/ . Embryol. exp. Morph., Vol. 16, 3, pp. 497-517, December 1966 4 9 7With 2 plates

Printed in Great Britain

The development ofthe chick embryo diencephalon and mesencephalon

during the initial phases of neuroblastdifferentiation

By KATHERINE M. LYSER1

Harvard Biological Laboratories, Cambridge, Massachusetts,and Hunter College of the City University of New York

The development of the nervous system presents many interesting problemsas a developing system with numerous parameters of differentiation as well asfrom the point of view of the establishment of adult structure and function. Withour growing understanding of developmental processes in general, and inter-actions at various stages of development in particular, it should be profitable tostudy more closely events of each period in a developing system, looking forinformation concerning their immediate control and their relation to events ofother periods. In the nervous system, one phase which should be investigatedmuch more thoroughly—especially from the point of view of the control ofcellular differentiation—is that of the initial appearance of neuroblast cellsand formation of the first nerve processes. Most studies of normal embryoswhich have included the period of initial differentiation have been primarilyconcerned with tracing the origins of definitive nuclei and fiber tracts, thoughpossible mechanisms controlling various aspects of their development have ofcourse been discussed.

The present study is concerned specifically with the period of initial differen-tiation of cells and fibers in the diencephalon and mesencephalon of the chickembryo. This region has been chosen because it is among the early areas ofdifferentiation, and it contains a number of different centers, which are notcontinuous with other areas of differentiation at first. This study was begunas part of a thesis (Lyser, 1960) and reconsidered in the light of recent work inrelated fields.

In the chick embryo, neuroblasts with processes appear first in the hind brainand shortly thereafter in the diencephalon and mesencephalon, where the firstneuroblasts with processes have been reported at 17- or 18-somite stages

1 Author's address: Department of Biological Sciences, Hunter College, New York,10021, U.S.A.

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(Tello, 1923; Windle & Austin, 1936). The initial differentiation of neuritesthus takes place quite early in the development of the central nervous system.In the spinal cord of the chick embryo (Hamburger, 1948), and presumablyin the brain also, initial neuroblast differentiation begins while or before pro-liferation has reached its peak (cf. Hamburger, 1948; Tello, 1923; Windle &Austin, 1936) and so overlaps this phase. It is of course continuous with thelater phases of development, including histological differentiation, but it beginswell before these become apparent. In the diencephalon and mesencephalonthe mantle layer does not become distinct from the inner cell layer nor can thelongitudinal columns of cells be distinguished until about the fourth day(Palmgren, 1921; Rendahl, 1924; Kuhlenbeck, 1937).

MATERIALS AND METHODS

Forty-five 13- through 30-somite chick embryos were studied. All the em-bryos were serially sectioned, either sagitally or transversely, and stained fornerve fibers with silver. Eighteen embryos were from the collection of ProfessorLeigh Hoadley. These embryos had been fixed in 95 % ethanol and stained withpre-war German Protargol by a modified Bodian (1936) method. The othertwenty-seven embryos were prepared for this study. A number of fixativesrecommended for the embryonic nervous system and several silver stains weretried in various combinations. Staining by a modified Holmes's (1942) methodwas most satisfactory. For these young embryos, the following fixatives werefound to be useful: 95 % ethanol, Bodian's fixative no. 2 or no. 4 (Bodian, 1937),Mahdissen's fixative as given by Gray (1954, p. 192), Lavdowsky's mixture asgiven by Guyer (1953, p. 236), or Lavdowsky's mixture modified by substitutingformic acid (1-6 ml) for acetic acid (2-0 ml). Embryos remained in ethanol forH h or in one of the other fixatives for approximately 24 h. They were storedin 70 % or 80 % ethanol, dehydrated in ethanol, cleared in cedar-wood oil, andembedded in 60-63 °C Tissuemat (Fisher). Serial sections were cut at 10 or 12 /*.

Graphic reconstructions of some of the younger embryos were made bydrawing neuroblast cells with processes and other segments of fibers on cameralucida tracings of each section and then tracing these on to an outline of thebrain (Text-figs. 1-3). All cells and fibers which could be seen were recorded.In addition, diagrams of the pattern in some of the older embryos were made bysketching representative cells and fibers on an outline of the brain as thesections were studied (Text-figs. 4-6). In these drawings the actual number ofcells present is not indicated; only a few are shown, illustrating the locationsand orientations of the neuroblasts and fibers observed.

Initial neuroblast differentiation 499

OBSERVATIONS

There is some variation in the development of the neuroblasts and nervefibers among embryos of the same stage as determined by somite count. Thisappears to be due to a difference in the time at which differentiation of axonsbegins in this area of the brain in relation to the development of the somites.However, development is fairly regular in general location and arrangement ofcells and fibers and in the order of their appearance. The embryos can bearranged in sequence by considering the numbers and distribution of cells andfibers together with the number of somites and incubation time.

The earliest stage at which fibers were identified in the diencephalon andmesencephalon in the group of embryos studied was the 14-somite stage. Forpurposes of description, development from the earliest appearance of nervefibers in this region through the 30-somite stage has been divided arbitrarilyinto five periods: (A) 14- to 16-somite embryos in which axon differentiation inthis region is just beginning, (B) 16-somite embryos in which differentiation isslightly more advanced, (C) 17- to 18-somite embryos, (D) 19- to 22-somiteembryos and (E) 23- to 30-somite embryos.

At the beginning of the period of development under consideration, the wallof the neural tube is essentially a pseudostratified columnar epithelium, thecells of which may be referred to as neural epithelial cells. Those which areundergoing division move toward the neurocoel; the nuclei of interphase cellsare at various levels (Sauer & Walker, 1959; Sidman, Miale & Feder, 1959;Fujita, 1963). At the outer edge is a nucleus-free zone, consisting of the outerends of the epithelial cells, where the marginal layer will subsequently form.This will be referred to here as the 'peripheral zone'; the area between theperipheral zone and the neurocoel will be called the 'nuclear zone'. Neuroblastsand fibers that are parallel to the surface of the neural tube and oriented in adorso-ventral direction or obliquely will be referred to as 'circumferential',those which are parallel to the longitudinal axis of the neural tube as 'longi-tudinal \ and those which are perpendicular to the margin of the neural tubeas 'radial'.

In these preparations, neuroblasts with processes stand out because thecytoplasm is more darkly stained than the cytoplasm of adjacent epithelial cells.Their processes and other segments of fibers are black. It usually is not possibleto trace a fiber that extends through several sections from one section to thenext, even in the younger embryos where there are only a few fibers. As withother methods for identifying nerve cells and fibers, the question of whether allneuroblasts are stained can be raised. It seems likely that with this methodmost neuroblasts are recognizable, but it cannot be definitely determined thatall are stained. Silver methods may demonstrate only nerve cells with neuro-fibrillae (Guillery, 1965; Gray & Guillery, 1966), but there is no evidence atpresent that there are nerve processes in the early embryo which do not contain

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neuro-fibrillae or neuro-tubules. In electron micrographs of motor neuroblastsof chick embryos, for example, all the axons which could be definitely identifiedcontained fibrillar structures (Lyser, 1964).

Two other problems encountered in studying silver-stained sections shouldbe remembered in regard to the intent and the basis of interpretation of observa-tions. As indicated above, in well-stained sections nerve fibers and the cellbodies of neuroblasts with processes are usually distinct. Sometimes, however,the edges of epithelial cells are dark and difficult to distinguish from axons, orfibers do not show up well and neuroblasts from which processes arise cannotbe clearly distinguished. In descriptions of individual embryos the lowernumber of fibers recorded includes only those which can be identified withcertainty; the higher number also includes those cellular structures which arethought to be neuroblasts or fibers but which are not clearly identifiable. Bothhave been included in the figures.

The neuroblasts which can be seen in any one embryo represent less thanthe total number present, since the plane of section must be nearly parallel tothe long axis of a neuroblast in order to see the origin of the process from thecell body. It is difficult to see cross-sections of individual fibers if they arescattered singly, even though groups of transversely sectioned fibers show upwell. To obtain an adequate picture of the pattern of nerve cell bodies, whichare oriented in various directions, both transversely and sagittally sectionedembryos must of course be studied. Also, deviation of the plane of section froma true sagittal or transverse plane must be taken into account. In 16- to 18-somiteembryos in particular, the plane of section is often at an angle to transverse orsagittal in part of the diencephalon and mesencephalon because the cranialflexure is beginning and the head of the embryo is turning to the right at thesame time.

Individual cells and fibers of each embryo, and the numbers present in eachcase, have been analysed in order to obtain as much information as possible onthe pattern of differentiation and on the way development proceeds. Specificnumbers, etc., are not intended to have any other significance per se. To repeat,it is felt that study of a series of embryos sectioned in different planes, andincluding several embryos of each stage, gives meaningful information ofthis sort.

A. 14- through 16-somite stage: initial appearance of neuroblasts with processes

The first group includes the least advanced embryos in which nerve fiberswere found in the diencephalon and mesencephalon. In each of these embryosa few neuroblasts with processes and a few additional segments of fibers couldbe seen on each side in the posterior half of the diencephalon. The embryoillustrated (Text-fig. 1), which was sectioned sagittally, has at least 2, andpossibly 4, neuroblasts with processes and 7 other nerve fibers on the right.There are at least 2 and possibly 5 fibers on the left, 3 of which seem to come

Initial neuroblast differentiation 501

from cells in the same sections. In other embryos studied a few more fibers arevisible; 1-4 neuroblasts and 4-20 other fibers were found on each side.

These neuroblasts and fibers are located within an area including the lowerpart of the dorsal half and the upper part of the ventral half of the lateral wall,and extending from the junction of the diencephalon and mesencephalon to

M

Text-fig. 1. Group A: 16-somite embryo, sagittal sections. Graphic reconstructionof neuroblasts with processes (a-d, f-h) and segments of fibers (e, others unlabelled)in the diencephalon and mesencephalon of one of the least-advanced embryos.O, Location of optic stalk. Arrows indicate boundaries of telencephalon (7),diencephalon (D) and mesencephalon (M). A, Right side; B, left side, x 110.

about the middle of the diencephalon. The area covered is not quite as large inthe least-advanced embryos (Text-fig. 1) as in those with slightly more neuro-blasts and fibers. The individual neuroblasts and fibers are scattered singlyamong the neural epithelial cells. There is no indication of a specific cell by cellpattern of distribution within this area. The more ventrally placed neuroblastsin this group appear comparable to those identified by other authors (Tello,

502 K. M. LYSER

1923; Windle & Austin, 1936) as belonging to the future nucleus of the mediallongitudinal fasciculus. The more dorsal neuroblasts, certainly in slightly olderembryos if not at the earliest stages, are farther dorsal than the early cells de-scribed by these authors, and apparently correspond to cells they identifyas thalamo-tegmental or thalamo-bulbar.

The neuroblasts in these embryos are oriented circumferentially with pro-cesses extending ventrally (Text-fig. \a:c, d; \b:f,g; Plate 1, fig. A), or radially,with their long axes parallel to adjacent epithelial cells and their processesextending laterally (Text-fig. 1 a:b; lb:h). There are also some neuroblasts at anangle between dorsal-ventral and medial-lateral orientations (Text-fig. I a:a).Due to the orientation of the cell body and axon arising from it relative to theplane of section, the radially oriented neuroblasts can be seen best in transversesections and the circumferential neuroblasts in sagittal sections. All of the cellbodies of the radially oriented cells and most of those of the circumferentialcells are in the nuclear zone. A few circumferential cells are just outside thenuclear zone.

The axons of almost all of the neuroblasts grow ventrally or slightly obliquelyin a ventral and posterior direction. Those of the radially oriented cells, whichinitially grow laterally, turn ventrally within the nuclear zone, at the borderbetween the nuclear zone and the peripheral zone, or in the peripheral zone(Text-fig, la: b). Fibers that extend ventrally within the nuclear zone tend toenter the peripheral zone eventually. The fibers in the peripheral zone lie in theinner or middle part; very few are found along the outer edge. Occasionallybranching fibers are seen (Text-fig. 1 b:e). In these embryos the fibers whose cellbodies are not seen appear as short, straight or slightly wavy segments, radiallyor circumferentially oriented. In the more advanced embryos some are slightlylonger than those of the younger embryos. Occasionally a few fibers orientedin a longitudinal direction are seen in a ventral position at the junction of thediencephalon and mesencephalon.

B. 15- through 16-somite stage: more advanced embryos

During this period more neuroblasts are added to the original area of differen-tiation and longitudinal fibers appear ventrally. In the more advanced embryosof this group (Text-fig. 2), cells with processes and fibers can be seen fartherdorsally, anteriorly and ventrally in the diencephalon than previously and alsoin the anterior mesencephalon, especially in the ventral part. The distributionof neuroblasts and fibers is more dense than before, particularly at the center ofthe area, where the first neuroblasts were located.

The neuroblasts in the lateral diencephalon are oriented in a radial or circum-ferential direction as the first ones were. The cell bodies are usually locatedin the nuclear zone, and most of the processes extend laterally, ventrally orobliquely in a posterior and ventral direction (Text-fig. 2; Plate, 1 fig. B). Someof these processes turn. An axon arising from the posterior-lateral side of the

Initial neuroblast differentiation 503

cell body may turn ventrally, immediately (Text-fig. 2: a) or at the outer edgeof the nuclear zone (Text-fig. 2:b, c). A ventral process may turn posteriorly(Text-fig. 2:d) or laterally (Text-fig. 2:e). A few axons can be seen to branch(Text-fig. 2:b,f, g). For example, one of these (Text-fig. 2:/) ends at the edgeof the nuclear zone in a triangular enlargement with what seem to be very finebranches curving out dorsally and ventrally. Another (Text-fig. 2:g) dividesjust before reaching the outer edge of the nuclear zone; the two branches curvearound the opposite sides of another cell. One cell (Text-fig. 2:b) extends

M

Text-fig. 2. Group B: 18-somite embryos, sagittal sections. Graphic reconstructionof neuroblasts with processes (a-g, i-j) and segments of fibers (h, others unlabelled)in the diencephalon and mesencephalon, right side. O, Location of optic stalk.Arrows indicate boundaries of telencephalon (T), diencephalon (D) and mesen-cephalon (M). x 150.

laterally to the outer edge of the nuclear zone, where it runs ventrally a shortdistance, then turns in a lateral direction and ends in a Y-shaped branch. Mostof the segments of fibers which can be seen in this area (Text-fig. 2) are orientedin a dorsal-ventral direction or obliquely from anterior and dorsal to posteriorand ventral.

At the ventral edge of the original area in the diencephalon and in theanterior mesencephalon, longitudinal fibers, as well as a few which are radiallyor circumferentially oriented, are seen. Some of the longitudinal fibers seemto be processes of neuroblasts located in the lateral part of the diencephalon.This is suggested by the fact that a few of the most ventral circumferential

504 K. M. LYSER

fibers turn posteriorly among the longitudinal fibers (Text-fig. 2: h) or occasion-ally anteriorly. Other longitudinal fibers can be seen to arise from neuroblastslocated at this level, often in the nuclear zone. The processes extend posteriorly,or posteriorly and laterally, from the cell bodies and pass into the peripheralzone, where they continue in a posterior direction (Text-fig. 2:i). A few of theneuroblasts in the mesencephalon have processes which extend in a ventraldirection (Text-fig. 2:j). As in other areas, a few of the fibers branch.

M

Text-fig. 3. Group C: 18-somite embryo, sagittal sections. Graphic reconstructionof neuroblasts with processes and segments of fibers in the diencephalon and mesen-cephalon, left side. O, Location of optic stalk. Arrows indicate boundaries oftelencephalon (T), diencephalon (D) and mesencephalon (M). x 150.

C. 17- through 18-somite stage

Between the 16- and 18-somite stages, there is further development of thefirst area of axon differentiation, and neuroblasts in a second area, the dorsalmesencephalon, begin to send out processes. The latter are tectal cells.

The number of neuroblasts in the lateral diencephalon continues to increaseand new cells with processes appear anterior and posterior to those seen pre-viously. By the 18-somite stage, nerve fibers and neuroblasts are found fromthe level of the optic stalk to the anterior edge of the mesencephalon (Text-fig. 3).In the anterior part of the diencephalon, short, scattered fibers are seen. Theseare oriented radially or circumferentially. In the posterior diencephalon somelonger fibers are seen and they are more densely distributed. Many of the fibersin the lateral part of the posterior diencephalon are oriented in an anterior anddorsal to posterior and ventral direction. Similarly oriented fibers are seen inthe anterior mesencephalon, constituting the posterior part of this area of

Initial neuroblast differentiation 505

differentiation. Neuroblast cells are oriented so that processes extend laterally,ventrally, or from those in the ventral diencephalon and mesencephalon,posteriorly, or occasionally anteriorly (Text-fig. 3). In some cases the processesextend ventrally and then turn posteriorly.

The number of longitudinal fibers in the ventral diencephalon and mesen-cephalon becomes greater and the area in which they are found increases in size.By the 18-somite stage, longitudinal fibers are present from the area justposterior to the base of the optic stalk to the middle of the mesencephalon

Text-fig. 4. Group D: 21-somite embryo, sagittal sections. Diagram of the patternof neuroblast cells and nerve fibers in the diencephalon and mesencephalon. Thepositions and orientation of representative cells and fibers are indicated; not all ofthe cells and fibers actually visible in the embryo are shown (see Plate 1, figs. C,D). O, Location of optic stalk. Arrows indicate boundaries of telencephalon (71),diencephalon (£>) and mesencephalon (M). x 40.

(Text-fig. 3). There are also circumferential fibers at this level in the diencepha-lon and a few in the ventral mesencephalon. The longitudinal fibers appear tobe processes of longitudinally oriented neuroblasts in the ventral diencephalonand mesencephalon and of neuroblasts whose processes extend ventrally andturn at this level.

Nerve fibers appear in the dorsal mesencephalon by the 18-somite stage. Atthis time the area covered by these fibers is discrete from the first area of differen-tiation. In the embryo in Text-fig. 3, segments of fibers can be seen scatteredalong and to each side of the midline from the posterior boundary of thediencephalon to the anterior end of the rhombencephalon.

D. 19- through 22-somite stage

The most striking feature of this period is the elaboration of the fiber patternwithin the lateral area of differentiation. Neuroblasts and processes becomeconsiderably more numerous, and the segments of fibers visible in each sectionare longer. In addition, a new center of differentiation develops just posteriorand ventral to the base of the optic stalk.

The lateral area of differentiation continues to expand. By the 21-somite

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stage (Text-fig. 4) it extends from just posterior to the optic stalk into theanterior part of the mesencephalon. At the anterior edge of the mesencephalon,in the synencephalon (approximately the posterior third of the diencephalon,which is demarcated for a time in the embryo from the anterior portion of thediencephalon, or parencephalon, by a transitory constriction) and in the pos-terior parencephalon, there are circumferential fibers from the dorsal part ofthe lateral wall to the level of the longitudinal fibers. Anterior to this they arenot found as far dorsally; just posterior to the optic stalk circumferential fiberscan be found from approximately the middle of the diencephalon to the levelof the longitudinal fibers. The most dorsal fibers in the posterior diencephalonand anterior mesencephalon have a dorsal-ventral orientation, axons extendingmore or less directly ventrally from the cell bodies (Plate 1, figs. C, D). Fartherventrally some are at an angle from anterior-dorsal to posterior-ventral andsome from posterior-dorsal to anterior-ventral. Most of the fibers in theanterior part of the parencephelon are oriented obliquely from anterior-dorsalto posterior-ventral.

The ventral longitudinal fibers begin to form a more clearly outlined fascicu-lus. At the 21-somite stage they are seen from the area posterior to the opticstalk to the posterior border of the mesencephalon, where they merge withthe longitudinal fibers of the hind brain. In the posterior parencephalon andin the synencephalon the fasciculus is quite wide, extending from the middleof the diencephalon to the ventral part of the lateral wall. The more dorsallongitudinal fibers curve ventrally as they approach the mesencephalon wherethey form a narrower fasciculus. There are fewer fibers in the fasciculus in themesencephalon than in the posterior part of the diencephalon; in the embryorepresented in the diagram (Text-fig. 4) the number decreases to about five atthe posterior end of the mesencephalon. In the anterior part of the parencephalonthe dorsal edge of the fasciculus also curves ventrally and the fibers decreasein number until there are just a few longitudinal fibers at the level about onefourth of the distance from the floor plate of the diencephalon to the roof.

PLATE 1

Fig. A. 16-somite embryo, group A. Ventral part of the posterior diencephalon, sagittalsection. A neuroblast cell with a process extending ventrally (AT) and two segments of fibers(F) can be seen, x 2000.Fig. B. 16-somite embryo, group B. Lateral diencephalon, dorsal to the middle, sagittalsection. The neuroblast (A0 has a process extending ventrally and slightly posteriorly alongthe border between the nuclear and peripheral zones, x 2000.Fig. C. 21-somite embryo, group D. The same embryo as in Text-fig. 4, mesencephalonand diencephalon, sagittal section. Fibers and neuroblasts in the lateral diencephalon (D)and longitudinal fibers in the ventral mesencephalon (M) can be seen in this section, x 240.Fig. D. 21-somite embryo, group D. The same section as fig. C. Neuroblasts in the posteriordiencephalon with processes extending ventrally and obliquely can be seen, x 1000.

J. Embryol. exp. Morph., Vol. 16, Part 3 PLATE 1

K. M. LYSER facing p. 506

/ . Embryol. exp. Morph., Vol. 16, Part 3 PLATE 2

facing p. 507

Initial neuroblast differentiation 507

Cells seen at the level of the longitudinal fasciculus have processes extendingventrally, at an angle anteriorly or posteriorly, or directly anteriorly or pos-teriorly. Sometimes two adjacent cells have processes extending in differentdirections; in a few instances the processes can be seen to cross near the cellbodies.

At the level of the optic stalk is another group of longitudinal fibers. Theseare oriented at an angle from anterior-ventral to posterior-dorsal. No fibersare visible between this group and the first longitudinal fibers and none can beseen crossing the mid line. No neuroblast cell bodies have been seen in this areain the embryos examined; it is not clear where the neuroblasts which give riseto these fibers are located.

The fibers in the dorsal mesencephalon have a pattern similar to that at theprevious stage; they do not extend much farther ventrally during this period.In the embryo illustrated (Text-fig. 4), a few short pieces of fibers are visibleon each side of the mid line.

E. 23- through 30-somite stage

During this period the areas of axon outgrowth which have appeared duringthe previous stages are enlarged and neuroblasts of the oculomotor nucleusbegin to send out processes.

In the lateral diencephalon more neuroblasts send out processes ventrally orobliquely, but their distribution does not change markedly (Text-figs. 5, 6;Plate 2, figs. A, B). At the end of this period (Text-fig. 6, 28-somite embryo)circumferential fibers can be seen throughout the lateral wall of the posteriordiencephalon. In the anterior diencephalon they are quite numerous in the ven-tral part of the lateral area, but much more sparsely distributed dorsally. At thedorsal edge of the lateral fiber area in the synencephalon and posterior part ofthe parencephalon there are a few fibers oriented longitudinally. There are alsoa few which cross the posterior part of the synencephalon at an angle from dorsaland posterior to anterior and ventral; that is, they seem to run from the dorsallongitudinal fibers to the longitudinal fasciculus. At the anterior end of thisarea, posterior to the optic stalk and just above the center of the lateral wall,

PLATE 2

Fig. A. 28-somite embryo, group E. The same embryo as in Text-fig. 6, lateral part of theposterior diencephalon, sagittal section. This section shows cells and fibers at the middle ofthe lateral wall, including oblique fibers which cross, x 800.Fig. B. 28-somite embryo, group E. This is the next section medial to that in fig. A, showingmore ventrally located cells and fibers in the diencephalon (D) and mesencephalon (M),including longitudinal fibers, x 800.Fig. C. 28-somite embryo, group E. The same embryo as figs. A and B, mesencephalon,sagittal section. This section is at the level of the oculomotor nerve. Its fibers can be seenemerging from and outside of the neural tube. Sections of longitudinal fibers (L) can be seenposterior to the oculomotor nerve, x 1000.

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there are also a few fibers oriented longitudinally. At the posterior end of thesynencephalon fibers oriented more or less transversely seem to cross thedorsal mid line. These fibers are not separated anteriorly and ventrally fromthose of the lateral area or posteriorly from the fibers of the dorsal mesencepha-lon. Neuroblasts that have processes extending ventrally or obliquely can beseen throughout the lateral diencephalon in sagittal sections; in transversesections, cells with processes extending laterally are apparent.

Fig. 5 Fig. 6

Text-fig. 5. Group E: 24-somite embryo, sagittal sections. Diagram of the patternof neuroblast cells and nerve fibers in the diencephalon and mesencephalon.O, Location of optic stalk. Arrows indicate boundaries of telencephalon (71), dien-cephalon (D) and mesencephalon (M). x 40.

Text-fig. 6. Group E: 28-somite embryo, sagittal sections. Diagram of the patternof neuroblast cells and nerve fibers in the diencephalon and mesencephalon.O, Location of optic stalk. Arrows indicate boundaries of telencephalon (T), dien-cephalon (D) and mesencephalon (M). x 40.

The number of fibers in the juxta-optic group increases also. In 24-somiteembryos (Text-fig. 5) more fibers than at the previous stages can be seen onboth sides posterior and ventral to the base of the optic stalk. As before, theyare oriented predominantly in an anterior and ventral to posterior and dorsaldirection. However, they are no longer separate from fibers of other areas.Circumferentially oriented fibers are found as far anterior as the level of thejuxta-optic fibers, just dorsal to them. Posteriorly, the juxta-optic fibers mergewith the longitudinal fasciculus. In this embryo fibers cannot be seen crossingthe ventral mid line. By the 28-somite stage (Text-fig. 6) the fibers are morenumerous still, especially just posterior to the optic stalk, and there are somefibers ventral and medial to it. In transverse sections, fibers crossing the midline at the level of the posterior edge of the optic stalk can be seen.

Initial neuroblast differentiation 509

At the 24-somite stage, the area of differentiation in the mesencephalon isa little more extensive than in embryos of the 19- through 22-somite group.In addition to circumferential fibers at the anterior edge of the mesencephalon,there are now fibers in the ventral half of the lateral wall as far posterior asthe middle of the mesencephalon. In this area some neuroblasts with fibersextending ventrally or obliquely posteriorly and ventrally can be seen. As before,there are circumferential fibers in the dorsal midbrain. Occasionally a fairlylong fiber extends ventrally to the middle of the mesencephalon. In the dorsalpart there are also a few longitudinal fibers.

By the end of this period (Text-fig. 6), circumferentially oriented fibers arepresent throughout the lateral wall of the midbrain. They form one con-tinuous group, which also includes transversely oriented fibers in the mid-dorsalregion. Most of the lateral fibers are dorsal-ventral or slightly oblique. Anothernew feature which appears during this period is longitudinally oriented fibersat the middle of the lateral area of the mesencephalon. The ventral ends of someof the circumferential fibers can be seen to turn posteriorly at this level, thuscontributing to the longitudinal group. Neuroblasts with processes extendingventrally or obliquely can be seen in the mesencephalon.

The number of fibers in the ventral longitudinal fasciculus and the numberof cells with processes located at this level also continue to increase. In theyounger embryos of this group (Text-fig. 5), there are only a few longitudinalfibers in the posterior mesencephalon and not many in the anterior diencephalon.By the 28-somite stage (Text-fig. 6), the ventral longitudinal fasciculus containsa considerable number of fibers throughout. The fibers are more or less parallelto each other, though there is some crossing. In the mesencephalon the fasciculusis located at the ventral-lateral corner of the neural tube. In the synencephalonand posterior parencephalon it is in a more dorsal position and is wider; themost dorsal fibers are at about the middle of the diencephalon. Anterior to thisthe fasciculus becomes narrower and is continuous with the fibers of the juxta-optic area. There are circumferential fibers at the level of the longitudinalfasciculus, that is, crossing the longitudinal fibers, particularly in the posteriordiencephalon. A few circumferential fibers which turn anteriorly or posteriorlyat the level of the longitudinal fibers are visible. Neuroblasts with processesextending posteriorly, or occasionally anteriorly, posteriorly and ventrally oranteriorly and ventrally, can be seen among the longitudinal fibers.

Fibers of the oculomotor nerve are first recognizable in 25-somite embryos.They are located near the anterior end of the mesencephalon at the medial edgeof the longitudinal fasciculus. The oculomotor area is not completely separatefrom other areas of differentiation, since by this time neuroblasts are recogniz-able at the level of the longitudinal fasciculus, just lateral to the oculomotorarea. No specific pattern of arrangement within the oculomotor area is dis-cernible during the period under consideration. In the youngest embryos inwhich oculomotor fibers can be identified, a few can be seen emerging from or

32 JEEM 16

510 K. M. LYSER

just outside the ventral mesencephalon on each side. For example, in one25-somite embryo, sectioned transversely, all the oculomotor fibers can be seenin four sections (six sections distant from the anterior edge of the mesencephalonand eighteen sections from the posterior edge). They are located at the medial edgeof the longitudinal fasciculus. In the first section there are three fibers ventralto but not touching the neural tube on the right side and one on the left whichis touching the edge of the neural tube but cannot clearly be seen to come fromwithin the mesencephalon. In the second section there are two fibers emergingfrom the mesencephalon on the right and one on the left. In the third sectionthere is one fiber emerging on the right. In the fourth section there is one fiberon the right which is touching the edge of the mesencephalon but not definitelyemerging in this section. The more lateral emerging fiber on the right in thesecond section turns out of the section just inside the edge of the neural tube.The other emerging fibers seem to come from within the nuclear zone in thesame section; the cell bodies cannot be identified. Just anterior to the fibers ofthe oculomotor nerve there are some radial fibers medial to or at the level of thelongitudinal fibers which may also be oculomotor. Some are in the nuclear zone,some pass from the nuclear zone into the peripheral zone.

In slightly older embryos, more fibers and some neuroblasts of the oculo-motor nucleus can be seen anterior and posterior to the first fibers as well asamong them (Text-fig. 6; Plate 2, fig. C). They are located at the medial partof the longitudinal fasciculus and just medial to it. A few of the neuroblastswhich give rise to the oculomotor fibers can be identified near the edge of theneural tube at the medial side of or just medial to the longitudinal fasciculus.They have processes that extend ventrally and emerge from the mesencephalon.In embryos sectioned sagitally, neuroblasts can be seen which have processesthat extend posteriorly and then turn ventrally to leave the neural tube.

In summary, during the period of initial neuroblast differentiation in thediencephalon and mesencephalon the following sequence has been observed.The first neuroblasts with processes were seen in 14-somite embryos in thelateral part of the posterior diencephalon. Their axons extend laterally orventrally from the cell bodies; the lateral fibers turn ventrally. By the 15- or16-somite stage the beginning of a ventrally located longitudinal group of fibersis indicated. The longitudinal fibers are apparently processes of laterally locatedcells which turn posteriorly at this level and of cells located among the longi-tudinal fibers. At about the 18-somite stage a new area of differentiation appearsin the dorsal mesencephalon with fibers extending circumferentially. The nextarea of differentiation is visible by the 21-somite stage in the juxta-optic region.All of these areas are enlarged so that by the 30-somite stage neuroblastsand fibers are present, fairly evenly distributed, over most of the lateral wall ofthe diencephalon and mesencephalon. The more dorsal and lateral fibers aremainly circumferential or oblique. A prominent group of ventrally locatedlongitudinal fibers extends from the juxta-optic area through the mesencephalon,

Initial neuroblast differentiation 511

and some longitudinal fibers are also seen dorsally in the posterior diencephalonand along the middle of the lateral wall of the mesencephalon. Oculomotorfibers, from neuroblasts in the ventral mesencephalon, are first recognizableat about 25 somites.

DISCUSSION

The general pattern of differentiation as described above agrees for the mainpart with previous studies which include early stages of nerve-fiber development(Tello, 1923; Windle & Austin, 1936; Van Campenhout, 1937). Observation offibers in slightly younger embryos in the present study may be due to differencesin staining procedures, as well as possible variations in embryos and determina-tion of stages. The present study is concerned primarily with details of develop-ment during this period and does not include study of older stages which wouldbe necessary for more extensive discussion of the identity of neuroblasts andfiber groups in terms of adult nuclei and tracts.

From these observations of the development of the diencephalon and mesen-cephalon in 13- to 30-somite chick embryos, the following generalizations canbe made about the way in which the initial phase of differentiation proceeds.(1) Several different areas of differentiation can be recognized in the diencepha-lon and mesencephalon, which appear in the embryo in regular sequence.(2) In each area the first neuroblasts are distributed in a scattered fashion, thearea is progressively enlarged by the differentiation of additional cells at itsedges, and at the same time new cells differentiate within the old area. (3) Pro-cesses of the cells in each area, or part of an area, grow out in a generallyconsistent and characteristic direction, with small irregularities and variationsof the courses being typical.

The sequence of initial neuroblast differentiation in the diencephalon andmesencephalon is more complex than the anterior-posterior, dorsal-ventralpattern seen in the development of the embryo in general. Differentiationbegins in the diencephalon and mesencephalon after it has started fartherposteriorly, in the hind brain. Within the mid- and forebrain it occurs first inthe lateral wall of the posterior diencephalon and anterior mesencephalon,followed by the more posterior and dorsally located dorsal mesencephalon, andthen by the more anterior juxta-optic area. Furthermore, in each area, differen-tiation spreads from the initial location in various directions. Such a charac-teristic pattern implies a specific regional differentiation within the neural tubeat the time of initial differentiation of neuroblasts. This organization may result,directly or indirectly, from the very early regional differentiation of the medul-lary plate and neural tube. The latter is demonstrated by the distinctive grossform of various parts of the brain and by developmental capacities underexperimental conditions, such as experimental regional induction or develop-ment of isolated regions, including development of characteristic patterns offunction (Corner, 1964). Separation of parts of the neural tube in vivo during

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512 K. M. LYSER

initial neuroblast differentiation demonstrates the ability of these regions todifferentiate further, morphologically and functionally, without continuity withone another (Rhines & Windle, 1944; Hamburger & Balaban, 1963; Hamburger,Balaban, Oppenheim & Wenger, 1965). This does not necessarily imply com-plete independence of differentiation in the neural tube. There is some evidencefrom organ-culture studies that differentiation in the central nervous system isinfluenced by the surrounding environment, including adjacent tissues (Szepsen-wol, 1940a, b; Lyser, 1966). Though certain adjacent tissues affect histologicalorganization in the spinal cord (Holtfreter, 1939; Holtfreter & Hamburger,1955), the effect could be somewhat non-specific in terms of cellular differentia-tion, comparable to certain cases of mesenchymal-epithelial interactions(Grobstein, 1953, 1962; McLoughlin, 1961). The influencing factor couldsupport development of a cell type determined by the specificity of the regionof the neural tube.

The characteristic, specific pattern observed pertains to the location andsequence of differentiation of groups of neuroblasts. No evidence of a specificpattern of individual cells has been found. This possibility cannot be ruled out byobservations such as those in this work, where all the cells cannot be seenbecause of their orientation relative to the plane of section, but it seems un-likely that such would occur.

The scattered distribution of the first cells within an area can probably becorrelated with their early differentiation. As regards each individual neuroblastcell, the sequence of proliferation followed by differentiation, and especiallythe absence of division once morphological differentiation has begun, is adheredto. Since proliferating cells are distributed all along the neural tube, and sincemuch proliferation is still to take place in all areas, it is not surprising thatscattered individual cells from among the neural epithelial cells differentiatefirst. This leaves the rest to divide until a sufficient number have been producedto form a mantle layer and an inner neural epithelial layer.

What is responsible for the initiation of differentiation in certain cells isa question still to be answered. The explanation must cover the localizationof the process in specific areas and also selection of a limited number of cellsin each area out of a large population, many of which would presumably beable to differentiate in the same way.

The sequence of differentiation in the earliest neuroblasts appears to bedifferent from that of the classical description, applicable to later stages; butit is consistent with the description of Windle & Austin (1936; see Windle &Baxter, 1936, and Lyser, 1964). The presence of radial cells, circumferentialcells and cells with an orientation intermediate between these suggests that theneuroblasts begin to send out processes while still having the position and orien-tation of neural epithelial cells. They subsequently shift to a position where themantle layer will form and sometimes, as in the lateral diencephalon, changetheir orientation.

Initial neuroblast differentiation 513

The characteristic pattern of development pertains to the orientation ofneuroblast cells and the courses of their axons as well as to the location andsequence of differentiation. Radial orientation of cell bodies is not necessarilyincluded in this category, but rather may be a reflexion of the sequence ofdifferentiation in the early neuroblasts. Otherwise, cell bodies generally havefairly consistent orientations which can be considered characteristic of eacharea. The courses of individual fibers suggest a specificity in regard to the generaldirection, with small variations probably due to a number of other factorsinfluencing the pathway at the same time. Small deviations in the courses ofindividual fibers suggest that they are following a path of least mechanicalresistance around various obstacles. Mechanical factors have been shown to beimportant in determining the pathways of nerve fibers under various experi-mental conditions, and could exert some effect on an outgrowing fiber in theembryo also. For example, nerve fibers need a surface or interphase along whichto grow (Lewis & Lewis, 1912; Harrison, 1914). Nerve processes grow alongthe fibrils in a plasma clot that have been oriented by stroking (Weiss, 1934).In regenerating tadpole tails, nerve fibers grow along the surface of fibroblastsand of other nerve fibers and also can be blocked by fibroblast cells and pro-cesses when these structures form obstructions in the paths of the fibers (Speidel,1933).

However, it seems that the overall pattern would be much more random,instead of fairly consistently the same for the fibers in a given area, if therewere not some more specific factor directing the fiber in a particular direction.No explanation of such a directive mechanism is apparent, but initial out-growth of fibers may be comparable to experimental situations, includingregeneration of fibers, where there is evidence of very specific selection of path-ways. For example, if the hind brain is reversed or if an obstruction is placedin its path, the axon of Mauthner's cell eventually assumes a position in thecord at, or fairly near, its normal location (Piatt, 1943, 1947; Stefanelli, 1950,1951). Also, after removal of spinal ganglia in tadpoles previous to the develop-ment of the hind limb there is an almost normal motor pattern and there areno nerves in the sensory pathways. After removal of ventral horn cells, a normalsensory pattern develops (Taylor, 1943, 1944). The most striking example ofthe growth of particular fibers to particular end structures is the regenerationof amphibian and teleost optic fibers from each part of the retina to a specificarea of the optic tectum, even after the eyeball is rotated (Sperry, 1945, 1948).

Since a consistent pattern of bifurcating fibers is not apparent, in the di-encephalon and mesencephalon the branching of axons observed once in a whilemay represent temporary branches from fine processes of the growth cone, oneof which will be established as the next segment of the fiber and the otherswithdrawn. The absence of many random fibers argues against formation andretraction of any but very short branches. Selection from among fibers reachingvarious other cells, as seen, for example, in regenerating tadpole tails, where

514 K. M. LYSER

cutaneous fibers sometimes grow toward muscles instead of toward the surfaceand are eliminated by retraction or degeneration (Speidel, 1942), is unlikelyto occur here.

The extent of cellular differentiation, or determination, with respect to neurontype at the time of initial outgrowth of axons, is not known. The morphologicalpattern suggests differentiation at least of cells in one major area of developmentas distinct from those in another, or as embarked on a different course ofdevelopment. The neuroblasts in the lateral diencephalon area, with processesextending ventrally and posteriorly, are distinct from those of the oculomotornucleus, with processes passing out of the neural tube. However, within eacharea cells may or may not be differentiated into more specific types, which willlater be included in various nuclei or parts of nuclei and have characteristicconnexions. One of the simpler examples of differentiation within a group ofneuroblast cells related to the present observations is the oculomotor nucleus.There is no indication of separate groups of cells at the beginning of differentia-tion, though the cells innervate four different extrinsic eye muscles, and henceare four functionally different groups of neurons, which are probably arrangedin a particular way in the adult. (This has not been demonstrated in birds butthere is some information on functional localization in mammals; see Warwick,1953).

Regardless of the extent of specificity present in the neuroblasts at the timethey begin to send out processes, the pattern of the location and the sequenceof initiation of differentiation does not parallel that of future nuclei. Forexample, the first neuroblasts in the diencephalon-mesencephalon area seemto be scattered so as to include thalamo-tegmental and thalamo-bulbar cellsas well as those of the future nucleus of the medial longitudinal fasciculus inone continuous area. This pattern suggests that the control of the initiation ofdifferentiation may be separate from factors determining particular pathwaysand connexions specific for each type of neuron; cells are somehow 'triggered'to differentiate but the particular type of differentiation depends on somemechanism already set within the cells, or on other factors influencing it at thesame time.

The pattern of differentiation of the first neuroblast cells and their processesin the diencephalon and mesencephalon of the chick embryo thus points outseveral problems: What is responsible for the initiation of differentiation inspecific locations? How is the specificity of each individual cell acquired? Whatis the mechanism of directional growth of a nerve fiber? Does the pattern ofinitial differentiation influence subsequent formation of nuclei? It is hoped thatthis study of the pattern in the normal embryo will be the basis of furtherinvestigations to obtain information on some of these questions.

Initial neuroblast differentiation 515

SUMMARY

1. The pattern of cells and nerve fibers in the diencephalon and mesen-cephalon during the initial stages of neuroblast differentiation has been studiedin silver-impregnated sections of 13- through 30-somite chick embryos.

2. The first neuroblasts were seen at the 14-somite stage, located in thelateral part of the posterior diencephalon with axons extending laterally andventrally. Ventral longitudinal fibers appear by the 15- or 16-somite stage.Centers of differentiation appear subsequently in the dorsal mesencephalon,and the juxta-optic area. Oculomotor fibers appear at about the 25-somite stage.

3. The differentiating neuroblasts are scattered; the initial areas are extendedby differentiation of additional neuroblasts among the first cells and at theedges of original areas.

4. The orientation of neuroblast cell bodies and the directions of fibers arecharacteristic for each area.

5. These observations demonstrate a specific pattern of development withinthe nervous system and emphasize the need for further investigation of the factorscontrolling the various aspects of differentiation.

RESUME

Le developpement du diencephale et du mesencephale d'embryon de pouletau cours des phases initiales de differentiation des neuroblastes

1. On a etudie la disposition des cellules et des fibres nerveuses du dien-cephale et du mesencephale au cours des stades initiaux de la differenciation desneuroblastes sur des coupes d'embryons de poulet de 13 a 30 somites, impreg-nees a 1'argent.

2. Les premiers neuroblastes ont ete observes au stade 14 somites, localisesdans la partie laterale du diencephale posterieur avec des axones s'etendantlateralement et ventralement. Des fibres longitudinales ventrales apparaissentaux stades 15 ou 16 somites. Des centres de differentiations apparaissent parla suite dans le mesencephale dorsal et la region juxta-optique. Les fibresoculomotrices apparaissent aux environs du stade 25 somites.

3. Les neuroblastes en differenciation sont disperses; les zones initialess'etendent par differenciation de neuroblastes additionnels au milieu despremieres cellules et sur les bords des zones d'origine.

4. L'orientation des corps cellulaires des neuroblastes et la direction desfibres sont caracteristiques pour chaque zone.

5. Ces observations mettent en evidence un plan de developpement specifiquedans le systeme nerveux et soulignent la necessite de nouvelles recherches sur lesfacteurs qui controlent les divers aspects de la differenciation.

The author wishes to express her appreciation to Professor Leigh Hoadley for all of hishelp in many ways. Part of this work was done during the tenure of a National ScienceFoundation Pre-doctoral Fellowship.

516 K. M. LYSER

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{Manuscript received 19 May 1966, revised 10 August 1966)