/ . Embryol. exp. Morph. Vol. 41, pp. 189-207,1977
Printed in Great Britain © Company of Biologists Limited 1977
\ gO,
The development of functional innervation in
the chick wing-bud following truncations
and deletions of the proximal-distal axis
By R.VICTORIA STIRLING 1 AND DENNIS SUMMERBELL 2
National Institute for Medical Research, London and
The Department of Anatomy, University of Otago Medical School
SUMMARY
Sections of the proximal-distal axis were removed so as to produce wings lacking entire
skeletal levels. The innervation of the resulting limb from the appropriate spinal nerves was
tested by electrophysiological recording, gross dissection and serial sections. The nerves
form repeatable and ordered patterns in the modified limbs which can be readily related to the
normal pattern. In general the segmental nerves only innervate their appropriate territory
irrespective of its location; the route followed by the nerves is characteristic for the skeletal
level they traverse. If a target is missing then so is the normal nerve branch to that region.
INTRODUCTION
What are the factors guiding nerves to their destination ? Many experiments
on this topic involve the regeneration of nerves to their original or surgically
altered destinations, on the assumption that the processes guiding regenerating
nerves are similar to those occurring during development. (See reviews by Gaze,
1970; Jacobson, 1970.)
Recently there has been increasing interest in the original development of the
innervation. For example, Jacobson and Hunt have been studying the retinal
organization of early amphibian embryos (see review by Hunt, 1975), and
Chung & Cooke (1975) and Gaze, Keating & Chung (1974) have been examining
the factors governing invasion of the optic tectum by axons from the retinal
ganglion cells (see review by Keating, 1976).
In the peripheral nervous system, Piatt (1942,1956,1957) suggested from anatomical observations on the innervation of aneurogenic limbs and those
innervated by transplanted cord segments in Ambystoma, that the innervation
pattern of the limb clearly involved interactions between the limb tissue and
the source of innervation. In these studies, however, the operations themselves
1
Author's address (reprints): National Institute for Medical Research, The Ridgeway, Mill
Hill, London NW7 1AA, U.K.
2
Author's address: The Department of Anatomy, University of Otago Medical School,
P.O. Box 913, Dunedin, New Zealand.
13
EMB 41
190
R. V. STIRLING AND D. SUMMERBELL
resulted in abnormalities of limb and cord structure so that it was difficult to
disentangle the relative contributions of these two factors.
We have chosen to study the innervation of the developing chick wing for
two reasons. Firstly, the segmental nerve roots innervate the limb in a definite
pattern which is remarkably invariant between animals of the same genetic
strain. The development of this pattern has been described for the chick forelimb by Roncali (1970). The whole process of laying down the adult pattern
takes about 120 h. In an elegant study using electrophysiological methods
Landmesser & Morris (1975) have described the appearance of the correct,
normal motor innervation in the chick hind limb as early as 5^ days of incubation. Secondly, it is experimentally easy to manipulate the developing chick
limb-bud so that it develops in an abnormal yet controlled and patterned manner. Using relatively simple operations on the wing-bud one can rapidly produce
large numbers of modified limbs with constant form: truncations (see, for
example, Saunders, 1948; Summerbell, 1974#), intersegmental deletions along
the proximal-distal axis (Summerbell & Lewis, 1975; Summerbell, 1977),
tandem reduplications in which parts of the proximal-distal axis are repeated
(Summeibell, Lewis & Wolpert, 1973), mirror image reduplications across the
cranial-caudal axis (Saunders & Gasseling, 1968; Summerbell, 1914b; Tickle,
Summerbell & Wolpert, 1975; Fallon & Crosby, 1975; Summerbell & Tickle,
1976), and many other forms. These operations are carried out so that the
tissues giving rise to the desired modification are laid down and determined
before the nerves have reached the abnormal portion.
Thus by changing the environment into which the invading axons have to
migrate, we are able to test their response when axons normally destined to
innervate the humerus region instead encounter forearm or hand. This kind of
approach provides a direct way of studying the factors which guide peripheral
nerves to their destination. We have used both anatomical and electrophysiological methods to trace the innervation pattern, since simply tracing the anatomical arrangement of fibres within the limb does not give any insight into the
functional connexions between a given segmental nerve root and a specific
muscle or region of skin.
Our general approach has been to prepare a number of limbs of the desired
type using 'cut and paste' methods. We wanted to examine the innervation of a
particular morphological type of operated limb rather than the effects of a
particular operation, so we studied only those limbs which were of a recognizable morphological type and discarded examples with intermediate or distorted
forms. This first paper concerns very simple operations in which whole segmental levels of the proximo-distal axis have been removed. A preliminary report
has already been given (Stirling, 1976).
The nomenclature of the nerves used in this paper is that suggested by Yasuda
(1960).
Limb-bud innervation
191
METHODS
Fertilized White Leghorn eggs were incubated at 38 °C and windowed on the
third, fourth or fifth day of development. The windows were sealed with Sellotape and returned to the incubator. Appropriately staged eggs (Hamburger &
Hamilton, 1951) were then selected for each operation. The basic technique
was to remove the distal tip of the wing bud using sharpened tungsten needles.
Removal of the tip of the wing bud of progressively older embryos results
in limbs having progressively more distal elements (Figures IB, C and D).
This simple operation produced the distal deletion series of limbs.
In the second series, proximal deletions, the tips removed from the embryos
were grafted back to the stumps of younger hosts so that an intervening segment or segments was missing (Figures 1E, F and G). Grafts were fastened to the
host stump with two platinum pins, as shown in the figures. In all cases a few
drops of Earles BSS (Paul, 1975) with 50i.u. penicillin, 50 peg streptomycin
and 2-5 i.u. Fungizone or Kanomycin (Bristol) were added to the eggs before
resealing with sellotape and returning to the incubator.
Serial Sections
On the 10th day of incubation some of the eggs were removed from the incubator, and fixed and stained by the method of Marsland, Glees and Erickson as
described in Ralis, Beesley & Ralis (1973). The embryos were embedded in wax
and serially sectioned at 20 /im. A three-dimensional reconstruction was made of
the innervation of the wing which was then mapped in two dimensions.
Electrophysiology
Electrophysiological recordings were made on embryos in ovo at 16-18 days
of incubation. The window used at operation was enlarged and the embryo
anaesthetised with 0-2 ml of 10 % Equithesin. The egg was kept warm with
previously heated packs containing polyethylene glycol. The skin above the
spinal column of the brachial region was held in a clip and a thread was tied to
the dorsal side of the scapula. Tension was applied between the clip and the
thread so that the muscles could be parted, exposing the spinal nerve roots, 13,
14, 15 and 16. The small 13th nerve root lies in close association with major
blood vessels and was not used for recording. The spinal nerves 14, 15 and 16
were dissected free and cut close to the spinal column. Recordings were made
using suction electrodes of pulled polyethylene catheter tubing. A map of the
sensory innervation territory of each nerve root was made by audiomonitoring
activity elicited by gentle stimulation of the wing skin. This map was then
checked by recording afferent volleys to small electrical pulses through a monopolar glass-coated tungsten needle placed on selected regions of the skin (Fig. 2).
Since the responses in the small diameter axons were small in amplitude, an
13-2
192
R. V. S T I R L I N G AND D. SUMMERBELL
A
B
21
D
19
Fig. 1. The types of operation used to produce each experimental wing type. Stage
of donor shown on left of each diagram. (A) Sham control: remove tip of stage 21 and
replace. (B) Loss of autopod: remove tip of stage 23 or 24. (C) Loss of zeugopod
and autopod: remove tip of stage 23 or 24. (D) Loss of stylopod, zeugopod, autopod:
remove whole of stage 19. (E) Loss of zeugopod: remove tip of stage 20, replace
with tip of stage 24 (from B). (F) Loss of stylopod: remove tip of stage 19, replace
with tip of stage 20 (from C). (F) Loss of stylopod: remove tip of stage 19, replace
with tip of stage 20 (from C). (G) Loss of stylopod and zeugopod: remove whole
of stage 19, replace with tip of stage 24 (from B).
average of 32 responses at each stimulus point was made using a Neurolog
NL 700 averager. The limb was then skinned, the nerve roots stimulated electrically and responses in the visible dorsal muscles of the wing were observed
and recorded.
Gross Dissection
Dissections were performed on at least six good examples of the seven limb
types described in the methods. In each group some were the formalin-fixed
Limb-bud inner vat ion
Rec. 14
193
Rec. 16
Stim. alar web
Stim. elbow
20 ms
B
Stim. 14
Rec. biceps
i ,' I
f \
Stim. 16
^S"**********
\ -•**
V
10 ms
Fig. 2. (A) Illustrates typical recordings from the 14th and 16th segmental roots
when the alar web and elbow are stimulated electrically. The upper trace in each
record shows the averaged responses to 32 stimuli; the lower trace a response to a
single stimulus. (B) Muscle potentials recorded from the surface of the exposed
biceps muscle to stimulation of the 14th and 16th nerves.
limbs which had been used for electrophysiological recordings, and others were
prepared separately and fixed on the 17th or preferably, the 18th day of incubation in 5 % trichloractetic acid. These were stained with 0-1 % alcian green 2
GX in 70 % alcohol with 1 % hydrochloric acid and then stored in 70 % alcohol.
The dissections were made under a Wild M8 microscope, and the nerve patterns
were displayed and drawn.
194
R. V. STIRLING AND D. SUMMERBELL
D
ad
b
i
A
B
C
14
+
+
+
+
O
O
16
O
O
+
+
+
+
Limb-bud innervation
195
RESULTS
The motor and sensory map of the innervation of the normal chick wing at
17 days of incubation is shown in Fig. 3C and 3D. The pattern of innervation
was identical in all our control animals. The 14th segmental nerve innervates
the anterior proximal part of the limb, the 16th, the posterior distal area. The
territory of the 15th lies in between but overlaps considerably those of both 14
and 16. Since the 14th nerve never innervates the hand nor the biceps region, we
therefore chose to concentrate on these nerves for our electrophysiological
studies.
There were no discrepancies between the results obtained from gross dissection and those obtained more precisely from serial sections. The gross
dissections provided our basic technique for establishing the range and the
representative results while the sections were used to confirm our impressions
gained from the dissections.
In all the experiments the modified limbs possessed recognizable peripheral
nervous systems, the individual nerves of which could be identified as homollogues of those found in normal limbs.
(I) Sham operations {normal limbs) (Figures 1A and 3).
The sham operations gave normal limbs, almost all of which were indistinguishable from unoperated controls. The only aberrations were occasional
minor kinks in the nerve tracts and/or the bones at about the level of the original
amputation. Our results confirmed those of Roncali (1970) and all the major
branches could be identified as those shown in Fig. 2 of her paper.
Fig. 3. The innervation of a typical normal chick wing or sham control. (A) Dorsal
nerves and brachial plexus. (B) Ventral nerves. Abbreviations used for all anatomical
diagrams: 13, 14, 15, 16 segmental levels of spinal nerves, ax, = N. axillaris,
bi = N. biceps, bli = N. brachialis longus inferior, bis = N. brachialis longus superior, cbi = N. cutaneous brachii inferior, cbs = N. cutaneous brachii superior,
/ = N. interosseus, lat — N. latissimus dorsi, m = N. metacarpales, pat = N.
patagiales, pect = N. pectoralis, rl = N. radialis lateralis, rp = N. radialis profundus, scap = N. subscapularis, scor = N. supracoracoideus, tri = N. triceps,
// = N. ulnaris. The stippled areas in these diagrams indicate the skeletal elements.
(C) Sensory innervation territory of the 14th nerve (light stipple) and 16th nerve
(dots). (D) Motor innervation of the main dorsal muscle blocks. + indicates contraction and O no detectable contraction in muscle when spinal nerve listed on
left is stimulated electrically. The same names and format are used in all figures. The
muscle blocks consist of (from Sullivan, 1962): ad-superficial pectoral and patagialis
longus, b - biceps, / - triceps, A - extensor metacarpi radialis, B - extensor digitorum communis, C - extensor metacarpi ulnaris. Muscles in the hand were too small
to record from reliably using surface electrodes.
196
R. V. STIRLING AND D. SUMMERBELL
|413
Oa
A
D
D
14
16
Fig. 4
ad
b
I
+
O
+
O
+
+
Fig. 5
Limb-bud innervation
197
(2) Autopod {hand) deleted (Fig. 1B and 4).
All three segmental nerve roots were present and the territories occupied by
the 14th and 16th nerves were exactly as expected from the innervation of a
normal wing. Similarly, the morphological pattern of nerves was quite normal
except that the hand was missing and all the distal nerve branches were thin
in the zeugopod and stopped at the wrist.
(3) Autopod and zeugopod {hand and forearm) deleted (Fig. 1C and 5).
All three nerves were present but the 15th and 16th nerves were reduced in
diameter when compared with those on the contralateral side. The innervation
territories of the 14th and 16th nerves were again as expected from their normal
innervation; the 14th nerve innervated the whole of the area found in normal
animals and the 16th a narrow strip of skin along the posterior ventral wing and
the triceps. There seemed to be some overlap of the sensory innervation territory
between these nerves at the distal face of the stump, which was also reflexly
extremely sensitive to gentle touch. All nerve branches which in controls arise
proximal to the elbow were present in all cases. In the dorsal bundle these
branches were all quite normal. The N. brachialis longus superior terminated
near the distal end of the humerus in a feathery plethora of small branches, all
apparently ending proximal to the tip of the stump. There was no web present
on the wing and the N. patagiales terminated at the mid-humerus level, again
ending in many fine branches. The proximal branches of the ventral bundle
also looked normal but in some cases our subjective impression was that the
branches to the flank were fatter than normal. The main branch, N. brachialis
longus inferior, could be traced to the tip of the humeius but did not have so
many obvious fine divisions distally as did the superior branch.
Our general impression was of rather meagre-looking nerves except for the
branches to the flank and shoulder. In some limbs the stylopod appeared to be
' hyper-innervated' with a profusion of slender branches to the distal end of the
limb; this perhaps correlates with the increase reflex sensitivity observed before
the roots had been cut for electrophysiological recording.
FIGURES 4 AND 5
Fig. 4. The innervation of a typical wing with autopod deleted. Labelling
as in Fig. 3. (A) Dorsal nerves and brachial plexus. (B) Ventral nerves. (C) Sensory
fields of the 14th and 16th spinal nerves. (D) Motor innervation of main dorsal
muscle blocks.
Fig. 5. The innervation of a typical wing with autopod and zeugopod deleted.
Labelling as in Fig. 3. (A) Dorsal nerves and brachial plexus. (B) Ventral nerves.
(C) Sensory fields of the 14th and 16th spinal nerves. (D) Motor innervation of
main dorsal muscle blocks.
198
R. V. STIRLING AND D. SUMMERBELL
,5\4
•
16
peel
Fig. 6. The innervation of a typical wing with the autopod, zeugopod and stylopod
deleted. Labelling as in Figure 3. (A) The anatomy of all nerves present in the
stump. (B) The sensory innervation territories of the 14th (light stipple) and 16th
(dots) spinal nerves. No good muscle recordings were made in these cases.
(4) Stylopod, zeugopod, autopod {whole limb) deleted (Fig. 1D and 6).
The spinal nerves innervated the flank, the cutaneous territory of 14 being
more anterior to the others.
The morphological pattern was more varied for this operation than for any
other. The spinal nerves were all very reduced, often the three spinal nerves
leaving the spinal column in one, rather than three, bundles. The pattern of
nerves to the 'limb girdle' and flank were as one would expect, with normallooking cutaneous branches.
In two cases which also lacked a scapula the nerves were extremely reduced
and serial sections were needed to demonstrate their presence. These nerves had
stout branches supplying the dorsal axial muscles and had a small ventral
branch joining in a small plexus close to the spinal column.
(5) Zeugopod {forearm) deleted (Fig. IE and 7).
The 14th nerve provided no motor or sensory supply to the hand region, but
did innervate biceps, triceps and the skin over the anterior aspect of the upper
arm. The morphological pattern looked exactly as if the appropriate region had
been cut out and the severed nerve ends had been joined together, except that
N. patagiales was either very reduced or absent.
(6) Stylopod deleted (Fig. 1F and 8).
The 16th nerve innervated the muscles of the forearm and the hand skin
dorsally and ventially. The 14th nerve only had a small share in the innervation
of the proximal anterior margin of the forearm.
The gross morphology followed the familiar pattern.
(7) Stylopod and zeugopod {upper and forearm) deleted (Fig. 1G and 9)
The 14th nerve in six animals did not innervate the hand region, although it
did supply the flank skin anterior to the limb as it does in normal animals. The
Limb-bud innervation
199
D
14
16
O
O
+
Fig. 7. The innervation of a typical wing with the zeugopod deleted. Labelling
as in Fig. 3. (A) Dorsal nerves and brachial plexus. (B) Ventral nerves. (C) Sensory
fields of the 14th and 16th spinal nerves. (D) Motor innervation of main dorsal
muscle blocks.
200
R. V. STIRLING AND D. SUMMERBELL
14
D
ml
14
16
B
C
O
O
e
Fig. 8. The innervation of a typical wing with the stylopod deleted. Labelling
as in Fig. 3. (A) Dorsal nerves and brachial plexus. (B) Ventral nerves. (C) Sensory
fields of the 14th and 16th spinal nerves. (D) Motor innervation of main dorsal
muscle blocks.
Limb-bud innervation
201
Fig. 9. The innervation of a typical wing with the stylopod and zeugopod deleted.
Labelling as in Fig. 3. (A) Dorsal nerves and brachial plexus. (B) Ventral nerves.
(C) Sensoryfieldsof the 14th and 16th spinal nerves.
morphological pattern was again normal for the segments present. The only
branches which aiose distal to the plexus were either those normally innervating
the flank or those branches of the N. brachial is longus superior and inferior
which normally arise in and innervate the hand. There was a tendency for the
origin of these branches to lie rather more proximally than usual, either in
among the carpals or even in the flank tissue. The 14th nerve was either very
Ieduced in size or in some cases did not even enter the plexus. In one case in this
group the 14th nerve supplied the anterior margin of the hand which was also
well supplied by the (16th and 15th) nerves. The pattern in this limb may be
202
R. V. STIRLING AND D. SUMMERBELL
explained on anatomical grounds for it seemed that an N. patagiales had developed and then looped back to form a distal plexus in the hand with the N.
brachialis longus superior. This wing was also remarkable for the number of
large branches reaching the hand from its normal supply.
DISCUSSION
We have examined innervation of the chick limb in both normal and abnormal development. Our work is based on the important observation that in
normal animals the pattern of innervation is constant. Each spinal nerve always
innervates the same region and the anatomy of the nerve tracts is largely predictable. Distortion of the gross anatomy prior to innervation does not result
in either chaos or in a badly misaligned peripheral nervous system. Instead, the
migrating axons are able in some way to interpret their changed environment so
as to develop a relationship with the available tissue which is as near as possible that achieved in the normal limb. The key mechanism, therefore, almost
certainly involves an interaction between axons and limb tissues and is not either
an autonomous function of the axons, or a result of simple contact guidance
with towing as suggested by Harrison (1935) and Weiss (1955).
(1) Sham controls and normal innervation (Fig. 10A)
The wiring diagram for a typical normal limb or sham control shown in
Fig. 10 A is representative of the anatomy that we found. The only common
variations were in the plexus region where occasionally there were small contributions by nerves 12 and 17; these we have ignored. In all the wiring diagrams we
have used the following conventions. Axons may only grow out from left to
right or vertically, they can never grow retrogressively, i.e. from right to left.
One should assume that all connexions shown in the diagram normally occur.
Thus the N. brachialis longus superior and inferior normally can receive
branches from spinal nerves 13, 14, 15 and 16, but N. subscapularis can receive
contributions only from spinal nerves 13, 14 and 15 and not from 16. This
scheme fits well with our observations of axon connexions within the limb
where major branches always travel proximal-distally (see Roncali, 1970). There
is one exception to this general rule; a significant branch of the N. radialis
lateralis migrates recurrently towards the elbow. In the diagram we maintain
a convention that nerves always travel proximo-distal ly (left to right) even in
the case of this recurrent branch (marked *).
(2) Deleted distal segments (Fig. 10 B, C and D)
The effect of these operations on the gross morphology of the limb and on
its nerves was simple. The limbs were truncated cleanly at wrist, elbow or
shoulder, All limbs possessed almost exactly those nerves and branches which
203
Limb-bud inner vation
Flank
Plexus
Stylopod
Zeugopod
Autopod
13
14
scap
bi
15
pat
bis
rp
cbs
Mi
16
pect
13
14
1
cbi
~
lat
pat
scap
bis
15
cbs
16
cbi
pect
13
14
1
i
lat
scap
•hi
15
bis
. pat
chs
•Mi
16
cbi
pect
f=
hit
D
13
14
1
scap
15
16
pect
r
r
Ms
Mi
cbi
i
lat
Fig. 10. Generalized wiring diagrams of limbs with distal segments deleted. Labelling as in Fig. 3. (*see text). (A) Normal wing. (B) Autopod deleted. (C) Autopod
and zeugopod deleted. (D) Autopod, zeugopod and stylopod deleted.
204
R. V. STIRLING AND D. SUMMERBELL
were normally present up to the region which was missing. The only modification was that in some cases the nerves had a number of slender branches
innervating the extreme tip of the limb. The electrophysiological evidence supported this view of simple truncation, the functional connexions to those parts
present were exactly what one would expect from the normal pattern of innervation.
The reduction in the size of the nerve plexus in limbs with large deletions was
extreme in cases where only the shoulder girdle remained. This reduction may be
the result of degeneration of nerves having no functional peripheral innervation
(Hamburger, 1958; Prestige, 1967).
These observations are important because they appear to rule out one possible class of explanation for the development of peripheral nerve pattern. The
system clearly did not develop with all the normal nerve branches present but
compressed into a smaller space, as if the normal wiring diagram in Fig. 10 A
had been reproduced at a smaller magnification with the whole pattern confined
to one compartment. Instead, it produces only those nerves which normally
innervate the bits of limb which are present in the stump.
(3) Intervening segments deleted (Fig. 11 A, B and C)
The results of all operations involving these deletions show that both the
functional and anatomical innervation of the wing is a simple mosaic. Within
segments, nerves follow the normal anatomical arrangement, and also functionally innervate only those areas which they normally innervate irrespective of
abnormal location. This is especially clear in animals where both the stylopod
° n i the zeugopod are missing. It is as if these regions had been completely
emoved using a pair of scissors and the autopod had been directly attached to
the shoulder.
These results rule out the model which postulates that nerves develop in a
simple proximal-distal sequence, forming all the branches in the correct sequence
until the nervous system 'runs out' of limb, thus preventing the formation of
the distal branches. Rather it seems likely that if a limb segment, e.g.
zeugopod, is missing then the nerve branches which innervate that level will
also be missing. The electrophysiological evidence is especially important in
these cases, since it shows that the nerves are actually selective in the innervation of their specific territories. Local factors in the environment encountered by the growing axon tip might control the branching within the limb,
but the specific distribution of the segmental nerves for particular territory
suggests a much more specific influence acting between the nerve fibre and the
area it will invade.
The only deviation from the simple patterns outlined above occurred where
both stylopod and zeugopod were missing, Usually fibres from the 14th nerve
are not able to invade the hand, but in this case they did so. The result is clearly
unusual and for the present we intend to ignore it.
205
Limb-bud innervation
A
Plexus
13
1
Flank
Stylopod
Autopod
scap
his
cbs
bli
peel
Plexus
1
cbi
—
hit
Flank
Zeugopod
scap
peel
Plexus
i
hit
Flank
Autopod
rp
chi
Autopod
met a
Fig. 11. Generalized wiring diagrams of limbs with intervening segments deleted.
Labelling as in Fig. 3 (*see text). Dashed line = branch not always present. (A)
Zeugopod deleted. (B) Stylopod deleted. (C) Zeugopod and stylopod deleted.
Although the results of these simple experiments do not suggest even a
tentative explanation of the control of limb innervation, they are successful in
disproving two possible, if rather naive, classes of hypothesis. They demonstrate
unequivocally that there is a specific pattern of nerve growth: each spinal nerve is
somehow restricted to a specific territory. They show also that the pattern
develops as a result of interactions between invading axons and limb tissue.
The system is clearly amenable to this kind of enquiry since it may be readily
and repeatedly manipulated, the nerves responding with manoeuvres which are
easy to detect and record and which create an interpretable pattern. In future
experiments we hope to develop a closer understanding of the interactions between the limb tissue and invading axons by looking at functional innervation
at much earlier stages and using recent neuroanatomical tools such as cobalt
chloride and horse radish peroxidase. Preliminary results using different
EMB 41
206
R. V. STIRLING AND D. SUMMERBELL
embryonic manipulations suggest that if certain operations are performed early
enough, then the segmental nerves do not innervate their normal, particular
territories (Stirling, 1976).
This work was supported by the Medical Research Council of Great Britain and the
Golden Kiwi Medical Research Distribution Committee. The authors would like to thank
Amata Hornbruch and Sarah Rendle for help and advice and Yvonne Joel for the drawings.
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{Received 15 February 1977)
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