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Development of the chick eyeRelation of ground substance change to organ growth.

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DEVELOPMENT O F T H E CHICK EYE: RELATION
O F GROUND SUBSTANCE CHANGE
TO ORGAN GROWTH1
J. G. ELCHLEPP2s3
Department o f Anatomy, The Univeisity of Chicago, Chicago, Illi?~ois
FIVE FIGUILES
TNTRODUCTION
Variations in the affinity of intercellular ground substance
for the intravital dye Evans blue (T-1824) have been correlated by Gersh and his associates ('49, '52) with tissue
changes during tumor growth, inflammatory reactions, and
hormonal stimulation. In these conditions, the dye, normally
restricted to intravascular channels, appears in the extravascular connective tissue, in sites corresponding to areas
of increased water solubility of ground substance components.
It was suggested that the ground substance changes thus
demonstrated might be a factor modifying organ growth
during normal embryonic development. The present study
was undertaken to test this.
The chick eye was selected as a suitable material for study
after consideration of Latimer's presentation ( '28) of embryo
organ weights measured by Schmaulhausen ( '26, '27). Latiweight y 100
body,r.eight
against the incubation age.
mer plotted the ratio :yrgKn
For the eyeball, this showed a great acceleration in relative
organ growth from the 4th to the 8th clay. From the 8th to
the 14th day, organ growth markedly slowed relative to the
'Aided by a grant from the Clara A. and Wallace ('. Abbott Memorial Research
Fund of the University of Chicago.
B a i t e r Laboratories Research Fellow.
8 Sational Cancer Institute, N. I. H., detached t o Department of Pathology,
Duke University School of Medicine, Durham, North Carolina.
425
426
J. G. E L C I ~ L E P l '
total body growth. Two successive periods of different growth
intensity are thus sharply demarcated for the chick eyeball,
a i d should be anienable to the proposed experimental ailalysis.
MhTERlALS A N D METHODS
White Leghorn chick embryos of known incubation agc
were used. Three to five embryos were taken daily from 3
to 16 days incubation age and one to three embryos daily
from 17 to 20 days. Each embryo was given an injection of
2 5 % Evans blue into the allaritoic vein, or one of its tributaries. Ten minutes after injection, the embryo was decapitated and selected tissues (eyeball a i d orbit, right wing bud
or wing, small intestine, kidney) were fixed by immersion
in isopentanc chilled to - 150" to - 160°C. in liquid nitrogen.
The tissues were then dried in vacuo at -30"C., and embedded in paraffin under reduced pressure. Sections werc
cut a t 40 p and affixed to albuminized slides by finger pressure.
Paraffin was removed from the sections with three to four
changes of warm Shillaber's oil, and the sections mounted
in this medium without further treatment. Sections cut at
10 ~1 were used f o r staining with periodic acid-Schiff (Gersh
and Catchpole, '49), hematoxylin and eosin, and the Mallory
triple stain. Similar preparations were made of tissues taken
from uninjected embryos at each incubation agc.
Estimated embryo body weight at each incubation age was
obtained by averaging measurements for white Leghorns made
by Murray ( 'as),Schmaulhausen ( '26, '27), and Needham
( '27). I n a preliminary series, the volume of dye injected
ranged from 0.1 to 0.9% of the estimated body weight. For
the orbital tissues, a dye volume of 0.2% of body weight or
greater gave consistent results in terms of extravascular dye
localization. I n absolute volumes, the injections ranged from
0.00024ml for a three-day enibryo to 0.16rril for a 20-day
embryo. For injection of these small vesscIs, capillary tubing
was drawn into calibrated needles.
I n the 10-minute interval between dye injection and sacrifice, dye appearing extravascularly remained extracellular in
GROUND SUBSTANCE A N D ORGAN G R O WTH
427
distribution. When tissue fixation was delayed or inadequate,
the dye might occasionally be seen intracellularly. Such tissues were eliminated from the series.
OBSEKVAT’IONS
The anatomical relations of the fully developed chick eye
and its associated structures are shown in figure 1. Morphologic development can be subdivided into tliree phases. First,
between 0 and 5 days, there is development of the general
form of the organ, with delineation of the retina, cornea,
arid lens. Second, between 6 and 9 days, differentiation of
the eyeball is completed, with marking-off of choroid, scleral
cartilage, iris and ciliary process areas and with the beginning
of development of accessory structures (eyelids, nictitating
Cornea -
Ey el id
-
Nictitating
Eye o s s i c l e ’
Muscla
tissue
Fig. 1 Diagram of fully-developed chick eye and its associated structures.
niembrane, Harderian aiid lacrimal glands, ossiclcs). Fiiially,
from 10 days t o Iiatching, dcvelopmcnt of the eyeball a i d
orbit is completed.
Localization of vital dye in the ground substance of orbital
connective tissue is summarized (figs. 2, 3). The morphological boundaries of this tissue are not well-defined until tllc
S c l e r a l procar-t//age
Harderion
Lacrimal
3
4
5
6
7
8
9
Fig. 2 Per cent increment iii eyeball weight aiid Evans hluc affinity iit orbital
coiiiicctive tissue from three to nine days incubation age.
7th or 8th day of development. Prior to this, orbital coiincctivc:
tissue (or mesenchyme) is arbitrarily defined as that tissue
surrounding the established outer layer of the eyeball at any
given stage of development. I n 6- and 7-day embryos, therci
was more variation in the degree of dye localizatioii at various
points around the eyeball. Although complete serial sections
of the whole organ were not studied, maximal amounts of
129
GROUND S U B S TA N C E AND ORGAN G R O W T H
the dye were consistently seen from the 4th through the 7th
day in the vicinity of the iris and in a central band encircling
the eyeball in a plane perpendicular to the optic axis. The
variations in dye localization at 14 days and from 18 to 20
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9
10
11
12
13
14
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16
17
Incubation age -days
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19
20
21
Fig. 3 Per cent increment in eyeball weight and Evans blue nfinity of orbital
connective tissue from 9 t o 20 days incubation age.
Fig. 4 Dye lornlizntion a t 6 days incubation age, camera luridn.
days were most prominent in areas of connective tissue near
the eyelids and nictitating membranes.
Illustrations of the dye distribution show tissues from a
6-day eye with minimal differentiation of scleral procartilagc
(fig. 4), and from a 7-day eye with maximal cartilage development (fig. 5) for that stage.
430
J. G . ELCHLEPP
Pig. 5 Dye localization at 7 days incubation age, cnibryo with m a s i r ~ n lsrlcr:ll
raitilage devcloynicrit foi tlint iiicnbntion age, c:tmc~a lucitln.
1,IS(’US810N
To relate the observed changes in orbital ground substance
to growth of the eyeball, some criterion of organ growth must
be selected. Latimer’s expression of organ weight as a percentage of body weight shows the greatest relative growth
of the chick eyeball t o occur between the 4th and 8th day
of incubation. Maximal dye affinity was observed to occur in
orbital ground substance at the 4th and 5th days ; this affinity
decreased at 6 and 7 days and was minimal by 8 days. L.q.ince
Latimer’s expression included the multiplc variables of total
hody weight, it was tiecitled to rc-grapll Schmaulhausen’s
data to show eye growth in terms of percentage weight increments of the cycball itself. I t can then he seen (figs. 2, 3 )
that ground substance affinity f o r the dye is actually increased
tlluriiig periods of accelerated eye growth. Also, during periods of slower change in size, (lye affinity decrrascd.
Changes in the organization of ground suhstance may hc
correlated with the degree of iritravital staining with Evans
blue (Catchpole and Gersh, ’49; Gel-sh, ’52). Dye affinity
npprtirs to he rclatcrl to increases in carbohychte, ~vatei*-
GROUND SUBSTANCE AND ORGAN GROWTH
431
soluble moieties of the ground substance in various physiological and pathological states, and it was inferred that certain connective tissue components had undergone a process
of disaggregation (depolymerization). I n the presence of
rapidly growing tumor transplants, such a process was postnlated to permit expansive growth of tumor parenchyma into
a less viscous, less resistant surrounding connective tissue
(Gersh and Catchpole, '49). The present observations suggest
that similar ground substance changes may occur during
phases of rapid growth in organogenesis, and, conversely,
that during phases of slow growth there may be an aggregation o r polymerization of ground substance. These properties of ground substance may significantly influence the
ultimate size and form of organs.
SIJMMARY
1. Increased localization of the intravital dye Evans blue
was shown to occur in orbital connective tissue during phases
of accelerated growth of the chick embryo eyeball.
2. During phases of slow growth of the chick embryo eyeball, little or no dye localization was seen in adjacent orbital
connective tissue.
3. During active organogenesis of associated eye structures,
increases in dye localization were noted consistently in adjacent connective tissue ground substance.
4. The changes in degree of dye localization are postulated to reflect changes in organization of certain watcrsoluble components of connective tissue ground substance.
5 . I t is suggested that these changes in the ground
substance may significantly affect the ultimate size and form
of organs.
LITERATURE CITED
CERSH, I.
1952 Ground substance and the plasticity of connective tissue. The
Harvey Lectures, Series XLV: 211-241.
GERSH, T., BND 11. R. CATCHPOLE 1949 The Organization of ground substance
end basement niernbrane and its significance in tissue injury, disease
:tnd growth. Am. J. Anat., S 6 : 457-522.
432
J. G. ELCHLEPI'
LATIMER, H. 1928 Growth changes in the body and some of the organs of the
chick at time of hatching. Anat. Rrc., 3 9 : 215-228.
MURRAY,H. A., JR. 1925 Chemical changes in fertile eggs during incubation;
selection of standard conditions. J. Gen. Physiol., 9 : 1-37.
NEEDHAM,
J. 1927 Energy sources in ontogenesis ; relation between absorption
and coinbustion of protein and f a t in the developing avian egg. Rr. .J.
Exp. Riol., 4: 258-279.
SCIIMAULHAUSEN,
J. 1926 Studien uber Wachstnm und Differenzierung. I1 1.
Die embrgonale Wnclistumknrve des Ruhnchens. Arch. f . Entwickl.,
108: 322-387.
1927 Beitragc zur quantitativen analyse der Formbildung. I. Uber
die gesetzmassigkeiten dcs emhryonale Wachstums. Arch. f . Entwirkl.,
1 0 9 : 455-512.
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