The Early Origin of Vertebral Anomalies, As Illustrated by a 'Butterfly Vertebra'

J. Anat. (1986), 149, pp. 157-169 157 With 7 figures Printed in Great Britain The early origin of vertebral anomalies, as illustrated by a 'butterfly vertebra'

F. MULLER, R. O'RAHILLY AND D. R. BENSON Carnegie Laboratories ofEmbryology, California Primate Research Centre and Departments ofHuman Anatomy, Neurology, and Orthopaedic Surgery, University of California, Davis, California 95616, U.S.A. (Accepted 30 January 1986)

INTRODUCTION Histological reports of vertebral malformations in the human embryo and early fetus are extremely rare, e.g. sagittal clefts at 17-25 mm (Orts Llorca, Ruano Gil & Jimenez Collado, 1962) and at 21 mm (Feller & Stemnberg, 1930). The present example of a 'butterfly vertebra' in an otherwise normal, early human fetus provided an opportunity to devise a developmental timetable for the appearance of this and similar congenital vertebral anomalies.

MATERIALS AND METHODS The anomaly, which had not been noted externally, was discovered in sections of the vertebral column in a fetus (listed as S6 in the Gardner-Gray-O'Rahilly Collection) of 63 mm in greatest length (and hence approximately 10 postovulatory weeks in age). The foot length was 11 mm and the weight was 25 g. As part of a project on vertebral development, the vertebral column had been isolated (Fig. 1), sectioned sagittally at 25 ,um and the sections stained by a modified Mallory azan technique. Graphic reconstructions of both the affected and the adjacent thoracic vertebrae were prepared from projection drawings of every second or every third section. Reconstructions similar to those of thoracic vertebrae 8 and 9 of the anomalous 63 mm fetus were made from horizontal sections of a normal 69 mm fetus (No. 4291 in the Carnegie Collection). In addition, sagittal sections of the vertebral column of another normal 69 mm fetus (S 34) were photographed and studied. In the account that follows cervical, thoracic, and lumbar vertebrae will be abbreviated to CV, TV and LV, respectively.

RESULTS Normal controls Reconstructions of normal thoracic vertebrae (TV8 and 9) of the affected fetus (S6) show that the centra are equal in height both ventrally and dorsally (Fig. 2A, B). The remains of the notochord are slightly anterior to the middle of the centra and are expanded in the region of the future nucleus pulposus (Fig. 2A). Laterally, segmental arteries are seen to be symmetrical and present at each vertebral level (Fig. 2B). The reconstruction of a control fetus (No. 4291) reveals the spinal nerve roots and ganglia as well as the normal vertebral anatomy. The cartilaginous neural processes have fused dorsally, so that the neural (future vertebral) arch is complete and a short spinous process is already present (Fig. 2 C). 158 F. MULLER, R. O RAHILLY AND D. R. BENSON

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Fig. 1. Radiograph of isolated vertebral column of fetus (S6). In the key drawing, the square surrounds TV10-12. Only the right hand hemicentrum of TVI1I is visible. C, an ossific locus within the cartilaginous costal element of CV7, superomedial to the first thoracic rib. This is potentially but not necessarily a cervical rib (Meyer, 1978). Anomalous vertebrae On radio'graphy (Fig. 1), the ossification centres of the neural arches of TV 10-12 are found to be slightly wider than normal, and the centre for the centrum of TV I11 is small and deviated to the right hand side. In a ventral view (Fig. 3A), the centrum is seen to be cleft sagittally, without cartilaginous continuity between the right and left sides. The right portion, which is considerably larger than the left, is joined by a cartilaginous bridge to the two adjacent vertebrae (Figs. 3A, SC). Origin of vertebral anomalies 159

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Fig. 2(A-C). Graphic reconstructions of normal thoracic vertebrae. (A) Normal thoracic vertebrae 8 and 9 of fetus S6, shown in median section. Spinal ganglia are included in the intervertebral foramina. (B) The same vertebrae on left lateral view. X indicates sectioned rib. Branches of the intercostal arteries are included. (C) Thoracic vertebra 11 of a normal fetus of comparable age, reconstructed from transverse sections and seen from above. The cartilaginous neural arch is complete. The eleventh ribs are short. The spinal cord and ganglia, as well as sympathetic ganglia, are included. N, the future nucleus pulposus. The contiguous vertebrae (TV 10 and 12) appear to be adapting to the lack of vertebral material at the TV 11 level (Fig. 3 A). As seen in a dorsal view (Fig. 3B), the neural arches are closed, as would be expected at this phase of development. A medial view of the left portion of TV 11 shows a ventral deficiency and wedging (Figs. 4 C, 5 B). The adjacent vertebrae are larger, thereby compensating partially for the lack of vertebral mass. A medial view of the right portion shows compression in the middle, accompanied by inadequate expansion ventrally and dorsally. The adjacent vertebrae have expanded into the deficient area. Lateral views indicate that the left side of TV11 is almost normal in height dorsally but is defective ventrally (Fig. 4D), whereas the larger, right hand side appears more nearly normal, although it is slightly wedged ventrally (Figs. 4A, 5A). Projections in a transverse plane reveal clearly the distribution of vertebral material between the right and left portions of the centrum (Fig. 3 C, D). The cartilaginous bridges to TV 10 (Figs 3 C, 5 C) and TV 12 (Figs 3D, 5 C) extend for a considerable distance dorsoventrally. A slight partition in the median plane is visible in TV 12. Notochord The notochord (Fig. 3 A) is very slightly deviated to the right hand side from TV9 to LV 1 and passes through the right hand component of TV 11 (Fig. 5 C). No signs 160 F. METLLER, R. O'RAHILLY AND D. R. BENSON

(D)

1 mm Fig. 3(A-D). Graphic reconstructions of thoracic vertebrae 1-12 offetus S6. (A) Ventral view. The butterfly vertebra (TV 1 1) is seen to consist of two parts, the larger of which is connected by cartilaginous bars with the suprajacent and subjacent centra. The right twelfth rib is continuous with the transverse process of TV 12. The pattern of the arteries is abnormal on the left side. (B) Dorsal view. All three neural arches are complete. (C) Inferior aspect of TV 11 projected onto TV10. The black area represents a section through the bar from TV 12. N, notochord. (D) Superior aspect of TV 11 projected onto TV 12. The black area represents a section through the bar from TV10. of notochordal duplication are present. The forerunners of the nuclei pulposi are small between TV11 and the adjacent vertebrae, and that between TV 12 and LV1 is branched (Fig. 3A).

Intervertebral discs The discs (Fig. 5 D) between the left portion of TV 11 and the adjacent vertebrae appear to be transversely placed rings and the upper and lower rings seem to be fused by a mass of fibrous tissue towards the median plane. Intervertebral discs in relation to the right portion of TV 11 are present only at the periphery and are separated by the centrum. In brief, TV 11 is in contact with the adjacent vertebrae without the intervention of disc material in the central areas of both the right and left portions of the upper and lower discs. (This deficiency of disc material might favour bony fusion of TV 10-12 at a later period, resulting in a block vertebra.)

Intervertebral foramina The openings are larger on the right side (Figs. 4A, B, SD) than on the left (Fig. 4 C, D) and, on both sides of the malformation, they are larger than in the Origin of vertebral anomalies 161

1 mm V Fig. 4 (A-D). Graphic reconstructions of thoracic vertebrae 10-12 of fetus S6. (A) Right lateral view. X indicates a sectioned rib. (B) Right portion of sectioned column, medial aspect. The notochord is outlined. Two vascular canals on the dorsal aspect of TV 10 are noticeable. (C) Left portion of sectioned column, medial aspect. (D) Left lateral view. X indicates a sectioned rib. normal spine at the level of TV 8/9 (Figs. 2A, B, 6F). The spinal ganglia lie close to the inferior vertebral notches. Ribs The ribs of TV 1 1 and 12 are abnormal. Rib 11 on the left side is short, articulates with the centrum of TV 11 only and fails to articulate with the transverse process. Rib 11 on the right side is shorter and articulates with centra 10 and 11 (Fig. 4A). Joint cavities have not yet appeared. The twelfth ribs are short and are not visible radiographically. That on the right side is fused with the centrum of TV 12 (Fig. 3A). Arteries The segmental (intercostal) arteries to the vertebrae show a normal pattern on the right hand side (Figs. 3 A, 4A). On the left side, however, intercostal arteries 10 and 11 share a common stem, whereas the subcostal artery is very narrow and extends no 162 F. MULLER, R. O'RAHILLY AND D. R. BENSON

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m m Origin of vertebral anomalies 163 further than rib 12 (Fig. 3A). In spite of these differences the number of branches (2 dorsally and 4 ventrally) entering the malformed centrum is the same as that for centra 10 and 12. Ossification Areas of calcification are present in the centra of TV 10 and 12 and are beginning to be vascularised. Although blood vessels also enter the two portions of TV 11, the cartilage there is less mature (Fig. 5A-D). Indeed, calcification is visible radiographically in only the right portion of TV 11. The pedicles include small areas of calcification and cartilage of Streeter's (1949) Phase 4 (Fig. 5 D), as in the normal (Fig. 5 E). Ossification here has begun on the internal wall, especially at the inferior and superior edges. Summary ofresults The main features are: (1) The notochord is deviated slightly to the right side at the level of TV9-LV 1; (2) the precursors of the nuclei pulposi are irregular and, at the level of the cleft vertebra, smaller; (3) the intervertebral discs are reduced in size, are annular and, on the left side, the two rings are connected rostrocaudally, thereby intervening between the right and left portions of the affected centrum; (4) vertebral bars connect the affected centrum with adjacent centra; (5) an arterial variation or malformation is present on the side where the butterfly vertebra is smaller.

DISCUSSION Normal development The normal development of the vertebral column has been described recently by O'Rahilly & Benson (1985) and the timing of the various events has been elucidated by O'Rahilly & Meyer (1979). After a succession of complicated changes that probably do not involve resegmentation (Neugliederung), the column at Stage 23 (30 mm in greatest length, 8 postovulatory weeks) comprises a series of cartilaginous centra and neural processes. The processes form incomplete neural arches dorsally, so that a general appearance of total spina bifida occulta is characteristic (O'Rahilly, Muller & Meyer, 1980). The cartilaginous neural processes, as shown in Figure 2 C, begin to unite at approximately 12 postovulatory weeks (70-80 mm). Data on the sequence of this process are unfortunately lacking. Ossification has usually already begun in some of the neural processes and centra by approximately 9 postovulatory weeks, although slightly later times are also noted, as in the specimens described here.

Fig. 5 (A-F). Photomicrographs of sagittal sections of butterfly vertebra (A-D) of fetus S6 and of normal thoracic vertebra 10 (E and F) of comparable normal fetus (S 34). The ventral aspect is at the right. (A) Left part of TV 11. (B) A more medial section showing ventral recession of the left part. (C) Right part of TV 1 1 showing cartilaginous bridges and parts of the notochord. (D) Right intervertebral notch of TV 11 showing imminent ossification (arrow). Parts of two intervertebral discs are visible ventrally. (E) Smaller control intervertebral notch showing imminent ossification nearby (arrow). The later development (at 101 mm) ofthis region has been nicely illustrated by Walmsley (1956). (F) A more medial section showing vascularisation of, and calcification in, the centrum (arrow). The upper disc shows a well marked nucleus pulposus. 164 F. MULLER, R. O'RAHILLY AND D. R. BENSON

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Fig. 6(A-D). Development of some important vertebral anomalies. (A) The normal development of the vertebral column in the embryonic and early fetal periods. Somites (s), the size and number of which are partly influenced by segmental arteries (a), form sclerotomes (s-t) around the notochord (N). Dense areas represent the future intervertebral discs while less dense areas, the centra (C), become chondrified. By the end of the embryonic period proper, the centra are united by a special, temporary tissue (*). By 10 weeks the intervertebral discs contain col- lagenous fibres. (B) Formation of a butterfly vertebra. A smaller somite (s), possibly caused by an anomalous segmental artery, would result in a defective sclerotome (s-t). A unilateral deficiency of sclerotomic substance would allow the ectopic development of disc material, as in the present case (B5). A bilateral deficiency would result in Feller & Steinberg's (1930) case 4 (B6). (C) Another example of a butterfly vertebra. Deviation of the notochord, possibly combined with (or producing) defective somites, may have been responsible for the case of Orts Llorca, Ruano Gil & Jimenez Collado (1962), shown in C3 and followed hypothetically by formation of bars. (D) Development of severe anterior spina bifida, as in the split notochord syndrome. Duplication of the notochord (N) is followed by bipartition of sclerotomes (s-t) and centra (C).

Sagittally cleft vertebrae In the orthopaedic and radiological literature, a sagittally cleft vertebra is generally known as a butterfly vertebra ('Schmetterlingswirbel'), a rare condition probably first described in 1844 in the twelfth thoracic vertebra of a 55 year old adult by Rokitansky (1850). Although it usually seems to be confined to the vertebral body, it is sometimes combined with posterior spina bifida, resulting in a complete sagittal cleft. Origin of vertebral anomalies 165 Butterfly vertebrae show a tendency to bar formation (Fig. 3 A), so that the distinction between cleft vertebrae and block vertebrae becomes blurred. Cleft vertebrae remain as such when the parts are united by disc material, whereas those joined by cartilage may proceed to a normal appearance (Theiler, 1952). Side-to-side osseous bridges have also been described in butterfly vertebrae. Most instances of butterfly vertebrae occur in the lumbar region, the notochord is probably not duplicated, and the spinal cord seems not to be affected. A more severe variety of spina bifida anterior that involves several vertebrae may be a different entity. It is most frequently cervicothoracic (Dodds, 1941), although its occurrence in lower regions is not unknown (Saunders, 1943). The notochord is duplicated, the neurenteric canal may be implicated in cervicothoracic cases, and the spinal cord may be affected to the extent of diplomyelia or diastematomyelia. Neural connections with the alimentary canal may be present, in which case a secondary diaphragmatic hernia is usually found. The complex, which may involve the skin of the back, the spinal cord, posterior and anterior (combined) spina bifida, and the intestine, has been termed the 'split notochord syndrome' (Bentley & Smith, 1960). Coronally cleft vertebrae Coronally cleft vertebrae are believed to be a different entity and have been attributed to " faulty vascularization and agenesis of either the anterior or posterior center of ossification" (Ehrenhaft, 1943). Ventral and dorsal centres as seen on radiography, however, are found histologically to be connected by one or more osseous bridges (Tondury, 1958). A coronal cleft sometimes visible in male fetuses disappears during the first year after birth (Fagerberg, 1963), although coronally cleft vertebrae may be encountered as a normal variation up to 4 years (Reichmann & Lewin, 1969; Tanaka & Uhthoff, 1983). Abnormal vertebrae in embryos A bipartite cartilaginous fourth sacral vertebra in a human embryo of 17-5 mm (Stage 19) was reconstructed by Orts Llorca, Ruano Gil & Jimenez Collado (1962). The notochord pursued an angulated course through the defect (Fig. 6C3). The authors believed that notochordal alterations arising at a very early stage were the responsible factor. A cartilaginous fourth sacral hemivertebra with a seemingly normal median notochord was found in an 18 mm embryo by Frets (1909) and was probably caused by unilateral absence of a somite. Also in the caudal region, a wedge-shaped vertebra in a 21 mm embryo was described by Feller & Sternberg (1930, their Fig. 18). An instructive case (their No. 4) was illustrated by Feller & Sternberg (1930). A boy aged 5 weeks presented inter alia with a butterfly vertebra T9), which showed histologically two ossific centres joined by a disc-like septum (bandscheibenahn- liche Scheidewand) that contained a single, median notochord (Fig. 6B6). The authors pointed out that the cartilaginous centrum must have already been abnormal before ossification commenced. Experimental studies In the golden hamster, administration of vitamin A maternally at the eighth day causes cellular necrosis of rostral somites within twelve hours (Marin-Padilla & Ferm, 1965). Within another twelve hours, focal necrosis of the notochord occurs. Somitic necrosis "is considered to be the primary defect induced by vitamin A 166 F. MULLER, R. O RAHILLY AND D. R. BENSON

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V.B. B ------__ Fig. 7. Timetable to show the origin of some important vertebral anomalies. The ordinate represents embryonic length in mm. The abscissa shows age in postovulatory weeks. The rectangles indicate Carnegie Stages from 7-23. The following periods are labelled: primary neurulation, the appearance of somites, the formation of sclerotomes, and vertebral chondrification. Abbreviations: B, butterfly vertebra and hemivertebra. M-C, meningocele. M-M, myelomeningocele, which, in the lumbosacral region, may be formed at a later time (secondary neurulation: Stages 13-20). M-S, myeloschisis, regarded by many as a forerunner of myelomeningocele. N.C., neurenteric canal. Not. pr., notochordal process. S.N.S., split notochord syndrome. Spina bif. occ., spina bifida occulta. V.B., vertebral bars. causing developmental malformations such as cranioschisis and sacral rachischisis". Moreover, "in experimental sacral rachischisis a notochordal abnormality is found in all embryos studied on the 14th day of gestation". Sagittally cleft vertebrae have been produced in the rabbit by maternal oxygen deprivation (Degenhardt & Kladetzky, 1955; Degenhardt & Knoche, 1959) at a time (9th day) corresponding to Stages 11-12 in the human. Abnormalities of the notochord were noted and butterfly vertebrae formed later. The thoracic region was most frequently involved. Sagittal clefts are well known in mutant mice (Gruineberg, 1963), for example in lumbar and sacral vertebrae of Danforth's short-tail mice (Theiler, 1954). Premature degeneration of the notochord was believed to cause delay in the median conden- sation of sclerotomic cells, with consequently reduced dorsoventral diameter of the Origin of vertebral anomalies 167 centrum. Two ossification centres are then formed. The importance of notochordal deformation in the production of vertebral anomalies has also been stressed in studies of the chick embryo (Strudel, 1971). In the mouse, the most sensitive teratogenic period for vertebral malformations is the 10th day, which would correspond to stages 11-13 in the human embryo (Muller & O'Rahilly, 1984). It has been found that " the later the treatment, the more caudal was the site of the malformation" (Murakami & Kameyama, 1963). This is in keeping with the rostrocaudal differentiation of the somites. It seems clear that butterfly vertebrae do not all arise in the same manner. Although abnormalities or degeneration of the notochord are a marked feature in experimental and mutant mice, no sign of such a process is evident in the present case. Although vascularisation is doubtless important in the development of normal vertebrae, for example in the formation and shaping of the ossific nuclei (Tondury, 1958), the malformations under discussion are likely already to have appeared in the much earlier mesenchymal phase of development. After administration of retinoic acid to hamsters on Day 8, however, vascular damage was thought to play "a significant role in the induction of the vertebral defects by disrupting somitogenesis" (Wiley, 1983). The formation of somites is now believed to be an extremely complicated process (Bellairs, 1985).

Timing ofabnormalities Based on detailed information available on normal staged embryos and on the results of experimental studies, it is possible to construct a tentative scheme (Fig. 7) of the timing of the appearance of various anomalies. As usual, the 'teratogenetische Terminationspunkt' has to be accepted with caution. The split notochord syndrome and combined (anterior and posterior) spina bifida may arise as early as Stage 7, when the notochordal process appears. Myelomenin- gocele, which involves the neural tube, probably appears at Stages 10-11 in the cervical and thoracic regions. Lumbosacral myeloschisis (regarded by many as a forerunner of myelomeningocele) cannot be attributed to a simple failure of neural closure but it also begins during primary neurulation (Stages 8-12). Lumbosacral lesions that are covered by intact skin appear during the period of secondary neuru- Jation (approximately Stages 13-20). Lesions in the transitional area of primary and secondary neurulation are not well understood. A butterfly vertebra or a hemivertebra may arise during somitogenesis (Stages 10-13) but a later origin is also possible. Chondrification, which normally begins at 6 postovulatory weeks (Stage 17), may, at least in some instances, take place in right and left centres within a mesenchymal centrum at 12 mm (personal observation), thereby giving the appearance of a median septum dorsal and ventral to the notochord. According to Ehrenhaft (1943), persistence of such a dorsoventral extension of the perinotochordal sheath "may prevent fusion of the laterally situated cartilaginous vertebral halves" and later produce a butterfly vertebra. The sensitive period for a butterfly vertebra, therefore, would seem to extend from 3 to 6 postovulatory weeks. The period for spina bifida occulta extends from the appearance of vertebrae at Stage 15 until completion of the neural arches early in the fetal period. Meningocele probably occurs towards the end of the embryonic period (Stages 18-23) or perhaps early in the fetal period. Vertebral bars are believed to be remnants of the peculiar 168 F. MPLLER, R. O'RAHILLY AND D. R. BENSON vertebral continuity that normally is seen from Stage 21 and into the early fetal period. It will be noted that the above-mentioned anomalies appear mostly within the embryonic period proper.

SUMMARY An anomalous (butterfly) eleventh thoracic vertebra in a fetus of 63 mm greatest length is described and graphic reconstructions (together with normal controls) are provided. The cartilaginous hemicentra are separated by disc-like material. Carti- laginous bars to adjacent vertebrae are present. The neural arch is complete. The notochord is not duplicated. Only one comparable case in the embryonic period has been described previously. After a discussion of cleft vertebrae in the human and in experimental animals, a developmental timetable of the appearance of several vertebral anomalies is provided. The sensitive period for butterfly vertebrae, depending on the mode of origin, seems to be 3-6 postovulatory weeks. More severe anomalies, such as the split notochord syndrome, appear earlier. It is concluded that most of the vertebral anomalies discussed arise during the embryonic period proper, although the timiing of a few, such as spina bifida occulta, extends into the early fetal period. This study was supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health, U.S.A.

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