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Differentiation in culture of pieces of the early chick blastoderm. I. The definitive primitive streak and head-process stages

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DIFFERENTIATION I N CULTURE O F PIECES O F THE
EARLY CHICK BLASTODERM
I. THE DEFINITIVE PRIMITIVE STREAK A N D HEAD-PROCESS STAGES
DOROTI-IEA RUDNICX
Department of Zoulogy, Uniaersity of Rochester
ELEVEN FIGURES
INTBODUCTION
In the course of exploration of various isolation techniques,
several fairly extensive series of short-time cultures of pieces
of the chick blastoderm have been prepared and studied. The
present communication deals with the definitive primitive
streak and head-process stages.
There have been many recent experimental investigations of
the organization of these stages in which isolation methods
have been used. Wetxel ('24, '36) and Hoadley ( '26) have
used an in situ sectioning technique. Waddington and his
collaborators ( '32, etc.) have combined explantation methods
with transplantation of embryonic parts. Waddington ( '32,
'35) has also at various times investigated the differentiation
of large pieces of the blastoderm on plasma clots, as well as
the morphogenesis of the whole blastoderm under these conditions ; Waterman ( '36) has briefly reported defect expcriments using this method. Hoadlev ( '26 b), Willier and Rawles
('31), Hunt ('31, '32)' Dalton ( ' 3 5 ) ' Rawles ('36), t o mention
oiily a few contributions, have tested various parts of blastoderms in the axis-forming stage by means of the chorioallantoic graft.
The preliminary question raised by the various results
obtained in such isolations seems to me to be the following:
In what degree do the structures differentiating from pieces
351
THB ANATOMICAL XICORD, VOJA. 70, NO. 3
352
DOROTHEA RUDNICK
correspond t o the normal axial layout of prospective areas as
determined by vital staining, and how far are they to be
ascribed to secondary reactions that may vaguely be designated
as reconstitutions or inductions? Only when this question is
answered is it possible to ask further ones concerning the
independence or dependence of various organ fields, levels
of the axis, germ layers, o r other units, and their developmental role ; or to evaluate the various experimental environments used.
The present experimental problem was to test in vitro
areas of the blastoderm comparable to those whose behavior
on the cliorio-allantois is already known. Since, with the technique used, very small pieces of the blastoderm do not differentiate in vitro, it was decided to use only transverse strips of
the pellucid area, the blastoderm being so divided into approximate fourths or fifths. In two of the series reported, the
entoderm was left intact; in the other two, the upper layer
(mesectoderm) was explanted separately. Pieces of this size
are large enough to undergo morphogenetic changes when
placed on plasma clots. S s will be seen, axial structures
arising under these conditions originate from their areas of
prospective significance.
TECHNIQUE
White Leghorn and Rhode Island Red eggs were used; 16
to 22 hours' incubation gave the range of stages used. Eggs
were opened in warm sterile Ringer, the blastoderm removed
from the yolk and dissected in Pannett-Compton fluid kept
at 38.5", under a binocular microscope. Measurements of the
length of streak, length of head process if present, o r the
distance from the pit to the anterior edge of the pellucid area,
were made with an ocular micrometer. Transverse marks
were made at the desired levels with a glass needle ; the distance of these from the pit was then measured. If the entoderm were t o be left intact, thesc marked out pieces were
simply separated one from another, the opaque area trimmed
off, and the piece transferred in a pipette to the surface of a
cover slip clot made of two drops of plasma and two drops
CULTURES O F C H I C K RLASTODERRL
I
353
of dilute embryonic extract. I n the series where it was
decided to remoire the entoderm, the marked blastoderm was
turned over in the dish. Almost invariably the light pressure
of needle necessary to make marks on the ectodermal side
had been sufficient t o sever the delicate splanchnopleure completely. The mesentodermal strips could then be removed
one by one, usually quite freely except at the streak or pit,
where dissection would be necessary. These strips, incidentally, were also explanted ; since their differentiation capacity
wa,s very slight and was limited almost entirely to mesodermal
components also found in mesectodermal cultures, they will
not be discussed here. The remaining heavy mesectodermal
strips were trimmed and transferred to clots.
The Naximow double cover slip method was used. Cultures
mere run for 2 to 6 days; if f o r more than 3 days, they were
washed in Pannett-Compton fluid and fed new plasma, or
transferred to entirely new clots every other day.
OBSERVATIONS
1. G a p a c i t i ~ ~of
s various levels. Figures 1 t o 3 show the
way the blastoderm was divided in the four experimental
series. In the first series, diagrammed in figure 1,lthe mescctoderm was explanted separately. The anterior cut was always
made directly through the pit. The second cut was made
one-fourth of the way back on the streak; this distance varied
from 0.27 to 0.59 mm. posterior to the first, as indicated. The
third cut was made halfway back on the streak, 0.59 to 1.00
mm. posterior to the pit.
Figure 2 shows a variation used in the second and third
series. I n the second, the anterior cut was made 0.14 t o
0.28 mm. anterior t o the pit, the second cut 0.14 to 0.28 mm.
posterior to the pit. The third was made 0.51 to 0.94 mm.
behind the pit-again halfway back on the streak. The posteriormost piece was not explanted, being entirely like piece 4
in figure 1. The entoderm was removed in this series.
The third series was cut as in figure 2, the first cut 0.17
to 0.34 mm. anterior to the pit, the second 0.14 to 0.41 mm.
* This series was the
subject of a preliminary report ( '37).
354
DOROTHEA RUDNICK
posterior to the pit, the third 0.51 to 0.97 mm. back. The
entoderm was included in this series, and all four levelsA, B, C, D-explanted.
It will be noticed that in the foregoing two series the node was left intact, whereas in the first
it was bisected transversely.
Figure 3 shows the cuts made on a small series of headprocess blastoderms. The first cut was made approximately
through the anterior tip of the head process. The second
cut was through the pit; in a few, it was varied to a position
Figs. 1and 2 Diagrams of cuts made on definitive primitive streak blastoderms;
series 1 to 3; see text. Figures t o the right represent distances from the primitive
pit in millimeters.
Fig. 3 Diagram of cuts made 0x1 head-process blastoderms (series 4).
as far as 0.34 mii. posterior to the pit. The third was about
one-fourth of the way back on the streak, the fourth halfway
back. Thc entoderm was left intact in these cases, its removal
being impractical.
Table 1presents a summary of the structures found in the
various types of piece explanted. It will be noted that the
piece is designated as in figures 1 to 3, the series to which it
belongs (vide supra) being indicated in the second column.
Thc measured limits of the pieces can be seen by reference
to the figures.
CELTURES O F C H I C K BLASTODEKM.
355
I
To take the levels in ordcr, in the dcfinitive primitive streak
and very early head-process stage : anterior material, cut at
least as f a r as 0.34mm. anterior to the pit, may form medullary
tube o r plate, though not invariably. If the cut be made
through the pit, the proportion of medullary differenti at'ion
TABLE 1
Showing distribution of dzffereictiated structures in definztive pmmzttve streak
and head process semes dangramrned in figures 1 t o 3 and desertbed in the text.
Figwres under pach heading show number of cases ~n which structure wus
identtfied; questtonable cases are also indicated. 'Mesenchyme' zs recwrded
only when formed i n d ' e a differentiated culture-not
when migrated as
fibroblasts. ' Epithelzum' refers t o oolwmnar and aubogal types; not to thin
membranes. .Endothelium i s recorded only when clear-cut blood tessels are found
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increases, and a few dubious cases of heart muscle occur. In
all this group, the presence of entoderm appears to have no
effect on the differentiation of the upper layers.
The node level (B), if thc node be left intact, produces
medullary material nearly always. Functional heart muscle
appears here rather strongly. Chorda is very frcqucnt. An
356
DOROTHEA RUDNICH
effect of the eiitoderni appears at this level: it will be noticed
in the second B group (series 3 ) that chorda is recorded in
practically all cases: 95 t o loo%, instead of 75 to 90% as in
the mesectodermal gronp. Also, body mesenchyme occurs in
100% of the former group, and is better developed in almost
every individual case. The removal of the entoderm in these
pieces undoubtedly has a mechanically disruptive effect: it
will be recalled that dissection was almost always necessary.
The post-nodal, anterior streak level (C) shows strikingly
diminishing capacity for formation of medullary plate. Only
the more inclusive C groups contain any of this tissue at all.
These are all of inferior grade, some dediffcrcntiating, some
very dubious. All are in pieces cut 0.21 mm. or less behind
the pit. The 3 group, where the anterior cut mas 0.27 mm.
or more posterior to the pit, contains none at all. Chorda,
as might be expected, is lacking in this group, as is sensory
epithelium. The series containing cntoderm ( 3 ) shows strikingly better heart differentiation; this of course is t o bc expected in view of the fact that the lower mesodermal layer
usually adheres to the entoderm; the only surprising thing
is that the difference did not appear previously, at the node
level.
The posterior (D, 4) piece produces erythroblasts uniformly; nothing else besides a little mesenchyme and epithelium. These pieces frequently do not differentiate at all, as
will be seen by comparing the number of cases in the 4 group
with other series 1 groups. All these levels were explanted
in equal numbers; the number of cases reported indicates
the proportion of successful differentiating cultures obtained.
The head-process cultures show the same picture, in slightly
expanded terms. The piece anterior to the process can form
medullary tube, as do process and node levels (1B). These
latter have strongly developed heart masses, frequently
double. Chorda, body wall epithelium, and mesenchyme are
regularly formed. The posterior half of the node (2) has
rather poor capacity f o r forming medullary tissue and chorda;
heart is more frequent. The posterior levels (3, 4) form
mesenchyme, some epithelia and erythroblasts.
CULTUBES O F CHICK BLASTODERM.
I
357
Thus a consistent picture is provided by the distribution of
differentiated structures in these series. Medullary tube or
plate is found regularly in node level strips, and for some
distance anteriorly. This corresponds with the prospective
significance of the anterior material ; and the relations of the
structures as they are observed differentiating coiifirm the
interpretation that these medullary tubes actually correspond to the prospective brain region. Posterior to the
node, however, the capacity for medullary plate formation
falls off rapidly; most of the prospective medullary field at
streak levels is incapable even of an attempt at forming medullary plate under these experimental conditions. The material
classified as ‘sensory epithelium’ will be discussed subsequently; here it may be noted that this epithelium is formed
with considerable frequency in the node and anterior levels in
streak stages ; relatively rarely in corresponding regions of
the head-process blastoderm. The notochord arises only from
the immediate vicinity of the primitive pit ; heart from lateral
material at and somewhat posterior to the node level. Body
wall ectoderm is found in all but posterior pieces ; mesenchyme
and indeterminate epithelia all through the levels tested ;
blood-forming tissue may arise almost anywhere, although
most strikingly in the posterior streak region.
2. Diferentiation. of the explaiated strips. The form assumed by the explanted pieces, always established in its essentials after 24 hours in vitro, was absolutely characteristic
for each level, although, of course, variations on the groundpattern mere abundant. Pieces anterior to the pit or process
tip, whether of the 1, A, o r 1A type, formed very large thinwalled vesicles, simple or complex. Usually such cultures
contained, in addition, small dense vesicles found subsequently
to be composed of mednllary tube, i.e., forebrain (fig. 9).
Strips containing all o r part of the node o r head process
behaved quite differently on the clot. The median region
would thicken considerably, remaining opaque, very rarely
forming a vesicular structure. In this mass, medullary tissue
and notochord could sometimes be distinguished in the living
358
DOROTHEA RUDNICK
culture. The lateral ends of the strip would swell, becoming
thin-walled vesicles which often contained pulsating masses
of heart tissue. This sort of culture is illustrated in figure 4,
where one lateral vesicle and a median one are shown; the
other lateral end had failed t o vesiculate. Fignre 6 illustrates
a section of such a culture.
Pieces of the 3 or C type underwent a rather striking
morphogenesis, of the sort shown in figure 5. The median
portion, instead of forming medullary plate, would become
a dense cord. This, on later days, might become extremely
long, thin and contorted. The lateral regions formed large
thin-walled vesicles, sometimes with a little heart tissue inside,
but usually quite empty except for a few delicate mesenchyme
strands, cell nests, tubules o r endothelial vessels (figs. 7, 8).
Posterior pieces (4,D) did not form vesicles in more than
half the cases. These pieces remain homogeneous ; the streak
disappears; usually the explant flattens out as a dense membrane, with slight peripheral migration. When vesiculation
does occur, the whole explant takes the form of a blister
rather than of a complete spherical structure. The interior
remains full of cells, which after a few days are seen to be
erythrocytes. Posterior pieces, whether vesicles do or do
not fopm, yield dense masses of erythroblasts, which acquire
haemoglobin usually on the third or fourth day.
The changes in form described above are well marked by the
end of the first day of cultivation. Variations in form usually
may be traced to the failure of one lateral portion to vesiculate :
in that case, migration sets in, and the region in question
becomes a membrane closely applied to the surface of the
clot (this is occurring in the culture shown in fig. 4). The
presence of the entoderm appears t o make no difference. The
fate of these structures is somewhat diverse. Compact vesicles
and masses of medullary tissue may persist through several
washings or transfers, and histological differentiation take
place, provided cell migration does not occur. Thin-walled
vesicles usnally collapse after a day of two, forming very
thin membranes on the clot. Rarely, they may shrink, become
CIJLTUBES O F C I I I C K BLASTOUERM.
1
359
dense, arid persist ; differentiation of tissues has not been
observd in these cases. Posterior pieces nsnally persist as
dense membraiies on tlie clot, with a minimum of migration.
As between strips from definitive primitive streak aiid hcadprocess stages, the differerices a r e about what rnight he ex-
4
5
Fig. 4 P h o t o g r a p h of a k i n g culture of a mesectoderma! strip o€ level 2
(tip. 1) a f t e r 24 hours explantalioii. Relow i s one lateral iTesicle; above t h e niedian
region, i ~ l i i c his iiow heyinning migration; tlie other lateral portion is cut off
at the top; this was not vrsieiilsterl. X 40.
Fig. 5 Photograph of living ciiltiire of a mesectoderimal Rtrip of lexel 3 (fig. 1)
aftcr 34 houru. The m d a n axial region has formed t h e dark pro.jection directcrl
toward the lower right hand corner; attached a r e seen the l a r g e thin walled
l a t e r a l vesicles, contniiimg dark mesenchyme aiid bluod cores, besides a delicate
membranous complex. X 40.
1)ected. The medidlary tnbes dereloping from head-process
explants are much more extensive than the others aiid are more
apt to be of recognizablc form, coiitaiiiing rclgions tori-esponding to optic vesicles, hrain segments, etc. T n goiicral, only tlici
360
DOXOTHE.4
RT-DNTCK
axis at and anterior to the node level iindergoes a normal
morphogenesis into axial structures under the coiiditions irnposed; in head-process stages this region is of course of greater
extent than in previous stages.
The features of primitive morphogenesis that remain unaltered by transverse section of the blastoderm may be summarized as follows : llediaii axial regions conclense in vitro
as they do in normal embryogenesis. Anterior and lateral
regions (i.e., ventral and extra-embrvonic) show marlied
vesiciilation ; thus the coelomic areas already have certain
innate capacities. Node and pre-nodal regions contain the
liead aiid heart already localizccl.
As for the primitive streak, thc following points seem significant: The second quarter of the streak shows its destined
capacity f o r regression by its marked anteroposterior sliortening in vitro. This level when left attached to the anterior
p a r t of the hlastoderm, mill show posterior growth of the
streak as a little ‘tail’ (Waddington, ’32; Wetzel, ’36) j when
this region is left attached to the posterior part of the blastoFigures 6 to 11 are photomicrographs of Pections through cultures.
Allen B-15; staiu, he id en ha in'^ iron hemntosylin.
Fixation,
Fig. 6 Rectioii through’ median and oiie lateral rrrass, series 1 culture, level 2,
mesectoderm, gromi 2 days. Right, central mass with thin riicdullary plate,
mesmchyme, aiid Eonie cords. Left, corlomicavrsicle coiitaiihg mass of iiicoiiipletely
differentiated henrt niusele and soine erpthroblasts. X 75.
Fig. 7 Section through one vesicle, I)PS niesectoderui, level 3, series 1, cultured
2 days. Note douhle structure, thick mesenrhynial wall. Insidu, epithelial tubule,
spaces and blood vessel. This vesicle coiitaiiis the iiiaximuiii structure yroduccd
by level 3. X 75.
Fig. 8 Section through vesicle of %day culture, I)YR mesectodenn, level 3,
wries 1. This i? the usual picture: double vesicle of meinbranous rpitheliuiu; a
little ineaeiichpme and blood inside. x 75.
Fig. 9 JIPS, series 1 , meseetodenn, level 1 ; cultured 3 days. Vesicle of hody
wall rrtoderin, containing lobed medullary tnbe. S o t e variations i n thiekucss of
mrrlullary wall, and ‘sensory epithelial’ character of upper lobe; also straiid of
~ ~ 1 eoniiecting
1s
medullary tube with outer layer. x 75.
Fig. 1 0 DPP, mesectoderm of level 4, series 1, cultured 2 tla3s. Blister of
thin epithelium, eoiitaiiiiiig fihiohlasts an11 erythroblasts. X 75.
Fig. 11 DPS, series 1 , inesectoderm o f level 2, eultnrrd 2 days. S o t e corving
medullar!: tube, partly underlaid 1)) chorcla (right) ; somite inasses a t lower left
of ehorda. Good drvrloprnent of meseiiehuiiic aiid entlothelial vessels. X 75.
CULTUIES O F C H I C K RLASTODERM.
I
361
derm, complete regression of the streak takes place (Waddiiigton, ’36). When the level is isolated, however, a sort of 90*
deflection of growth takes place in the streak region. The
condensed streak material becomes a cord connecting lateral
vesicles; t o do this it stretches tremendously in a lateral
direction. This is accomplished, i t seems from sections, much
more by shape changes thaii by proliferation; it is not a direct
resnlt of tension in the cultures, since the cord may tx-ist and
ereiz double on itself 011 the second or third day of explantation.
The posterior half of the streak, evidently, possesses no such
morphogenetic urge ; it soon loses its identity in culture.
3. Microscopic difereuttintiow. Figures 9 and 11 show the
sort of medullary material developing in the cultures during
the first 2 days of explantation. They are ixsnally closed-ofi
tubes, irregular in shape, with out-pocketings that may or may
not be of morphological significance. In one case, such an
evaginatioii was found to be in coiitact with the outer ectoderm,
and the contact was marked by a definite spherical thickening.
This is undoubtedly an optic vesicle with lens ; other similar
formations are less sure of interpretation.
The medullary tubes or plates may vary in thickness, as comparison of the illustrations shows. There is a well-marked
ependymal layer ; on subsequent clays of culture, ganglionic
and fibrous zones may differentiate, although not in any regular pattern. This differentiation takes place only if the cnlture is transferred regularly, and at the same tinie prevented
from migrating. Usually, a disorganization process sets in
after 2 to 6 days; the medullary plates are broken up apparently by two processes: migration of cells from the plate,
and over-proliferation within the plate itself-sometimes clediffercntiatiiig masses are found black with mitoses. I n
nnhealthy cultures, o r where the tissue mass is excessively
bulky, necrosis may of course set i n ; but in the period under
discussion the migration and proliferation factors are the
major disrupting ones.
Frequently continuous with typical medullary plates (compare fig. 9) is a high pseudostratified epithelium very like that
CULTURES O F CHICK BLASTODERM.
I
363
normally forming the primordia of the sense organs-nose
o r ear. Some of this may actually be of such an extra-medultary origin; however, the relations of most of this epithelium,
and the fact that it predominates in primitive streak stages
and is relatively rare in the head-process series, makes one
incline to interpret it as low-grade medullary plate in most
cases.
hIedu3lary and sensory epithelia, especially where tubes
have formed, grow within vesicles of epithelium that resembles
body wall ectoderm: low cuboidal type, with vacuolated cells.
This differs from the thin membranes surrounding non-axial
vesicles (compare figs. 9, 8) : in section these latter are extremely thin, with almost no cytoplasm. More (fig. 11) o r
less (fig. 9) mesodermal structure may be interposed between
the medullary tubes and the body mall. Figure 11 shows a
rather extensive development of mesenchyme, notochord,
somite masses, and blood spaces.
The notochord at first develops as a solid cord, which may
or may not follow the course of the medullary tube, but which
is almost always contorted. I n some older cultures-after
3 days-the notochord may take on its characteristic vacuolated appearance; in others it never does this, but remains
in its primitive condition.
Heart develops as little masses of muscle. In its earliest
form, this tissue cannot be distinguished from mesenchyme ;
such masses may pulsate in vitro. Usually, a typical though
minute mass of recognizable primitive myocardium is present.
Interlacing myoblasts distinguish these masses ; striations
may develop after 3 days’ culture. Figure 6 shows a typical
general picture of a culture containing heart. To the left is
the original median region of the strip, containing f r a p e n t s
of medullary plate. To the right is one lateral vesicle, containing the heart mass. Another heart vesicle, not shown in
this section, is present on the opposite side.
Some sporadic formation of isolated erythrocytes seems to
occur in certain anterior pieces ; there are cases containing
only a few cells of this type, usually in endothelial channels
THE ANATOYICAL BJWORD, VOL. 70, NO. 3
364
DOROTHEA GUDNICK
or sacs. Posterior cultures tend to give a typical blood-island
picture : dense masses of erythroblasts in endothelial vessels,
although the latter may be absent. These masses are not
always surrounded by an epithelial membrane, as in figure 10 ;
but the position of the erythropoeitic masses suggests that
their origin is mesodermal. The yellow color of haemoglobin
is usually detected in the living explant during the third day
of culture-a little later than its normal first appearance,
but still while the erythroblasts are in their primitive form.
Subsequently they may differentiate completely, and become
dispersed in any space available.
The entodermal layer, in the series where it was retained,
has shown no true differentiation. Occasionally a thin epithelial layer, or even a closed vesicle, is found, which has most
probably been derived from the entoderm; hut no structure
beyond this.
DISCXJS8ION
The organization o€ the definitive primitive streak blastoderm takes on different aspects as different methods of analysis are applied. If the blastoderm be left on the yolk, and one
or more cuts be made in it (Wetzel, '36), the isolated parts will,
in general, develop according to their original intention ; the
movements of material in the streak region, and some other
features of morphogenesis, are suppressed ; but the main
axial primordia develop in the position they held at the time
the operation was performed. The same result is obtained
if the whole blastoderm be explanted to a plasma clot (Waddington, '32) or if large pieces, as half or a third, be isolated
on a clot (ibid., '35; Waterman, '36) : an embryo, or parts,
arises from the material which would normally have formed
these structures, even if the material is prevented from
executing its usual movements. The posterior, or streak
region of the axis is at a definite disadvantage when isolated
from the node j nevertheless it may form primitive trunk parts
independently. When the intact blastoderm is explanted,
trunk and tail parts form very Ii-ell. The movements of in-
CULTURES O F C H I C K BLASTODERX.1.
I
365
vagination and regression of the streak, while not absolutely
essential f o r the formation of the posterolateral medullary
plate and somites, must account for the precision with which
these events occur in the intact blastoderm. With the formation of the head process, there is no essential change in the
behavior of the blastoderm when transected. Node and
process levels form axis freely; streak levels do so with some
difficulty when isolated, although streak movements are more
uniform (Rndnick, '38).
When the blastoderm is tested by cutting it into pieces and
grafting these to the chorio-allantois, a different picture is
obtained. I n the definitive streak stage, the head regionnode level and anterior-consists of overlapping fields, of
definite histogenetic potency, but more extensive and less
definite in form than the prospectire areas corresponding to
the organs in question. The immediate vicinity of the node
can form almost all axial tissues unaided. Behind the node,
the prospective medullary fieId is weak in histogenetic potency
(Hunt, '31, '32; Stein, '33; Dalton, ' 3 5 ; Rndnick, '38). I n
head-process stages, the fields previously so concentrated in
the node vicinity become spread out to a position more nearly
corresponding to their final site : lateral and posterior organs,
in particular, become localized in this and later stages (Rudnick, '32, ' 3 5 ; Clarke, '36; Rawles, '36). The post-nodal
prospective medullary area, however, does not acquire any
further independence (Hunt, '31 ; Rawles, '36). Thus this
analysis gives a picture of the blastoderm as a system localized
only in the most general way in the node field and anteriorly,
becoming progressively more specific during the head-process
and subsequent stages (Willier and Bawles, ' 3 5 ) .
The present experiments, like the other culture experiments
and the in situ sections, show in the definitive primitive streak
stage a localized axis in the anterior portion of the pellucid
area, with the heart material, at least, already bilaterally
situated. As in the chorio-allantoic analysis, capacity for
medullary or nerve differentiation falls off very sharply behind
the node. A transverse strip of the blastoderm at the level
366
DOROTHEA RUDWICK
of the second quarter of the streak, f o r example, will never
form medullary plate in vitro, whereas the same material left
attached t o the whole posterior portion will form medullary
plate in culture (Waddington, '35) and may even, infrequently
(Dalton, '35 ; Rudnick, '38), form nerve tissue when grafted.
Dalton finds that the presence of the entoderm is necessary
for such differentiation; the present results suggest that the
entirety of the posterior piece is also of importance, at least
for the formation of medullary plate.
The present cultures also suffer restrictions in morphogenesis of mesodermal and entodermal derivatives ; indeed,
the dissection of the blastoderm appears to entail all the
developmental limitations of all other methods. This is due
apparently to mechanical block of movements in and of the
streak.
In spite of a strong tendency to migrate and disorganize,
the primitive structures formed in culture-medullary plate,
heart, notochord, erythroblasts-are capable of completing
their differentiation in vitro under suitable conditions. Burrows ('11) and Olivo ('27) have described the migration and
differentiation of neurones from young medullary plate explants ; Grigorieff ( '31) describes the differentiation of nerve
elements remaining in the original explant. The present
eultures, if transferred, form fibrous areas distinct from
ganglionic ones. No migration of nenroblasts has been observed: the cultures carried on to differentiation were selected
because the outer layer remained intact.
Olivo ('28) has likewise observed, among other things, the
differentiation of pulsating areas, and finally of striated heart
muscle, from the prospective heart areas of the stages used
here. Murray ('32) has described the differentiation of
erythrocytes from all parts of the blastoderm except the
anterior quarter of the streak. The histological differentiation
of notochord in cultured total embryos has of course been
described by Waddington ('32). Thus potencies for differentiation of cell types and small tissue units appear quite
complete even in the early stages studied here.
CULTURES OF CHICK BLASTODERM.
I
367
SUMMARY
1. Short-time cultures of transverse fourths or fifths of
definitive primitive streak and head-process blastoderms,
either with or without the mesentodermal layer, mere studied
with reference to morphogenesis of early embryonic structures.
2. The node level and anterior region, in the stages studied,
form medullary plate, body wall, notochord, and some mesoderm from their axial (median) portion, which undergoes
relatively typical morphogenesis. Anterior and lateral nonaxial regions form coelomic vesicles. Heart is localized
laterally. Removal of the mesentoderm reduces the incidence
of chorda and heart.
3. The second quarter of the streak forms no axial structures
in vitro, although the streak tissue behaves in a striking and
unique manner. Lateral regions at this level form vesicles.
Presence of the mesentoderm makes no difference in the
behavior of this region.
4. The level including the posterior half of the streak,
whether entoderm is present or no, forms large masses of
erythroblasts and a few non-specific cell types.
5 . The structures formed may be histologically similar to
those of a corresponding normal stage, or they may present
deficiencies. The best cases, at least, are capable of complete
histogenesis in vitro, although the tendency to dediffercntiation is strong.
6. Like the results obtained by the in situ sectioning method
at the same stage, the present ones show a localized axis at
the node and anterior levels; unlike the former, but like
the results from chorio-allantoic grafts, they show complete
lack of axial organization posterior to the node level.
LLTERATURE CITED
BURROWS,
M. T. 1911 The growth of tissues of the chicken embryo outsine
of the animal body with special reference t o the nervous system.
J. EX^. Zool., v01. 10, pp. 63-53.
CLARKE,L. F. 1936 Regional differences in eye-forming capacity of the early
chick blastoderm a s studied in chorio-allantoic grafts. Physiol. Zool.,
VOI. 9, pp. 102-128.
DALTON,A. J. 1935 The poteiicies of portions of young chick blastoderms as
tested in chorio-allantoie grafts. J. Exp. Zool., vol. 71, pp. 17-52.
368
DOROTHEA RUDNICK
GRIGORIBFF,
L. M. 1931 Differenzierung des Nervengewebes ausserhalb des
Organismus. 1 Mitt. Arch. Exp. Zcllf., Bd. 11, S. 483-519.
HOADLEP,
L. 1926a The in situ development of sectioned chick blastoderms.
Arch, d e Biol., vol. 36, pp. 225-309.
1926 b Developmental potencies of parts of the early blastoderm
of the chick. 1-111. J. Exp. Zool., vol. 43, pp. 151-224.
HUNT,T. E. 1931 An experimental study of the indepenclent differentiation of
the isolated Henseu’s node and its relation to the formation of axial
a n d non-axial parts in the chick embryo. J . Exp. Zoijl., vol. 59,
pp. 395-427.
_____ 1932 Potencies of transverse levels of the chick blastoderm in the
definitive-streak stage. Anat. Rec., vol. 55, pp. 41-70.
MURRAY,P. D. F. 1932 The development in vitro of the blood of the early
chick embryo. Proc. Roy. Soe. Lond., B, vol. I l l , pp. 4 9 7 5 2 1 .
OLIVO, 0. M. 1927 Rfigrazione di elementi nervosi coltivati in vitro. Arch.
Exp. Zcllf., Bd. 4, S. 43-63.
1928 ~
o b e r die friihzeitige Deterniinierung der Herzanlage beim
Huhner-embryo und dercn histologische und physiologische Differenzieruiig in vitro. Anat. Aiiz., Bd. 66, Erg. Heft, s. 108-118.
RAWLES,M. E. 1936 A study i n the localization of organ-forming areas in the
chick blastoderm of the head-process stage. J. Exp. Zoiil., vol. 72,
pp. 271-315.
R L ~ N I C KD., 1932 Thyroid-forming potencies of the early chick blastoderm.
J. EX^. ZOO]., v01. 62, pp. 287-313.
1935 Regiond restriction of potencies in the chick during embryogenesis. J. Exp. Zoo]., vol. 71, pp. 83-99.
-___1937 Differentiatioil in vitro of transverse strips of the mesectoderm
of the chick blastoderm. Anat. Rec., vol. 67, sup. no. 3, p. 43.
1938 Contribution to the problem of neurogenic potency in postnodal isolates from chick blastoderms. J. E X ~ZOO~.
.
( I n press.)
STEIN,K. F. 1933 The location and differentiation of the presumptive ectoderm
of the forebrain and hypophysis as shown by chorio-allantoic grafts.
Physiol. Zool., vol. 6, pp. 205-235.
WADDINGTON,
C. H. 1932 Expcrimentv on the development of chick and duck
embryos cultivated in vitro. Phil. Tram. Roy. SOC.B, vol. 221, pp.
179-2 30.
1935 The development of isolated parts of the chick blastoderm.
J. EXP.ZoOl., V O ~ .71, pp. 273-288.
WATERMAN,
A. J. 1936 Experiments on young chick embryos culturd in vitro.
Proc. Nat. Acad. Sci., vol. 22, pp. 1-3.
WETZEZ,R. 1924 Uber dem Primitivknoten des Huhnchens. Verh. phys. med.
Ges. Wurzburg, Bd. 49, 8. 227-233.
_ _ _ 1936
. ~
Primitivstreifen und Urkorper naeh Storungsrersuchen a m 1-2
Tage bebriiteten Huhnehen. Arch. Entwmech., Bd. 134, S. 357-465.
WILLIER,B. H., AND M. E. RAWLES 1931 The relation of Hensen’s node to the
differentiation capacity of whole chick blastoderms a s studied in
chorio-allantoic grafts. J. Exp. ZoOI., vol. 59, pp. 429-465.
1935 Organ-forming a r e w in the early chick blastoderm. Proc.
Soe. Exp. Biol. and Med., vol. 32, pp. 1293-1296.
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