[ 174] THE ACTIVITY OF THE TRIGEMINAL PLACODE IN THE SHEEP EMBRYO. Giglio-Tos (1902) and Wen (1928). The investigations of Campenhout (1935, 1936,

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1 [ 174] THE ACTIVITY OF THE TRIGEMINAL PLACODE IN THE SHEEP EMBRYO BY E. H. BATTEN Department of Physiology, University of Bristol INTRODUCTION It is widely accepted in the literature that the trigeminal ganglion differentiates principally from the neural crest in all the vertebrate classes. In fish and amphibian embryos the neural crest rudiment of the ganglion is usually augmented by a moderate contribution of cells which detach in a mass from one or several placodal thickenings of the surface ectoderm. There is abundant evidence that the neuroblasts of the trigeminal ganglion may differentiate from both these sources. Knouff (1927) has summarized the details of the pattern of development of the trigeminal nerve, particularly in the lower forms, and Ariens Kappers (1941) has reviewed the extensive literature on the problem of placodal activity in the vertebrates. The question of the participation of placodal cells in the establishment of the trigeminal ganglion among mammals has attracted little attention and many of the available descriptions are incomplete owing to the difficulty of collecting sufficient material and the impracticability of experimental verification. As early as 1885 Froriep noted the intimate fusion of thickened epibranchial ectoderm with the ganglia of nerves VII, IX and X in bovine embryos, but failed to find any similar sites in contact with the ganglion of nerve V. Chiarugi (1897) described the intervention of ectodermal cells in the formation of the trigeminal ganglion in the guineapig, while in the same species Da Costa (1931) finally reached a negative conclusion after re-examining his earlier results (1923, 1925). The reality of the process of placodal budding in the squirrel has been accepted by Volker (1922), but denied by Weigner (1901), who considered that the ganglion developed exclusively from the neural crest. Further denials of placodal participation have been expressed in the rat by Adelmann (1925), in the cat, roe-deer and man by Holmdahl (1928) and in man by Weigner (1901). In contrast to these views a definite ectodermal contribution to the developing trigeminal ganglion in the human embryo has been claimed by Atwell (1930), Bartelmez (1924), Bartelmez & Evans (1926), Campenhout (1948), Giglio-Tos (1902) and Wen (1928). The investigations of Campenhout (1935, 1936, 1937) in the pig, Codrs (1946) in the rabbit and Halley (1955) in the cat have all shown a migration of placodal cells from a series of small thickenings scattered within the broad 'area V' of the lateral head ectoderm. The only reference to the sheep was made by Froriep (1885), who found no evidence of placodal sites over the trigeminal ganglion in a small number of embryos. In the present study, which provides a more detailed account of the behaviour of the trigeminal placode in this species, an attempt has been made to visualize the pattern and extent of placodal activity in space and in time by recording the presence of migration points and by counting their cells in each embryo examined.

2 The activity of the trigeminal placode in the sheep embryo 175 MATERIALS AND METHODS This report is based on the examination of fifty-three sheep embryos ranging in size from 14-somites to 9*5 mm. crown-rump length (15-23 days). Many of the younger specimens are the property of Prof. J. D. Boyd, who kindly allowed me to examine his collection of sheep embryos of known age; all these embryos were cut serially at 6/z and stained with Heidenhain's haematoxylin alone or with Weigert's haematoxylin and orange G. The remaining material was collected in the Bristol abattoirs and was fixed in Bouin's fluid, dehydrated and stored temporarily in 2 % celloidin before infiltration with paraffin wax. Serial sections were then cut at 7 or 1Ou and stained with Ehrlich's haematoxylin and eosin Y. Profile reconstructions of the head region were prepared by the graphic method, using selected embryos which were cut serially at 1Ot. Alternate sections were drawn at a magnification of fifty diameters using an 'Edinger' projector and the detail was transferred to an enlarged outline traced on millimetre squared graph paper. This method will reconstruct fine detail such as the pattern of placodal contacts with an acceptable degree of accuracy. Text-figures 1-3 are tracings of the trigeminal nerve taken from reconstructions of the entire head region prepared by the graphic method. OBSERVATIONS Somite stage, days The trigeminal neural crest is already present in the 15-day embryo in which the neural tube is still open at the mid- and hind-brain levels. As in the rat (Adelmann, 1925) it is quite independent of the facial crest, from which it is separated by a distinct gap. At first the trigeminal crest is an extremely diffuse mass of cells lying beneath the low cuboidal epithelium of the surface ectoderm. Later, in the 23-somite embryo of 18 days, the crest cells become grouped in a more compact mass except along the distal edge where they come into intimate contact with the epidermis. By the 26-somite stage the trigeminal crest has grown into an oblong mass which presents a well-defined outline at only two of its borders: dorsally, where the cells lie in close apposition to the hind-brain and caudally, where they are clearly delimited from the mesenchyme (Text-fig. 1). Rostrally the margin of the crest rudiment is indefinite since the cells are more loosely arranged and appear to be growing forwards. Distally the crest is apparently continuous with the mesenchyme of the mandibular arch and here, too, a distinct margin is lacking. The greater part of the lateral surface of the trigeminal crest now lies close beneath and almost in contact with the epidermis; at isolated points this bears minute cellular thickenings which produce local irregularities of the deep surface of the epithelium. These placodal thickenings are distributed not only within the present limits of the crest, but also ahead of it in an area below which the rostral tip will shortly grow to form the anlagen of the ophthalmic lobe of the ganglion. The placodal sites are produced by local mitotic proliferation and appear in section either as low cushion-shaped thickenings or as short free ending spurs which project obliquely downwards towards the crest. In the 26-somite embryo which was reconstructed nine placodal sites occur on the left side (Text-fig. 1) and seven on the right. On either side four of

3 176 E. H. Batten these sites are connecting spurs which project down to fuse with the crest (Table 4). The total number of cells in all the placodal thickenings of this embryo is sixtyfive on the left and fifty-seven on the right side; further details of the shape and size of individual thickenings are summarized in Table 1. In the present series of embryos the 23-somite stage marks the onset of placodal activity, since with only one exception placodal thickenings are present regularly on both sides of six embryos of this stage (Table 4). Dorsal n.c. V Rostral / 7n.c CI4p. i A.2 A. 1 L 1 mm. Text-fig. 1. Profile reconstruction of the left trigeminal nerve in a 26-somite sheep embryo (no. 10). Magnification x 50. The placodal sites are numbered 1-9 and brief details of their shape and number of cells are given in Table 1. Table 1 Site 1 10-cell spur 2 7-cell spur 3 7-cell cushion 4 9-cell cushion in contact with the neural crest 5 6-cell cushion in contact with the neural crest 6 8-cell cushion 7 6-cell cushion in contact with the neural crest 8 6-cell cushion in contact with the neural crest 9 6-cell spur Total 65 placodal cells Somite stage, 5*5-7*0 mm. (crown-rump length) embryos, days In the 6 mm. (32-somite) embryo the neural crest cells have condensed into a more compact mass of young preneuroblasts so that the external shape of the ganglion is more precise than in the earlier stages (Text-fig. 2). A flat band of fine nerve fibres growing out from the dorsal edge of the caudal part of the ganglion marks the appearance of the dorsal sensory root. From the rounded ventral border a short outgrowth of fibres foreshadows the development of the maxillary and

4 The activity of the trigeminal placode in the sheep embryo 177 mandibular rami. The most striking advance, however, is the development of the tapering ophthalmic lobe in which the present compact arrangement of preneuroblasts contrasts with the diffuse character of this portion in the earlier embryos. Externally a low maxillary bud forms a new feature alongside the more prominent and lengthened mandibular process. During the 6 mm. stage the proliferation of placodal cells by the epidermis reaches the climax of activity (Table 4). A reconstruction of a 6 mm. embryo shows Dorsal Rostral Ophth. lobe I 1 mm. I... Text-fig. 2. Profile reconstruction of the left trigeminal nerve in a 32-somite sheep embryo (no. 13). Magnification x 50. The placodal sites are numbered 1-17 and brief details of their shape and number of cells are given in Table 2. Site Total Table 2 10-cell cushion 5-cell cushion 7-cell cushion 9-cell file 10-cell cushion 5-cell file connecting with the ganglion 6-cell cushion 15-cell cushion connecting with the ganglion 7-cell spur 8-cell cushion 5-cell file 8-cell spur 8-cell cushion connecting with the ganglion 5-cell file 7-cell spur connecting with the ganglion 5-cell cushion 8-cell spur 128 placodal cells

5 178 E. H. Batten seventeen placodal sites distributed over the ganglion and ahead of the ophthalmic lobe (Text-fig. 2). Some of these sites are low cushions, others are short spurs and the remainder are narrow files of cells projecting towards the outer face of the ganglion (Table 2). Four of these sites consist of streams of epidermal cells connected with the ganglion and may be interpreted as placodal cells fixed during the course of their detachment, migration and incorporation into the ganglion. The total number of cells counted in all the thickenings in this embryo reached 128 for the left and 123 for the right side; in another embryo of 6-8 mm. crown-rump length 114 and 116 cells were counted (Table 4). The proximity of the trigeminal neural crest to the thin ectoderm in most early embryos makes the placodal thickenings difficult to identify without a careful scrutiny. In a favourable section (PI. 1, fig. 1) from a 33-somite embryo three placodal sites are easily recognizable with the x 10 objective. Both the upper and the lower spurs consist of streams of twelve cells which have been fixed during the course of their migration toward the ganglion. The middle site is a flat cushionshaped plaque which is not yet connected to the ganglion. In the extreme rostral part of the placodal field the epidermal thickenings are more easily seen, since the ophthalmic tip of the ganglion has not yet grown forward to this level (PI. 1, figs. 2, 3). The upper site in PI. 1, fig. 2, is a typical cushion-shaped thickening which in this section contains eight nuclei out of a total of ten cells. The lower conical site shows three nuclei and a single mitotic figure out of a total of five cells. On the right side of the same embryo (PI. 1, fig. 3) the upper site contains five nuclei out of a total of ten cells for the whole spur. A pinched attenuated nucleus present at the base of this spur probably represents an early stage in the detachment of placodal cells. In the smaller lower site four nuclei are visible, but the whole thickening contains six cells mm. (crown-rump length) embryos, days By the 8 mm. (42-somite) stage the definitive pattern of the trigeminal nerve has been established (Text-fig. 3). The ganglion possesses a short, but broad, sensory root in which separate fascicles are not apparent until the 10 mm. stage. Although it is larger and more rounded the present shape of the Gasserian ganglion reflects the triangular form seen in the previous stage; the rostral angle becomes clearly identified as the ophthalmic lobe for it now sends forward a fibrous ophthalmic ramus in the direction of the eye. The maxillary ramus penetrates deep into the maxillary bud before terminating in a spray of fine nerve bundles. Slightly caudal to this the broad sensory root of the mandibular ramus emerges from the ganglion and after a short independent course receives a slender motor root; beyond this union the mandibular ramus passes down the axis of the arch as a mixed nerve. Placodal activity continues in the 8 mm. embryos although at a reduced pace, which is reflected in the smaller number of spurs present and in a lower total of placodal cells. Of the seven sites present on the left side of the reconstructed 8 mm. embryo two connect with the ganglion and the total number of cells is eightysix (Table 3). The right side of the same embryo bears nine sites with a total of eighty-six placodal cells (Table 4). In all embryos of this stage the field of placodal activity is characteristically

6 The activity of the trigeminal placode in the sheep embryo 179 displaced over the rostral part of the ganglion and even minute thickenings over the maxillo-mandibular part are infrequently found. As the epidermis becomes lifted away from the surface of the ganglion by an increasing thickness of mesenchyme the placodal sites become more conspicuous than in the earlier embryos. Dorsal Rostral 6? 1 mm. Text-fig. 3. Profile reconstruction of the left trigeminal nerve in an 8 mm. (crown-rump length) sheep embryo (no. 15). Magnification x 50. The placodal sites are numbered 1-7 and brief details of their shape and number of cells are given in Table 3. Table 3 Site 1 25-cell spur 2 25-cell spur connecting with the ganglion 3 6-cell cushion 4 8-cell file connecting with the ganglion 5 7-cell cushion 6 7-cell spur 7 8-cell Mfie (PI. 1, fig. 4) Total 86 placodal cells Some placodal sites appear as narrow files of cells (P1. 1, fig. 4) which are about to detach from the epidermis and migrate to the ganglion, while others are of the minute conical type. A further type of placodal thickening, which is frequently present over the ophthalmic lobe of the trigeminal ganglion during this stage, is I

7 180 E. H. Batten Table 4. The incidence ofplacodal thickenings in relation to the trigeminal ganglion in 50 sheep embryos. The number of placodal sites which make connection with the ganglion is given in brackets. The letter ' C' preceding the serial number distinguishes embryos of Prof. Boyd's collection. Total no. of Crown-rump No. of placodal sites placodal cells Embryo Age length no. (days) (mm.) Left Right Left Right C C IV 18 C C 4 18 C C 6 19 C *C 1 21 *C 2 21 C C C C C C 12 20i A lib C C C C C C C *0 6* * * * * *0 9* *2 9*2 9* Somite count 14 s. 23 s. 23 s. 26 s. 30 s. 31 s. 32 s. 32 s. 33 s. 33 s. 35 s. 42 s. I (1) (1) 3(-) (1) 8 (1) (-) 9 (2) (2) 7 (1) (1) 5 (1) (4) 7 (2) (Text-fig. 1) 4 (-) 5 (-) (-) (-) (-) 7 (-) (2) 9 (4) (1) 3(-) (1) 2 (1) (1) 4 (2) (-) 42 2 (1) 3(-) (3) 8 (3) (2) 5 (-) (2) 7 (3) (4) 11 (4) (Text-fig. 2) 10 (4) 8 (-) (4) 7 (3) (-) 3 (1) (5) 11 (4) (1) 5 (2) (1) 2 (1) (2) 9 (3) (Text-fig. 3) 7 (2) 5 (1) (3) 5 (3) (1) 5 (-) (4) 8 (2) (1) 1 (-) (1) 4 (2) (2) 3 (1) (1) 5 (2) (-) 6 (3) (2) 3 (1) (2) 4(-) (-) 2(-) (-) 2(-) (-) 1 (1) 1 (-) (-) (-) 11 I (-) 5 1 (1) 1 (1) 6 28 * C 1 and C 2 are twins, but have the developmental structure of a 19-day embryo.

8 The activity of the trigeminal placode in the sheep embryo 181 illustrated in PI. 1, fig. 5. It is similar to sites 1 and 2, Text-fig. 3, and consists of a massive stream of from twenty-five to thirty cells. Similar massive spurs of up to thirty-six cells are present in the ectoderm over the ophthalmic lobe in several embryos of between 8-0 and 9-2 mm. in length (Table 5). The majority of these spurs are connecting streams similar to that shown in PI. 1, fig. 5, and the remaining examples are about to begin migrating to the ganglion. It is also evident from Table 5 that this massive proliferation forms a significant fraction of the total number of placodal cells present in any embryo of this stage. Table 5. The incidence of large placodal thickenings related to the ophthalmic lobe ofthe trigeminal ganglion during the final phase of placodal activity. The cell count for each large placodal site is given in brackets. Crown-rump Total number of placodal cells Embryo Age length no. (days) (mm.) Left Right C (-) 19 (15) (25, 25) 86 (16, 25) (15) 34 (10) (18) 84 (20, 18, 23) (-) 41 (20) (-) 62 (15) C (10) 5 (-) (15) 37 (18) (12, 36) 26 (-) C (12, 17) 65 (26, 16) C (20) 75 (19, 20, 16) C (21) 17(-) (22, 25) 31 (-) (-) 34(-) (-) 10(-) (-) (-) 5(-) (-) (-) C (-) C (-) 28 (28) The 9 0 mm. stage marks the end of the phase of active placodal proliferation. Beyond this stage there is a marked decline in the number of placodal thickenings encountered and fewer of these have the form of connecting strands. The epidermis changes to a cubical epithelium which, for a short time, may bear occasional small nodules of rarely more than six cells; these are residual and senile placodal sites which usually disappear without trace by the 9-2 mm. stage. The exception given by the right side of the 9-5 mm. embryo no. 23 is due to the laggard detachment of a large placodal spur related to the ophthalmic lobe of the ganglion; this type of placodal thickening is always the last to disappear. DISCUSSION In the sheep the greater part of the Gasserian ganglion originates from the neural crest, but there is convincing evidence that this material is augmented by cells which proliferate from a wide area of the overlying ectoderm. Bartelmez (1924) introduced the term 'area V' to distinguish the same territory in the early human 12 Anat. 91

9 182 E. H. Batten embryo. Unlike the dorso-lateral and epibranchial placodes, which consist of distinct localized thickenings showing central proliferation, the trigeminal placode has a diffuse form; it appears as isolated and irregular thickenings from which short spurs of cells are proliferated towards the ganglion. During the early phase of placodal activity these thickenings are distributed in an area extending over the whole ganglionic rudiment and also rostral to it, but after the 8 mm. stage they are restricted to the more rostral district beneath which the ophthalmic ramus differentiates. An examination of these placodal sites in a graded series of embryos reveals three different types of thickening. The first type is a minute cellular nodule of up to twelve cells arranged as a flat cushion, or as a conical papilla, or as a short spur projecting obliquely into the mesenchyme. Such thickenings are produced by local mitotic activity and may be interpreted as sites of impending cellular detachment and migration. In the second type of thickening the epidermal cells traverse the mesenchyme as a continuous strand which fuses intimately with the outer face of the ganglion. These connecting strands represent streams of placodal cells which have been fixed at the moment of their migration and entry into the ganglion. The third type of placodal thickening predominates in the rostral area covering the ophthalmic lobe of the ganglion and is a massive stream of between twenty and thirty-six cells. Several important conclusions about the pattern of activity of the trigeminal placode may be drawn from the results presented in Table 4. First, there is the marked regularity with which the evidence of epidermal proliferation appears in almost every embryo between the 23-somite and the 9 0 mm. stages. If embryos larger than this may be disregarded, since placodal activity shows a sharp decline after this stage, then it is evident that unmistakable placodal thickenings have been found in eighty-one of eighty-six placodes examined, i.e. in 94 % of the total. It seems reasonable to infer from this high value that the proliferation may be a continuous activity. In this respect the trigeminal placode stands in marked contrast to the epibranchial placodes, certainly of the VII and IX nerves, where the pattern is one of bursts of proliferation alternating with periods of inactivity (Batten, 1954). Secondly the fact that in every case the number of connecting spurs is fewer than half the total number of placodal sites suggests that each thickening may rest in the epidermis for a short time before it begins to detach cells, and also that once this has started the actual migration is rapidly completed. Additional support for the last idea comes from the fact that only eight examples (9.3 %) were observed of short cellular spurs projecting from the ganglion in such a way as to be accepted as incompletely incorporated placodal contributions. If this interpretation of placodal activity as a process of continuous proliferation followed by intermittent detachment and rapid migration is valid, then one should not expect to find a large number of sites in which a connexion between the ectoderm and the ganglion afforded irrefutable evidence of a placodal contribution. Since, however, one or more connecting spurs are found in fifty-five of the eighty-six placodes examined, representing 63*9 % of the total, the reality of the process can hardly be held in doubt.

10 The activity of the trigeminal placode in the sheep embryo 183 The present observations suggest that the peak of activity, as indicated by numerous placodal sites and the high total count of their cells, is reached about the 6-7 mm. stages. During this period the placodal cells emigrate to all parts of the ganglionic rudiment. From the 8 mm. stage onwards the placode is still moderately active, but the placodal sites are now mainly of the massive type which are contributed to the ophthalmic lobe during the phase when the fibrous ophthalmic ramus is developing. The relative importance of these massive contributions to the ophthalmic lobe during the closing phase of placodal activity is expressed in Table 5. Although the trigeminal placode is already active in the 23-somite sheep embryo it is probable that the process may commence at a still earlier stage, as Bartelmez & Evans (1926) and Campenhout (1948) have found in the human embryo. The discrepancy between the conflicting opinions mentioned in the Introduction involves the evidence for the actual migration of placodal cells into the trigeminal ganglion. Both Holmdahl (1928) and Weigner (1901) have denied the existence of placodal contacts. Adelmann (1925) admits that the trigeminal ganglion in the rat has a broad area of contact with the epidermis, but is firmly convinced of the complete absence of placodal proliferation. In 200 embryos he found only a few cases of epidermal papillae which he explained as fortuitous adhesions of mesenchymal or ganglionic cells. It is perhaps significant that his observations end at the 34-somite stage, which, if the embryos of the rat and sheep may be broadly compared, is only half-way through the period of activity of this placode in the sheep and well before the phase when the ophthalmic lobe receives its major placodal contribution. This fact may explain Adelmann's statement (1925, p. 65) that 'a placodal origin for the ramus ophthalmicus profundus trigemini in the mammal... is untenable'. While the short spur form of most placodal sites in the sheep may suggest the detachment of single, or at the most a few, cells into the mesenchyme, the only positive evidence of migration is the presence of cellular strands connecting the ectoderm with the ganglion. These connexions are essentially transient features which may not be present in every embryo and, even when present, may easily be overlooked. It must be admitted that the evidence for migration is circumstantial, but nevertheless deserves serious consideration since it occurs repeatedly in a closely graded series of embryos. The most reasonable conclusion is that the short conical thickening of the ectoderm and the long connecting spur represent the extreme stages of a cellular proliferation and migration which is discontinuous both in space and in time. Except for a few minor points the derivation of the trigeminal placode is fundamentally similar to the descriptions given by Campenhout (1935, 1936, 1937, 1948) for the pig, chick and human, Coers (1946) for the rabbit and by Halley (1955) for the cat. As in the pig the ventral migration of the placodal field down the surface of the head in the sheep embryo precedes the parallel downgrowth of the neural crest V. Campenhout has attempted to explain this by postulating a chemical induction of placodal sites in the overlying ectoderm by an agent traceable to degenerating nuclear fragments found among the cells of developing ganglion. Similar pycnotic debris has been reported in the cat (Halley, 1955), but despite a careful scrutiny there is no evidence of any cellular degeneration within the trigeminal 12.2

11 184 E. H. Batten ganglion in the sheep during the period of placodal activity. An apparent exception occurs during the 19th day at a time when the ventral border of the neural crest V is becoming clearly delimited from the mesenchyme of the mandibular arch. Similar fleeting evidence of degeneration at this interface has also been recorded by Adelmann (1925) and Davis (1923) in rat and human embryos of the corresponding stage. The conclusion that both the maxillo-mandibular and the ophthalmic portions of the trigeminal ganglion receive a placodal accession in the sheep is in harmony with the findings of Campenhout (1937, 1948) in the pig and human, and Halley (1955) in the cat. There is, however, no indication of the formation of a separate profundus ganglion of placodal origin as Coers (1946) found in the rabbit. Yet the several massive placodal sites which contribute to the ophthalmic part of the trigeminal ganglion in the sheep resemble the single site of the rabbit in that they are the last to disappear. In every vertebrate class the profundus ganglion, and to a lesser extent the Gasserian ganglion, are associated during their development with placodal thickenings, and an excellent review of the comparative development of this nerve is given by Knouff (1927). The peculiar position of the trigeminal placode has misled several writers, notably Campenhout (1936, 1948), Da Costa (1923), and Giglio-Tos (1902), into classifying it as dorso-lateral or even as epibranchial. As early as 1920, however, Landacre pointed out that although the trigeminal nerve contained general somatic sensory fibres it invariably lacked special somatic sensory or lateral line fibres, so that the use of the term 'dorso-lateral placode' was inaccurate. In the same way de Beer (1924) emphasized the incongruity of accepting the profundus placode in the selachian as dorso-lateral when the definitive nerve was known to contain exclusively somatic sensory fibres. Landacre (1920, p. 304) further suggested that the placodal source of the profundus ganglion represented 'the most marked and constant displacement of the neural crest in the head region'. This conforms closely with the opinion of Herrick (1910) that the whole of the lateral surface of the embryonic head is potentially nervous. According to Landacre (1910, 1914, 1920) the general somatic sensory component of the cranial nerves is derived from the neural crest either directly or, in the single exception of the trigeminus, also by later placodal contribution from the overlying ectoderm. Knouff (1927) reached the same conclusion in the frog. Moreover, the experimental studies of Stone (1924, 1928) have clearly demonstrated the functional specificity of the trigeminal placode in Amblystoma. And in the chick similar extirpation procedures have yielded evidence of a dual origin of the cells of the trigeminal ganglion (Yntema, 1942). It is unfortunately not possible to determine the fate of the placodal cells in the mammal after they are incorporated into the trigeminal ganglion owing to the absence of cytological features by which they might be distinguished from crest cells. Since, however, there is in the sheep no evidence of cellular degeneration within the ganglion one may postulate that the placodal cells differentiate either into neuroblasts or into sheath cells. It is significant that the epibranchial placode of the vagus nerve in the sheep provides convincing evidence of the transformation of migrant placodal cells into neuroblasts (Batten, 1956). When the present observations are considered in the light of the embryological and experimental studies

12 The activity of the trigeminal placode in the sheep embryo 185 on the Anamniota it seems logical to accept the trigeminal placode of the mammal as a general cutaneous placode, as Ariens Kappers (1941) and Coers (1946) have done. SUMMARY 1. A trigeminal placode is active in sheep embryos from the 23-somite to the 9*2 mm. stages. It is present as an extensive, but ill-defined, ectodermal area overlying and reaching ahead of the developing ganglion. 2. Within this area local proliferation of the epidermis produces small thickenings of up to twelve cells and, more especially in the rostral region, larger thickenings of from twenty to thirty-six cells. 3. The presence of placodal sites in the form of streams of cells connected with the ganglion is accepted as evidence of a placodal contribution to the latter. Placodal activity is visualized as an intermittent process of cellular detachment followed by rapid migration and incorporation into the ganglion. 4. The maxillo-mandibular portion of the trigeminal ganglion arises principally from neural crest V, but receives a limited contribution from placodal thickenings. 5. The ophthalmic lobe of the ganglion similarly has a dual origin, but here the placodal contribution is significantly greater than in the maxillo-mandibular portion. 6. Although there is no certainty as to their exact fate, there is a strong suggestion that the placodal cells received by the trigeminal ganglion differentiate either into sheath cells or into neuroblasts. This investigation is an extension of part of a thesis submitted for the London Ph.D. degree, under the supervision of Prof. E. C. Amoroso, to whom I wish to express my gratitude for his advice and guidance. I am also grateful to Prof. J. D. Boyd for allowing me access to his valuable collection of sheep embryos and to Dr R. N. Smith for reading and criticising the manuscript. REFERENCES ADELMANN, H. B. (1925). The development of the neural folds and cranial ganglia of the rat. J. comp. Neurol. 39, ARIENS KAPPERS, J. (1941). Kopfplakoden bei Wirbeltieren. Ergebn. Anat. EntwGesch. 33, ATWELL, W. J. (1930). A human embryo with seventeen pairs of somites. Contr. Embryol. Carneg. Instn, 21, BARTELMEZ, G. W. (1924). Ectodermal areas of the head in young human embryos. Anat. Rec. 29, 109. BARTELMEZ, G. W. & EVANS, H. M. (1926). The development of the human embryo during the period of somite formation, including embryos with 2-16 pairs of somites. Contr. Embryol. Carneg. Instn, 17, BATrEN, E. H. (1954). The establishment of the cranial nerves in the sheep embryo. Thesis approved for award of degree of Ph.D., University of London. BATTEN, E. H. (1956). The activity of the placodes during the development of the mixed cranial nerves in the sheep embryo. J. Anat., Lond., 90, 585. DE BEER, G. R. (1924). Note on placodes and the ophthalmic nerves. Quart. J. micr. Sci. 68, CAMPENHOUT, E. van (1935). Sur l'origine des ganglions craniens chez le porch et le poulet. C.R. Soc. Biol., Paris, 118,

13 186 E. H. Batten CAMPENHOUT, E. VAN (1936). Contribution a 1'e6tude de l'origine des ganglions des nerfs craniens mixtes chez le pore. Arch. Biol., Paris, 47, CAMPENHOUT, E. VAN (1937). Le r6le de placodes epiblastiques au cours du developpement embryonnaire du pore et du poulet. Bull. Acad. Med. Beig. 6, CAMIPENHIOUT, E. VAN (1948). La contribution des placodes epiblastiques au developpement des ganglions des nerfs craniens chez l'embryon humain. Arch. Biol., Paris, 59, CHIARUGI, G. (1897). Contribuzioni allo studio dello sviluppo dei nervi encefalici nei mammiferi in confront con altri vertebrate. IV. Sviluppo dei nervi oculomotore e trigemello. Publ. Ist. Stud. Sup. Firenze Med. Chir. pp COERS, C. (1946). La formation des nerfs mixtes craniens chez le lapin. Arch. Biol., Paris, 57, DA COSTA, C. (1923). Os problemas morf6logicos da cabeca dos vertebrados e a formacao dos ganglios cranianos nos mamiferos. Arch. Anat. Antrop, Lisbon, 8, DA COSTA, C. (1925). Sur la formation des ganglions nerveux de la tete chez les mammiflres. C.R. Ass. Anat. 20, DA COSTA, C. (1931). Sur la constitution et le developpement des ebauches ganglionnaires craniennes chez les mammiferes. Arch. Biol., Paris, 42, DAVIS, C. L. (1923). Description of a human embryo having twenty pairs of somites. Contr. Embryol. Carneg. Instn, 15, FRORIEP, A. (1885). Cber Anlagen von Sinnesorganen am Facialis, Glossopharyngeus und Vagus, uber die genetische Stellung des Vagus zum Hypoglossus und uber die Herkunft der Zungenmuskulatur. Arch. Anat. Physiol., Lpz., pp GIGLIO-TOS, E. (1902). Sull'origine embrionale del nervo trigemino nell'uomo. Anat. Anz. 21, HALLEY, G. (1955). The placodal relations of the neural crest in the domestic cat. J. Anat., Lond., 89, HERRICK, C. J. (1910). The relations of the central and peripheral nervous system in phylogeny. Anat. Rec. 4, HOLMDAHL, D. E. (1928). Die Entstehung und weitere Entwicklung der Neuralleiste (Ganglienleiste) bei Vogeln und Saugetieren. Z. mikr.-anat. Forsch. 14, KNOUFF, R. A. (1927). The origin of the cranial ganglia of Rana. J. comp. Neurol. 20, LANDACRE, F. L. (1910). The origin of the sensory components of the cranial ganglia. Anat. Rec. 4, LANDACRE, F. L. (1914). Embryonic cerebral ganglia and the doctrine of nerve components. Folia neuro-uiol. 8, LANDACRE, F. L. (1920). The origin of the cerebral ganglia. Ohio J. Sci. 20, STONE, L. S. (1924). Experiments on the transplantation of placodes of the cranial ganglia in the amphibian embryo. I. Heterotopic transplantation of the ophthalmic placode upon the head of Amblystoma punctatum. J. comp. Neurol. 38, STONE, L. S. (1928). Experiments on the transplantation of placodes of the cranial ganglia in the amphibian embryo. II. Heterotopic transplantation of the ophthalmic placode upon the head and body of Amblystoma punctatum. J. comp. Neurol. 47, VOLKER, 0. (1922). Normentafel zur Entwicklungsgeschichte des Ziesels (Spermophilus citellus). Normentafeln zur Entwicklungsgeschichte der Wirbeltiere, 13. Jena, Gustav Fischer. WEIGNER, K. (1901). Bemerkungen zur Entwicklung des Ganglion acustico-faciale und des Ganglion semilunare. Anat. Anz. 19, WEN, I. C. (1928). The anatomy of human embryos with pairs of somites. J. comp. Neurol. 45, YNTEMA, C. L. (1942). Experiments on the origin of the sensory cranial ganglia in the chick. Anat. Rec. 82, 455.

14 Journal of Anatomy, Vol. 91, Part 2 Plate 1 Ft* - at* 2 AO, ** a ^ *' am# fa. e t,6 I.5 13ATTEN-THE ACTIVITY OF TIHE TRIGEMINAL PLACODE IN TILE SHEEP EMBRYO (Facing p 187)

15 The activity of the trigeminal placode in the sheep embryo 187 LIST OF ABBREVIATIONS A. 1 pharyngeal arch 1 m.t. motor root of mandibular ramus A. 1 (max.) maxillary bud of arch 1 n.c. V trigeminal neural crest A. 1 (mand.) mandibular process n.c. VII facial neural crest A. 2 pharyngeal arch 2 ophth.r. ophthalmic ramus cl.pl. 1 closing plate of first pharyngeal ophth. lobe ophthalmic lobe of Gasserian pouch ganglion mand.r. mandibular ramus s.r. sensory root of trigeminal nerve max.r. maxillary ramus V Gass.g. Gasserian ganglion EXPLANATION OF PLATE PLATE 1 Fig. 1. Coronal section through the trigeminal ganglion in a 33-somite sheep embryo no. 1i A. The thin epidermis lies close to the ganglion and bears three placodal spurs (arrows). Fig. 2. Section through the left side of a 32-somite sheep embryo no. 12 showing two placodal sites (arrows) in the ectoderm rostral to the tip of the trigeminal ganglion. Fig. 3. Section through the right side of the same embryo as fig. 2 showing two similar placodal sites (arrows). Fig. 4. Section through the trigeminal ganglion in an 8 mm. sheep embryo no. 15. The young neuroblasts are separated from the epidermis by a zone of mesenchyme. A narrow file of eight placodal cells (arrow) is about to detach from the epidermis; this file is site 7 in Textfig. 3. Fig. 5. Section through the ophthalmic part of the trigeminal ganglion in the same 8 mm. sheep embryo showing a massive spur of about thirty cells (arrow) streaming into the ganglion. This spur is similar to sites 1 and 2 in Text-fig. 3.