Development of the chick eyeRelation of ground substance change to organ growth.код для вставкиСкачать
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 t++ - I CJU 22 30 tt n Y ' 20 t t $0 L U CO 1 9 10 11 12 13 14 IS 16 17 Incubation age -days I8 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.