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Mapping of the oxygen consumption in the gastrulating chick embryo
Respiration Physiology (1983) 51,153-166
153
Elsevier Biomedical Press
MAPPING OF THE OXYGEN CONSUMPTION CHICK E M B R Y O
ERIC RADDATZ
IN THE GASTRULATING
and PAUL KU(~ERA
Institute of Physiology, 7 Rue du Bugnon, 1011 Lausanne CHUV, Switzerland
Abstract. The spatial variations of the 0 2 uptake in the area pellucida (AP) and area opaca (AO) of the chick embryo were measured in vitro by scanning microspectrophotometry. In the AP, the 02 fluxes (stage 4: 20-90, stage 6-7:30-110 n l . h - l . m m - 2 ) as well as the oxidative activity (stage 4: 14-25, stage 6-7:10-28 n l . h - l . ~ g protein)-1) show distinct regional differences. The most active regions are not those corresponding to the axial embryonic structures but those located in the latero-posterior, relatively undifferentiated zone of the AP. The total 02 uptake increases during 6 h of development from 0.3 to 0.6 pl •h - l but the average oxidative activity and 02 uptake per cell remain constant (18 nl .h -1. (pg protein)-l and 2.5 pl-h -1 .cell-x). In the AO, the 02 uptake is rather homogeneous and all respiratory parameters increase during the development: total 02 uptake from 1.6 to 5.5/~1 .h -l, oxidative activity from 28 to 45 nl .h -t .(/~g protein) -l and cell respiration from 7 to 16 pl .h -l cell-1. The described metabolic variations are discussed with respect to the known morphogenetic activities taking place in the chick blastodisc during the studied period of development. Area opaca Area pellucida Cell respiration
Chick embryo Growth Tissue respiration
T h e m o r p h o g e n e t i c activities o c c u r r i n g in the very y o u n g chick e m b r y o d e p e n d to a g r e a t extent o n the o x i d a t i v e c a t a b o l i s m o f c a r b o h y d r a t e s , especially glucose (see e.g. S p r a t t , 1952c, for review). H o w e v e r , m u c h c o n t r o v e r s y exists a b o u t the q u e s t i o n w h e t h e r the r e s p i r a t o r y activity within the e m b r y o is everywhere the same o r varies a c c o r d i n g to i n d i v i d u a l e m b r y o n i c regions so r e m a r k a b l y different in the relative i m p o r t a n c e o f the o b s e r v a b l e m o r p h o g e n e t i c events, e.g., cell m i g r a t i o n s , histogenetical differentiation. On the one h a n d , r e g i o n a l differences ( b o t h q u a l i t a t i v e a n d q u a n t i t a t i v e ) have been d e s c r i b e d in e x p e r i m e n t s using v a r i o u s e x o g e n o u s o x i d o - r e d u c t i o n indicators, i.e., vital dyes ( R u l o n , 1935; Miller, 1941; Spratt, 1951, 1952a,b, 1958). T h e s e Accepted for publication 6 November 1982
0034-5687/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
154
E. RADDATZ AND P. KUCERA
findings, although obtained in long-lasting anoxic conditions unfavourable to normal development, and although rather qualitative because of difficulty to correct the observed colour changes for the tissue quantity, have been recently strongly supported by studies of the intracellular N A D redox state in an intact and normally developing embryo in vitro (KuEera and de Ribaupierre, 1980). On the other hand, the respiratory rates of isolated fragments of the chick embryo were measured manometrically and no significant regional variations have been found (Philips, 1941, 1942). In these experiments, however, the long-range cell interactions, so important for the normal differentiation, have been completely abolished. We have developed a non-invasive and very sensitive technique allowing continuous measurements of the oxygen uptake of minute regions o f the intact chick embryo normally developing in vitro under controlled conditions (KuEera and Raddatz, 1980). The first results clearly indicated the presence, within the neurulating embryo, of spatio-temporal variations of the oxidative activity. This paper presents the respiratory patterns found in the whole gastrulating Chick embryo. List FL : FR : Q : Qp : QD : Qc :
o f symbols and units Oxygenflux measured locally at a given point of the blastodisc: nl •h - l. mm -2 AverageFL value for one defined embryonicregion: nl. h-1. mm-2. Absolute oxygenconsumption of a givenembryonicregion of known area S (Q = FR •S): nl •h - 1. Q corrected for the protein content, i.e., the oxidativeactivity: nl. h-I. (~g protein)-1. Q corrected for the DNA content: nl .h -1 .~g DNA) -l. Oxygenuptake per cell, calculated from QD: pl-h- 1 . cell-l.
Materials and methods CULTURE OF THE EMBRYO The embryos (see fig. 1) at the stage 4 (definitive primitive streak) and at stage 6-7 (head-fold) according to Hamburger and Hamilton (1951) were obtained by preincubation o f fertilized eggs (White Leghorn) at 38 °C and 80% relative humidity. The entire blastodiscs with the vitelline membrane were carefully removed from the yolk under sterile conditions according to the technique of New (1955) and cleaned of yolk particles. As described in previous papers (KuEera and Raddatz, 1980; Raddatz, 1980), the preparation is covered by a second very thin and transparent silicone membrane and mounted in a special incubation chamber. The embryo with the membranes separates the internal volume of the chamber into two compartments tightly closed by two windows. The blastodisc faces the lower compartment containing a defined nutritive medium. The upper compartment is perfused by a solution of purified human hemoglobin saturated at Ps0 (54 mm Hg) with oxygen representing the only source of oxygen available for the embryo. During a steady-state perfusion, the oxygen passes easily through the silicone and vitelline
REGIONAL02 REQUIREMENTS IN CHICKEMBRYO
155
membranes down its concentration gradient, i.e., from the hemoglobin solution to the respiring tissue. The chamber is fixed on the stage of a scanning microspectrophotometer and thermostabilized at 37 °C.
SCANNING MICROSPECTROPHOTOMETRICMEASUREMENTS OF THE OXYGEN CONSUMPTION As the absorption spectrum of the hemoglobin changes during its deoxygenation, this protein can be used as the oxygen donor as well as the indicator of the oxygen uptake (Barzu and Borza, 1967). Thus, under the stop-flow condition, the time variation of the absorbance measured in the linear domain of the HbO2 dissociation curve (around Ps0) is proportional to the deoxygenation rate of the hemoglobin, i.e., to the respiratory rate of the adjacent tissue. Therefore, if the regional variations of the absorbance in the layer of hemoglobin are recorded over the entire embryo during 10 sec periods of stop-flow, it is possible to establish the spatial pattern of its respiration. Such spectrophotometric measurements have been done by scanning the preparation at a speed of 0.5 m m . s -~ between the left and the right margins of the area pellucida through various levels of the embryo. In order to investigate the opacal region, two additional scans have been extended up to the edge of each blastodisc; the first one from the right end of the scan passing through the Hensen's node, and the second from the right end of the most posterior scan. All the measurements have been done at 435 nm using a light beam of a crosssection of 0.0025 mm2 covering approximately 25-80 ceils according to the place within the embryo. Such a technique allows us to determine the oxygen flux (FL) at any point of a given scan with a precision of + 2 nl .h -~ .mm -2. In order to minimize the error due to a lateral diffusion of oxygen within the hemoglobin layer, the measurements must be done as quickly as possible. The desaturation slopes at the maximally and minimally respiring points of the same embryo have been systematically recorded as a control to the scanning measurements. Up to 10 sec virtually no deviation from linearity has been found and the FL values obtained by both methods for the same points were identical. This indicates that, within this interval, the scanned profiles of hemoglobin absorbance are not yet significantly smoothed by the lateral diffusion. In addition, t h e same values of FL have been obtained for the same points of the embryo using different initial Hb saturation levels allowed by the linearity domain of the method. This indicates that no region of the embryo suffers from hypoxic conditions during the stop-flow period and confirms the same conclusion derived from theoretical calculus based on three different models of penetration of oxygen into the tissue (KuEera and Raddatz, 1980; Raddatz, 1980). The local values of FL have been plotted with respect to their scanning coordinates. Such FL maps display the intensity of the oxygen flux within the embryonic territory
156
E. RADDATZ AND P. KU(~ERA
(see fig. 2). O n the o t h e r h a n d , the F R values o f the a r e a pellucida, the a r e a o p a c a a n d the entire b l a s t o d i s c have been o b t a i n e d b y i n t e g r a t i o n o f all the FL values over the c o n s i d e r e d zones.
NORMALIZED TOPOGRAPHY OF THE EMBRYOS T h e r e exists for a given d e v e l o p m e n t a l stage a large i n d i v i d u a l v a r i a b i l i t y in the f o r m a n d hence the d i m e n s i o n s o f the e m b r y o . Therefore, a c o m p a r i s o n o f the FL values f r o m o n e e m b r y o to a n o t h e r necessitated the definition o f c o m p a r a b l e regions. A s the e m b r y o at the e a r l y stages is r a t h e r u n d i f f e r e n t i a t e d , we have defined these regions as follows.
Fig. 1. The scanned part of the chick embryos photographed at the same magnification in the incubation chamber. The central and more transparent area pellucida containing the axial embryonic structures is surrounded by the area opaca heavily charged with yolk. The letters and numbers refer to the embryonic regions defined in the text. For the protein determinations the borders containing yolk were dissected off.
REGIONAL 02 REQUIREMENTS IN CHICK EMBRYO
157
Each scanned area was subdivided by longitudinal and transversal lines, the intersections of which defined 12 (stage 4) or 15 (stage 6-7) normalized embryonic regions (see figs. 1 and 2). Thus, the investigated area was divided into three longitudinal strips of equal width: one axial (A), and two lateral - left (L) and right (R). Then, it was divided transversally into anterior and posterior parts with respect to the Hensen's node: the anterior one was represented by one strip at stage 4 (labelled 4) and by two strips at stage 6-7 (labelled 4 and 5); the posterior one was divided into three strips (labelled 3, 2, 1) so that the primitive streak was divided into anterior (including the node), median and caudal segments, i.e., the regions labelled A3, A2 and A1, respectively.
DETERMINATION OF THE PROTEIN AND DNA CONTENTS The embryonic regions as defined above were carefully dissected and pooled and their protein content determined using the Coomassie Blue method (Bradford, 1976) and bovine serum albumin as reference. The regional protein content (metabolically active and inactive fractions mixed) serves to calculate the QP. The DNA content of the area pellucida and area opaca was determined according to Hinegardner (1971). Knowing the average cellular DNA content (2.3 pg, McMaster and Modak, 1977) we can evaluate the number of cells in each area and thus calculate the cellular respiration Qc.
STATISTICALEVALUATION The analysis of variance (Fisher's method) within and between the regions of the individual embryos, of the embryos of the same stage and of the both stages were done for all the measured and calculated parameters. Whenever this test had allowed it, the significance of all differences found was evaluated using the studentized range Q-test. This latter test, more severe than the one of least significant difference, has the property that the probability of making an erroneous claim of significance is <0.05. All the statistics were done according to Snedecor and Cochran (1971).
Results
MAPS OF THE LOCALOXYGEN FLUXES (FL) They were obtained in 22 normally developing embryos: 14 at the definitive primitive streak stage (1248 measurements) and 8 at the head-fold stage (1172 measurements).
158
E. R A D D A T Z A N D P. KU(~ERA
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Fig. 2. Examples of the individual maps of the oxygen uptake within the area pellucida and adjacent parts of the area opaca. The values of the local 02 fluxes (nl .h -1 . m m -2) are represented by the dimensions of the black dots calibrated at the bottom. Each value is plotted with respect to its scanning coordinates. Stage 4 : 1 5 4 data; stage 6 - 7 : 2 1 3 data. n: Hensen's node; ps: primitive streak (compare with fig. 1).
The variations of the values of FL in both investigated stages are illustrated by two representative maps (fig. 2). At stage 4 (fig. 2, left) the most elevated FL values are situated at the Hensen's node and in the anterior part of the primitive streak (A3). Sometimes, a relatively active region appears also posteriorly (A1). The FL values determined in the adjacent parts of the area opaca are rather homogeneous and lower than those in the area pellucida. At stage 6-7 (fig. 2, right) the most elevated FL values in the area pellucida are observed along and on both sides of the regressing streak, the head process and the neural plate. The lowest FL values are found over the mesoderm-free region of the proamnion. The maps established for each individual embryo thus show clear cephalocaudal and medio-lateral gradients of oxygen uptake per unit area. These differences
159
REGIONAL O 2 REQUIREMENTS IN CHICK EMBRYO
are highly significant. From one embryo to another, however, the FL profiles vary considerably, as illustrated by the extreme values found so far: 20 and 90 at stage 4 and 28 and 110 nl .h -) .ram -2 at stage 6-7. The variability is due to different steepness of the gradients but also due to different mean levels of the oxygen consumption. These variations, roughly corresponding to the local cell packing density, are more pronounced at the younger stage which shows also a greater variability in form and which is more difficult to define at the time scale.
THE REGIONAL DIFFERENCES IN THE POOLED VALUES
All the measured and calculated parameters are reported in table 1. As we .,4~a~,e never observed significant variations between the left and right halves of the area pellucida, the lateral regions were grouped together. Table 2 gives the results of the statistical analysis within each stage and between both stages. For each parameter, the difference between any two regions is significant if equal or greater than the value D. At each stage, the FR values show practically the same pattern as in the individual FL maps, although the differences are smaller due to the averaging of the highly dispersed values as mentioned before. Nevertheless, at stage 4, the FR of the region containing the Hensen's node is significantly higher if compared to all other regions. At stage 6-7, almost all longitudinal and transversal variations are significant. The QP values are consistently more elevated in the lateral regions of the embryo. The differences are significant at stage 6-7 but just not significant (P = 0.07) at stage 4 where the errors in the protein determinations are greater. TABLE 1 Morphometric, chemical and metabolic parameters for each embryonic region at the two developmental stages. SD: standard deviation. EMBRYONIC STAGE H,H. Area
± SD
mm 2
6-7
Protein content
4
ug
6-7
FR ± SD
4
al,h=~mm-2
6-7
Q ± SD
4
nl.h -I Qp ± SD nl.h-~pg -I
LR5
AS
4
6-7
0.5±0.1
1.5±0.4
53±7
27±7
0.7±0.1
4.5±0.6
60±13
43±9
4 6-7
19±5
10±1
REGIONS
DR4
A4
LR3
0.3±0.2
0.7±0.1
0.6±0.1
0.7±0.1
A3
0.5±0.I
LR2
0.7±0.1
A2
0.2±0.1
LRI
0.7±0.
AI
0.7±0.1
0.7±0.1
0.7±0.1
0.7±0.1
0.6±0.2
0.7±0.1
0.4±0.2
0.7±0.
1.0±0.4
2.7±0.9
1.4±0.1
3.0±0.4
1.2±0.03
2.7±0.611.0±0.2
2.3±0.
2.0±0.01
4.2±0.1
1.7±0.1
4.1±0.1
1.4±0.01! 3.8±0.1
55±12
56±13
58±10
62±16
58±9
57±9
71±16
S6~8
76±17
17±9
36±7
33±10
40±6
Sl±ll
18±7 20±0.01
1.4±0.2
3.6±0.(
59±12
57±11
59±12
62±8
7S±16
63±9
71±12
43±14
31±9
41±11
15±7
40±11
41±6
56±12
40±9
55±10
23±9
52±7
14±5
23~2
15±2
25±i
16±3
16±4
19±7
12±0.3
24±1
14±0.3
28±0.01
IS±0.2
17±2
IS±2
160
E. RADDATZ A N D P. KU(~ERA
TABLE 2 The results of the statistical analysis within and between the stages, d.f. : degrees of freedom; F : Fisher's ratio of variances; D: threshold value of significance (P <0.05) for any regional difference within or between the stages. D values have the units of the corresponding parameters (see text). N.S.: not significant. STAGE
4
STAGE
6 - 7
!PARAMETERS
d.f.
F
D
d.f.
AREA
11/156
27.7
0.14
14/105
1.8
14/15
42.5
11/12
PROTEIN
6.1
BETWEEN
F
9.0
STAGES
D
d.f.
F
0.20
1/11
16.4
0.I
i.i
1/11
30.8
0.3 4.1
FR
11/1241
4.7
2.2
14/1157
47.6
2.3
1/11
11.9
Q
11/156
17.2
ll.9
14/105
12.8
15.1
1/22
59.6
Qp
11/12
2.2
N.S.
14/15
14.5
8.1
1/11
1.4
D
3.6 N.S.
The comparison between the two stages shows a clear increase in all the parameters directly linked to the growth o f the embryo. However, we do not observe any significant change in the oxidative activity during the studied period of development.
OXYGEN UPTAKE OF THE AREA PELLUCIDA A N D AREA OPACA BETWEEN STAGES 4 A N D 6-7 (TABLE 3)
Area pellucida During the 6 h of development the following parameters increase significantly: the area by 6 7 ~ , protein content by 71~o, D N A content by 6 4 ~ , number of cells from 1.4.105 to 2.3 • 105 ( + 6 4 ~ ) , FR by 9 ~ and the total oxygen need by 7 1 ~ . On the other hand, the QP, QD and Qc values remain practically the same (about 18 nl •h - z. pg- l, 1100 nl • h - ~ •/~g- ~ and 2.5 pl • h - ~ • cell - l, respectively). TABLE 3 Morphometric, chemical and metabolic parameters for the area pellucida, area opaca and whole blastodisc ( + SD). Blastodisc St 4 Area mm 2 Protein content
51 ~ ii 117 ± 19 80
gg
DNA c o n t e n t FR:
~g
n l . h - l . m m -2
Q: n l . h -I Qp:
St 6-7
nl.h-~(~g
QD: n l . h ' ~ ( ~ g
prot) -I DNA) -1
0.86
158 1.34
Area
Pel l u c i d a
Area
Opaca
St 4
St 6-7
6 ± I
i0 ± 1
43 + ii
104 ± 18
21
36
58 ± 2
122 ± 25
0.33
0.54
0.53
0.80
St 4
38 ± 12
St 6-7
53 + 15
39
52
2000
6100
25
39
18
17
28
45
2300
4600
ii00
ii00
3000
6900
58 + 15
63 ± 15
350 -+ 150 600 + 250
1600
± i100
5500
± 2800
REGIONAL 0 2 REQUIREMENTS IN CHICK EMBRYO
"
161
Area opaca
169 and 103 FL measurements were collected from stages 4 and 6-7. During the 6 h all the parameters increase significantly: the area by 142~, protein content by 110~o, DNA content by 51~o, cell number from 2.3.105 to 3.5.105 (+52~), FR by 39~o and the total oxygen need by 244~. The most interesting findings concern the corrected values which are all initially much higher than those in the area pellucida and which all increase considerably: the QP from 28 to 45 nl-h-1 ./~g-1 (+61~), the QD from 3000 to 6900 nl .h -1 .#g-~ (+ 130~o) and the Qc from 7 to 16 pl .h -l -cell -1 (+ 129~o).
Discussion EMBRYONIC AND EXTRAEMBRYONIC OXYGEN REQUIREMENTS
Area pellucida
In spite of differences in the methods used in this work and those of Philips (1942) and Bartels and Baumann (1972) a quantitative comparison of the obtained results is possible (table 4). Philips measured the oxygen consumption manometrically in several dissected embryonic fragments. Bartels and Baumann measured the oxygen consumption polarographically in isolated entire embryos cleared of membranes and placed in a stirred medium. The fact that our culture technique approximates closely the in ovo situation could explain that our Q values for the area pellucida are the highest found. The oxidative activity (QP) remains constant between the two stages (about 18 nl .h -~ .#g-l). Philips reached the same conclusions but found a lower value (about 13 nl .h -~./~g-i at stage 5-6). Bartels and Baumann found about 18 nl .h -~./ag -j at stage 8 (estimated from the data corrected for the dry weight TABLE 4 Comparison of the total oxygen consumption ~1. h -l) of the young chick embryo as determined by different authors. Stage H.H.
Philips (1942)
Bartels and Baumann (1972)
4 5+
This work
0.35 0.25-0.30
6
0.25
7
0.36
8
0.50
0.61
162
E. RADDATZ AND P. KUCERA
assuming that 70~ of the dry weight are proteins). In addition, the invariability of the embryonic oxidative activity is perfectly corroborated by the values of QD and Qc during the same period of development. Area opaca To our knowledge, no data about the oxygen uptake of the area opaca have been so far available. Rulon (1935), using an intracellular indicator of oxido-reduction (Janus Green) in anoxic conditions, noted that the cells of the area opaca became highly active at the head-process stage. We have found that the values of QP, QD and Qc increase indeed by 60~o, 130~o and 130~, respectively (table 3). From these data it may be deduced that some important metabolic variations occur in the opacal tissue during its intense expansion between the definitive primitive streak stage and the head-fold stage, i.e., a period including the head-process formation. In summary, with respect to growth, the oxygen uptake of the area pellucida seems to increase in parallel whilst the oxygen uptake of the area opaca increases much faster. The following section tries to correlate the two parameters in some detail.
EVALUATION OF THE METABOLIC COST OF THE GROWTH
The metabolic cost of the growth can be estimated both for the embryonic and extraembryonic parts of the blastodisc. In each of these parts we know: (1) the net gain of the total proteins (P) during the 6 h of development, i.e., 15 and 64 #g in the pellucid and opacal areas, respectively (see table 3); (2) the total quantity of oxygen consumed (T) during the same time interval, i.e., 2.9 and 21.2 pl; (3) the 'maintenance rate' (M), i.e., the quantity of oxygen that should have been consumed if the oxygen consumption had remained that of stage 4, i.e., 6 x0.35 /~1 and 6 x 1.6 /~1. Thus, assuming that the additional aerobic energy expenditure ( T - M) is assigned mainly to the protein synthesis and turnover, the ratio ( T - M)/P gives a good approximation of the oxygen requirement for the net protein gain. The values calculated for the area pellucida and area opaca are 52 and 181 ml 02 consumed per gram of gained protein, respectively. Using the caloric equivalent of the oxygen of 5 kcal/L 02 (respiratory quotient RQ = 1), the corresponding metabolic costs are 0.3 and 0.9 kcal per gram of gained protein, respectively. A theoretical calculation of the maximal metabolic cost of the growth shows that 0.5 kcal are necessary for the gain of one gram of protein if the following hyptheses are considered: all the proteins are condensed only from individual amino acids; the energy requirement for the formation of one peptidic bound is about 30 kcal (Lehninger, 1975); the aerobic efficiency is 0.5; the RQ is 1. It is interesting that the value found for the embryonic area (0.3 kcal per gram of gained protein) is very close to this theoretical value and, moreover, that it is in good agreement with that found by Hommes et al. (1975) for the human infant (0.4 kcal).
REGIONAL O2 REQUIREMENTSIN CHICK EMBRYO
163
The elevated value found for the area opaca (0.9 kcal) indicates that, in addition to the proteosynthesis, this area ensures other important energy dependent functions such as massive yolk digestion or transport of materials required by the embryonic area (in a preliminary study we have shown that the gain of protein in the area pellucida during development takes place at the expense of the protein pool of the area opaca). Interestingly, high rates of proteosynthesis and/or accumulation of endogenous proteins in the opacal cells are supported by the increase of the protein/ DNA ratio (from 109 to 152) in the area opaca between stage 4 and stage 6-7 (see table 3). On the contrary, in the area pellucida this ratio remains constant (about 66). Moreover, an important energy could be spent on mechanical work. There exist within the blastodisc quite important forces resulting from the imbalance between the cell migration and proliferation rates, specially at stage 5-6 (Downie, 1976). The drop of the DNA content per mm 2 (roughly proportional to the cellular density; see table 3) could indeed reflect the flattening of the opacal cells stretched by the pulling of the migrating edge cells involved in the genesis of mechanical tensions during the late gastrulation (Downie, 1974, 1975 and 1976). These tensions seem to be necessary for the normal development of the area pellucida (Bellairs et al., 1967). The cell contraction state has been recorded within the embryo using a new technique (KuEera and de Ribaupierre, 1982) and its modifications due to anoxia or withdrawal of glucose (unpublished results) indicate that these forces are active. It is noteworthy that Spratt (1958) observed a stimulation of dehydrogenase activity in response to changes in mechanical tension in the blastodisc.
SPATIO-TEMPORALPATTERNSOF THE REGIONALOXYGEN CONSUMPTION At the two studied developmental stages, the pattern of QP indicates clearly that regional differences of the oxidative activity exist and confirms the spatial variations of the oxygen flux corrected for the tissue optical density (Ku~era and Raddatz, 1980). The most important variations of QP are perpendicular to the embryonic axis and the highest values (up to 28 nl .h -~ .#g-l) are found exclusively in the lateral regions (LR2 and LR3) in the vicinity of the limit between the embryonic and extraembryonic areas. These regions show also a high oxido-reducing capacity as demonstrated in studies of the dehydrogenase systems in the young chick embryo (Spratt, 1958). Philips (1942), the sole author who has tried to demonstrate quantitatively the regional differences in oxygen consumption in the chick embryo, observed that the isolated posterior streak and lateral regions of area pellucida had higher but not significantly different rates (for comparison of his data and our results, see table 5). He has, however, been well aware of the fact that cutting the embryo in pieces might eliminate the respiratory differences arising from the normal morphogenetic activity in vivo. To what specific functions then Could be related the high oxidative metabolism
164
E. RADDATZ AND P. KUCERA
TABLE 5 Comparison of the regional oxidative activities (QP, nl •h-l ./tg- 1) at stage 6-7 with those determined by Philips at stage 5-6. The indicated regions are not exactly superimposable but their relative positions in the area pellucida are similar. Present
Regions
A4, A5
A3, A4
A2
A1
LR2, LR3
work
Qp
10-12
12-14
15
15
24-28
Philips
QP
12.3
12
11.8
13.6
14.5
(1942)
Corresponding regions
Node & anterior streak
Middle streak
posterior streak
Right & left lateral
Headprocess
of the relatively undifferentiated posterior and lateral parts of the embryo at the late gastrulation ? Mitotic activity seems to be unequally distributed through the embryo. Emanuelsson (1961), D o n d u a et al. (1966) and Stern (1979) have shown that the mitotic index at stage 4-5 is higher in the posterior part of the primitive streak, at the Hensen's node and, interestingly, also near the boundary between the area opaca and the area pellucida. Cell migration. Nicolet (1970) studied the destination of the invaginating ectoblastic cells with respect to different levels of the primitive streak. He has demonstrated that, at stage 4, the invagination stops at the most anterior part of the streak including the Hensen's node but remains particularly active in the middle and posterior parts. Moreover, important cell movements near the lateral margins of the area pellucida have been repetitively observed in films made in our experimental conditions. As all cell activities coexist in the embryo, experiments allowing their relative importance in the energy requirements to be distinguished are necessary. For this purpose carefully chosen inhibitors of the specific cellular functions could be used. The analysis of the metabolic patterns obtained in such experiments could provide more precise information about the functions that are locally and transiently dominant in the early morphogenesis.
Acknowledgements We are grateful to Professor M. Dolivo for his constant interest and support. We thank Miss M.-B. Schraner for her skilful technical assistance, Miss B. Giintert for the D N A determination and Miss J. Braissant for typing the manuscript.
REGIONAL 02 REQUIREMENTS IN CHICK EMBRYO
165
References Bartels, H. and F. Baumann (1972). Metabolic rate of early embryos (4-22 somites) at varying oxygen pressure. Respir. Physiol. 16: 1-15. Barzu, O. and V. Borza (1967). Spectrophotometric method for assay of mitochondrial oxygen uptake using oxyhemoglobin as indicator and oxygen donor. Anal. Biochem. 21: 344-357. Bellairs, R., D. R. Bromham and C. C. Wylie (1967). The influence of the area opaca on the development of the young chick embryo. J. EmbryoL Exp. Morphol. 17: 195-212. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248. Dondua, A. K,, V.I. Efremov, E.B. Krichinskaya and I.P. Nilolaeva (1966). Mitotic index, duration of mitosis and proliferation activity in the early phases of the development of the chick embryo. Acta Biol. Hung. 17: 127-143. Downie, J.R. (1974). Behavioural transformation in chick yolk-sac cells. J. Embryol. Exp. Morphol. 31 : 599-610. Downie, J.R. (1975). The role of microtubules in chick blastoderm expansion: a quantitative study using cotcbicine. J. Embryol. Exp. Morphol. 34: 265-277. Downie, J.R. (1976). The mechanism of chick blastoderm expansion. J. Embryol. Exp. Morphol. 35 : 559-575. Emanuelsson, H. (1961). Mitotic activity in chick embryos at the primitive streak stage. Acta Physiol. Scand. 52: 211-233. Hamburger, V. and H.L. Hamilton (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92. Hinegardner, R.T. (1971). An improved fluorometric assay for DNA. Anal. Biochem. 39: 197-201. Hommes, F.A., Y. M. Drost, W. X. M. Geraets and M. A. A. Reijenga (1975). The energy requirement for growth: an application of Atkinson's metabolic price system. Pediat. Res. 9: 51-55. Ku~era, P. and Y. de Ribaupierre (1980). Evaluation of the NAD redox states in a living chick embryo using a computer-controlled double-beam scanning microfluorometry. Microsc. Acta Suppl. 4: 283-287. Kurera, P. and E. Raddatz (1980). Spatio-temporal micromeasurements of the oxygen uptake in the developing chick embryo. Respir. Physiol. 39: 199-215. Kurera, P. and Y. de Ribaupierre (1982). In situ recording of the mechanical behaviour of cells in the chick embryo. In: Embryonic Development. New York, Alan R. Liss, Inc., pp. 433-444. Lehninger, A. L. (1975). Biochemistry. 2nd ed. New York, Worth Publ., 949 p. McMaster, G. and S.P. Modak (1977). Cellular and biochemical parameters of growth of chick blastoderms during early morphogenesis. Differentiation 8: 145-152. Miller, J.A. (i941). Janus Green reduction by localized areas of the developing chick, demonstrated by time-lapse microcinematographs in color. Anat. Rec. 79: suppl, p. 47. New, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3: 326-331. Nicolet, G. (1970). Analyse autoradiographique de la localisation des diffrrentes 6bauches pr~somptives dans la ligne primitive de l'embryon de poulet. J. Embryol. Exp. Morphol. 23: 79-108. Philips, F. S. (1941). The oxygen consumption of the early chick embryo at various stages of development. J Exp. Zool. 86: 257-289. Philips, F.S. (1942). Comparison of the respiratory rates of different regions of the chick blastoderm during early stages of development. J. Exp. Zool. 90: 83-100. Raddatz, E. (1980). La consommation d'oxygrne d'un centre organisateur de l'embryon de poulet, mesur6e par microspectrophotomrtrie. Thesis, University of Lausanne. Rulon, O. (1935). Differential reduction of Janus Green during development of the chick. Protoplasma 24: 346-364. Snedecor, G.W. and W.G. Cochran (1971). Statistical methods. Iowa, Iowa State University Press, pp. 258-338.
166
E. RADDATZ AND P. KUCERA
Spratt, N.T. (1951). Demonstration of spatial and temporal patterns of reducing enzyme systems in early chick blastoderms by neotetrozolium chloride, potassium tellurite and methylene blue. Anat. Rec. 109: 384-385. Spmtt, N.T. (1952a). Reducing enzyme (deshydrogenase) systems in unincubated and one day old chick embryo. Anat. Rec. 112:434. Spratt, N.T. (1952b). Differentiation in reducing enzyme systems in the early chick blastoderm. Anat. Rec. 113: 602. Spratt, N.T., Jr. (1952c). Metabolism of the early embryo. Ann. N.Y. Acad. Sci. 55: 40-50. Spratt, N.T. (1958). Analysis of the organizer center in the early chick embryo. IV. Some differential enzyme activities of node center cells. J. Exp. Zool. 138: 51-79. Stern, C.D. (1979). A reexamination of mitotic activity in the early chick embryo. Anat. Embryol. 156: 319-329.
153
Elsevier Biomedical Press
MAPPING OF THE OXYGEN CONSUMPTION CHICK E M B R Y O
ERIC RADDATZ
IN THE GASTRULATING
and PAUL KU(~ERA
Institute of Physiology, 7 Rue du Bugnon, 1011 Lausanne CHUV, Switzerland
Abstract. The spatial variations of the 0 2 uptake in the area pellucida (AP) and area opaca (AO) of the chick embryo were measured in vitro by scanning microspectrophotometry. In the AP, the 02 fluxes (stage 4: 20-90, stage 6-7:30-110 n l . h - l . m m - 2 ) as well as the oxidative activity (stage 4: 14-25, stage 6-7:10-28 n l . h - l . ~ g protein)-1) show distinct regional differences. The most active regions are not those corresponding to the axial embryonic structures but those located in the latero-posterior, relatively undifferentiated zone of the AP. The total 02 uptake increases during 6 h of development from 0.3 to 0.6 pl •h - l but the average oxidative activity and 02 uptake per cell remain constant (18 nl .h -1. (pg protein)-l and 2.5 pl-h -1 .cell-x). In the AO, the 02 uptake is rather homogeneous and all respiratory parameters increase during the development: total 02 uptake from 1.6 to 5.5/~1 .h -l, oxidative activity from 28 to 45 nl .h -t .(/~g protein) -l and cell respiration from 7 to 16 pl .h -l cell-1. The described metabolic variations are discussed with respect to the known morphogenetic activities taking place in the chick blastodisc during the studied period of development. Area opaca Area pellucida Cell respiration
Chick embryo Growth Tissue respiration
T h e m o r p h o g e n e t i c activities o c c u r r i n g in the very y o u n g chick e m b r y o d e p e n d to a g r e a t extent o n the o x i d a t i v e c a t a b o l i s m o f c a r b o h y d r a t e s , especially glucose (see e.g. S p r a t t , 1952c, for review). H o w e v e r , m u c h c o n t r o v e r s y exists a b o u t the q u e s t i o n w h e t h e r the r e s p i r a t o r y activity within the e m b r y o is everywhere the same o r varies a c c o r d i n g to i n d i v i d u a l e m b r y o n i c regions so r e m a r k a b l y different in the relative i m p o r t a n c e o f the o b s e r v a b l e m o r p h o g e n e t i c events, e.g., cell m i g r a t i o n s , histogenetical differentiation. On the one h a n d , r e g i o n a l differences ( b o t h q u a l i t a t i v e a n d q u a n t i t a t i v e ) have been d e s c r i b e d in e x p e r i m e n t s using v a r i o u s e x o g e n o u s o x i d o - r e d u c t i o n indicators, i.e., vital dyes ( R u l o n , 1935; Miller, 1941; Spratt, 1951, 1952a,b, 1958). T h e s e Accepted for publication 6 November 1982
0034-5687/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
154
E. RADDATZ AND P. KUCERA
findings, although obtained in long-lasting anoxic conditions unfavourable to normal development, and although rather qualitative because of difficulty to correct the observed colour changes for the tissue quantity, have been recently strongly supported by studies of the intracellular N A D redox state in an intact and normally developing embryo in vitro (KuEera and de Ribaupierre, 1980). On the other hand, the respiratory rates of isolated fragments of the chick embryo were measured manometrically and no significant regional variations have been found (Philips, 1941, 1942). In these experiments, however, the long-range cell interactions, so important for the normal differentiation, have been completely abolished. We have developed a non-invasive and very sensitive technique allowing continuous measurements of the oxygen uptake of minute regions o f the intact chick embryo normally developing in vitro under controlled conditions (KuEera and Raddatz, 1980). The first results clearly indicated the presence, within the neurulating embryo, of spatio-temporal variations of the oxidative activity. This paper presents the respiratory patterns found in the whole gastrulating Chick embryo. List FL : FR : Q : Qp : QD : Qc :
o f symbols and units Oxygenflux measured locally at a given point of the blastodisc: nl •h - l. mm -2 AverageFL value for one defined embryonicregion: nl. h-1. mm-2. Absolute oxygenconsumption of a givenembryonicregion of known area S (Q = FR •S): nl •h - 1. Q corrected for the protein content, i.e., the oxidativeactivity: nl. h-I. (~g protein)-1. Q corrected for the DNA content: nl .h -1 .~g DNA) -l. Oxygenuptake per cell, calculated from QD: pl-h- 1 . cell-l.
Materials and methods CULTURE OF THE EMBRYO The embryos (see fig. 1) at the stage 4 (definitive primitive streak) and at stage 6-7 (head-fold) according to Hamburger and Hamilton (1951) were obtained by preincubation o f fertilized eggs (White Leghorn) at 38 °C and 80% relative humidity. The entire blastodiscs with the vitelline membrane were carefully removed from the yolk under sterile conditions according to the technique of New (1955) and cleaned of yolk particles. As described in previous papers (KuEera and Raddatz, 1980; Raddatz, 1980), the preparation is covered by a second very thin and transparent silicone membrane and mounted in a special incubation chamber. The embryo with the membranes separates the internal volume of the chamber into two compartments tightly closed by two windows. The blastodisc faces the lower compartment containing a defined nutritive medium. The upper compartment is perfused by a solution of purified human hemoglobin saturated at Ps0 (54 mm Hg) with oxygen representing the only source of oxygen available for the embryo. During a steady-state perfusion, the oxygen passes easily through the silicone and vitelline
REGIONAL02 REQUIREMENTS IN CHICKEMBRYO
155
membranes down its concentration gradient, i.e., from the hemoglobin solution to the respiring tissue. The chamber is fixed on the stage of a scanning microspectrophotometer and thermostabilized at 37 °C.
SCANNING MICROSPECTROPHOTOMETRICMEASUREMENTS OF THE OXYGEN CONSUMPTION As the absorption spectrum of the hemoglobin changes during its deoxygenation, this protein can be used as the oxygen donor as well as the indicator of the oxygen uptake (Barzu and Borza, 1967). Thus, under the stop-flow condition, the time variation of the absorbance measured in the linear domain of the HbO2 dissociation curve (around Ps0) is proportional to the deoxygenation rate of the hemoglobin, i.e., to the respiratory rate of the adjacent tissue. Therefore, if the regional variations of the absorbance in the layer of hemoglobin are recorded over the entire embryo during 10 sec periods of stop-flow, it is possible to establish the spatial pattern of its respiration. Such spectrophotometric measurements have been done by scanning the preparation at a speed of 0.5 m m . s -~ between the left and the right margins of the area pellucida through various levels of the embryo. In order to investigate the opacal region, two additional scans have been extended up to the edge of each blastodisc; the first one from the right end of the scan passing through the Hensen's node, and the second from the right end of the most posterior scan. All the measurements have been done at 435 nm using a light beam of a crosssection of 0.0025 mm2 covering approximately 25-80 ceils according to the place within the embryo. Such a technique allows us to determine the oxygen flux (FL) at any point of a given scan with a precision of + 2 nl .h -~ .mm -2. In order to minimize the error due to a lateral diffusion of oxygen within the hemoglobin layer, the measurements must be done as quickly as possible. The desaturation slopes at the maximally and minimally respiring points of the same embryo have been systematically recorded as a control to the scanning measurements. Up to 10 sec virtually no deviation from linearity has been found and the FL values obtained by both methods for the same points were identical. This indicates that, within this interval, the scanned profiles of hemoglobin absorbance are not yet significantly smoothed by the lateral diffusion. In addition, t h e same values of FL have been obtained for the same points of the embryo using different initial Hb saturation levels allowed by the linearity domain of the method. This indicates that no region of the embryo suffers from hypoxic conditions during the stop-flow period and confirms the same conclusion derived from theoretical calculus based on three different models of penetration of oxygen into the tissue (KuEera and Raddatz, 1980; Raddatz, 1980). The local values of FL have been plotted with respect to their scanning coordinates. Such FL maps display the intensity of the oxygen flux within the embryonic territory
156
E. RADDATZ AND P. KU(~ERA
(see fig. 2). O n the o t h e r h a n d , the F R values o f the a r e a pellucida, the a r e a o p a c a a n d the entire b l a s t o d i s c have been o b t a i n e d b y i n t e g r a t i o n o f all the FL values over the c o n s i d e r e d zones.
NORMALIZED TOPOGRAPHY OF THE EMBRYOS T h e r e exists for a given d e v e l o p m e n t a l stage a large i n d i v i d u a l v a r i a b i l i t y in the f o r m a n d hence the d i m e n s i o n s o f the e m b r y o . Therefore, a c o m p a r i s o n o f the FL values f r o m o n e e m b r y o to a n o t h e r necessitated the definition o f c o m p a r a b l e regions. A s the e m b r y o at the e a r l y stages is r a t h e r u n d i f f e r e n t i a t e d , we have defined these regions as follows.
Fig. 1. The scanned part of the chick embryos photographed at the same magnification in the incubation chamber. The central and more transparent area pellucida containing the axial embryonic structures is surrounded by the area opaca heavily charged with yolk. The letters and numbers refer to the embryonic regions defined in the text. For the protein determinations the borders containing yolk were dissected off.
REGIONAL 02 REQUIREMENTS IN CHICK EMBRYO
157
Each scanned area was subdivided by longitudinal and transversal lines, the intersections of which defined 12 (stage 4) or 15 (stage 6-7) normalized embryonic regions (see figs. 1 and 2). Thus, the investigated area was divided into three longitudinal strips of equal width: one axial (A), and two lateral - left (L) and right (R). Then, it was divided transversally into anterior and posterior parts with respect to the Hensen's node: the anterior one was represented by one strip at stage 4 (labelled 4) and by two strips at stage 6-7 (labelled 4 and 5); the posterior one was divided into three strips (labelled 3, 2, 1) so that the primitive streak was divided into anterior (including the node), median and caudal segments, i.e., the regions labelled A3, A2 and A1, respectively.
DETERMINATION OF THE PROTEIN AND DNA CONTENTS The embryonic regions as defined above were carefully dissected and pooled and their protein content determined using the Coomassie Blue method (Bradford, 1976) and bovine serum albumin as reference. The regional protein content (metabolically active and inactive fractions mixed) serves to calculate the QP. The DNA content of the area pellucida and area opaca was determined according to Hinegardner (1971). Knowing the average cellular DNA content (2.3 pg, McMaster and Modak, 1977) we can evaluate the number of cells in each area and thus calculate the cellular respiration Qc.
STATISTICALEVALUATION The analysis of variance (Fisher's method) within and between the regions of the individual embryos, of the embryos of the same stage and of the both stages were done for all the measured and calculated parameters. Whenever this test had allowed it, the significance of all differences found was evaluated using the studentized range Q-test. This latter test, more severe than the one of least significant difference, has the property that the probability of making an erroneous claim of significance is <0.05. All the statistics were done according to Snedecor and Cochran (1971).
Results
MAPS OF THE LOCALOXYGEN FLUXES (FL) They were obtained in 22 normally developing embryos: 14 at the definitive primitive streak stage (1248 measurements) and 8 at the head-fold stage (1172 measurements).
158
E. R A D D A T Z A N D P. KU(~ERA
STAGE 8 - 7
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Fig. 2. Examples of the individual maps of the oxygen uptake within the area pellucida and adjacent parts of the area opaca. The values of the local 02 fluxes (nl .h -1 . m m -2) are represented by the dimensions of the black dots calibrated at the bottom. Each value is plotted with respect to its scanning coordinates. Stage 4 : 1 5 4 data; stage 6 - 7 : 2 1 3 data. n: Hensen's node; ps: primitive streak (compare with fig. 1).
The variations of the values of FL in both investigated stages are illustrated by two representative maps (fig. 2). At stage 4 (fig. 2, left) the most elevated FL values are situated at the Hensen's node and in the anterior part of the primitive streak (A3). Sometimes, a relatively active region appears also posteriorly (A1). The FL values determined in the adjacent parts of the area opaca are rather homogeneous and lower than those in the area pellucida. At stage 6-7 (fig. 2, right) the most elevated FL values in the area pellucida are observed along and on both sides of the regressing streak, the head process and the neural plate. The lowest FL values are found over the mesoderm-free region of the proamnion. The maps established for each individual embryo thus show clear cephalocaudal and medio-lateral gradients of oxygen uptake per unit area. These differences
159
REGIONAL O 2 REQUIREMENTS IN CHICK EMBRYO
are highly significant. From one embryo to another, however, the FL profiles vary considerably, as illustrated by the extreme values found so far: 20 and 90 at stage 4 and 28 and 110 nl .h -) .ram -2 at stage 6-7. The variability is due to different steepness of the gradients but also due to different mean levels of the oxygen consumption. These variations, roughly corresponding to the local cell packing density, are more pronounced at the younger stage which shows also a greater variability in form and which is more difficult to define at the time scale.
THE REGIONAL DIFFERENCES IN THE POOLED VALUES
All the measured and calculated parameters are reported in table 1. As we .,4~a~,e never observed significant variations between the left and right halves of the area pellucida, the lateral regions were grouped together. Table 2 gives the results of the statistical analysis within each stage and between both stages. For each parameter, the difference between any two regions is significant if equal or greater than the value D. At each stage, the FR values show practically the same pattern as in the individual FL maps, although the differences are smaller due to the averaging of the highly dispersed values as mentioned before. Nevertheless, at stage 4, the FR of the region containing the Hensen's node is significantly higher if compared to all other regions. At stage 6-7, almost all longitudinal and transversal variations are significant. The QP values are consistently more elevated in the lateral regions of the embryo. The differences are significant at stage 6-7 but just not significant (P = 0.07) at stage 4 where the errors in the protein determinations are greater. TABLE 1 Morphometric, chemical and metabolic parameters for each embryonic region at the two developmental stages. SD: standard deviation. EMBRYONIC STAGE H,H. Area
± SD
mm 2
6-7
Protein content
4
ug
6-7
FR ± SD
4
al,h=~mm-2
6-7
Q ± SD
4
nl.h -I Qp ± SD nl.h-~pg -I
LR5
AS
4
6-7
0.5±0.1
1.5±0.4
53±7
27±7
0.7±0.1
4.5±0.6
60±13
43±9
4 6-7
19±5
10±1
REGIONS
DR4
A4
LR3
0.3±0.2
0.7±0.1
0.6±0.1
0.7±0.1
A3
0.5±0.I
LR2
0.7±0.1
A2
0.2±0.1
LRI
0.7±0.
AI
0.7±0.1
0.7±0.1
0.7±0.1
0.7±0.1
0.6±0.2
0.7±0.1
0.4±0.2
0.7±0.
1.0±0.4
2.7±0.9
1.4±0.1
3.0±0.4
1.2±0.03
2.7±0.611.0±0.2
2.3±0.
2.0±0.01
4.2±0.1
1.7±0.1
4.1±0.1
1.4±0.01! 3.8±0.1
55±12
56±13
58±10
62±16
58±9
57±9
71±16
S6~8
76±17
17±9
36±7
33±10
40±6
Sl±ll
18±7 20±0.01
1.4±0.2
3.6±0.(
59±12
57±11
59±12
62±8
7S±16
63±9
71±12
43±14
31±9
41±11
15±7
40±11
41±6
56±12
40±9
55±10
23±9
52±7
14±5
23~2
15±2
25±i
16±3
16±4
19±7
12±0.3
24±1
14±0.3
28±0.01
IS±0.2
17±2
IS±2
160
E. RADDATZ A N D P. KU(~ERA
TABLE 2 The results of the statistical analysis within and between the stages, d.f. : degrees of freedom; F : Fisher's ratio of variances; D: threshold value of significance (P <0.05) for any regional difference within or between the stages. D values have the units of the corresponding parameters (see text). N.S.: not significant. STAGE
4
STAGE
6 - 7
!PARAMETERS
d.f.
F
D
d.f.
AREA
11/156
27.7
0.14
14/105
1.8
14/15
42.5
11/12
PROTEIN
6.1
BETWEEN
F
9.0
STAGES
D
d.f.
F
0.20
1/11
16.4
0.I
i.i
1/11
30.8
0.3 4.1
FR
11/1241
4.7
2.2
14/1157
47.6
2.3
1/11
11.9
Q
11/156
17.2
ll.9
14/105
12.8
15.1
1/22
59.6
Qp
11/12
2.2
N.S.
14/15
14.5
8.1
1/11
1.4
D
3.6 N.S.
The comparison between the two stages shows a clear increase in all the parameters directly linked to the growth o f the embryo. However, we do not observe any significant change in the oxidative activity during the studied period of development.
OXYGEN UPTAKE OF THE AREA PELLUCIDA A N D AREA OPACA BETWEEN STAGES 4 A N D 6-7 (TABLE 3)
Area pellucida During the 6 h of development the following parameters increase significantly: the area by 6 7 ~ , protein content by 71~o, D N A content by 6 4 ~ , number of cells from 1.4.105 to 2.3 • 105 ( + 6 4 ~ ) , FR by 9 ~ and the total oxygen need by 7 1 ~ . On the other hand, the QP, QD and Qc values remain practically the same (about 18 nl •h - z. pg- l, 1100 nl • h - ~ •/~g- ~ and 2.5 pl • h - ~ • cell - l, respectively). TABLE 3 Morphometric, chemical and metabolic parameters for the area pellucida, area opaca and whole blastodisc ( + SD). Blastodisc St 4 Area mm 2 Protein content
51 ~ ii 117 ± 19 80
gg
DNA c o n t e n t FR:
~g
n l . h - l . m m -2
Q: n l . h -I Qp:
St 6-7
nl.h-~(~g
QD: n l . h ' ~ ( ~ g
prot) -I DNA) -1
0.86
158 1.34
Area
Pel l u c i d a
Area
Opaca
St 4
St 6-7
6 ± I
i0 ± 1
43 + ii
104 ± 18
21
36
58 ± 2
122 ± 25
0.33
0.54
0.53
0.80
St 4
38 ± 12
St 6-7
53 + 15
39
52
2000
6100
25
39
18
17
28
45
2300
4600
ii00
ii00
3000
6900
58 + 15
63 ± 15
350 -+ 150 600 + 250
1600
± i100
5500
± 2800
REGIONAL 0 2 REQUIREMENTS IN CHICK EMBRYO
"
161
Area opaca
169 and 103 FL measurements were collected from stages 4 and 6-7. During the 6 h all the parameters increase significantly: the area by 142~, protein content by 110~o, DNA content by 51~o, cell number from 2.3.105 to 3.5.105 (+52~), FR by 39~o and the total oxygen need by 244~. The most interesting findings concern the corrected values which are all initially much higher than those in the area pellucida and which all increase considerably: the QP from 28 to 45 nl-h-1 ./~g-1 (+61~), the QD from 3000 to 6900 nl .h -1 .#g-~ (+ 130~o) and the Qc from 7 to 16 pl .h -l -cell -1 (+ 129~o).
Discussion EMBRYONIC AND EXTRAEMBRYONIC OXYGEN REQUIREMENTS
Area pellucida
In spite of differences in the methods used in this work and those of Philips (1942) and Bartels and Baumann (1972) a quantitative comparison of the obtained results is possible (table 4). Philips measured the oxygen consumption manometrically in several dissected embryonic fragments. Bartels and Baumann measured the oxygen consumption polarographically in isolated entire embryos cleared of membranes and placed in a stirred medium. The fact that our culture technique approximates closely the in ovo situation could explain that our Q values for the area pellucida are the highest found. The oxidative activity (QP) remains constant between the two stages (about 18 nl .h -~ .#g-l). Philips reached the same conclusions but found a lower value (about 13 nl .h -~./~g-i at stage 5-6). Bartels and Baumann found about 18 nl .h -~./ag -j at stage 8 (estimated from the data corrected for the dry weight TABLE 4 Comparison of the total oxygen consumption ~1. h -l) of the young chick embryo as determined by different authors. Stage H.H.
Philips (1942)
Bartels and Baumann (1972)
4 5+
This work
0.35 0.25-0.30
6
0.25
7
0.36
8
0.50
0.61
162
E. RADDATZ AND P. KUCERA
assuming that 70~ of the dry weight are proteins). In addition, the invariability of the embryonic oxidative activity is perfectly corroborated by the values of QD and Qc during the same period of development. Area opaca To our knowledge, no data about the oxygen uptake of the area opaca have been so far available. Rulon (1935), using an intracellular indicator of oxido-reduction (Janus Green) in anoxic conditions, noted that the cells of the area opaca became highly active at the head-process stage. We have found that the values of QP, QD and Qc increase indeed by 60~o, 130~o and 130~, respectively (table 3). From these data it may be deduced that some important metabolic variations occur in the opacal tissue during its intense expansion between the definitive primitive streak stage and the head-fold stage, i.e., a period including the head-process formation. In summary, with respect to growth, the oxygen uptake of the area pellucida seems to increase in parallel whilst the oxygen uptake of the area opaca increases much faster. The following section tries to correlate the two parameters in some detail.
EVALUATION OF THE METABOLIC COST OF THE GROWTH
The metabolic cost of the growth can be estimated both for the embryonic and extraembryonic parts of the blastodisc. In each of these parts we know: (1) the net gain of the total proteins (P) during the 6 h of development, i.e., 15 and 64 #g in the pellucid and opacal areas, respectively (see table 3); (2) the total quantity of oxygen consumed (T) during the same time interval, i.e., 2.9 and 21.2 pl; (3) the 'maintenance rate' (M), i.e., the quantity of oxygen that should have been consumed if the oxygen consumption had remained that of stage 4, i.e., 6 x0.35 /~1 and 6 x 1.6 /~1. Thus, assuming that the additional aerobic energy expenditure ( T - M) is assigned mainly to the protein synthesis and turnover, the ratio ( T - M)/P gives a good approximation of the oxygen requirement for the net protein gain. The values calculated for the area pellucida and area opaca are 52 and 181 ml 02 consumed per gram of gained protein, respectively. Using the caloric equivalent of the oxygen of 5 kcal/L 02 (respiratory quotient RQ = 1), the corresponding metabolic costs are 0.3 and 0.9 kcal per gram of gained protein, respectively. A theoretical calculation of the maximal metabolic cost of the growth shows that 0.5 kcal are necessary for the gain of one gram of protein if the following hyptheses are considered: all the proteins are condensed only from individual amino acids; the energy requirement for the formation of one peptidic bound is about 30 kcal (Lehninger, 1975); the aerobic efficiency is 0.5; the RQ is 1. It is interesting that the value found for the embryonic area (0.3 kcal per gram of gained protein) is very close to this theoretical value and, moreover, that it is in good agreement with that found by Hommes et al. (1975) for the human infant (0.4 kcal).
REGIONAL O2 REQUIREMENTSIN CHICK EMBRYO
163
The elevated value found for the area opaca (0.9 kcal) indicates that, in addition to the proteosynthesis, this area ensures other important energy dependent functions such as massive yolk digestion or transport of materials required by the embryonic area (in a preliminary study we have shown that the gain of protein in the area pellucida during development takes place at the expense of the protein pool of the area opaca). Interestingly, high rates of proteosynthesis and/or accumulation of endogenous proteins in the opacal cells are supported by the increase of the protein/ DNA ratio (from 109 to 152) in the area opaca between stage 4 and stage 6-7 (see table 3). On the contrary, in the area pellucida this ratio remains constant (about 66). Moreover, an important energy could be spent on mechanical work. There exist within the blastodisc quite important forces resulting from the imbalance between the cell migration and proliferation rates, specially at stage 5-6 (Downie, 1976). The drop of the DNA content per mm 2 (roughly proportional to the cellular density; see table 3) could indeed reflect the flattening of the opacal cells stretched by the pulling of the migrating edge cells involved in the genesis of mechanical tensions during the late gastrulation (Downie, 1974, 1975 and 1976). These tensions seem to be necessary for the normal development of the area pellucida (Bellairs et al., 1967). The cell contraction state has been recorded within the embryo using a new technique (KuEera and de Ribaupierre, 1982) and its modifications due to anoxia or withdrawal of glucose (unpublished results) indicate that these forces are active. It is noteworthy that Spratt (1958) observed a stimulation of dehydrogenase activity in response to changes in mechanical tension in the blastodisc.
SPATIO-TEMPORALPATTERNSOF THE REGIONALOXYGEN CONSUMPTION At the two studied developmental stages, the pattern of QP indicates clearly that regional differences of the oxidative activity exist and confirms the spatial variations of the oxygen flux corrected for the tissue optical density (Ku~era and Raddatz, 1980). The most important variations of QP are perpendicular to the embryonic axis and the highest values (up to 28 nl .h -~ .#g-l) are found exclusively in the lateral regions (LR2 and LR3) in the vicinity of the limit between the embryonic and extraembryonic areas. These regions show also a high oxido-reducing capacity as demonstrated in studies of the dehydrogenase systems in the young chick embryo (Spratt, 1958). Philips (1942), the sole author who has tried to demonstrate quantitatively the regional differences in oxygen consumption in the chick embryo, observed that the isolated posterior streak and lateral regions of area pellucida had higher but not significantly different rates (for comparison of his data and our results, see table 5). He has, however, been well aware of the fact that cutting the embryo in pieces might eliminate the respiratory differences arising from the normal morphogenetic activity in vivo. To what specific functions then Could be related the high oxidative metabolism
164
E. RADDATZ AND P. KUCERA
TABLE 5 Comparison of the regional oxidative activities (QP, nl •h-l ./tg- 1) at stage 6-7 with those determined by Philips at stage 5-6. The indicated regions are not exactly superimposable but their relative positions in the area pellucida are similar. Present
Regions
A4, A5
A3, A4
A2
A1
LR2, LR3
work
Qp
10-12
12-14
15
15
24-28
Philips
QP
12.3
12
11.8
13.6
14.5
(1942)
Corresponding regions
Node & anterior streak
Middle streak
posterior streak
Right & left lateral
Headprocess
of the relatively undifferentiated posterior and lateral parts of the embryo at the late gastrulation ? Mitotic activity seems to be unequally distributed through the embryo. Emanuelsson (1961), D o n d u a et al. (1966) and Stern (1979) have shown that the mitotic index at stage 4-5 is higher in the posterior part of the primitive streak, at the Hensen's node and, interestingly, also near the boundary between the area opaca and the area pellucida. Cell migration. Nicolet (1970) studied the destination of the invaginating ectoblastic cells with respect to different levels of the primitive streak. He has demonstrated that, at stage 4, the invagination stops at the most anterior part of the streak including the Hensen's node but remains particularly active in the middle and posterior parts. Moreover, important cell movements near the lateral margins of the area pellucida have been repetitively observed in films made in our experimental conditions. As all cell activities coexist in the embryo, experiments allowing their relative importance in the energy requirements to be distinguished are necessary. For this purpose carefully chosen inhibitors of the specific cellular functions could be used. The analysis of the metabolic patterns obtained in such experiments could provide more precise information about the functions that are locally and transiently dominant in the early morphogenesis.
Acknowledgements We are grateful to Professor M. Dolivo for his constant interest and support. We thank Miss M.-B. Schraner for her skilful technical assistance, Miss B. Giintert for the D N A determination and Miss J. Braissant for typing the manuscript.
REGIONAL 02 REQUIREMENTS IN CHICK EMBRYO
165
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166
E. RADDATZ AND P. KUCERA
Spratt, N.T. (1951). Demonstration of spatial and temporal patterns of reducing enzyme systems in early chick blastoderms by neotetrozolium chloride, potassium tellurite and methylene blue. Anat. Rec. 109: 384-385. Spmtt, N.T. (1952a). Reducing enzyme (deshydrogenase) systems in unincubated and one day old chick embryo. Anat. Rec. 112:434. Spratt, N.T. (1952b). Differentiation in reducing enzyme systems in the early chick blastoderm. Anat. Rec. 113: 602. Spratt, N.T., Jr. (1952c). Metabolism of the early embryo. Ann. N.Y. Acad. Sci. 55: 40-50. Spratt, N.T. (1958). Analysis of the organizer center in the early chick embryo. IV. Some differential enzyme activities of node center cells. J. Exp. Zool. 138: 51-79. Stern, C.D. (1979). A reexamination of mitotic activity in the early chick embryo. Anat. Embryol. 156: 319-329.