the presence of a
notochord is necessary for the normal elongation of the embryo. However,
Kitchin (1949), who removed definitive notochords from Amblystoma neurulae
at the stage of the fully formed plate, found that stretching of the embryos immediately after the operation was normal, though shortening was obvious by
the time the tail buds were fully formed. This he interpreted as indicating that
the elongation in the early period was 'the result of activity which is intrinsic
to the differentiation of these individual structural rudiments and it is not at all
affected by the removal of the notochord' (p. 403). The somites in these amphibians segmented normally even though they fused in the mid-line. Spratt (1955)
has also observed neural-plate elongation in posterior pieces from which the
notochord centre has been removed and which have, as a consequence, no notochord. The somites appear to be unaffected. Rothfels (1954) reported that a
marked shortening of the notochord, produced in chicks in vitro by the addition
of an amino-acid analogue to the culture medium, was not accompanied by
extensive somite fusion. Occasional neural-tube abnormalities, including both
'zigzag' and open neural tubes, occurred, however, in some shortened embryos,
typically in explants treated with /8-2-thienylalamine, and the majority of these
embryos showed somite blocks, i.e. fusion of two or three adjacent somites.
In any case, the primary defect in the Lp/Lp embryos cannot be easily
explained in terms of either an increased or decreased number of notochord or
neural-tube cells. Although the diameter of the posterior region of the notochord and the amount of neural tissue in individual cross-sections of Lp/Lp
embryos is greater than in normal embryos, the body-length is clearly shorter
and the total number of cells in both normal and abnormal embryos may be
similar.
Whatever the original cause of the failure of the neural plate to elongate, its
AXIAL ELONGATION IN LOOPTAIL MICE
85
failure to close appears to result from the failure of elongation. An examination
of the photographed sections shows an apparently normal floor plate and basal
portions elevated in normal fashion. The alar portions of the plate seem to
receive no support from the relatively much too small somites and thus to be
unable to fold upward and fuse. In the head region, the brain may close, not
because of any difference in adhesive properties of the neural-plate cells in this
region, but because the disproportion between neural and mesenchymal cells
may not be as great, or because lengthening in this region is clearly not due to
notochord stretching.
No other mouse mutation produces just this array of effects. In his description of Brachyury which results in a kinky and shortened tail in heterozygotes
(77+), Griineberg (1958, p. 428) says that 'the growing end of the notochord
tends to be larger than normal and is often very bulky'. He further states that
the notochord is slow in separating from the overlying neural plate or tube, that
it divides frequently into two parts, the more dorsal of which eventually merges
with the neural tissue while the ventral portion becomes the definitive notochord.
This ventral portion is slow in separating from the gut roof, which is therefore
retarded in fusing beneath it. Although in most respects this description is
different from that for Lp/Lp embryos, Griineberg illustrates 9 | T/+ embryos
which appear to have neural plates somewhat larger than those of the normal
embryos, and he states that the tubes close later. He believes that an abnormality
of the notochord which is part of a more general primitive-streak abnormality
accounts for the defects of these mice.
Splotch is another mutation which like Looptail results in an open neural
plate in homozygotes. In all Sp/Sp embryos the plate is open in the hind-limb
region and in 56 per cent, of the cases in the region of the hind brain. Auerbach
(1954, p. 310) reported that the rachischitic area remains proportional to the
length of the embryo but ' the degree of neural overgrowth shows a relative
increase when compared with the growth of normal tissues in the same region'.
These embryos, however, also show a reduction in size or absence of spinal
ganglia and their derivatives and it is suggested that difficulty in closing results
from the incomplete separation of neural crest from neural tube rather than
from overgrowth of neural tissue. Overgrowth is not demonstrable prior to the
time of normal neural-fold closure (Gluecksohn-Waelsch, 1955). In Lp/Lp
embryos the size of the ganglia appears normal but no attempt to count their
number has been made.
Another example of a mutation in mice in which the neural folds occasionally
fail to close is Fused (Theiler & Gluecksohn-Waelsch, 1956). In homozygotes
for this mutation, overgrowth of neural tissue was observed but was usually
manifest either as a greater thickening of the walls of the closed neural tube or
as multiple closed neural tubes. No relative reduction in size of other tissues in
the open region of the tube was reported and no defect in notochord was observed in Fused embryos.
86
L. J. SMITH AND K. F. STEIN
SUMMARY
1. Mice homozygous for the mutation Looptail have shorter bodies than
normal homozygous or heterozygous littermates. Shortening is relatively greater
in the posterior portion of the embryo.
2. The regression or the shortening of the primitive streak is retarded but the
separation of notochord from gut occurs prematurely in the abnormal embryos.
3. Umbilical cords are shorter in Lp/Lp mice than in normal embryos or in
heterozygotes.
4. The possible significance of these facts in relation to the open neural folds
in the homozygous Lp/Lp mice is discussed.
RESUME
U allongement axial du corps de la Souris et son retard chez les
Souris homozygotes Looptail
1. Les souris homozygotes pour la mutation Looptail ont un corps plus court
que chez les souris normales homo- ou heterozygotes de la meme portee.
2. La regression ou le raccourcissement de la ligne primitive est retardee,
mais la separation de la notochorde et de l'intestin a lieu prematurement chez
les embryons anormaux.
3. Les cordons ombilicaux sont plus courts chez les souris Lp/Lp que chez
les normales ou les heterozygotes.
4. La discussion porte sur une relation possible entre ces faits et la persistance
de l'ouverture des plis neuraux chez les souris homozygotes Lp/Lp.
ACKNOWLEDGEMENTS
The authors are indebted to Sandra Durick for photomicrographs and to
Rosemary Lee and Susanne Foster for technical assistance.
This investigation was supported by a research grant, B-684, from the
National Institute of Neurological Diseases and Blindness of the National
Institutes of Health, Public Health Service.
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/ . exp. Zool. Ill, 305-30.
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(1956). A ventral ectodermal ridge of the tail in mouse embryos. Nature, Lond. 177, 787-8.
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L. J. SMITH and K. F. STEIN
AXIAL ELONGATION IN LOOPTAIL MICE
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EXPLANATION OF PLATE
FIG. A. Six 19-day embryo sibs of line 8. From left to right one normal ( + / + ) , 4 abnormal (Lp/Lp),
and one Lp/ + . In abnormal embryos note short body posterior to forelimbs, crick in back, x 1-7.
FIGS. B, C. Abnormal embryo (Lp/Lp) and normal 9-day littermate. x 17.
FIGS. D, E. Successive cross sections of lOJ-day Lp/Lp embryo 499-3. x 100.
FIG. F. Cross-section of normal 9J-day embryo 491—7. Notochord cells form part of roof of gut.
X533.
FIGS. G, H, I. Sections 50, 40, 30 from tip of tail of normal 9i-day embryo 491-7. x 100.
FIGS. J, K, L. Sections 50, 40, 30 from tip of tail of abnormal 9i-day littermate 491-6. x 100.
(Manuscript received 14: vii: 61)