On the Development of Chicken Embryos*

Home , Vitelline membrane

J. Anat. (1984), 139, 3, pp. 491-512 491 With 27 figures Printed in Great Britain The effects of mesencephalic neural crest cell extirpation on the development of chicken embryos*

G. J. MCKEE AND M. W. J. FERGUSON Department ofAnatomy, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland (Accepted 8 March 1984)

INTRODUCTION All skeletal and connective tissues of the vertebrate face, apart from the striated muscle cells of branchial arch, extrinsic eye and hypoglossal musculature, are derived from ectomesenchymal cells of the neural crest (Horstadius, 1950; Weston, 1970, 1982; Johnston, 1975; Le Douarin, 1975, 1976, 1982; Morriss & Thorogood, 1978; Le Lievre, 1978; Noden, 1978, 1980, 1982, 1983). These cells disrupt the ectodermal basement membrane after neural fold closure, and migrate towards the tissues as mesenchyme (Di Virgilio, Lavernda & Worden, 1967; Tosney, 1982). Study of the migratory pathways of neural crest cells from their dorsal position in the midline to their terminal location is by indirect means, as crest cells are microscopic- ally indistinguishable from underlying mesodermal cells. These techniques include orthotopic grafting of cells labelled with tritiated thymidine (Johnston, 1966; Noden, 1973, 1975), and heterospecific chimaeric grafting experiments (Le Douarin, 1973a, b) which exploit the structural difference between the nuclear heterochromatin of Japanese quail (Coturnix coturnix japonica) and that of the chick (Gallus gallus). Such indirect techniques have also been used to elucidate the subsequent differentiation of neural crest cells and to map the derivatives of defined regions of neural crest (Noden, 1975, 1978, 1980, 1982, 1983; Le Lievre & Le Douarin, 1975; Le Douarin, 1975, 1976,1980,1982; Le Lievre, 1978; Nakamura, 1982). Thus, it is known that crest cells emigrating at the level of the mesencephalon (mesencephalic neural crest cells) migrate to the middle of the face and differentiate into neural, skeletogenic, connective tissue and glandular derivatives. These include the bones of the upper and lower jaws, Meckel's cartilages, the trigeminal ganglia, the connective tissue of the first branchial arch muscles, vascular and dermal smooth muscle, melanocytes and facial adipose tissue. Heterotopic chimaeric grafting experiments also suggest that regions of premigratory neural crest cells are uncommitted as to eventual fate, and unpatterned as to postmigratory terminal localisation (Noden, 1975; Thorogood, 1981; Nakamura, 1982). However, recent experiments (Noden, 1983) indicate that the basis for patterning of branchial arch skeletal and connective tissues resides within the neural epithelium and not within thepharynx or pharyngeal pouches as previously suggested. Such a finding appears to conflict with a wealth ofdata (e.g. Smith &Thorogood, 1983) which indicate that differentiation of cranial neural crest cells is dependent upon specific tissue interactions either during their migration and/or at the point of their * Reprint requests to M. W. J. Ferguson, Department of Basic Dental Sciences, Turner Dental School, University of Manchester, Higher Cambridge Street, Manchester M15 6FH. 492 G. J. MCKEE AND M. W. J. FERGUSON final localisation. A model for patterned organisation ofmesenchymal cell differentiation exists for the limb (Wolpert, Lewis & Summerbell, 1978), but the mechanisms which regulate the laying down of specific craniofacial patterns of mesenchymal differentiation are unknown (Thorogood, 1981; Noden, 1983). Neural crest cell migration, differentiation and patterning have also been studied by experiments involving extirpation of specific regions of the crest. These studies have generated contradictory and ambiguous results. Johnston (1964) found that unilateral extirpation (surgical removal of the lips of the approximating neural folds) of premigratory chick mesencephalic crest results in characteristic midface deformities, for example, wide palatal clefts. However, others (Hammond & Yntema, 1964; Le Lievre, 1974) comment that more extensive extirpations are required to produce facial deficiencies, as compensation from neighbouring crest cell levels masks the effects of the selective lesions. The existence of such putative compensation mechanisms is of great interest since it is widely believed (Poswillo, 1975; Johnston, Morriss, Kushnur & Bingle, 1977) that specific regional deficiencies of neural crest cells are an important factor in thepathogenesis ofmany craniofacial malformations, forexample, deficient prosencephalic crest cells in cyclopia (Toernien, 1975; Johnston, 1975; Lieuw Kie Song & Been, 1980) and deficient mesencephalic crest cells in cleft palate (Johnston et al. 1977). It is surprising, therefore, that no carefully conducted, systematic study of the short term (compensation) and long term (malformation) effects of regionally specific neural crest cell extirpation exists; so that the capacity of the neural crest to compensate is largely unknown. The general compensatory abilities of very early embryos are well documented (Snow & Tam, 1979; Tam & Snow, 1981). Therefore the effects of precise, regional extirpation of mesencephalic neural crest cells have been studied in order to investigate (1) whether specific craniofacial malformations are generated by such surgical extirpations; (2) whether premigratory neural crest cells from adjacent levels of the neuraxis can compensate either com- pletely or partially for a large regional loss; (3) whether premigratory crest cells are either patterned or determined before they migrate from the immediate vicinity of the neural tube; and (4) the effects (if any) of such interferences and putative compensation on general (i.e. non-craniofacial) embryonic development.

MATERIALS AND METHODS Eggs of domestic fowl (Gallus gallus), strain RI from Ross's Hatcheries, Lisbum, were incubated at 38 °C and 70-80 % relative humidity. At approximately 32 hours of incubation, 4 ml of albumen was removed from each egg and the development of each embryo noted by removing a small oval piece of shell, recording the number of somites present and then resealing the egg with sellotape. Eggs containing embryos at Stages 9-11 (Hamburger & Hamilton, 1951) (i.e. 6-13 somites) were selected and prepared for operation by the subendodermal injection of a solution of sterile black Pelikan ink in tyrode solution to act as a background contrast medium below the semidiaphanous embryo. Antibiotics, antimycotic agents and fetal calf serum were not used at any stage of the experimental procedures as such agents are known to be teratogenic (Shepard, 1980). Operations were carried out in a sterile environment under a Nikon dissection microscope using electrolytically sharpened tungsten needles. At H.H. Stages 9-11 of development, mesencephalic neural crest cells are located in a large kidney shaped Mesencephalic crest cell extirpation 493 Table 1. Unilateral extirpations Hamburger- Developmental stage at termination or death Hamilton, -A, stage 14 17 20 23 26 29 32,33, at 12 15 18 21 24 27 30 34 operation 9 10 11 13 16 19 22 25 28 31 35 Totals Totalnumberoflive <10 6 2 2 0 2 2 33 4 6 1 31 embryos recovered > 10 2 8 4 2 - 2 2 4 3 4 31

Totalnumberofdead <10 - - - 1 10 18 7 12 7 - 55 embryosrecovered >10 -- - 1 15 4 44 3 31 Totalnumberof <10 6 2 2 112112 20 9 21 11 6 1 86148 operations > 10 - 2 8 50 6 15 6 6 7 3 4 621 performed Combined mortality <10 0 0 0 22 82 94 73 59 55 0 0 58% ratio for operations and performed (%) > 10 The average mortality ratio after unilateral extirpation = 58%. Table 2. Unilateral controls

Developmental stage at termination or death Hamburger-Hamilton, A- stage 17 20 23 26 29 32,33, at 18 21 24 27 30 34 operation 9 19 22 25 28 31 35 Totals Total number oflive < 10 1 - - 1 2 2 1 7 13 embryosrecovered >10 - - 2 - 1 1 2 6 Totalnumberofdead <10 - 1 1 - - 2 3 embryos recovered >_ 10 1 1 Total number ofoperations < 10 1 1 2 2 2 1 916 performed > 10 - 1 2 - 1 1 2 7 Combined mortality ratio 0 100 0 50 0 0 0 19% as a percentage The average mortality ratio after unilateral control operations = 19 %. clump adjacent to the mesencephalic portion of the neural tube. Therefore a stall hole was made in the vitelline membrane directly overlying the cephalic portion of the embryo, the dorsal ectoderm was incised and reflected laterally, mesencephalic neural crest cells were surgically removed, after which any remnants of the ectodermal flap were reflected (Figs. 1-3) to lie on the exposed endoderm. The operated eggs were coded and the windows sealed with sellotape. The vitelline membrane was moistened prior to operation by running albumen over it from the ends of the egg, kept moist during operation by dripping albumen on to it and maintained moist after operation by immediate incubation of the eggs in an atmosphere of 100 % humidity where they were manually twisted to prevent dehydration and adhesion of the vitelline membrane (Silver, 1960). The reliability of this surgical extirpation technique was checked by histological section of appropriate control embryos immediately after operation in order to determine the extent and completeness of neural crest cell removal (Figs. 1-3, 6, 8). In all cases, none or few (n < 20) mesencephalic crest cells remained. The mesencephalic neural crest cells of chick embryos with 6-13 somites (Stages I7 ANA I39 494 G. J. MCKEE AND M. W. J. FERGUSON Table 3. Bilateral extirpations Developmentalstageat termination or death Hamburger-Hamilton - stage 17 at 18 20 22 24 26 operation 19 21 23 25 27 Totals

Total number oflive embryos <10 - - 1 - 2 4 recovered > 10 1 1 2 Total number ofdead embryos < 10 - 1 - - 1 2 4 recovered > 10 1 - 1 - 2 Total number ofbilateral operations < 10 - 1 1 1 4 =8 performed > 10 1 - 1 1 1 4 Combined mortality ratio as a 100 100 50 0 50 50% percentage The average mortality ratio after bilateral extirpation = 50%.

9-11) were extirpated either unilaterally (n = 148; Table 1) or bilaterally (n = 8; Table 3). Sham operations (consisting of an identical surgical procedure but with no crest cell removal) were performed unilaterally on 16 control embryos (Fig. 4; Table 2), and bilaterally on one control embryo (terminated at Stage 30). The developmental stages at operation and at termination or death for operated and control embryos are given in Tables 1-3. The average mortality ratio of normal eggs incubated under identical conditions was 20 %. Forty seven normal embryos were also studied. After operation, all embryos, operated, control and normal, were incubated at 100 % relative humidity in a ventilated incubator. Embryos were fixed in 10 % formal saline at 0, 1, 2, 3, 4, 6, 8, 12, 15 and 20 hours postoperatively and then at 2, 3, 4, 5, 6, 7 and 8 days of incubation. Normal embryos were fixed for comparison and all specimens were analysed by macroscopic examina- tion, light microscopy or scanning electron microscopy. Specimens for light microscopy were dehydrated through ascending grades of alcohol, cleared in xylene, embedded in Fibrowax (Raymond A. Lamb, London) and serially sectioned at a thickness of 8 ,um. Early (up to Stage 13) embryos were sectioned intact in the transverse plane, whilst later embryos were either decapitated at the lower cervical level and the head and neck sectioned in the coronal plane, or else the whole embryo was sectioned in the sagittal plane. Sections were stained with Harris' haematoxylin and eosin. After operation and up to Stage 13 of development, the number of neural crest cells lying lateral to the neural tube and dorsal to the dorsal extremity of the foregut were counted and the average value of 5 or more consecutive slides obtained on the operated and unoperated sides at levels corresponding to the rostral part of the mesencephalon, mesencephalon, metencephalon, otic placode, myelencephalon, somite 2 and somite 6. The mean, standard deviation and 95 % confidence limits of cell counts at each level on the operated and unoperated sides are shown in Table 4 and represented graphically in Figure 26. Specimens (whole embryos, heads and transverse sections at various levels of the Mesencephalic crest cell extirpation 495 neuraxis) for scanning electron microscopy were dehydrated through ascending grades of alcohol, critical point dried from amyl acetate, mounted on aluminium stubs, sputter coated with gold and viewed in a Cambridge S 180 Stereoscan at an operating voltage of 18 kV.

RESULTS Unilateral extirpation of mesencephalic neural crest cells obviously produced a deficiency of crest cells lateral to the mesencephalon on the operated side (Figs. 1-3, 8). Equal numbers of crest cells were again present lateral to the mesencephalon on the operated and unoperated sides 7 hours after operation (Table 4; Fig. 26). This repopulation in place of the extirpated cell mass was associated with compensatory hyperplasia of contiguous populations of migrating neural crest cells from adjacent neuraxial levels. The important findings at each level of the neuraxis for the first 12 hours after operation are documented below. Rostral mesencephalon Intrinsic hyperplasia of remaining crest cells was observed but this depended on the stage at which the operation was performed. Operations before Stage 10 of embryonic development were associated with hyperplasia due to intrinsic proliferation and continued recruitment from the midline (Fig. 5). By Stage 10, emigration of neural crest cells from the dorsal midline has ceased. After this stage, the contribu- tion of rostral mesencephalic neural crest cells to repopulation at mesencephalic levels was limited and there was frequently an insignificant deficiency of crest cells on the operated side until 7 hours after operation (Fig. 26). Mesencephalon On the operated side there was a statistically significant gross deficiency of neural crest cells for 6 hours after operation (Figs. 1-3, 6, 8, 26; Table 4). The cell numbers on operated and unoperated sides had equalised by 7 hours after operation (Table 4; Fig. 26). The mesencephalic crest cell deficit was repopulated by migration of crest cells from adjacent levels of the neuraxis (principally the metencephalon). These cells arose by increased emigration from the dorsal midline and by intrinsic hyperplasia of migrating crest cells (Figs. 5, 7, 10, 11, 26). Emigration of crest cells from the midline of the mesencephalic neural tube had ceased by Stage 10 and did not contribute significantly to the repopulation (Fig. 6). The repopulating crest cells reached the cell- free space lateral to the mesencephalon by migrating along the basement membranes of the neural tube and of the regenerating epithelium. Re-epithelialisation of the surgical lesion was incomplete 3 hours postoperatively (Fig. 10) but was complete by 4 hours after operation (Fig. 9). Metencephalon A statistically significant hyperplasia of crest cells lateral to the metencephalon on the operated side was observed for 7 hours after operation (Figs. 7, 26; Table 4). This hyperplasia contributed to the repopulation of the mesencephalic deficit (Fig. 26). No hyperplasia of underlying mesodermal cells was apparent (Fig. 26). Otic placode There was no evidence of increased recruitment of placodal cells on the operated side (Fig. 26; Table 4). I7-2 496 G. J. MCKEE AND M. W. J. FERGUSON

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Fig. 1. Scanning electron micrograph of a Stage 11 embryo in which the left mesencephalic neural crest was extirpated at Stage 10+ (i.e. one hour after operation). Note the surgical extirpation site (E) in the region of the mesencephalon (M) and the patent posterior neuropore (N). Fig. 2. Higher power view of Fig. 1. Note the incised margins of ectoderm (EC) and the neural tube (NT). The mesencephalic crest cells have been removed. Fig. 3. Dorsal view of a chick embryo one hour after operation (Fig. 1). Note the excised mesencephalic crest cells (E) and the flap of ectoderm (EC) on the operated side compared to the normal appearance on the opposite side. P, prosencephalon; M, mesencephalon; MT, metencephalon. Fig. 4. Histological section through the mesencephalon of a Stage 9+ unilateral control (sham operated) embryo one hour after operation. Note the paramedian incision line (I). Migration of crest cells from the dorsal midline of the neural tube is evident and the edges of the incised ectoderm are mitotically active. Haematoxylin and eosin. Mesencephalic crest cell extirpation 497 Myelencephalon There was a slight hyperplasia of neural crest cells lateral to the myelencephalon on the operated side but this was not statistically significant (Fig. 26; Table 4). This slight imbalance was due to greater emigration of crest cells toward the operated side (Fig. 11).

Somitic neural tube There was no statistically significant difference between the numbers of neural crest cells present on operated and unoperated sides at the levels of either somite 2 or 6 (Fig. 26; Table 4). Considerably fewer neural crest cells are formed from somitic levels (less than half the number formed at the mesencephalic level) so that there was much greater variation in the crest cell counts (Fig. 26). The incongruity seen in Figure 26 during the period of time 8-12 hours after operation was probably due to the fact that the embryo had reached Stage 13 of development, at which time the telencephalon and mesencephalon undergo normal embryonic rotation through 90°. This rotation proceeds along the craniocaudal axis of the embryo by a mechanism which is unknown but which is thought to involve differential proliferation of the mesenchymal cell mass and overlying epithelial cells at, and immediately below, the level of axial rotation (Miller, 1982). Thus the unequal cell numbers on right and left sides could have been due to differential mesenchymal proliferation prior to axial rotation of the mesencephalic level at Stage 13. The effects of experimental and sham operations on later embryonic development are described below.

Unilateral extirpations (Table 5) Development of maxillae and mandibles (or their respective primordial processes) in experimental embryos was identical to that of normal embryos in 23 out of 24 cases (Figs. 13-16). There was no histological evidence of agenesis or hypoplasia of any mesencephalic crest cell derived structures on the operated side (Fig. 17). Moreover, facial symmetry was present in 21 out of 24 cases (Figs. 13-16) with asymmetry in the remaining 3 cases being due to unilateral or bilateral microphthalmia. The aetiology of the latter may have been either spontaneous or caused by undetected, accidental surgical damage to the early embryo.

Fig. 5. Section through the rostral mesencephalon of a Stage 9 + embryo two hours after a left sided extirpation of mesencephalic crest cells. Note the increased density of crest cells on the operated side (0) caused by increased emigration from the dorsal midline and by increased division of the migrating crest cells. These cells assist in repopulating the mesencephalic deficit. No hyperplasia of the underlying mesodermal cells (M) is evident. Haematoxylin and eosin. Fig. 6. Section through the mesencephalon of a Stage 11 + embryo two hours after a left sided extirpation of crest cells. Note the deficiency of crest cells on the operated side (0) but the commencement of repopulation by the migration of crest cells(C)fromadjacent neuraxial levels along the basement membrane ofthe neural tube. Haematoxylin and eosin. Fig. 7. Section through the metencephalon of a Stage 9+ embryo two hours after a left sided extirpation. Crest cells on the operated side (0) are undergoing compensatory hyperplasia and increased emigration from the dorsal midline so that their density is increased compared to the unoperated side. Haematoxylin and eosin. 498 G. J. MCKEE AND M. W. J. FERGUSON

^"Pr ,- "F ..- 1'' 100p Mesencephalic crest cell extirpation 499 Scoliosis of the cervical spine (with the concavity on the operated side) was present in 20 out of 23 experimental unilateral extirpation embryos (Figs. 20, 22, 24, 25, 27). Scoliosis included lesions at one or more sites (single or multiple). In multiple scoliosis, the most cephalad lesion was invariably toward the operated side (Figs. 20, 22, 24, 25, 27). Rotation of the cervical spine with the head toward the side of the lesion was observed in 4 out of 23 cases. Unilateral control operations (Table 6) Facial morphology was identical to normal in all cases. Histological development of all mesencephalic crest cell derived structures appeared to be normal at all stages studied and facial symmetry was present in all 9 cases. The spine was generally neither bent nor rotated (Fig. 18) but appeared to be normal (Fig. 19). However, in 2 embryos a slight single scoliosis of the cervical spine (again with the concavity toward the operated side) was present. Multiple scoliosis was never observed whilst mild rotation of the cervical spine was only present in one out of 9 cases. Bilateral extirpation (Table 7) Facial morphology was identical to the normal in 4 out of the 5 bilaterally extirpated embryos. The left mandibular process was slightly hypoplastic in the remaining specimen. Again, normal development of all mesencephalic crest cell derived structures was observed and facial symmetry was present in 4 of the 5 cases. Scoliosis of the cervical spine occurred in all 5 embryos of which 3 showed multiple scoliosis

Fig. 8. Rostral view of the caudal mesencephalon (ME) of a Stage 11- embryo three hours after a right sided extirpation. Neural crest cells are absent lateral to the mesencephalon on the operated side (0) where strands ofmatrix (M)can be seen. MC, mesocardium. Fig. 9. View of the caudal mesencephalon (ME) of a Stage 12- embryo four hours after a left sided extirpation. There is a large deficiency of crest cells on the operated side (0) but some (NC) are present adjacent to the basement membrane of the neural tube. Regeneration of the overlying ectoderm (EC)is proceeding. Fig. 10. Section through the mid-mesencephalon (ME)of a Stage 10 embryo three hours after a right sided extirpation. Note the repopulation of crest cells on the operated side (0). These cells are fewer in number but greater in density than on the unoperated side. The incised ectoderm is thickened (arrows) and regenerating. Emigration of crest cells from the midline has ceased. Haematoxylin and eosin. Fig. 11. Section through the caudal metencephalon (M) of a Stage 11 embryo six hours after a right sided extirpation. On the operated side (0) emigration (arrow) of crest cells from the midline between the otic placode (OP) and neural tube (NT) is still occurring, but migration has ceased on the unoperated side. Fig. 12. View of a Stage 14 embryo twenty hours after a right sided extirpation. The trunk is abnormally bent at the levels of the 6th and 14th somites and rotated so that the neural tube (NT) and somites (S) are visible. This embryo is illustrated in relation to a normal counterpart in Fig. 27. OP, otic pit; NP, nasal pit. Fig. 13. View of the face of a Stage 24 embryo which underwent a right sided control operation at Stage 9-. Facial symmetry and development are normal. NP, nasal pits; MN, median nasal process; LN, lateral nasal process; MX, maxillary process; M, mandibular process. Fig. 14. View of the face of a Stage 24 embryo which underwent a left sided extirpation at Stage 10+. Facial symmetry and development are normal. NP, nasal pits; MN, median nasal process; LN, lateral nasal process; MX, maxillary process; M, mandibular process; E, developing eye. Fig. 15. View of a Stage 24 embryo which underwent bilateral extirpation at Stage 9+. The heart has been removed (H) to reveal normal facial development. A 120° left sided scoliosis of the midcervical spine is present (arrow). 500 G. J. MCKEE AND M. W. J. FERGUSON

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(Figs. 15, 21, 23). Moreover, rotation of the cervical spine was present in 4 of the 5 embryos: in 3 specimens toward the right and one toward the left side. Bilateral control operation (Table 8) A bilateral control operation was successfully performed on only one embryo and resulted in both normal facial and spinal development. Table 9 summarises the frequency of facial malformations, scoliosis and rotation after various operations. The embryonic stages at which operations were performed did not influence the development either of normal faces or of single or multiple cervical scoliosis (Table 10). However, rotation of the cervical spine was three times more frequent (6 cases out of 14:43 %) when operations were performed at or after Stage 10, compared with operations performed before Stage 10 (2 cases out of 14:14 %) (Table 10). The embryonic stage at operation might have been expected to influence the subsequent development of the embryo more profoundly as crest cell emigration from the midline of the mesencephalic neural tube ceases at Stage 10.

Fig. 16. View of the face of a Stage 35 embryo which underwent a right sided extirpation at Stage 9. Facial symmetry and development are normal. Fig. 17. Oblique coronal section of a Stage 32 embryo which underwent a right sided extirpation at Stage 10. Derivatives of the mesencephalic neural crest appear histologically normal in size and shape, for example, Meckel's cartilages (M), palatal shelves (P), nasal septal cartilages (N). C, ceratobranchial cartilages; MA, masseter muscle; PT, pterygoid muscles. Haematoxylin and eosin and alcian blue. Fig. 18. Dorsal view of a Stage 33 embryo which underwent a left sided control operation at Stage 10. Development is normal and no spinal deformity is present. Compare with Figs. 19, 20 and 21. Fig. 19. Dorsal view of a Stage 30 normal embryo. Compare with Figs. 18, 20 and 21. Fig. 20. Dorsal view of a Stage 27 embryo which underwent bilateral extirpation at Stage 10-. Multiple scolioses at the branchial and upper cervical levels, together with a 90° rotation of the head to the right side, are present. Compare Figs. 18, 19 and 21. Fig. 21. Dorsal view of a Stage 30 embryo which underwent a left sided extirpation at Stage 9+. Note the 90° bend (scoliosis) to the left in the midcervical spine. Fig. 22. Dorsal view of a Stage 22 embryo which underwent a right sided extirpation at Stage 9. Note a scoliosis of 120° to the left in the brachial region and of 45° to the right in the upper cervical region. There is also a 45° rotation to the right. Fig. 23. Dorsal view of a Stage 25 embryo which underwent a bilateral extirpation at Stage 10. Multiple scolioses are present in the brachial and upper cervical regions. Fig. 24. View of a Stage 26 embryo which underwent a right sided extirpation at Stage 10+. Right sided scoliosis is present in the cervical region. Fig. 25. View of a Stage 25 embryo which underwent a left sided extirpation at Stage 10-. Multiple scolioses have occurred in the mid and upper cervical regions. From caudal to rostral the angulations are 45° to the left (A), 90° to the right (B), and 90° to the left (C). Mesencephalic crest cell extirpation 505

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'1 2 3 4 5 6 7 8 9 110 1 2 Time after operation (h) Fig. 26. Graphical representation of the data in Table 4.The pecked lines represent the means and 95% confidence limits of the crest cell counts on the unoperated side in five specific histological slides at each of the various levels of the neuraxis. The means for the unoperated sides are calculated to be 100%. The solid lines represent comparable means and 95 % confidence limits of the crest cell counts on the operated side. The mesencephalic region is repopulated with crest cells within approximately 7 hours of operation. Also note the compensatory hyperplasia in the metencephalic region, but no significant hyperplasia elsewhere along the neuraxis. The numbers of crest cells at the level of somite 2 at these stages of development are extremely small, hence the large variation.

DISCUSSION Extirpation of migrating neural crest cells lateral to the mesencephalon either unilaterally or bilaterally during Hamburger-Hamilton Stages 9-11 of development does not disrupt or delay normal facial morphogenesis. This absence of facial malformations apparently conflicts with the results of earlier extirpation experiments by Johnston (1964) and Hammond & Yntema (1964). Johnston found that removal Mesencephalic crest cell extirpation 507

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Fig. 27. Outline of a Stage 14 embryo twenty hours after a right sided extirpation (solid line) superimposed on the normal outline (dotted) of a Stage 14 embryo. The levels of various somites are given for reference. Note the abnormal bends in the operated embryo (scoliosis) and the overall foreshortening of the latter.

Table 9. The frequency (expressed as a percentage) of morphological characteristics following unilateral or bilateral extirpations or control operations Unilateral Unilateral Bilateral Bilateral extirpation controls extirpation controls Normal face 23/24 = 95-8 9/9 = 100 4/5 = 80 1/1 = 100 Cervical scoliosis 20/23 = 87-0 2/9 = 22-2 5/5 = 100 0/1 = 0 Single scoliosis or 16/20 = 80-0 2/2 = 100 2/5 = 40 multiple scolioses 4/20 = 20-0 3/5 = 60 Cervical rotation 4/23 = 17-4 1/9 = 11.1 4/5 = 80 0/1 = 0

Table 10. Thefrequency (expressed as apercentage) ofvarious morphological characteristics following unilateral or bilateral extirpations or control operations as a function of the stage at operation (i.e. < or > 10 somites) Stage at Unilateral Unilateral Bilateral Bilateral operation extirpation controls extirpation controls Normal face < 10 11/12 = 91-6 4/4 = 100 1/2 = 50 >10 12/12= 100 5/5= 100 3/3= 100 1/1 = 100 Cervical scoliosis < 10 11/12 = 91-6 1/4 = 25 2/2 = 100 >10 9/11 = 75 1/5 = 20 3/3 = 100 0/1 = 0 Single scoliosis <10 9/11 = 75 1/1 = 100 1/2 = 50 >10 7/9 = 77 1/1 = 100 1/3 = 33-3 Multiple scolioses < 10 2/11 = 18-2 1/2 = 50 > 10 2/9 = 22-2 2/3 = 66.6 Cervical rotation < 10 1/12 = 8-3 0/4 = 0 1/2 = 50 >10 3/11 = 27-3 1/5 = 20 3/3 = 100 0/1 = 0 508 G. J. MCKEE AND M. W. J. FERGUSON of the mesencephalic neural crest and adjacent neural folds prior to cell migration (before Stage 9) results in deficiencies in mesencephalic neural crest cell derivatives, such as the maxillary and mandibular structures. However, the difference between Johnston's (1964) results and the present findings probably resides in differences in experimental surgical technique. In the present study, only the mesencephalic neural crest cells were excised, whereas in Johnston's study both the premigratory crest cells and the lips of the neural folds were removed. The latter technique probably removes much of the source for crest cell compensation which is clearly documented in the present study. In the absence of such compensation, facial malformations are to be expected. Clearly, account must now be taken ofdifferences between interferences with crest cells per se and interferences with crest cells and the compensatory mechanism. Although Hammond & Yntema (1964) obtained facial malformations after crest cell extirpation, these were not as severe as would have been expected, probably due to compensation via rostral migration from the caudal end of the operative field. However, these investigators do not document the source, speed or extent of this compensation whilst others do not even speculate on its existence. This study demonstrates that there are two principal sites of hyperplastic activity which compensate for the mass of extirpated mesencephalic cells. There is increased formation of neural crest cells within the neural tube, principally at the level of the metencephalon, which migrate toward the operated side. There is also intrinsic compensatory hyperplasia of migrating neural crest cells at adjacent levels of the neuraxis on the operated side, principally at metencephalic levels. Compensation is rapidly achieved with the crest cell number at the operative site becoming equal to that on the unoperated side within a period of 6-8 hours after operation. This replenishment originates mainly from metencephalic neural crest cells and has no significant effect on the numbers of crest cells formed at more caudal levels of the neural tube. Given this rapid regulation of crest cell numbers, it is not surprising that there is normal facial development after 95 % of unilateral and 80 % of bilateral extirpations. The mechanism of regulation generates a number of intriguing questions. What triggers the compensation? Is the rate of crest cell division regulated by density- dependent factors such as the number of contacts with adjacent crest cells? How and why do adjacent metencephalic neural crest cells migrate in an abnormal, cranial, direction, given that they normally migrate laterally on specific substrata of extracellular matrix and are perhaps oriented towards concentrations of the matrix (Tosney, 1982; Noden, 1980, 1982)? What factors signal that the compensation is complete and how do they operate? Future experiments, such as replacing the excised mesencephalic crest cell mass with inert substances like agar or latex beads, are required to answer some of these questions. The existence of the compensatory mechanism also casts doubt on the suggestion that the pathogenesis of facial malformations involving deficient crest cell derivatives, for example, cyclopia (Toerien, 1975; Lieuw Kie Song & Been, 1980) or cleft palate (Johnston, 1975; Johnston etal. 1977; Johnston & Sulik, 1979; Morriss & Thorogood, 1978), is due to local deficiency in initial crest cell number, unless the compensatory mechanism is also disturbed. It is more likely that these malformations are caused by abnormal crest cell migration (perhaps due to disruption in the extracellular matrix along the pathway of migration), division and differentiation. Crest cell compensation also suggests a possible mechanism whereby repeated small doses of a teratogen, which might otherwise be considered trivial, could exert a severe, cumulative, effect Mesencephalic crest cell extirpation 509 on facial development. The first dose could decrease crest cell numbers (by arresting crest cell formation, arresting crest cell division, killing crest cells, etc.) and activate the compensatory mechanism. The second dose might then act during the period of increased cell division and migration, and hence of increased susceptibility, when its effects are likely to be much more severe than usual. This hypothesis requires testing but is a logical extension of a similar suggestion by Snow & Tam (1979) based on their observations of extensive compensatory growth in early (primitive streak stage) mouse embryos. These workers (Snow & Tam, 1979; Tam & Snow, 1981) have demonstrated that the cell mass of a mouse embryo at the primitive streak stage can be reduced to around 10% of normal, yet a morphologically normal individual is eventually produced. As in the present study, much of the compensatory regulation is accomplished rapidly, within 48 hours of damage, and morphogenesis of the brain and head takes place in half the normal time. However, this compensatory growth disrupts sequential developmental programmes within the embryo and fetus, so producing subtle malformations such as sterility, impaired motor and sensory function and mental retardation. Likewise, the spinal scoliosis observed after extirpation of mesencephalic neural crest cells may be related to the compensatory mechanism. Thus the compensatory mechanism may disrupt the normal craniocaudal sequential developmental programmes which occur along the neural tube, for example, prolonged emigration of crest cells may delay the development of longitudinal bundles of microtubules (Sadler, Greenberg & Coughlin, 1982) or affect the concentrations ofcatecholamines (Newgreen, Allan, Young & Southwell, 1981) within neural tube cells. These non- muscle contractile systems have been implicated in the mechanism of normal axial flexure (Newgreen et al. 1981). Scoliosis is first observed approximately 10 hours after operation, i.e. 2-3 hours after the projected time for total repopulation of the extirpated crest cell mass. The cessation of neural crest stem cell activity within the tube may result in altered tensile forces within the tube, especially about the fulcrum of attachment of the embryo to the underlying yolk sac. Thus, cervical scoliosis may be caused at that level. This attachment, which corresponds to the anterior intestinal portal of the chick embryo, moves caudally and underlies the cervical spine at Stages 14-15. Therefore, an association might be postulated between the effects of compensation for a large local loss of neural crest cells and the rare malignant progressive variant of infantile idiopathic scoliosis. It is noteworthy in man that neurofibromatosis, which is a neural crest lesion present from birth, is associated in 39 % of cases with inexplicable severe angular scoliosis ofthe spine (Vinken & Bruyn, 1982). It is possible that the scoliotic lesions are experimental artifacts produced by adhesion of the embryo to the surgically incised vitelline membrane as the latter dehydrates at some stage postoperatively (Silver, 1959, 1960). However, this is considered to be unlikely for several reasons. (1) All precautions against dehydration and adhesion were taken (Silver, 1960) and, in any case, the embryos were operated upon in the late stages ofsusceptibility to suchabnormalities. (2) In unilateral extirpations, single scoliosis was frequent and the concavity of the lesion was always toward the operated side, despite the fact that the hole in the vitelline membrane was approximately equal on both sides. (3) Scoliosis of the cervical spine at more than one site (i.e. multiple scoliosis), usually involving an additional rotation of the spine, was frequently observed after bilateral extirpations despite the fact that the hole in the vitelline membrane was comparable to that made in unilateral extirpations. 510 G. J. McKEE AND M. W. J. FERGUSON (4) It is particularly significant that cervical scoliosis was not usually observed after unilateral or bilateral control operations. The few cases that did result were slight and may have been caused by unintentional removal of mesencephalic crest cells whilst making the incisions during the control operations. Eggs used for each of the operative procedures (extirpation, control, etc.) were controlled so that all were open for approximately the same time (four minutes). This strongly suggests that the number of crest cells removed, rather than the duration ofthe operation, governs the nature and extent of the spinal lesion. Finally, the level and severity of the scoliosis were unrelated to the stage of operation, in contrast to malformations induced by dehydration and adhesion of the vitelline membrane (Silver, 1959, 1960). The compensation for mesencephalic crest cell loss also suggests that neural crest cells are neither patterned nor determined before they migrate extensively from the neural tube, as it is obvious that metencephalic crest cells which replenish the mesencephalic region migrate and differentiate into normal mesencephalic derivatives. This conclusion differs from that of Noden (1980, 1982, 1983) and earlier workers on amphibia (reviewed by Noden, 1982, 1983) who found that, when chick crest cells destined to enter the second or third branchial arches are excised and replaced en masse with presumptive first arch crest cells from quail embryos, the latter cells differentiate into first arch skeletal structures. Thus they appear to develop in a fashion characteristic of their origin rather than of the host site, suggesting an intrinsic patterning determined by their original position along the neuraxis. The apparent disparity between these two sets of results may reside in the fact that Noden's (1980, 1982, 1983) experiments employed transplantations where there was presumably little compensatory activity and that the grafts were en masse, so that neural crest cells were grafted together with their surrounding microenvironment. It may be that the ability of metencephalic crest cells, which are replenishing a mesencephalic deficit, to differentiate into normal mesencephalic derivatives is either a property of their origin by compensatory hyperplasia, or is more probably a result of their interactions with extracellular matrix, basement membranes, etc. as they migrate cranially to fill the surgical deficit. It is therefore possible that patterning of neural crest cells is not an intrinsic premigratory property but rather a result of interactions which they undergo whilst in the vicinity of the neural tube. Clearly these possibilities require further experimental testing.

SUMMARY The mesencephalic neural crest cells of Hamburger-Hamilton Stage 9-Stage 11 chick embryos were surgically extirpated unilaterally in 148 embryos and bilaterally in 8 embryos. Sham operations were performed unilaterally on 16 control embryos and bilaterally on one control embryo. Embryos were fixed at various time intervals after operation, studied macroscopically, and by light and scanning electron microscopy, and their development compared with that of 47 normal embryos. The extirpated mesencephalic region was repopulated by crest cells within 6-8 hours after operation. These 'new' crest cells migrated from adjacent neuraxial levels (principally the metencephalon and prosencephalon) along the basement membrane of the neural tube and the regenerating ectoderm. At prosencephalic and metencephalic levels, both intrinsic hyperplasia of migrating crest cells and prolonged migration of crest cells from the dorsomedian part of the neural tube contributed the additional cells required to repopulate the mesencephalic region. Morphogenesis and differentia- Mesencephalic crest cell extirpation 511 tion of all crest cell derivatives were normal and craniofacial malformations were absent. Thus the neural crest and neural tube can compensatefor an extensiveregional loss, premigratory crest cells are neither regionally patterned nor determined (as prosencephalic and metencephalic cells give rise to normal mesencephalic derivatives) and regional failure of crest cell formation is an unlikely facial pathogenetic mechanism. Previous workers who observed facial malformations following crest cell extirpations performed the latter by removing the lips of the neural tube which not only removed the crest cells but also the compensatory mechanism. Cervical scoliosis was observed in extirpated embryos but not in controls. The pathogenesis of this scoliosis may be related to the process of compensation, which could disturb the sequential differentiation of the neural tube and so disorganise the mechanisms of normal axial flexion. These observations may be relevant to the pathogenesis of some forms of congenital and infantile idiopathic scoliosis; such scoliosis in man is frequently present in neurofibromatosis - a neural crest lesion. We are grateful to Messrs G. Bryan, C. Rix, R. Reed and Mrs S. Higgins for valuable technical assistance. Miss J. M. Smith kindly typed the manuscript. This work was supported by MRC grant no. G 8113610 CB, EHSSB grant EB 109/74/75 and NIH grants DEO 2848 and DEO 3569.

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