tangential neuronal migration in the avian tectum: cell type identification and mapping of regional...

Developmental Brain Research, 66 (1992) 153-163 © 1992 Elsevier Science Publishers B,V. All rights reserved. 0165-3806192/$05.00

BRESD ';1421 Research Reports

153

Tangential neuronal migration in the avian tectum: cell type identification and mapping of regional differences with quail/chick

homotopic transplants

S a l v a d o r M a r t f n e z a'b, Lu is Pue l l e s b a n d R o s a M. A l v a r a d o - M a l l a r t a

aiNSERM UI06, H6pital de la Salp#tridre, Paris (France) and b Department of Morphological Sciences, University of Murcia, Murcia (Spain)

(Accepted 26 November 1991)

Key words: Optic tectum; Histogenesis; Cell migration; Transplant; Stem cell population

This paper is a sequel to a previous report, using quaiVchick chimeras with partial tectal transplants, in which a tangential invasion of host (chick) tectal territories by cells originating in the quail graft was demonstrated. The cells displaying this secondary tangential migration appeared restricted to two strata (stratum griseum centrale (SGC) and stratum griseum et fibrosum superficiale (SGFS)). Here we describe the morphology of the tangentially displaced neurons, as well as their overall distribution in the host tectal lobe, by means of an antibody that specifically recognizes quail cells, staining them in a Golgi-like manner. Neurons that migrated into the SGC are identified as multipolar projection neurons, typical of this stratum. The majority of cells that migrated into the SGFS correspond to horizontal neurons, as was also corroborated by observations in Golgi-impregnated material. These horizontal cells are concentrated in laminae b, d and f, where their processes form well delimited axonai plexuses. In confirmation of previous results, SOC neurons have a limited range of migration, whereas SGFS cells translocate across much longer distances. In reconstructions of appropiate cases, a remarkable polarity was noted. Significant invasion of chick tectum by quail cells mostly occurred in the rostral half of the host rectum. The long-range migration of superficial horizontal cells frequently reached, but did not cross, the rostral tectal boundary. Conversely, tangential migration in the caudal half of the host tectum was scarce and coincided with a typical arrangement of quail-derived radial columns interdiglted with chick-derived columns. These findings are discussed in relation to existing data on immature neuronal populations, molecular marker distribution and polarity of the avian optic tectum.

INTRODUCTION

Descriptive analyses of avian tectal neuronal migration patterns have placed emphasis on radial cell migration. Information was drawn from reduced silver and Golgi preparations 6'7'19'2°'2~'4°, thymidine autoradiographic experiments 1s'24, chick/quail chimeras 1'36 and lineage analysis after infection with recombinant retrovi- ruses 1°. Radial translocation of postmitotic tectal neurons is massive and spatially complex, as suggested by the mixed outside-in and inside-out layering patterns that have been reported 1s'24'26.

A study using quaiVchick chimeric embryos with partial tectal transplants disclosed that cells originating in the transplanted area invade tangentially the stratum griseum centrale (SGC) and the uppermost laminae of the stratum griseum et fibrosum superficiale (SGFS) of the contiguous host territory 36. In that study cytoarchitectonic techniques and Feulgen staining were used for the identification of quail cells, so that the morphology of

tangentially migrated cells could not be accurately analysed.

Gray et al. ~°, and, more recently, Gray and Sanes 12, studied retrovirus-labeled clones, together with several immunocytochemical markers, to examine further radial and tangential migratory phenomena in the chick tectum. They characterized deep tangential migratory cells as multipolar SGC neurons, and interpreted most superficial tangential migratory cells as glial cells.

In the present study we used chimeric embryos with various types of partial tectal grafts, employing an antiserum that specifically recognized cell surface epitopes of quail cells ~6, and studied correlative Golgi-impreg- hated material, to analyse the morphology and overall distribution of tangentially migrated cells. Our observations support and extend previous data, highlighting remarkable differences in the extent of migrations in the rostral and caudal territories of the tectum. In addition, superficial migratory cells are shown to include a major contingent of horizontal neurons.

Correspondence: S. Marffnez, Department of Morphological Sciences, Fac. Medicine, Murcia, 30100, Spain.

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MATERIALS AND METHODS

White Leghorn chick and Japanese quail eggs were incubated in a forced-draft incubator at 38 _ I°C and prepared for heterospe- cific transplantation at the stage of 10-14 somites (stage 12, according to Hamburger and Hamilton '5. for the chick, and stages 8-10,

.4-# according to Zacche~ -, for the quail). Quail embryos were gener- ally used as donors and chick embryos as hosts, but control experiments inverting these roles were also performed.

The detailed microsurgical protocol .has been previously described'. Here, in order to obtain chimeric tecta with quail territory located at different rostrocaudal levels, we removed either the restral, middle or caudal third from the host tectal anlage (sometimes affecting also rostrally or caudally adjacent territories; see extent of tectal anlage in Marffnez and Alvarado-Mallart22). A homotopic donor graft was inserted in substitution, keeping the orig- inal anteroposterior and inside-out orientation. The transplants ex- tended from the dorsal midline to an undefined lateral level within the prospective area tectalis. That is. they did not carry any basal plate material. After grafting, the eggs were sealed with tape and incubated until the chick embryos reached stages 43-44 (17-18 days of incubation).

The chimeric embryos were perfused transcardially with 4% paraformaldehyde in phosphate buffer. The brains were removed, cryoprotected in 30% buffered sucrose overnight, and frozen-sectioned into 50/em-thick serial sections. The sectioning plane was roughly parallel to the optic tract.

Immunocytochemical detection of quail cells was performed on floating sections, using a polyclonal chick antiserum against quail tissue (1:500), kindly provided by C. Lance-Jones (see character- ization in Lance-Jones and Lagenaur'~). The secondary antibody was a biotinylated goat anti-chicken IgG (Vector), and the visual- ization of the reaction was obtained with diaminobenzidine in 0.05 M "Iris buffer, after treating with a standard ABC kit (Vector). The stained sections were mounted on subbed slides and covered. Some sections were counterstained with Cresyl violet, or with the fiuo- rochrome Hoechst 33258 (1% solution, 2-3 h), in order to corre- late stained cells with tectal cytoarchitecture. Ten cases showing perfect integration of the graft into the host tectum were selected for the present study.

Several Golgi-impregnated chick embryos (stages 24=35) were also studied for comporison, aiming particularly to detect cell forms migrating superficially after stage 31. This material was peepared according to the Golgi-Stensaas procedure, as previously described =¢~,

RESULTS

Transplant morphology In accord with previous experience ''36, the grafted and

host territories of the operated tectum are perfectly fused together, but basically develop independently one from another. The grafted area appears heavily labeled by the antiquail antibody, and the surface extent of each graft is congruent at the ependymal and pial surfaces. Since host territories are unlabeled, it is striking that a relatively high number of immunopositive graft-derived cells are outside of the grafted area. The reverse is true in quail tecta that received a chick graft,

This is illustrated in Fig. 1, showing 12 sections taken at regular intervals through a representative chimeric tectum that received a graft (shaded area) of the middle tilird of the mesencephalic vesicle (see inset, Fig. 1, in the upper right corner). The small and large dots repre-

sent quail cells that have invaded host territory. This panel allows a comparison of the quaiVchick cell mixing patterns obtained at the rostral and caudal host/graft in- terfaces. The rostral limit of the transplant is relatively smooth, representing a rather clearcut transition of radial structures derived from the graft to similar structures derived from the host (Figs. 1, 2a and 3a). Con- versely, the caudal interface between transplant and host, • ,hat lies in this case in the caudal tectum, shows a tran- sitional zone in which quail radial columns intercalate with others of chick origin. These columns appear sectioned radially in the upper and middle rows of Fig. 1 (see also Fig. 3b), whereas they appear as tangentially- sectioned patches in the lower row of Fig. 1, due to the relative change in the plane of section. Dorsally, the transplant thins out towards the intertectal commissure (Fig. 1, upper row; inset). Ventrally, it becomes broader approaching the isthmic nuclei (Fig. 1, lower row).

The cellular components stained in the quail-derived columnar territory correspond to various neurons inserted in a framework of radial and free glia cells ex- tending from the ventricular lining to the pial surface. Due to intense staining of the central port ion of the graft, only the borders of the graft can be accurately analysed (Figs. 2b and 3). As expected, a full set of morphological cell types known from other studies is present t7,26.

Tangential migration In accord with previous data t2'36, extensive tangential

migration of quail cells occurs within the upper SGFS and the SGC of the chick host tectum. Most quail neurons found outside of the graft have sizes and shapes that are characteristic of the neurons of the host strata that they have invaded (Figs. 2 and 3).

The morphology of cells which have migrated into the SGC closely corresponds to Golgi-stained multipolar cells of this layer. Large, polygonal or fusiform cell bodies give rise to several divergent main dendrites. Their axons converge in the tectothalamic and tectobulbar tracts (Mart |nez et al., manuscript in preparation). A small number of much smaller, possibly glial, cells are mixed with them (Fig. 2b).

The upper SGFS is invaded by quail cells at levels that were identified in counterstained sections as the plexiform laminae b, d and f. Laminae b and d are also filled by a dense net of fine immunopositive fibers (Figs. 2 and 3a). The plexus of lamina d is slightly thicker and denser

than that of lamina b. Since neurons from other tectal laminae are not known to project laterally within the tectum itself, it may be supposed that these plexuses are

mostly produced by axonal branches of the neurons ly- ing within them. These are medium- and small-sized,

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Fig. 1. Drawings of a series of sections through a representative case of midtectal homotopic quaiVchick transplant. Inset at upper right shows a schema of the operation and a lateral view reconstruction of resulting grafted area; quail donor tissues appear stippled; the orientation and dorsoventrai range of the sections drawn is indicated by the bars and directional symbols (D,V,R,C); asterisks show the host area invaded by tangential migration from the graft. The drawings illustrate the immunoreactive grafted tectal sector (shaded), and the disuibu- tion of ectopic quail cells which have migrated tangentially. Superficial migrated neurons are marked as fine dots, whereas deep migrated neurons appear as large dots. Extensive tangential migration occurs within the rostral part of the tectum. Cell displacement is considerably more extensive for cells migrating within stratum griseum et fibrosum superficiale (SGFS), but stops at the boundary of the tectum with the griseum tectalis (GT). Note columnar arrangements of quail-derived clones at the caudal edge of the graft, coinciding with scarce tangential

migraiion.

156

horizontal neurons (Fig. 2b). Slightly larger horizontal (and sometimes multipolar) neurons are found within lamina f. Very few cells invade the laminae a, c, e, and g.

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Fig. 2. Illustration of the distribution of quail ectopic cells within the rostral rectum of the host. The rostral e0ge of t.he graft appears at the right (marked with arrowheads in a), The alphabetic lamination of the SGFS is indicated (b, at left), a: at low magnification numerous migrated cells are detected within the upper SGFS laminae and within the stratum griseum centrale (SGC), Bar - 50 l~m, b: at higher magnification, superficial cells are seen to have mainly horizontal shapes, and are restricted to the immunoreactive nearo- piles present within laminae b and d of SGFS and to lamina f. A few smt, ll glial cells occupy the stratum opticum, The characteristic multipolar morphology of SGC cells is also evident, Abundant radially oriented cells are immunoreactive within the quail grafted sector (right hand edge of the micrograph). Bar = 50 ~+m.

Sparse medium- and small-sized quail neurons were also found within the plexiform lamina h of SGFS (Figs. 2 and 3). These mostly had horizontal shape, although others showed rounded perikarya, with several dendritic processes lacking a preferred orientation. Finally, very few medium-sized or small radial neurons were immunoreactive within the cellular lamina i of SGFS (Figs. 2 and 3a).

Regional differences in cell migration Tangential migration is much more extensive within

SGFS than within SGC, as already observed by Senut and Alvarado-Mallart a6. Our preparations disclosed additional spatial anisotropy with regard to overall extent of tangential migration. Much larger tangential displacement of quail neurons occurred within the rostral portion of the tectum than elsewhere.

After grafting the middle third of the tectal anlage, tangential migration within SGFS laminae ly'cl~ of the rostral tectum frequently covered the remaining sector of the host tectum up to its boundary with the griseum tectalis (Figs. 1 and 2a). In these cases, the tangential migration of SGC neurons stopped at about 400 #m of the graft/host interface (large dots in Figs. 1 and 2a).

On the contrary, at the caudal graft/host interface of these tectal transplants only sparse SGC or SGFS quail cells were found invading the caudal half of the tectum in any directioa outside of the graft-derived patches or columnar domains (Figs. 1 and 3b).

Other experiments were performed with the aim to resolve the question whether this difference in the amount of cell migration into the rostral and caudal regions of the host rectum depends on a directional mechanism or on regional differences between these tectal areas.

When the graft occupies the rostral-most tectum, extensive tangential migration occurs in the SGC and the SGFS, particularly in vential direction. The case represented in Figs. 4a,b has a graft occupying the whole griseum tectalis and a small adjacent part of the rostralmost tectum. In addition to the massive invasion of adjacent SGC and SGFS, some quail SGFS horizontal neurons and glia appeared dispersed at larger distances caudal-wards (not shown). Within the grafted quail tectal t~tritory, a number of non-immunoreactive spots were observed in the SGC and the upper SGFS laminae. They correspond to unstained host cells, as indicated by coun- terstaining with the fluorochrome Hoechst 33258. Cells from the host territory thus also invade tangentially the rostrally adjacent graft. It is of interest to note that, contrary to what occurs in the previously described transplants, such cases as this, having a caudally-oriented graft/host interface within the rostral tectal region, show

no mixing of quail- and chick-derived radial columns (Fig. 4b).

Conversely, cases with grafts placed at the caudalmost tectum show scarce tangential migration across the rostraUy oriented interface, and the interdigited radial columnar pattern is present (Fig. 4c,d). According to these experiments the amount of tangential migration is higher in the rostral tectum, and interdigited columns of quail- and chick-derived cells only appear in the caudal tectum, irrespective of the rostro-caudal orientation of the host/graft interface.

An additional case (chick donor/quail host), in which the caudal boundary of the graft coincided with the limit between griseum tectalis and tectum, served to corrob- orate the finding that tectal neurons do not invade tangentially the griseum tectalis. The chick-derived griseum tectalis showed only a few quail glial cells.

Golgi-impregnated material We have re-examined Golgi-impregnated material

from a previously prepared collection 26, in order to further characterize the cell shapes of cells migrating tangentially, with emphasis on the superficial horizontal cells. Aiming not to repeat previously published d-~ta, we have summarized the differentiation sequence of multipolar type I cells of the SGC and of horizontal type lI cells of the SGFS in Fig. 5 (terminology and data taken from Puelles and Bendala26).

Prospective multipolar neurons first grow their tectof- ugal axon while the cell bodies still lie within the ventricular zone (E4 in Fig. 5), After the somata have moved radially into the level occupied by the respective axons in the stratum album c~ntrale (SAC), these cells are transiently recognized as tangential young neurons (E6, E8 in Fig. 5, in black).

This period apparently coincides with tangential, ventral ward migration t2. Subsequently, the cells grow ob- lique or vertical ascending dendritic processes, and then gradually translocate their somata into them, thus +,ort- ing themselves out of the SAC and becoming multiv~olar neurons within the SGC (E8; El0 in Fig. 5, in t~,iack).

Prospective SGFS horizontal cells first appear ~t wo or three days later than the earliest multipolar prec~r~ors They detach immediately from the ventricle after mito- sis and migrate radially to the tectal surface. They have a rather simple shape, consisting of a small cell body and an apical, digitiform leading process (E6 in Fig. 5, striped). Many of these type II young neurons differentiate into various radial and stellate neuronal SGFS cell types, but prospective horizontal cells reorientate their leading processes in parallel with the tectal surface (E6, E8 in Fig. 5, striped; Fig. 6a). They transiently show similar immature shapes in this location, possibly corre-

157

sponding to their active tangential migration (Fig. 6b). Later their axons sprout from the trailing pole of the cell bodies, and the leading processes mature as dendrites (El0 in Fig. 5, striped; Fig. 6b,c).

DISCUSSION

Insofar as the ependymal and pial extents of the grafts are congruent, there can be no doubt that quail cells detected individually within adjacent host-derived domains of the tectum must have reached these positions by active tangential migration. As described by Senut and Al- varado-Mallart a6, and recently corroborated by Gray and Sanes ~2, these cells first follow normal radial courses within the grafted tectum, before they find conditions suitable for lateral displacement into host tissue.

Identity of tangentially migrating neurons Immunocytochemical staining of tectal grafts with the

chicken antiquail antibody provides cytologic resolution sufficient for satisfactory identification of most tangentially migrating cell types as either type I multipolar cells, within the SGC, or type II horizontal cells, within the upper SGFS laminae (see Fig. 5), thus supporting previous preliminary conclusions a6.

Tangentially migrated multipolar neurons (or 'ganglion cells') were also clearly recognized with retroviral clonal labeling ~2. However, concerning the cells migrating within the SGFS, these were identified as being mostly glial cells, This interpretation is at variance with our own and should be discussed more closely.

We base our identification of horizontal neurons on the following findings: (a) immunoreactive migrated SGFS neurons clearly have similar shapes, and are iden- tically distributed as horizontal cells in normal chick embryos and adults; this includes that such neurons are rarely found lower than in lamina f of St3FS t7'26 (excep- tional cases are found within lamina h, like in the present study); (b) innermost horizontal cells are larger than more superficial ones t7 (see our Figs. 2b and 3a); (c) our Golgi-impregnated material shows immature young neurons adopting a tangential superficial position, presum- ably migrating tangentially, and differentiating into characteristic horizontal neuronal shapes (Fig. 6); (d) the small number of tangentially-migrated cells that we did identify as possible glial cells had cell bodies that are rounded and smaller than those of fusiform horizontal neurons. Several thin cytoplasmic processes radiated in all directions from the somata, ramifying in astrocyte- like fashion. These cells did not occupy fixed positions relative to SGFS lamination.

In our hands, the Golgi-Stensaas technique impreg- nates only neurons, ventricular cells and radial glia at the

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Fig. 3. a: high magnification detail of graft/host rostral interface, showing clearcut border and related radial neurons. Bar = 50 #m. b: Mi- crograph showing columnar quail cell arrangements present at the caudal graft/host interface. Note much reduced number of tangentially migrated neurons, both at SGFS and SGC. Bar = 50 #m. Fig. 4. a: schematic illustration of homotopic transplant of the rostralmost tectum, as well as resulting graft and extent of migration, seen in lateral view. Spatial directions are indicated as D,V,R,C. Quail donor tissues are stippled, and asterisks mark the area invaded by tangential migration, b: micrograph showing section through level marked with a bar in a, illustrating straight border of the graft and migrated cells in SGC and SGFS. Bar = 50 #m. c: schematic illustration of homotopic transplant of the caudalmost tectum (symbols as in a). d: micrograph of a section through the level marked with a bar in c, showing columnar arrangements and scarce tangential migration. Bar = 50 #m.

stages analyzed. Although we do not exclude that glial cells also participate in the superficial taagential migration, as described by Gray and Sanes t2, we think that the cells illustrated in their Fig. 5d are similar to the horizontal cell population described above.

Cell type specification and tangentially migrating cells Tectal cell types were previously proposed to be clas-

sifiable into type I and type II groups, according to their distinctive postmitotic sequences of differentiation and migration 26. Our present data on Golgi-impregnated material also support differential postmitotic sequences of differentiation of the neurons migrating tangentially either in the SGC or the SGFS (Fig. 5).

The hypothesis was advanced 26 that cells maturing according to type I or type II sequences might be born from separate stem cell lineages. Multipolar cells (earliest type I cells) are born between E2.5 and E4 ~7'24, whereas type II neurons are born later, first appearing at ES.5-E6, together with other type I ne,,rons, like the characteristic shepherd's crook neurons 26. It is thus only after 5 days of incubation that type I and type II neurons are produced simultaneously. The clonal analysis performed by Gray and Sanes n marks a mixed population of type I and type II cells, as well as cells of glial derivation. However, these authors introduced the retrovirus at E3, well before the two hypothetic ventricular stem population,s generating type I or type II cells start to behave differentially. Other experiments with retrovirus injection at stage 24 (E4) produced much smaller clones that do not include all tectal cell types ~°, as pre- dicted by the double stem cell hypothesis. Labeling of ventricular clones at E4-E6 may be necessary to resolve whether the observed differences in migration and differentiation between these two kinds of neurons are related to their stem lineage or to epigenetic transforma-

tion.

Mechanism and topography of migration Quail neural primorO~a mature at a quicker rate than

those of the host species 3s'36. It might be asked whether

the normal tangential migration of chick horizontal and multipolar cells is largely comparable to the heterospe- cific case. The fact that some chick neurons invade a

grafted quail tectal territory when the latter is placed rostrally, and that control experiments transplanting chick donor tissue into quail hosts give essentially similar results, leads us to conclude that the tangential migration is not strongly affected by heterochronic aspects of the chimeras.

The different range of the superficial and deep tangential migrations may be related to differential migratory properties inherent in the type If, free migratory mode of movement of young horizontal cells, compared to the type I, secondary somatic translocation of the big- ger tangential/multipolar neurons u'26. In both cases, migrating neurons seem to behave in a so-called neurophilic way a°, since they move orthogonally to the main radial glial substrate.

Retroviral labeling achieves precision in the identification of the origin and direction of cell migration ~2, due to the restricted cell sources obtained from isolated clones. However, the random distribution of many labeled clones in the same tectum (on average 40 clones, rectum 1°) may bias the observer towards disre- garding neurons having moved very far away from the

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Fig. 6. Panel illustrating Golgi-Stensaas-impregnated type II horizontal neurons arriving at the tectal superficial laminae (a), presamably migrating tangentially (b) and differentiating into various shapes (c) in chick embryos at stages 31 (a,b) and 32 (c). Notice that the simple, immature appearance of these freely migrating type II cells does not change much as they exchange radial guidance cues (a) for tangential ones (b). Compare with scheme in Fig. 5, Differentiating cell shapes in c are comparable to quail cells migrated into the host SGFS. At these stages no glial cells were impregnated, excepting radial glia. Magnification as in Fig. 2b.

clone being analyzed. Our grafting approach is equiva- lent to labeling multiple adjacent clones (all those derived from the graft,~d tissue). It allowed us to choose the location of the marked tissue within the tectum, and to obtain amplification of the observable migrated cell populations. Unequivocal species-specific identification of migrated cells, whatever their distance from the graft, helps in the study of regional differences in cell displacement,

Whereas muitipolar neurons initially move ventral- ward, apparently guided by the topologically dorsoven- tral tectobulbar axonal bundles ~2, there is some indica- tion that there may be a later, rostrally-directed com- ponent of their displacement. This is suggested in our material by the predominance of SGC migrated cells

within the rostral half of the tectum, as well as by the presence of migrated chick cells inside the quail region grafted at the rostral most part of the tectal vesicle. The mapping of this migration on tectal flat-mounts reported by Gray and Sanes t2 (their Fig. 3c) also reveals a slight rostralward drifting of the SGC cells that have advanced most ventrally (most 'laterally' according to their terminology). It should be noticed that we examined the distribution of migrated cells at a much later stage (stages 43-44) than these authors did in that study (stage 32). The inferred rostralward movement may have been only incipient at stage 32. This secondary drifting may coin- cide with the period in multipolar cell differentiation in which the latter translocate their somata out of the SAC into the SGC (cf. Fig. 5).

On the whole, our tectal transplants revealed a polarity of chimeric tecta, such that significant tangential migration only occurred in the rostral tectal regions. Lack of tangential migration caudally might be due to a different molecular environment. Dramatic changes of tectal cell adhesive specificity occur between E5 and E8 in the chick, including a marked preference of posterior tectal cells to adhere on anterior tectal ceUs 14. More specifically, Cox et al. 4 have recently observed that membranes extracted from the posterior chick tectum cause temporal retinal axons to collapse their growth cones. Perhaps the same activity (or a related phenomenon) participates in the failure of neurophilic tangential migration within the caudal tectum. It could be asked whether heterotopic grafts of rostral tectum into caudal tectum, or viceversa, might alter the observed polarity relationships. According to our experience however, such experiments are not instructive, since prospective tectal neuroepithelium has a strong tendency to regulate its fate specification relative to positional cues. Rostrocau- dally inverted grafts do not keep their primary specification properties and transform into 'homotopic- like' grafts 23. There are various reports on the expression of different specific molecules in the tectum in temporal coincidence with tangential migratory phenomena 5'~3'32' 33,38

The apparent rostralward bias of superficial tangential migration, occurring at the time that optic axons first invade superficially the tectum 29'4~, might be related to preferential adhesion between optic fibers and the immature horizontal cells. Retinal axons are known to ex- press specific surface glycoproteins 3~'34'37. A possible af- finity of tectal-derived cells for optic terminals was suggested after studying heterotopic tectal grafts placed inside the diencephalon; ectopic tectal cells selectively colonize all retinorecipient neuropiles of the host 2~.

It is interesting that rostraUy migrating horizontal neurons do not normally cross the limit of the tectum with the griseum tectalis, although the previously cited heterotopic transplants showed that they are able to migrate inside the diencephalon. Curiously enough, such ectopic tectal cells within the diencephalon never move caudally across the same limit into the host tectum 2~. This boundary is apparently impermeable in both directions. It is perhaps significant that this border is the place where the retinotopic mapping of the temporal-nasal retinal axis

ABBREVIATIONS

EM GLV GT lpc

n.ectomammillaris n.geniculatus lateralis ventralis n.griseum tectalis n.isthmi principalis, pars parvocellularis

161

displays a specular inversion 8'9'27'28. Molecular epitopes selecting for preferent arborization of peripheral temporal retinal fibers apparently abut on each side of it 2'3'2s.

Fluidity of the caudal tectal neuroepithelium Patterns of quail cells developing at the host/graft in-

terface in the caudal tectum are similar to radial arrays of clonally-related tectal cells 1°-~2. Such columns are not formed when the interface lies in the rostral tectum. The predominance of this pattern caudally may be related to the well-known rostrocaudal proliferation gradient across the tectal primordium TM. Since the caudal half of the mesencephalic vesicle sustains dynamic proliferative activity for a longer time than the rostral half, conditions may be favourable there for lateral mixing of quail and chick ventricular cells at the host/graft interface, leading to the isolation of quail ventricular clones inside nearby host tissue. Alternatively, blocks of cells may shift within the plane of the proliferative epithelium. Gray et al. ~°ax found some clones that evolved as separate radial cell strands, and they also reported that clones are larger caudally in the tectum than rostrally. All this suggests considerable fluidity of the caudal mesencephalic neuroepithelium, and agrees with experimental results demonstrating the early transitory location of the matrix neuroepithelium for the prospective isthmus and rostral cerebellum within the caudal third of the 'mesencepha- lie' vesicle 22 (Martfnez-de-la- Torre and Puelles, in preparation). Evidence of such neuroepithelial 'fluidity' might have implications for certain cell dispersion phenomena observed with retrovirally labeled lineages in the fore- brain II.

In conclusion, two neuronal cell types are shown to migrate tangentially at two different levels of the tectal cortex. The overall distribution of the migrated populations indicates differential properties of the rostral and caudal tectal regions as substrates for cell migration.

Acknowledgements. The main part of this work was carried out while S.M. was successively recipient of an INSERM fellowship, a MEC,MRT fellowship (U106, Paris) and a Beca, de Reincorpora- ci6n en Espafia (MEC). The latter part of the work was supported by DGICYT Grant PB87/0688-C01 to L.P. The gift of specific antiquail antibody by C. Lance-Jones is gratefully acknowledged.

IPSP PP PV R SAC SGC SGFS

n.interstitialis tractus pretecto-subpretectalis n.precommissuralis principalis n.posteroventralis n.rotundus stratum album centrale stratum griseum centrale stratum griseum et fibrosum superficiale

162

SLU SO SP SPL

n.semilunaris stratum opticum n.subpretectalis n.spiriformis lateralis

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tangential neuronal migration in the avian tectum: cell type identification and mapping of regional...

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