J. Anat. (1968), 102, 3, pp. 407-414 407With 5 figuresPrinted in Great Britain

The influence of the peripheral field on the morphogenesis ofHofmann's nucleus major of chick spinal cord

P. N. DUBEY, D. K. KADASNE AND V. S. GOSAVI

Department of Anatomy, Medical College, Nagpur

Scattered groups of nerve cells at all the segments of the vertebrate spinal cord havebeen reported by various workers and are termed 'paragriseal nerve cells'. Theseneurons are situated outside the grey matter. Historical accounts of these cells havebeen given by Streeter (1904), Ariens Kappers, Huber & Crosby (1960) and Romanoff(1960). Various names are given to these groups of neurons as lobi accessorii ofLachi, marginal nuclei major and minor and Hofmann's nuclei major and minor.

Streeter (1904) has studied them in the ostrich and divided them into threegroups: (i) marginal nucleus major, (ii) marginal nucleus minor, (iii) scatteredgroups of cells. The major and minor nuclei are subpial cells situated just dorsal tothe attachment of the denticulate ligament with a definite segmental arrangement.Major nuclei are found in the lumbosacral region in birds and produce 6-7 pairs ofswellings on the external surface of the cord, each of which consists of a few cellslying in a loose matrix of neuroglia. So far, no conclusive evidence is available abouttheir function. Their prominence in the reptiles and birds and their relative absence inmammals (Anderson, Meadows & Chambers, 1964) is a feature which needs furtherinvestigation. Concerning the functions ascribed to them, Ariens Kappers et al. (1960)have mentioned the following possibilities: (i) that they are preganglionic neurons, (ii)that they are somatic motor neurons, (iii) that they are commissural cells. Schnitzlein,Hoffman, Hamlett& Howell (1963) have listed them as preganglionic parasympatheticneurons.

This investigation is limited to Hofmann's nuclei major. The morphogenesisof the cells of these nuclei is studied and efforts are made to group them withfunctionally similar cells. An attempt has been made to find out whether removalof the peripheral field has any influence on the morphogenesis of these nuclei. Themorphology of the cell group in adult fowl is also studied.

MATERIAL AND METHOD

White Leghorn chick embryos from stage 7 to 45 (Hamburger & Hamilton, 1951)were serially sectioned at 10 tm thickness. Two such series were prepared. Oneseries was stained with haematoxylin and eosin and another was impregnated withsilver by the Holmes pyridine method. The differentiation of cells in Hofmann'snucleus major was traced in both these series. The lumbosacral region of the spinalcord of one embryo at stage 40 was sectioned serially in a longitudinal plane andimpregnated with silver by Holmes's method.To study the effect of the influence of the peripheral field on the morphogenesis of

these cells, limb buds of right side were extirpated in a series of 34 embryos of stage

P. N. DUBEY, D. K. KADSNE AND V. S. GOSAVI

18-19. In another series of 23 embryos of stage 18-19, limb buds were transplantedto the flank of the host embryo according to the method described by Hamburger(1960). Sixteen of these flank grafts took and were used for the present study. Sevenother embryos were discarded because the transplants were either rudimentary orgrew in the abdomen. The embryos were sacrificed from the 13th day of incubationto hatching. The lumbosacral region of the spinal cords of the extirpation serieswere serially sectioned and stained with hematoxylin and eosin to note if there wereany decrease in the number of cells in these columns. For comparison the cells of thelateral motor column of segments 26, 27 and 28 were counted on the normal and onthe operated side, because maximum hypoplasia was seen in these segments. Cells ofthe Hofmann's nucleus situated between segments 26 and 27 were counted on bothsides. Only cells with the nucleoli were counted. Similarly the brachial and thoracicregion of spinal cords of the graft series were serially sectioned and stained, to note ifthere was any attempt on the part of the peripheral group of cells in the brachialand thoracic regions to organize into masses like Hofmann's nuclei major.For a quantitative study one adult cord and one cord of an embryo of stage 40

were stained by block staining with the thionin formalin method. The total numberof cells in every section of 8,um was counted in the adult fowl and in the chickembryo. Only cells with nuclei containing at least one nucleolus were counted. Inthe adult the greatest diameter and the widest diameter at right angles to this weremeasured by means of a micrometer in all the cells present in every section. Cross-sectional areas of cells were calculated by using the formula A x B/2, where A and Brepresent the above diameters (Tomasch & Ebnessajjade, 1961). The cross-sectionalarea gives a reasonable estimate of cell size. No corrections were applied to compen-sate for shrinkage caused by histological procedures, and about 58 0% higher figuresare to be expected in fresh untreated tissue (Brattgard, Edsterom & Hyden, 1958).

NORMAL DEVELOPMENT

The seperation of neuroblasts destined to become the cells of the peripheral nucleibegins at stage 29. Apolar neuroblasts separate from the ventrolateral aspect of thebasal lamina. This separation of the outermost layer of the ventrolateral group of thebasal lamina does not occur by migration. As is seen in Fig. 1, the mantle layer ofcells at stage 29 lies near the surface of the neural tube. Subsequently when the mnantlezone recedes from the surface as a result of an increase in the thickness of thefibrous layer, the outermost group of its cells remain at the periphery immediatelydeep to the differentiating pia mater.Between stages 29 and 30 these cells are situated between the ventral grey column

and the subpial lateral funiculus. At stage 31 two groups of neurons are seen at alllevels. One is situated between the ventral horn and the subpial white matter,particularly round the axis cylinders of ventral horn cells. The second group is acompact mass of neurons immediately deep to the pia mater dorsal to the ligamentumdenticulatum. At this stage no external bulging is visible. These cell groups showsegmental arrangement. Between stage 32 and 36, 25-27 pairs can be counted on thetwo sides of the cord. There is no evidence available to show that these cells migratefrom the dorsolateral aspect of the basal lamina or secondarily from the column of

408

Morphogenesis ofHofmann's nucleus major 409Terni which subseqently differentiates into the preganglionic cells of the visceralmotor component. Neuroblasts of the column of Terni are also derived from theventrolateral group of the basal lamina (Levi-Montalcini, 1950). It is suggested that

St. 29 St. 30 St. 31

St. 34 St. 35

St. 36 St. 37

St. 39 '-'

000

St. 38

St. 40

/'S~~~~~~Fig. 1. Development of Hofmann's nucleus major from stages 29 to 40 (Hamburger &

Hamilton).

P. N. DUBEY, D. K. KADASNE AND V. S. GOSAVI

the site of origin of the cells of the column of Terni and the peripheral paragrisealcell group is the same.

After stage 34 the cell masses of the segment of the spinal cord corresponding tothe sinus rhomboideus show very marked and rapid development. At stage 34, thesecell columns start bulging from the surface of the cord (Fig. 1) and starting from thefirst lumbar segment there are eight pairs of such bulging protrusions from thewhite matter of the cord. By stage 39 and 40 all these eight pairs of distinctly markedgroups of cells labelled as Hofmann's nuclei major have differentiated. Betweenstages 34 and 40 the nuclei become prominent and the number of cells, which areclosely packed in earlier stages, is reduced. The maximum reduction in number ofcells is seen at stages 36 and 37. The cells of these major nuclei are not as tightlypacked as those of the minor nuclei which are situated in the same segments but lieseparately in the loose matrix of neuroglia and longitudinally running myelinatednerve fibres.At stage 40 the cells are round or oval in transverse and in longitudinal section.

Their nuclei are centrally placed. Most of them are multipolar. The formation ofthe processes could not be ascertained.

EXPERIMENTS

Limb-bud extirpation and transplantation experiments in chick embryos weredone to study the influence of the periphery on the differentiation of Hofmann'snucleus major. In pyridine silver-stained preparations the axons arising from theseneurons pass out of the spinal cord with the ventral nerve root fibres. This suggeststhat these cell groups may belong to the somatic efferent component and their sizeand prominence at the lumbosacral level suggests that they are innervating the limbmusculature. If they belong to the somatic motor component then, according toRomanoff (1960), removing the peripheral field (i.e. limb bud) and transplanting thelimb bud may cause hypoplasia and hyperplasia respectively. In this series limb budswere extirpated as early as stage 18 (Hamburger & Hamilton, 1951) or about thespinal cord stage 3 n 3 to 3 n 10 (Hamburger, 1948) or about 72 h of incubation. Atthis stage the spinal cord is in preregional stage of differentiation and none of the cellgroups can be identified. Average cell count of the lateral motor column of segments26, 27 and 28 in 34 limbless embryos was 10200 cells on the normal side and 2754cells on the operated side, thereby showing 73 0% hypoplasia of cells of lateral motorcolumn on the operated side. The average number of cells in one Hofmann's nucleuson the operated side was 284 and on the normal side 268. We have not observedany hypoplasia of cells in Hofmann's nucleus major following limb extirpation.Scattered cells were also counted at the brachial and thoracic levels, but there was noincrease in the number in these peripheral groups of cells of the spinal cord followinglimb transplantation to this region. These negative results suggest that this group ofcells does not take part in the innervation of limb musculature as suggested byAriens Kappers et al. (1960).

410

Morphogenesis of Hofmann's nucleus major

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Fig. 2. Chick embryo of stage 39 (13 d). Extirpation of leg bud at stage 19.Fig. 3. Spinal cord, at the level of segment 27 of stage 39 embryo. Lateral motor column andHofmann's nucleus major on the normal side. H. and E.Fig. 4. Same as Fig. 3. Absence of majority of cells ot lateral motor column; and absence ofhypoplasia in Hofmann's nucleus major on the operated side. H. and E.

411

26 Anat. I02

P. N. DUBEY, D. K. KADASNE AND V. S. GOSAVI

ADULT CONDITION

In the adult bird macroscopic examination shows only five external protuberancesin the spinal cord at the level of the sinus rhomboideus. Each swelling occupies thespace between two ventral roots on the ventrolateral aspect. Microscopic examina-tion shows eight groups of nuclei on each side. There is a considerable difference of

278

205

65

L42

56

57

265

159

234

222

85

116

6561

L25

1 2 3 4 5 6 7 8Hofmann's nuclei major

Fig. 5. Comparison of the number of cells in the adult (hatched) cord and the embryonic (dark)cord of stage 40, from first to eighth nuclei. Note the uniformity in number of cells in the secondto sixth nuclei.

opinion about the number of nuclei as reported in the literature. Streeter (1904),reviewing the work of Terni, has reported their numbers from 9 to 11 in dove, chickand other birds. In chick embryos as well as in the adult fowl, eight nuclei aredetected in the lumbosacral region of the spinal cord, each situated between theemergence of the rootlets of the two successive spinal nerves. In serial sections of

412

6

Morphogenesis of Hofmann's nucleus major 4138 ,um thickness in the adult cord the extent of each nucleus is, from 1st to 8th nucleus,460, 640, 640, 640, 640, 640, 384, 384 and 256,m respectively. As the length ofsuccessive segments of the spinal cord gradually declines in the caudal part, thelongitudinal extent of the caudal three nuclei and the distances between them arereduced.The total number of cells in the chick embryo and in the adult hen in all these eight

nuclei is shown in Fig. 5. The cranial as well as caudal nuclei contain fewer cells thanthe middle nuclei in both instances. The maximum number of cells in the embryo isfound in the third nucleus whereas in the adult it is found in the fifth nucleus. There isa marked reduction in the number of cells in each nucleus in an adult as compared tothe corresponding nucleus in an embryo. The reduction is more marked in thosenuclei in which the original number of cells was higher, and with the exception of the1st and 8th nuclei, there seems to be a definite tendency for the number of cells tobecome uniform. In the embryonic as well as in the adult cord the number of cells inall nuclei is more or less the same except in the first and the last group in either case.

Table 1

Diameters of No. of Cross-sectionalthe cells cells area (,um2)$

A* Bt

36 32 4 57636 20 6 36032 32 13 51232 28 4 44832 24 18 38432 20 11 32032 16 2 25628 28 21 39228 24 14 33628 20 36 28028 16 2 22424 24 85 28824 20 65 24024 16 14 19220 20 138 20020 16 18 160

* Greatest diameter.t Widest diameter at right angles to A.I Cross-sectional area of cells derived by formula A x B/2 in one adult hen.

Most of the cells in the adult are round or oval with a centrally placed nucleus.Medium size Nissl granules are distributed throughout the cytoplasm. The cells(Table 1) have diameters varying from 20 to 36,tm and 16 to 36 ,tm. Of 431 cells,324 cells measured 20-28 by 20-24,m. Of 50 cells traced in their longitudinal extent,most could be found in three successive sections of 8,m thickness. The cross-sectional areas of the cells is derived by the formula A x B/2, where A is the greatestdiameter and B the largest diameter at right angles to A. The cross-sectional areasvary from 160 to 576 ,Mm2: 327 out of 431 cells fall in between 200 and 300,m2.The stock of cells from which the neurons of Hofmann's nuclei major are derived

26-2

414 P. N. DUBEY, D. K. KADASNE AND V. S. GOSAVI

and the pattern of distribution of Nissl granules suggest that probably these cells aremotor neurons rather than sensory or association or commissural cells. Theirspherical or oval shape and size indicate that probably they contribute to thevisceral motor component. The cells do not innervate muscles of the limb as is seenby extirpation and transplantation experiments, and hence it is suggested that theydo not belong to the somatic motor group, which might have been suspected becauseof the enormous size of the major marginal nuclei. However, further investigation isnecessary to prove that these cells do not belong to sensory, association, or com-missural groups.

SUMMARY

1. Two series of white leghorn chick embryos were serially sectioned from stage 7to 45, and stained with haematoxylin and eosin and with the silver pyridine method.Cells of Hofmann's nuclei major differentiate from ventrolateral cells of the basallamina at stage 29 and come to lie in their normal situation at stage 40.

2. There is no hypoplasia of cells of these nuclei following limb-bud extirpationnor hyperplasia in either Hofmann's nuclei minor or other peripheral cells followinglimb-bud transplantation. This finding suggests that Hofmann's nuclei major do notinnervate limb muscles.

3. A reduction in number of cells at stage 37, during differentiation and a furtherreduction between hatching and the adult, is observed.

4. Spinal cords of one adult chick and one embryo of stage 40 were stained bythionin formalin method en bloc. Serial sections of 8 ,um thickness were obtained.Eight major marginal nuclei were counted on each side. Out of 431 cells, 324 cellsmeasured 20-28 by 20-24 ,um in diameters A and B, respectively. The cross-sectionalarea varies from 160 to 576,am2. Most of the cells fall within the range of 200-300Qm2.

5. In the adult cord the cell size, shape and pattern of Nissl granule distributionsuggest that they may belong to the visceral motor component.

The authors wish to express their gratitude to Dr P. L. Powar, Dean, MedicalCollege, Nagpur, for giving facilities to conduct this study.

REFERENCES

ANDERSON, F. D., MEADOWS, I. & CHAMBERS, M. M. (1964). The nucleus marginalis of the mammalianspinal cord. Observation on the spinal cord of cat and man. J. comp. Neurol. 123, 97-110.

ARIENS KAPPERS, C. U., HUBER, G. C. & CROSBY, E. C. (1960). The Comparative Anatomy ofthe NervousSystem of Vertebrates, including Man, vol. i. New-York: Hafner.

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HAMBURGER, V. (1948). The mitotic patterns in the spinal cord of the chick embryo and their relation tothe histogenetic process. J. comp. Neurol. 88, 221-284.

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