The Prootic Somites of Heterodontus and of Amia.

The Prootic Somites of Heterodontus and of Amia. By G. R. dc Beer, B.A., B.Sc, F.L.S., Fellow of Merton College, Demonstrator in Zoology and Comparative Anatomy, University Museum, Oxford. With 16 Text-figures. 1. HETERODONTUS. THAT the prootic somites in the head of the vertebrate give rise to the muscles actuating the eyeballs is a fact so well known as to require no comment. As the result of the work of Marshall (7) it was understood that the eye-muscles, of which there are six, arise in three groups, each group from one prootic somite. The first group, consisting of the superior, internal, and inferior recti and the inferior oblique, arise from the first or premandibular somite, and are innervated by the oculomotor nerve. The second, to which the superior oblique alone belongs, is produced by the second or mandibular somite, its nerve being the pathetic. To the third, consisting of the external rectus, the third or hyoid somite gives rise, and it is innervated by the abducens. Neal (8), in a recent and careful work on S qua Ins, has, however, slightly modified this scheme. According to him the external rectus muscle has a double origin. Part of it is formed from the hyoid somite and part from the mandibular. The element contributed by the mandibular is in his opinion the posteriorly directed process of the mandibular somite named muscle E by Platt (10), its discoverer. While it is generally supposed to disappear, Dohrn (3) was of the opinion that muscle E persisted, and NeaJ NO. 209 C

IS 0. H. DE BEER asserts that it is actually incorporated in and forms part of the external muscle. The eye-muscles of Squalus have also been, studied by Lamb (6), according to whom muscle E vanishes at a stage when the embryos are 26 mm. long. The external rectus would therefore be independent of the mandibular somite in its development. I have examined preparations of the appropriate stages of Squalus, but failed to be convinced that muscle E persists. Nevertheless I regarded the matter as being in an unsatisfactory condition, and when through the generosity of Mr. P. D. P. Murray I came into possession of a series of embryos of Heterodontus philippi, I investigated the development of the eye-muscles in this interesting and comparatively little-known form. In my earliest stage (11 mm.) the prootic somites are still large and contain spacious cavities. The mandibular somite retains connexion with the general coelom through the mandibular arch. Posteriorly the mandibular somite runs obliquely ventrally,so that the hyoid somite overlaps it for a short distance from behind and above ; the two somites are just in contact. Immediately lateral to the point of overlap of the somites is the large ganglion of the trigeminal nerve. All these structures can be discerned in a transverse section (Text-fig. 1). A reconstruction as seen from the left side is given in Text-fig. 7, a. The premandibular somite lies ventral to the main body of the mandibular somite, which position it owes to the curvature of the head. Not much attention will be paid to its later history, since its further development is typical and there is no controversy as to the muscles to which it gives rise. At the next stage (14 mm.) the mandibular somite has lost connexion with the general coelom, and forms an elongated structure lying horizontally. Posteriorly it is still overlapped by the hyoid somite, the cavity of which has shrunk as the walls become thickened. The two somites are in contact but the limiting membranes separating them are plain (Textfigs. 2 and 7, b). Lateral to the somites at this point there is, as before, the

PROOTIC SOMITES 19 ganglion of the trigeminal. The profundus ganglion is joined to the trigeminal by a connexion which passes down median to the mandibular somite. This somite is therefore external and dorsal to the profundus, a condition which persists in the adult where the superior oblique to which the mandibular somite gives rise is dorsal to the ramus ophthalmicus profundus (Text-fig. 3). The hyoid somite at this stage is still TEXT-FIO. 1. Heterodontus philippi, 11mm. transverse section, showing the hyoid somite overlapping the mandibular. Explanation of lettering of Text-figs. 1-7. ab, abducens ; exr, external rectus ; /, facial; hy, hyoid somite; infr, inferior rectus ; inob, inferior oblique ; inr, internal rectus ; m, mandibular somite; oc, oculomotor; opn, optic nerve; p, profundus ganglion ; pa, pathetic ; pm, premandibular somite ; top, ramus ophthalmicus profundus ; ro>, combined superficial ophthalmic nerves; ros V, ramus ophthalmicus superh'cialis trigemini; ros VII, ramus ophthalmicus superfieialis facialis ; sudb, superior oblique; sur, superior rectus ; tm, thick mesenchyme ; tr, trigeminal. largo, and the greater part of it is still posterior to the trigeminal ganglion. The superior ophthalmic branches of the trigeminal and facial nerves are formed, but there are as yet no ventral roots. c 2

20 G. R. DE BEER TEXT-FIG. 2. lmm. m -^=r^r Hetorodontus, 14 mm. transverse section. _ f O,-v- ft-r- tr--u- - -ros V Heterodontus. Reconstruction of 14 mm. stage seen from above. The brain has been cut horizontally and the upper part removed.

PROOTIC SOMITES TEXT-FIG. 4. tr hb 21 me sob Heterodontus. Longitudinal vertical section, 21 mm. stage. TEXT-FIG. 5. Heterodontus. Transverse section, 22 mm. stage. The muscle E is very small.

22 G. R. DE BEER A vertical longitudinal section at a stage 21 mm. long (Text-fig. 4) shows the mandibular somite extending forwards close to the profundus ganglion. The cavity of the somite is lateral to the section ; the walls are beginning to take on the form of muscle. The anterior portion of the somite is made up of cells densely packed together and staining deeply. This TEXT-FIG. G. hb-- - ros VII Heterodontus, 25 mm. transverse section. Muscle E has disappeared. is the rudiment of the superior oblique muscle. The posterior portion is composed of cells which are looser, and constitutes muscle E. This can be seen to underlie the hyoid somite for a short distance. The cavity in the hyoid somite has become much reduced, and the rudiment of the external rectus muscle is forming. It is not easy to decide whether there is a mixture of the elements of the two somites at the point of overlap. The membrane surrounding the hyoid somite is still visible, and muscle E appears to show signs of degenerating. Neal contests that no proof of any such degeneration has been given,

PROOTIC SOMITES 23 and indeed it is not easy to give, but its cells are less tightlypacked than in the external rectus rudiment, and they are hot orientated in any particular direction. In transverse section a very similar stage (22 mm., Text-fig. 5) shows muscle E as a thin plate of cells lying beneath and slightly to the outside of the hyoid somite, and just touching it. The mandibular and hyoid somites are during this time moving forwards. The rudiment of the superior oblique muscle extends anteriorly over the ramus ophthalmicus profundus and over the eyeball (Text-fig. 7, c). The hyoid somite no longer lies behind the trigeminal ganglion, but the posterior ends of both are at about the same level. The abducens has arisen slightly behind the facial, very close to the middle line, and extends forwards to the hyoid somite. The oculomotor, passing ventral and median to the ramus ophthalmicus profundus and the superior oblique, innervates the premandibular somite. From now onwards comes the critical point in the elucidation of the problem of the fate of muscle E. Already small at 22 mm. r I cannot find it at later stages at all. The hyoid somite, which must now be called the external rectus muscle, appears to be homogeneous all through. There is no trace of the incorporation of foreign elements, which should be discernible if present. The anterior extremity of the.external rectus lies close to the eyeball and the layer of denser mesenchyme which surrounds it. As this is the region which muscle E occupied at previous stages, it is my opinion that it breaks down into mesenchymo here. This mesenchyme, cut tangentially in a transverse section of a 25 mm. stage, is shown in Text-fig. 6. Whether mesenchyme cells originally forming part of muscle E are, after disappearance of the latter as such, incorporated in the external rectus I cannot tell; but even if this were so it would not justify the statement that the external rectus arises from the somatic musculature of two segments. After breaking down into mesenchyme the elements of muscle E must lose their somitic value. The superior oblique continues to move forwards until it is

24 G. R. DE BEER TEXT-FIG. 7. inob Hctcrodontus. Reconstructions seen from the loft side showing the development of the eye-muscles in embryos of 11 (A), 14 (B), 21 (c), 37 (D), and 70 (E) mm.

PHOOTIC SOMITES 25 the most anterior of all the eye-muscles (Text-fig. 7, d, 37 mm. stage). It is innervated by the pathetic, which passes down beneath the superficial ophthalmic. The premandibular somite, which at a previous stage had developed a pair of dorsally and a pair of ventrally projecting buds, has now completely split up into its muscles. The superior and internal recti extend dorsally over the optic nerve and the ramus ophthalmicus profundus, the inferior oblique and inferior rectus extend forwards ventrally beneath the optic nerve. In Text-fig. 7, e, a reconstruction is given of a 70 mm. stage in which the conditions cannot be very different from what they are in the adult. The external rectus, which extends laterally in front of the trigeminal, has been followed by its nerve, the abducens. The abducens follows the wanderings of the hyoid somite, for which reason it passes forwards median to the facial and trigeminal nerves and emerges from the skull anterior to the maxillo-mandibular branches of the latter. Thus, although it is the ventral root of the third segment, it passes in front of the dorsal root of the second segment. The oculomotor has divided into two branches, of which one supplies the internal and superior recti, the other passes round behind the superior rectus, under the ramus ophthalmicus profundus, in front of the external rectus, and innervates the inferior rectus. Passing still further forward beneath the optic nerve it innervates the inferior oblique. The relations of the eye-muscles to the ophthalmic nerves are not constant in Selachians. In Heterodontus the internal rectus passes over the profundus and thus lies dorsal to it. In Squalus the profundus passes dorsal to the internal rectus (Norris and Hughes, 9) which is precisely the opposite. In ScyIlium the profundus while it persists is ventral to the internal rectus, and similarly in Amia (Allis, 1). In Mustelus and Galeus (Allis, S) the profundus passes through the internal rectus. With regard to the superior rectus, the profundus passes ventral to it in Heterodontus, Squalus, Polypterus,

26 O. R. DB BEER and A mi a ; it passes dorsal to it in Galeus, and through it in Scyllium. The internal reetus passes above the optic nerve in Heterodontus, below it in A mi a and Salmo. The only constant relations are those between the superior oblique and the profundus, the latter being always ventral to the former. That this should be so is understandable from the development, for the mandibular somite lies over the profundus from the earliest stages. The positions of the other muscles are not fixed until much later, and there is no reason why the rudiments of the superior or internal recti should not grow and extend to one side or the other of the profundus, or straight towards the nerve so that the latter comes to be enclosed by the muscle. Variations in relations between muscles can be accounted for in this way without violating the principle that ventral roots remain faithful to their segment's muscles and follow them about in changes in ontogeny. It is precisely this principle which would be violated if the external reetus were partly composed of muscle E. This would mean that some musculature belonging to the second segment had been abandoned by the pathetic nerve and captured by the abducens, for there is no question of the pathetic or any branch of it having anything to do with the innervation of the external reetus. Pacts must of course not be made subservient to principles, but in such a case as this the persistence of muscle E in the external reetus must be established by strong evidence. I have not been able to find such evidence in Heterodontus. The inclusion in the external reetus of isolated mesenchyme elements derived from the degeneration of muscle E is another matter. It is possible that this occurs, but even then, in the absence of evidence to the contrary, I believe that in Heterodontus at any rate the external reetus is not made up of two somites. 2. AMIA. In young specimens of Amia in which the trunk somites have not yet specialized to form muscles, the line of somites is not continuous right up to the front of the head, as it is in

PROOTIC SOMITES 27 Selachians for example. The earliest stage investigated was that in which the embryo lies completely on the surface of the yolk and occupies about 220 degrees of the circumference of the yolk. There are visible at this stage one prootic headcavity, a line of postotic somites starting just behind the rudiment of the vagus nerve, and the adhesive organ. The TEXT-FIG. 8. ado Longitudinal vertical section through an embryo of A m i a c a 1 v a ; early stage showing adhesive organ and the single head-cavity. Explanation of lettering of Text-figs. 8-10. <xh, abducens ; adm, adductor muscle; ado, adhesive organ; cs, connecting stalk of head-cavities ; dw, dorsal wall of gut; ep, epithelium ; epart, efferent pseudobranchial artery; exr, external rectus ; fb, fore-brain ; fg, fore-gut; g, gut; k, hypophysis ; lib, hind- brain ; he, head-cavity; ;', infnndibulum ; ig, isolated ganglion; infr, inferior rectus; inob, inferior oblique; inr, internal rectus ; mb, mid-brain ; n, notochord ; oc, oculomotor ; ol, occipitale laterale cartilage; opn, optic nerve; ov, optic vesicle ; pa, patheticus ; pm 1, first permanent myotome ; rsur, rudiment of the superior rectus ; tm 1, first transient myotome ; tr, trigeminal; sg 1, first spinal nerve ; suob, superior oblique ; sur, superior rectus ; v, vagus ; vr 1, first ventral root; vr 2, second ventral root ; y, yolk. space between the head-cavity and the postotic somites is occupied by mesenchyme not showing a trace of segmentation. The head-cavity and the adhesive organ are shown in Text-

28 G. R. DE BEER It is puzzling that there should be only one head-cavity,, and it is improbable that earlier stages, had they been available, would have revealed other somites in this region, for Eeighard and Phelps (11), who have studied the development of the adhesive organ from the earliest stages, state that in their TEXT-FIG. 9. -y A m i a. Reconstruction of a o-o mm. embryo. The head is represented as cut transversely, and the observer is looking forwards at the posterior face of the anterior part. The other organs are seen by transparency. investigations no trace could be found of any other prootic somites. The question arises as to which somite the head-cavity of Ami a represents. A pair of sacs lying behind and between the optic vesicles and lateral to the infundibulum, they fill up most of the space between these structures and the mid-brain at a stage 5-5 mm. in length. A diagrammatic reconstruction

PROOTIC SOMITES 29 of the front portion of the head as seen from behind is shown in Text-fig. 9, a transverse section through the head-cavity in Text-fig. 10, and a reconstruction from beneath in Text-fig.14, a. Immediately posterior to the infundibulum the two cavities, one on each side, communicate by a cord of cells which becomes hollowed out into a tube. TEXT-FIC. 10. ~adm A m i a. Transverse section, 5-5 mm. stage, showing the head-cavity and the oculomotor nerve. In other chordates, the only segment in which the right and left somites communicate is the premandibular or first. This communication is in a precisely similar position, viz. behind the infundibulum and in front of the end of the notochord. It looks then as if the head-cavity of Amia were the premandibular somite, and this conclusion is confirmed by the fact that in the stage referred to above the oculomotor nerve can be seen clearly, issuing from the brain and going to the headcavity (Text-fig. 10).

30 G. R. DE BEER It is clear then that the remaining prootic somites, i.e. mandibular and the hyoid, must not be looked for in front of the head-cavity. Two possibilities are open. Either they occurred behind the head-cavity and have broken down early into mesenchyme, or else the single head-cavity, as well as representing the premandibular somite, contains the mandibular and hyoid somites also, all fused together. In this connexion I may quote from Reighard and Phelps : ' In one specimen of this stage, although the cavity was continuous on the left side, it consisted on the right side of three isolated cavities contained in the common sheet of mesoblast.' I have also observed this, and in Text-figs. 11 and 12 are diagrams of some sections through the heads of two specimens, with reconstructions of the head-cavities represented as seen from beneath with the ventral walls removed. It is at once apparent that not only are the subdivisions incomplete, but they differ in the two specimens and on both sides of the same specimen. In one of them there is apparent subdivision into three, and in the other into two. In the majority of specimens there is no subdivision at all. Further, the connecting stalk from one side to the other is not associated with the anterior subdivision, but with the second. If the anterior subdivision alone represents the premandibular somite, the connecting stalk should be found there. The conclusion is, therefore, that the head-caa'ity represents only one somite, and that the rnandibular and hyoid somites have broken down into mesenchyme and are not recognizable as such. The apparent subdivisions of the head-cavity have no significance. The proof of this conclusion is to be found in the development of the eye-muscles. For if the head-cavity represents only the premandibular somite, only four of the six muscles can arise from it, viz. recti superior, inferior, internal, and the inferior oblique. The earliest differentiation to appear is that of the rudiment of the superior rectus (Text-fig. 13) at the posterior end of the head-cavity. At 6 mm. in length the superior rectus rudiment is distinct

T-FIG. 11. A m i a. a to h, longitudinal vortical sections through early stage, showing apparent subdivision of the head-cav i, Reconstruction of the latter seen from below with the ventral surface removed.

TEXT-FIG. 12. The same as Text-fig. 4 in another specimen. The subdivisions of the head-cavity are not constant. 1mm.

PBOOTIC SOMITES and the rest of the head-cavity extends forwards. Behind it and separate from it the external rectus has appeared, innervated by the abducens (Text-fig. 14, b). There is no evidence of the external rectus having been budded off from the head-cavity, and there is no visible somite from which it TEXT-FIG. 13. 3S Amia. Transverse section, 6 mm. stage, showing the rudiment of the superior reotus muscle arising from the head-cavity. arises. It must therefore be formed by condensation of the mesenchyme which exists in that region, and which represents the broken-down hyoid somite. At 7 mm. (Text-fig. 14, c) the rudiments of the previous stage have become more distinct, and the inferior oblique is separate. The remainder of the head-cavity, apart from the superior rectus, represents the rudiments of the internal and inferior recti. These two muscles are distinct at 11 mm. (Text-fig. 14, d), NO. 269 D

34 G. R. DE BEER and also the superior oblique has appeared, innervated by the pathetic. It also has no connexion with the head-cavity. Of the six eye-muscles, therefore, four (innervated by the oculomotor and arising from the premandibular segment) are formed from a definite somite ; the other two, representing the somites of the mandibular and hyoid segments, are formed from mesenchyme. In those forms in Teleosts where no TEXT-FIG. 14. A m i a. Reconstructions of the head-cavity and eye-muscles, a 5-0 mm.. 6 6 mm. from below ; c 7 mm., d 11 mm. from the left side. prootic somites can be distinguished, presumably all the eyemuscles must arise by the latter method. It is interesting that Amia should retain the primitive method only for one somite, the premandibular. It is of interest to compare the relations of the eye-muscles of Amia, a Teleostome, with those obtaining in Selachians such as Heterodontus. The relations of the

PROOTIC SOMITES 35 muscles of both groups to the optic nerve are constant except for the internal rectus. This in Heterodontus (and Squalus and presumably all Selachians) extends forwards dorsal to the optic nerve, whereas in Arnia (and Salmo and Teleosts) it lies beneath the optic nerve. There is also a variation in the distribution of the oculomotor nerve. In Heterodontus it divides into two branches, the dorsal of which innervates the internal and superior recti; the ventral branch goes to the inferior rectus and oblique. In Ami a I find, in agreement with Allis (1), that the oculomotor divides into two branches, the dorsal of which supplies only the superior rectus ; the internal rectus is not innervated by this branch but by the ventral one which passes in front of the external rectus, beneath the inferior rectus which it innervates, and then forwards to the inferior oblique supplying the internal rectus on its way. The eye-muscles of Amia and Heterodontus cannot have been independently developed, but must have been derived from those of a common ancestor. Whatever the relations in this ancestor may have been, they must have been such as to be capable of modification into either of these two types, if one was not derived from the other. The possibility of these modifications lies in the facts that the muscles grow and extend during their development, and so may come to bear different relations to the relatively fixed structures such as the optic nerve, and that the nerve is not connected with them from the beginning but grows freely towards them. The change need not be attended by any functional discontinuity, because the fibres supplying the internal rectus may change their position of exit from the oculomotor nerve and yet retain connexion with the internal rectus without having to pass through any other structures. Intermediate stages in the transition from Heterodontus to Amia, or vice versa, or from an intermediate condition to either, are shown in Text-fig. 15. The fact that a number of somites has broken down into mesenchyme prevents the possibility of counting the number D 2

36 G. R. DE BEER TEXT-FIO. 15. sur infr Diagrams of the relations to the oculomotor nerve of the superior, internal, and inferior recti muscles in A, Heterodontus; B and c, hypothetical stages in the passage from one to the other or from an intermediate condition to either showing that the transition can be effected without functional discontinuity; D.Amia.

PROOTIO SOMITES 87 of segments included in the head of A mi a. In the occipital region, with Schreiner (12), I find four somites anterior to the one innervated by the first ventral root behind the occipitale laterale cartilage, which marks the posterior limit of the skull. This is one more somite than Allis (1) describes. Of these four somites the first two, which have no ventral roots, degenerate. There are then two permanent myotomes in front of the occipitale laterale. These segments are z and «, TEXT-PIG. 16. A m i a. Reconstruction of the occipital region from the right side, 11 mm. according to Fiirbringer (4). Actually which segments these are cannot be told without the complete series of somites between the prootic and postotic regions. In discussing the prootic somites of Ami a it is perhaps necessary to mention the adhesive organ. This organ (Textfig. 8) arises as a pair of lateral pouches of the fore-gut. There is no doubt that it is a structure which develops muscle anterior to and separate from the premandibular somite, but this does not prove that it is of somitic value. It is regarded by Reighard and Phelps as homologous with the anterior head-cavity of Squalus, but the somitic value of that has not been demonstrated satisfactorily (see Goodrich, 5).

38 G. E. DE BEER SUMMARY. 1. The external recfcus of Heterodontus is not partly composed of the mandibular somite. 2. The only prootic somite recognizable as such in A mi a is the premandibular. 3. The muscles representing the mandibular and hyoid somites arise from mesenchyrne in A m i a. 4. The relations of the eye-muscles to the optic nerve and the distribution of the oculomotor differ in Amia and Heterodontus. LIST OF LITERATURE. 1. AUis, E. P. " The cranial muscles and nerves in Amia ", ' Journ. Morph.', 12, 1897. 2. " Lateral sensory canals of Muatelus ", ' Quart. Journ. Micr. Sci.', 45, 1901. 3. Dohrn, A. " Die Mandibularhohle ", ' Mit. Zool. Stat. Neapel', 17, 1904. 4. Fiirbringer, M. " Uber die spino-occipitalen Nerven ", ' Festschr. von Gegenbaur.' Leipzig, 1896. 5. Goodrich, E. S. " Proboscis pores in Vertebrates ", ' Quart. Journ. Micr. Sci.', 62, 1917. 6. Lamb, A. B. " The development of the eye-muscles in Acanthias ", ' Am. Journ. Anat.', 1, 1902. 7. Marshall, A. M. : ' On the head-cavities and associated nerves ", ' Quart. Journ. Mier. Sci.', 21, 1881. 8. Neal, H. V. " The histoiy of the e3^e-muscles ", ' Journ. Morph.', 30, 1918. 9. Norris, H. W., and Hughes, S. R. "Nerves of Squalus ", 'Journ. Comp. Neur.', 25, 1920. 10. Platt, J. B. " A contribution to the study of the vertebrate head ", ' Journ. Morph.', 5, 1891. 11. Reighard and Phelps. " The development of the adhesive organs of Amia ", 'Journ. Morph.', 19, 1908. 12. Schreiner, K. E. " Einige Ergebnisse iiber den Bau, etc., von Amia "', ' Zeit. wiss. Zool.', 72, 1902.