differentiation of neuroblasts in the chick optic tectum up to eight days of incubation: a golgi...

DIFFERENTIATION OF NEUROBLASTS IN THE CHICK OPTIC TECTUM UP TO EIGHT

DAYS OF INCUBATION: A GOLGI STUDY

L. PUELLES and MARIA C. BENDALA Department of Anatomy, Faculty of Medicine of Cad& and

Department of Anatomy, Faculty of Medicine of Sevilla, Spain

Ahtract-Ted histogencsis up to the 8th day of incubation is described, according to adze in Golgi-impregnated material. Emphasis is placed on the mode of initial differentiation of the different types of neuroblasts and evidence has been obtained that there are essentially two kinds of neurobhtsts within the optic tectum.

Type I cells initiate their differentiation process, starting from an interphase epithelial morphology, by losing their ventricular attachment while they keep the subpial one. This subpial attachment subsequently transforms into a sprouting for~ti~, whose growth produces the axon of the neuroblast. The cell body is transported by translocation of the nucleus within the peripheral cylinder of cytoplasm. Periventricuhu, multipolar and shepherd’s crook neurons of the tectum are shown to differentiate out of type I neuroblasts.

Type II cells lose their ventricular attachment shortly after mitosis, and they are thus free unattached cells. They migrate radially to the superEcial tectal strata, forming the cortical plate of the tectum. Their leading processes transform into dendrites, and the axons grow finally out of the inner poles of the cell bodies. These neuroblasts ditTerentiate into a set of known adult neuronal types (~~forrn radial, horizontal and stellate neurons of the stratum griseum et fibrosum super&ale).

IN HIS studies on the histogenesis of the spinal cord and of the axebellar and cerebral vortices, RM&N Y CAJ~. (1909; 1911) introduced the use of the Golgi method in developmental brain research. Immature neuronal and glial cells can be visualized on a clear background alter suazessful impregnation. This effect allows a classification of the cells impregnated at each stage of development on a double basis: their tridi- mensional morphology and their relative position within the neuroepithelium. An analysis of such material from embryos of gradually increasing age produces a quasidynamic picture of neuronal development-a picture which must be taken into account in any explanation of the mechanisms of neurogenesis.

The optic tectum of birds has attracted consider- able attention in the past. It is organized as a complex multilayered cortex, and it represents a choice area for the study of nervous connectivity, given the mass- ive topographically ordered projection of the contra- lateral retina upon it (HMntI & Wan 1954; MCGILL, PCBWELL & COWAN, 1966). According to the most widely used terminology, derived from the scheme of Huentr & CROSBY (1933), the adult tectum is divided into six basic layers from the surface in-

Abbreuiutions: HH, stage of development of chick embryo according to -URGER & HAMILTON (1951); SAC, stratum album centrale; SFP. stratum fibrosum periventricukxe; SGC, stratum griseum centrale; SGPS, stratum griseum et fibrosum superflciale; SGP, stratum griseum periventriculare.

wards: stratum opt&m; stratum griseum et Jibroswn ~~~c~ (SGFS); stratum gristwn central (SGC); stratum uZbwn centrule (MC); stratum grisewn periefrn- triealme (SGP) and stratum jbrosum periventrMure (SFP). The SGFS can be subdivided into ten alternat- ing cellular and plexiform laminae.

Several distinct types of adult tectal neurons are widely recognized (&+x&4 Y CAJAt, 1889; 1911; VAN Gus, 1892; LB-, 1957; 1958; LA VAIL & COWAN, 19714. The SGFS contains piriform or fusiform radial cells, as well as small horizontal and stellate neurons. The SGC contains mostly big multipolar neurons, although some radial cells may be also found in it. The SGP has relatively few multipolar, radial and horizontal neurons.

The pioneer developmental studies of TELLO (1923) in chick embryos were done with the reduced silver method of Cajal, which has not proved to be very successful in developing cortical areas. L~HISSA (1957; 1958) only succeeded in obtaining Golgi impregnations in embryos older than 9 days of incubation. The scanty info~tion based on the Golgi method contrasts with the detailed studies reported by LA VAIL & COWAN (1971o,b), who used aniline stains and the autoradiographic technique. However, shortly before this paper was submitted for publica- tion, DoMEsic~ & MOREST (1977&b) reported Golgi observations on the developing multipolar cells (ganglion cells) and shepherd’s crook cells of the optic tee- turn.

We report here the results of a thorough Golgi study of the developing tectum in the chick up to

307

308 L. PWI.LES and MARIA C. BENUALA

the 8th day of incubation. Our purpose has been to obtain a coherent picture of the mode of appearance of the different neuronal types, without placing emphasis in any one specific type. This study was pursued, therefore, until every cell form encountered could be assigned to a definite developmental sequence. At the 8th day stage, the fundamental strata of HUBER & CROSBY (1933), although still incomplete,

already are demonstrable as primordia. Further de-

velopment of the tectum, especially of the SGFS. will be dealt with in a subsequent paper.

EXPERIMENTAL PROCEDURES

Chick embryos were incubated at 38”C, staged according to the tables of HAMBURGER & HAMILTON (1951), and fixed by immersion in the solution of STENSAAS (1967). After 24 h, they were transferred to a 0.75% (w/v) silver nitrate solution, after brief washing in tap water. Two days later, the impregnated embryos underwent rapid dehydration and were embedded in low viscosity nitrocellulose. The blocks were cleared in cedarwood oil, and sectioned in a plane transverse to the long axis of the midbrain. The sections (60 pm thick) were collected serially in a dish with toluol, and were mounted under coverslips with Cover- bond (Harleco). In our hands this technique gives first quality impregnations at all stages of development studied. Table 1 shows the data on the number and stage of development of the embryos employed for this work. They constitute a closely graded series from stage HHl6 to stage HH34.

Although the whoie extent of the optic tectum was scanned in each embryo, descriptions and drawings are based on the structures present at the most advanced zone, located ventrolaterally in the rostra1 third of the tectal vesicle (LA VAIL & COWAN, 197la.b). The photomicrographs are a representative selection from more than I500 photo- graphs taken during this study. Drawin@ were made with a camera lucida attachment. Besides the Golgi material, chick embryo series of our collection were at hand, includ-

TABLE 1 -

Stage Embryonic Number of (HAMBURGER & age embryos HAMILTON, 1951) (Ex) studied

16 E2 1 17 E2-S 2 18

::t I

19 1 20 E% 21 E3i_ 2 22 E3+4 3 23 E4 5 24 1 2 25 Z&S 3 26 3 27 ::-5+ 1 28 ES&6 3 29 E6 7 30 E6+-7 3 31 4 32 ::-74 4 33 E7+-8 4 34 E8 4

ing material stained with cresyl v101e1 and Embryos impregnated in the block with Cajal’s reduced silver method,

The nomenclature of the BOULI~ Cnwm-rr:~ (1970) ii followed, using the term ‘neuroblast’ :I\ ;vnonymous with ‘immature neuron’. The following critrri;r have bcrn MZLI

to distinguish between ventricular ~11s and postmltoric neuroblasts during the early stages of differentiation H hen neurites are still absent: (a) k’m~ricth c :,ii.\. irrcspectw:

of the moment of the cell cycle in whrch they become em- pregnated, alwuys display a rwr~rrc.rd~ ~.I~~u~/wI~vI! tit I hc inner surface of the neuroepithelium. ibt &~ntirr~r~ W~W(I blasts may be considered to he arrrsicri in rhe Crl phase of the cell cycle. and this facr is used :.> characterlzc them in autoradiographic studies. Employing the Golgi method. however, we can only detect the transformation of a \-cry young cell into a neuroblast when it r, II’IIC~S thy ~ntrrcular process characteristic of the ventricular cell<. A> will he seen in this paper, this moment may l:ccur early ‘1~ ~;IW in the Gi phase of the cell cycle.

RESULTS

Cytoarchitectural dewlopmcnt

Up to stage HH21 of development (E34 embryos),

the structure of the tectal wall, as seen in our material, is that of a simple pseudostratified neuroepithelium (Figs 1 and 10). It contains proliferating ventricular cells, which are attached at the pial and ventricular surfaces. Intermediate stages of the cell cycle. when the ventricular cells round up for mitosis at the ventricular surfa= (HINDS & RUFFE~. I971; DERER,

1974), and when postmitotic cells grow a new pial process, were present in our material (Fig. 1, v).

During the next few stages of development (HH22-HH26) cresyl violet staining detects the appearance of a primitive marginal zone. This coin- cides with differentiation of the first neuroblasts, which send their axons circumferentially towards the tegmentum (see below and Figs 2 4). Stages HH27 and HH28 show incipient migration of cell bodies into the zone occupied by the circumferential fibers, and we may thus speak of marginal. intermediate and ventricular cytoarchitectural zones (Fig. 5). At 6 days of incubation (stage HH29). accumulation of postmitotic neuroblasts just peripheral to the ventricular zone, before they migrate outwards, allows us to distinguish a subventricular zone (Fig. 6). We have not detected cell bodies of proliferating ventricular cells at this level.

A cortical plate appears within the developing tec- turn in E7 embryos (HH31). A rapid increase in over- all thickness of the tectal vesicle occurs between the 6th and the 8th days of incubation. This growth, and especially that of the cortical plate, continues during the following stages. E8 embryos (HH33, HH34) di+ play further progress in cytoarchitectural differentiation. The intermediate zone subdivides into an inner, cell-poor portion, the anlage of the SCA, and an outer, cell-rich portion, the an&e of the SGC. The ventricular and subventricular ~zones thin out, contributing to the growth of the SAC. A distinct

Tectal neuroblasts in the chick embryo 309

stratum opticurn appears at the SUM of the m&urn (Figs 9 and 36). In Golgi-impregnated preparations, the upper half of the cortical plate is seen to be occupied by a dispersed, circumferential fiber plexus. These fibers are more compact at the middle level of the cortical plate. We shall refer to this compact portion of the plexus as the ‘intracortical plexiform layer’ (Fig. 36). Our findings in older embryos indicate that this layer represents the forerunner of the plexiform lamina ‘h’ of the adult SGFS (b VAL & COWAN, 1971u).

Two types of neurobhst

Our observations have disclosed the existence of two basic types of neuroblasts in the developing optic tectum. The essential differences relates to the moment of the Gl phase of the cell cycle in which they detach from the ventricle, thus becoming identifiable as neuroblasts. They are distinguished throughout this work as type I and type II neuroblasts. It will be shown that, in both cases, differential growth of neurites leads to the appearance of a characteristic set of adult neuronal types.

Differentiation of type I neuroblasts

The earliest cells which may be classitied as very young neuroblasts were detected in E3t4 embryos (HH22, HH23) (Fig 2, I and Fig. lla-f). Their cell bodies are mixed with those of the ventricular cells, but they are characterized by being attached at the pial surface only, having lost the ventricular attachment. A short remnant of the retracting ventricular process may be present, but it never contacts the ventricular surface. The most interesting feature of these cells is found at their subpial attachment. This structure has a variable configuration: in its simplest form it is a tiny rounded terminal enlargement of the pial process (Fig 1 la, b); in other cases it has a sprouting appearance, with several knobby lateral outgrowths (Fig. 1 ld,e); finally, a number of them display a short digitiform outgrowth which courses tangentially under the external limiting membranei (Fig llf). Some of these cells which have detached from the ventricle have double subpial attachments (Fig llc), corresponding, probably, to those ventricular cell precursors which have bifurcated pial processes.

Observations in later stages of development allow us to assume that the different conformations of the subpial attachments of these cells represent different stages of the sprouting of an axon. We have classified these cells as neuroblasts, therefore, on the basis of the loss of the ventricular attachment and the appearance of a polarization of the soma, as indicated by

I The term ‘external limiting membrane’ refers to the basal lamina of the neural epithelium. Although it is invis- ible itself in Golgi-impregnated material, its location may be detected as a sharp boundary, using phasecontrast optics, or manipulating the iris diaphragm of the micro- scope (see Figs 11, 13, 15, 26, 27 and 29).

the htcipient growth of an axon. All cells displaying the &aracteristics described above will be called ‘type I postmitotic neuroblasts’ in this paper. Subsequent developmental configurations of this type of cell will be referred to as ‘type I neuroblasts’ or, later on, as ‘type I neurons’.

E4&5 embryos (HH24-HH26) display elongation of the incipient axons of type I neuroblasts. Near- tangential sections to the tectum show that they group together in fascicles and course circumferentially towards the tegmentum. All the cells which display such an axon have a similar conformation: the more or less piriform cell bodies each have a radially disposed process, which bends just beneath the external limiting membrane and continues as a circumferential axon (Figs 3, I; 4, I; 12a,b; 13a,b). Those neuroblasts which have the longer axons display the bending of their process deeper within the marginal zone. This seems to indicate that contact with the basal lamina is lost at the moment when the axon starts to grow. As the tectal wall continually gains in thickness, and given that new axons sprout always just beneath the external limiting membrane, the older axons are left at the inner levels. The cell bodies of these neuroblasts lie within the ventricular zone, mixed with the ventricular cells and with numerous type I postmitotic neuroblasts which do not have axons yet (Figs 3, I;

4, I). E5-5f embryos (HH27-HH28) already have an

intermediate zone which contains mostly circumferential axons of type I neuroblasts (Figs 5, I; 14a,b). Axonal growth cones are frequently seen throughout this zone. Numerous type I postmitotic neuroblasts are detected, showing the transformation of their subpial attachments through sprouting into axonal growth cones (Figs 5 ; 1 Sa, b). The cell bodies of some type I neuroblasts are now found outside the ventricular zone. They lie tangentially, having translocated up to the level where their original processes adopted a circumferential course (Figs 5, It; 14a,b).

In E6 embryos (HH29) many type I neuroblasts still have their cell bodies in the subventricular zone (Fig. 6). Their ascending process bends at a certain level of the intermediate zone and continues as a circumferential axon (Fig 6, I). Within the inner levels of the intermediate zone, type I neuroblasts are seen, the cell bodies of which lie just below the bending of their processes. Other elements already have their somata oriented tangentially (Fig 6, It). At this stage we detect an increased number of type I postmitotic neuroblasts sprouting an axonal growth cone under the external limiting membrane. Their cell bodies lie within the subventricular zone (Fig. 6).

Subdivisions of type I neuroblasts. In the E7 embryos (HH31) the type I neuroblasts begin to differentiate into definite neuronal types. This population of neuroblasts lies dispersed throughout the subventricular and intermediate zones. They do not participate in the formation of the cortical plate, which contains only type II cells at this stage of development.

310

Ftr.~ l-9. Camera lucida drawings of representative cell types ui the tndicated d~v~l~pn~~n~i~~ rtage>. as seen in transverse sections of the tectum. Ma~i~~tjon is the same throughout this series of Figures. The chief ~ytoarchitectonic borders are marked by interrupted lines. The striped areas ~~~~pond

to axon-rich plexiform or white matter WLL~~. Abbreviations: C. Cortical zone; IN, Intermediate zone; M. Marginal Lone; SV. Subventrrculdr ,~~)nc:

V. Ventricular zone; OC, IC, Outer and inner cortical zone: OIN. IIN. 0uter aad inner j~l*~rm~~ii~~~

zone; if%, IRtracorticaI plexiform layer: I, Type I postmjtoii~ nemoblast; Ia_ Ar&‘ornl type 1 fr’cUio- blast; h. Multipolar type I neuroblast; Ip. Periventricular type I neuroblast: it. ~anger~~~~~~ tFpe 1 nemoblast; II, Type II postmitotic neuroblast = freety migrating neuroblast; IIe. t:,longured 1.v~~ 11

mKObk&: IIh, Horizontal type II nemoblast; IIm, Mitra! type I1 neuroblilst; IjF. R&d tpe 11

neuroblast: Ifs. Prospective stetlate type II neurobjast : \. Ventrjcu]ar ceil

h. $0. Tad neuroepitheii~~m impregnated at 31 days of inoubation. I tf&

I%. 1 k-f. Type I postmitotic neuroblasts of tke .&4th day stage. drspiaying their subp&i atra~~m~nts~ The smait arrow at ‘f” shows an incipient axonai process. x 432.

FIG. 12% b. Type I postmitotic neuroblasts at “If 5 days of incubation, The small arrow at ‘;I* shows the growing axon. Double suhpiai attachments are seen at ‘h’. x 432

FIG. 13&b. Typical early type I neurobiasts at 5 days of incubation” Arrowhead = axonal growth cone. The ceils are detached from the externaf Limiting membrane. x 432.

FIG. 14a,b. Nearly tangential and tangential type f neurobfasts at 5.54 days of incubation. The interrupted line marks the border between the ventricular zone and the prospective intermedtate zone.

” t 08

FOG. 1%. b. Type I postmitotic neurobiasts at 5-S) days of incubation. Small arrows indicate &be subpial attachments tr~sforming into axonat growth cones. x 108.

FIG. l&e. Type II (freely mi~ating) neuroblasts at 551 days of incubation. (a,@ dells oear the ventricle (arrowheads): (c) cefl approaching the intermedjate zone: (d.e) cells entering the intermediate

)rone. x 43-1.

FIG. 17a-i. Developmental sequence of piriform radial type II neuroblasts at 7 days of incubation. x432. (a) postmitotic cell retracting its ventricular process (small arrow) from the ventricle (arrowheads); (b,c) type II neuroblasts migrating freely through the intermediate zone (notice the typical constriction of the Ieading processes); (d) freely migrating neurohlasf within the cortical plate: (e-i)

dendritjc sprouting on the former Ieading process and growth of the descending axon.

FIG. 1ga-c. DeveIoping mitrai ceils of the cortical plate at 7 days of incubation. x 4.Q.

FOG. tgd. Displaced radial neuroblast. lying below the cortical. plate. x 432.

FIG. 19a,b. Axonal growth cones of type II piriform radial neuroblasts at 7 days of incuharion. x 432.

f%G. 2Oa-c. Developing horizontal type iI neuroblasts at 7 days of incubation. x432.

FZG. 21. Panoramic view of the ted wall at 7 days of incubatjon. Ventricufar (V), intermkdjate (IX) and cortical (CT) zones are easily distingujsh~d. The intermediate zone contains the tangentiaI type

1 neuroblasts and many circumferential fibers. x 216.

FIG. 2&b. Typical tangential nemoblast of the 7th day stage. Inset ‘b’ shows a detail of the growing oblique process. a: x 171. h: x 540.

FIG. 23a-e. Growth of a vertical process on the upper side of tangential neuroblast z.eZi bodies. The indicated borderline has the same meaning ihrou~hout Figs 23a-,e and 24a-e. 7th day stage. x432.

Fro, 24a-e. Secondary tramlocation of the ce!t body of tangential nenroblasts to the upPer intermediate zone, and appearance of the descending Portion of their axons. Arrows at ‘a’ and ‘b’ show the o~&ond

stalked configurations which are detected during this process. x 432.

FIG. 2%~ Differentiation of periventricular type I nemoblasts at 7 days of incubation. a,b: x 432; c: x 216. (a) Incipient dendritic outgrowth (big arrow) in the subventricolar zone (SV); small arrows: freely ~~at~~g tyPe II neuroblast; arrowheads: ventricular lining: (b) More advanced dendritic growth in a ~r~vent~cu1~ type I nentoblast (big arrow); (cl Panoramic view of the subven~~cu~r zone with

wveral periventricular ceils. Notice the ascending axons entering the intermediate zone.

FIG. %a-+. Details of the axonal growth cones of type I postmitotic nenrobiasts at 7 days of incubation. The subpi& attachment transforms into a growth cone -a--, which subsequently adopts a descending course across the cortickl plate -b.c,d-, untjt it reaches the jnterm~ia~ zone -e-. Notice the fame&r and i%podjaf protrusions of the type I growth cones, and how eontact with the externaf

~irni~ng membrane is Iost as soon as the growth c0ne appears. X f%.

Fro, 27a-d. Arciform type I nemoblasts at 7 days of incubation. a,e: x 270; b,d: x X!@.

FK,& 27e. ‘I% figure displays an especially convoluted case of the disorietitni arcifofm XQX& Processes whjch are sometimes found at this stage (7 days). The small arrow indicates the Frovlrth cn~‘~. x 270.

FIG. 28sr-c. Appearance of demiritic outgrowths at the convex edge of the arciform a%Onal irimwtion

(small arrows). Notice the simultaneous transloeation of-the cell body upwards into thk cortkal Plate !‘e’), x 270.

Y

h

i Y

n

114

__..

315

Fro. 29a-c. Details of the subpial attachments of the last type I postmitotic neuroblasts found at 7-74 days of incubation. Transformation into axonal growth cones occurs as in earlier stages. x 675.

FIG. 30. Arciform neuroblast at 7-74 days of incubation. The small arrows shows the apical dendritic outgrowths at the axonal incurvation. x 432.

FIG. 31. This is another a&form cell at 7-74 days of incubation which has its cell body within the inner cortical zone and displays a growing basal dendrite. Small arrows: apical dendritic outgrowths.

x 270.

FIG. 32. This figure shows several cells of the intermediate zone at 7-74 days of incubation. The small arrow points out the axon of a multipolar type I cell which grows from a descending dendrite

as a consequence of the secondary translocation process. x 270.

FIG. 33a, b. These are characteristic piriform radial type II neuroblasts at 7-74 days of incubation. The axon at ‘a’ descends to the lower intermediate zone levels. The axon at ‘b’ returns to the cortical

plate after a brief descending trajectory and enters the marginal zone. x 270.

FIG. 34. A mitral cell at 7-7) days of incubation. x 750.

FIG. 35. This figure allows a comparison between a typical type II horizontal neurobhrst, with its dendritic (small arrow) and trailing (thick arrow) processes, and a typical tangential type I neuroblasts

of the intermediate zone. Seven to seven and a half days of incubation. x432.

FIG. 36. Panoramic view of tectal strata at 8 days of incubation. Notice the identification of the SAC anlage (IIN) and the intracortical plexiform layer (IPL). x 108.

FIG. 37a-c. Multipolar type I cells at 8 days of incubation. The small arrows show the axonal hillocks, frequently found on a descending dendrite. x 270.

FIG. 37d. Periventricular type I cells at 8 days of incubation. Notice the ascending axons. x270.

FIG. 38. Two nearly mature arciform type I cells at 8 days of incubation. Notice the long apical and basal dendritic processes, and the emplacement of the cell bodies within the inner cortical zone.

The initial segments of the axons lie within the IPL. x 270.

FIG. 39. Freely migrating type II neuroblasts as it crosses the outer intermediate zone at 8 days of incubation. x 270.

FIG. 40. Mitral cell at 8 days of incubation. Its dendritic arbor ramifies within the IPL. The cell body occupies the uppermost level of the inner cortical zone. Notice the type II elongated neuroblast at its side, whose spiny appearance indicates the transformation of the leading process into a dendrite.

x 270.

FIG. 41. Piriform radial cell of the outer cortical zone at 8 days of incubation, displaying a typical descending axon. x 270.

FIG. 42. This piriform radial cell of the uppermost cortical strata is just growing its axon, a bifurcated one in this case. x 270.

FIG. 43. Horizontal neuroblast found within the intracortical plexiform layer at 8 days of incubation. Notice the characteristic beaded appearance of its trailing (axonal) process. x 270.

FIG. 44a, b. Details of the axonal growth cones of type II neuroblasts. These processes have practically no lamellar or pilopodial protrusions. Eighth day stage. x 675.

FIG. 45a,b. These figures display two planes of focus of the same cell, a radial neuroblast of the outer cortical zone whose axon + grows out of a descending and recurrent dendrite at the level of

the intracortical plexiform layer. x 270.

FIG. 46. Tangential section of the rostra1 tectal pole, showing the ingrowing retinal fibers and their axonal growth cones. Tectal sphericity prevents a sharp focus throughout the image. x 171.

FIG. 47. Panoramic view of tangential and multipolar type I cells at three tectal rostra-caudai levels, showing their gradual absolute displacement relative to the axon-rich portion of the intermediate zone (indicated between interrupted lines). A,B,C: Rostral, middle and caudal thirds of the tectum. dm,

vl: dorsomedial and ventrolateral.sectors. Eight days of incubation.

FIG. 48. Tangential section of the rostra1 tectal pole, showing the orthogonal disposition of the horizontal type II ceils relative to the ingrowing retinal fibers. Big arrows = direction of growth of the retinal axons. Most cells tie either in parallel or perpendicular to these fibers. Eight days of incubation.

V

- 200 I

Tee&l aeuroblasts in the chick embryo 319

WC distinguish three separate groups of type I neuroblasts:

(a) The 6rst one consists of those cells which were generated during the 3rd and the 4th days of incubation and have had time to translocate their somata up to the intermediate zone. They are the tangential neuroblasts, which are now numerous in that zone (Figs 7, It; 21). Most of them grow a cytoplasmic process from the pole of the cell opposite the axon (Fig. 22a,b), in the tangential plane. Al&wards, other processes appear which are oriented vertically or obliquely towards tbe surface. Figures 23a-e and 24a-e illustrate two peculiar cell forms adopted by some of the tangential neuroblasts at this stage. The first one is represented by the appearance of a vertical cytoplasmic outgrowth with a smooth surface at the superficial side of the fusiform cell bodies (Fig 23a-e). The second one consists in the appearance of certain rather strange configurations, where the cell body lies outside of the main tangential axis of the cell and remains connected to it by a thin cytoplasmic pedicle (Fig. 24a,b,c). After careful revision of the material we have come to interpret these conjurations as transitional stages of a process which extricates the cell bodies of these neuroblasts progressively out of the inner levels of the intermediate zone, causing them to accumulate near the upper boundary of this zone within the next stages of development. This process seems to require a secondary translocation of the cell bodies within the vertical or oblique cytoplasmic outgrowths. The advance of the translocating soma may sometimes proceed more quickly than the growth of the vertical or oblique process, causing the appearance of the pediculated con@urations. Once the cell bodies have attained a new emplacement higher up, a set of prove dendritic processes are grown (Fig 24d,e). The axonal hillock of these cells appears now at the lower pole of the somata.

(b) The second group of type I neuroblasts is formed presumably by those cells which were generated during the 5th day and during the first half of the 6th day of incubation. This gumption rests on the observation that their cell bodies still lie within the subventricular zone and their axons adopt a circumferential course within the intermediate zone higher up than those of the tangential neuroblasts. These cells are distinguished by having dendritic buds which sprout from the somata into the intercellular space of the ventricular and subventricular zones (Figs 7, Ip; 25a,b,c). These signs of differentiation reduce the probability of a continued peripheral translocation of the cell bodies. This assumption is corroborated by our analysis of the subsequent stages. D~er~~tion of these neurobl~ts within the subventricular zone gradually builds up the an& of the SGP. Therefore, we call these cells ~&twtr~ct&r net&dusts.

(c) The third group of type I neuroblasts is formed by those cells which were generated during the second half of the 6th day of incubation plus those which

become postmitotic during the 7th day. The most immature elements of this group, the type I postmitotic wroblasts, have their cell bodies within the ventricular or the subventricular zones. Their subpial attachments are seen to be sprouting an axonal growth cone beneath the external limiting membrane (Figs 7, I; 26a-e). At variance with the behavior of type I axonal growth cones in previous stages, at 7 days of incubation most of them do not elongate laterally in the circumferential plane. The vast majority of these growing processes are detected following more or less precise descending courses across the cortical plate, until they reach the intermediate zone. There they become oriented along with the other circumferential axons (Figs 7, Ia; 27a-d). The more di&rentiated neuroblasts of this group (arci&wrn neuroblusts) have thus an arciform process which follows a vertical trajectory to the marginal zone, bends suddenly there, and traces a haphazardly curving course as it descends again to the intermediate zone. One gains the distinct impression that the growth cones are forced to descend by the predominantly radial organization of the incipient cortical plate, and that this happens in a state of partial ‘d~rientation’. Some extreme examples were found, in which these a&form processes describe turns about themselves and even ‘figures-of-eight’ before they rejoin the orienting fibers of the intermediate zone (Fig. 27e). A small proportion of these late type I neuroblasts send their axons a certain distance in the ~r~~~~tial plane, before they descend to the intermediate zone (Fig. 7).

Finally, numerous examples were found of arciform neuroblasts that have translocated their somata a certain distance towards the cortical plate. These cells display another sign of advanced differentiation in the presence of one or two slender ascending out~owths sprouting from the convexity of the arciform process (Fig 28a, b,c). These outgrowths are of variable length and always end peripherally with a thin, rounded tip.

In the E7-74 embryos (HH32) there are no more transitional forms which can be interpreted as prospective tangential neuroblasts. We find either rungen- rial cells or periwntricuhr cells; each one of these groups has its characteristic pattern of dendritic growth (see above and Fig. 8, It, Ip). Those primi- tively tangential neuroblasts which have translocated their cell bodies into one of their ascending cytoplasmic processes, grow several long obliquely ascending dendrites. They will be called herein rnulti~~ neurons from now on (Figs 8, Im; 32)

If one compares the predominant location of the mciform neuroblasts’ somata at this stage (Figs 8, Ia; 30; 31) and the preceding one (Fig 7, Is), it becomes evident that they are tr~sl~t~g their cell bodies into the cortical plate. In E7-74 embryos many of them he within the intermediate zone, and some somata have reached already the lower cortical plate levels (Figs 8, Ia; 30; 31). The number of a&form neuroblasts displaying filiform outgrowths at the convexity of their axons has increased (Figs 8, Ia; 30;

320 L. PUELLES and MARIA C. BENDAI i\

31). Some of these processes cross the whole cortical plate and show small spiny appendages. This presom- ably indicates that they differentiate as dendrites. This stage of the differentiation sequence of arciform neuroblasts allows an identification of these cells with the well-known shepherd’s crook neurons of the adult tectum. which were described originally by RAM~N

Y CAJAL (1889). The last type I postmitotic neurobiasts are found

at this stage. The manner in which they begin to differentiate is exactly the same as that shown by earlier type I neuroblasts. The only exception consists in the considerably greater length of the pial processes, which keep the postmitotic cells attached to the external limiting membrane. While the cell bodies lie within the subventricular zone, the subpial attachment transforms by sprouting into an axonal growth cone (Fig. 29a, b,c).

In E8 embryos (HH33, HH34) we did not find type I postmitotic neuroblasts. At this stage, thus. the whole type I population consists of differentiating tangential, multipolar, periventricufar and arciform neuroblasts.

Tangential and multipolar cells seem to concentrate themselves within the upper half of the intermediate zone, giving rise to the distinction of the SAC and SGC anlagen. The relative displacement of these cells may be due in part to preferential growth of efferent and a&rent fibers into the lower intermediate zone levels. It is accentuated, however, by an active translocation of many cells to the upper levels, as detected already at 1 days of incubation. Two kinds of observations support this conclusion. First. tangential neuroblasts have their axonal hillock at one of the poles of their fusiform somata up to the 7: day stage (Fig. 35), whereas at 8 days of incubation, most of them have transformed into multipolar cells, whose axons are seen to originate from the lower side of the cell bodies or from a descending dendrite (Fig. 37a. b,c). Secondly, a tracing of the position of all tangential and multipolar neuroblasts which were impregnated in single 60 pm thick sections, at three different rostra-caudal levels of the tectum, suggests that these cells translocate out of the zone occupied by the circumferential fibers (Fig. 47). This approach clearly discloses the existence of rostro-caudal and ventro-dorsal gradients in this translocation process (Fig. 47), which parallel the earlier, proliferative ventrolateral to dorsomedial gradient (LA VAIL Br COWAN, 197 la, h). Most tangential neuroblasts thus differentiate into multipolar neurons of the developing SGC at this stage. Only a small number of them remain at the border between the SAC and the SGP (Fig. 47), mixed with the periventricular type 1 neuroblasts occupying this zone (Figs. 9. It, Ip, Im; 37d).

’ It is to be understood that ‘free migration’ of these units is an assumption based only on indirect evidence. Arguments supporting this assumption are presented in the discussion.

The arcdbrm type I neuroblasts lie within the inner half of the cortical plate (Fig. 9. la). The origins of their axons are seen higher up. at the level of :hc dense intracortical plexiform layer. Most of these cells have ascending dendritic outgrowths at the convexit! of the axonal curvature (Figs 9, la: 3X). Some of them also display a descending basal dendrite. which can be followed down to the SAC level (Fig. 3X). Thosr, late type I neuroblasts whose axons have a long trajectory, running within the cortical plate in the circumferential plane, before they descend to the inters

mediate zone (Figs 7; 8; 9), arc now seen to participate in the intracortical plexiform layer. At this stage. they display short dendritic outgrowths ascending from the convexity of their axonal initial segment (Fig. 9). As this type of dendritic sprouting parallels that of the arciform neuroblasts. we consider these cells as a subgroup of the arciform population. Their cell bodies also become included into the inner cortl- cal plate.

Djfjhrentiation of type II neurohiu.w

The first type II neuroblasts appear in our material at 5-5: days of incubation (HH27). Most of these cells have their cell bodies very near the ventricular surface. They are easily distinguished from the sur- rounding ventricular cells and from type I neuroblasts, because they are completely detached from the inner and also from the outer surfaces of the tectum (Figs 5. II; 16a-e). They have an oval-shaped soma. and it is continued peripherally by a short thick cyto- pfasmic process of variable length. At the root of this short process (leading process), a constriction is frequently found. This morphology is typical of these type II neuroblasts throughout subsequent stages of development. We assume that these neuroblasts are early postmitotic cells which have detached from the ventricular surface shortly after the end of mitosis. This stands at variance with the observed behavior of type I neuroblasts, which attain an epithefial morphology, identical to that of interphase ventricular cells, before they detach from the w~ntricle. Type II neurobfasts have to migrate freely within the neuroepithelium to reach the superficial levels of the tectal wall, whereas type I cells trandocate their cell bodies within a pre-existent cylinder of cytoplasm. We call these postmitotic forms of the type II neuroblasts @ely migrating neuroblusts’. as long as they keep the characteristic morphology herein described.’ The term ‘type II neuroblasts’ is meant to include both the freely migrating forms and the subsequent immature stages of their differentiation sequence, once they finish their migration.

Radially disposed, freely migrating neuroblasts are detected at this stage not only near the ventricle, but also at various levels of the ventricular zone (Figs 5, II; 16~). Some of them have reached the layer of circumferential fibers and may even touch with the tip of their leading processes the pial surface (Figs


5, II; lfjd,e). A small number of these cells have a short gliform trailing process.

In Et5 etirycs (HH29), freely migrating neuroblmts are eucou&rEd also at all levels of the tectal Wall (Fig 6, II). Those that have arrived at the marginal zone now display signs of incipient dendritic growth.

This process is preluded by a spiny appearance of the leading process. Afterwards, two or more slender branches grow from its sides and apical tip. Shortly thereafter, an axon grows vertically into the intermediate zone from the inner pole of the cell body (Fig. 6, II@.

In E7 embryos (HH31), freely migrating neuroblasts detach from the ventricle and migrate outwards as in previous stages (Figs 7, II; 17a-d). THOSE cells which migrated during the 5th and 6th days of incubation accumulate now in the growing cortical plate. They are seen to differentiate into two types of neuroblasts: the piriform, radial type II neuroblasts (Figs 7, IIr; 17e-i) and the horizontal type II neuroblasts (Figs 7, IIh; 2@b,c)

The piriform radial n~r~~ts develop by dendritic and axonal sprouting of radially disposed type II neuroblasts once these become fixed within the cortical plate. As found in the E6 embryos, the leading processes of these postmigratory cells first becomes spiny, and then it sprouts a sparsely arborized dendritic tuft, which grows into the marginal zone (Fig. 17e-i). These tufts are not attached to the external limiting membrane, but end freely, just below it. An axon grows vertically into the intermediate zone (Fig. 7, IIr). The axons of the piriform radial neuroblasts do not have growth cones as striking in appearance as those of type I neuroblasts. The growing extremity is a more or less rounded, small terminal enlargement of the axon (Fig 19a,b). Generally, these axons course vertically, until they reach the lower boundary of the intermediate zone, where they adopt a circumferential course. Their continued accumulation at this level contributes to the appearance of the SAC in later stages of development.

cortjcal plate. ‘the most advanced portion of the leading process now shows a growth cone-like appearance, sprouting numerous radiating short @odia (Fig. 20b). Intermediate configurational exmpl@s iu- dkate that these cells gradually acquire a horizontal position within the marginal zone. At the trailing pole of these cells, an axonal process becomes visible, which has a characteristically beaded appearance (Figs 2Ck; 35; 43).

In E7-7* embryos (HH32) we detect again freely twang neuroblasts at all levels of the tectal wall (Fig 8, II). The thickened cortical plate contah the same cell types described in E7 embryos: piriform radial, mitral and horizontal neuroblasts The pirifirm radial neuroblasts display an inside-out maturation gradient. Most of their axons descend into the grow-

ing SAC (Figs 8, IIr; 33a); a small proportion of them, however, adopt a recurrent course towards the marginal zone (Fig. 33b) or grow circumferentially within the cortical plate. Deep to the cortical radial cells lie the big mitral neurom Their wide dendritic arbor extends oblique branches through an ample conic segment of the cortical plate (Figs 8, IIm; 34). The ho& zontai neuroblasts and their respective transitional forms display the described typical morphology (Figs 8, IIh; 35). Some of these cells lie within the upper levels of the cortical plate, although they are most numerous in the marginal zone.

Some immature type II neuroblasts seem not to be able to migrate upwards across the cortical plate; they seem to be detained at its lower border or nearby in the intermediate zone. We cau ~s~~i~ this new form of neuroblasts from all other ones, because they have extraordinarily elongated leading processes (Fig. 8, IIe). Observations in later stages indicate that these elongated type II cells differentiate in situ without further migration. We shall call them henceforth elongated neuroblasts.

The most immaturel~~g radial ne~obl~~ occupy a higher position within the cortical plate. This indicates that successive waves of neuroblasts become layered according to an inside-out pattern. Some of the more mature-looking radial cells have big obliquely ascending dendrites, which sprout just out of the outer pole of the cell bodies (Fig. 18a-d). As our observations in later stages support that they are an ,especial group of type II neuroblasts, we call them @it& cells. Possibly due to their small number and;their dispersed disposition, this mo~holo~~l type has not been recognized in other studies of avian tectal cytology.

In E8 embryos (HH33, HH34), generation of type II cells still goes on; &eZy migrating neuroblasts are found throu~out the tectal wall (Figs 9, II; 39). Those which are detected at the border between the cortical plate aud the marginal zone are characterized by having a very short leading process. As this shortened configuration is not seen elsewhere in the tectal wall, we do not know whether they have migrated through the intermediate zone with this configuration (and have escaped detection) or whether this results from a postmigratory retraction of the leading process (Fig 9,IIs). Observations in more advanced embryos (M. C. BENDALA & L PUELIES, unpublished observations) indicate that these short type II neuroblasts may be the precursors of the small stellate neurons of the upper SGFS laminae.

The horizontal type II neuroblasts make their first The mitral &ifs are present, irregularly spaced, at a appearance at this stage. Their most immature form constant depth. This characteristic level is more or less is represented by freely migrating neuroblasts, the at the middle of the cortical plate, just below the in- leading processes of which lose their typical radial tracortical plexiform layer (Fig. 40). The initial seg- orientation (Figs 7, IIh; 20a). The loss of the radial ments of the a&form cells’ axons lie all above this orientation occurs exactly at the upper border of the level, as well as all the horizontal type II neuroblasts.

322 1.. PUELLES and MARIA C. &.NI).AI A

Most piriform radial neuroblasts lie in the upper half of the cortical plate, generally above the dense part of the intracortical plexiform layer (Figs 9. IIr; 41). The axons descend vertically to the SAC. or, alternatively, enter the plexiform layer. Many of the more superficial, immature ones display the initial stage of the growth of an axon. As before. we find that their axonal growth cones are smoothly con- toured, small, rounded enlargements of the axon tips (Figs 42; 44a, b), being thus completely different from the much bigger velamentous growth cones of type I neuroblasts (Figs 26 and 27). A number of piriform radial neuroblasts, however. are encountered below the intracortical plexiform layer at this stage. Their somata are level with those of the mitral neurons, The dendrites of this group arborize within the intracortical plexiform layer. At this stage of development. most of them were axonless. Observations in older embryos (M. C. BENDALA & L. PUELLES. unpublished observations) indicate that still another neuronal type evolves out of these type II cells.

The horizontal nuuroblusts, located superticially below the ingrowing retinal fibers or within the upper levels of the cortical plate, keep their characteristic morphology (Figs 9, IIh; 43). Some of them have an additional dendrite. An interesting feature of these neuroblasts, which is observable only in sections which pass tangential to the tectum, is that they are disposed as an orthogonal array relative to the retinal fibers. The horizontal neuroblasts lie either in parallel or perpendicular to these afferents (Fig. 48). This orientation of the cells appears for the first time in the ES embryos.

An increasing number of elongated neuroblasts appear within the inner half of the cortical plate, that is. below the mitral cell level, lying intermingled with the cell bodies of the a&form type 1 neuroblasts (Fig. 9, Be). It is remarkable that while these cells are able to elongate their leading processes up to the marginal zone, their cell bodies cannot traverse the cortical plate. Some of these cells are detected even within the SGC and the SAC. The spiny appearance of some of the elongated leading processes indicates their differentiation into dendrites (Fig. 40). In those few cases where an axon is detected. it grows from the soma; the majority sprout from the stalk of the former leading process and grow upwards, entering either the intracortical plexiform layer or, most frequently, the marginal zone. These neuroblasts are thus identifiable with certain neurons described by RAM~N f CAJAL (1889) which have ascending axons that enter into the optic fiber stratum. RAM~N Y CNAL (1891; 1911) thought these were efTerent fibers to the retina, but recent work by HUNT & KC~NZJ.E (1976) indicates that they end in the pretectum and in the ventral thalamus.

Finally, the data on the ingrowing retinal fibers merit only brief mention. They appear at the rostra1 pole of the optic &turn since the 6th day of incubation. In E8 embryos their course is still limited to

the stratum opticum. Their growth cones are of the membranous type and display lateral and termina~ filopodia (Fig. 46).

DISCUSSION

The evidence for the existence of two separate sequences of differentiation within the developing population of the optic tectum depends on two sets of facts. First, all intermediate stages postulated for these sequences have been found (Figs IO 45). Secondly, we have been unable to detect any transitional forms which could account for a hypothetical transformation of type I cells into type II ones. or vice versa.

Type 1 neuroblasts are distinguished primarily by the fact that the postmitotic elements only detach from the ventricular lining when they already possess a subpial attachment foot. On the other hand. type 11 cells detach from the ventricle before the postmitotic element has had time to grow a new peripheral process. Type I neuroblasts are produced uninterrup- tedly between 34 and 7i days of incubation. and type II nemoblasts. which begin to appear at 5: days. are still being produced at 8 days of incubation. These periods of generation overlap during the 5th. 6th and 7th days of incubation. This raises the question whether there are two genetically different subpopuia- tions of ventricular cells. Seeing that both type I and type II postmitotic neuroblasts differentiate into specific neuronal cell types, we must conclude that either these elements are already determined to their respective maturation sequences before they detach from the ventricle, or the cell configurations obtained at the moment of detachment themselves determine the differentiation pattern of the neuroblasts.

It may prove profitable to seek a hetter onderstand- ing of how and why postmitotic neuroepithehai elements detach from the ventricle. Are only unspecific pressure effects implied (SMUT, 1972u,h) or is this a more or less determined event’? Smart’s hypothesis on the role of pure mechanical stress within the ventricular zone may be correlated partially with our observations. The earliest tectal neuroblasts, which are produced during the 3rd and 4th days of incubation, when proliferation is not yet maxima1 (COWAN,

MARTIN & WENGER, 1968; LA VAIL & (IOWAN.

1971h), have time to grow a new peripheral process before they detach (or are expelled) from the ventricle. As proliferation becomes maximal during the 5th and 6th days of incubation, an increasing number of type II cells is produced. Smart’s hypothesis would predict such a shortening of the delay between the end of mitosis and detachment. The same hypothesis does not explain, however, why type I neuroblasts continue to be produced up to the 79 day stage.

Type I neuroblasts

me next peculiar feature of type I neuroblasts is the sprouting of an axon superficially beneath the

external limiting membrane. This is preluded by transformation of the pre-existent subpial foot into an axonal growth cone. At the same time that lilopo- dial and foliopodial outgrowths are extended laterally, the contacts with other subpial feet and the external limiting membrane are loosened. During the 3rd, 4th and 5th days of incubation, these axons grow circumferentially within the primitive marginal zone, or prospective intermediate zone.

Fig, 3A. If it is a representative example of their material, the discrepancies between the two sets of observations may be explained by the fact that the dense precipitate on the marginal zone of the tectum makes it rather difficult accurately to assess fine cellular details in 80-160~ thick sections.

hfultipoh neurons. The early type I neuroblasts translocate their cell bodies upwards within the radial portion of their process. Once the perikarya arrive at the origin of the circumferential portion of these processes (6th day stage), they become oriented tangentially within the intermediate zone. These cells, the tangential neuroblasts, grow tangential and obliquely or vertically ascending cytoplasmic processes during the 7th day of incubation. Subsequently the ccl1 bodies translocate again, this time into one of the ascending processes. This places these type I neuroblasts out of the inner, fibrillar portion of the intermediate zone, and establishes the anlage of the SGC. Definitive dendrites then grow from the translocated cell bodies, which become now multipolar. Their axons are seen to descend from the lower side of the perikarya or from one of the dendrites into the SAC.

The maturational sequence of the multipolar neurons, as found in our material, is identical in certain aspects to that one described recently by DOME- SICK & McREST (1977a; they use the name ‘ganglion cells’). Our tangential neuroblasts, secondary translocation process and multipolar neurons of the SGC anlage are exactly equivalent to the ‘tangential’, ‘oblique’ and young ganglion cell’ phases of their sequence (DoWIcK BE Mores 19774. Our results thus corroborate these observations.

Besides the discrepancy concerning the mode of initial growth of the axon, there is another point where our results differ from those of DIXUBICK & MOREBT (19774. This is the question on the stage at which the earliest prospective multipolar neuroblasts are detected in Golgi preparations. The last round of DNA synthesis for these cells occurs during the 3rd day of incubation (LA VAIL & COWAN, 1971b), and we can estimate a delay of 10-12 h (HART, 1970) between autoradiographic labeling and the appearance. of detached cells in the Golgi material. Our earliest type I neuroblasts in the E3j-4 embryos appear thus more or less on schedule. This is not the case with Domesick and More&s cells, which are said to appear only at 5 days of incubation. These authors have to assume a long waiting period between the end of the S phase of the cell cycle and morphological differentiation of these neuroblasts. This discrepancy could also be due to technical prob- lems, related to the failure of the rapid Golgi method to give useful impregnations in the younger embryos.

Periventricdar neurons. An intermediate group of type I neuroblasts do not translocate their cell bodies into the intermediate zone. They start to grow dendrites while located in the subventricular zone (7th day stage) and thus become ‘anchored’ at this level. They constitute the most characteristic neuronal element of the SGP anlage, having been described only by HART (1970) and LA VAIL. & COWAN (19714.

This is not the case, however, for the earlier ‘primitive epithelial’ and ‘radial’ phases of that sequence. DoM@WZK & MORBT (1977a,b) have not observed the subpial attachments of the postmitotic type I neuroblasts, nor their transformation into axonal growth cones at the tectal &face. According to their data, initial axonal growth occurs at the interface between the ventricular and marginal zones. They speak rather vaguely of tangential processes or axons growing by elongation of lateral excresences present on the peripheral processes of radially oriented immature cells. We con&m that a certain number of oentricular cells have occasional lateral spikes on their peripheral processes. However, these were never seen to elongate and develop into tangentially oriented axons. The transitional cell types shown in Fig. 4B (2, 3) and Fig. 4C (2, 3) of DWBICK & MOREW (19774 were not found in our material of the corresponding stages. The shortened remnants of the former external process- said to be often seen in the radial stage neuroblasts (DOMESICK & MO-T, 19774, are nonexistent in our preparations. The only photographic evidence on the earIy developmental stages that is shown in the. study of DoblWCK L MEREST 11977a.b) is their

Taken together, the multipolar neurons of the SGC, the small number of tangential neurons remaining in the SAC and the periventricular neurons of the SGP are the substrate of the outside-in layering gradient described for these strata in an autoradiographic study by LA VAIL &K COWAN (1971b). It seems evident that this gradiental layering does not result from simple, uniform radial migration of neuroblastic waves, but from a rather complex process featuring different degrees of primary and secondary nuclear translocations in successively differentiating neuroblasts.

Arciform neurons. The last group of type I neure blasts to become postmitotic begin to sprout their axons at a moment when the first type II cells already accumulate in the marginal zone forming the cortical plate (647th day stage). The subsequent behavior of these axons, descending across the cortical plate into the intermediate zone, may be explained in two ways: either they are ‘attracted’ by the intermediate zone, or, axonal elongation occurring at random, the predominantly radial organization of the immature cortical plate guides a substantial number of axons down- wards. We propound the random growth mechanism,

1 , ~...- since it can account for those axons which have long


324 L. PUELI.ES and MARIA C. BENDALA

circumferential courses within the marginal zone, as well as those other axons which trace ‘turnabouts or ‘figures-of-eight’ before they reach the intermediate zone. It is remarkable that all these early events in the sequence of differentiation of the arciform neurons have escaped detection by DOMJZXK & M~KEST (19776). in their study of this particular cell group. In our preparations, practically any field of view displays two or three arciform neuroblasts, showing pro- gressive stages of axonal growth (see for example our Figs 26 and 27).

Up to the 7th day stage, the cell bodies of the arciform neuroblasts lie within the ventricular or the subventricular zones. Subsequently, they are translocated upwards within the ascending portion of their processes. Already at 7: days of incubation some of them reach the lower cortical plate levels, and at 8 days of incubation many cell bodies lie within the inner cortical plate (Fig. 38). This time-table differs from that given by DtXEXCK & M~KFZX (197%) since they state that these cell bodies do not enter into the cortical plate before 10-11 days of incubation. This discrepancy can be explained if these authors have been studying a tectal level lying caudal to the rostral, ventrolateral sector, which is most advanced in development (LA VAIL & COWAN, 1971a,b).

There are also differences between the time-table and the sequence of differentiation given by us and those of LEGHI~~A (1957; 1958). We would explain them by the fact that he does not seem to have had useful Golgi impregnations before the 9th day of incubation, and thus depended on his reduced silver preparations for his data on the early stages.

During the 7th and 8th day stages the adendritic arciform neuroblasts gradually transform into those elements which display ascending dendrites sprouting from the convexity of their arciform process. We believe that it is clearly documented in our illus- trations that the apical dendrites, as well as the basal ones, are grown after elongation of the axon. There- fore, the arciform neurons should not be presented as an example of neurons which grow an axon from a dendrite, as seems to be implied in the study of DOME~ICK & MEREST (1977b). This is rather a case of dendritic sprouting near the origin of an axon, a phenomenon which is not infrequent in neurogenesis.

Type II neuroblasts

Our data on type II neuroblasts favour the hypothesis that the immature postmitotic forms of these cells migrate freely upwards during the late 5th and the 6th days of incubation to accumulate under the tectal surface in the incipient cortical plate at 7 days of incubation. Up to the 74 day stage, all the type II cells found below the cortical plate have the same simple configuration. Since they appear first in the ventricular zone (ES-54 embryos), then in the intermediate zone (E6 embryos) and finally in the cortical plate (E7 embryos), and given that cell proliferation occurs in the ventricular zone, it follows that these

cells migrate radially outwards. Another argumcnn can be presented which supports this conclusion: if the postmitotic elements found at 5 and 6 days of incubation in the ventricular and intermediate zones are not migrating, then they should be seen to differ-

entiate within these strata in subsequent stages of development. This is not the case. since only type 11 cells located in the cortical plate sprout dendrites and axons. And, furthermore. if these cells do not migrate. which cellular group forms the cortical plate’! Cer- tainly not the type I neuroblasts; the arciform neur_o- blasts are the only ones of this group tha.t participate in the formation of the cortical plate, and they arc only a minor component of its population.

Free migpution or perikaryal trcmslocurion’? DOME-

SICK & MOREST (1977b) discussed the mode in which neuroblasts attain their definitive locations in the neural tube. They concluded that there is no evidence as yet of free migration of neuroblasts. We agree with them in the conclusion that the developmental sequence of tectal multipolar and arciform neurons provides strong evidence of perikaryal translocation Our type II neuroblasts. however, seem to represent a clear example of what these authors describe as ‘classical amoeboid neuroblasts’. They are certainly similar to the telencephalic freely migrating neuroblasts which have been described b! RAKIC (1971 j and

SIDMAN & RAKIC (1973) Careful verifications on our preparations allow us to assert thnt all our photographic examples of freely migrating neuroblasts depict whole cells. As far as our Golgi material shows, therefore, we believe that there occurs both free migration of type II neuroblasts and perikaryal translocation of type I neuroblasts simultaneously in the tectum. We have additional evidence of free neuroblastic migration in the chick embryo retina. telence- phalon, diencephalon and spinal cord (L. PIJELL~. unpublished observations).

Cortical phtr neuron. Up to the 8th day stage. differentiating type II neuroblasts within the cortical plate diversify into horizontal, piriform radial, mitral and elongated neuronal types. The radial neuronal group is possibly the most abundant in the upper cortical plate. and the elongated c-ells tire predominant in the inner cortical plate. The mitral cells are the biggest type II cells (and probably the oldest ones also). They are scarce and lie dispersed at a characteristic level just below the intracortical plexiform layer. These characteristics may explain why they have not been distinguished as a separate group of neurons before. The horizontal cells appear throughout the upper cortical plate, but are most numerous .iust beneath the stratum opticurn. At the 8th day stage prospective cortical cells are still being produced in the ventricular zone (LA VAIL & COWAN, 1971b). We have seen postmitotic type II cells within the inner- most tectal strata at this stage. Observations in embryos up to 12 days of incubation (M. C BENDALA & L. PUELLES. unpublished observations) indicate that additional neuronal cell types differentiate within the


cortical plate out of these. late migrating neuroblasts. stm& by LA VAIL de COWAN (1971b). At the 8th day

As a full examination of the histogenesis of the tectal stage, however, an increasing number of neuroblasts

SGFS up to the 12th day stage will he presented in are no longer able to migrate all the way to the sur-

a subsequent paper, we do BOt discuss these data face, and become detained below the characteristic

further in the present report. mitral cell level. They are ~s~~ish~ by their

We wish to point out, however, that those type greatly elongated leading processes. These Cells, II cells that reach the cortical plate during the 7th together with the arciform type I neurons, populate day and 7+ day stages become layered in an inside-out the inner cortical plate and are the morphologic sub- manner. This correlates with the layering gradient de- strate of the outside-in layering gradient reported by scribed autora~o~aphi~ally for the outermost SGFS LA VAIL & COWAN (197%) for the inner SGFS strata.

REFERENCES BOULDER C0~~ri-n~ (1970) Embryonic vertebrate central nervous system: revised terminology. Anat. Rec. 19% 259-262.

COWAN W. M., MARTIN A. H. & WENGER E. (1968) Mitotic patterns in the optic tectum of the chick during normal development and after early removal of the optic vesicle. J. exp. Zool. 169, 71-92.

DERER p. (1974) Histogenhfs du n&cortex du rat albinos durant la p&iode foctale et ntonatale. J. Hirnforsch. 15(l), 49-74.

~XJMGS~CK V. B. & ~&REST D. K. (1977u) Mi~ation and differentiation of ganglion cells in the optic tectum of the chick embryo. Neuroscience 2,459-475.

D~MESICK V. B. & MJRE~T D. K. (19776) Migration and differentiation of shepherd’s crook cells in the optic tectum of the chick embryo. Neuroscience 2, 477-491.

HAMBURGER V. & HAMILTON H. L. (1951) A series of normal stages in the development of the chick embryo. J. Morph. 88, 48-92.

HAMLW F. A. & WHIITERIDGE D. (1954) The representation of the retina on the optic tectum of the pigeon. Q. J1 exp. ~hys~o~. 39, 111-119.

HART J. R. (1970) Some observations on the development of the avian optic tectum. Thesis, Univ. of Wisconsin. HINDS J. W. & RUFFE~T T. L. (1971) Cell proliferation in the neural tube: an electron microscopic and Golgi analysis

in the mouse cerebral vesicle. Z. Zelforsch. mikrosk. Anat. 115, 226-264. HUGER G. C. & CROSBY E. C. (1933) A phylogenetic consideration of the optic tectum. Proc. natn. Acad. Sci., U.S.A.

19, 15-22. HUNT S. P. & UJNZLE H. (1976) Observations on the projections and intrinsic organization of the pigeon optic tectum:

an autor~io~aphic study based on anterograde and retrograde axonal and dendritic flow. J. camp. Neural. 170, 153-f72.

LA VAIL J. H. & COWAN W. M. (19X$ The development of the chick optic tectum-I. Normal morphology and cytoarchitectonic development. Brain Res. 28, 391-419.

LA VAIL J. H. & COWAN W. M. (197lb) The development of the chick optic tectum-II. Autoradiographic studies. Brain Res. 28, 421-441.

LEGHLSA S. (1957) II d~ere~~ento ontogenetico e istogenetico de1 tetto ottico nell’embrione di polio. Archs Sci. biol., St. Pete&. 41, 601-628.

LEGHISSA S. (1958) Sviluppo e struttura de1 tetto ottico nel polio. Z. Anat. EntwGesch. 12@, 247-273. biCGILL J. I., POWELL T. P. S. & COWAN W. M. (1966) The retinal representation upon the optic tectum and isthmo-optic

nucleus in the pigeon. J. Anat. 100, S-33. RAKIC P. (1971) Guidance of neurons migrating to the fetal monkey neocortex. Bruin Res. 33, 471-476. RAM~N Y CUAL S. (1889) Estructura de1 16bulo bptico de las aves y origen de 10s nervios bpticos. Rev. trim. Histol.

norm. Pa~hol. Nos 3 and 4* 1 March. RAM~N Y CAIAL S. (1891) Sur la fine structure du lobe optique des oiseaux et sur l’origine rkelle des nerfs optiques.

Int. Mschr. Anat. Pkysiol. 8, 337-366.

RAM~N Y CAIAL S. (1909) Histologic du SystPme Nerueux de 1’Homme et des Vertebrb, Vol. I (1952 reprint). Instituto Rambn y Cajal, Madrid.

RAM~N Y CAJAL S. (1911) Histologic du Systlme Nerveux de l’hlomme et des Vertebrhs, Vol. II (1955 reprint). Institute Ram& y Cajal, Madrid.

SIDMAN R. L. & RAKIC P. (1973) Neuronal mi~ation, with special reference to developing human brain: A review. Brain Res. 42, l-25.

SMART I. H. M. (1972a) Proliferative characteristics of the ependymal layer during the early development of the spinal cord in the mouse. J. Anat. 111, 365-380.

SMART I. H. M. (1972b) Proliferative characteristics of the epeadymal layer during the early development of the mouse diencepbalon, as revealed by recording the number, location, and plane of cleavage of mitotic figures. J. Anat. 113, loel29.

STEN.SA* L. J. (1967) The development of hip~pal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits-I. Fifteen millimeter stage, spongioblast morphology. J. camp. Ne~ol. 129, 59-70.

TELu) F. (1923) LJS diff&nciations neuronales dans l’embryon du poulet, pendant les premiers jours de l’incubation. Trav. Lab. Rech. Biol. Univ. Madrid 21, l-93.

VAN GEHUCHTEN A. (1892) La structure des lobes optiques chez l’embryon de poulet. Cell& g, I-46.

(Accepted 28 October 1977)

differentiation of neuroblasts in the chick optic tectum up to eight days of incubation: a golgi...

Documents