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The midbrain/hindbrain (MH) territory containing the mesencephalic and isthmocerebellar primordial is characterized by the expression of several families of regulatory genes including transcription factors (Otx, Gbx, En, and Pax) and signaling molecules (Fgf and Wnt). At earlier stages of avian neural tube, those genes present a dynamic expression pattern and only at HH18-20 onwards, when the mesencephalic/metencephalic constriction is coincident with the Otx2/Gbx2 boundary, their expression domains become more defined. This review summarizes experimental data concerning the genetic mechanisms involved in the specification of the midbrain/hindbrain territory emphasizing the chick/quail chimeric experiments leading to the discovery of a secondary isthmic organizer. Otx2 and Gbx2 co-regulation could determine the precise location of the MH boundary and involved in the inductive events characteristic of the isthmic organizer center.

This paper summarizes chick/quail transplantation experiments performed in the INSERM U106 by Alvarado-Mallart's group from 1989 to 2002. First, it will present the various steps leading us to demonstrate that, at stage 10 of Hamburger and Hamilton, the avian neuroepithelium is still competent to change its fate influenced by environmental inductive factors and that these factors emanate from the cerebellar neuroepithelium; then, it will be briefly reported, experiments aimed to characterize the genetic cascade involved in the formation of the midbrain/hindbrain boundary and the specification of the meso-isthmic-cerebellar domain.

Correlative in situ hybridization of Otx2, Pax2, Gbx2, and Fgf8 mRNA probes in adjacent serial sections through the chicken midbrain and isthmus at early to intermediate stages of development served to map in detail the area of overlap of Otx2 and Pax2 transcripts in the caudal midbrain. The neuronal populations developing within this preisthmic domain made up a caudal part of the midbrain reticular formation, the interfascicular nucleus, and the magnocellular (pre)isthmic nucleus, plus the corresponding part of the periaqueductal gray. The torus semicircularis-the inferior colliculus homolog-expressed Otx2 in its ventricular lining exclusively, but it never expressed Pax2. The parvicellular isthmic nucleus, although placed inside the midbrain lobe, never expressed Otx2, and its cells rapidly down-regulated an early transient Pax2 signal; this pattern is consistent with its reported isthmic origin and forward tangential translocation. This analysis reveals the existence of four distinct midbrain histogenetic domains along the longitudinal axis, at least for the alar plate. These presumably result from step-like isthmic organizer effects on Otx2-expressing midbrain neuroepithelium at different distances from a caudal FGF8 morphogen source (isthmic Fgf8-positive domain). The final phenotypes of these domains are histologically diverse and make up the griseum tectale (rostrally), the optic tectum, the torus semicircularis, and the presently characterized preisthmic domain (lying closest to the isthmic organizer). Available comparative data for reptiles and mammals suggest the general validity of this scheme., ((c) 2005 Wiley-Liss, Inc.)

Transplantation of prosomeres 1-2 into the cerebellar plate were used, by using chick/quail chimeras, to analyse the temporal sequence of the genetic cascade leading the graft to develop a midbrain/hindbrain phenotype. Our results show that (1) at Hamburger and Hamilton (HH) stage 13, Pax2 and En2 are already induced within the graft, before all other genes of the cascade, whereas misexpression of Fgf8 is also observed within the contiguous host cerebellar plate; (2) within the graft, Otx2 repression and Gbx2 induction (see Hidalgo-Sánchez et al. [1999] Development 126:3191-3203) are secondary events that affect, from stages HH14-15, the areas in contact with the host Gbx2/Fgf8-expressing cerebellar plate; (3) at these stages, the repressed Otx2 territory extends beyond the areas induced to express Gbx2, with the two territories not abutting before HH17-18; (4) Fgf8 expression becomes progressively induced within the Otx2-repressed/Gbx2-induced territory, starting at HH15-16. Our results support the hypothesis that the host-Gbx2/graft-Otx2 interface could trigger the genetic cascade induced within the graft and that the Gbx2-induced domain could play a key role during the establishment of the induced intragraft midbrain/hindbrain boundary., (Copyright 2001 Wiley-Liss, Inc.)

We have studied the neuromeric organisation of the mesencephalic-metencephalic (mes-met) territory of the avian neural tube using chick/quail transplantation experiments and analysing the expression of various regulatory genes in chimeric and normal embryos. Homotopic grafts demonstrate the presence of an interneuromeric boundary separating the mesencephalic and cerebellar territories (the mes-met or midbrain/hindbrain boundary). This boundary is characterised from HH10 onwards by the confrontation of the Otx2-Wnt1 and Gbx2-Fgf8 expressing domains, while En2 and Pax2 genes are expressed at both sides of the mes-met boundary. The evolution of the position of the Otx2/Gbx2 boundary with respect to the vesicles and constriction observed within the mes-met domain between stages HH10 and HH20, allows us to redefine the fate map of this region and to propose a new nomenclature for HH10. Transplantation between the prosencephalic neuroepithelium and the mes-met domain shows the possibility of inducing a mes-met phenotype within the two caudal-most prosomeres, preceded by its characteristic genetic cascade. The induction selectively takes place along the boundary between the graft (Otx2 positive) and the host cerebellar territory (expressing high levels of Gbx2); this includes the induction inside the graft of a new Otx2/Gbx2 boundary. Conversely, no induction is ever observed when the graft is confronted to the host Otx2 expressing domain. Although Fgf8 may be involved in the inductive events, our data strongly suggest that confrontation between Otx2 and Gbx2 is essential as an organiser of the mes-met domain.

Chick/quail transplantation experiments were performed to analyse possible factors involved in the regionalisation of the midbrain-hindbrain domain. The caudal prosomeres, expressing Otx2, were transplanted at stage HH10 into rostrocaudal levels of the midbrain-hindbrain domain, either straddling the intra-metencephalic constriction (type 1 grafts), or at rostral and medial levels of pro-rhombomere A1 (type 2 and 3 grafts, respectively); thus, in all situations, one border of the graft was in contact with the host Gbx2- and Fgf8-expressing domains. The area containing the graft, recognised by QCPN immunohistochemistry, was first analysed 48 hours after transplantation for Otx2, Gbx2, En2 and Fgf8. Although in all three situations, a large part of the graft maintained Otx2 expression, another part became Otx2 negative and was induced to express Gbx2 and Fgf8. These inductive events occurred exclusively at the interface between the Otx2-positive transplanted domain and the ipsilateral host Gbx2-positive rhombomere 1, creating a new Otx2-Gbx2 boundary within the grafted territory. In type 1 and 2 grafts, the induced Fgf8 domain is in continuity with the host Fgf8 isthmic domain, whereas for type 3 grafts, these two domains are separate. High levels of En2 expression were also induced in the area expressing Gbx2 and Fgf8, and Wnt1 and Pax2 expressions, analysed in type 3 grafts, were induced at the intragraft Otx2-Gbx2 new boundary. Moreover, at later embryonic stages, the graft developed meso-isthmo-cerebellar structures. Thus, gene expressions induced in the grafted prosencephalon not only mimicked the pattern observed in the normal midbrain-hindbrain domain, but is followed by midbrain-hindbrain cytodifferentiation, indicating that not only Fgf8 but also confrontation of Otx2 and Gbx2 may play an essential role during midbrian-hindbrain regionalisation.

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.

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 chick/quail chimeric system is now extensively used to study the development of the central nervous system. Here we discuss data obtained by this powerful experimental approach by which to study several issues of the cerebellar ontogenesis. We first discuss experiments which have allowed redefinition of the localization of the cerebellar primordium in the early neural tube and which suggest that the cerebellum could originate from different morphogenetic units. Then, we discuss experiments testing the possible role of the homeobox containing gene En-2 in cerebellar specification and showing that the En-2 expressing cerebellar neuroepithelium can act as an organizer. Finally we discuss data obtained in chimeric embryos with partial cerebellar grafts used to reexamine the origin and settling of several types of cortical cerebellar cells, in particular granule cells, molecular layer interneurons and Purkinje cells.

Segmentation of the neural tube has been clearly shown in the forebrain and caudal hindbrain but has never been demonstrated within the midbrain/hindbrain domain. Since the homeobox-containing gene Otx2 has a caudal limit of expression in this region, we examined, mainly in chick embryos, the possibility that this limit could represent an interneuromeric boundary separating either two cerebellar domains or the mesencephalic and cerebellar primordia. In situ hybridisation with chick or mouse Otx2 probes showed the existence of a transient Otx2-negative area in the caudal mesencephalic vesicle, between stages HH10 and HH17/18 in chick, and at embryonic day 9.5 in mice. The first post-mitotic neurons of the mesencephalon sensu stricto, as labelled with an anti-beta-tubulin antibody, overlay the Otx2-positive neuroepithelium with a perfect match of the caudal limits of these two markers at all embryonic stages analysed (until stage HH20). Chick/quail homotopic grafts of various portions of the midbrain/hindbrain domain have shown that the progeny of the cells located in the caudal mesencephalic vesicle at stage HH10 are found within the rhombomere 1 as early as stage HH14. Furthermore, our results indicate that the cells forming the HH20 constriction (coinciding with the caudal Otx2 limit) are the progeny of those located at the caudal Otx2 limit at stage HH10 (within the mesencephalic vesicle). As a result, the Otx2-positive portion of the HH10 mesencephalic vesicle gives rise to the HH20 mesencephalon, while the Otx2-negative portion gives rise to the HH20 rostral rhombomere 1. Long-survival analysis allowing the recognition of the various grisea of the chimeric brains strongly supports the view that, as early as stage HH10, the caudal limit of Otx2 expression separates mesencephalic from isthmo/cerebellar territories. Finally, this study revealed unexpected rostrocaudal morphogenetic movements taking place between stages HH10 and HH16 in the mediodorsal part of the caudal Otx2-positive domain.

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.

The expression of the homeobox-containing gene En-2 was analysed with the monoclonal antibody 4D9 in the chick central nervous system throughout embryogenesis. Confirming previous studies, early expression of the En-2 protein [beginning at stage 9 of Hamburger and Hamilton (HH9)] is restricted to a portion of the neural tube containing the primordia of the cerebellum, the isthmic region and the mesencephalic grisea, and forms a double gradient decreasing both caudally and rostrally from a high point located around the midbrain-hindbrain constriction. This mes-isthmo-cerebellar region contains all the En-2-positive germinative cells and the great majority of the En-2-positive postmitotic neurons throughout embryogenesis. Nevertheless, as the postmitotic neurons appear, En-2 expression also occurs outside this region: in two columns of non-motoneuron cells in rhombomeres two to four (between HH20 and HH30) and, from HH24 onwards, throughout the grey matter of the lumbar and thoracic spinal cord, with the exception of the ventral motoneuron columns. Here, a detailed description of En-2 expression is provided for the mes-isthmo-cerebellar region at stages HH30-32 [embryonic day (E) 7], HH37 (E11) and HH46 (E21, hatching). This allows the visualization of cellular groups with heterogeneous patterns of En-2 expression, which are specific for each group in the intensity of En-2 expression, the distribution of the labelled cells and the temporal regulation of the gene. The use of tyrosine hydroxylase antiserum shows coexpression of the tyrosine hydroxylase enzyme and En-2 protein in the caudal part of the nuclei tegmenti pedunculo-pontinus, the area ventralis of Tsai and the substantia grisea centralis, but not in the locus coeruleus. In the cerebellum, the first expression, which is located in the deep nuclei and parasagittal bands of Purkinje cells, is down-regulated when the molecular layer interneurons and the granular cells begin to express the gene, at the end of embryogenesis. Finally, at hatching, En-2 expression permits the visualization in the cerebellum of a population of small En-2-negative cells located around the Purkinje cells that may correspond to those described in chick/quail chimaeras as having an origin different from that of the bulk of granular neurons.

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)

At early phases of development (stage HH12 of chick and quail embryos) the rostral part of the cerebellar anlage extends into the caudal third of the mesencephalic vesicle. This region is characterized by its high level of expression of the Wnt-1 and engrailed genes, which encode, respectively, for a secreted molecule and for a homeodomain protein. In order to analyze the potentialities of various parts of the cerebellar anlage, pieces of quail or mouse embryonic neural tubes have been transplanted heterotopically in the prosencephalon of HH12 chick embryos. These experiments indicated that signals operating in the plane of the neural tube are still operating in the donor avian and murine embryos. The met- mes- and prosencephalic territories of HH12 chick embryos are still competent to change their fates under the influence of these signals. It was also found that the Wnt-1 expression domain, the caudal mesencephalic vesicle, has striking organizing properties until late stages of development in avian and rodent embryos. The exact role of Wnt-1 in these inductive interactions is still unknown. Wnt-1 influence on cell fate could be mediated by the engrailed genes. Observations suggesting a later role of en-2 in the regionalization of the cerebellar cortex are also reported.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.

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.

Despite Purkinje cell replacement, leading to the repair of the cortical circuit of the pcd mouse cerebellum grafted with E12 cerebellar primordium, the reestablishment of the corticonuclear projection only occurs for some Purkinje cells and in a small percentage of grafted mice. In order to assess the importance of: (1) competition between host and grafted deep nuclei, and (2) the distance between the implants and the host deep nuclei, new grafted experiments have been performed. In the latter, solid grafts were taken from E13 or E14 donor embryos after removal of the region containing the postmitotic deep nuclear neurons, and randomly positioned at various cerebellar depths. With cortical implants, the absence of donor nuclear neurons is not sufficient to allow the axons of the grafted Purkinje cells that have invaded the host molecular layer to escape the confinement of this layer. The molecular/granular layer interface appears as an almost impassable obstacle, and the granule cell layer as a nonpermissive milieu. With grafts located between the host deep nuclei and the 4th ventricle (deep grafts), the grafted Purkinje cells project massively to the host nuclei, but they are unable to leave the implant and, therefore, they are not integrated in the deficient cortical circuit. Finally, when the grafts positioned in the central white matter (intermediate grafts) disrupt the integrity of the host granule cell layer, some of the grafted Purkinje cells invade the host molecular layer, while most of them remain within the implant. Some axons of the cortically integrated Purkinje cells, using the nearby graft as a bridge, seem able to innervate the host deep nuclei. The latter, in addition, receive a massive projection from the nonintegrated Purkinje cells. These results emphasize the ability of grafted Purkinje cells to specifically innervate their target host neurons, when either there is proximity, or when a permissive microenvironment for their axonal outgrowth is created by embryonic grafted cortical cerebellar neurons, filling the gap between the molecular layer and the deep nuclei of the host.

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.

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.

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 homeobox gene Chick-en, sharing homologies to the engrailed gene of Drosophila, is expressed, during early steps of development, in a restricted area of the chick embryo including mes-metencephalic neuroepithelia. The expression of the Chick-en gene has been analyzed in chick/quail chimeric embryos in which a portion of the 2-day-old mes-metencephalic neuroepithelium has been transplanted in an inverted position. By means of a monoclonal antibody, "Mab 4D9," recognizing engrailed proteins, it is shown that the expression of the Chick-en gene is regulated in the inverted neuroepithelium according to its new position in the host neural tube. The regulation takes place within 20 hr after transplantation. These results, together with previous data demonstrating that the phenotypic expression of the inverted neuroepithelium depends, also, on its new position in the host neural tube, strongly suggest that the engrailed protein could play an important role in the positional specification of the mes-metencephalic neuroepithelium.

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)

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.

In a previous study using chick/quail chimeric embryos with homotopic transplants (Martinez & Alvarado-Mallart, 1989b), we have delimited in the 2-day-old avian embryo the areas of the neural tube giving rise to optic tectum and mesencephalic grissea as well as to isthmic grissea and cerebellum: respectively, "mesencephalic" and "metencephalic" alar plates. To investigate the determination or the competence of these areas, portions of these germinative neuroepithelia from a quail embryo were transplanted in substitution for other areas of the chick neural tube. The analysis of the chimeric brains was done by comparing alternating transverse sections stained for cytoarchitecture and with two different techniques to recognize transplanted versus host cells: either the Feulgen and Rossenbeck DNA histochemical reaction and/or immunohistochemical methods with a monoclonal antibody recognizing quail but not chick cells. The eventual visual innervation of the quail graft was analyzed in many cases by injecting anterograde axonal tracers in the eye contralateral to the graft. The results are as follows: (1) caudal metencephalon transferred to mesencephalon maintained in all cases its presumptive cerebellar phenotype, whereas (2) rostral metencephalon transferred to mesencephalon changed its fate to a tectal phenotype but maintained its cerebellar fate when transferred to diencephalon; (3) caudal mesencephalon maintained its tectal fate in 65% of the cases when transferred to diencephalon, whereas (4) rostral mesencephalon transferred to a cerebellar domain changed its fate and became influenced by the surrounding structures in all cases, but only in 85% of the cases when it was transplanted to diencephalon; (5) the in situ host diencephalon, isolated from its normal environment by a mesencephalic graft, is competent to change its fate and express a mesencephalic phenotype. These results demonstrate that at least some regions of the germinative neuroepithelium from either metencephalon, mesencephalon, and diencephalon are still pluripotent in the 2-day-old avian embryo and that their fate seems to be under the influence of the surrounding structures. Rostral mesencephalon and rostral metencephalon have been more easily influenced by environmental factors than their caudal counterparts, suggesting that regions providing instructive positional factors exist within the 2-day-old germinative neuroepithelium. These regions might play an important role in the determination of the various segments of the neural tube.

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.

Cell suspensions from cerebellar primordia of 12-day mouse embryos were grafted into the cerebellum of 4-month-old Purkinje cell degeneration (pcd) mutant mice and examined 2-3 months later. In contrast to those of nontreated mutants, all of the grafted cerebella exhibited Purkinje cells that had migrated into the molecular layer, where they were clustered over its superficial two-thirds. These Purkinje cells develop flattened dendritic trees perpendicular to bundles of parallel fibers. Ultrastructural examination of their synaptic inputs and outputs disclosed that (i) as in normal cerebella, climbing fibers and axons from basket and stellate cells synapse on thick dendrites, whereas parallel fibers almost exclusively contact the distal spiny branchlets, and (ii) a substantial number of Purkinje cell axons reach their appropriate targets in the deep cerebellar nuclei, where they establish synaptic connections on large and small neurons. These results indicate that embryonic Purkinje cells grafted into the cerebellum of adult mice with heredodegenerative ataxia integrate themselves very specifically into the cerebellar circuitry of the recipient mouse, where they can replace the missing Purkinje cells. They also provide a morphological basis favoring the notion of functional restorative capabilities of neural grafts in systems in which neurons are connected in an almost point-to-point manner.

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.

This paper examines the organization of host afferents within cerebellar grafts implanted into kainic acid lesioned cerebellum. Our selection of a cerebellum, a prime example of a 'point-to-point' system, permits precise determination of the degree and the specificity of host-graft interactions. One month after a cerebellar injection of kainic acid, the lesion produced can be divided into two concentric regions: (i) a central necrotic zone, totally depleted of neurons (zone 1), and (ii) a peripheral zone which lacks all Purkinje cells but preserves its cortical lamination (zone 2). Two months after the implantation of solid pieces of embryonic cerebellum, the graft has evolved into a minicerebellar structure, occupying most of zone 1. The grafted minicerebellum consists of a highly convoluted trilaminated cortex with a core containing deep nuclear neurons. Purkinje cells are positioned between the molecular and granular layer with their short and irregular dendrites branching within the former. Donor foetal Purkinje cells migrate into the contiguous portion of the molecular layer of the host zone 2. These embryonic neurons set up within the upper three-quarters of the host molecular layer, and develop monoplanar dendritic trees that span the whole width of the layer. The organization of host-graft interactions was studied by autoradiography of anterogradely transported tritiated leucine, injected in the host bulbar region containing the caudal half of the inferior olivary complex (origin of all vermal climbing fibres) and the dorsally adjacent paramedian reticular nucleus (origin of a few mossy fibres). Numerous labelled fibres cross the host-graft interface from the white matter of the host cerebellum, and provide innervation to the minicerebellar structure. The vast majority of these labelled axons terminate in the molecular layer, forming axonal arborizations that follow the shape of the Purkinje cell dendrites. The labelled climbing fibres are organized into uneven sagittally aligned strips, which mimic that of olivocerebellar projections in control rats. Only a small proportion of host labelled fibres end in the donor granular layer, forming typical mossy fibre rosettes. The latter are present in the region of the graft close to the host-graft interface. In addition, labelled axons are observed climbing over the dendritic trees of grafted Purkinje cells that have invaded a portion of the host molecular layer of zone 2. In all regions containing grafted Purkinje cells and labelled climbing fibres, the density of the innervation is close to normal with practically all Purkinje cells receiving a climbing fibre. The extensive integration of the grafted cells into the deficient neuronal networks of the host clearly illustrates the positive neurotropic effect exerted by immature cerebellar neurons on adult extracerebellar afferent fibres. The hodological integration, allowing a possible restoration of the impaired cerebellar circuitry, takes place respecting the specificity and topographic distribution which characterize the 'point-to-point' arrangement of normal cerebellar circuitry.

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.

Responses to passive stretch applied to the rectus lateralis muscle (RL) were recorded with a microelectrode from the mesencephalic nucleus of the fifth nerve (Mes V) in cat encéphale isolé preparation. The necessary conditions to attribute the unit response to an eye muscle stretch are the followings: i) respond neither to jaw movements nor to pressure applied to the eye ball: ii) be activated with a short latency, following stretch of the eye muscles; iii) be excited by a light touch applied to a small area of one eye with a glass rod. The characteristics of the Mes V responses following stretch of the eye muscle are similar to those obtained in a primary afferent fibre from a skeletal muscle. Horseradish peroxidase was injected in the RL muscle of young and adult cats. As a consequence of fast retrograde axonal transport, the tracer was found to accumulate in the corresponding motoneurones of the VI nucleus. In addition, the enzymes accumulation was also found in cells of Mes V corresponding to the somata of RL sensory terminals. Units responding to RL stretchs, and primary sensory neurons labelled by the tracer, were found in the same area of Mes V.

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.

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.

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.

To study the adaptative capabilities of the retinotectal system in birds, the primordium of one optic tectum from 12-somite embryos of Japanese quail was transplanted either homotopically , to replace the ablated same primordium, or heterotopically, to replace the ablated dorsal diencephalon in White Leghorn chick embryos of the same stage. The quail nucleolar marker was used to recognize the transplants. The cytoarchitecture of the tecta and the retinal projections from the eye contralateral to the graft were studied on the 17th or 18th day of incubation in the chimeric embryos by autoradiographic or horseradish peroxidase tracing methods. Morphometric analysis was applied to evaluate the percentage of the tectal surface receiving optic projections. It was observed that: (i) quail mesencephalic alar plate can develop a fully laminated optic tectum even when transplanted heterotopically; (ii) retinal ganglion cells from the chick not only recognize the tectal neurons of the quail as their specific targets in homotopic grafts, but the optic fibers deviate to innervate the heterotopically grafted tectum; (iii) in the presence of a graft, the chick retina is unable to innervate a tectal surface of similar or larger size than that of the control tectum; (iv) tectal regions devoid of optic projections, whether formed by donor or by host cells, always present an atrophic lamination; (v) the diencephalic supernumerary optic tectum competes with and prevails over the host tectum as a target for optic fiber terminals.

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)

It has often been proposed that one way of replacing degenerating neurons in the brain is to implant embryonic neurons of the same type. However, in the case of so-called 'point-to-point' systems, as opposed to the 'paracrine' systems which mainly involve local release of neurotransmitter, functional recovery requires a precise re-establishment of the missing circuitry. We recently showed that in one point-to-point system, the cerebellum of adult mice homozygous for the mutation Purkinje cell degeneration (pcd)2, missing Purkinje cells can be replaced by grafting cerebellar primordia from normal mouse embryos. Here, we present studies of the cellular mechanisms underlying this successful replacement. Grafted Purkinje cells leave the graft to migrate along stereotyped pathways to their final position in the deficient molecular layer, where they receive synaptic contacts from adult host neurons. Both the detailed timetable and the precise cellular interactions observed are remarkably similar to those occurring during normal development. Our results suggest that the deficient molecular layer exerts a selective neurotropic effect on neurons of the missing category, and that the embryonic neurons are able to respond to this signal during a period defined by their own internal clock. We also raise the possibility that embryonic Purkinje cells can induce in adult neural cells a new type of plasticity, that of recreating a permissive microenvironment for the integration of embryonic neurons.

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.

Homotopic and isochronic transplantations of the right dorsal half of the mesencephalic vesicle have been performed between chick and quail embryos at the stage of 10 - 14 somites. Analysis of the extension of the graft, by means of the quail nucleolar marker, combined with cytoarchitectonic analysis has disclosed that the transplanted neuroepithelium gives rise to isthmic nuclei and to a portion of rostral cerebellum, in addition to the optic tectum and mesencephalic dorsal grisea. These results show that the rostral portion of the cerebellar primordium is located in the so-called 'mesencephalic' alar plate, thus considerably more rostrally than previously supposed. This has been confirmed by two other types of chimeric embryos resulting from homotopic transplantation of either: (i) the quail right alar plate of the first rhombencephalic vesicle, which gives rise to caudal but not rostral cerebellum in the operated side, or (ii) the right alar portion of a segment of the quail neural tube including both the caudal third of the mesencephalic vesicle and the rostral half of the first rhombencephalic vesicle, which gives rise to the whole hemicerebellum in the operated side. Moreover, in chimeric embryos with transplants restricted to the mesencephalic alar plate, the grafted portion of the cerebellar primordium gives rise both to deep cerebellar neurons and to all types of cortical neurons. Among the quail cortical neurons, the Purkinje cells, although intermingled with host Purkinje cells, are organized, at E18, in a characteristic longitudinal band which is strongly reminiscent of the longitudinal functional and morphological organization of the cerebellum. Other types of quail cortical neurons, that is, Golgi cells, granule cells, and molecular layer interneurons, are also observed within this sagittal band. In addition, quail granule cells and molecular layer interneurons as well as quail glial cells, extend over a larger territory on both sides of the longitudinal band containing quail Purkinje cells and even cross the midline and invade the contralateral hemicerebellum. In all types of chimeric embryos, the proliferation, migration, and differentiation of quail transplanted neurons, both in the isthmic region and in the cerebellum, evolve asynchronously from the host homologous ones, following a more precocious and faster developmental schedule. This asynchrony in the development of grafted and host isthmic and cerebellar homologous areas confirms and extends previous findings concerning the proliferation and migration of quail tectal cells in chick quail chimeric embryos (Senut and Alvarado-Mallart, 1987).

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)

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.