/ . Embryol. exp. Morph. Vol. 58, pp. 35-61, 1980
Printed in Great Britain © Company of Biologists Limited 1980
35
A gradient of morphogenetic information
involved in muscle patterning
By G. J. A. WILLIAMS 1 AND S. CAVENEY 1
From the Cell Science Laboratories, Department of Zoology,
University of Western Ontario, London
SUMMARY
The results of grafts performed in the larva of the beetle Tenebrio molitor reveal that the
position at which certain adult muscle attachments form is specified by a regulative, rather
than a mosaic, mechanism. The results of grafts involving the rotation of squares of integument through 180° or 90°, or the antero-posterior transposition of two adjacent rectangles
of integument, show that the site of muscle attachments in the antero-posterior axis of an
abdominal sternite is specified by an epidermal segmental gradient of positional information.
This gradient is presumed to be identical to the gradient which specifies cuticular patterns in
this insect. There is a good correlation between the effect of grafts on adult muscle morphology and cuticular patterns.
INTRODUCTION
Many of the elements involved in the control of morphogenesis remain to be
determined. However, Wolpert (1977) has suggested that three basic components
can account for the spatial control of interactions between tissues during animal
development: cell movement, cell contact and gradients of positional information. The gradients of positional information form a spatial map within the
animal which governs and coordinates the activities of individual cells.
At present the rules and nature of positional information are incompletely
understood, as there appear to be few developing systems which are simple
enough to analyse. However, studies of insect development have proved particularly fruitful in yielding information about the control of pattern development. Insects have the following advantages for this type of analysis: (1) Changes
in body form are largely the result of changes in the epidermis which is an unfolded, or simply folded, monolayer of cells. (2) Patterns in the epidermis are
reflected in the cuticle that the epidermal cells secrete. It is therefore possible to
analyse epidermal patterns by looking at the polarity and patterns of cuticular
structures. (3) Extensive changes in body form occur at metamorphosis. These
post-embryonic changes, by virtue of the size of the insect, are accessible to
surgical manipulation. (4) Insects are segmentally repetitive, or metameric.
1
Authors' address: Department of Zoology, University of Western Ontario, London,
Ontario N6A 5B7, Canada.
36
G. J. A. WILLIAMS AND S. CAVENEY
Consequently, by using single segments, or limbs, it has proved possible to deduce
rules of development applicable to the whole insect.
These advantages have been exploited by Locke (1959, 1960) and Lawrence
(1973) (amongst others), who have demonstrated that an axial gradient of positional information controls the development, and also the polarity, of epidermal
cells in the insect segment. The gradient repeats itself in each segment; and the
segmental boundaries separate the high point of one segmental gradient from
the low point of the next.
It is of fundamental interest to discover whether Locke's unidimensional
segmental gradient could be used to govern the development of the overall,
three-dimensional, form of an insect segment. That is, can tissues which interact
with the epidermis utilize epidermal positional information ? The development
of insect muscle attachments provides a suitable system to try to answer this
question as insect muscles attach directly to the epidermis. Furthermore, the
positions of some muscle attachments change at metamorphosis. These features
enabled us to narrow the problem to a more specific question. That is, does the
epidermal gradient govern the position at which new muscle attachments form
during metamorphosis ?
Our approach to answering this question has been to use grafting experiments
similar to the pioneer experiments of Locke on Rhodnius. We have repeated and
extended the work of Caveney (1973) using the abdominal sternite integument of
the beetle, Tenebrio molitor. This insect was chosen because the ventral abdominal
retractor muscles undergo extensive changes in position within the abdominal
sternites during metamorphosis (Williams & Caveney, 1980). The system is
particularly convenient as muscle relocations take place in two dimensions, so
the need to use sectioned material is obviated.
The effect of reorientating and transposing grafts of larval integument on the
position of two adult muscles in Tenebrio is described. Non-regulative mechanisms governing the position at which adult muscle attachments are formed do
not adequately explain the results of these grafting experiments. Instead the
results support our hypothesis that an epidermal gradient determines the position
in the antero-posterior axis of the sternite at which adult muscle attachments are
formed.
MATERIALS AND METHODS
Beetle culture
Last-instar larvae of Tenebrio molitor were taken from a culture reared on a
wholewheat flour mixture as previously described (Williams & Caveney, 1980).
Operational technique
All operations were performed on abdominal sternite 4 of last-instar Tenebrio
larvae, between eyestages 3 and 8. (Staging technique of Stellwaag-Kittler, 1954.)
Larvae were anaesthetized for grafting operations by immersion in distilled water
37
Gradient control of muscle patterning
Ant.
Zone I
'•*•*
Zone II
Post.
Ant.
Zone I
Zone II
Post
Fig. 1. (A) Diagram of the larval sternite showing the position of the retractor
muscles Rl, R2, R3 and R4, and of graft area A (used in 90°- and 180°-rotated
grafts). The muscles are shown in their entirety on one side of the sternite, and the
position of their anterior attachments are shown as black islands in the Zone-I (ZI)
cuticle on the other side of the sternite. Ant., Anterior; Post., Posterior; ZII, Zone II a flexible region of cuticle between the abdominal segments; M, mid-line of the
sternite; b, marker bristle. (B) Diagram of the larval sternite showing the position of
graft area B (used in antero-posterior exchange grafts). (C) Micrograph of a typical
graft at graft-area A. There is a rim of melanized blood around the graft margin.
(D) Micrograph of a typical graft at graft-area B. Scale bar is 1 mm.
for a period of about 6 h at room temperature, as previously described (Williams
& Caveney, 1980). During operations, the larvae were held in place in a bed of
plasticine with two soft plasticine strips.
Integumental grafts were excised under a low-power dissecting microscope
using a razor blade broken to a point. Grafts were removed with watchmakers'
forceps, rotated or transposed, replaced in the same larva, and the wound gently
dried with filter paper. After grafts became sealed in place with dried haemolymph, operated larvae were placed individually in petri dishes and allowed to
undergo metamorphosis. No food or water was provided.
38
G. J. A. WILLIAMS AND S. CAVENEY
Two different graft areas were used in these operations (Fig. 1). (a) Graft area
A (Fig. la) was used for grafts involving 180° and 90° rotations. The position
and size of the graft square was established by the position of a marker bristle
(b) and the mid-line of the larva. The square was approximately 0-8 x 0-8 mm,
and composed of approximately 8000 epidermal cells, (b) Graft area B (Fig. 1 b)
was used for grafts involving the antero-posterior exchange of two adjacent
rectangles of integument (A <-> P grafts). Each rectangle was approximately
0-8 x 0.5 mm and composed of approximately 5000 epidermal cells.
As grafts were performed on only one side of the sternite, the unoperated side
of the sternite served as an internal control for each operation. This served to
compensate for the natural variability of muscle patterns in different insects.
The lateral position of both graft areas A and B also prevented damage to the
ventral nerve cord, and extrusion of a centrally located ribbon of fat body during
operations.
Controls
Controls for 180°-rotated and A *? P grafts consisted of sham operations in which
integumental grafts were removed and replaced on the same larva without
rotation, or transposition. Homograft operations between larvae of opposite sex
(Williams & Caveney, 1980) also provided a source of controls for 180°-rotated
grafts. Controls for 90°-rotated grafts consisted of sham operations in which
integumental grafts were rotated in situ through 90° before being returned to
their original position.
Quality control
Larval exuvia from operated animals were mounted in glycerol, and scrutinised using phase-contrast microscopy. In order to reduce the contribution of
wound-induced effects, adult muscle patterns were only analysed in insects
which showed a minimal level of dark (melanized) blood around the graft on
the larval exuvium (Fig. 1 c, d). Adult insects which had patches of untanned
cuticle on the operated sternite, indicating damage to the underlying epidermal
cells, were also eliminated from the study.
Analysis
Adult beetles were killed 24-30 h after eclosion. At this time the sternite
epidermis has deposited a single unidirectional layer of endocuticle which is
oriented parallel to the body axis (Caveney, 1973). Adult beetles were dissected
from the dorsal surface immersed in ethanol, as previously described (Williams
& Caveney, 1980). Preparations consisting of the ventral abdominal integument
and associated retractor muscbs were then dehydrated, cleared and mounted in
glycerol for preliminary observation in polarized light. Selected preparations
were subsequently rinsed in 100 % ethanol, mounted in Canada Balsam and
photographed in polarized light.
Use of polarization microscopy made it possible to relate graft-induced changes
Gradient control of muscle patterning
39
in the position of retractor muscle fibres to changes in the orientation of the
birefringent endocuticle. A Senarmont compensator was inserted between the
specimen and the analyser to intensify the contrast of birefringence patterns in
the endocuticle. By slightly rotating the analyser from the 'crossed-polars'
position, the birefringence due to endocuticle microfibrils oriented at 90° to
the segment axis could be extinguished (Figs. 2d, 3d).
A more detailed analysis of cuticular patterns in operated insects was performed using endocuticle peels, examined in phase contrast (technique in
Caveney, 1973). The sites of attachment of individual muscle fibres can be seen
in these peels as small distortions in the normal orientation of the endocuticle
(Fig. 4d, arrow). Also, the position of the graft margin can be determined, in
adults, using these peels. The graft margins are delineated by slight distortions in
the cuticle, and a reduced density of pit glands (Caveney, 1973).
RESULTS
There are two principal alternative mechanisms by which the epidermis could
determine the position at which adult retractor muscle attachments are formed
in Tenebrio. (a) Non-regulative, or mosaic, mechanisms. These require the presence
of two quite distinct types of epidermis in each sternite: pre-determined muscle
attaching epidermis composed of 'tendinous epidermal cells' (Lai-Fook, 1967)
and 'general' epidermis, (b) Regulative mechanisms. These require that all
epidermal cells have the capacity to become tendinous cells, but will only do so
if certain conditions are fulfilled. The specific hypothesis which we have been
testing is that the capacity for an epidermal cell to become a tendinous cell is
defined by its position in an epidermal segmental gradient.
In order to test the gradient hypothesis we have performed grafts which have
predictable results in terms of the disruptions they produce in the topography
of the gradient. Subsequently graft-induced changes in the position of the anterior
attachment sites of the adult Rl or R2 muscles (Williams & Caveney, 1980) are
analysed in terms of these predicted changes, and compared with graft-induced
changes in the gradient-controlled cuticular patterns. In unoperated insects the
tendinous cells to which the anterior ends of the Rl and R2 muscle fibres
attach form narrow strips oriented approximately at right angles to the body
axis (Fig. 6a and la, control Rl and control R2).
Theory behind graft-induced changes in the topography of the gradient
Grafts involving rotation through 180°
In a model of a unidimensional gradient this operation generates a valley and
a peak in the gradient (Fig. 2a), which are represented on a contour map as two
whorls: one at the anterior and one at the posterior of the graft region (Fig. 2b).
Furthermore, three horizontal levels of contour value identical to the level
normally used for adult Rl muscle attachments are generated by 180° grafts
40
G. J. A. WILLIAMS AND S. CAVENEY
Ant.
Ant.
Post.
Ant.
Post
Post.
The gradient contour maps shown in the following threefigures(Figs. 2 b, 3 b, 4 b)
are based on computer simulations of gradient-controlled integumental patterns in
Rhodnius (Lawrence, Crick & Munro, 1972). They show an early stage in pattern
relaxation.
Fig. 2. Diagrams illustrating the effects of 180°-rotated grafts according to the
gradient model. (A) The segmental gradient is represented by a wedge. The base of
the wedge (/) is the length of the segment, and the height of the wedge Qi) is the
height of the gradient. A 180°-rotated graft produces a valley and a peak in the
gradient. The gradient level specifying for the attachment of the adult Rl muscle is
indicated by a heavy line. Ant., Anterior of the sternite; Post., Posterior of the sternite.
(B) Contour diagram showing the effect of a 180°-rotated graft. The position of the
peak and the valley in the gradient are indicated by dashed lines. The gradient level
specifying for the attachment of the adult Rl muscle is indicated by heavy lines. The
three horizontal levels of identical value specifying for Rl muscle attachment are designated x, y and z, although y and z are actually one continuous contour line. Ant.,
Anterior; Post., Posterior. (C) Diagram of an endocuticle pattern resulting from a
180°-rotated graft, drawn from a phase-contrast photo-montage. The graft area lies
within the brackets. The endocuticularmicrofibrils are oriented at 90° to the contours
of the gradient. Small arrows indicate the polarity of the endocuticle. Large arrows
indicate the position of the peak and valley in the gradient produced by the 180°
rotated graft, where the endocuticular microfibrils are oriented at 90° to the body
axis. The gradient level specifying for adult Rl muscle attachment is indicated by
dashed lines. In this example all Rl muscle attachments (Rl, black islands) are at level
x. M, Midline of the sternite. Ant., Anterior sternite boundary; Post., Posterior
sterniteboundary. (D) Polarized light micrograph of the endocuticle peel diagrammed
in (c). Arrows indicate the dark areas where endocuticular microfibrils are oriented
at 90° to the body axis, showing the position of the peak and valley in the gradient.
Ant., Anterior sternite boundary; Post., Posterior sternite boundary.
Gradient control of muscle patterning
Ant.
'Post.
Ant.
Post.
Post.
Fig. 3. Diagrams illustrating the effect of A <-» P grafts, according to the gradient
model. (A) The segmental gradient is represented by a wedge. The base of the wedge
(1) is the length of the segment, and the height of the wedge (h) is the height
of the gradient. An A ^ P graft produces a valley and a peak in the gradient.
The gradient level specifying for the attachment of the adult Rl muscle is indicated
by a heavy line. Ant., Anterior of the stemite; Post., Posterior of the sternite.
(B) Contour diagram showing the effect of an A <-> P graft. The position of the peak
and the valley in the gradient are indicated by dashed lines. The gradient level
specifying for the attachment of the adult Rl muscle is indicated by heavy lines.
The three horizontal levels of identical value specifying for Rl muscle attachment are
designated x', y' and z', although y' and z' are actually one continuous contour line.
Ant., Anterior; Post., Posterior. (C) Diagram of an endocuticle pattern resulting from
an A «-> P graft, drawn from a phase-contrast photo-montage. The graft area lies
within the brackets. The endocuticle microfibrils are oriented at 90° to the contours
of the gradient. Small arrows indicate the polarity of the endocuticle. Large arrows
indicate the position of the peak and valley in the gradient produced by the A <-> P
graft, where the endocuticular microfibrils are oriented at 90° to the body axis. The
gradient level specifying for adult Rl muscle attachment is indicated by dashed lines.
In this example all Rl muscle attachments (Rl, black islands) are at level x'. M, Midline of the sternite; Ant., Anterior sternite boundary; Post., Posterior sternite boundary. (D) Polarized light micrograph of the endocuticle peel shown in (c). Arrows
indicate the dark areas where endocuticular microfibrils are oriented at 90° to the
body axis, showing the position of the peak and valley in the gradient. Ant.,
Anterior sternite boundary; Post., Posterior sternite boundary.
42
G. J. A. WILLIAMS AND S. CAVENEY
(Fig. 2b, c). These three levels have been designated x, y and z; where x is
the most anterior, y an intermediate, and z the most posterior level. (It is not
important from the point of view of interpreting the results of grafts that levels
y and z actually represent one continuous contour.)
In Tenebrio the orientation of the microfibrils in the first unidirectional layer
of the endocuticle is controlled by an epidermal gradient (Caveney, 1973). These
microfibrils are normally laid down parallel to the body axis, and at right angles
to the contours of the gradient; 180° grafts therefore produce characteristic
changes in the orientation of the endocuticle. The position of the peak and valley
in the gradient are indicated by regions where the endocuticular microfibrils
are oriented at right angles to the body axis (Fig. 2c, d, arrows). Therefore, as
the endocuticle is birefringent, the position of the valley and peak in the gradient,
and hence the approximate position of levels x, y and z, can be established by
examining the endocuticle in polarised light (Fig. 2 d).
Grafts involving antero-posterior exchange of two adjacent rectangles
This operation generates changes in the topography of the gradient which are
similar, though not identical, to those produced by 180° grafts. A<->P grafts
produce a peak and a valley in the gradient (Fig. 3 a, b) without graft rotation.
This operation also produces three horizontal levels of identical value to the
level normally used for attachment of adult Rl muscle fibres. These three levels
have been designated, from anterior to posterior, x', y' and z' (Fig. 3b, c). The
position of the peak and valley in the gradient, from which the approximate
positions of levels x', y' and z' can be inferred, are indicated by regions where
the endocuticular microfibrils are oriented at right angles to the body axis
(Fig. 3 c, d, arrows).
Grafts involving rotation through 90°
This operation generates rather different distortions in the gradient, where the
contours follow an S-shaped curve (Fig. Aa-c). The level corresponding to the
level normally used for adult R2 muscle attachments is illustrated (Fig. Aa-c,
dashed line). A typical cuticular pattern resulting from 90° grafts is shown in
Fig. Ac.
Relaxation of graft-induced changes in the topography of the gradient
Before presenting results of particular grafting experiments, further discussion of the theory underlying these experiments is required. In particular,
relaxation effects make an important contribution to the contour pattern generated by any particular graft.
Relaxation effects cause the initial rather angular topography which is generated by the grafts to be smoothed out with time. Relaxation is of two distinct
Gradient control of muscle patterning
43
• Ant.
Ant."
Post.
Ant.
Post
Fig. 4. Diagrams illustrating the effect of 90°-rotated grafts, according to the gradient
model. (A) The segmental gradient is represented by a wedge. The base of the wedge
(/) is the length of the segment, and the height of the wedge (h) is the height of the
gradient. The effect of a 90°-rotated graft on the gradient topography is indicated.
The gradient level specifying for the attachment of the adult R2 muscle is indicated
by a dashed line. Ant., Anterior of the sternite; Post., Posterior of the sternite. (B)
Contour diagram showing the effect of a 90° graft. The contours followed a tilted
S-shaped curve. The gradient level specifying for the attachment of the adult R2
muscle is indicated by a dashed line. Ant., Anterior; Post., Posterior. (C) Diagram of
an endocuticle pattern resulting from a 90°-rotated graft. The graft area lies within
the brackets. The endocuticle microfibrils are oriented at 90° to the contours of the
gradient. Small arrows indicate the polarity of the endocuticle. The gradient level
specifying for adult R2 muscle attachment is indicated by a dashed line. M, Mid-line
of the sternite; Ant., Anterior; Post., Posterior. (D) Phase-contrast micrograph of a
small region of an endocuticle peel. The white slits are pore canals oriented parallel
to the endocuticle microfibrils. The arrow indicates a region of distorted endocuticle
at the site of a muscle attachment.
EMB 58
44
G. J. A. WILLIAMS AND S. CAVENEY
Post.
Post.
Fig. 5. Diagrammatic representation of gradient relaxation after 180°-rotated and
A <-> P grafts. The abscissa represents the length of the sternite, and the ordinate
represents the height of the gradient. Ant., Anterior; Post., Posterior. Rl, The
gradient level specifying for adult Rl muscle attachment. Dashed lines represent
successive stages of relaxation after each operation. Relaxation after 180°-rotated
and A <-> P grafts is due to two processes: intercalary cell proliferation; and re-setting
of gradient values. (A) Relaxation after a 180°-rotated graft. The anterior sternite
boundary is pushed forward as a result of intercalary regeneration. Relaxation at
the peak (P) involves re-setting of gradient values, which results in the fusion and
subsequent loss of levels y and z as the peak drops below the Rl muscle attachment
level. Relaxation occurs more rapidly at the peak (P) than at the valley (V) as anterior
cells undergo more relaxation than cells from posterior regions. (B) Relaxation after
an A <-> P graft. There is more intercalary regeneration than in a 180°-rotated graft
due to the greater total extent of graft-induced vertical slopes. Relaxation at the peak
(P) is more extensive than relaxation at the valley (V). However, as the peak has
further to fall than in the case of 180°-rotated grafts, this relaxation does not result in
the loss of levels y' and z' in our experiments. (Given sufficient time we would predict
the loss of levels y' and z', after A <-• P grafts, in the same way that y and z are lost
after 180°-rotated grafts.)
types: relaxation resulting from wound-induced cell division, which occurs at
the graft margins; and gradient-induced relaxation, or regulation. (A schematic
diagram of the effects of regulation after 180° graft rotation and A<-> P grafts is
presented in Fig. 5.)
Gradient control of muscle patterning
45
The mechanisms underlying the relaxation effects which lead to pattern reconstitution in Tenebrio have not been clearly established. Relaxation may occur
by epidermal cell migration, by cell proliferation, by cells re-setting their positional values, or by a combination of these mechanisms. In some insects, relaxation after 90°-rotated grafts involves bulk re-rotation of the entire graft (Bohn,
1974; Lawrence, 1974; Niibler-Jung, 1974). However, in Tenebrio, because there
are no appropriate integumental markers, it is not possible to distinguish
between relaxation due to cells re-setting their positional values, and rerotation after 90°-rotated grafts. An empirical observation is that, after any
particular grafting operation, cells that were originally anterior in the segment
undergo more relaxation than those from posterior regions.
Regulation occurs rapidly at the regions of greatest induced discontinuities in
the gradient (i.e. at vertical slopes). Regulation at vertical slopes involves the
intercalation of missing gradient values by means of cell proliferation and resetting of positional values; and the extent of this intercalary regeneration will
depend on the number of missing values. That is, it will be great when there is a
large discontinuity between apposed values at the top and bottom of a vertical
slope. (Under our grafting conditions it will be slightly greater after A «-> P, than
after 180°-rotated grafts, due to the greater number and extent of graft-induced
vertical slopes, Fig. 5.) In Tenebrio the results of intercalary regeneration can
be seen after 180°-rotated and A<-+ P grafts. The anterior sternite boundary is
pushed forward on the operated side of the insect (Figs. 6 a, Id), reflecting an
overall increase in the length of the sternite on this side. Also, prominent
integumental ridges are often present at the graft boundaries. These are comparable to the wrinkles which are found after certain grafting operations in
Dysdercus (Niibler-Jung, 1977), which have been convincingly attributed to
intercalary regeneration.
In Tenebrio, regulation (as opposed to wound-induced relaxation) does not
occur in the pupal stage. This may be due to the absence of proliferative epidermal cell divisions in the pupa (Caveney, 1973), or because epidermal cells are
not free to migrate. Nonetheless, relaxation is proportional to the time which
elapses after an operation and will therefore be maximal in larvae operated upon
well before pupation. In choosing a time to perform grafting operations we aimed
to compromise between obtaining a maximal effect in terms of changing the
gradient topography and minimal wounding effects on muscle development.
Muscle patterns after 180° grafts
The adult Rl muscle was used for analysis of the results of 180°-rotated grafts.
The origin of this muscle lies outside the graft area (graft area A in Fig. 1 a) in
the larva, but inside the graft area in the adult (see fig. 5, Williams & Caveney,
1980). Sham operations performed at graft area A invariably result in normal
adult Rl muscle patterns (n = 28). In contrast, sham operations often produce
abnormalities in the arrangement of the adult R2 fibres. The origin of the R2
4-2
46
G. J. A.WILLIAMS AND S. CAVENEY
Ant.
Ant.
Fig. 6. Three categories of adult Rl muscle patterns in sternites with 180°-rotated
grafts. (A) Polarized light micrograph of an adult sternite showing Rl and R2 muscles
on both control and operated sides of the sternite. The 180°-rotated graft has resulted
in an Rl muscle with attachments at levels x and y, and there are bridging muscle
fibres between levels x and y. Arrows show regions where the endocuticular microfibrils are oriented at 90° to the body axis, indicating the position of the peak and
valley in the gradient. The anterior sternite boundary is bowed forward on the
operated side due to intercalary regeneration. Ant., Anterior; Post., Posterior; M,
Mid-line of the sternite. Scale bar is 0-5 mm. (B) Polarized light micrograph of the
Gradient control of muscle patterning
47
muscle lies within graft area A in the larva, and the R2 muscle was always broken
during sham operations and 180° grafts. Therefore we have not attempted to
analyse gradient-controlled changes in the arrangement of adult R2 muscle
fibres after 180°-rotated grafts. In some adult insects, particularly when the adult
R2 muscle fibres are abnormally arranged, it is not at first obvious whether
individual muscle fibres belong to the Rl or R2 muscle. We have designated
them as belonging to R l or R2 on the basis of the position of their insertions,
on the segment boundary.
Table 1. Frequency of attachment ofRl muscle fibres to the three attachment levels
(x, y and z) predicted to be generated by 180° rotations of larval integument
in = 37)
Attachment level of Rl muscle fibres
Cuticle pattern*
Unrelaxed pattern
Partially relaxed pattern
Totally relaxed pattern
Level x
Level y
Levels x and y\
Levels y and z
10
8
3
1
—
—
11
3
—
1
—
—
* Unrelaxed patterns = patterns where the two lines due to endocuticle oriented at 90° to
the body axis (Fig. 2d, arrows) are distinct. Partially relaxed patterns = patterns where these
two lines are indistinct. In totally relaxed patterns these lines are not visible; and there is no
cuticle with reversed polarity in the sternite (Fig. 2 c). The same criteria have been used for
analysis of A <-> P grafting operations.
t Includes bridging patterns.
In 21 larvae with 180°-rotated grafts which were dissected 1-2 days after the grafting operation, 6 showed severed Rl muscles on the operated side of the segment. See text p. 22.
The results of 37 180°-rotated grafts are shown in Table 1; and selected
examples of some of the different Rl muscle patterns produced by 180°-rotated
grafts are shown in Fig. 6. Table 1 shows that Rl muscle fibres may attach at
any of the equivalent gradient levels x, y and z that are predicted to be generated
by rotating a graft through 180°. Various patterns are possible due to muscle
operated side of the sternite from another beetle with a 180°-rotated graft. All Rl
muscle fibres attach at level x. Arrows indicate the position of the peak and valley
in the gradient. (C) Diagram of the endocuticle from the half-sternite shown in 5 (b).
The graft outline is indicated by brackets. Small arrows show the polarity of the
endocuticle. The gradient level specifying for adult Rl muscle attachment is shown as
dashed lines. All Rl musclefibres(Rl, black islands) attach at level x. (D) Polarized
light micrograph of a third half-sternite with a 180°-rotated graft. The Rl muscle has
attachments at levels x and y. A few muscle fibres bridge between levels x and y.
Arrows indicate the position of the peak and valley in the gradient. (E) Diagram of
the endocuticle from the half-sternite shown in 6(d). Conventions are as in 6(c).
Rl muscle fibres (Rl black islands) can be seen to attach at levels x and y.
48
G. J. A. WILLIAMS AND S. CAVENEY
Table 2. Relationship of" bridging' Rl muscle patterns to the cuticle pattern
following 180° rotation of larval integument
Attachment level o f bridging'Rl
Attachment level of' non-bridging'
fibres
fibres
xtoy
y
Cuticle pattern:
Unrelaxed pattern
Partially relaxed patterns
4
—
xtoy
x and y
1
—
fibres attaching in different combinations at these three levels, but the commonest
result of 180°-rotated grafts is to find all the Rl muscle fibres attached at level x.
In some sternites 'bridging' muscle fibres extend between contours x and y
(Table 2). These fibres, unlike the other R l muscle fibres, attach within the
sternite at both ends and are therefore non-functional in the sense that they
cannot bring about the relative movement of integumental plates. In all other
respects they are identical to the other Rl fibres.
Muscle patterns after A <-> P grafts
The adult Rl muscle was used for analysis of the results of A «-»P grafts. The
origin of this muscle lies outside the graft area (graft area B, Fig. 1 b) in the larva,
but inside the graft area in the adult. Nineteen out of 26 sham operations performed at graft area B resulted in normal adult Rl muscle patterns, while seven
sham operations resulted in the anterior attachments of the adult Rl fibres being
slightly more posterior than those on the control side of the insect. These slight
deviations from the normal arrangement probably result from cutting the R l
muscle during grafting. They are less extensive than the changes seen after
A *-* P grafts. In contrast, sham operations often produce considerable abnormalities in the arrangement of the adult R2 muscle fibres, and so we have not
Table 3. Frequency of attachment ofRl muscle fibres to the three attachment levels
(x\ y' and z') predicted to be generated by antero-posterior exchanges of
larval integument (n = 29)
Attachment level of Rl muscle fibres
K
~~
i
Level x'
Level/
Level z'
Levels
x'and/
2
3
—
—
3
—
—
•»
Levels
/andz'
Levels xf,
/andz'f
7
1
—
7
1
—
Cuticle pattern
Unrelaxed pattern
3
Partially relaxed pattern 1
Totally relaxed pattern —
1
—
t Includes bridging patterns.
In 21 larvae with A <->• P grafts which were dissected 1-2 days after grafting, 13 showed
severed Rl muscles on the operated side of the segment. See text p. 22.
Gradient control of muscle patterning
49
attempted to analyse gradient-controlled changes in the adult R2 muscle after
A <-> P grafts. The results of 29 A «-»P grafts are shown in Table 3; and selected
examples of some of the different Rl muscle patterns are shown in Fig. 7.
Table 3 shows that Rl muscle fibres may attach at any of the equivalent gradient
levels x', y' and z' predicted to be produced by A <-* P grafts. Table 4 shows the
distribution of A <-» P grafts which produce bridging fibres in operated insects.
Bridging fibres usually extend between levels x' and y', but in one insect they
were also seen to extend from level x' to level z'.
Table 4. Relationship of'bridging'' Rl muscle patterns to the adult cuticle pattern
following antero-posterior exchanges of larval integument
Attachment level of Rl muscle fibres
Bridging between levels . . .
Other Rl fibres at level...
Cuticle pattern:
Unrelaxed pattern
Partially relaxed pattern
x' and y'
z'
3
x' and y'
y' and z'
1
1
-v
x' and y'
x', y' and z'
1
x and y'; x' and z'
z'
1
Muscle patterns after 90° grafts
In order to demonstrate gradient control of muscle patterns using 90° grafts,
a slightly different approach was adopted to that described for 180° and A <-> P
grafts. The results of 90° grafts have been analysed in terms of their effect on
the adult R2 muscle, as we predicted that the arrangement of this muscle would
be maximally affected by such grafts. As the origin of the R2 muscle lies within
the graft area (graft area A, Fig. 1 a) in the larva, care was taken not to snap the
muscle during operations; the graft square was rotated in situ, and not lifted
from the animal.
All 90°-rotated grafts were performed by rotating the graft square in an anticlockwise direction. Two considerations discouraged the use of 90°-clockwiserotated grafts. (1) As cells which are originally anterior in position undergo more
relaxation than those from posterior regions of the sternite, it seemed likely that
grafts rotated 90° anticlockwise would produce more pronounced changes in
gradient topography in the region of R2 attachment than grafts rotated 90°
clockwise. (2) Grafts rotated 90° clockwise cause the larval R2 and R3 muscles
to twist round one another. It seemed likely that this by itself might have an
effect on migration of the R2 muscle during metamorphosis, and consequently
might mask gradient-controlled effects. In fact preliminary experiments with
90°-clockwise rotations resulted in the R2 muscle fibres attaching outside the
graft area in the adult insect, and were therefore not useful for the purpose of
determining graft-induced changes in the R2 muscle fibre patterns.
Two difficulties were encountered with 90°-anticlockwise-rotated grafts.
50
G. J. A. WILLIAMS AND S. CAVENEY
Ant.
Post.
Fig. 7. Three categories of adult Rl muscle patterns in sternites with A <-> P grafts.
(A) Polarized light micrograph of an adult sternite showing Rl and R2 muscles on
both control and operated sides of the sternite. The A <-> P graft has resulted in an
Rl muscle with attachments at levels x', / and z', and there are bridging muscle fibres
between levels x' and y'. Arrows show regions where the endocuticular microfibrils
are oriented at 90° to the body axis, indicating the position of the peak and valley
in the gradient. The anterior sternite boundary is bowed forward on the operated side
due to intercalary regeneration. Ant., Anterior; Post., Posterior; M, Mid-line of the
sternite. Scale bar is 0-5 mm. (B) Polarized light micrograph of the operated side of
the sternite from another beetle with a 180°-rotated graft. Rl muscle fibres attach at
Gradient control of muscle patterning
51
(1) Due to the curvature of the larval sternite, these grafts were never flush with
the surrounding integument. Consequently, the quality of 90°-rotated grafts was
not as high as that of 180°-rotated and A <-* P grafts. (2) Due to relaxation, it is
difficult to induce sufficient distortions in the gradient topography, to produce
detectable effects on the adult R2 muscle morphology. Therefore in order to
produce detectable changes in the adult R2 muscles, the majority of 90°-rotated
grafts were performed on larvae at a late eyestage (eyestage 7).
Sham operations for 90°-rotated grafts were performed at graft area A (Fig.
1 a), and consisted of rotating the graft in situ through 90° anticlockwise before
returning it to its original position. Care was taken not to snap the R2 muscle
during these operations. Fifteen out of 20 sham operations resulted in normal
adult R2 muscle patterns; while five sham operations resulted in adult R2
muscles which had a few fibres extending in an anterior, or antero-lateral,
direction. These slight abnormalities are attributed to wounding produced by
the grafting operations. Their exact cause is not known.
The results of some 90°-rotated grafts are shown in Fig. 8. Using the position
of the insertions of individual muscle fibres on the segment boundary to determine
whether the fibres belong to muscle Rl or R2, it becomes obvious that the origins
of the Rl muscle fibres are at separate sites from those of R2 fibres. The results
of 90°-rotated grafts (n = 19) show a good correlation between the attachment
sites of R2 muscle fibres and the pattern of gradient contours as determined from
an examination of cuticular patterns (Figs. 4, 8). Results presented in Fig. 8 show
a temporal series of relaxation patterns produced by 90°-rotated grafts. These
results show the correlation between the position of R2 muscle attachments and
the orientation of endocuticular microfibrils at different stages of pattern
regulation.
As in sham operations, 90°-rotated grafts produce adult R2 muscles with a
few anomalous fibres which extend in an anterior, or antero-lateral, direction
(Fig. 8 c, arrow). These fibres are presumed to be wound-induced. They occur
at a higher frequency after 90°-rotated grafts (49 % of operated insects) than
after sham operations (25 % of operated insects). This difference is attributed to
the unavoidably higher level of wounding in insects with 90°-rotated grafts,
because the curved grafts do not fit flush with the surrounding integument.
levels y' and z'. Arrows indicate the position of the peak and valley in the gradient.
(C) Diagram of the endocuticle from the half-sternite shown in 6(6). The graft outline is indicated by brackets. Small arrows show the polarity of the endocuticle. Level
x' is shown as a dashed line. Rl muscle attachments (black islands) appear at the
positions of levels y' and z'. (D) Polarized light micrograph of third half-sternite
with an A *-*• P graft. All Rl muscle attachments are at level z'. Arrows indicate the
position of the peak and valley in the gradient. (E) Diagram of the endocuticle from
the half-sternite shown in 7 (d). Conventions are as in 7 (c). The gradient level for Rl
muscle attachment is shown as dashed lines. All Rl muscle fibres attach at level z'.
52
G. J. A. WILLIAMS AND S. CAVENEY
Ant
Ant.
Post
Fig. 8. Examples of adult R2 muscle patterns in sternites with 90°-anticlockwiserotated grafts. The diagrams show a temporal sequence of the relaxation patterns
produced by 90°-rotated grafts. (A) Polarized light micrograph of an adult halfsternite showing Rl and R2 muscles on the operated side of the insect. The 90°rotated graft has produced an R2 muscle that tilts at an unusual angle within the
graft area. This insect was killed before the adult endocuticle was laid down. Ant.,
Anterior; Post., Posterior. (B) Diagram of the endocuticle from another half-sternite
showing similar muscle patterns to those of the insect shown in 8 (a). The graft outline is indicated by brackets. Small arrows show the polarity of the endocuticle. Rl
muscle attachments are grey islands. R2 muscle attachments are black islands.
Gradient control of muscle patterning
53
DISCUSSION
Although much work has been done on the internal differentiation of single
muscle fibres, there is a paucity of information on the control of the spatial
arrangement of fibres during muscle morphogenesis. In a previous paper
(Williams & Caveney, 1980) we have described the extensive, yet precise, rearrangement of retractor muscles that occurs during metamorphosis in Tenebrio.
What are the factors which control these muscle rearrangements ?
Morphogenetic factors influencing the development of
adult retractor muscles
On a priori grounds, it seemed likely that the epidermis might determine the
position at which adult retractor muscle attachments are formed. Epidermal
patterns are established before muscle patterns during metamorphosis; and
muscle rearrangement in the pupal stage involves migration of muscles over the
epidermis (Williams & Caveney, 1980). Furthermore, the formation of an insect
muscle attachment involves specialization in the epidermis which results in the
formation of tendinous epidermal cells. Therefore the development of muscles
and epidermis must be interrelated. Some observations from the literature
support the idea that the epidermis has an important morphogenetic influence
on muscle development in insects. Fournier (1968) has shown that when the
development of apodemes in the legs of Carausius is suppressed, using microsurgery, this interferes with the differentiation and segregation of the muscles
which would normally insert upon these apodemes. In separate studies of insect
muscle morphogenesis during metamorphosis, Sahota & Beckel (1967 c, b), and
Deak (1978), using Galleria and Drosophila respectively, have come to the
conclusion that the orientation of adult muscle fibres is defined by the topographical relationship between muscle precursors and muscle attachment sites
on the epidermis.
The results of grafting operations (to be discussed further below) support the
idea that in Tenebrio the epidermis plays a dominant role in determining the
Muscle attachments are oriented at 90° to the orientation of endocuticular microfibrils. M, Mid-line of the sternite. (C) Polarized light micrograph of the operated
side of the sternite from another beetle with a 90°-rotated graft. The graft has produced an R2 muscle that is tilted, within the graft area. Arrow indicates R2 fibres
which extend in an antero-lateral direction to attach outside the graft area. These
fibres are wound-induced. (D) Diagram of the endocuticle from the half-sternite
shown in 8 (c). Conventions are as in 8 (b). Dashed lines illustrate that the R2 muscle
attachments are oriented at 90° to the orientation of the endocuticular microfibrils.
Arrow indicates outlying Rl muscle attachments which were formed by myoblasts
which failed to traverse the graft boundary. (This is the result of wounding at the
graft margin). (E) Diagram of the endocuticle from another beetle with a 90°-rotated
graft. Conventions are as in (8 b). The endocuticular pattern is more relaxed than
the endocuticle patterns shown in 8(6) and 8(J). However, muscle attachments are
still oriented at 90° to the orientation of the endocuticular microfibrils.
54
G. J. A. WILLIAMS AND S.CAVENEY
position at which adult retractor muscle attachments are formed. However, other
factors also have morphogenetic effects on the development of these muscles.
One important morphogenetic influence is the larval muscles from which the
adult muscles are derived. During normal development the myoblasts which
give rise to the adult muscles are closely associated with the degenerating larval
muscles. In Drosophila the dorsal longitudinal muscles of the thorax, which are
thought to form in close association with preexisting larval muscles, have been
found to be less plastic in their ability to utilize muscle attachment sites on the
epidermis than the dorsoventral muscles, which form independently of preexisting larval muscles (Deak, 1978). In Tenebrio the state of integrity of the
larval retractor muscles during metamorphosis probably determines whether
myoblasts are free to interact with all available prospective adult muscle attachment sites on the epidermis. When the larval muscles remain intact throughout
metamorphosis myoblasts are constrained in their movements by their close
association with the larval muscles. Therefore, both the epidermis and larval
muscles influence the morphology of the adult muscles, and the contribution
made by both must be considered when analysing the results of grafting operations.
The determination of muscle attachment sites is regulative
Several general observations on the results of different grafts support the idea
that a regulative, rather than a mosaic, mechanism determines the position of
adult retractor muscle sites in Tenebrio.
(1) Grafts which involve rotating the prospective adult attachment sites
through 90° do not result in adult muscles which attach at right angles to their
normal position, as would be expected if the attachment sites were rigidly
determined.
(2) The majority of 180°-rotated grafts, and some A <-> P grafts, result in the
Rl muscle attaching anterior to the graft margin in the adult. Such results cannot be explained by a mosaic mechanism.
(3) Two very different types of grafts - 180° rotation and A <-> P exchanges produce adult Rl muscle patterns in which muscle fibres attach at one or more
of three different levels in the sternite. It is not possible, according to a mosaic
mechanism, to generate three attachment levels from a single one using 180°rotated and A <-» P grafts. There is no evidence for preferential attachment at
the original adult Rl muscle attachment site as would be predicted on a mosaic
mechanism.
(4) The most persuasive evidence in favour of a regulative mechanism for the
determination of the sites of muscle attachment is that graft-induced disruptions
of adult muscle patterns have variable effects according to the age of the larva
at the time that the graft is performed. That is, grafts in early eyestage larvae
often result in less pronounced disruptions of adult muscle patterns than grafts
in late eyestage larvae. This can be explained by relaxation of graft-induced
Gradient control of muscle patterning
55
changes in the topography of the epidermal gradient. Relaxation effects are not
predicted by a mosaic mechanism.
Grafts that generate three equivalent muscle attachment levels
Two different kinds of grafts- 180°-rotated grafts and A <->P grafts which
involve transposition but no rotation - generate similar disruptions in gradientcontrolled cuticular patterns, and also generate similar types of disruptions in
the adult Rl muscle patterns. These results support the hypothesis that a gradient
of morphogenetic information, present in the epidermis, specifies the position at
which adult muscle attachments are formed.
Both A <-> P and 180°-rotated grafts are predicted to generate three gradient
levels which are equivalent in value to the single original level for Rl muscle
attachment (Figs. 2, 3). The results of 180°-rotated and A <-> P grafts (Tables 1
and 3) show that all of these three levels can be used by Rl muscle fibres. However, despite general similarities between the types of muscle patterns found after
180°-rotated and A<-> P grafts, the frequency of particular Rl muscle patterns
is different in the two groups. This is due to two factors:
(A) 180°-rotated grafts are more subject to relaxation than A <-> P grafts
Adult cuticle patterns reveal that, whereas two distinct whorls of contours are
present after A <-> P grafts, even in insects operated upon at early eyestages,
this is often not true after 180°-rotated grafts. In 180°-rotated grafts the posterior
whorl may be reduced or lost during relaxation, and therefore there may be
fusion and loss of Rl muscle attachment levels y and z (Fig. 5).
Relaxation effects, coupled with the low probability of severing the larval Rl
muscle during grafting (see further below), explain why Rl fibres commonly
attach at level x, and rarely at level z, after 180°-rotated grafts (Table 1). (In fact
the frequency of attachment at level z is probably an underestimate, due to the
difficulty of establishing the position of levels y and z in relaxed patterns. As
levels y and z fuse during relaxation it would perhaps be more realistic to regard
muscle attachment in some animals as occurring at a single level yz. This
difficulty is compounded by the slight, natural variation in the sites of attachment of individual Rl fibres.) The development of muscle attachments outside
the graft boundary, at level x, is not a wound effect. The use of unrotated marked
integument at graft site A shows that the graft margin does not present a barrier
to the posterior migration of the Rl muscle (Williams & Caveney, 1980). Furthermore, attachment at level x does not simply reflect the fact that the Rl muscle
has failed to migrate at all, as the original (larval) muscle attachment is anterior
to level x. Therefore, attachment at level x can best be explained by the gradient
hypothesis. It reflects the loss or blurring of positional information at levels y and
z during relaxation, as well as the fact that level x is the first muscle attachment
level encountered by myoblasts at the anterior end of the intact Rl muscle
during posterior migration.
56
G. J. A. WILLIAMS AND S. CAVENEY
(B) The larval Rl muscle is more likely to be severed by A *->P
than by 180°-rotated grafts
This is because the larval Rl muscle lies closer to the integument near its origin,
so it is more likely to be severed during operations at graft site B (Table 3, footnote), where the anterior margin of the graft is more anterior than at graft site
A (Table 1, footnote). Therefore, it is more likely that myoblasts will be released
from their association with the larval muscles after an A<->P than a 180°rotated graft, and that they will interact with all the muscle attachment levels
generated by the grafting operation.
Relaxation after A <-> P grafts does not result in the loss of Rl muscle attachment levels, and an impressive vindication of the gradient hypothesis is that all
three Rl muscle attachment levels are used by Rl muscle fibres after A <-> P
grafts. (Rl muscle patterns in which fibres attach at all three levels represent
28 % of all A <-» P grafts, Table 3.) The type of Rl muscle pattern seen in any
particular sternite after an A <-» P graft is probably largely dependent upon
whether the Rl muscle was severed, partially severed, or left intact, during
grafting.
The combination of an increased probability of severing the Rl muscle, and
the presence of all three muscle attachment levels, explains why bridging muscle
patterns are more common after A <-> P, than after 180°-rotated grafts (24 % of
A «-> P grafts compared with 14 % of 180°-rotated grafts, Tables 2 and 4). It also
explains another common result of A <-> P grafts. That is, muscle patterns in
which muscle fibres attach at levels y' and z\ Such patterns may be the result of
severing the Rl muscle in such a way that the anterior stump of the muscle
degenerates. In this case myoblasts derived from the posterior (contracted)
stump of Rl attach at levels y' and z', while no myoblasts are in a sufficiently
anterior position to interact with level x'.
The results of 180°-rotated and A<->P grafts taken together support the
hypothesis that a gradient governs the position at which new Rl muscle attachments form. These results demonstrate that when new levels for Rl attachment
are generated by these grafts, and are not lost during subsequent relaxation they
can indeed be recognized and used for the development of new muscle attachments.
One further interesting feature of these grafts is that where relaxation occurs,
it tends to be more marked in the cuticle patterns than in the Rl muscle patterns
of the same sternite. Therefore, it appears that the muscle patterns reflect
positional information present in the epidermis at an earlier, and less relaxed,
stage than the position information that is expressed in the cuticular patterns.
Muscle R2 is also under gradient control
The results of 90°-rotated grafts support the hypothesis that a gradient also
controls the position at which adult R2 muscle attachments form. However, it is
Gradient control of muscle patterning
57
more difficult to demonstrate gradient control of muscle patterns using 90°rotated, than 180°-rotated or A <-> P, grafts. This is due to the theoretical
difficulty of inducing sufficient distortions in the gradient topography to produce
detectable effects on adult R2 muscle morphology. Also, unavoidable graftinduced wounding produces small abnormalities in the adult R2 muscles, as
discussed above (see Results).
The effect of 90°-rotated grafts on adult R2 muscles varies from negligible, as
illustrated in Fig. S(e) (compare the pattern of attachment sites of the R2 muscle
in Fig. 8(e) with those of control R2 muscles in Figs. 6 (a) and 7 (a)), to patterns
where the R2 fibres are tilted at an angle (Fig. 8a, c). However, no insect shows
an R2 muscle attaching at 90° to the normal position, as would be predicted by a
simple mosaic mechanism.
Analysis of the endocuticle patterns of selected insects with 90°-rotated grafts
shows that R2 muscle attachments are at right angles to the orientation of the
endocuticular microfibrils (Fig. 8). They follow the S-shaped curve of the
gradient contours (Fig. 4 b). (This analysis ignores fibres with very anterior or
antero-lateral attachments, as these fibres are the result of wounding.) Furthermore, there is a good correlation between the endocuticle patterns and the position of the R2 muscle attachments at different stages of pattern relaxation. The
results of 90°-rotated grafts therefore fully support the hypothesis that the
epidermal gradient which governs cuticle patterning also governs the position at
which new R2 muscle attachments form.
Myoblasts discriminate between attachment sites for different muscles
Both Rl and R2 attachment levels are triplicated by A «-> P and 180°-rotated
grafts. Therefore these operations each generate a total of six potential muscle
attachment levels if myoblasts cannot distinguish between the attachment levels
for the Rl and R2 muscles. However, although the resolution possible in this
system is limited due to the natural variability in the sites of attachment of Rl
muscle fibres, it appears that a maximum of only three contour levels are ever
used after these grafting operations - levels x, y and z after 180°-rotated grafts
and levels x', y' and z' after A <-> P grafts. The results of 180°-rotated and A <-> P
grafts therefore indicate that gradient levels may specify qualitatively distinct
sites of attachment for the Rl and R2 muscles; and also that myoblasts from
the two muscles are distinct.
Does the gradient slope govern the direction ofmyoblast migration?
The results of grafting experiments show that certain gradient levels are used
to determine adult retractor muscle attachment sites. However, the mechanisms
by which myoblasts find these sites is at present unknown. Myoblasts may
extend processes randomly until they establish contact with prospective tendinous
epidermal cells; or alternatively the slope of the epidermal gradient may direct
myoblast migration. For example, Millen (1975) has suggested that migration
58
G. J. A. WILLIAMS AND S.CAVENEY
of the dorsal scutellar longitudinal muscle in Galleria is governed by a gradient
of adhesiveness present in the overlying epidermis. He proposes that this muscle
advances up the adhesive gradient until it encounters prospective tendinous cells
which are distinguished by their ability to form strong adhesions with the muscle.
A similar mechanism could also explain the directed migration of myoblasts in
Tenebrio. However, it is important to realize that an epidermal gradient that
dictates the position of tendinous cells does not necessarily also express its
polarity to migrating myoblasts. Regions of muscle attachment may simply be
specified in what, as far as myoblast recognition is concerned, is an otherwise
undifferentiated epidermal field.
Analysis of the results of 180°-rotated and A <-» P grafts have revealed that
there are restrictions on the gradient levels which are used as attachment sites
for bridging muscle fibres. While x-y, or x'-y', fibre bridges are often found
(Tables 2, 4), x'-z' bridging has only been observed in one insect, and y-z or
y'-z' bridging is never seen. One plausible explanation for these results is that
the slope of the epidermal gradient determines the net direction of myoblast
migration. That is, using the conventions established in Figs. 2^4, myoblasts
will normally migrate down the gradient. Bridging fibres between gradient levels
x and y, or x' and y', could be the product of myoblasts derived from an anterior
muscle stump migrating posteriorly down the gradient slope and fusing with
myoblasts which had migrated anteriorly, from a posterior muscle stump, down
the reversed gradient slope in the centre of the graft region (Fig. 9 illustrates the
mechanism in the case of an A <->• P graft). Bridging between levels y and z, or
y' and z', would be forbidden in this mechanism as such fibre patterns could only
result from myoblasts migrating up gradient slopes. The one result showing
fibres bridging between levels x' and z' would have to be regarded as an anomaly
which could perhaps have resulted from partial severing of the larval Rl muscle,
so that myoblasts migrated along the larval muscle fibres as well as over the
epidermis.
Some (26 %) of sham operations at graft area B (Fig. 1 b) result in adult Rl
muscles attaching slightly more posteriorly than the Rl muscles on the control
side of the insect. These muscle patterns may result from severing the Rl muscle
during grafting in such a way that the anterior muscle stump degenerates. Under
these conditions myoblasts would have to migrate in an anterior direction (i.e. up
the gradient) from the contracted posterior muscle stump, to get to the gradient
level appropriate for Rl muscle attachment. We suggest that, rather than migrating up the gradient, myoblasts form attachment sites in the vicinity of the anterior
end of this muscle stump, even though this epidermis is actually inappropriate
for Rl attachment.
At present there is little direct evidence in support of these mechanisms.
Consequently the concept of gradient-directed myoblast migration remains
speculative. Even if myoblasts normally migrate down the gradient slope, this is
clearly not an inviolable rule as myoblasts can under certain circumstances
59
Gradient control of muscle patterning
Ant.
Post.
Post stump Rl
Ant.
Post.
Rl
C
| Epidermis
Ant.
Post.
Fig. 9. Diagrams illustrating a proposed mechanism for the formation of x'-y' muscle
fibre bridges after anA<->P graft, where myoblasts migrate down the slope of the
gradient. (A) Profile showing a relaxed pattern resulting from an A <-> P graft. The
abscissa represents the length of the stemite, and the ordinate represents the height
of the gradient. Rl, The gradient level for Rl muscle attachment. Ant., Anterior of
the sternite; Post., Posterior of the sternite. (B) A diagrammatic representation of a
longitudinal section through the sternite epidermis and muscle Rl. The Rl muscle
is severed at the anterior graft margin to give an anterior contracted stump (Ant.
stump Rl), and a posterior contracted stump (Post, stump Rl). Myoblasts are
released from the cut surfaces of the muscle stumps, and migrate down the gradient
slopes until they encounter prospective muscle attachment cells (stippled) at levels
x', y' and z'. Arrows indicate the direction of myoblast migration. (C) Diagram of a
longitudinal section through the sternite epidermis and adult Rl musclefibres(Rl).
Myoblasts have fused to form a muscle fibre bridge between levels x' and /, while
other musclefibresattach at level z' and extend to the posterior margin of the segment.
migrate up the slope of the gradient to form muscle fibres which attach at the
anterior segment boundary. This occurs when there is substantial wounding at
the anterior graft margin, and myoblasts are presumably frustrated in their
attempts to move in a posterior direction due to the presence of a barrier of
damaged epidermis. The importance of this observation in terms of the normal
mechanism of migration remains doubtful.
5
EMB 58
60
G. J. A. WILLIAMS AND S. CAVENEY
CONCLUSION
The results of grafting operations show that a regulative, rather than a mosaic,
mechanism determines the position at which certain adult retractor muscle
attachments form in the abdominal sternite of Tenebrio. The results support the
hypothesis that the epidermal gradient which controls cuticular patterns in this
insect also dictates the position, in the antero-posterior axis, of muscle attachment sites. Attachment sites for different retractor muscles are specified independently and are probably qualitatively distinct.
The results of grafting operations suggest that the establishment of correctly
positioned muscle attachments results from a 'conversation' between myoblasts
and epidermis, involving the following events. (1) Myoblasts extend processes
from the degenerating larval muscles and sense the epidermis. Cutting the larval
muscles releases myoblasts from their close association with these muscles. The
direction of migration of free myoblasts may be dictated by the slope of the
epidermal gradient. (2) Myoblast processes encounter and recognize epidermal
cells at a level in the gradient which specifies for muscle attachment. (3) The
interaction between myoblasts and epidermis at the appropriate muscle attachment level results in the formation of a new muscle attachment.
We would like to thank Drs M. Locke and J. Nardi for their helpful criticism of the manuscript, and J. Locke for assistance with photography. This work was supported by National
Research Council of Canada grant A6797 (S.C.), University of Western Ontario Special
Scholarships and an Ontario Graduate Scholarship (G. J. A.W.).
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