Extracerebellar donors were transplanted to P1 rat hosts. At this age, glial types as well as GABAergic interneurons of the cortex and deep nuclei are being generated in the PWM, whereas granule cells are produced by progenitors located in the external granular layer (Ramón y Cajal, 1911; Altman & Bayer, 1997; Carletti & Rossi, 2008). Although grafted cells yielded both neurons and glia (Carletti et al., 2004; Milosevic et al., 2008), our analysis was restricted to neuronal types. In addition, because donor cells did not engraft in the external granular layer and never acquired the phenotypic traits of granule cells, we primarily assessed whether they can differentiate into cerebellar GABAergic interneurons.
Progenitors for cerebellar inhibitory interneurons proliferate in the PWM, where they become specified to different mature identities (Zhang & Goldman, 1996; Maricich & Herrup, 1999; Leto et al., 2009). To determine whether this neurogenic environment can direct the fate choice of extracerebellar progenitors towards local identities, we examined the behaviour of donor cells from the LGE, SVZ, VM and DSC, which are the origin of GABAergic interneurons destined to populate different regions of the mature forebrain, midbrain and hindbrain. To elucidate whether the transplanted cells acquired cerebellar identities, we applied a set of concurrent criteria, including: (i) expression of region-specific transcription factors, (ii) position occupied in the host cerebellar architecture, (iii) expression of mature type-distinctive markers and (iv) acquisition of type-distinctive morphological features. Furthermore, given the precise spatio-temporal sequence by which different categories of cerebellar neurons are generated and assigned to specific laminar positions (Altman & Bayer, 1997; Carletti & Rossi, 2008), we assessed whether transplanted cells shared the same fate and placement as endogenous elements generated during the same developmental period.
Expression of region-specific regulatory genes by donor cells exposed to the host cerebellar environment
In the developing CNS, regional identities are defined by the activity of specific combinations of transcription factors. Therefore, as a first index of the phenotype adopted by donor cells, we investigated whether they retained the expression of transcription factors typical of their site of origin or turned on regulatory genes characteristic of cerebellar development.
Dlx homeobox genes are crucial for the specification of forebrain GABAergic interneurons (Anderson et al., 1997; Eisenstat et al., 1999; Panganiban & Rubenstein, 2002). Among these genes, Dlx2 is expressed by cells derived from the medial and lateral ganglionic eminences, the origins of GABAergic interneurons of neocortex, hippocampus, basal ganglia and olfactory bulb (Panganiban & Rubenstein, 2002). Dlx2 is switched on at early developmental stages (Eisenstat et al., 1999) and is maintained up to adulthood (Saino-Saito et al., 2003), being strictly confined to forebrain derivatives.
Expression of Dlx2 in LGE or SVZ progenitors grafted to the postnatal cerebellum was assessed by immunocytochemical labelling at 2 or 30 days after transplantation to evaluate the activity of the gene in donor cells at different maturation stages. In no instance did we observe any transplanted cell labelled by anti-Dlx2 antibodies (Fig. 1A), including both immature cells in the PWM and mature neurons throughout the recipient parenchyma [2 days after grafting (DAG), n = 158 cells/two transplants; 30 DAG, n = 267 cells/four transplants].
Figure 1. Expression of region-specific transcription factors in extracerebellar cells in vitro and in vivo. LGE/SVZ-derived cells do not express Dlx2 (A) or Pax2 (C) 2 days after transplantation, but they maintain expression of Dlx2 when placed in vitro (B). VM- (D) and DSC-derived cells (G) express Lhx1 in vitro and maintain this expression in the recipient cerebellum (E and H respectively; 2 days after grafting). Some of these transplanted cells (indicated by arrowheads in F and I) are also labelled by anti-Pax2 antibodies. LGE, lateral ganglionic eminence; SVZ, subventricular zone; VM, ventral mesencephalon; DSC, dorsal spinal cord. Scale bars: 25 μm in E, F and I; 50 μm in A–D, G and H.
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To rule out the possibility that Dlx2 expression was modified by cell dissociation, LGE/SVZ cells were plated in vitro and examined after 24 h. In this condition, most of the cells were labelled with anti-Dlx2 antibodies (Fig. 1B; 80.1%, n = 602/three cultures). Furthermore, to determine whether Dlx2 expression could be suppressed as a consequence of transplantation per se, we performed homotopic grafts in the postnatal forebrain. In line with previous reports (Fishell, 1995; Olsson et al., 1997; De Marchis et al., 2007), donor cells acquired morphologically identifiable phenotypes: LGE cells generated medium-sized spiny neurons in the striatum and interneurons in the olfactory bulb, whereas SVZ cells almost exclusively produced olfactory bulb interneurons (Supporting information, Fig. S1A and B). Donor cells were Dlx2-positive in the SVZ of the lateral ventricle (supporting Fig. S1C), along the rostral migratory stream (supporting Fig. S1D) and in the granule cell layer of the olfactory bulb (supporting Fig. S1E), but they were Dlx2-negative in the striatum (supporting Fig. S1F; Panganiban & Rubenstein, 2002). Therefore, homotopically transplanted LGE/SVZ cells express Dlx2 in an appropriate region-specific manner and acquire local phenotypes in different structures of the forebrain.
To assess whether LGE/SVZ cells grafted to postnatal cerebella upregulated host-specific genes, we examined the expression of Pax2, a selective marker of maturing cerebellar interneurons (Maricich & Herrup, 1999; Weisheit et al., 2006). Also in this case, none of the grafted cells at any survival time was ever labelled by anti-Pax2 antibodies (Fig. 1C; 2 DAG n = 147 cells/two transplants; 30 DAG, n = 197 cells/three transplants).
The LIM-homeodomain transcription factor 1, Lhx1, is expressed during the genesis of inhibitory interneurons in the mesencephalon and spinal cord (Gross et al., 2002; Müller et al., 2002; Pillai et al., 2007; Nakatani et al., 2007). Moreover, Lhx1 and Pax2 interact when these interneurons acquire GABAergic identities (Pillai et al., 2007). In the postnatal cerebellum, Lhx1 identifies granule cell progenitors and developing Purkinje neurons, which remain positive in adulthood (Furuyama et al., 1994; Hayes et al., 2001; Zhao et al., 2007). VM and DSC cells maintained expression of these markers in vitro (Fig. 1D and G, VM 43.9%, n = 1345 cells/three cultures; DSC 82%, n = 1228 cells/three cultures). Two days after transplantation to the cerebellum, Lhx1 was expressed by a fraction of VM (Fig. 1E; 8.9%, n = 212 cells/two transplants) and DSC cells (Fig. 1H; 22.5%, 256 cells/two transplants), but only a few of the latter were still positive at 30 days (VM, 0%, n = 154 cells/three transplants; DSC 3.6%, n = 166 cells/three transplants). On the other hand, at any time point anti-Pax2 antibodies only labelled rare VM- or DSC-derived neurons (Figs 1F and I; 2 DAG, DSC 12.7%, n = 267 cells/two transplants; VM 4.7%, n = 213 cells/two transplants; 30 DAG, VM 2%, n = 200 cells/two transplants, DSC 7.8%, n = 108 cells/two transplants).
Placement of extracerebellar interneurons in the recipient cerebellum
Cerebellar cells heterochronically transplanted to the developing cerebellum incorporate in the recipient PWM and acquire the same mature phenotypes and positions of the endogenous interneurons that are generated at the time of transplantation (Leto et al., 2006, 2009). Therefore, exogenous progenitors that switch their fate towards cerebellar types are expected to comply with this ontogenetic schedule.
Regardless of their extracerebellar origin, 2 days after grafting the vast majority of GFP-positive cells were located in the PWM surrounding the deep nuclei or along the axis of the folia (Fig. 2A–C). Rare cells were present on the pial surface of the cerebellar cortex, but they were never seen within the external granular layer. The donor cells displayed morphological features of immature neurons and glia, undergoing migration or initial phases of differentiation (Fig. 2A–C). Thirty days after transplantation, mature neurons were present in wide areas of the recipient parenchyma, including cerebellar cortex, white matter and deep nuclei, with no clear differences among the different donor cell populations (Table 2). Concerning the laminar position in the cortex, transplanted cells were scattered throughout the whole granular layer, but totally absent from the molecular layer (Fig. 3A–C).
Figure 2. Initial placement of donor cells in the recipient cerebellum 2 days after transplantation. Regardless of their origin, LGE/SVZ (A), VM (B) and DSC (C) progenitors are preferentially located in the host PWM and in the adjacent granular layer (gl). High-magnification images (insets) illustrate the morphological features of donor cells. DAPI, blue. Scale bars: 100 μm.
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Neurochemical phenotype of extracerebellar interneurons in the recipient cerebellum
The placement of donor neurons in the deep nuclei and cortical granular layer, but not in the molecular layer, suggests that they may be only able to acquire certain cerebellar phenotypes. Thus, we examined the neurochemical profile and morphology of mature transplanted neurons (30 days after transplantation), relative to their position in the host cerebellum. In particular, we studied the expression of a panel of well-established markers that, alone or in specific combinations, are distinctive of different categories of cerebellar inhibitory interneurons (Fig. 3D–H; Bastianelli, 2003; Singec et al., 2003; Leto et al., 2008).
Concerning donor cells in the granular layer of the recipient cerebellar cortex, the majority of LGE-donors expressed NG (65.6% of 270 cells/eight transplants; Fig. 3D) or NeuN (61.6% of 239 cells/eight transplants; Fig. 3D), whereas PV- and CR-positive cells were less represented, being 14.4% (of 250 cells/eight transplants; Fig. 3D) and 8.7% (of 81 cells/eight transplants, Fig. 3D), respectively. SVZ-derived neurons showed a similar pattern of marker expression: NG-positive cells, 73.1% (of 91 cells/five transplants, Fig. 3D), NeuN-positive cells, 60% (of 116 cells/five transplants, Fig. 3D); CR-positive cells, 11.3% (of 79 cells/five transplants, Fig. 3D). PV-expressing cells were absent (no positive cells out of a sample of 98 cells/five transplants, Fig. 3D).
VM-derived cells yielded considerable amounts of NG- (62.3% of 108 cells/four transplants), NeuN- (67.9% of 103 cells/four transplants) and CR-positive neurons (35.3% of 48 cells/four transplants), plus a minor fraction of PV-expressing cells (8.5% of 70 cells/four transplants). By contrast, DSC donors also generated numerous NeuN-positive cells (53.1% of 92 cells/four transplants), whereas NG- (23.6% of 233 cells/four transplants), CR- (20.2% of 81 cells/four transplants) and PV-positive neurons were less frequent (11.7% out of 145 cells/four transplants). In spite of the differences among the various donor cell populations, many of them expressed CR and NG, markers of granular layer interneurons (CR is also expressed by a subset of the glutamatergic unipolar brush cells), whereas expression of PV, which is distinctive of molecular layer interneurons, was less frequent. On the other hand, expression of NeuN, which was common among the different types of donors, is unusual in granular layer interneurons (Weyer & Schilling, 2003; Leto et al., 2008).
The morphology of donor interneurons is influenced by cerebellar cortical architecture
The different categories of cerebellar GABAergic interneurons are characterized by highly distinctive morphological features (Ramón y Cajal, 1911; Lainé & Axelrad, 2002), particularly relating to the dendritic and axonal patterns. A salient feature of granular layer interneurons is the orientation of their main dendrites relative to their position deep within the layer (Golgi cells, Fig. 4B), or close to the row of Purkinje cell somata (Lugaro cells, Fig. 4A). This peculiar arrangement can be highlighted by mapping the dendritic orientation of endogenous interneurons (Fig. 4D–E): dendrites of Golgi neurons radiate from the cell body in all directions, whereas those of Lugaro cells are typically aligned to the Purkinje cell layer (χ22 = 7.965, n = 96, P = 0.0186).
We investigated whether extracerebellar donors also adopted the same structural arrangement. As shown in Fig. 4F and G, GFP-positive neurons in the granular layer developed the generic morphology of small to medium-sized multipolar neurons, which is consistent with, though not distinctive of, granular layer interneuron phenotypes. Accordingly, evaluation of cell body sizes showed no correspondence between transplanted cells and their endogenous counterparts (Fig. 5A and B; LGE/SVZ vs. Golgi, χ24 = 18.37, n = 151, P = 0.001; VM vs. Golgi, χ24 = 10.01, n = 125, P = 0.0403; DSC vs. Golgi, χ24 = 15.58, n = 134, P = 0.0036; LGE/SVZ vs. Lugaro, t125 = 2.695, P = 0.008; VM vs. Lugaro, t141 = 10.51, P < 0.0001; DSC vs. Lugaro, t117 = 9.727, P < 0.0001). To analyse dendritic orientation (Fig. 4H–P), grafted neurons were subdivided into two groups according to the distance of their cell bodies from the Purkinje cell layer (cut-off being at 50 μm). The result was clear-cut and consistent within all donor populations: neurons positioned deep in the layer had randomly orientated dendrites, whereas those located close to the Purkinje cell bodies displayed a clear preferential orientation along the direction of the layer (LGE/SVZ, χ22 = 14.01, n = 181, P = 0.0009, Fig. 4H–J; VM, χ22 = 8.677, n = 59, P = 0.0131, Fig. 4K–M; DSC, χ22 = 8.970, n = 94, P = 0.0113, Fig. 4N–P).
Figure 5. Distribution of cell body sizes of endogenous and transplanted interneurons located in the superficial regions of the granular layer (A), in the depth of the same layer (B) or in the deep cerebellar nuclei (C). Endogenous interneurons, green; LGE/SVZ, red, lateral ganglionic eminence/subventricular zone; VM, yellow, ventral mesencephalon; DSC, blue, dorsal spinal cord; PCL, Purkinje cell layer; GL, granular layer; DCN, deep cerebellar nuclei (see colour graphics on-line).
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Finally, we examined the axons of transplanted neurons, although this analysis was hampered by the faint GFP staining of this neuronal compartment. Donor neurites terminated in the vicinity of the parent cell body, as expected for local interneurons, but their axonal fields appeared highly variable and never displayed the distinctive features and distribution of the axons of Golgi and Lugaro neurons. Of a sample of 50 GFP-positive axons, 31 comprised scarcely branched slender processes (Fig. 4Q), whereas 19 formed extensive networks of varicose chains, either extended throughout the granular layer (Fig. 4R) or restricted along the Purkinje cell layer (Fig. 4S).
Morphological and neurochemical phenotype of extracerebellar donors located in the deep cerebellar nuclei
The deep cerebellar nuclei (DCN) contain a population of GABAergic interneurons that share the same lineage with their cortical counterparts, and can be distinguished by their small size, multipolar shape and expression of NeuN and CR (Leto et al., 2006). Donor cells from any of the different extracerebellar sources generated moderate amounts of neurons that settled in the deep nuclei (Table 2), and bore morphological features similar to those of their local counterparts. Indeed, in this position cell body size of VM or DSC donor cells matched that of endogenous interneurons, whereas LGE/SVZ cells remained significantly smaller (Fig. 5C; LGE/SVZ vs. DCN, χ24 = 69.79, n = 180, P < 0.0001; DSC vs. DCN, χ24 = 9.944, n = 188, P = 0.0414; VM vs. DCN, χ24 = 1.563, n = 210, P = 0.8155). Similar to what was observed for cortically positioned cells, the vast majority of donor neurons expressed NeuN, regardless of their origin (Fig. 6A–C; Table 2). By contrast, expression of CR varied significantly among the different donor cell populations, being present in the vast majority of VM cells (94.1%), in 37% of DSC cells and only in 3.3% of LGE/SVZ cells (Fig. 6A and D–G; Table 2). Finally, a few cells also expressed PV and NG (Table 2), markers that are unusual for nuclear interneurons.