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Intensities of incandescent light and the development of chick embryos in ovo and in vitro
Comp. Biochem. Physiol., 1970, Vol. 35, pp. 229 to 235. PergamonPress. Printed in Great Britain
I N T E N S I T I E S OF INCANDESCENT L I G H T AND THE DEVELOPMENT OF CHICK EMBRYOS I N OVO AND IN VITRO S H E I L A T. ISAKSON, B E T T Y JO H U F F M A N and P. B. S I E G E L Biology Department, Radford College, Radford, Virginia 24141; and Poultry Department, Virginia Polytechnic Institute, Blacksburg, Va. 24061 (Received 16 December 1969)
A b s t r a c t - - 1 . Developmental differences were observed among White Rock embryos (GaUus domesticus) incubated in darkness and under various intensities of incandescent light. 2. A highly significant difference in the developmental success of embryos explanted to glucose medium was found between those incubated in light and those incubated in darkness. Exposure to light interfered with normal morphogenesis in vitro. 3. Temperatures within the culture dishes were the same for incubation in light and dark suggesting that the phenomenon observed in vitro was due to light rather than temperature.
INTRODUCTION ENVIRONMENTAL factors, such as relative humidity, light and temperature, influence the rate of development of the chick embryo. Eggs incubated under white light hatch approximately 1 day earlier than those incubated in darkness, and 8 hr earlier than those incubated under colored light (Shutze et al., 1962). Siegel et al. (1969) found that light during the first week of incubation had a more pronounced effect than light during the second or third weeks, and that the acceleration of development was apparent by 10 hr of incubation. Kallen & Rudeberg (1964) observed abnormal development of embryos exposed to light, through a window in the shell, during the first and second day of incubation. Abnormal embryogenesis and delayed hatching occurred when incubation was conducted in either continuous or intermittent light (Tamimie & Fox, 1967). I n vitro studies showed glucose was the only essential nutrient for normal development of the chick during the first 2 days (Harrison, 1960; Harrison et al., 1965). They found that glucose provided the necessary energy for the extensive cell migrations occurring during this period. Therefore, the early acceleration of development observed by Siegel et al. (1969) may be linked with an effect of light on the glucose metabolic system. The present study was designed to observe the effects of various intensities of incandescent light in ovo and in vitro during the first 48 hr of incubation. 229
230
SHEILA T . ISAKSON, BETTY Jo HUFFMAN AND P. B. SIEGEL
MATERIALS AND METHODS Eggs from low-weight lines of White Rocks (Siegel, 1962) were incubated at 37-5°C and 75% r.h. in a forced-draft incubator. Light was supplied from incandescent bulbs located 13 cm above the middle region of the incubator trays. Light intensities were varied by using bulbs of different wattage. Treatments consisted of: (1) complete darkness, (2) low intensity illumination (ranging from 54 to 108 luxes), and (3) high intensity illumination (ranging from 215 to 430 luxes). Illumination was measured at the opposing end-sections of the trays where the eggs were incubated. I n order to provide approximately equal amounts of diffuse light to all embryos, eggs were never placed directly under the bulbs. After 24 hr of incubation, in ovo blastoderms were removed from the yolk by the method of Harrison (1967) and staged according to the Hamburger & Hamilton process (1951). All blastoderms were from primitive streak through 10-somite stages. Staged blastoderms were then explanted to watch glasses containing the buffered glucose-saline-agar medium of Harrison (1960). This glucose culture method was chosen to provide the same frame of reference for comparison of effects of light on the glucose metabolic system in vitro rather than some of the more recent vitelline membrane culture techniques. Light regimens of explants were the same as those given during in ovo incubation. After 24 hr explants were classified as normally developed, abnormally developed or not developed. Explants exhibiting axiation with indistinct morphology, Fig. 1 (1), were classified as not developed; those having a brain, cord, somites, heart and vitelline vessels, Fig. 1 (2), were classified as normally developed; explants with either an open neural cord, Fig. 1 (3), an open neural cord and indistinct somites, Fig. 1 (4), indistinct somites and edema, Fig. 1 (5), or lacking somites, Fig. 1 (6), were classified as abnormally developed. Air temperatures in the incubator and within the culture dishes were measured at 4, 16 and 24 hr of incubation with a thermometer probe (Tele-thermometer; Yellow Springs Instrument Co.) placed either in the incubator or directly over the explant, i.e. immediately under the lid of the culture dish, for approximately 1 rain. Data were analyzed by Xa utilizing the correlated test statistics of Jensen et al. (1968). This procedure allows data to be used in more than one X2 analysis for simultaneous tests of hypotheses. Correction for this lack of independence was made by using the Bonferroni X2 tables (Jensen et al., 1968).
RESULTS Classification of 279 embryos for somites after in ovo incubation in darkness or under various light intensities is summarized in Table I. There was a highly significant difference among treatments with development greater for embryos incubated in light than in darkness. The percentage of embryos that developed somites was similar for both high and low light intensities. TABLE 1--PERCENTAGE OF EMBRYOS WITH SOMITES AFTER 24 hr in OR DARKNESS
ovo
INCUBATION IN LIGHT
Incubation r~gime
No. embryos With somites (%) Lacking somites (%)
Darkness
54-108 luxes
215--430 luxes
140 11 89
82 28 72
57 26 74
FIG. 1. Photomicrograph of: (1) explant exhibiting axiation, but indistinct morphological structure; (2) explant exhibiting normally developed brain, cord, heart and somites; (3) explant with an open neural cord; (4) explant with an abnormal brain, open neural cord and small somites; (5) explant with small and indistinct somites and edema; (6) explant with somites lacking. Photomicrograph 1 was classified as not developed, photomicrograph 2 as normally developed, and photomicrographs 3-6 as abnormally developed.
INCANDESCENT L I G H T AND THE DEVELOPMENT OF CHICK EMBRYOS
231
T h e developmental success of 281 explants is presented in Table 2. Comparisons of various in vitro treatments after in ovo incubation in darkness showed highly significant differences (X~ = 91.3). For low intensity in ovo incubation no significant T A B L E 2 - - C L A S S I F I C A T I O N OF EXPLANTS INCUBATED FOR
24 hr
I N L I G H T OR DARKNESS ON
S Y N T H E T I C GLUCOSE MEDIUM
Treatment In ovo
In vitro
Darkness
Darkness 54-108 luxes 215-430 luxes 54-108 luxes Darkess 54-108 luxes 215-430 Darkness luxes 215-430 luxes
No. explants
Normal development (%)
Abnormal development (%)
No development (%)
51 55 60 36 30 26 23
76 14 0 33 30 42 9
18 64 58 39 53 38 43
6 22 42 28 17 19 48
differences (X2 = 1.7) were observed when the explants were provided darkness or low intensity treatments. Likewise, no significant differences (X2=8.33) were observed when explants were provided darkness or high intensity treatments following in ovo incubation under high intensity. These results suggest that developmental success in vitro is related to the effect of light on the embryo during in ovo incubation. T o test this hypothesis more closely, the data were rearranged and analyzed again using the correlated test statistics (Table 3). T h e r e was a highly significant difference (X2= 19-2) in developmental success among in ovo T A B L E 3 - - C L A S S I F I C A T I O N OF EXPLANTS INCUBATED UNDER L I G H T AND DARKNESS
Treatment In vitro
Darkness
In ovo
Darkness 54-108 luxes 215-430 luxes 54-108 luxes Darkness 54-108 luxes 215-430 Darkness luxes 215-430 luxes
No. explants
Normal development (%)
Abnormal development (%)
No development (%)
51 36 26 55 30 60 21
76 33 42 14 30 0 9
18 39 38 64 53 58 43
6 28 19 22 17 42 48
treatments when in vitro incubation was in darkness. Explants incubated in darkness following in ovo incubation in darkness had the greatest developmental success. N o significant differences (X~ = 2-9) in developmental success of explants were observed between in ovo treatments when the in vitro treatment was under low
232
SHEILA T , ISAKSON, BETTY
Jo
HUFFMAN AND P. B. SIEGEL
intensity nor were there significant differences (X~ = 0.7) between in ovo treatments when the in vitro treatment was under high intensity. The most profound differences in developmental success of explants are found when comparisons are made between embryos from eggs incubated in darkness and subsequently exposed to light in vitro with explants incubated in darkness following exposure to light in ovo. Since elevation of temperature is known to induce developmental abnormalities, temperatures were recorded from inside the culture dishes. Results of 120 observations are presented in Table 4. Air temperature in the culture dishes was lower than the incubator air temperature of 37.5°C. This probably resulted from evaporation of water from the cotton rings in the culture dishes. TABLE 4
MEAN AIR TEMPERATURES WITHIN CULTURE DISHES JUST ABOVE THE EXPLANT WHEN INCUBATOR TEMPERATURE WAS 37.5°C
Treatment Darkness 54-108 luxes 215-430 luxes
No. observations
~ temp. (°C)
42 42 36
36"7 36"7 36"6
DISCUSSION Results from the in ovo experiment show that light accelerates development of the chick embryos, substantiating the observations of Shutze et al. (1962), Lauber & Shutze (1964) and Siegel et al. (1969). Our results also show that, within a range, light intensity affects development in ovo. Shutze et al. (1962) observed that developmental acceleration is not due to a particular wavelength. The abnormal development observed by Kallen & Rudeberg (1964) resulted perhaps because the embryo did not receive the benefit of reflection of light by the shell at the ske of the window. Tamimie & Fox (1967) also observed abnormal development in ovo with incubation condkions of twice the intensity used in this investigation and by Siegel et al. (1969). Perhaps the intensity of light used by Tamimie & Fox was so great that the shell could not reflect enough of the light to prevent deleterious effects. It appears that light within a range operates as an accelerating factor, i.e. below a low intensity threshold absorbed light resuks in acceleration, while at a high intensity threshold absorbed light resuks in abnormal development. Bursian (1964) reported that patterns of response to light by chick embryos are dependent upon wavelength and intensity of illumination as well as on the age of the embryo. He suggested that the chick embryo, at early developmental stages, possesses a primitive form of photosensitivity like that of certain invertebrates and lower vertebrates. The developmental stages of embryos at explantation were equivalent to those used in Harrison's (1960) investigation. He concluded that embryos which have attained the head fold or early somite stages are equally capable of continuing normal morphogenesis on synthetic glucose medium. The developmental success
I N C A N D E S C E N T L I G H T AND THE DEVELOPMENT OF C H I C K EMBRYOS
233
of embryos incubated in darkness during both the in ovo and in vitro treatments had relatively the same success as did those reported by Harrison (1960). A comparison of the developmental success of embryos following exposure to 48 hr of light, i.e. 24 hr in ovo followed by exposure at the same intensity in vitro, is presented in Fig. 2 (1). As the intensity of light increased, a reduction in the incidence of normal development was observed. Seventy-six per cent of the explants developed normally in darkness, while 30 per cent developed normally under low intensity regimens, and only 9 per cent under high intensity regimens. The greatest effect of light in arresting development was found in the high intensity range where 48 per cent of the explants did not develop. It would seem that the differing percentages
IOO
I
2 [] []
80
3
Normal Abnormal
[ ] Not developed 60
40 0 20
0 Dark
5 4 - 1 0 8 :>15-450 Luxes Luxes
Dark
54-108 215-430 Luxes Luxes
Dark
54-108 Luxes
215-430 Luxes
FIG. 2. Comparison of the developmental success of explants incubated under: (1) equivalent in ovo and in vitro conditions of light or darkness; (2) darkness following in ovo incubation conditions of light or darkness; (3) conditions of light or darkness following in ovo incubation in darkness.
of abnormally developed embryos within the high intensity treatment are a result of the cumulative effect of light. At the low intensity, 53 per cent developed abnormally while 43 per cent developed abnormally at high intensity. The lower percentage in the latter group is masked by the threefold increase in embryos that did not develop when compared with those which did not develop in the low intensity group. Comparisons between explants exposed to various intensities following in ovo incubation in darkness are shown in Fig. 2 (3). Only 14 per cent of the exp!ants developed normally when exposed to low intensity, while none of those exposed to high intensity developed normally. Since more than half of the explants developed abnormally with either light treatment, it is conceivable that light interferes with the glucose metabolic system. Glucose has been shown (Harrison, 1960; Harrison
234
SHEILAT. ISAKSON,BETTYJo HUFFMANANDP. B. SIEGEL
et al., 1965) to provide the necessary energy for the massive cell migrations that
occur at this time of development. The abnormal development shown in Figs. 1 (3) through 1 (6) indicate interference with development of the neural cord and somites. These same abnormalities were reported by Kallen & Rudeberg (1964) who made histological sections of malformed embryos and observed heavy cell degeneration in the lateral folds of the central nervous system and tripcar mitotic figures with a preponderance of abnormal anaphases. Furthermore, these investigators observed that after 2 days of incubation the embryos became resistant to the effects of light. The neural crest is the point of origin of melanophores in chick embryos (Dorris, 1936; Eastlick, 1939; Rawles & Willier, 1939). Perhaps light is absorbed by the neural crest cells which, during normal morphogenesis, migrate ventrolateraUy during closure of the neural tube. More light may be absorbed by embryos developing in vitro than those in ovo because the former do not have the reflection of light by the shell. Absorption of additional amounts of light by neural crest cells may cause disturbed mitoses which result in delayed migration and the subsequent closure of the neural tube. Failure of the neural tube to close could, in turn, interfere with subsequent cephalogenesis. Hillman & Hillman (1965) observed extensive proliferation of mesoderm during the developmental period studied here. Fusion of the neural tube precedes detachment of the notochord, which subsequently differentiates at different times along its length. This sequence of differentiation could be related to the metabolic gradients observed by Hymen (1927) and Rulon (1935). The hypothesis that the shell reduces the intensity of light absorbed by the developing embryo in ovo could be expanded to explain the observed acceleration of development. Perhaps the shell reduces light to an intensity threshold which stimulates mitosis of the neural crest cells resulting in an earlier closure of the neural tube, or stimulates proliferation of mesoderm causing an acceleration of somite development. Bellairs (1963) noted that the size of individual somites or the number of somites is related both to the speed of development during the period of incubation and to the amount of available mesoderm. She speculated that the lateral plate mesoderm which lies lateral to the developing somites and the regression movements of the primitive streak play a decisive role in somite formation. REFERENCES
BELLAII~SR. (1963) The development of somites in the chick embryo. ~. Embryol. exp. Morph. 11,697-714. BURSlANA. V. (1964) The influence of light on the spontaneous movement of chick embryos. Bull. exp. Biol. Med. (Russian) 58, 767. DoRms F. (1936) The production of pigment by chick neural crest in grafts to the 3-day limb bird. s~. exp. Zool. 80, 315-345. EASTLICKH. L. (1939) The pigment forming capacity of the blastoderm of Barred Plymouth Rock embryos as shown by transplants to White Leghorn hosts. Anat. Rec. (Suppl.) 73, 64-65.
I N C A N D E S C E N T L I G H T A N D T H E D E V E L O P M E N T OF C H I C K EMBRYOS
235
HAMBURGERV. & HAMILTON H. C. (1951) A series of normal stages in the development of the chick embryo. 3t. Morph. 88, 49-92. HARRISON J. R. (1960) In vitro utilization of glucose by the chick embryo at early stages. Physiol. Zo61. 33, 68-77. HARRISONJ. R. (1967) Studying morphogenesis. Am. Biol. Teach. 29, 103-109. HARRISON J. R., HAYES V. E. & TOYE S. K. (1965) Developmental relationships of chick primitive-streak and head process stages explanted to glucose media. Physiol. Zo6l. 38, 138-148. HILLMAN N. W. & HILLMAN R. (1965) Chick cephalogenesis. The normal development of the chick cephalic region of stages 3 through 11 chick embryos, ft. Morph. 116, 357-370. HYMEN L. H. (1927) The metabolic gradients of vertebrate embryos--III. The chick. Biol. Bull. 52, 1-39. JENSEN D. R., BEES G. B. & STORM G. (1968) Simultaneous statistical test on categorical data. ft. exp. Educ. 36, 47-56. KALLEN B. &; RUDEBERG S. I. (1964) Teratogenic effects of electric light on early chick embryos. Acta morph, neer. scand. 6, 95-99. LAUBERJ. K. & SHUTZEJ. V. (1964) Accelerated growth of embryo chicks under the influence of light. Growth 28, 179-190. RAWLES M. E. & WILLIER B. H. (1939) The localization of pigment producing potency in presomite chick blastoderms. Anat. Rec. (Suppl.) 73, 43-64. RULON O. (1935) Differential reduction of Janus green during development of the chick. Protoplasma 24, 346-364. SHUTZE J. V., LAUBERJ. K., KATO M. & WILSON W. (1962) Influence of incandescent light and colored light on chick embryos during incubation. Nature, Lond. 96, 594-595. SIEGELP. B. (1962) Selection for body weight at eight weeks of age---1. Short term responses and heritabilities. Poult. Sci. 41,954-962. SIEGEL P. B., ISAKSONS. T., COLEMANF. N. & HUFFMAN B. J . (1969) Photoacceleration of development in chick embryos. Comp. Biochem. Physiol. 28, 753-758. TAMIMIE H. S. • Fox M. W. (1967) Effect of continuous and intermittent light exposure on embryonic development of chicken eggs. Comp. Biochem. Physiol. 20, 793-799. Key Word Index--Development of chicks; light-accelerated embryology; photoacceleration of development.
I N T E N S I T I E S OF INCANDESCENT L I G H T AND THE DEVELOPMENT OF CHICK EMBRYOS I N OVO AND IN VITRO S H E I L A T. ISAKSON, B E T T Y JO H U F F M A N and P. B. S I E G E L Biology Department, Radford College, Radford, Virginia 24141; and Poultry Department, Virginia Polytechnic Institute, Blacksburg, Va. 24061 (Received 16 December 1969)
A b s t r a c t - - 1 . Developmental differences were observed among White Rock embryos (GaUus domesticus) incubated in darkness and under various intensities of incandescent light. 2. A highly significant difference in the developmental success of embryos explanted to glucose medium was found between those incubated in light and those incubated in darkness. Exposure to light interfered with normal morphogenesis in vitro. 3. Temperatures within the culture dishes were the same for incubation in light and dark suggesting that the phenomenon observed in vitro was due to light rather than temperature.
INTRODUCTION ENVIRONMENTAL factors, such as relative humidity, light and temperature, influence the rate of development of the chick embryo. Eggs incubated under white light hatch approximately 1 day earlier than those incubated in darkness, and 8 hr earlier than those incubated under colored light (Shutze et al., 1962). Siegel et al. (1969) found that light during the first week of incubation had a more pronounced effect than light during the second or third weeks, and that the acceleration of development was apparent by 10 hr of incubation. Kallen & Rudeberg (1964) observed abnormal development of embryos exposed to light, through a window in the shell, during the first and second day of incubation. Abnormal embryogenesis and delayed hatching occurred when incubation was conducted in either continuous or intermittent light (Tamimie & Fox, 1967). I n vitro studies showed glucose was the only essential nutrient for normal development of the chick during the first 2 days (Harrison, 1960; Harrison et al., 1965). They found that glucose provided the necessary energy for the extensive cell migrations occurring during this period. Therefore, the early acceleration of development observed by Siegel et al. (1969) may be linked with an effect of light on the glucose metabolic system. The present study was designed to observe the effects of various intensities of incandescent light in ovo and in vitro during the first 48 hr of incubation. 229
230
SHEILA T . ISAKSON, BETTY Jo HUFFMAN AND P. B. SIEGEL
MATERIALS AND METHODS Eggs from low-weight lines of White Rocks (Siegel, 1962) were incubated at 37-5°C and 75% r.h. in a forced-draft incubator. Light was supplied from incandescent bulbs located 13 cm above the middle region of the incubator trays. Light intensities were varied by using bulbs of different wattage. Treatments consisted of: (1) complete darkness, (2) low intensity illumination (ranging from 54 to 108 luxes), and (3) high intensity illumination (ranging from 215 to 430 luxes). Illumination was measured at the opposing end-sections of the trays where the eggs were incubated. I n order to provide approximately equal amounts of diffuse light to all embryos, eggs were never placed directly under the bulbs. After 24 hr of incubation, in ovo blastoderms were removed from the yolk by the method of Harrison (1967) and staged according to the Hamburger & Hamilton process (1951). All blastoderms were from primitive streak through 10-somite stages. Staged blastoderms were then explanted to watch glasses containing the buffered glucose-saline-agar medium of Harrison (1960). This glucose culture method was chosen to provide the same frame of reference for comparison of effects of light on the glucose metabolic system in vitro rather than some of the more recent vitelline membrane culture techniques. Light regimens of explants were the same as those given during in ovo incubation. After 24 hr explants were classified as normally developed, abnormally developed or not developed. Explants exhibiting axiation with indistinct morphology, Fig. 1 (1), were classified as not developed; those having a brain, cord, somites, heart and vitelline vessels, Fig. 1 (2), were classified as normally developed; explants with either an open neural cord, Fig. 1 (3), an open neural cord and indistinct somites, Fig. 1 (4), indistinct somites and edema, Fig. 1 (5), or lacking somites, Fig. 1 (6), were classified as abnormally developed. Air temperatures in the incubator and within the culture dishes were measured at 4, 16 and 24 hr of incubation with a thermometer probe (Tele-thermometer; Yellow Springs Instrument Co.) placed either in the incubator or directly over the explant, i.e. immediately under the lid of the culture dish, for approximately 1 rain. Data were analyzed by Xa utilizing the correlated test statistics of Jensen et al. (1968). This procedure allows data to be used in more than one X2 analysis for simultaneous tests of hypotheses. Correction for this lack of independence was made by using the Bonferroni X2 tables (Jensen et al., 1968).
RESULTS Classification of 279 embryos for somites after in ovo incubation in darkness or under various light intensities is summarized in Table I. There was a highly significant difference among treatments with development greater for embryos incubated in light than in darkness. The percentage of embryos that developed somites was similar for both high and low light intensities. TABLE 1--PERCENTAGE OF EMBRYOS WITH SOMITES AFTER 24 hr in OR DARKNESS
ovo
INCUBATION IN LIGHT
Incubation r~gime
No. embryos With somites (%) Lacking somites (%)
Darkness
54-108 luxes
215--430 luxes
140 11 89
82 28 72
57 26 74
FIG. 1. Photomicrograph of: (1) explant exhibiting axiation, but indistinct morphological structure; (2) explant exhibiting normally developed brain, cord, heart and somites; (3) explant with an open neural cord; (4) explant with an abnormal brain, open neural cord and small somites; (5) explant with small and indistinct somites and edema; (6) explant with somites lacking. Photomicrograph 1 was classified as not developed, photomicrograph 2 as normally developed, and photomicrographs 3-6 as abnormally developed.
INCANDESCENT L I G H T AND THE DEVELOPMENT OF CHICK EMBRYOS
231
T h e developmental success of 281 explants is presented in Table 2. Comparisons of various in vitro treatments after in ovo incubation in darkness showed highly significant differences (X~ = 91.3). For low intensity in ovo incubation no significant T A B L E 2 - - C L A S S I F I C A T I O N OF EXPLANTS INCUBATED FOR
24 hr
I N L I G H T OR DARKNESS ON
S Y N T H E T I C GLUCOSE MEDIUM
Treatment In ovo
In vitro
Darkness
Darkness 54-108 luxes 215-430 luxes 54-108 luxes Darkess 54-108 luxes 215-430 Darkness luxes 215-430 luxes
No. explants
Normal development (%)
Abnormal development (%)
No development (%)
51 55 60 36 30 26 23
76 14 0 33 30 42 9
18 64 58 39 53 38 43
6 22 42 28 17 19 48
differences (X2 = 1.7) were observed when the explants were provided darkness or low intensity treatments. Likewise, no significant differences (X2=8.33) were observed when explants were provided darkness or high intensity treatments following in ovo incubation under high intensity. These results suggest that developmental success in vitro is related to the effect of light on the embryo during in ovo incubation. T o test this hypothesis more closely, the data were rearranged and analyzed again using the correlated test statistics (Table 3). T h e r e was a highly significant difference (X2= 19-2) in developmental success among in ovo T A B L E 3 - - C L A S S I F I C A T I O N OF EXPLANTS INCUBATED UNDER L I G H T AND DARKNESS
Treatment In vitro
Darkness
In ovo
Darkness 54-108 luxes 215-430 luxes 54-108 luxes Darkness 54-108 luxes 215-430 Darkness luxes 215-430 luxes
No. explants
Normal development (%)
Abnormal development (%)
No development (%)
51 36 26 55 30 60 21
76 33 42 14 30 0 9
18 39 38 64 53 58 43
6 28 19 22 17 42 48
treatments when in vitro incubation was in darkness. Explants incubated in darkness following in ovo incubation in darkness had the greatest developmental success. N o significant differences (X~ = 2-9) in developmental success of explants were observed between in ovo treatments when the in vitro treatment was under low
232
SHEILA T , ISAKSON, BETTY
Jo
HUFFMAN AND P. B. SIEGEL
intensity nor were there significant differences (X~ = 0.7) between in ovo treatments when the in vitro treatment was under high intensity. The most profound differences in developmental success of explants are found when comparisons are made between embryos from eggs incubated in darkness and subsequently exposed to light in vitro with explants incubated in darkness following exposure to light in ovo. Since elevation of temperature is known to induce developmental abnormalities, temperatures were recorded from inside the culture dishes. Results of 120 observations are presented in Table 4. Air temperature in the culture dishes was lower than the incubator air temperature of 37.5°C. This probably resulted from evaporation of water from the cotton rings in the culture dishes. TABLE 4
MEAN AIR TEMPERATURES WITHIN CULTURE DISHES JUST ABOVE THE EXPLANT WHEN INCUBATOR TEMPERATURE WAS 37.5°C
Treatment Darkness 54-108 luxes 215-430 luxes
No. observations
~ temp. (°C)
42 42 36
36"7 36"7 36"6
DISCUSSION Results from the in ovo experiment show that light accelerates development of the chick embryos, substantiating the observations of Shutze et al. (1962), Lauber & Shutze (1964) and Siegel et al. (1969). Our results also show that, within a range, light intensity affects development in ovo. Shutze et al. (1962) observed that developmental acceleration is not due to a particular wavelength. The abnormal development observed by Kallen & Rudeberg (1964) resulted perhaps because the embryo did not receive the benefit of reflection of light by the shell at the ske of the window. Tamimie & Fox (1967) also observed abnormal development in ovo with incubation condkions of twice the intensity used in this investigation and by Siegel et al. (1969). Perhaps the intensity of light used by Tamimie & Fox was so great that the shell could not reflect enough of the light to prevent deleterious effects. It appears that light within a range operates as an accelerating factor, i.e. below a low intensity threshold absorbed light resuks in acceleration, while at a high intensity threshold absorbed light resuks in abnormal development. Bursian (1964) reported that patterns of response to light by chick embryos are dependent upon wavelength and intensity of illumination as well as on the age of the embryo. He suggested that the chick embryo, at early developmental stages, possesses a primitive form of photosensitivity like that of certain invertebrates and lower vertebrates. The developmental stages of embryos at explantation were equivalent to those used in Harrison's (1960) investigation. He concluded that embryos which have attained the head fold or early somite stages are equally capable of continuing normal morphogenesis on synthetic glucose medium. The developmental success
I N C A N D E S C E N T L I G H T AND THE DEVELOPMENT OF C H I C K EMBRYOS
233
of embryos incubated in darkness during both the in ovo and in vitro treatments had relatively the same success as did those reported by Harrison (1960). A comparison of the developmental success of embryos following exposure to 48 hr of light, i.e. 24 hr in ovo followed by exposure at the same intensity in vitro, is presented in Fig. 2 (1). As the intensity of light increased, a reduction in the incidence of normal development was observed. Seventy-six per cent of the explants developed normally in darkness, while 30 per cent developed normally under low intensity regimens, and only 9 per cent under high intensity regimens. The greatest effect of light in arresting development was found in the high intensity range where 48 per cent of the explants did not develop. It would seem that the differing percentages
IOO
I
2 [] []
80
3
Normal Abnormal
[ ] Not developed 60
40 0 20
0 Dark
5 4 - 1 0 8 :>15-450 Luxes Luxes
Dark
54-108 215-430 Luxes Luxes
Dark
54-108 Luxes
215-430 Luxes
FIG. 2. Comparison of the developmental success of explants incubated under: (1) equivalent in ovo and in vitro conditions of light or darkness; (2) darkness following in ovo incubation conditions of light or darkness; (3) conditions of light or darkness following in ovo incubation in darkness.
of abnormally developed embryos within the high intensity treatment are a result of the cumulative effect of light. At the low intensity, 53 per cent developed abnormally while 43 per cent developed abnormally at high intensity. The lower percentage in the latter group is masked by the threefold increase in embryos that did not develop when compared with those which did not develop in the low intensity group. Comparisons between explants exposed to various intensities following in ovo incubation in darkness are shown in Fig. 2 (3). Only 14 per cent of the exp!ants developed normally when exposed to low intensity, while none of those exposed to high intensity developed normally. Since more than half of the explants developed abnormally with either light treatment, it is conceivable that light interferes with the glucose metabolic system. Glucose has been shown (Harrison, 1960; Harrison
234
SHEILAT. ISAKSON,BETTYJo HUFFMANANDP. B. SIEGEL
et al., 1965) to provide the necessary energy for the massive cell migrations that
occur at this time of development. The abnormal development shown in Figs. 1 (3) through 1 (6) indicate interference with development of the neural cord and somites. These same abnormalities were reported by Kallen & Rudeberg (1964) who made histological sections of malformed embryos and observed heavy cell degeneration in the lateral folds of the central nervous system and tripcar mitotic figures with a preponderance of abnormal anaphases. Furthermore, these investigators observed that after 2 days of incubation the embryos became resistant to the effects of light. The neural crest is the point of origin of melanophores in chick embryos (Dorris, 1936; Eastlick, 1939; Rawles & Willier, 1939). Perhaps light is absorbed by the neural crest cells which, during normal morphogenesis, migrate ventrolateraUy during closure of the neural tube. More light may be absorbed by embryos developing in vitro than those in ovo because the former do not have the reflection of light by the shell. Absorption of additional amounts of light by neural crest cells may cause disturbed mitoses which result in delayed migration and the subsequent closure of the neural tube. Failure of the neural tube to close could, in turn, interfere with subsequent cephalogenesis. Hillman & Hillman (1965) observed extensive proliferation of mesoderm during the developmental period studied here. Fusion of the neural tube precedes detachment of the notochord, which subsequently differentiates at different times along its length. This sequence of differentiation could be related to the metabolic gradients observed by Hymen (1927) and Rulon (1935). The hypothesis that the shell reduces the intensity of light absorbed by the developing embryo in ovo could be expanded to explain the observed acceleration of development. Perhaps the shell reduces light to an intensity threshold which stimulates mitosis of the neural crest cells resulting in an earlier closure of the neural tube, or stimulates proliferation of mesoderm causing an acceleration of somite development. Bellairs (1963) noted that the size of individual somites or the number of somites is related both to the speed of development during the period of incubation and to the amount of available mesoderm. She speculated that the lateral plate mesoderm which lies lateral to the developing somites and the regression movements of the primitive streak play a decisive role in somite formation. REFERENCES
BELLAII~SR. (1963) The development of somites in the chick embryo. ~. Embryol. exp. Morph. 11,697-714. BURSlANA. V. (1964) The influence of light on the spontaneous movement of chick embryos. Bull. exp. Biol. Med. (Russian) 58, 767. DoRms F. (1936) The production of pigment by chick neural crest in grafts to the 3-day limb bird. s~. exp. Zool. 80, 315-345. EASTLICKH. L. (1939) The pigment forming capacity of the blastoderm of Barred Plymouth Rock embryos as shown by transplants to White Leghorn hosts. Anat. Rec. (Suppl.) 73, 64-65.
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HAMBURGERV. & HAMILTON H. C. (1951) A series of normal stages in the development of the chick embryo. 3t. Morph. 88, 49-92. HARRISON J. R. (1960) In vitro utilization of glucose by the chick embryo at early stages. Physiol. Zo61. 33, 68-77. HARRISONJ. R. (1967) Studying morphogenesis. Am. Biol. Teach. 29, 103-109. HARRISON J. R., HAYES V. E. & TOYE S. K. (1965) Developmental relationships of chick primitive-streak and head process stages explanted to glucose media. Physiol. Zo6l. 38, 138-148. HILLMAN N. W. & HILLMAN R. (1965) Chick cephalogenesis. The normal development of the chick cephalic region of stages 3 through 11 chick embryos, ft. Morph. 116, 357-370. HYMEN L. H. (1927) The metabolic gradients of vertebrate embryos--III. The chick. Biol. Bull. 52, 1-39. JENSEN D. R., BEES G. B. & STORM G. (1968) Simultaneous statistical test on categorical data. ft. exp. Educ. 36, 47-56. KALLEN B. &; RUDEBERG S. I. (1964) Teratogenic effects of electric light on early chick embryos. Acta morph, neer. scand. 6, 95-99. LAUBERJ. K. & SHUTZEJ. V. (1964) Accelerated growth of embryo chicks under the influence of light. Growth 28, 179-190. RAWLES M. E. & WILLIER B. H. (1939) The localization of pigment producing potency in presomite chick blastoderms. Anat. Rec. (Suppl.) 73, 43-64. RULON O. (1935) Differential reduction of Janus green during development of the chick. Protoplasma 24, 346-364. SHUTZE J. V., LAUBERJ. K., KATO M. & WILSON W. (1962) Influence of incandescent light and colored light on chick embryos during incubation. Nature, Lond. 96, 594-595. SIEGELP. B. (1962) Selection for body weight at eight weeks of age---1. Short term responses and heritabilities. Poult. Sci. 41,954-962. SIEGEL P. B., ISAKSONS. T., COLEMANF. N. & HUFFMAN B. J . (1969) Photoacceleration of development in chick embryos. Comp. Biochem. Physiol. 28, 753-758. TAMIMIE H. S. • Fox M. W. (1967) Effect of continuous and intermittent light exposure on embryonic development of chicken eggs. Comp. Biochem. Physiol. 20, 793-799. Key Word Index--Development of chicks; light-accelerated embryology; photoacceleration of development.