Paper - Studies on the area vasculosa of the embryo chick 2 (1937)
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Studies on the Area Vasculosa of the Embryo Chick
II. The Influence 0f the Circulation on the Diameter of the Vessels
By A. F. W. Hughes
Sir Halley Stewart Research Fellow
From the Strangeways Research Laboratory, Cambridge
The first paper of this series (Hughes, 19385) was an attempt to continue Thoma’s work of the effects of the blood stream on the vessels of the chick area vasculosa. In general, arteries and veins are formed from smaller vessels, usually in a capillary network. The circulation of blood is believed to be responsible for the enlargement of the capillary vessels, and Thoma suggested a relationship between the flow of blood in a vessel and the diameter of the vessel, which is as follows: The increase in size of the lumen of a vessel, which is proportional to the growth in surface of the vessel wall, is dependent upon the rate of the blood flow.
The evidence which he offered for this assertion is concerned with the manner in which the vitelline artery is first formed in the chick blastoderm (Thoma, 1898). This evidence is briefly as follows: After the heart has begun to beat effectively, all the blood flowing from the aortae of the embryo to the area vasculosa runs through a narrow band of the vascular network on each side, in which the rate of flow is therefore much more rapid than elsewhere. These regions are soon transferred into the vitelline arteries by the enlargement and fusion of the meshes of the network.
The first paper of the present series (Hughes, 1935) is chiefly concerned with the detailed development of the vitelline artery, but neither in this paper, nor in those of Thoma are there any measurements of the comparative rates of flow in different regions of the area vasculosa, and thus no direct proof of Thoma’s principle is attempted. Recently, the rate of flow of the blood in different regions of the area vasculosa has been measured, and quantitative data concerning the validity of Thoma’s hypothesis have been obtained, which it is the purpose of this present paper to describe.
Text-fig. 1. Diagram of apparatus, showing egg chamber mounted on indirectly illuminating microscope, which is enclosed by the hot box.
There are two methods for observing living chick blastoderms, one is to remove the blastoderm from the yolk and grow it in vitro in a suitable culture medium. In the other method, the blastoderm is left inside the yolk; part of the shell is removed, and the egg is incubated in a moist atmosphere (Romanoff, 1931). The second of these methods has been chosen for the present work, since it was necessary to observe a single blastoderm over long periods. Eggs opened during the second day of incubation and placed in a suitable moist chamber, frequently continue to develop for a further 7 days, and occasionally longer. By this means, therefore, it is possible to study a wide range of blood vessels, in which growth and differentiation are taking place extremely rapidly, and the method thus offers considerable possibilities in the study of vascular development.
(2) Estimation of the velocity of the blood stream
The rate of blood flow to which Thoma’s hypothesis refers is expressed in volumes of blood flowing in unit time. Cannulation methods which are inapplicable to embryonic vessels are necessary in order to measure directly the rate of blood flow. In this work, the velocity of the corpuscles seen in motion under the microscope has been measured. To calculate the rate of flow of the blood from the velocity of the corpuscles, two further sets of data are required: one, the general relationship between the position of the corpuscles along the radius of the vessel and their velocity, and secondly, the actual position on the radius of the vessel of the corpuscles whose velocity we are able to measure. For a homogeneous fluid flowing in a tube under certain circumstances, the first of these relationships can be derived from Poiseuille’s law connecting the rate of flow and the viscosity of a fluid flowing in a tube with the difference of pressure at the two ends. It would not be justifiable to apply the Poiseuille formula to the vessels of the area vasculosa without previous demonstration of its validity in these circumstances.
Viewing the vessels by indirect illumination, the corpuscles which we see in motion are the outermost ones in the vessel. In the larger vessels, these corpuscles appear very close to the vessel wall, and no layer of corpuscle-free plasma lining the vessel wall can be seen; under the magnifications which have been used in this work it is not possible to measure the actual distance between these corpuscles and the vessel wall, and small differences in this quantity would make very large differences to the rate of flow calculated on Poiseuille’s formula.
For these reasons, we have used the velocity of the superficial corpuscles itself as our index of rate of flow and have called it “marginal velocity”. The exact manner in which blood flows through the vessels of the area vasculosa is now being investigated by means of slow-motion cinema pictures, but the present paper describes only the correlations which have been observed between rate of change in diameter of the vessels, and the marginal velocity.
The marginal velocity is measured by means of an apparatus in which an external point of light reflected into the field of the microscope by means of a camera lucida is made to traverse the field at such a speed that the point appears to travel at the same rate as the corpuscles in the blood vessel. The point source of light is attached to a pencil which is moved across a band of paper, which itself moves at right angles to the direction of motion of the pencil. A series of oblique lines on the paper are thus drawn, and from the slope of these lines, the velocity of the paper band, and the magnification of the microscope, the velocity of the corpuscles can be calculated.
About ten tracings of corpuscle movement were made at a time for each vessel, and the resulting velocity values were averaged. Table I gives the results of a series of measurements of corpuscle velocity in a single vessel made at intervals of a few minutes, with two different magnifications. Under the higher of these magnifications, the blood stream appears so rapid that it is difficult to follow it accurately with the pencil point. Accordingly, the values for corpuscle velocity tend to be lower and more divergent with the higher magnification. The lower magnification has therefore been used generally for measuring corpuscle velocity in this work. The degree to which such results are comparable may be judged from Table I, in which the lowest and highest average values for corpuscle velocity obtained with the lower magnification differ by 32 per cent.
Table I. Measurements of marginal velocity on one vessel
Average marginal Percentage of Time Heart rate Objective velocity Range mean 8.55 136 3-8 416 390-460 94-110 9.12 136 3-8 423 360-500 88-118 9.30 150 3-8 416 350-490 84-118 9.45 144 3-8 476 445-540 93-114 10.5 —_— 11-0 270 220-290 82-108 10.25 —_— 11-0 446 374-540 84-120 10.30 136 11:0 360 290-420 81-116 10.40 — 3-8 503 480-540 95-108 10.55 150 3-8 551 400-630 73-114
Average of nine series: 423
Measurement of Diameter and Marginal Velocity
(1) Arterial branches
The marginal velocity and the diameter of each of the main branches of the vitelline artery of one side of the area vasculosa were measured at intervals; observations were begun early in the second day of incubation, and the differences between the various branches were used to study the relationship between marginal velocity and rate of change in diameter.
The changes in relative importance of the branches of a vitelline artery follow certain lines which are determined by the general changes which take place within the area vasculosa. During the first 10 hours of circulation, blood is returned to the embryo mainly through the only veins which have so far developed, namely the anterior pair which are part of the marginal sinus. Consequently, the greater part of the blood issuing from the vitelline arteries flows anteriorly, in the anterior branches of the main artery. The main flow from the vitelline artery does not run directly anterior, however, for the vessels along the direct path nearest the embryonic axis remain in a contracted state for the first few hours after the circulation has begun (Hughes, 1935) and must therefore offer a greater resistance to the blood flow than a less direct path through wider vessels. In A V,} (Plate I, fig. 2), the second of the three branches of the left vitelline artery (LVA,) carries most of the flow throughout the whole period of observation; in AV, (Text-fig. 3), where the left vitelline artery has four branches, branch (LVA,) is the largest in diameter at the time when observation begins. Up to this period, therefore, those vessels in which the flow has been greatest have acquired the larger diameter.
Text-fig. 2. Relations between marginal velocity and diameter of the branches of the left vitelline artery and of the posterior vein of AV,. The points for the diameter of LVA, are omitted for the sake of clearness.
Text-fig. 3. Relations between marginal velocity and diameter of the branches of the left vitelline artery of AV,. The same symbols are used as in text-fig. 2.
The development of other return paths soon modifies these relationships. A direct path is formed from the posterior region of the area vasculosa along the left side of the embryonic axis, and over the left vitelline artery. This is the posterior vein. This path is formed by capillaries already present on either side of the artery, but it is not completed until these are connected dorsal to the artery by newly formed vessels, which originate in the manner described in the previous paper of this series (Hughes, 1935).
1 The following abbreviations are used in this paper. The series of individual area vasculosae examined in the living state are referred to as AV,, AV, etc. In each specimen the branches of the left vitelline artery are numbered antero-posteriorly, as LV.A,, LV Ag, ete. 6 A. F. W. Hughes
The establishment of a direct return path from the posterior region of the area vasculosa tends to increase the arterial flow to that region by diminishing the peripheral resistance. Accordingly, a smaller share of the total arterial flow on each side is distributed through the anterior branches. In AV, the posterior venous path had formed after 5 hours of observation, and at this time the marginal velocity in the posterior branch, LVA, increased sharply, as shown in Text-fig. 4.
Text-fig.4. Percentage change in diameter plotted against average marginal velocity for veins and arteries in six individuals, over the periods indicated. Points referring to arteries and veins of the same individual joined by dotted lines; points referring to similar vessels of the same individual joined by continuous lines.
By the time the posterior venous flow has begun, a further series of veins are differentiating, which, like the posterior vein, are formed through the isolation of the arteries from the surrounding capillaries, which then join together across the dorsal surface of the arteries. A lateral system of veins develops over the branches of the vitelline arteries, which ultimately completely cover the main branches. In low-power photographs, it is possible to see the lateral veins only where they do not follow the same course as the pre-existing arteries. Thus in figs. 7 and 8 of Plate I, the vessels (vv) which develop rapidly during the latter part of the period of observation are part of the lateral vein system. The venous network which runs alongside each artery provides a more direct return path for the capillary area supplied by that artery than the main anterior and posterior veins. A rapid flow in this venous network begins in the reverse direction to that in the underlying arteries, and the capillaries of the network rapidly enlarge to form a system of venae comitantes which largely follow the same path as the arteries. The greater the flow carried by an artery at the time of the formation of this venous network, the more rapidly will an accompanying vein differentiate alongside the artery.
In both AV, and AV, the main branches of the arteries were obscured by veins 20 hours after observation had begun, with the exception of the smallest vessels, which were LV.A, and L VA, respectively. Consequently, it was only possible to measure the marginal velocity in all the branches over a total period of 12 hours, and hence comparison of marginal velocity with rate of change in diameter is only possible over this period.
In order to compare the ranges of values in these two quantities with both the biological variation and the size of the errors involved in these measurements, the correlation diagram shown in Text-fig. 5 was constructed in which percentage change in diameter over the period of observation is plotted against average marginal velocity. For the arteries alone, the points show a general trend of increase in rate of enlargement with increase in average marginal velocity, but this correlation becomes far clearer when the venous points are considered in addition, since veins show a much wider range in these values than do the arteries. For this reason, veins received the greater share of attention during the course of this work.
In order to compare the rate of enlargement and of marginal velocity in different veins in the same blastoderms, three veins were chosen, namely the posterior vein of which the development has already been described, and the two anterior veins, whose subsequent development is particularly suitable for this purpose.
During the second and third days of incubation, the gap between the anterior veins of the area vasculosa gradually narrows, and is finally bridged by means of anastomoses between these vessels (Plate III). At this stage the anterior region of the area vasculosa is a continuous network, and the vessels Diameter in p
Text-fig. 5. Relations between marginal velocity and diameter of main veins AV,;: (i) diameter only for the three veins; (ii) diameter and marginal velocity for the posterior vein; (iii) diameter and marginal velocity for the two anterior veins..
Marginal velocity in per sec.
which carry most blood back to the heart from the anterior region are those which offer least resistance, irrespective of whether these are the original right or left anterior veins. Usually one path offers less resistance than all others, and in carrying a greater flow of blood than other paths, gradually enlarges to form the final anterior vein, which in common with other vessels which differentiate from a vascular network does not originate in an identical manner in different individuals. Popoff (1894) states that the left anterior vein becomes the final anterior vein, but Grodzinski (1935), who has followed these changes in the living area vasculosa, found that out of ten blastoderms, the final anterior vein was formed from the left vein in four, from the right in three, and from parts of both in the other three. In the photographs of A V, (Plate III), the proximal part of the original left anterior vein becomes the proximal part of the final anterior vein, but the distal part of the latter is formed from an anastomosing vessel, between the original anterior veins, both of which ultimately become part of the general area vasculosa in this region. In these veins therefore, there is considerable scope for the study of rate of change in diameter,
Text-fig. 6. Relations between marginal velocity and diameter of main veins of A V,: (i) diameter only, for the three veins; (ii), (iii) and (iv) diameter and marginal velocity for posterior, left anterior, and right anterior veins respectively. The same symbols are used as in Fig. 5.
Two sets of results are given here of measurements of all three veins in the same area vasculosa. In AV, (Text-fig. 5), both the diameter and marginal velocity of the posterior vein increased rapidly for the first 12 hours of observation. Then the marginal velocity suddenly decreased, and the diameter of the vessel began slowly to diminish, until at 25 hours, the diameter again began to increase following a temporary increase in marginal velocity. The course of events was similar for the left anterior vein, which became the final anterior vein, but here the initial increase in diameter was less sudden, and was followed by a less strongly marked period of decrease. In the right anterior vein, the marginal velocity was less than in the other veins when observations began, and the diameter of the vein slowly decreased, as the marginal velocity tended to drop still further.
In AV,, where observations extended only over 12 hours, the earlier of these events are seen extremely clearly. Both marginal velocity and diameter at first increased rapidly for both posterior and left anterior veins and the drop in marginal velocity clearly preceded the change in direction of the diametertime curve. The right anterior vein did not begin to decrease in diameter until the marginal velocity had been diminishing for at least 4 hours.
The exact relationship between marginal velocity and rate of change in diameter is clearly not a simple one. In the first phase of increasing diameter, the vein continues to enlarge only as long as the marginal velocity is itself increasing. When the marginal velocity no longer continues to increase, instead of increasing at a much slower rate, the vein decreases in diameter. Thus for the posterior vein of 4V,, the marginal velocity and diameter are the same respectively 16 and 40 hours after observations begin, yet at the former time the vein is decreasing in diameter, and increasing at the latter time.
(8) Comparison of arteries and veins
It remains now to consider the correlation between marginal velocity and rate of change in diameter for both arteries and veins. The values for the percentage increase in diameter of veins over a whole period will largely depend upon how great a proportion of this period is occupied by the initial rapid increase in diameter; during this initial stage, the percentage rate of increase per hour may be as much as 40; and this value will diminish progressively as more of the subsequent stages fall within the period considered. On the other hand, the arteries change in diameter more gradually, and no constant phases in their development over this period can be distinguished. In Text-fig. 4 most of the points relate to a period of about 12 hours, although some are calculated for a 20-hour period. For AV;, points are given for the three veins for both periods.
Two main conclusions can be drawn from Text-fig. 4. First, that the numerical relationship between average marginal velocity and percentage change in diameter may be quite different for corresponding vessels in different blastoderms. If we compare the anterior veins of 4V, and AV, with those of AV, and AV,, we see that they all increase approximately equally in diameter over the whole period, but the average marginal velocity in the former group is about 1300, per sec., and in the latter about 2400, per sec. The same average marginal velocity resulted in rapid decrease in diameter of the right anterior veins in the latter group, and increase in diameter of the left anterior veins in the former group.
These biological variations, however, do not prevent a further conclusion being drawn, namely that in comparing veins and arteries generally, for a given percentage change in diameter, the average marginal velocity is higher for veins than for arteries. An average marginal velocity of between 400 and 1000 per sec. in the arteries results in no change in diameter; the corresponding value for veins is between 1600 and 2200» per sec. In AV, and AV,, observations were made in each blastoderm for both veins and arteries which increased in diameter at similar rates. In AV,, these rates were almost the same for both veins and arteries, and the average marginal velocity for the former was about double that of the latter; in 4V,, the rate of increase in diameter of the artery was higher than that of the vein and the average marginal velocity was lower.
The Growth of Endothelium
(1) Increase in vessel diameter
During the first phase of the development of a vein from a capillary, the rate of increase in diameter may be as much as 40 per cent of the original diameter per hour. This rate is far 00 greater than the rates at which embryonic organs 240r generally increase in size. The area of the endothelium —_ 4,9. of a vessel increases at the same rate as the diameter. It is first necessary to know whether this rate is also 200° the rate of increase in number of the endothelial cells, 180+ or whether the increase in area of the vessel wall is due 160 Diameter to any extent to increase in the size of the endothelial => fixation cells.
To investigate this point, the diameter of a living : 120 vessel was measured over a certain period, which ended F at fixation. The size of the cells in the vessel wall was 100 estimated subsequently from serial sections, and expressed as the average area of endothelium to each endothelial nucleus.
The first area vasculosa which was studied in this 40 way was AV,, in which the increase in diameter of the 2b posterior vein is shown in Text-fig. 7 and in Plate II. og
In the fixed state the diameter of the vein is 1604 and oo 51015 the value for the area of endothelium per nucleus is Hours 303 sq. ». For a capillary on the opposite of the Text-fig. 7. Diameter of embryo, 40 » in diameter and similar to that from which _ posterior vein of AV, the posterior vein originated, the value is 470 sq. » per _Plotted against time. nucleus. These figures indicate that the vein had not enlarged merely by dilation of the capillary endothelium, and the observations tabulated in Table II confirm this for a number of vessels in different blastoderms. The values for area of endothelium per nucleus are all remarkably close, and we may conclude that even for the most rapid rates of increase in diameter of a vessel, the increase in the number of endothelial nuclei approximately keeps pace with the increase in surface area of the vessel wall.
Table IT Average Rate of circum- Percentage increase ference Sq. per ofnuclei from Observed Vessel (fixed) nucleus. indivision mitosis rate Obs./Cale. 25 S, anterior region 135 405 3-6 _ _ _ 168 430 0-0 _ _ _ 185 340 4-0 _ _ _ 278, anterior vein 625 520 5-0 _— — — AV,. 368, anterior region 148 436 3-06 6-1 4:8 0-79 235 435 1:0 2-0 9-9 5-0 - 435 290* 6-6 13-0 39-6 3-05 AV,. 358, anterior vein 576 415 2-23 4-56 18-2 4-0 RVA, 710 508 2-3 4-6 4-3 0-94 RVA, 495 350. =. 2-04 4-1 35 0-85 AV,. 378, left posterior vein, 505 303* 3-84 7 44-4 5-8 6 hours after differentiation Capillary on opposite side 146 470 0-0 _ _ _
Rate of increase calculated as percentage increase per hour.
The two lowest values for sq. » per nucleus are those where percentage increase per hour is greatest.
The increase in diameter of the vessels therefore represents roughly the rate of increase in the number of endothelial nuclei. There are two possible ways in which new endothelial nuclei may arise, by division of existing endothelial nuclei, or by the entry of new cells into the endothelium from outside.
To decide which of these two alternatives is the correct one, we must calculate the rate of increase in area of the vessel wall which corresponds with the number of endothelial cells in division.
The relationship between the rate of increase in numbers of cells and the percentage of cells in division is given by the formula:
Percentage of cells seen in division = percentage rate of increase per hour ‘ x mitotic time in hours.
To apply this formula therefore, it is necessary to know the time occupied by the process of mitosis, and since cell division has not yet been followed within the living chick embryo, data obtained from chick tissue cultures must be used for this purpose. Strangeways (1922) states that “the time occupied for complete division of a cell, from the beginning of the prophase to the daughter nuclei being clearly seen, varied in thirteen cells, from 23 to 65 minutes, the average being 84 minutes”. These results agree closely with the earlier work of Lewis and Levi.
For the purpose of the present work, an average mitotic time of half an hour has been assumed. It will probably not be possible to make a closer approximation until observations have been made on living intact chick embryos, and the variability of the time taken for cell divisions within the embryo estimated. In Table II therefore, the values for the percentage of nuclei seen in division has been multiplied by two to give the estimates for the rate of increase by mitosis. These are then compared with the observed rate of increase in the living state before fixation, and the last column gives the quotient obtained by dividing the calculated rate by the observed rate. ~ It can be seen that for the smaller rates of increase that were observed, namely from 3-5 to 4-8 per cent per hour, the calculated rate agrees closely with the observed, but for the higher observed rates of increase (19-44 per cent), these are from 4 to 5:8 times as high as the calculated values. This implies that this number of times more nuclei are being added to the vessel wall from outside than are being formed within the endothelium by division.
There are two main possibilities of error in these calculations. The first is in the estimation of the mitotic time, and the second is in the tacit assumption that the rate of cell division within the endothelium is constant not only during the half hour occupied by one division, but also over the whole preceding period, when the rate of increase in the diameter of the vessel was observed for comparison with the rate calculated from mitosis. However, even if we allow . & generous margin for these two sources of error, it is hardly possible to escape the conclusion that nuclei enter the endothelium from outside when a vessel increases in diameter rapidly. The most likely source of these nuclei is the mesenchyme surrounding the vessel, and a histological study was made to find whether there was evidence of such a relationship between endothelium and mesenchyme.
In counting endothelial nuclei in sections, it is extremely difficult. to decide exactly which nuclei are endothelial, and which belong to the mesenchyme bordering the vessel. Plate HI, fig. 7 is a photomicrograph of a section through the posterior vein of AV,, to illustrate this point. In counting, the line of the endothelial membrane was used as the criterion; those nuclei which lie within this line, or are cut by it, are counted as endothelial, and those outside it as mesenchymal. It is clear that every intermediate condition exists between those which definitely belong to one group and those which belong to the other. This fact provides substantial indirect evidence that endothelial cells are supplied from the mesenchyme, but this view cannot be regarded as proved until the entry of mesenchyme cells into the endothelium has been observed in the living tissue.
In the previous paper of the series (Hughes, 1985), a hypothesis of arterial growth is given, based on cell division within the endothelium. The relationship of this hypothesis to the findings described in this section must now be discussed. In the first place, the greatest observed rate of enlargement of an artery which we have noted in this work is 5 per cent of the original diameter per hour, whereas the greatest observed rate for a vein is 40 per cent. In Table II, the rates of increase per hour by mitosis have been calculated for two arteries, and are 4-6 and 4-1 per cent per hour, the corresponding observed rates being 4-3 and 3-5 per cent respectively. Their rates of increase are thus fully. accounted for by mitosis. It may therefore be that vessel walls which increase comparatively slowly in area do so entirely by mitosis, possibly in the way suggested in the earlier paper, and that only the more rapid rates of increase depend on the incorporation of the mesenchyme.
(2) Increase in vessel length
The proximal part of the posterior vein does not increase very greatly in length. In the anterior region of the area vasculosa, there are vessels which are changing in diameter and increasing in length at the same time. To examine the effects on the growth of endothelium in vessels which were simultaneously changing in both length and diameter, the anterior region of 4 V3 was studied (Text-fig. 8 and Plate ITI).
In AV,, the final anterior vein was formed not from either of the two original anterior veins, but from a capillary in between them.
Text-fig. 8 shows graphically the changes in length, diameter and area of wall of the two original anterior veins and of the vessels which became the single anterior vein. In Table I the rates of mitosis in the endothelium of these three vessels are given, and also their corresponding rates of increase in area by cell division. The values obtained for these latter rates range from 2 to 18 per cent of the original diameter per hour; the observed rates on increase in length of the same vessels are 8-5, 9-7 and 12 per cent. It is therefore doubtful if the mitotic rates of these peripheral vessels are sufficiently high to keep pace with the actual growth of the area vasculosa, for the vessel with the highest calculated rate of increase by mitosis, namely 13 per cent, is the one which steadily increases in diameter, and in which the endothelium actually increases in area at the rate of 39-6 per cent per hour. Conversion of mesenchyme into endothelium therefore may well be part of the normal process of extension of the vascular area, apart from the influence of the circulation on the diameter of the vessels.
Thoma’s Hypothesis in Relation to these Results
The results described in the present paper show that there is a general correlation between marginal velocity and rate of change in diameter for vessels of the area vasculosa from the second to the fourth day of incubation. Since a relationship must exist between marginal velocity and rate of flow, there must also be a correlation between rate of change of diameter and rate of flow.
This correlation is not a simple mathematical relationship for the following reasons. The relationship between marginal velocity and rate of flow depends on the diameter; the relationship between marginal velocity and rate of change in diameter depends also on the diameter. A given marginal velocity does not produce the same rate of change in diameter at two different diameters. The rate of change in diameter depends also on the amount of change in diameter during the preceding few hours.
A Right anterior vein 4F WV Final anterior vein —f- Surface area in sq. mm.
Text-fig. 8. Growth of three vessels in the anterior part of A4V,. The data are derived from the photographs shown in Plate III. (ii) gives the diameter and length of the three vessels marked in Plate III, and (i) gives the surface area of these vessels, obtained from the data in (ii). 16 A. F. W. Hughes
Thoma’s explanation of his hypothesis was that the rate of flow of the blood determined the rate of supply of nutritive substances to the vessel wall from the blood stream, and that this rate of supply controlled the rate of growth of the wall. Arguments against this hypothesis were discussed in the preceding paper (Hughes, 1935). To them we can now add that growth in the area of vessel wall is only one way in which an embryonic vessel increases in diameter. Cells from the surrounding mesenchyme are incorporated into a vessel wall which increases rapidly in area, a process to which Thoma’s nutritive hypothesis does not apply.
With these reservations, the present work confirms the hypothesis as applied to the early embryonic vessels of the chick area vasculosa.
Summary of Results
- A description is given of a method by means of which a chick embryo, growing in an opened egg, can be observed continuously with an indirect microscope over a period of several days.
- During the third and fourth days of incubation, the maximum rate of increase in diameter of the arteries of the area vasculosa is 5 per cent per hour. The maximum velocity of the superficial corpuscles in the arteries is about 1-2 mm. per sec. During the first 10 hours of the formation of the posterior vein, the rate of increase per hour may be as much as 40 per cent per hour, and the maximum velocity of the superficial corpuscles is about 4 mm. per sec.
- Increase in number of endothelial nuclei is found to keep pace with increase in diameter of the vessels, and no increase in cell size was found in the most rapidly enlarging veins.
- It is found that the mitotic rates in the endothelium of the arteries are sufficient to account for their rates of increase in diameter. In veins increasing rapidly in diameter, cell division may account for only one-fifth of the actual increase in number of endothelial cells. The remaining four-fifths of the cells appear to be derived from the mesenchyme bordering the vessel.
- The growth of endothelium in the outward extension of the area vasculosa probably takes place by the conversion of mesenchyme into endothelium, as well as by cell division within the vessel walls.
- The bearing of these results on Thoma’s hypothesis of vessel growth is discussed.
I wish to thank Dr H. B. Fell for her continued interest and encouragement throughout the progress of this work, and also Dr D. E. Lea for his generous assistance in many matters. The expenses of the investigation were defrayed by the Medical Research Council.
Gropzinsk1, Z. (1935). ‘‘ Die Entwicklung der Venen in der Keimscheibe des Hithnchens.” Bull. int. Acad. Cracovie, Ser. B, Sci. Nat. (2), p. 305.
Hucuss, A. F. W. (1935). ‘Studies on the area vasculosa of the embryo chick. I. The first differentiation of the vitelline artery.” J. Anat., Lond., vol. Lxx.
Poporr, D. (1894). Die Dottersack-Gefasse des Huhnes. Wiesbaden.
Romanorr, A. (1931). ‘Cultivation of the chick embryo in an opened egg.” Anat. Rec., vol. XLVII.
Srranceways, T. S. P. (1922). “Observations on the changes seen in living cells during growth and division.” Proc. roy. Soc. B, vol. xctv.
Tuoma, R. (1893). Untersuchungen iiber die Histogenese und Histomechanik des Gefassystems. Stuttgart.
EXPLANATION OF PLATES
Photographs of the left vitelline artery and itasurroundings of A V,. The diameters and marginal velocity of the three branches of the artery and of the posterior vein are plotted in Text-fig. 1. The vessels marked vv are part of the lateral vein system. x14. Time of photographs: fig. 1, 10 a.m.; fig. 2, after 44 hours; fig. 3, after 74 hours; fig. 4, after 9 hours; fig. 5, after 1] hours; fig. 6, after 13 hours; fig. 7, after 194 hours; fig. 8, after 24 hours.
Figs. 1-6. Photographs of the posterior vein of AV;, in the region immediately posterior to the vitelline artery. x41. Time of photographs: fig. 1, 3 p.m.; fig. 2, after 2} hours; fig. 3, after 3} hours; fig. 4, after 53 hours; fig. 5, after 7} hours; fig. 6, after 9} hours.
Fig. 7. Section through the posterior vein A V,, to illustrate the difficulty in distinguishing between endothelial and mesenchymal nuclei bordering the vessel. A dot is placed opposite the eleven nuclei which were counted as endothelial in this section. x 470.
Photographs of the anterior region of AV,, from which the data in Text-fig. 8 were obtained by measuring diameter and length of the left and right anterior veins, and of the vessels between them which became the final anterior vein. The length of the vessels was measured between the points marked. x14. Time of photographs: fig. 1, 3-25 p.m.; fig. 2, after 5 hours; fig. 3, after 8 hours; fig. 4, after 14 hours; fig. 5, after 19 hours; fig. 6, after 26 hours. :
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