THE DEVELOPMENT OF GRAFTED EMBRYONIC FRAGMENTS OF THE CHICK

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					THE DEVELOPMENT OF GRAFTED EMBRYONIC
       FRAGMENTS OF THE CHICK.*
             BY P. D. F. MURRAY, B.A., B.Sc, University of Sydney, and
                      J. S. HUXLEY, M.A., Oxford University.

                                             WITH PLATE.

                                             CONTENTS
                                              PAGE                                                          PAGE

i. I n t r o d u c t i o n   .   .   .   .      9    4. R e f e r e n c e s                                  32
                                                     5. List of A b b r e v i a t i o n s e m p l o y e d
2. M e t h o d s                                9                , „.
                                                v         in t h e F i g u r e s         .  .   .       .
                                                          in t h e F i g u r e s                             33
                                                     6. D e s c r i p t i o n of P l a t e . . .             34
3. Discussion                                  29

                          1. Introduction.
T H E method of embryonic grafting has proved of considerable
value. Murphy (1913), Danchakoff (1916), and others have
extended the method to the chick, by utilising the chorio-allantois
of other embryos as host-tissue for the grafted pieces.
     In the following pages some account is given of experiments
designed especially to test the capacity for differentiation of
whole portions of the body of early chick embryos. Some other
experiments with organ-rudiments are also recorded.

                                             2. Methods.
    The methods used were essentially those of previous
investigators. 6- to 12-day embryos were used as hosts, the
most frequent age being 7 to 9 days. The eggs were " candled "
and the position of the embryo marked. The shell was swabbed
with 90 per cent, alcohol over the site of operation. A
triangular cut was made in the shell a short distance away
from the position of the embryo. The cut was made with a
small triangular file or else a fret-saw blade. Care was taken
to avoid injury to the shell-membrane.
    The graft had been meanwhile prepared. The embryo was
dissected out aseptically and placed in warm sterile Ringer.
                                     * Received July 29th, 1924.
                                                  9
        P. D. F. Murray and J. S. Huxley
With fine instruments the required dissection was performed
under a binocular microscope.
    The host egg was then placed close below a strong light.
With an iridectomy needle a V-shaped incision was made in
the shell-membrane and the resulting tongue turned back;
when this is successfully done, the chorio-allantois sinks down
a little, but is undamaged. The piece to be grafted is then
taken up, either in a pipette or else on a needle (a convenient
way is to lift it out of the fluid on a closed forceps, and pick
off with a needle). It is then squirted on to or laid on the
chorio-allantois, if possible near a large blood-vessel, and not
immediately below the site of operation. The shell-membrane
flap is then turned back, the piece of shell replaced, and sealed
with shell-membrane moistened with albumen from another
egg. Sometimes an additional sealing of the edges of the
piece of shell-membrane was undertaken later with collodion
solution. The egg is then replaced in the incubator, and
removed for examination after a suitable time.
    The best method of examination is to hold the egg with the
site of operation downwards, open the egg on the opposite side,
and carefully remove the embryo and yolk-sac into a small
vessel. The chorio-allantoic membrane should remain in place
over the lower half of the egg. If there is a reaction at the
site of operation, this is readily visible. A small piece of shell
and membrane is cut out surrounding the reaction, and the
whole preserved, the chorio-allantois being later stripped off
before clearing. Most of the specimens were examined as
toto mounts in clove oil before embedding: this often gave
valuable information. Bouin's fluid was chiefly used as fixative.
IO-M sections were cut and stained in various ways. Recon-
struction models were in some cases made by the wax and
blotting-paper method. In other cases graphic reconstructions
were plotted on co-ordinate paper. This method serves admir-
ably for comparatively simple structures, and takes far less
time than the construction of models.
     A large number of operations produced no visible result,
or the hosts died. The visible result in the apparently suc-
cessful ones was often a mere reaction of the host-tissue
(Huxley and Murray, 1924). A number of successful grafts,
                                10
   Grafted Embryonic Fragments of the Chick
however, were obtained. Those described in this paper are
as follows:—
    (1) No. 21. Head region (anterior third) of a 24-hour
          embryo : was allowed to differentiate for four days in
          its new position.
     (2) No. 45. A piece of about four somites from the middle
          region of a 2-day embryo. Four days' differentiation
          as graft.




                                                                                        rhomb.
                                                                                                 eel.




FIGS. A 1 and A 2.*—Drawings of grafted head No. 21 as seen in oil of cloves before sectioning:
    A I, from above ; A 2, from the left side. In A 2 all blood-vessels are omitted ; in A I some
    of the more prominent are inserted. In A I, note communication between fore- and mid-brain.

       (3) No. 50. From a 24-hour embryo; a piece from the
            middle of the body, one cut having passed a little
            in front, the other a little behind the vitelline vein.
            Two days' differentiation in new host.
    The Head (No. 21).—In order to arrive at a correct under-
standing of the anatomy of this specimen, it is necessary
to describe the distortions of form which it has undergone
(figs. 2, A, B, C). The brain, which is the predominant feature in
the sections, is divided clearly into telencephalon, diencephalon,
    • Note.—All text-figures refer to grafted head No. 21. For the meaning of the abbreviations
see the list on p. 33.
                                              II
            P. D. F. Murray and J. S. Huxley
mesencephalon, and rhombencephalon. The entire brain, in all
parts, is expanded laterally so as to be far broader than in
the normal chick, presumably on account of pressure. This
expansion is everywhere markedly greater on the left side.
Partly as a result of this, the eyes have been forced into a
ventral position. They, of course, start normally with the optic
stalk attached ventro-laterally to the diencephalon, so that the
position of the eye is at first ventro-lateral. In this specimen,




                                                                            -mi




                                                                  -•aucC.    v.




FIG. B.—Reconstruction model, from above, indicating outline of brain-cavities (the model was
    unfortunately reversed in construction, apparent right being real left). The arrows in this
    and fig. C indicate the approximate morphological axes. The communication between fore-
    and mid-brain is seen.

owing to the great extension towards the left of the telencephalon
and diencephalon, the left eye is very far ventral, so far that it
is really on the right side of the brain and ventral. In other
words, the more dorsal and left latero-dorsal regions of the
brain have grown out laterally and have left the left eye behind,
so that it has come to occupy this ventral position.* Like the
    * The ventral position of the eyes in specimens showing tendencies to cyclopia
after treatment with depressants should also be borne in mind. Doubtless growth-
inhibition has here contributed to their ventral situation.
                                              12
   Grafted Embryonic Fragments of the Chick
eyes the epiphysis is far to the right of the mid-line of the
specimen, and the same applies to the hypophysis. The mid-
brain shares in this same distortion ; it is expanded to some
extent in both lateral directions, as though forming optic lobes,
but especially and far more to the left. The extension of the
mid-brain to the left is shown by the point of continuity with
the hind-brain, which is far over on the right side.




            mb"



                    aud


                            (e   .
                                                            V




        FIG. C.—The same reconstruction model, from below ; cavities indicated
                              only in gut and heart.

    This lateral left extension of all regions of the brain is
probably due to the position of the graft on the chorio-allantoic
membrane. The graft was found with its dorsal surface facing
inwards towards the centre of the egg, but with a tilt over
to the right. Thus the right side would have had less room
to grow in and so the left side expanded at its expense.
    Another distortion of form has involved the establishment
of a new and abnormal brain flexure. In a normal chick, the
head, as a consequence of the cranial flexure, is in such a
position that it faces backwards. In this specimen the graft
                                         13
         P. D. F. Murray and J. S. Huxley
was made before the cranial flexure appeared, when the brain
 was a straightforward continuation of the spinal cord, without
any flexures. The graft was made in such a way that the
 ventral surface of the fore-brain region became closely attached
 to the chorio-allantoic membrane, as shown in figs. 2, A2, etc.
 It was thus impossible for the cranial flexure to influence the
 telencephalon, on account of its firm attachment: it could,
however, influence the diencephalon and mesencephalon, and
has done so, because they were not in such close and direct
connection with the chorio-allantoic membrane, but separated
from it by a considerable space of more or less loose mesenchyme,
and also by the gut region. The result of this has been that,
while there is a cranial flexure having the apex of its arch
in the mid-brain, the telencephalon, instead of constituting one
of the limbs of the arch, has maintained its original antero-
posterior position, and so it has introduced a new upward
flexure as seen in the figures.
     Before proceeding to the more detailed description of the
brain of this specimen, it is necessary to mention the main
features of the brain of a normal 24-hour chick. In such a
chick the neural groove is just closing, but the closure is not
complete until thirty-three hours. The outlines of the three
primary brain-vesicles are barely indicated, but the fore-brain
has large lateral expansions, the optic cups, and a constriction is
appearing separating it from the mid-brain. A little later,
at about twenty-six hours, the mid-brain becomes marked off
from the hind-brain. There are no brain-flexures at this time,
the cranial flexure beginning at twenty-nine hours.
     In the graft, fixed four days after grafting, the neural tube
is completely closed, and the brain has increased greatly in
size and histological differentiation, and is clearly divided into
telencephalon, diencephalon, mesencephalon, and rhomben-
cephalon.
     The telencephalon is large and roughly approximates to
the normal form at a stage prior to the formation of cerebral
hemispheres, except for the two features mentioned above:
the lateral expansion to the left, and the fact that it points
forward, as it did before the formation of any cranial flexure
in the brain, instead of downwards and backwards as is normally
                                14
   Grafted Embryonic Fragments of the Chick
the case. Cerebral hemispheres are entirely absent, there not
being any trace of them at all. As these are strongly indicated
in a normal chick even as early as seventy-two hours, it is clear
that there has been considerable retardation in the development
of the telencephalon. Retardation appears to be a general
feature of grafts such as these. A peculiar abnormality in
the floor of the telencephalon will be described later.
     The diencephalon has also advanced considerably. It is
expanded laterally to the left like the rest of the brain, but
somewhat less so than the other regions. This expansion is,
however, sufficient to put the epiphysis well on to the left side.
The two eyes are both present. Their position is ventral, as
described above.
    The mid-brain has a flat floor and longer arched roof (figs.
2, A 2) and, as described, is much expanded to the left, and
also, though less, to the right, as though it had been trying
to form optic lobes (o.l.). It is marked off posteriorly from
the rhombencephalon by a constriction, the isthmus.
    The hind-brain is present in part, the posterior part of the
medulla oblongata being absent. The roof of the anterior end
is thinner than elsewhere, being in fact epithelial, and marking
the position of the myelencephalon.
    As regards the flexures of the brain, the cervical flexure
does not come into the discussion, because none of the spinal
cord was" included in the graft. The pontine flexure is not well
marked, but is present. It would have been clearer but for the
fact that at the posterior end of the graft where the brain was
cut it has closed again and taken a downward curve (figs. 2, A 2),
thus obscuring the pontine flexure a little farther forward.
The cranial flexure, on the other hand, is marked, the brain
forming an arch of which the mesencephalon forms the apex
and the diencephalon and rhombencephalon the two limbs..
The relations of the telencephalon to this flexure have already
been described.
    It should be pointed out that the normal turning of the
embryo on to its side has been prevented by the fixation of the
brain to the chorio-allantoic membrane by its ventral side.
    Histologically the brain has reached a high stage of differ-
entiation, as shown by the development of grey and white
                               15
           P. D. F. Murray and J. S. Huxley
matter and of cranial nerves. As there are a number of mitoses
among the germinal cells lining the ventricles, it is clear that
development had not by any means stopped when the specimen
was fixed.
    Turning to the organs of special sense, we find both eyes
developed, but very abnormal. Both are very small as compared
with the eyes of a normal 5-day chick. Each has an optic
cup and a lens. The optic stalks are narrow, the process of
narrowing down the original wide aperture from brain to optic
cups having been complete. The lenses show signs of con-
siderable retardation, and one of them of abnormality of develop-




  FlG. D.—Drawing of section of right eye (the pigment has been omitted in this and fig. E).

ment. In the more normal of the two (the left—figs. 1, E) the
lens is well formed, except that the thinning out of the external
layer of cells has not proceeded nearly as far as it should have
done in a 5-day chick. The lens of the right eye, however
(fig. D), is of abnormal form, being in places triangular in
section, with but little difference between the two layers. The
optic cups are both very abnormal. Instead of being large,
approximately spherical sacs, they are without any regular
shape, and the walls are thrown into a number of large folds,
somewhat as though an unsuccessful attempt had been made
to grow into a large sphere of normal size. They are also very
much smaller than they should be in a 5-day chick. The
explanation of this may perhaps lie in the position of the eyes
                                             16
   Grafted Embryonic Fragments of the Chick
relative to the other structures of the graft. It is possible that
the eyes, growing, as a consequence of the expansion of the
lateral expansion of the brain, under pressure, may have
attempted to reach the normal size and form of the eyes of
a 5-day chick, but, there being insufficient space for their
expansion, were unable to do so, so their continued growth
led to folding of the walls of the optic cup. Apart from the
question of size and form of the cup, there are two other signs of
considerable retardation. The difference between the thickness
of the inner and outer walls of the optic cup is much less than
in a normal chick of this age. There is, in fact, hardly any




                                                                         f.b.




                                                                   ~-opt St. ap.



                     FIG. E.—Graphic reconstruction of left eye.


difference at all, while in a normal specimen the inner wall is
considerably thicker than the outer. The optic nerve seems
not to have begun developing in the graft, while in a normal
chick it reaches nearly or quite to the brain during the sixth
day. A small amount of pigment has been developed in the
optic cup, but not as much as normally. Barfurth and Dragen-
dorff (1902) also found abnormal folding in the eyes of operated
chick embryos, as did Hoadley (1924) in the eyes arising from
grafted rudiments. In all recorded operative cases, the lens is
much more normal than the optic cup.
     Fig. F shows an organ which may be the otic sac. As,
however, some of the sections in this part of the series have
unfortunately been lost from the slide in staining, and as part
of this organ is involved in the loss, it is impossible to be
  VOL. III.—NO. I.                      17                                      B
        P. D. F. Murray and J. S. Huxley
certain. The structure in question is a sac occupying approxi-
mately the position expected of the left otic sac. It has walls
in part thick, in part thinner, and a cavity with an irregular
shape. This cavity gives off two tubes which run dorsally
and end close to the mid-brain. One of these may be the
endolymphatic duct, but not both. Neither can either of them
be the lagena or the sacculus, as the former does not develop
till the seventh day and the latter is by then barely indicated.



                                                 :•••. • •'   lendd.'




           biv.


                   audv.

            FIG. F.—Drawing of section of presumed auditory vesicle.

The matter must therefore remain in doubt. Neither can they
be semicircular canals as both are blind ducts, not re-entering
into the sac. This is the only structure that has been found
in any way resembling an otic vesicle labyrinth, and if it is
that, its fellow is completely absent. It could hardly be among
the missing sections as there are too few of these, and all are
close to the structure just described.
    It is doubtful if olfactory pits are present. Just anterior
and lateral to the mouth on each side is a small pit which
may possibly represent the pits. As, however, one of these
is very small and the other rather a groove than a pit the
                                      18
   Grafted Embryonic Fragments of the Chick
matter must remain in doubt, although since they are in
the correct position, the probability is that they do represent
retarded olfactory organs. Hoadley (1924) has shown the
capacity for differentiation of olfactory organs in grafts con-
sisting of the front end of the head grafted prior to the
appearance of visible olfactory rudiments.
    The epiphysis (fig. G) is present in its usual position and
is a long tube. This tube normally lies flat over the roof
of the telencephalon, thus
lying forward from its base.                    ect fP.
In the graft, however, it
has grown in a sideways
direction, and lies stretching
towards the right.       It is
possible that this occurred
through the base being
pulled to the left through
the marked growth of the
brain to that side.
    The hypophysis is re-
presented by a small mass
of tissue applied to the
dience'phalon floor (fig. H),
and still attached (in another
section) to the gut by a
Stalk.   T h e infundibulum is   FIG- G.—Drawing of section showing epiphysis.
a deep depression reaching
out posteriorly from the fore-brain. It is much larger than
would be expected, both in depth (figs. 1, 2, H) and lateral
extension (fig. C). Very probably an explanation of this is
to be sought in mechanical causes. Owing to the abnormal
upward flexure of the telencephalon, a kink is bound to occur
at the juncture of telencephalon and diencephalon on their
ventral surface—a kink which will produce a fold of just such
a nature as the one described, which would incorporate the
normal infundibulum in itself. Details of this somewhat aberrant
pituitary complex are being investigated by Mr de Beer.
    In connection with the brain, there should be mentioned
a peculiar bar of tissue (fig. 2) which extends across the cavity
        P. D. F. Murray and J. S. Huxley
of the brain from the ventral wall of the telencephalon to the
posterior (morphologically ventral) wall of the diencephalon.




                                                                                                                                 eel.



                                                                                 •••'. • • ' • ' • • • . ; . 4 i s g v • . / U


              bl. v. '"•••'.'•••                                  \           •'.'•:'•
                          •       -       .   -   .   .   •   .       \




                              •       •                                   '




                                      -• • •"•'hyp.                               '
                 FlG. H.—Drawing of section showing hypophysis.


This bar consists of neuroblast tissue similar to that of the
future grey matter of the brain, but includes no fibres. It
is traversed by a blood vessel. Just behind its ventral end
                                                                                         ves.
                                                                                      •'/ V ^




                                                                          br.car.
           FIG. J.—Abnormal vesicles in the wall of the mesencephalon.


is a slight depression in the brain-floor. Its interpretation is
exceedingly difficult. Obviously it corresponds to no normal
structure in the brain of the chick.
                                                                                      20
  Grafted Embryonic Fragments of the Chick
    Another abnormal feature is the existence in several places
in the brain, but especially on the left ventro-lateral region
of the wall of the mesencephalon, of groups of small vesicles
or tubes (figs. J, K). Most of these small vesicles or short
tubes have small cavities. They are usually embedded in the
brain wall, external to the neuroblasts. Sometimes, as in fig. K,
they protrude slightly from the general brain surface. Their
walls are composed of neuroblasts with often several mitotic
figures in the central layer, as though these were germinal
cells. Usually there is a clear area without nuclei immediately




                                                   rues'.
  FlG. K.—A very small and a large abnormal vesicle in the wall of the mesencephalon, the
                       larger one protruding and partly separated.


surrounding the central space, and this is suggestive of the
narrow clear area internal to the germinal area of the brain-
wall itself. It differs, however, in having taken a lighter stain.
    A still more striking abnormal development of the same
general type is illustrated in figs. 3, 4, C, L, M. Two quite
large " organs " of brain-tissue have been produced and make
prominent features on the ventral surface of the fore-brain.
They are completely continuous with the brain proper in certain
regions, but elsewhere protrude freely over large areas. Else-
where again they are closely applied to the brain-wall in such a
way that it is often hard to say whether continuity exists or no.
This partial continuity is brought about by the fact that in
  VOL. III.—NO. I .                        21                                   B2
             P. D. F. Murray and J. S. Huxley
several places both they and the brain-wall proper are not
sharply delimited in the normal manner, but show an irregular
margin with cells apparently proliferating outwards in a loose
way into the surrounding mesenchyme.




               mtti.F.                             ect'.      ps.oy.
FlG. L.—Section showing larger pseudo-organ on ventral side of fore-brain produced by pro-
    liferation of brain-tissue, (fn this and fig. M, the histology of the brain-wall has not been
    indicated.)


    In other parts, however, the tissue both of these pseudo-
organs and of the brain is dense and sharply delimited against
the mesenchyme. Numerous vesicles or tubules are to be seen




                             ie.                           e'ct.       py
            FIG. M.—Drawing of the pseudo-organ shown in fig. L ; another section.


in these "organs." Often the vesicles are identical with those
just described as existing in the brain-wall. Sometimes they
are very minute, with only a virtual cavity; or even only an
indication of radial cell-arrangement. Some, however, are large
and spacious, looking like definite morphological features
                                              22
   Grafted Embryonic Fragments of the Chick
    In some places the " organs" are in contact with the
ectoderm ; but no modification of either tissue is visible in these
regions.
    It is clear that these large pseudo-organs result from a
further extension of the process which gives rise to the embedded
vesicles: as to the particular reason for their production, how-
ever, we are quite in the dark. It may be noted that the
smaller (left) of the two " organs " is much elongated laterally




          rhomb




    ect
                    xp.

                                                                  mth rec.
                  FlG. N.—Drawing of longitudinal section showing gut.

(fig. C). It looks as if the primordium of this formation had
been produced early, and had then become involved in the great
lateral extension of the whole of this side of the graft.
     The undefined margin of the nervous tissue in certain areas
is interesting, as is the fact that in such regions the cells appear
to be migrating irregularly out into the surrounding tissue, and
form a much less compact tissue than usual. We have here
something which reminds us of the conditions obtained by
Hoadley (1924, 1925) in some of his mesencephalon grafts,
although the details are different. Other authors have noted
extra proliferation of the embryonic central nervous system of
                                          23
           P. D. F. Murray and J. S. Huxley
the chick as a result of interference, etc. ; e.g. Alsop (1919)
(temperature); Harman (1918) (abnormal specimens found in
nature); the folded optic cups above discussed, etc. However,
so far as we are aware, such extensive abnormalities as the bar
and the pseudo-organs have not hitherto been described.
    The Alimentary Canal (figs. N, O).—There is present a gut
(pharynx), mouth, and anterior intestinal portal. The mouth
opens into the gut. No trace can be found of lungs. These
                             should be well marked at forty-eight
                             hours, but it is doubtful if the site
                             of the laryngo-tracheal groove would
                             have been included in the graft. A
                             solid mass of cells is found attached
                             to the floor of the pharynx. This
                            presumably represents the thyroid,
                            although it is considerably retarded.
                                 Of visceral pouches (v.p., fig. O)
                             there is one on the left side, two on
                             the right. Those on the right side
                             are large with prominent openings,
                             while that on the left is small and
                             leads to the exterior through a
                             narrow tube. The external aper-
                             tures of the two right pouches are
FIG. 0.—Graphic reconstruction of gut, more or less confluent.           In a normal
   from ventral   side.   The   recesses   u - t i - t - ' U         J      J I _ - J
   round mouth and anterior intestinal <&^        b o t h t h e Second a n d t h i r d
   portal are indicated by hachures ; p o u c h e s h a v e d o u b l e Openings, b u t
     ^^^OTt«rOTrthofri«ht                     s d0rSal

                             There is apparently no trace of
this division here. Another peculiarity of the right pouches
is that there is given off from their united wall, close to
the external opening, a short prominence containing a cavity
(fig. O (x)). There is nothing to show to what, if anything,
this corresponds in the normal chick. There are no traces of
visceral arches, and in the absence of both these and of the
other pouches it is impossible to tell to which pouches of the
normal chick those present in the graft correspond. The
presence of three open visceral pouches is another feature
indicating retardation, since the third pouch, the last to close,
                                             24
   Grafted Embryonic Fragments of the Chick
is normally closed during the fifth day. The absence of the
others, indeed the absence of any trace of them, indicates that
they never formed. Normally they would close on the third,
fourth, and fifth days, and one would expect that some trace of
them would remain in a chick showing considerable retardation
of at least most of its organs such as the one under considera-
tion. Even in a normal chick the epithelial tubes representing
the second, third, and fourth gill-pouches remain till the sixth
day. That the left visceral pouch in the graft has already
reached the condition of an epithelial
tube, argues that it is further advanced
than those on the right. So we are
forced to the somewhat curious con-
clusion that one p6uch is slightly
retarded, two are much retarded, and
the remainder have not developed.
There is no trace of a tubotympanic
cavity, part of which is of course
normally derived from the dorsal
division of the first visceral pouch.
    Attached to the dorsal side of the
gut just anterior to the anterior in-
testinal portal is an apparent Organ FlG- P-—Graphic reconstruction o       f
                                            tubular organ arising from gut.
consisting of a number of tubules and       The arrow shows the entrance
vesicles (fig. P). It seems impossible      from the gut cavity. The por-
                                            tions marked * are closed vesicles;
to regard this as having any corre-         the rest are tubular and in open
spondence with any organ in a normal        communication with the gut.
chick except the liver or pancreas. It
can, however, hardly be the former, as it arises from the wrong
side of the gut, or rather of the anterior intestinal portal, being
on its dorsal side instead of ventral. It is therefore impossible
to say what it may be, except just possibly the pancreas, which
of course is a dorsal structure. The organ in question, while
arising from the dorsal side, lies mostly laterally. In a normal
chick both liver and pancreas arise about the end of the second
day. Now the graft was made at about twenty-four hours.
Further, the liver and pancreas are both very close to the
opening of the anterior intestinal portal when they arise. Since
a considerable portion of the gut must become closed in during
                                      25
          P. D. F. Murray and J. S. Huxley
 the second day, it is highly improbable that the region destined
 to form liver and pancreas could be included in the region
closed off in the graft, since it is hardly possible that, being
affixed to the chorio-allantoic membrane ventrally, any more of
the gut became closed off after grafting. It is thus improbable
                                          that this body can be either liver
                                          or pancreas, and so it is impos-
                                          sible to say what it is, unless one
                                          or other of these has been abnor-
                               v,.bdb.m. mally formed more anteriorly than
                                          usual.
                                              The blood vessels of the
                                          graft have not been worked out
                                      ect thoroughly. It, of course, receives
                                          its blood supply from the vessels
                                          of the chorio-allantoic membrane.
                                          The heart is present, but in part
                                          only (figs. Q, R). The piece
                                          which is present consists of a
                                          bulbus arteriosus and at least
                                          part of the ventricle, but there is
                                          no auricle or sinus. Evidently
                                          the cut which isolated the head
                                          passed through the heart in such
                                          a way that the future ventricular
                                          region or a large part of it was
     v'                                   included in the graft, while the
                                          auricular region was not; the
                                          part contained in the graft con-
                                          tinued its differentiation, but there
                                          was no regeneration of the auricle.
   FlG. Q.—Longitudinal section of heart.
                                          Thus in the heart rudiment at
                                          twenty-four hours the auricular
and ventricular regions are predetermined, although not visibly
differentiated, as otherwise the part included in the graft should
have made at any rate some attempt at the formation of both
these regions. In a normal chick the constrictions between the
chambers only appear during the third day. The septa between
the auricles and ventricles only begin forming on the fifth day in
                                      26
   Grafted Embryonic Fragments of the Chick
a normal chick, and in this specimen are entirely absent. The
cardiac muscle, on the other hand, seems to be well-developed
histologically, there being thick muscular walls. As usual,
however, with the organs of the graft, the entire organ is small
compared to the normal size.
    The mesenchyme of the surrounding region is continuous
with mesenchyme within the heart, there being an open collar
                                        bulb

                                                           mes
                                                           '   bulb, m




                             V.'




FIG. R.—Graphic reconstruction of heart, from below. The mesenchyme is dotted, the muscular
                          and partly muscular tissue cross-hatched.


of muscle around the large area of entrance (fig. R). From the
centre of this area springs a vessel which continues the cavity
of the bulbus. It gives off a short blind prolongation, and
continues outward to link up with blood vessels in the vicinity.
The ventricle is honeycombed with irregular spaces (not shown
in fig. R). At one place (see fig. Q) its wall is incomplete,
so that here too the external mesenchyme penetrates into the
interior of the organ The muscular tissue of the bulbus is
much better differentiated than that of the ventricle.
                                           27
        P. D. F. Murray and J. S. Huxley
     As regards skeleton, there is no cartilage in the specimen,
and the presence of procartilage is very doubtful. Chondrifica-
tion begins in the normal chick only on the sixth day, so the
absence of cartilage cannot be taken as a sign of retardation.
Examination of the notochord (nch., fig. C) reveals a very
curious condition. It is very minute. It does not extend as
far forwards as the diencephalon as is normally the case, but
comes to an abrupt end on the ventral side of the hind-brain,
petering out into a small mass of what appears to be degenerate
tissue. As this mass of degenerated tissue is very small, it
is improbable that any conclusions can be drawn from it as
to the fate of the rest of the notochord. Until more is known
about the origin of the notochord, and especially of its anterior
end, in the normal chick, speculations as to the reasons for
its absence in this specimen have little value.
      The next two specimens to be described, Nos. 45 and 50, do
 not require detailed description as they both show in essentials
 the same general features as the previous one, i.e. continued
growth and differentiation after grafting; retardation of both
growth and differentiation; and distortion. Both were pieces
 taken from the middle of the body, in the case of No. 45 of a
 2-day embryo and in the case of No. 50 of a 24-hour embryo.
      In both, the spinal cord has undergone differentiation, and
 there are dorsal and ventral spinal roots. In both, the vertebrae
 are well developed and chondrified (fig. 6). The notochord,
 prominent in No. 45 (fig. 6), is altogether absent in No. 50. This
 is curious, especially as No. 45 represents a 7-day, and No. 50 a
 6-day chick. Normally chondrification of the vertebrae begins on
  the fifth day. In a normal chick, the notochord is prominent in
  the centre of at least many vertebrae up to eight days. There
 is, of course, no ossification in either.
       No. 45, which was taken from the region of the body
  including the omphalo-mesenteric arteries, shows on each side
 a region of thickened mesenchyme, resembling procartilage
  (h.l.b., fig. 6). This is probably the rudiment of the skeleton of
  the hind limb bud. The four projections, representing the four
  regions, axis of limb, ilium, pubis, ischium, are not as yet
 distinguishable.     There is no true cartilage in this mass of
  dense mesenchyme, and even procartilage is doubtful.
                                 28
   Grafted Embryonic Fragments of the Chick
     There is no alimentary canal in either specimen. This is
presumably because both specimens, when grafted, were taken
 from regions in which the gut had not been closed off. When
they were grafted they became attached and spread-eagled
out on the chorio-allantoic membrane, so that closure of the
gut became impossible. There is a gut present in the above
described head-region (No. 21) because in that case it had
been closed off before grafting.
     No. 45 (fig. 6) has, on each side, a mesonephros, lying in its
usual position. The ccelome is present and the mesonephros lies
against it as usual. No. 50 has a mesonephros on one side
but none on the other, and has no ccelome. As the meso-
nephros only begins to develop at about forty-eight hours, it is
clear that in the case of No. 50, which was grafted at twenty-
four hours, the entire mesonephros must has developed since
grafting; while in the case of No. 45 it might have been laid
down as vesicles before grafting, but certainly has undergone
most of its development since then.
     In No. 50, the embryo has been distorted obliquely; this
is especially noticeable in the spinal cord. Myotomes are
developed, especially well in No. 50.

                          3. Discussion.
    The chief point of interest lies in the power of independent
differentiation of a whole region of the body, such as the head
or a section of. trunk, when supplied with food by the blood-
vessels of the host chorio-allantois. Thi§ is combined with
an apparently total lack of regeneration. This finding confirms
those in many other vertebrate groups, both as regards single
organs and large fragments of the embryo.
    To mention only the work on the chick, the self-
differentiating capacity of various organs and regions has been
demonstrated by Barfurth and Dragendorff (1902—eye),
Lillie (1903, 1904—large anterior regions of embryos),
Spurling (1923—limbs and girdles), Minoura (1921—gonads),
Atterbury (1923—metanephros), Hoadley (1924—eye, olfactory
region, auditory organ, mesencephalon), Agassiz and DanGhakoff
(1923—spinal cord). In particular Danchakoff (1923) has
                                29
        P. D. F. Murray and J. S. Huxley
recently published a preliminary account of experiments
essentially similar to ours both in material and results. We
may perhaps mention that much of our work had already been
accomplished before this note was published.
     It thus appears that in all vertebrates there comes a period
after which the potentialities of each region of the germ are
rigidly fixed, as opposed to an earlier period during which
complete regulation is possible.         Spemann (references in
Spemann, 1921) has shown in Triton that the time when the
first phase passes into the second is about the middle of
gastrulation. One of us (Huxley, 1924) has proposed the
term chemo-differentiation for the process of differentiation
which then sets in, since it appears to make of the germ a
mosaic of chemically different regions, each one capable of
pursuing its own development independently of the rest (provided
suitable conditions are provided) up to the onset of the third
period, that of functional or co-ordinated differentiation, when
nervous and endocrine co-ordination, as well as functional
modification, come to play a part.
     The three stages, and especially the second and third,
overlap considerably as regards different organs and parts of
organs. For any one organ or part of an organ, however,
they seem to succeed each other invariably in this order. This
can be regarded as one of the most important empirical laws
discovered in recent times as regards the developmental
physiology of vertebrates.        It seems valuable to have the
existence of the same stages demonstrated for the highest,
homothermic vertebrates, and these observations provide some
evidence helping to establish this.
     Grafts like these, however, throw certain new light on the
question of early differentiation. They show (1) that retardation
and inhibition of development accompany any unfavourable
circumstances; (2) that the self-differentiation above described
is essentially chemical, leading to the production of specific
tissues in definite amounts; but that (3) the assumption of
particular morphological form must be largely determined by
physical (mechanical) influences.
     As regards (1) effects of inhibition and retardation due to
operation or to depressants, etc., are of course well known. It
                                   3°
  Grafted Embryonic Fragments of the Chick
is worth while stressing the fundamental similarity of result
obtained by very different methods. The total absence of
certain organs in certain grafts only (see Hoadley, 1924), or
of one only of two paired organs {e.g. the auditory vesicle
of our No. 21) can only mean that although chemical pre-
determination is complete from a certain stage, the realisation of
its potentialities will not occur in certain unfavourable conditions.
E.g. immersion in various salts will produce cyclopia and failure
of certain anterior median regions to develop. Pressure and
insufficient blood supply, presumably, have brought about the
failure to develop in certain organs in our grafts—e.g. some of
the gill-slits, the R. auditory sac, etc. Retardation is thus a
partial inhibition.
     As regards (2), there are two points to be considered:
(a) Spemann (1918) has shown that the parts destined to
produce retina, tapetum, and optic stalk are not only
qualitatively but quantitatively predetermined, within a definite
area, in the neural plate stage. However, any part of this
area, even though it may possess abnormal proportions of the
 different substances, will form an optic cup with reasonable
approach to normal shape, i.e. the shape of the optic cup is
 determined quite differently from the histological differentiation
 of its various parts.
     {p) Purely mechanical considerations of available space are
 also of great importance. This was first pointed out by
 Cotronei (references in Cotronei, 1921) whose work, however,
 does not seem to be well known. The same point has recently
 been emphasised by Hoadley (1924, 1925). In many of
 Hoadley's grafts, the histological appearance was normal or
 nearly so, while grave distortions of morphological form had
 been mechanically brought about.
      This distinction between chemical and physical, between
 histological and morphological differentiation, is clearly one of
 some theoretical importance.
      The extreme asymmetry of our grafted head affords another
 interesting example of the influence of mechanical causes upon
 morphological differentiation, as does the reversed flexure of the
 telencephalon. Clearly the provision of adequate space and
 growth unhampered by adhesion and adjacent structures is as
                                 31
             P. D. F. Murray and J. S. Huxley
 important in development as the relative timing of chemical
 processes.
     Finally, the extraordinary proliferations of the brain tissue,
 leading in extreme cases to the production of new "organs," is
 very interesting. Hoadley (1924, 1925) has shown that the
 absence of the normal limiting membrane on the outside of the
 brain in a graft leads to abnormal fibre-outgrowths simulating
 nerves, together with abnormal immigration of blood vessels.
{We are reminded of the importance of bounding membranes in
 the thyroid : higher forms possess a capsule to the organ, which
if it enlarges, forms a localised goitre; but e.g. Teleost fish
have no capsule, and if a goitrous condition arises in them, the
thyroid cells proliferate irregularly, and actually invade muscle
and bone—Marine, 1911.)
    The work was carried out with the aid of grants from the
Department for Scientific and Industrial Research and from
the Royal Society, to both of which we desire to make grateful
acknowledgment.

                                   4. References.
Agassiz, A., and Danchakoff, V. (1922), "Growth of the Medullary Tube grafted into
     the Allantois," Anat. Rec, 28 (Proc), 7 (abstract).
Alsop, F. M. (1919), "The Effect of Abnormal Temperature upon the Developing
     Nervous System in Chick Embryos," Anat. Rec, 16, 307.
Atterbury, R. (1923), "Development of the Metanephric Anlage of the Chick in
    Allantoic Grafts," Amer. Journ. of Anat., 81, 409.
Barfurth, D., and Dragendorff, O. (1902), " Versuche iiber Regeneration des Auges
    und der Linse beim Hiihnerembryo," Anat. Am. (Verh. Anat. Ges., 16), 185.
Cotronei, G. (1921), "I processi di inibizione differenziale nel vestibolo boccale degli
    Anfibi Anuri," Riv. di Biol., 8 (4).
Danchakoff, V. (1916), "Equivalence of Homopoietic Anlages," Amer. Journ. Anat.,
    20, 255.
Danchakoff, V. (1922), "Grafts in the Allantois of Embryonic Anlages of the Chick,"
    Anat. Rec, 28 (Proc), 14 (abstract).
Harman, M. T. (1918), "Abnormalities in the Chick Embryo," Science, 48, 476.
Hoadley, L. (1924, 1925), "The Independent Differentiation of Isolated Chick
   Primordia in Chorio-allantoic Grafts, I.," Biol. Bull., 46, 281 ; " I I . and III.,"
     / . Exp. Zool., 42, 143.
Huxley, J. S. (1924), "Early Embryonic Differentiation," Mature, 118, 276.
Huxley, J. S., and Murray, P. D. F. (1924), "A Note on the Reactions of Chick
      Chorio-allantois to Grafting," Anat. Rec, 28, 385.
Lillie, F. R. (1903 and 1904), "Experimental Studies on the Development of the
     Organs in the Embryo"of the Fowl, I and II," Biol. Bull., 6, 92, and 7, 33.
Marine, D. (1911), " Further Observations and Experiments on the so-called Thyroid
      Carcinoma of the Brook Trout, etc,"/. Exp. Med., 18,455.
                                           32
    Grafted Embryonic Fragments of the Chick
Minoura, T. (1921), "A Study of Testis and Ovary Grafts on the Hen's Egg, etc.,"
   Journ. Exp. Zool., 88.
Murphy, J. B. (1913), " Transplantability of Tissues to the Embryo of Foreign Species,"
   Journ. Exp. Med., 11, 483.
Spemann, H. (1912), "Zur Entwicklung des Wirbeltierauges," Zool. Jahrb., 82.
Spemann, H. (1918), "Ueber die Determination der ersten Organanlagen des
    Amphibienembryo, I. to VI., Arch. Entw. Mech., 48, 448.
Spemann, H. (1921), " Die Erzeugung tierischer Chimaren, etc.," Arch. Entw. Mech.,
    48, 533-
Spurling, R. G. (1923), "The Effect of Extirpation of the Posterior Limb Bud on the
    Development of the Limb and Pelvic Girdle in Chick Embryos," Anat. Rec,
    26, 41.


           5. List of Abbreviations employed in the Figures.
   Note.—All figures except fig. 6, plate I., refer to grafted head No. 21. For the meaning
             of the abbreviations, consult the list below. The small arrows indicate the
             morphological axis.

a. i. p.     . Anterior intestinal                    mth . . . Mouth.
                      portal.                         tnth. f. . . Fold of ectoderm ex-
 aud. v. . . Auditory vesicle.                                       tending forwards
 b. . . . . Bar across fore-brain                                    from mouth-recess.
                 cavity.                              mth. rec.. . Mouth-recess.
 bl. v. . . . Blood-vessel.                           ntyot. . . . Myotome.
 br. cav. . . Brain cavity.                           n. a. . . . Neural arch.
 bulb. . . , Bulbus.                                 nch. . . . Notochord.
 bulb. m. . . Muscle fibres of                       opt. c . . . Optic cup.
                 bulbus.                             opt. st. . . Optic stalk.
 b. v. ap. . . Aperture from ven-                    opt. st. ap. . Aperture from brain-
                 tricle into bulbus.                                  cavity into optic
 c. .          Centrum.                                               stalk.
 coel. . . . Coelom.                                 p.fl. . . . Pontine flexure.
 di. . . . . Diencephalon.                           ps. org. . . Pseudo-organs pro-
e. .           Eye.                                                  duced by prolifera-
eel.. . . . Ectoderm.                                                tion of brain-tissue.
end. d. . . Endolymphaticduct.                       r. e. . . . Right eye.
 ep. . . . . Epiphysis.                              rhomb. . . Rhombencephalon.
f.b.    . .    Fore-brain.                           sp.c. . . . Spinal cord.
&. .           Gut.                                  t. . . . . Tubular organ at-
g.c. . . . Gut cavity.                                               tached to gut.
h.b. . . . Hind-brain.                               tel.. . . . Telencephalon.
h.l.b.. . . Hind-limb bud.                           v. . . . . Ventricle.
ht. . . . . Heart.                                   ves. . . . Abnormal vesicles in
hyp. . . . Hypophysis.                                               brain-wall.
in/./.. . . Infundibular fold.                       v.p..     . . Visceral cleft or
I. . . .       Lens.                                                 pouch.
I.e. . . . Left eye.                                 v. r. . . . Ventral root of
m. . . . . Mouth.                                                    spinal nerve.
m. b. . . . Mid-brain.                               x. . . . . Tubular prominence
mes. . . . Mesenchyme.                                               on right wall of
tnes. n. . . Mesonephros.                                            pharynx.
  VOL. III. —NO. I.                           33
          P. D. F. Murray and J. S. Huxley
                         6. Description of Plate.
FIG. I.—Photograph of section of left eye ; medium power.
FIG. 2.—Photograph of longitudinal section of the head ; low power.
FIG. 3.—Photograph of a section of the pseudo-organ, of which another section is
      shown in fig. L.; medium power.
FIG. 4.—Photograph of the smaller pseudo-organ of the same type ; high power.
FIG. 5.—Photograph of section of right eye, showing markedly folded optic cup ;
      high power.
FIG. 6.—Photograph of transverse section of graft No. 45 ; low power.




                                      34
T H E DEVELOPMENT OF GRAFTED EMBRYONIC FRAGMENTS
OF THE CHICK.—P. D. F. MURRAY AND J. S. HUXLEY.




                                                                                     F.b



           opt. st.
    opt.




      ect.

                       I.




                                                   h.Lb.   y.r.   sp.c   n.a   ect   myot




                                                     C.      n.ch.       mes.n.