Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en.

The homeobox gene en, homologous to the gene en-grailed of Drosophila, is expressed in the metencephalic-mesencephalic segment of the vertebrate neural tube. Using quail-chick chimeras, an antibody against en proteins, and cytoarchitectonic techniques, we demonstrate that metencephalon transplanted to prosencephalon, at E2, maintains a high level of en proteins and its presumptive cerebellar fate. The ectopic metencephalon induces in the contiguous host prosencephalon the expression of en and, subsequently, a mesencephalic phenotype. These related genetic and phenotypic expressions indicate that the transcriptional regulatory en gene is involved in cerebellar and mesencephalic cyto-differentiation. The expression of en can also be induced in chick prosencephalon by a mammalian metencephalic graft, indicating that the factors regulating the transcription of en are phylogenetically well conserved.

Cajal-Retzius cells regulate the radial glia phenotype in the adult and developing cerebellum and alter granule cell migration.

Studies on the reeler mutation have shown that pioneer Cajal-Retzius (CR) cells are involved in neuronal migration in the developing cortex. Here, we use grafting and coculture experiments to investigate the mechanisms by which CR cells govern migration. We show that transplantation of embryonic CR cells, but not other cortical neurons, into adult cerebella induces a transient rejuvenation of host Bergmann glia into a radial glia phenotype. Similarly, CR cells sustain the phenotype of developing radial glia in postnatal cerebellar slices and induce the organization of a glial scaffold inside the CR cell explants. Studies with semipermeable inserts show that these effects are mediated by diffusible signals. We also show that CR cells adjacent to the surface of cerebellar slices reverse the direction of the migration of granule cells. Finally, CR cells from reeler mutant embryos elicited similar effects. These observations imply a role for CR cells in the regulation of the radial glia phenotype, a key step for neuronal migration, and suggest that these pioneer neurons may also exert a chemoattractive influence on migrating neurons.

The reconstruction of cerebellar circuits.

Repair of adult 'point-to-point' systems by neural grafting is possible only when grafted neurons succeed in synaptically replacing the host's missing neurons, thus re-establishing the anatomical and functional integrity of the impaired circuits. Grafting experiments carried out on the cerebellum of the adult pcd (Purkinje-cell-degeneration) mutant mouse (an animal model of hereditary degenerative ataxia) reveal that embryonic Purkinje cells, by some unknown sorting mechanism, selectively invade the deprived cerebellar cortex. These neurons migrate to their proper domains and, inducing axonal sprouting of specific populations of host neurons, they become integrated synaptically within the pcd cerebellar cortex. However, the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit.

Further observations on the susceptibility of diencephalic prosomeres to En-2 induction and on the resulting histogenetic capabilities.

It has been previously shown by chick/quail heterotopic grafts that En-2 expression and a mesencephalic phenotype can be induced within the avian primordial prosencephalic vesicle, although the induction appeared restricted to the caudal forebrain. The present experiments were aimed at further analyzing the competence of the prosencephalic neuroepithelium. Different types of grafts were performed between chick and quail embryos: (i) caudal forebrain grafts positioned in the midbrain/hindbrain junction (the En-2-positive domain); (ii) En-2-positive grafts integrated at different levels of the forebrain. In both cases, the grafts were transplanted either with a normal orientation or after inversion of their rostro-caudal axis. The chimeric embryos were analyzed at stages HH19-24 for expression of En-2 and Pax-6 homeobox-containing genes, normally expressed in the meso-isthmo-cerebellar and prosencephalic domains, respectively. A cytoarchitectonic analysis of grafted and surrounding host tissue was also performed at later developmental stages in chimeric embryos with caudal forebrain grafts. Our results show that the caudal diencephalon, including the prospective territories for prosomeres 1 and 2, is competent to express En-2 when in close contact to the En-2 polarizing region, whereas the more rostral neuroepithelium, including the prospective territories for the third prosomere and telencephalon, does not change its fate under similar conditions. The ectopic-induced neuroepithelium can develop mesencephalon, but also isthmus and cerebellum according to its site of integration rostrally or caudally to the mesencephalic/isthmo-cerebellar boundary. Our data also show that within the competent diencephalon, the induced En-2 expression can be arrested at the P1/P2 interneuromeric boundary. This arrest appears to be directionally oriented as it only takes place when the induction is produced within prosomere 1 but not when it comes from prosomere 2. These data can be considered as resulting from either a possible oriented permissiveness of cells which form the boundary separating prosomeres 1 and 2, or of a different permissiveness of the cells composing these two caudal prosomeres.

Comparative analysis of Otx2, Gbx2, Pax2, Fgf8 and Wnt1 gene expressions during the formation of the chick midbrain/hindbrain domain.

The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centred on the MH boundary with a double decreasing gradient. We propose a new nomenclature to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary.

Differentiation of cerebellar anlage heterotopically transplanted to adult rat brain: a light and electron microscopic study.

Pieces of cerebellar primordia from (days 14 or 15 of gestation) E14 or E15 rat embryos were dissected out and transplanted into a cavity of the occipital cortex and underlying hippocampus, over the superior colliculus of 2-month-old rats. The host animals were allowed to survive for 2 to 3 months. The cytoarchitectonic and the synaptic organizations were analyzed in 16 of such transplants. Only 4 of the implants established connections with the host brain through several thin peduncles composed of myelinated fibers. The remaining 12 implants survived in an extraparenchymal situation. Independently of its partial linking to the host brain, the graft grew and developed a cerebellar structure composed of nuclear and cortical regions. The latter exhibited normal lamination and foliation, and contained the five categories of neurons which characterize normal cerebellar cortex. Electron microscopic examination disclosed that the synaptic connections normally present in the cerebellar cortex were also formed in the implants with the exception of climbing fibers, which were absent. The cerebellar interneurons kept their normal topographic distribution and gave origin to numerous synapses which maintained their own specificity. Some mossy fibers were present in the granule cell layer at the center of typical glomeruli. However, abnormal synaptic arrangements were also observed within the neuropil of this granule cell layer. They consisted of pseudoglomerular formations composed of clusters of tightly packed small axon terminals covered by granule cell dendrites. The origin of these boutons was not established. Since they did not correspond to the classes of presynaptic elements normally synapsing on these dendrites, they constitute a new example of cerebellar heterologous synapses. Their presence could be related to changes in the cellular environment due to the rarity of mossy afferents. HRP tracing experiments, carried out in extraparenchymal transplants, have allowed us to determine that the corticonucleocortical loop of normal cerebellum is also developed in the implants. Nuclear neurons are at the origin of the mossy fibers involved in glomerular formations, whereas Purkinje cells project to the nuclear region. The establishment of these reciprocal connections could determine the functional stabilization of both kinds of cerebellar neurons and thus the long survival of extraparenchymal grafts. These results allow the conclusion that the presence of extracerebellar afferents is not necessary for the organotypic and synaptotypic differentiation of cerebellar anlage.

Compensatory climbing fiber innervation after unilateral pedunculotomy in the newborn rat: origin and topographic organization.

In neonatal rats the unilateral transection of the cerebellar peduncles causes a fast and complete degeneration of the contralateral inferior olive. Axons from the remaining olive recross the cerebellar midline and partially innervate the deprived hemicortex. Analysis of the topographic organization of this compensatory projection studied with the axonal tracing method provided the following results: Retrograde tracing experiments revealed that the bulk of compensatory afferents originates from neurons in the ipsilateral medial accessory olive, especially from its medial region, whereas afferents from the principal olive and the dorsal accessory olive contribute to a much lesser degree. In case of incomplete neonatal pedunculotomy, neurons with a similar location in the ipsilateral intact olive still contribute to the innervation of the partially deprived hemicortex, along with the atrophic contralateral olive. Moreover, these experiments revealed important information about the organization of the compensation. Although its specificity was not totally maintained, the mediolateral distribution of sprouted afferents in the cerebellum matched the caudorostral disposition of parent neurons in the olive, as in the case in normal olivocerebellar projection. Anterograde studies showed that compensatory fibers recrossing the cerebellar midline spread throughout the whole extent of the deprived cortex and terminate solely in the molecular layer as typical climbing fibers. The latter were not homogeneously distributed, their density being markedly reduced according to a mediolateral gradient. Compensatory projection followed a sagittal striped pattern, as does the normal climbing fiber projection. Moreover, if the cortex is divided broadly into vermal, intermediate, and hemispheral regions, an apparent reciprocity seems to exist concerning the relative involvement of the various cortical subdivision in both hemicerebella. Our present results indicate that the immature olivocerebellar system is capable of anatomical plasticity, although to a limited extent. More important, they suggest that a certain degree of specificity is maintained during the process of sprouting, resulting in a topographical arrangement of the transcommissural climbing fiber projection. This indicates, in turn, that cues which guide the growth of olivocerebellar fibers during normal development could also direct the compensatory innervation.

Ultrastructural evidence for compensatory sprouting of climbing and mossy afferents to the cerebellar hemisphere after ipsilateral pedunculotomy in the newborn rat.

Unilateral section of the inferior and middle cerebellar peduncles was performed in rats at postnatal days 1 or 2. The ultrastructure of the cerebellar hemispheric cortex ipsilateral to the lesion was examined 3 months later. The absence of contralateral inferior olive and of ipsilateral middle peduncle, together with a marked regression of the contralateral pontine gray, were indicative of successful pedunculotomy. In spite of a relative atrophy of the hemisphere, its cytological structure was qualitatively normal. Mossy and climbing fibers were present and their terminal varicosities disclosed normal features. The density of climbing fiber terminals was reduced compared to control cerebellum, whereas the density of mossy terminals seemed unchanged. subsequent to the reduction of climbing afferents two subclasses, or types, of Purkinje cells were present: A "normal" type characterized by its climbing fiber innervation and a "hyperspiny" type devoid of climbing fiber. In some of the adult rats pedunculotomized at birth, section of the contralateral peduncles was performed 24 hours before fixation. Terminal degeneration of climbing and mossy fibers was observed in the neonatally deprived hemisphere, providing the proof that these fibers result from a compensatory transcommissural sprouting of afferents destined to the contralateral hemicerebellum. These results demonstrate that the cerebellar cortex neonatally deprive of its main afferents can be innervated by climbing and mossy fibers through a process of transcommissural sprouting. Although the newly formed synapses maintain their target specificity, a functional reorganization must occur because of the altered distribution of both systems of afferents.

Chick/quail chimeras with partial cerebellar grafts: an analysis of the origin and migration of cerebellar cells.

Chick/quail chimeras with partial cerebellar grafts have been performed to obtain further information about the origin and migratory movements of cerebellar cortical neurons. The grafts were performed by exchanging between these two species a precise, small portion of the E2 cerebellar primordium, as defined in Martinez and Alvarado-Mallart (Eur. J. Neurosci. 1:549-560, 1989). All grafts were done unilaterally. The chimeric cerebella, fixed at various developmental stages, were analyzed in serial Feulgen-stained preparations to map the distribution of donor and host cells in the ependymal layer (considered to be reminiscent of the primary germinative neuroepithelium) and in the various cortical layers. In some of the oldest cases, we also used antiquail immunostaining to recognize quail cells. In the ependymal layer, it has been possible to conclude that each hemicerebellar primordium undergoes a morphogenetic rotation that changes its rostrocaudal axis to a rostromedio-caudolateral direction. However, important individual variations were observed among the chimeric embryos with respect to the ependymal area expected to be formed by donor cells. These variations cannot be explained solely on the basis of microsurgical procedure; however, they suggest the existence of important reciprocal interaction between host and grafted neuroepithelia. Therefore, it was not possible to draw a precise fate map of the E2 cerebellar primordium. Nevertheless, the dispersion of grafted cells in the cerebellar cortex, when compared to the real extent of the ependymal grafted area in each particular case, provided important data: (1) The external granular layer (EGL), the secondary germinative epithelium, seems not to originate exclusively from the "germinative trigone," as is usually considered the case. It emerges from a larger but restricted portion of the primary cerebellar matrix extending about the caudal fourth or third of the ventricular epithelium, as defined after its morphogenetic rotation. (2) The Purkinje cells (PCs) develop from all areas of the cerebellar epithelium. Although the distribution of donor PCs parallels the grafted ventricular layer mediolaterally, donor PCs extend more in the rostrocaudal dimension. The PC layer is formed mainly by donor cells in the lobules underlain by the grafted ependymal layer. However, donor PCs are also observed in cortical lobules surmounting the host ventricular layer. In these lobules, the donor PCs form clusters of various widths interrupting the host PCs. Reciprocally, clusters of host PCs are also found in the lobules formed mainly by donor PCs. The alternate small clusters of donor or host PCs are surrounded by Bergmann fibers of the other species' origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. I. Development of reciprocal graft-host interactions.

In this paper, we have morphologically studied the developmental events underlying the neuronal replacement, 3-21 days after grafting. Despite their abnormal environment, Purkinje cell progenitors proceed with their proliferation in the grafted neuroepithelium, with a time window similar to that characterizing proliferation of this neuronal class in control mouse embryos. Only postmitotic Purkinje cells leave the grafts and migrate to the host molecular layer following stereotyped pathways. These neurons invade the host molecular layer, either through a tangential migration under the pial basal lamina from the graft/host interface or breaking locally the latter, and passing directly from the lateral swellings of the graft lying on the surface of the host folia. Whatever the pathway for host invasion, the migrating Purkinje cells penetrate radially and/or obliquely into the host molecular layer until their inward-oriented processes attain the molecular/granular layer interface, which occurs about 7 days after grafting. At the end of their migration, the grafted Purkinje cells with bipolar shapes and long and smooth processes begin to build up their ultimate dendritic trees. This dendritogenesis proceeds with constructive and regressive processes, passing through the same three developmental phases described by Ramón y Cajal (Trab. Lab. Invest. Biol. Univ. Madrid 24:215-251, 1926) for control Purkinje cells (phase of the fusiform cell, phase of the stellate cell with disoriented dendrons, and phase of orientation and flattening of the dendrites). In the grafted cerebella, the duration of the second and third phases is somewhat shorter than during normal cerebellar ontogenesis. Synaptogenesis between adult host axons and grafted Purkinje cells starts when the latter attain their second phase of dendritic development. Somatic filopodia emerging from grafted Purkinje cells begin, 10-11 days after grafting, to be synaptically contacted by axonal sprouts of the host climbing fibers resulting, 2 days later, in the formation of pericellular nests. Synaptogenesis between slender dendritic spines and host parallel fibers, together with that of axon terminals from host molecular layer interneurons and the smooth surface of the grafted Purkinje cell somata, begin earlier than in control mouse development, being almost simultaneous with climbing fiber/Purkinje cell synaptogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. II. Development of synaptic responses: an in vitro study.

Solid pieces of cerebellar primordia from 12-day-old C57Bl embryos were implanted in the cerebellar vermis of 3-4-month-old "Purkinje cell degeneration" mutant mice. Ten to 22 days after grafting, mutant mice were sacrificed, and synaptic responses of grafted Purkinje cells were studied by intracellular recordings performed in 400 microns thick sagittal slices in vitro. As early as 10 days after transplantation, grafted Purkinje cells have already completed their migration from the implant into the host molecular layer. Accordingly, inhibitory as well as excitatory responses were already elicited in these cells by electrical stimulation of the host subcortical white matter. Furthermore, a transient stage of multiple innervation of Purkinje cells by climbing fibers exists between 10 and 15 days after grafting, as revealed by the stepwise variation in amplitude of the climbing fiber-mediated excitatory postsynaptic potentials recorded before 15 days after grafting. Thirteen days after transplantation, typical all-or-none climbing fiber-mediated responses, parallel fiber-mediated excitatory postsynaptic potentials, and inhibitory postsynaptic potentials were also already present. Finally, normal adult-type synaptic responses were observed in all tested cells 15 to 17 days after grafting. Together with the companion paper (Sotelo et al., 1990), these results demonstrate that grafted Purkinje cells are able to impose on host afferents a pattern of synaptogenesis which closely follows that occurring during normal development, in particular, the transient stage of multiple innervation of Purkinje cells by climbing fibers.

Reconstruction of the defective cerebellar circuitry in adult Purkinje cell degeneration mutant mice by Purkinje cell replacement through transplantation of solid embryonic implants.

Solid pieces of cerebellar primordia taken from 12-day-old C57BL embryos were implanted into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice and analysed 2-3 months later. Purkinje cell replacement was followed by means of immunocytochemistry with antisera against either cyclic guanosine monophosphate-dependent protein kinase or vitamin D-dependent calcium-binding protein, which allows the complete staining of these neurons. Although all solid graft implants survived, their fate within the mutant cerebellum varied in three ways: Often, a more or less large fragment of the solid graft remained in the white matter, close to the cortex or even partially replacing it. These remnants contained a few distorted Purkinje cells and a region corresponding to the transplanted deep nuclei, composed of numerous immunostained axons and axon terminals surrounding immunonegative neurons. Less frequently remnants of the graft were extruded to an extracerebellar location, between two adjacent folia. They contained a few Purkinje cells intermixed with granule cells and other neurons. In a few cases corresponding to superficial deposition, the implants developed lobulated and trilaminated minicerebella which were located outside the mutant cerebellum but integrated into it. In all three situations, a large number of grafted Purkinje cells succeeded in moving out of the implants and in invading the host molecular layer. These Purkinje cells develop flattened dendritic trees perpendicular to host bundles of parallel fibres. Ultrastructural examination of the synaptic investment of Purkinje cells which have reached the host molecular layer revealed that they acquire normal synaptic inputs although complex pericellular baskets and pinceau formation do not develop. Axons from molecular layer interneurons synapse on perikaryal and smooth dendritic membranes, climbing fibres synapse on stubby spines emerging from thick dendritic branches, and parallel fibres contact almost exclusively the long-necked spines of the distal spiny branchlets. Finally, Purkinje cells which succeed in migrating to molecular layer regions no further than 0.6 mm from the host deep nuclei are able to grow axons which reach appropriate target areas and establish synaptic connections on nuclear neurons. The results obtained from this series of long-term survival cerebellar transplantations point to the possibility of fulfilling most of the conditions necessary for functional restoration of neural grafts in systems in which neurons are connected in a point-to-point manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological demonstration of a synaptic integration of transplanted Purkinje cells into the cerebellum of the adult Purkinje cell degeneration mutant mouse.

After implantation of solid pieces of cerebellar primordia from 12-day-old C57BL embryos into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice, Purkinje cells from the donor leave the implant and differentiate while migrating into the host molecular layer. Electrophysiological studies were performed using in vitro cerebellar slice preparations from "Purkinje cell degeneration" mutants 1-2 months after grafting, when grafted Purkinje cells have reached their final location in the host molecular layer and have completed their morphological differentiation. Intracellular recordings obtained from 45 Purkinje cells in mutant mice demonstrated that such grafted neurons have normal bioelectrical properties including sodium and calcium conductances and inward rectification. Moreover, all grafted Purkinje cells responded to electrical white matter stimulation by a typical all-or-none climbing fiber response. Responses mediated through the activation of mossy and parallel fibers, as well as inhibitory postsynaptic potentials, were also recorded in a significant number of grafted Purkinje cells. On the whole, all these excitatory and inhibitory responses in grafted "Purkinje cell degeneration" mutant mice have characteristics comparable to those in control mice. After electrophysiological studies, Purkinje cells were further characterized by their positive staining by calbindin antibody. Neurons of this class were dispersed throughout the molecular layer of the host folia in which the electrophysiological recordings had been performed. The ectopic location of their perikarya, the presence of dendritic trees spanning most of the molecular layer (without entering the granular layer), and the occasional presence of axons emerging from the ectopic neurons and forming loose bundles at the white matter axis of the folia, corroborate the grafted nature of the Purkinje cells studied. Therefore, these experiments demonstrate that embryonic Purkinje cells from the graft can complete differentiation in the adult host cerebellum, and establish specific synaptic contacts with the presynaptic elements previously impinging on the missing neurons of "Purkinje cell degeneration" mutants. This process leads to a qualitative functional synaptic restoration of the cortical cerebellar network.

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation.

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.

Cytodifferentiation of quail tectal primordium transplanted homotopically into the chick embryo.

The development of the retinotectal system in the quail embryo starts earlier and evolves faster than in the chick embryo. In order to establish whether the mesencephalic alar plate (i.e., the primordium of the optic tectum) of a quail embryo maintains its own rate of cytodifferentiation after transplantation into a chick embryo or whether this rate could be influenced by the host, we performed homotopic transplantations of the tectal primordium between the two species on day 2 of incubation (E2) by removing the mesencephalic alar plate in the chick and replacing it with that of the quail embryo. Graft extension was evaluated by means of the well-known quail nucleolar marker, and cytodifferentiation of both operated and unoperated tecta was analyzed from E3 to E12. It was found that: in most cases, the operated tectum is a chimera formed by a large dorsal territory consisting solely of grafted quail cells and a smaller ventral territory almost entirely made up of host chick cells. A clear boundary exists at the interface between these two territories. In the host, the temporal sequence of appearance of the various laminae, following a well-established rostroventral-caudodorsal developmental gradient, is comparable in both the operated tectum and the host territory of the chimeric tectum to that of a control chick tectum. In the graft, the migration of postmitotic cells starts earlier than in the host. However, in the former there is about a 12-h delay with respect to a control quail tectum. Proliferation and migration of cells take place in the graft much faster than in the host. Thus, the formation of the 8 deepest layers occurs according to the normal quail schedule, indicating that the early delay is quickly recuperated. This process of lamination follows the normal quail rostroventral-caudodorsal developmental gradient. The post-mitotic neurons originating in the grafted neuroepithelium follow a normal radial migration. Nevertheless, a few grafted cells occupy the host tectal territory far from the host/graft interface. These cells have been observed in both the stratum griseum centralis and the uppermost tectal layers, indicating that some tectal neurons are able to displace themselves tangentially. Contrary to what happens in the 8 deepest layers, which in the graft follow the normal quail cytodifferentiation schedule independently of the host, cytodifferentiation in the upper tectal layers is partially influenced by the host.(ABSTRACT TRUNCATED AT 400 WORDS)

Transplanted mesencephalic quail cells colonize selectively all primary visual nuclei of chick diencephalon: a study using heterotopic transplants.

A portion of the quail mesencephalic alar plate (10-12 somites embryos, second day of incubation) was heterotopically transplanted to replace a portion of the diencephalic alar plate of a similar stage chick embryo. Analysis of the chimeric embryos on day 18 of incubation was performed both by Feulgen and Rossenbeck histochemical staining to recognize the transplanted cells, and by cytoarchitectonic methods. The heterotopically transplanted neuroepithelia were integrated in the host pretectal area, although their precise location, substituting some missing host pretectal nuclei, varied slightly from case to case. The cytoarchitecture of the graft and its extension allowed to distinguish two types of transplants: in 50% of the cases the graft developed a laminated, tectal-like structure appearing as a supernumerary optic tectum, whereas in the other 50% of the cases it gave rise to a smaller, not well-defined, non-laminated structure, which could not be recognized as tectal. Independent of the extension and cytoarchitecture of the grafts, in all cases numerous transplanted quail cells were observed beyond the limits of the graft spreading along the optic tract, into all the retino-recipient diencephalic nuclei and into the mesencephalic tectal gray. Conversely, the host optic tectum and the non-primary visual nuclei, even those in close apposition to the transplant, were always devoid of transplanted cells. Analysis of 5- to 10-day-old chimeric embryos has shown that the ectopically located mesencephalic quail cells start migrating from the transplant on day 7 of incubation and follow a tangential pathway at the surface of the diencephalon, throughout the optic tract and between the optic tract and the incipient primary visual nuclei. On day 10, many of these cells have already invaded most of the host retino-recipient nuclei. These observations are discussed with respect to both the phenotypic expression of the transplanted primordium and the tangential migration of tectal cells previously observed in homotopically transplanted chimeric embryos. The possible significance of these results is also discussed.

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

This paper is a sequel to a previous report, using quail/chick 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 axonal plexuses. In confirmation of previous results, SGC neurons have a limited range of migration, whereas SGFS cells translocate across much longer distances. In reconstructions of appropriate cases, a remarkable polarity was noted. Significant invasion of chick tectum by quail cells mostly occurred in the rostral half of the host tectum. 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 interdigited 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.

Nerve endings on the intramuscular tendons of cat extraocular muscles.

The extraocular muscles of the cat have been studied with silver-impregnated, teased preparations. Short and long muscle fibers have been found among the multi-innervated as well as among the focally-innervated muscle fibers. The attachments between the short fibers are made by intramuscular tendons. Longer fibers may have myomyal bridges. A peculiar kind of nerve ending has been found on the intramuscular tendons of short multi-innervated fibers. These endings are supplied by myelinated axons which also give endings to the muscle fibers. The significance of these nerve endings is discussed.

Retinal and tectal connections of embryonic nucleus superficialis magnocellularis and its mature derivatives in the chick.

In a companion paper (Puelles et al, this issue), the cytoarchitectonic development of the thalamic primordium called nucleus superficialis magnocellularis (SM) and its adult configuration in the chick were studied, correcting the misinterpretations that have impeded proper study of this neuronal group. Given its superficial position in the diencephalon, in contact with the optic tract and neighbouring retinorecipient grisea (SS, GV), as well as with the tecto-recipient n. rotundus, SM was suspected to have connections with centers of the visual pathway. In this paper we report the existence of a non-topographic retinal projection over the superficial adult derivate of SM (n. interstitialis tractus opticus, ITO) and a non-topographic, diffuse projection of the whole SM-derived population (area perirotundica, ApR, and ITO) onto the optic tectum. The latter was demonstrated throughout the late embryonic period in which SM loses its embryonic unitary character and becomes dispersed into its ill-defined, definitive adult portions (ITO, ApR). Golgi-like HRP- or DiI-labeling of SM cells showed a protracted immature appearance of their dendrites, expressed coincidently with a capacity to translocate superficially into the optic tract.

The palisade endings of cat extraocular muscles: a light and electron microscope study.

The palisade endings (PEs), a particular type of nerve ending found only in extraocular muscles of mammals, have been studied using both silver-stained teased preparations and electron microscope techniques. They have been found, in act, in both the proximal and distal muscle insertions of the four recti and the two oblique mucles. PEs are exclusively associated with some of the mitochondria-poor, multiply-innervated muscle fibres present in the globar layer os these muscles, and consist of a multitude of terminal branches embracing the extremity of the muscle fibre and penetrating the infoldings formed by the muscle fibre at its tendinous attachment. The whole formation is surrounded by a thin capsule. These nerve endings present striking similarities to the developing Golgi tendon organ; the terminal branches lying among the collagen fibrils and occasionally making 'sensory-like' close contacts with the muscle fibre are disposed in such a way that they could easily have a sensory role. It was concluded that PEs present sufficient morphological evidence to be considered as sensory, encapsulated, myotendinous receptors, each related to a single multiply-innervated muscle fibre.