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Waddington CH. Principles of Embryology (1956) The MacMillan Co., New York

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Chapter XII Organ Development in Vertebrates

ir is clearly impossible, in a book of this size, to do more than sketch in very broad outline the extremely complex processes by which the details of the adult anatomy come into being. There are, moreover, already many excellent accounts of them; for more extended comparative treatments, Dalcq and Gerard’s revision of Brachet (1935), and particularly Nelsen’s recent work (1953) can be recommended.

In the present chapter, an attempt has been made to single out those aspects of organ development in which new advances have recently been made, or which are particularly important in connection with various general principles of development; and two organs, the limbs and the kidneys, which provide very interesting illustrations of a number of points, have been dealt with in rather fuller detail.

1. The general form of the embryo

At the end of gastrulation, the amphibian embryo—often known at this stage as a neurula—is still a solid, approximately spherical object. The neural system runs along its dorsal meridian, at first in the form of an open neural plate, which however rapidly closes up into a neural groove and finally a tube. It also stretches considerably in the anterior-posterior direction, continuing that process of elongation which we have seen to be characteristic of the dorsal region through gastrulation. At an early stage in this stretching, the head and tail curl round the opposite ends of the egg, which has now become somewhat ovoid, so that the embryo seems to be on the point of biting its tail; but as the elongation becomes still greater, particularly in the more posterior regions, the dorsal axis straightens out again and the creature begins to assume the form of a tadpole, with a long thin tail stretching out behind its body. During all this time, there has been a tubular gut, formed directly by the invaginating mesoderm and endoderm, and opening out to the exterior through the blastopore, which lies at its posterior end in the region where the definitive anus will eventually be. At first the tube has a floor and walls of endoderm and a roof of mesoderm, but the lateral edges of the endoderm soon grow in from each side to mect in the midline and from then on the gut-tube is lined entirely by endoderm; we shall see that this situation is not so easily brought about in the flat blastoderm of the birds. The wall of the amphibian gut is at first rather thin on the dorsal side, but very thick on the ventral, where the cells are still swollen by the large stores of yolk. From the exterior, it appears as though little is happening on the ventral side while the tail is growing out, but beneath the skin the first blood cells are forming, and the mesoderm is beginning to form a pulsating tube, the rudiment of the heart.

The chick embryo, when it reaches the neural plate stage, is not, to speak crudely, a solid lump like the amphibian, but is instead a flat circular plate, on which the embryo proper is linear in form, the remainder of the plate being no part of the final body but concerned only with the absorption and digestion of the yolk and with the respiratory exchanges between the embryonic blood and the outside air. One of the most striking features of the embryo proper is the very great difference in the stage of development reached by the anterior and posterior parts. In the Amphibia the whole neural plate folds up more or less simultaneously to form a neural tube, which stretches from the anterior right to the posterior end, where there is only a small region of ‘tail-bud’ at which new additions of neural tissue and mesoderm continue to be formed. In the chick on the other hand, at a time when the anterior neural plate has folded up into a completely closed tube in the head region, it is still only a shallow groove at the level of about the tenth somite, while posterior to this there is still a remnant of the primitive streak at which mesoderm is being formed and the primary organiser is active. Thus although differentiation progresses roughly from anterior to posterior in both forms, the differences are much more marked in the chick than in the frog or newt.

Fairly soon after the closure of the neural tube in the anterior region of the chick, one can see the beginning of a process by which the embryo proper eventually becomes separated from the remainder of the blastoderm. Along a crescent-shaped line in front of the head, the blastoderm is tucked downwards and backwards under the neural tube. The fold so formed is known as the head fold. As it gets deeper and is pushed further backwards, the head region is gradually left isolated on a projection above it. At a considerably later stage, when the embryo has some thirty or more pairs of somites, a similar fold (the tail fold) cuts under the posterior end of the embryo; and eventually these two folds each progress so far towards the centre of the body that the whole embryo is left attached to the non-embryonic blastoderm only by a narrow stalk (Fig. 12.1).

As the blastoderm is tucked downwards in the head fold, a pocket lined with endoderm appears on the under side of the head. This is the first rudiment of the gut to appear, and is known as the foregut, to distinguish it from the hindgut which appears later in connection with the tail fold.

Bellairs (1953, 1954) has recently studied its development in detail, with the aid of vital stained marks. The foregut opens posteriorly on to the space between the blastoderm and the yolk, and its posterior edge is a clearly defined landmark in early chick embryos. As the head fold is pushed further and further backwards under the embryo, so the opening of the foregut moves posteriorly; in fact it does so even faster than the head fold progresses, and it is probable that its movement is not as simply dependent on that of the head fold as appearances might at first suggest (Waddington 1952, p. 149). After the opening to the foregut has progressed some distance towards the posterior, the mesoderm in the edge of the fold develops into a hollow tube, which at first extends some distance along each side of the crescent-shaped opening. This tube is the rudiment of the heart. It is really a double structure, since the mesoderm which forms the tube on the left side originally lay well out at the left of the blastoderm, and a considerable distance away from that which forms the tube on the right side; it is only the backward progress of the edge of the foregut, which takes place by a sort of zip-fastener process along the midline, which has brought them together.

FIGURE 12.1

Diagrammatic longitudinal section through a chick embryo, to show the relation of the embryo to the yolk.

This obvious doubleness in the early rudiment of the heart is one of the points in which the chick embryo seems to differ most radically from the amphibian type. Even so, the two systems are not completely different. We can relate them by the imagining what would happen if the central part of the chick blastoderm were wrapped round a spherical core of yolky endoderm; if the two presumptive heart regions were at the edges of this part of the blastoderm, they would come into contact on the ventral side of the endoderm and no further folding would be needed to bring them together; we should in fact have arrived at the amphibian condition. The reverse of this can actually be achieved experimentally. Nicuwkoop (1946) has slit an amphibian neurula up the ventral midline, removed the endoderm and flattened out the mesoderm and ectoderm on a plate of stretched silk, so that it is forced into a sort of ‘blastodermic’ configuration. It then develops two heart rudiments, one on each side, which fail to fuse so as to form a single heart, since the embryo is not provided with the foregut-forming mechanism which brings this about in the chick.

In the stage immediately after its formation, the heart of the chick embryo continues to have an important influence on the general form of the body. In the first place, it becomes extremely large. This is necessary because it has to pump blood not only through the embryo itself, but for very much longer distances through the blood vessels which run into the non-embryonic blastoderm. It becomes so disproportionate to the body proper that it bulges out to one side. At the same time, the whole body rotates, so as to lie with its left side down against the yolk; and the head also arches round so that the anterior end of the brain is bent down towards the chest, with the heart protruding between them. It is the development of the heart and its associated structures which is the cause of most of these foldings and rotations, and if it is removed, the main part of the neural system remains straight, only the anterior tip of the brain bending downwards.

2. The development of the head In both the amphibian and the bird, the neural plate is wider in the anterior than in the posterior. This is very clear in the newt, in which the whole plate is marked out on the surface of the egg at the time the blastopore is closing; it is definitely pear-shaped. As the edges of this area fold together to form the neural tube, the future brain, developing from the anterior end, has from the beginning a wider lumen than the hinder parts of the nerve cord. At first the tube remains open at its anterior tip, the small hole connecting the interior of the tube with the outside being known as the anterior neuropore; it closes gradually during the later development of the brain.

The comparatively slight initial difference in the width of the brain and of the main nerve tube soon becomes greatly increased by the appearance of swellings in the former. At first there are three, which form the primary brain vesicles which give rise to the forebrain, midbrain and hindbrain. Fairly soon the first and third swellings each become differentiated into two, so that the brain comes to consist of five vesicles, whose names are shown in Fig. 12.2.

The division of the forebrain into two starts early, but progresses slowly. Even before it has properly started, the formation of the eyes begins. They are developed from the region which will eventually form part of the second forebrain vesicle (the diencephalon), and are originally merely exaggerated lateral swellings of the brain tube. These bulge out through the mesenchyme which lies between the brain and the ectoderm (Fig. 1.1, p- 7), and come up against the latter. At this stage, two things happen. The first is the formation of the ‘optic cup’; the swelling begins to be transformed into a roughly mushroom-shaped structure, with a rather narrow stalk leading up to the brain while at the other end the part which comes into contact with the ectoderm is folded inwards rather as one pushes in the foot of a sock before putting it on. The cavity thus formed is the eye-cup, and is approximately circular except for a groove, the choroid fissure, on the ventral side. The layer of tissue which lines the cavity of the cup becomes the light-sensitive retina, and the outer layer or tapetum develops into part of the pigmented and protective coverings of the eye.

FIGURE 12.2

Diagram of the various regions of the brain, the eye-cups, and the nasal, lens and ear placodes.

The second ingredient in the developing eye is the lens. This originates from an infolding of ectoderm at the point where the optic swelling touches it. Its inception normally depends on a process of induction. Some time before Spemann discovered that the appearance of the main axis of the embryo depends on induction by the primary organisation centre, he had found that the eye-cups of newt embryos, transplanted so as to come in contact with the ectoderm of the belly, caused the appearance there of an induced lens (Reviews: Mangold 1931, Spemann 1938, Needham 1942).

The experiment reveals a classical example of a secondary organiser, that is, one which is effective after the primary organiser activity is completed. Very many of these operate during the period in which the earliest organ rudiments are appearing (Fig. 12.3). In fact it is probable that in the vertebrates every rudiment owes its origin to a secondary organiser or to a complex of organising influences; they are found in all the vertebrate classes which have been studied. For instance, lens induction by the eyecup has been demonstrated by Waddington, van Deth and others in the chick (Review: Waddington 19522). And to give further examples, two other organs which require notice at this point also depend on secondary organiser complexes. The nervous component of the nasal organ arises as two ectodermal thickenings or placodes near the anterior end of the forebrain; and it can be shown by transplantation experiments that the forebrain induces them. Again, the ears originate near the hindbrain from similar lens-like ectodermal thickenings, around which, after they have been folded inwards, the mesodermal structures are later formed. Experiments have shown that in this case the inducing system is complex; the hindbrain plays an important part in calling forth the thickenings, but so does the local mesoderm, and a fully normal ear can only develop when both components induce together in a harmonious way.

FIGURE 12.3 Diagram of secondary organisers in the newt. (After Holtfreter.)

There are in reality whole sequences of ‘secondary’ organisers, acting one after the other; perhaps they should be classed as secondary, tertiary, quaternary organisers and so on, but it would be difficult to do this in any very sensible way. For example, after the eye-cup has induced the lens, the whole complex then induces the skin lying above it to become transparent and to differentiate into the cornea, and the mesoderm which clothes it to become the sclerotic coats of the eyeball. Again the original ectodermal placode of the ear induces the mesoderm to form the other parts of the ear structure.

Within its ectodermal covering, the head of course contains not only the brain but also an infilling of mesoderm and some endoderm. The latter forms the pharynx, or anterior part of the gut, and will be discussed below in connection with the latter. It remains here to say something about the mesoderm. This tissue has a double origin. The greater part of it is formed by invagination through the blastopore or primitive streak, and this is the part to which we have so far paid most attention; but some mesoderm is also formed from cells which follow quite a different path. When the two neural folds finally come together and fuse to form the neural tube, some of the cells at the two fusing edges break loose and move down between the tube and the overlying ectoderm. These have been given a variety of names by various authors; sometimes they are alluded to as ‘mesectoderm’, a word which is also used for the epiblast of a blastoderm before the mesoderm has invaginated and thus become separated from the ectoderm; a somewhat better name is ‘ecto-mesoderm’, which is not so ambiguous, but probably it is simplest and best to speak of this second contribution to the mesoderm as the ‘neural crest material’, a phrase which clearly describes what is meant (Fig. 12.7, p. 266).

In most of the trunk, the neural crest material is scanty compared with the whole bulk of the mesoderm; it eventually forms the pigmented cells of the skin and contributes to the spinal ganglia. In the head it plays a much more important part, and forms large masses of tissue, which develop not only into some of the cranial nerves but also give rise to many parts of the cartilaginous skeleton (Hérstadius and Selman 1942, de Beer 1947).

3. The gut: anterior portion

In the Amphibia, it looks at first sight as though the formation of the gut is extremely simple, since a complete closed tube is formed during the process of gastrulation (but see p. 261). At first the endoderm forms only a trough, which is covered dorsally by the mesodermal layer of which the notochord is a part, but fairly soon the edges of the trough grow round to meet under the notochord, so as to form a closed tube made wholly of endoderm. The anterior part of this tube reaches forward under some of the head structures which have just been described. At its tip, the tube is at first closed, and in this region there is no mesoderm lying between it and the ectoderm. In the mesoderm-free area, the gut first fuses with the ectoderm, and then both layers break down, so that an opening appears leading from the exterior into the lumen of the gut; this is the rudiment of the mouth.

Just posterior to this, the gut becomes swollen into a large cavity, which is known as the pharynx. The wall of this becomes thrown into a series of deep folds, which run from top to bottom on each side. These folds, five in number, eventually reach through the mesoderm to the ectoderm, and fuse with it. Again, the combined ectoderm and endoderm breaks down and forms an opening. In this case, the openings correspond to the gill openings of fish; in the higher groups of vertebrates they make only a transitory appearance during early embryonic life, before becoming transformed into something else; in some cases they never open completely at any stage. Their fate in the various groups is too complex to be followed in detail here. (For instance, the most anterior forms part of the Eustachian tube and the tympanic cavity of the middle ear.) Between cach of the gill slits (which are also known as pharyngeal or visceral clefts) is a gill ‘arch’. In these there is a core of mesoderm between the ectoderm and endoderm, and in the centre of this core, an important blood vessel, one of the so-called aortic arches, will eventually run.

Essentially the same structures are formed in the chick, but by somewhat different processes, since the anterior part of the gut, as we have seen (p. 252) appears not during gastrulation but in connection with the head fold. The formation of the mouth and pharynx, however, involves the same processes of the local fusion of ectoderm and endoderm, followed by their breaking down to give place to an opening.

In the chick, another derivative of the pharynx makes its appearance at a fairly early stage. A pocket of endoderm pushes out from the floor towards the posterior end of the swollen pharyngeal region, and rapidly elongates and extends backwards; it soon branches into two. This is the rudiment of the trachea leading to the two lungs. In the Amphibia it arises in the same region and in a similar way, but at a considerably later stage.

4. The trunk: ectodermal organs

As might be expected, it is in the main body of the embryo that the three fundamental layers, of ectoderm, mesoderm and endoderm, are most typically developed and most clearly defined.

The ectoderm produces only three main organs or organ-systems. The mid-dorsal part forms the central nervous system, which in the trunk is in the shape of a tube with thick walls, a thin floor and roof, and a cavity which is high and narrow in transverse section. From this tube the ventral motor roots grow out in a series corresponding to the somites. The dorsal sensory roots, with the accompanying ganglia, although they are eventually so closely associated with the main central nervous system, have a different origin in that they arise from the neural crest material. This, the second of the three ectodermal systems, also comprises the sympathetic nervous system and contributes to the mesodermal sheaths of the spinal cord; moreover the greater part of the pigmented cells of the body, whether they lie in the skin or in the linings of the gut and other internal organs, arise from the same source (Rawles 1948, 1953) (the most important pigmented tissues with another origin are those of the iris and outer layers of the eyeball). Finally the third system formed from the ectoderm is the outer layer of the skin, the epidermis. The skin as a whole is a composite structure, a major part of its thickness being contributed by the mesodermal layer known as the dermis, which originates from the upper layers of the somites.

The skins of different classes of vertebrates are by no means simple structures, but contain several sorts of sweat glands, oil glands and so on. Two of these structures are of particular importance, and are worth mentioning very briefly, namely feathers and hairs. Feathers are formed from a so-called feather germ or follicle. This is a slight hillock on the skin, from the tip of which a canal extends down through the thickness of the follicle. At the base of the canal lies a conical papilla, and it is from this that the feather actually grows. The papilla is a double structure, with an epidermal (ectodermal) cap fitting over a dermal (mesodermal) core. Many experiments have been performed on this structure, since it is one of the few organs in a bird which will continue to go through its developmental performance late into adult life; when a feather is plucked it will be regrown from its original follicle. Wang (1943) was able to peel the epidermal cap away from the core, and transplant the latter into follicles from which their own papillae had been removed. He showed that the dermal core can induce such a follicle to produce a new epidermal cap, and eventually a feather. This is an example of a ‘secondary’ organiser acting at a very late stage. The induction is not effective on non-follicular ectoderm; and it is very remarkable that when a feather is induced in this way, the details of its structure (for instance, whether it is typical of the breast or the back) is determined not by the dermal core which induced it but by the place of origin of the follicular epidermis which responded to the inducing stimulus. (For a longer discussion of feather formation see Waddington 1952a).

Hairs also are formed from follicles which possess both ectodermal and mesodermal components. Much less is known about their structure and the inducing actions, if any, which go on within them; but since one special type of hair, namely wool, is one of the major raw materials of industry, a great deal is known about its development in other respects. Perhaps the aspect which is of most interest to general embryology is the study of the various shapes which may be taken by wool fibres, a subject which has been largely opened up through the pioneer investigations of Dry (1933-34). There are many different types of wool fleeces; each of them is characterised by a particular array of fibre-types, which occur with particular frequencies and can be distinguished by their length, thickness and the sequence of curves along them. It has long been known that the follicles occur in groups in the skin; within each group, the follicles develop in series, first the central primary, then the lateral primaries, finally one or two waves of secondaries. Fraser (1952) has recently presented evidence that each type of fibre is formed from some particular type of follicle; and he has elaborated a theory which shows how one could account for the shapes of the fibres, which differ in the series of curvatures along their length, by the interaction between a varying growth rate and a regular periodic chance in the direction in which the fibre is pushed out. If all the fibres grew at a constant rate, they would all have a regular wavy form. But Fraser suggests that actually they grow at a rate which is dependent on the efficiency of their follicle in competing with other follicles for a limited quantity of available substrate; and this efficiency is supposed to change according to the time of origin of the follicle. In the various breeds of sheep there are differences, not only in the relative numbers of the primary and secondary follicles, and in their density per unit area, but also in the curve which relates the efficiency to the time of origin of follicle (Fig. 12.4). In this way, the different shapes of the final structures can be accounted for in terms of physiological processes. The theory, although still in need of further testing, is a good example of the kind of mechanism one has to search for in the attempt to explain the facts of structure in terms of functional activities.

The formation of hairs, which usually grow very rapidly in comparison with the cells nearby, would seem to provide a good opportunity for studying the processes of protein synthesis in embryos. Some histochemical work on the growth of wool has been published by Hardy (1952), who finds that, as usual, RNA is present in high concentration in the cells in which growth is occurring most rapidly. The distribution of some other substances thought to be important for protein synthesis, such as sulphydryl-containing proteins, is also interesting, but the whole range of data cannot yet be fully interpreted (Fig. 12.5). Investigations by Lees and Picken on the influence of genetic factors on the rate of synthesis and the kind of protein produced in Drosophila hairs are referred to on p. 337.

FIGURE 12.4

The rate of growth of the wool fibre from a follicle depends on (1) the amount of available substrate, which is proportional to the area of skin, (2) the efficiency of the follicle divided by the sum of the efficiencies of all the other follicles with which it is competing. In the figure on the left, the time of initiation of the primary (Pc and Pl), and secondary (S) follicles is shown below. The dotted line shows the increase in skin area. The group of dashed lines show various relationships between the efficiency of a follicle and its time of initiation—the upper curve being that characteristic of a coarse fleece, the lower one of a fine fleece. The stepped line shows the increase in the total efficiencies of all the follicles. The figure on the right shows the growth curves of a series of fibres calculated on this basis (medium fleece). (After Fraser 1952.)

5. The trunk: endodermal structures

It is, of course, the endoderm which forms the innermost structures, namely the gut and its annexes. In fact it produces only the central core of these, since the adult organs include an investment of mesoderm which clothes the original endodermal rudiments.

We have seen that in the Amphibia the process of gastrulation itself produces a tubular primitive gut reaching from the blastopore, which occupies the position of the anus, to the head. It was for long believed that the cavity of this remained as the lumen of the adult gut, and that the various organs associated with the gut developed as outpocketings from its walls. The most striking of these pockets develops just posteriorly to the pharynx, and is usually considered to be the rudiment of the liver. According to recent studies by Balinsky (1947), however, this conventional interpretation is in error. He claims to have shown, by vital staining experiments, that the so-called liver pocket continues to elongate bac wards until it eventually reaches right back to the blastopore region. In fact, it constitutes the whole of the definitive gut, and outfoldings which will give rise to the liver, the pancreas and other organs appear in its walls, not in those of the primitive gut, which eventually disappears completely. These conclusions relate to Urodela. Nakamura and Tahara (1953) find that in Anura the situation is more like the conventional picture.

FIGURE 12.5

Semi-diagrammatic longitudinal sections of hair follicles in the mouse. On the left, stained to show sulphydryl groups, on the right to show cytoplasmic RNA. S.G., sebaceous gland; D.P., dermal papilla. (From Hardy 1952.)

In the chick, the main stretch of intestine (the midgut) is formed by the continued backward extension of the pocket of the foregut. A similar pocket, to be mentioned later, is formed under the tail, originating at a later stage than the foregut. It slowly extends forwards, and the two gradually come together near the middle of the body, leaving a narrow connection through which the gut opens out on to the underlying yolk; this is the umbilical cord. It is not till near the end of incubation that the chick gut becomes completely closed, and this is achieved, not by closing off the umbilical cord, but by the small remaining piece of yolk being drawn in inside the body, when the ectoderm and mesoderm close the hole through which it has entered.

6. The trunk: mesodermal structures

The mesoderm makes up the greater part of the bulk of the adult body, between the outer ectodermal layer of the skin (the epidermis) and the inner endodermal layer of the gut (the intestinal lining).

The first organs which become separated out from the rest of the mesoderm are those lying along the dorsal side. Immediately under the centre of the neural tube, a long rod is formed; this is the notochord, which acts as the first element in the skeleton, providing a longitudinal stiffening of the axis of the body. Its stiffness is not due in any large part to the production of hard or inflexible substance, but to the fact that the cells become swollen with fluid, so that the whole rod becomes turgid and stiff. The principle is one which Nature has made use of in other cases, where a temporary stiffness is required for physiological functioning. In this case the turgor is more permanent since the notochord induces the neighbouring mesoderm: to secrete around it a thin but inelastic sheath (Mookerjee 1953).

On each side, the layer of mesoderm is thicker at its median edges, where it abuts on to the notochord. Very soon, this thicker portion becomes more or less separated from the more lateral parts by the formation of a thinner longitudinal strip; this is known as the intermediate mesoderm, and from it the kidneys will develop. The thicker medial strips of mesoderm soon become cut up by a series of transverse grooves into separate blocks, known as the somites, which lie in pairs on each side of the notochord. The transverse grooving, and thus the appearance of the distinguishable somites, begins at the anterior end, and gradually progresses posteriorly, until there may be forty or more pairs of somites; the number differs in different species. One or more of the most anterior pairs may break up and disappear fairly shortly after their formation. The remainder of the somites persist and gradually give rise to the main segmental organs of the trunk, particularly the vertebrae and the associated segmental muscles; they also contribute to the dermal layer of the skin. Somewhat unexpectedly at first sight, the boundaries between the vertebrae do not correspond to the grooves between the somites; in each vertebra of the adult the posterior half has been produced by the anterior part of one somite while the anterior half is derived from the posterior part of the somite next forwards in the series. This arrangement ensures that the muscles arising within each somite are from the Pes joined to two contiguous vertebrae.

In Amphioxus, and in the primitive vertebrates such as cartilaginous fishes, the somites are hollow (Fig. 12.7). The cavity within them (the myocoe)) is part of the general body-cavity, and is continuous with the larger and better-developed space (the splanchnocoel) which forms within the lateral mesoderm. Both cavities together are known as the coclome. In higher vertebrates the myocoels are small and not always easy to detect. It is within the-lateral parts of the mesoderm that the main bodycavity of the adult develops; the mesoderm lying above the space becomes closely applied to the ectoderm, and forms the dermal layer of the skin, while that below lies against the endoderm and produces the muscular layer of the gut. The connection between the upper and lower layers of mesoderm persists in certain places, and provides the mesenteries by which the gut and its derivatives are attached to the main part of the body.

7. The tail and hind part of the body

At the stage when, in the amphibian, the neural plate becomes clearly delimited and the neural folds appear, the greater part of the plate is destined to form the brain and the nervous system of the anterior region of the body. The material for the whole posterior part of the trunk, and for the tail, is concentrated in a small region near the remains of the blastopore. In the chick, the material for the brain and anterior end arrives in place, and begins to differentiate, still more in advance of that destined to build the posterior end; and by the time only five or six pairs of somites have appeared, the primitive streak has already become quite short, although the greater part of the trunk is still to be produced. These facts have suggested to some authors, of whom the most authoritative in recent years was Holmdahl (1939), that the gastrulation process as we normally conceive it is responsible only for the formation of the anterior part of the animal and that the posterior part is produced by some radically different process which goes on within the small remnant of blastopore or primitive streak. This region, from which the posterior part forms, is known as the tail-bud. The authors who argue that the processes going on within it are quite different from those of gastrulation nevertheless do not agree on the position of the dividing line between the anterior part and the posterior part for which it is responsible.

The existence of this theory has led to an intense study of the development of the region in the neighbourhood of the blastopore of the neuralplate stage amphibian. Bijtel (1931) first showed that invagination is still proceeding at the blastopore even after the appearance of the neural folds; and further that some of the material between the posterior ends of the folds (i.e. material of the neural plate itself) will actually form mesoderm (Fig. 9.10, p. 166). More recent authors, particularly Pasteels (1939), Nakamura (1942) and Chuang (1947) fully confirm this, and demonstrate conclusively that the processes going on in the late blastopore are essentially invagination processes broadly similar to those of gastrulation proper. The main differences are two. The first is relatively trivial. Throughout the earlier phases of gastrulation, the dorsal midline above the blastopore is occupied by presumptive notochord. In the neurula stage, however, the last piece of presumptive notochord invaginates before all the somite mesoderm has moved into the interior; and in its final stages, therefore, the dorsal lip of the blastopore consists of presumptive somite material. The second difference is perhaps more important. Combined with the normal inrolling movement of gastrulation, there is in the late blastopore a stretching by which the tail is thrust out as an elongated structure. This produces a rather complicated system of movements, but it does not alter the essential fact that each region of tissue moves in a precisely defined way and reaches a definite final position. There is no reason to believe, as we are urged to do by Holmdahl, that the tail-bud is a mass of indifferent tissue from the general undifferentiated bulk of which the posterior neural tube, somites and chorda appear.

In the chick, the precise movements occurring in the late primitive streak are not so fully known, but there seems no reason to doubt that here again the posterior part of the body is produced by gastrulation processes essentially similar to those which give rise to the anterior end (cf. Pasteels 1939, Waddington 1952a).

Associated with the tail-bud is the hind end of the gut. This opens to the exterior through the anus, or cloaca, which is formed near the site of the blastopore, but not directly out of it. In forms such as Amphibia, which during gastrulation possess an open blastopore leading in to the cavity of the gut, this opening is closed by the fusion of its lips as invagination terminates. From the hind end of the gut, two pockets are then pushed out. One extends from the ventral side of the gut, and this reaches the ectoderm (which may fold inwards to meet it); the endoderm and ectoderm first fuse, and then break down to give an opening which becomes the definitive cloaca and anus. The other endodermal pocket starts from the dorsal side of the gut and extends backwards into the tail, where it is known as the tailgut. The extent to which it is developed varies greatly in different species even of the same group of animals; thus in the frog it is inconspicuous, while in some toads it forms a rather long tube, which extends right round the posterior tip of the notochord, its lumen becoming confluent with the cavity of the neural tube (the junction being known as the neurenteric canal) (Fig. 12.6).

FIGURE 12.6

On the left, a diagrammatic longitudinal section through a newt neurula. Closely lined neural plate, showing the future regions into which it will develop; darkly dotted, notochord; lightly dotted, endoderm; cross-hatch ed, ventral mesoderm. On the right, section through the tail region at a later stage.

8. The kidneys

There are three main types of ‘kidney’ developed in vertebrate embryos. First, the pronephros forms in a more or less anterior position; later the mesonephros appears further posteriorly; and finally the metanephros differentiates still further back. All these organs are paired, one appearing on each side of the body in the so-called ‘intermediate’ mesoderm, 1.¢. that lying between the somites and the lateral plate (Fig. 12.7). The basic unit, from which the kidneys are constructed can be thought of as a tube, one end of which opens into the coelome, while the other is connected with a duct-which leads the secretion away towards the posterior; and somewhere between these two ends, the tube becomes closely apposed to a blood vessel. In functional kidneys, this simple unit becomes highly modified and complicated; we shall not discuss these details which belong rather to the province of comparative morphology than that of general embryology. But it is necessary to say something about the duct.

This is a tube which leads, in the early stages, from the pronephros to the posterior where it opens into the cloaca. It is at that time known as the pronephric duct. Later the mesonephric tubules become connected with it; and in the higher vertebrates the pronephros degenerates and disappears; the duct is then entitled to be called the mesonephric duct. (It is also referred to as the Wolffian duct.) Finally, a diverticulum is pushed out from it, starting from the region near the cloaca; this makes contact with the metanephros, and is known as the metanephric duct (Fig. 12.8). Closely associated with it is another duct, the Mullerian duct; this leads from the exterior to the gonads, which originate near the kidneys, though they may later shift into another region of the abdomen.

FIGURE 12.7

Diagrammatic section through the anterior trunk of a vertebrate, showing the relations of the mesonephric tubules.

The comparative study of kidney formation in the different classes 0. vertebrates, besides being of great interest from the anatomical point of view, illustrates one or two points of importance for general embryology.

In the first place, we may note that the pronephros, which is an actively functional excretory organ in amphibian tadpoles, is formed opposite a well-defined group of somites; numbers 2, 3 and 4 in Anura, and numbers 3 and 4 in urodeles (cf. Cambar 1949). In the chick, in which the pronephric tubules are rudimentary, and never function as excretory organs, they are developed transitorily opposite somites 5 to 16. This is a good example of an organ being moved along the length of the body during evolution, a phenomenon which is of fairly widespread occurrence.

The epigenetics of kidney development has been rather extensively studied; recent reviews are those of Fraser (1950) for the vertebrates in general, Cambar (1948) for Amphibia and Waddington (19522) for birds.

In the Amphibia, the capacity to develop into pronephros appears at a certain level of a gradient which runs from a high point in the chorda toa low in the lateral plate. This was demonstrated by Yamada (1940), who showed that if lateral mesoderm is taken from a position in the neurula some distance away from the embryonic axis and cultivated in isolation, it will develop only into tissues normally formed from such lateral regions, such as blood, while if chorda is added to the isolate, pronephric tubules appear (see Fig. 10.10, p. 191). It is to be presumed that the first step in the development of the more posterior intermediate mesoderm (which will form mesonephros) is taken in the same way; and it seems not unlikely that a similar process occurs in the bird embryo, though this has not been definitely proved (cf. Waddington 1952a).

There is no evidence that the amphibian pronephros requires any further stimulus, after its position on the medio-lateral gradient is fixed, before being able to complete its development. For the more posterior kidneys (mesonephros and metanephros), however, something further is necessary, namely an inductive influence which is normally exerted by the pronephric duct. As we have seen, this duct grows backwards from the region of the pronephros. If its backward extension is prevented (e.g. by a transverse cut which fails to heal completely) no sign of the duct appears in the posterior region, and only minor traces of the mesonephros develop. The dependence of the amphibian mesonephros on an inductive stimulus from the pronephric duct was first suggested by Miura (1930) and later work by many authors has fully confirmed it (O’Connor 1939, Cambar 1948). In birds, Boyden (1927), Griinwald (1937) and Waddington (1938) have found a similar situation (Fig. 12.8).

In birds the pronephros never functions as an excretory organ, and Waddington suggested that it had been retained in the ontogeny of the animal simply because it is an essential step in the formation of the pronephric duct which is itself necessary as the inducer of the mesonephiros. Cambar (1948) has criticised this suggestion on the grounds that in the Amphibia the pronephric duct arises from a mass of tissue which is distinct from, although it lies immediately in contact with, that which gives tise to the pronephros itself; moreover he states that the growth of the duct is independent of the continued presence of the pronephros, whence he concludes that that organ is quite unnecessary for the production of the mesonephros. However, it is not at all clear that even in the Amphibia the original production of the rudiment of the duct is quite independent of the presence of the pronephros, and in birds the two components are so intimately associated that it is difficult to imagine the duct being produced without at least some transitory appearance of the tubules of the nephros. There seems, therefore, no good reason to doubt that we have, in the appearance of the pronephros in birds, an example of the retention during evolution of an organ for the sake of its function as a component of the epigenetic system, rather than for any contribution it makes to the physiological functioning of the embryo as a metabolising organism. Although one may probably conclude that the pronephric duct is an important factor in the epigenetic system, it is by no means the only actor on the stage. The competence of the intermediate mesoderm plays an important part. Even in the absence of the duct, accumulations of what one may consider as “pre-mesonephric’ cells may put in a transitory appearance. Further, the inductive influence of the duct is only effective if it operates on intermediate mesoderm (which has probably been brought to a state of competence by its position in the medio-lateral gradient); and this mesoderm can, in the chick at least (Griinwald 1942, 1943), react successfully to abnormal inductors, such as transplanted pieces of neural tissue.

FIGURE 12.8

Figure 1 shows, on the left a diagram of the normal urogenital system, comprising the adrenal (A), gonad (G), mesonephros (M), Mullerian duct (MD), Wolffian (or mesonephric) duct (WD), ureter (U) and definitive kidney (metanephros) (K). The parts which develop independently of others are shown in solid black, those resulting from inductive actions in outline. On the right is the result of preventing the mesonephric duct from growing posteriorly beyond the arrow; the posterior mesonephros and other organs fail to appear. (From Gruenwald 1952.) Figure 2 is a section through a chick embryo in which the posterior growth of the pronephric duct was prevented on the right side. There is not only no sign of the duct (WD) but the mesonephros (Af) has also failed. (From Waddington 19524.)

Griinwald also showed that an inductive reaction is concerned in the production of the metanephros of the chick. Here the inductor is the diverticulum which pushes out from the region where the pronephric duct joins the cloaca. He discovered the remarkable fact that if a piece of the main (mesonephric) region of the duct is substituted for this diverticulum, and allowed to act on the presumptive metanephric tissue, it succeeds in inducing kidney, but mesonephros rather than metanephros. This is one of the comparatively few cases in which the character of an induced organ is determined by the nature of the inducer rather than by the competence of the reactant.

Grobstein (19534, b) is analysing the inductive reaction between the duct diverticulum (uretic bud) and the metanephric mesoderm in the mouse, which is probably rather similar. He finds that the mesoderm can be induced to form tubules by a variety of different types of tissue, all of which are epithelial in character and to that extent at least similar to the uretic bud which is the normal inducer. An important result is that the inducing agent given off by embryonic spinal cord, for example, can pass through a 20y thickness of an artificial porous membrane; this is one of the most direct proofs that induction may be carried out by diffusible chemical substances. Grobstein has used the same methods for investigating other examples of the development of glands which contain both epithelial and mesencliymal components, particularly the submandibular (salivary) gland. By careful exposure to trypsin solutions, he can separate the epithelial and mesenchymal tissues; the epithelium can then be cultured in combination with mesenchyme from its own type of gland or with that from some other organ. He finds that the epithelium differentiates typically only when combined with its own type of mesenchyme; other mesenchymal tissues may partially inhibit the spreading tendency which is usually seen in isolated epithelium, but do not induce the formation of normal tubules. If the epithelium and mesenchyme are separated by a fine-grain filter membrane, the inhibitory action of foreign mesenchyme passes through, and the specific effect of like mesenchyme also does so to some extent, though not completely; it induces the formation of tubules but not quite typical ones (Fig. 12.9).

The reproductive organs of vertebrates develop in rather close association with the kidneys and their ducts. We shall not deal with them here; recent summaries of the literature may be found in Nelsen (1953) and Nieuwkoop (1946). The germ-cells themselves originate at considerable distances from the glands in which they eventually lie, and reach them after performing a peculiar migration, as isolated mobile cells, through the intervening tissues of the embryo. The place of their origin seems to be surprisingly different in different groups; probably the lateral mesoderm in urodeles, the posterior dorsal endoderm in Anura, endoderm lying anterior to the head in birds, yolk-sac endoderm and mesoderm from the posterior end of the primitive streak in mice. An introduction to the literature can be found in Nieuwkoop (1949), Willier (1950) and Chiquoine (1954).

FIGURE 12.9

A shows the epithelium of the submandibular gland of the mouse growing in tissue culture combined with mesoderm from the same source: the epithelium is forming typical tubules. In B the epithelium is lying on top of a porous membrane, below which is mesoderm from another source (maxillary region): no morphogenesis. C is a similar culture but the mesoderm below the membrane is from the submandibular gland: tubule formation has been induced, although the morphogenesis is somewhat abnormal. (After Grobstein 1953.)

9. The limbs

The limbs first appear as slight external swellings which fairly rapidly elongate. In their early stages they consist of condensations of mesoderm covered by epidermis which does not differ from that of the rest of the body. The mesoderm cells at first show little sign of particular differentiation, but form a rather loosely aggregated mass of mesenchyme. As the limb elongates, the cells towards the centre of it become more tightly packed, forming a number of condensations within the mesenchyme (Fig. 20.5, p. 429). These group themselves into the pattern of the skeletal elements of the normal limb, and gradually differentiate, first into cartilage and then into bone. Meanwhile the remaining mesenchyme develops into the muscles.

The development of the limbs has been very extensively studied, and provides examples of a number of points which cannot be so well illustrated in any other field.

At about the time of the First World War, Ross Harrison (1918) began a long study on the polarity and asymmetry of the limbs of urodeles. The subject was also pursued by a number of his students, such as Detwiler and Swett. The most recent summary of the extensive literature of this group of workers is that of Swett (1937) and the main contribution since then is an extensive and important work by Takaya (1941).

It is clear that a fully developed limb must be either a right or a left limb and that these two have essentially different asymmetry, being, in fact, mirror images of one another. The genesis of this asymmetry can be studied by excising the presumptive limb region from an embryo and grafting it back in such a way as to change the relation between the polarity of the graft and that of the host body. Consider, for instance, the forelimb of a newt. In the tail-bud stage the region from which this limb will develop is represented by a circular area on the side of the body just below somites 3 and 4. Suppose that the limb area on the left side of a newt embryo was cut out, then pushed up to the dorsal midline and down the other side, and eventually grafted in place of the right limb area, which had been previously removed. Then it is clear that its anterior end would still point towards the anterior end of the whole body, and its exterior side would still lie towards the exterior, but its dorsal side would now be below and its original ventral side uppermost. Such an orientation is described by saying that we have reversed the dorso-ventral axis of the graft but left its antero-posterior and medio-lateral axes unchanged. We could, of course, reverse both the antero-posterior and dorso-ventral axes leaving the medio-lateral one unchanged, by making a circular cut around the limb-forming area on one side and then rotating the area through 180 degrees about an axis perpendicular to its surface before allowing it to heal in again. By a variety of such methods one can, in fact, reverse at will any particular axis or combination of axes. After such operations one allows the limb to develop and examines whether a disc whose antero-posterior axis was reversed actually develops back to front, in which case the axis may be said to have been already determined, or whether influences from the host’s body succeed in causing a reversal of polarity within the limb-disc (Fig. 12.10).

It was soon discovered that the various axes are determined at different times. The antero-posterior one always becomes fixed first. According to Detwiler it is already determined by the middle gastrula stage, but Takaya gives reasons for doubting this, and suggests that the determination does not actually occur until the neural plate stage. Even so, this is much earlier than the determination of the dorso-ventral axis, which does not occur till the fairly late tail-bud stage, by which time the limb-buds have already become slightly elevated from the side of the body. It is shortly after this that the medio-lateral axis of the mesodermal component of the limb-bud becomes also determined.

FIGURE 12.10

On the left, an axolotl embryo into which a limb-bud has been grafted with its dorso-ventral and anterior-posterior axes reversed. The transplant (Tr) has developed into a limb with reversed anterior-posterior axis but normal dorso-ventral axis. (After Harrison.) On the right, a newt embryo showing the direction of the anterior-posterior field in different regions of the flank. The circles mark the normal positions of the limb-buds. (From Takaya 1941.)

The determination is certainly brought about by the tissues in the immediate neighbourhood of the disc. This can be shown by rotating a fairly large area in the limb region and then grafting into this region a limbdisc, with one or more of its axes reversed. In such cases it is the orientation of the immediately surrounding area rather than that of the whole host embryo which is decisive over the future development of the limb.

The nature of the influence which fixes the polarity of the limb-disc is still imperfectly understood. It clearly falls into the general category of what have been referred to as ‘field characters’, but that terminology does not tell us much about what it actually is. Harrison (1936) thought that the polarity might depend on some fine-grain structure of the tissue of almost molecular dimensions and perhaps comparable to the oriented arrangements found in liquid crystals, but x-ray microscopy was not able to detect any such structure (Harrison, Anthony and Rudall 1940). Takaya, on the other hand, suggests that the fundamental factor is capacity for growth, which is highest in the antero-dorsal part of the early limb-disc. In his view it is the fixing of gradients in growth capacity which determines the polarity which the limb will exhibit.

If an early limb-disc is excised and grafted into a new position in such a way that its polarity is opposed to that of the immediately surrounding area, a frequent result is the development of a pair of duplicated limbs. These are nearly always mirror images of one another, one having the symmetry of a right limb and the other that of a left. The frequency with which this mirror-imaging occurs suggests that two limbs developing close to one another influence each other’s polarity. This is confirmed by the results of grafting two limb-buds into each other’s neighbourhood. It is found that even if they are grafted so that their polarities are concordant, nevertheless the two limbs which form often turn out to be mirror images, one of the two having had its original polarity reversed by its neighbour (Fig. 12.11).

It is a remarkable fact that the polarity of a normal embryo does not run in a constant direction throughout the whole flank of the animal. Opposite the anterior somites, from somite 1 to about somite 7, the polarity is such that it tends to cause the pre-axial side of the limb to develop on the side nearest the head of the embryo. The same is true of posterior regions where the hindlimb forms, from about somite 14 backwards, but in the midflank region, from the level of somite 7 to that of somite 14, the polarity of the flank is in the opposite direction (Takaya 1941) (see Fig. 12.10).

This reversal of polarity in the flank is clearly expressed in the experiments which have been made on the induction of limbs. This was first successfully accomplished by Balinsky (1925). He found that if an ear vesicle is transplanted into the flank of a young tail-bud embryo it induces the formation of a supernumerary limb. Later work (Balinsky 1933) showed that the same effect could be produced even more regularly by the implantation of a nasal placode. The organs used in these grafts can not, of course, be the normal inducer of the limb. It is not at all clear what organ or tissue fulfils this function in normal development. Attempts to induce limbs by the implantation into the flank of the pronephros, which lies near the site of the normal forelimb, have so far been unsuccessful.

The induction of limbs by such ‘foreign’ tissues as the auditory and nasal vesicles, raises in an acute form the problem of the specificity of the inducing stimulus. Do the inducers give off some specific substance which is essential for limb formation, or do they only activate potentialities already fully present in the flank? Needham (1942) argues that the former alternative is the more likely on general grounds and is the better guide to future experimental work. Balinsky (1937), on the other hand, suggests that the inductors owe their power to a rather ill-defined quality which he speaks of as a ‘high physiological activity’, and he believes that it is this which sets in motion the inherent capacity of the flank mesenchyme to develop into a limb. The question needs much further study; no serious attempt has yet been made to investigate the phenomenon at a biochemical level. It seems likely that it will turn out to be very similar to the situation with which we are confronted in the induction of the neural plate: that is to say, that the induction can be performed by relatively unspecific implants, but that these may act in a secondary way, their first effect being the release of specific substances within the reacting tissues. One single case has been described [Balinsky 1927] of the induction of a limb following the implantation of a fragment of celloidin into the flank. This may probably be compared with the induction of neural plate by treatments which produce ‘sub-lethal cytolysis’ (see p. 196).

FIGURE 12.11

Mirror imaging when limb-buds develop near one another. In a the ap axes are concordant; one of the limbs frequently has its polarity reversed. In b the ap axes point away from each other, and both limbs retain their polarity. In c the ap axes point towards one another, and a supernumerary limb, of ill-defined polarity, often appears. (From Takaya 1941.)

Whatever the position as regards the specificity of the inducing stimulus, there is no doubt that the competence of the reacting material plays a large part in the production of the limb. One evidence of this is the asymmetry of the induced limb in the various regions of the flank. Back to the level of somite 7 the pre-axial side develops anteriorly, but between somites 7 and 14 the asymmetry is reversed (Takaya 1941). Again Balinsky (1933) showed that the frequency with which limbs are induced falls off fairly steadily from the region of the forelimb- to that of the hindlimb-bud (Fig. 12.12). This gradient is probably not a straightforward reflection of the intrinsic capacities of the flank mesenchyme, but is partially a result of the general influence of the body as a whole. Thus Takaya has shown that if material from the lowest point in the gradient (just in front of the hindlimb-bud) is transplanted to a more anterior position, and then submitted to the influence of an implanted inductor, the frequency of limb induction is much higher than it would have been if the material had been left in its original location.

FIGURE 12.12

Limb induction in the newt. The graph above shows the frequency with which limbs (thick line) or pelvic girdles (thin line) were induced by the implantation of nasal placodes in the corresponding region of the flank indicated on the drawing below. In the drawing the position of the normal limbs is shaded, that of the pronephros dotted. (From Balinsky 1933.)

Another manifestation of the field of competence can be seen in the frequency with which the induction produces forelimbs or hindlimbs. Forelimb-like structures can be induced in the anterior region back to the posterior margin of somite 8, while hindlimbs can be induced in the posterior region, which extends forward to the anterior margin of segment 8. Thus there is a small region of overlap between the forelimband hindlimb-producing regions. Shoulder girdles are usually not induced, but the pelvis can be evoked very regularly in the posterior end of the field near somites 13 and 14. These three lines of evidence—the asymmetry of the induced limbs, the frequency of the induction and the character of the limbs induced—clearly demonstrate the existence of a field of competence in the flank, but do not show how far this is a property of the localised areas of tissue, or how far it is dependent on factors which act in a general way over the whole region.

In the period immediately after the limb-bud is formed, the various individual parts of it are still incompletely determined. A single bud may give rise to a duplicated pair of limbs, or, in other cases, two anterior half-buds may fuse to give a single limb. We shall not carry any further the discussion of the gradually increasing determination of the amphibian limb-bud (see Review of Mangold 1929), but instead turn to a consideration of the limb-buds in the chick, which illustrate certain other points of general significance.

In the chick, technical difficulties have made it impossible to investigate the symmetry relations of the early limb-discs as has been done in the Amphibia, nor is anything known about limb induction in birds. It is in connection with the later stages of development of the limbs that investigations on bird embryo have been most informative. The first point we shall notice is one which emerges from the investigations of Saunders (1948). He showed that the bulk of the material which forms the limb-bud at the earliest stage at which it can be recognised will eventually form the proximal parts of the limb. More distal parts are added later as the limb-bud elongates. During this process a specially important part is played bya thickened cap of ectoderm which forms the actual apex of the elongating bud. If this apical cap of ectoderm is removed, the laying down of further distal regions of the limb ceases, and the limb remains as a stump from which the distal parts are missing (Fig. 12.13). This does not occur in the Amphibia, where the proximal parts can form a complete limb; but in that group, of course, proximal stumps can regenerate their missing distal parts till quite a late stage, while such regeneration does not occur in the chick.

Young limb-buds of the chick can be excised from their normal site and planted into the coelom just lateral to the somites, where they will continue their development in a remarkably normal manner. In such situations the limb-buds are often very incompletely innervated. In the nerveless limbs the muscles atrophy nearly completely, and the growth rate is somewhat less than normal, but although no movement at the joints occurs, the structure of the skeletal elements is exceedingly normal, even to the formation of the joint surfaces (Hamburger 1939) (Fig. 12.14). It is well known that if, owing to injury, a functional limb has to be moved in an abnormal manner, the joint surfaces may become modified in such a way as to facilitate this. It is clear, then, that function can play a part in moulding the structure of the skeleton, but the development of these isolated and functionless limbs shows that this structure can also develop with a considerable degree of perfection, solely under the influence of factors inherent within the developing tissues themselves. The interaction of intrinsic and extrinsic factors in the architecture of the skeleton has been discussed particularly by Murray (1936). The development of limbs in abnormal sites in the body has provided the opportunity for a large number of investigations on the way in which the peripheral nerves make contact with the various parts of the limb, and also on the influence of an excess or deficiency of peripheral organs on the central nervous system. There is no space here to do more than mention this subject as one which provides evidence of epigenetic interactions between parts of the body continuing into the later stages of development. Recent reviews of the field will be found in such works as Piatt (1948), Weiss (1941, 1950a) and Detwiler (1936).

FIGURE 12.13

On the left, two stages (in section) of the limb-bud of the chick, showing the presumptive areas. C, coracoid; S, scapula; oblique shading, upper arm: dashes, forearm; horizontal shading, wrist and hand; A, apical ectoderm. On the right, the results of excising anterior (A above) or posterior (B below) regions of the apical ectoderm; the parts developing are shaded. (After Saunders 1948.)

FIGURE 12.14

The development of a limb-bud of the chick, transplanted to the coclome and badly innervated (b), compared with that of a normal limb (a). (From Hamburger and Waugh 1940.)

Waddington1956 12

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