Development of Axon-Target Specificity of Ponto-Cerebellar Afferents

The function of neuronal networks relies on selective assembly of synaptic connections during development. We examined how synaptic specificity emerges in the pontocerebellar projection. Analysis of axon-target interactions with correlated light-electron microscopy revealed that developing pontine mossy fibers elaborate extensive cell-cell contacts and synaptic connections with Purkinje cells, an inappropriate target. Subsequently, mossy fiber–Purkinje cell connections are eliminated resulting in granule cell-specific mossy fiber connectivity as observed in mature cerebellar circuits. Formation of mossy fiber-Purkinje cell contacts is negatively regulated by Purkinje cell-derived BMP4. BMP4 limits mossy fiber growth in vitro and Purkinje cell-specific ablation of BMP4 in mice results in exuberant mossy fiber–Purkinje cell interactions. These findings demonstrate that synaptic specificity in the pontocerebellar projection is achieved through a stepwise mechanism that entails transient innervation of Purkinje cells, followed by synapse elimination. Moreover, this work establishes BMP4 as a retrograde signal that regulates the axon-target interactions during development.


Introduction
The specificity of synaptic connectivity in the central nervous system is a prerequisite for brain function. The neuronal circuits in the vertebrate cerebellum represent a remarkable example of wiring specificity. This was first recognized by Santiago Ramón y Cajal when he chose cerebellar circuits as revealed by the Golgi method for his early studies on brain organization (discussed in [1]). In its simplest form, the cerebellar microcircuit integrates input from two afferent classes-climbing and mossy fibers. Climbing fibers selectively innervate Purkinje cells. By contrast, mossy fiber afferent activity is relayed to Purkinje cells via granule cells in the inner granular layer of the cerebellum (IGL) [2][3][4]. In the IGL, mossy fibers also form synapses on Golgi cells, a class of inhibitory interneurons that provide feed-forward inhibition in the cerebellar circuit. Climbing and mossy fiber information is then integrated in Purkinje cells and transduced via cerebellar efferent projection neurons in the deep cerebellar nuclei. Despite the apparent simplicity of the cerebellar circuit, it is unknown how the specificity of synapse formation emerges during development for each of the principal cerebellar afferent systems. Indeed, the molecular mechanisms regulating synapse specificity for most circuits in the mammalian brain have remained obscure.
Two key steps determining the incipient pattern of synaptic connectivity during development are axon-target contact formation and synaptic differentiation. Ultrastructural reconstruction of mature neuronal circuits suggests that only a subset of contacts differentiates into bona fide synapses [5]. The fraction of actual synapses compared to cellular contacts (potential synapses) has been termed ''filling fraction'', with a filling fraction of 1.0 representing a case where all contacts are synaptic structures [6]. In vertebrate and invertebrate systems several attractive and repulsive factors have been identified that contribute to synaptic specificity [7][8][9][10][11][12][13]. However, pinpointing whether these specificity factors regulate primarily selective contact formation, synaptic differentiation, or both has been challenging, given the limited resolution of light microscopy in assessing direct cellular contacts in vivo. One possibility is that some signaling pathways regulate primarily contact formation, whereas other factors drive the synaptic differentiation process after axon-target contacts are established.
The ponto-cerebellar projection represents an excellent model system to explore mechanisms of synaptic specificity in the mammalian brain [14]. Mossy fiber axons emerging from the basilar pons (PGN) in the ventral brain stem form a major projection to the cerebellar cortex which relays information from sensory and motor cortex. Structurally, mossy fiber afferents exhibit synaptic specificity at two levels: Mossy fiber axons elaborate synapses exclusively with granule and Golgi cells but not Purkinje cells. At the subcellular level, mossy fiber synapses are restricted to the proximal regions of Golgi cells within the IGL but are excluded from the molecular layer where distal Golgi cell dendrites arborize. Cell culture studies indicated that immature granule cells provide a stop-signal for mossy fiber growth [15]. Maturing granule cells, in contrast, contribute positive signals for the differentiation of mossy fiber synapses and the elaboration of mossy fiber glomeruli [16][17][18][19]. However, it is unknown how contact and synapse specificity emerges for mossy fibers and their granule and Golgi cell targets. Moreover, negative signals that suppress contact of mossy fibers with inappropriate target cells in vivo have not been identified.
Previous anatomical studies indicated that axons with the appearance of mossy fibers do not exhibit strict targeting specificity but form broader projection patterns during early postnatal stages [20][21][22]. Fibers with mixed mossy fiber and climbing fiber morphology (''combination fibers'') were observed to contact Purkinje cells during development though the extent of these interactions remained unknown. A developmental gene expression analysis in pontine nuclei revealed distinct transcriptional programs for axonal growth and synaptic differentiation of pontine mossy fibers [23]. Surprisingly, the termination of the axonal growth program did not require the presence of granule cells in the cerebellar cortex but was perturbed in mutant mice with Purkinje cell degeneration accompanied by granule cell death [23]. In combination, these studies raised the question of whether Purkinje cells may provide signals for the development of mossy fiber projections.
To define the cellular nature of mossy fiber-Purkinje cell interactions and the rearrangements resulting in the specific synaptic wiring pattern, we undertook a systematic analysis of axon-target interactions in the mouse ponto-cerebellar system.
Using correlated light-electron microscopy analysis we quantitatively mapped physical contacts and synaptic structures formed between identified pontine mossy fibers and Purkinje cells. Using this methodology, we observed extensive transient mossy fiber contacts and synapses on Purkinje cells that are subsequently eliminated. Patterning molecules such as WNTs, FGFs, and BMPs have been shown to exert novel neuronal signaling functions at the Drosophila neuromuscular junction and in the mammalian central nervous system [24][25][26]. Of this class of molecules, the BMPs have been extensively studied as retrograde signals at the Drosophila neuromuscular junction [27][28][29][30][31][32] and as trophic factors in mammalian neurons [33][34][35]. However, their signaling functions in vertebrate axon-target interactions have not been determined. We explored a role for BMP signaling in mossy fiber transient target interactions in the developing cerebellum. Using expression analysis, in vitro assays, and conditional knock-out mice we identify BMP4 as Purkinje cell-derived signal that specifically controls mossy fiber-target contact selectivity during development.

Establishment of Mossy Fiber Target Specificity during Development
To examine the emergence of synaptic specificity of pontocerebellar mossy fibers we adopted an in utero electroporation approach [36,37]. Pontine precursor cells are selectively electroporated by injection of DNA constructs into the 4 th ventricle at embryonic day 14.5 ( Figure S1). Following differentiation and migration, these cells settle into the pontine gray nucleus ( Figure 1A,B) [36,37]. Pontine axons labeled by electroporation of an EGFP expression plasmid project to the cerebellar cortex assume typical mossy fiber morphology and are restricted to the IGL at postnatal day 21 (P21), consistent with the selective elaboration of mossy fiber-granule cells synapses in the adult cerebellar circuitry ( Figure S1). To examine how this target specificity emerges, we examined earlier developmental time points. At P7, we identified a significant number of GFP-positive mossy fiber extensions projecting beyond the IGL into the Purkinje cell layer (PCL) ( Figure 1C-E). Using three-dimensional analysis of high resolution confocal stacks, mossy fiber varicosities were found in direct proximity with calbindin-positive Purkinje cell somata and axons, suggesting direct mossy fiber-Purkinje cell contacts ( Figure 1F,G). These contacts contained synaptic markers, as they concentrated the endogenous synaptic vesicle protein VAMP2 or a synaptophysin-fluorescent protein fusion that was introduced by electroporation into the pontine projection neurons ( Figure 1H,I).
Mossy fibers emerge not only from the PGN but multiple precerebellar nuclei. The elaboration of mossy fiber-Purkinje contacts was not unique to PGN-derived mossy fibers as it was also observed in GFP-O transgenic mice where multiple other mossy fiber populations are marked by EGFP ( Figure S1) [38]. Some of these contacts concentrated the postsynaptic scaffolding protein Shank1a (Shank1a-positive: 16% of somatic contacts, n = 50 contacts; 44% of contacts with proximal PC axon segments, n = 167; Figure 1J). In sum, these findings suggest that mossy fiber afferents establish transient synapse-like contacts with Purkinje cells during postnatal development.

Correlated Light-Electron Microscopy Reveals Transient Mossy Fiber-Purkinje Cell Synapses
In order to visualize the developmental progression of mossy fiber-Purkinje cell contacts and their differentiation into synapses we undertook a systematic light-electron microscopy analysis of

Author Summary
Brain functions rely on highly selective neuronal networks which are assembled during development. Network assembly involves targeted neuronal growth followed by recognition of the appropriate target cells and selective synapse formation. How neuronal processes select their appropriate target cells from an array of interaction partners is poorly understood. In this study, we have addressed this question for the axons emerging from the pontine gray nucleus, a major brainstem nucleus that relays information between the cortex and the cerebellum, a brain area responsible for the control of skilled movements but also emotional processing. Using advanced microscopy techniques, we find that developing mossy fibers establish synaptic contacts rather promiscuously, and elaborate extensive synapses with Purkinje cells, an inappropriate target. These contacts are subsequently eliminated, and proper synaptic connectivity is then restricted to granule and Golgi neurons. We identify bone morphogenetic protein 4 (BMP4) as a regulator of these inappropriate mossy fiber-Purkinje cell contacts. BMP growth factors are best known for their functions in cell specification during embryonic development, and our results support an additional retrograde signaling function between axons and their target cells in early postnatal stages. In summary, we show that the specificity of the synaptic connections in the ponto-cerebellar circuit emerges through extensive elimination of transient synapses. PGN-derived mossy fiber axons. Pontine projections were labeled by DiI tracing followed by photoconversion of the dye ( Figure 2). Mossy fiber projection patterns and Purkinje cell interactions in the cerebellar hemispheres (Crus1, Crus 2, and Simplex lobules) were quantitatively examined by light microscopy and apparent contacts were subsequently analyzed by electron microscopy. By light microscopy, mossy fiber rosettes and the thin protrusions extending from them are seen in great detail, and somata of Purkinje and granule cells of the cerebellar cortex can be clearly identified by DIC microscopy (Figures 2B,C, S2A-D). To further confirm the identity of the Purkinje cell territory, some sections were additionally labeled with antibodies to calbindin ( Figure  S2I,J). Camera lucida drawings of mossy fiber terminals from 50 mm sections at P0, P7, P14, and P21 revealed that the mature mossy  [20]. Scale bars: middle panel 50 mm, right panel 10 mm. EGL, external granule cell layer; PCL, Purkinje cell layer; IGL, inner granule cell layer; WM, white matter; Sim, simplex lobule; mcp, medial cerebellar peduncle; v-IV, fourth ventricle; Pf, paraflocculus. (C) High magnification photograph of 7 mm semithin section from cerebellar cortex at P7, next to camera lucida drawing reconstructing mossy fiber axonal arborizations and their cellular relationships with Purkinje cell somata from 50 mm thick section of the same area. Several Purkinje cell somata are highlighted in blue. Since only a single plane of focus can be visualized in the photomicrograph, mossy fibers appear as discontinuous segments (delineated by asterisks). Reconstruction from multiple focal planes using camera lucida allows us to visualize continuous axonal arbors. Red arrowheads point at putative contacts on Purkinje cell somata. Mossy fiber segments and Purkinje cells that are not on the same focal plane as the photograph are grayed out in the camera lucida drawing. Scale bar: 10 mm. (D) Camera lucida drawings of the upper IGL and the PCL of DiI-calbindin double-labeled 50 mm sections from P0, P7, P14, and P21 time points. Purkinje cells are shown in blue. Red arrows point at putative contacts between mossy fibers and Purkinje cell somata. Scale bar: 10 mm. (E) Quantitative assessment of mossy fiber invasion into Purkinje cell territory from camera lucida drawings at P0, P7, P14, and P21 time points. Drawings encompass all of the mossy fiber segments and Purkinje cell outlines reconstructed from an area 175 mm horizontal6120 mm vertical6approximately 30 mm deep (thickness of one Purkinje cell soma), oriented parallel to the PCL, and recorded with a 1006 objective. The percentage of all mossy fiber segments that enter the PCL out of all mossy fiber segments was scored (.1,000 segments from .25 areas obtained from 5-9 animals per time point, .3 50 mm sections per animal were analyzed). Statistical significance was determined by Mann-Whitney t test. Two-tailed p values were used with 95% confidence intervals, ** p,0.01, *** p,0.001. doi:10.1371/journal.pbio.1001013.g002 fiber projection pattern emerges from a series of transformations during postnatal development that includes an extensive invasion of and eventual withdrawal from the Purkinje cell territory ( Figure 2D,E).
At P0 mossy fibers extend far into the developing cerebellar cortex where Purkinje cells are unevenly distributed and intermixed with migratory and maturing granule cells of the emerging IGL ( Figures 2D, S2). At P7, Purkinje cells form a recognizable monolayer above the IGL. However, pontine mossy fibers substantially invade this Purkinje cell territory with close to 30% of all labeled segments in the IGL penetrating into the PCL ( Figure 2E,D, see Methods and figure legends for details on quantitative analysis). This invasion of the PCL was significantly reduced at postnatal days 14 and 21, yielding the mature mossy fiber projection pattern. At postnatal days 7 and 14 mossy fibers in close apposition to Purkinje cell somata often exhibited marked varicosities, resembling the presumptive mossy fiber-Purkinje cell synapses identified using the in utero electroporation approach ( Figure 2C,D).
Fifty-five putative contacts identified at the light microscopy level in P0, P7, P14, and P21 tissues were examined by electron microscopy. Tissue sections (50 mm) were re-sectioned into 7 mm semithin sections and re-examined again by light microscopy. Sections encompassing the putative mossy fiber-Purkinje cell contacts were then thin-sectioned (70 nm) and processed for ultrastructural analysis ( Figure S2E-H). Cytological characteristics defined in previous studies allowed unambiguous identification of Purkinje and granule cell somata in electron micrographs, as well as other relevant cellular components of the cerebellar cortex ( Figure S3) [3,39]. Over 90% of putative contacts between mossy fibers and Purkinje cell somata identified by light microscopy at P0 and P7 indeed represent direct cellular appositions ( Figure 3A,B,E). At P0, none of the mossy fiber-Purkinje cell (or mossy fibergranule cell) contacts in the developing IGL/PCL had synaptic features, representing a filling fraction (synapses per contacts [6]) of 0.0 ( Figure 3A,F). However, at P7 a substantial number of mossy fiber-Purkinje cell contacts exhibited ultrastructural characteristics of synapses ( Figure 3B,E,F; several consecutive sections shown in Figure S4A, filling fraction = 0.37). At P14, direct contacts were still observed (5 direct contacts verified by EM Mossy fiber-Purkinje cell contacts at P0, P7, P14, and P21 time points. Each panel consists of camera lucida drawing (top left), corresponding electron micrograph (EM), and a schematic drawn to scale highlighting major ultrastructural elements representing mossy fibers (orange), Purkinje cells (blue), glial processes (yellow), unlabeled terminals (presumably from PC collaterals, white), synaptic vesicles (small circles), and PSDs (demarcated by blue arrowheads). Contacts are highlighted by red arrowheads. Scale bars: camera lucida drawings 10 mm, electron micrographs 100 nm. (A) At P0 electron micrographs from the contact region verify direct cellular appositions between the mossy fibers and the Purkinje cells. By light microscopy mossy fibers form extensive putative contacts throughout the entire length of Purkinje cell somata. Even though mossy fiber segments apposed to Purkinje cell somata are densely filled with synaptic-like vesicles, no synaptic specializations can be seen by electron microscopy. (B) At P7 electron micrographs from the contact region verify direct cellular appositions between the mossy fibers and the Purkinje cells. These contacts occur en passant, are densely packed with synaptic vesicles, and exhibit ultrastructural characteristics of synapses: synaptic vesicles polarized towards PSDlike structures on the Purkinje cell (blue arrowheads). (C) At P14 mossy fiber segments typically do not invade more than 15 mm into PCL. By electron microscopy the diameter of mossy fibers at the sites of contact with Purkinje cell soma is smaller compared to P7. Purkinje cell somata become surrounded by thin glial process, identified by very light cytoplasm and presence of glycogen particles. (D) At P21, none of the potential mossy fiber-Purkinje cell contacts identified by LM could be verified by EM as direct contacts, even though in some cases mossy fiber terminals were positioned as close as 150 nm from Purkinje cell soma. (E) Quantitation of correlated light-EM analysis. More than 90% of putative mossy fiber-Purkinje cell contacts identified by LM are direct cellular appositions as verified by serial EM at P0 and P7. At P14, only 40% of putative contacts are direct cellular appositions, and at P21 mossy fiber-Purkinje cell contacts are completely removed. Total of 55 potential contacts analyzed by serial EM analysis (P0, n = 13; P7, n = 21; P14, n = 13; P21, n = 8; taken from 34 areas, from 12 animals). (F) The filling fraction was calculated for somatic mossy fiber-Purkinje cell contacts by dividing the number of synapses by the number of direct contacts identified in the EM analysis. Synapses were defined by the presence of clustered synaptic vesicles docked at the plasma membrane opposite a postsynaptic density in the Purkinje cell. Since no direct mossy fiber-Purkinje cell contacts are detected at P21 no filling fraction can be calculated for this developmental time point. doi:10.1371/journal.pbio.1001013.g003 of 13 putative contacts analyzed) but only one of them was synaptic ( Figure 3C,F, filling fraction = 0.2). Finally, at P21 no direct mossy fiber-Purkinje cell contacts or synapses could be identified ( Figure 3D).
During the apparent removal of contacts and the elimination of synapses between P7 and P14 we frequently observed mossy fibers separated from the Purkinje cell soma and/or ensheathed by glial processes ( Figure S4C-F), reminiscent of pruning processes with axosome shedding observed in peripheral axons [40][41][42]. In addition, we observed instances where mossy fiber axons were engulfed by Purkinje cells (Figure S4C,D). Glial process ensheathing of Purkinje cells became even more prominent at P21. Some mossy fibers were positioned as close as 150 nm from the Purkinje cell soma ( Figure 3D) but glial processes separated mossy fiber endings and the Purkinje cell soma. In summary, during the first 10 postnatal days approximately 30% of all labeled IGL mossy fibers derived from the PGN establish direct contacts with Purkinje cells. The quantitative analysis uncovers remarkable developmental changes in the ''filling fraction'', i.e. the differentiation of direct mossy fiber-Purkinje cell contacts into synapses, rising from 0.0 at birth to 0.37 at postnatal day 7 ( Figure 3F). In the second to third postnatal week, these synapses are eliminated and the contacts withdrawn, resulting in the selective innervation of granule and Golgi cells in the IGL. Notably, not all mossy fiber axons contact Purkinje cells. Therefore, specific signaling mechanisms must exist, first, to limit the invasion of pontine mossy fiber axons into the Purkinje cell territory during the first postnatal days and, second, to promote the removal of pontine mossy fiber-Purkinje cell synapses in the second postnatal week of development.

Identification of Bone Morphogenetic Proteins as Candidate Retrograde Signals for Pontine Mossy Fiber Afferents
In Drosophila melanogaster, growth factors of the bone morphogenetic protein (BMP) family regulate synaptic growth, axon arborization, and synaptic homeostasis [27,28,[43][44][45]. To explore whether a comparable signaling function might be conserved in the mouse cerebellum, we surveyed the expression of BMP signaling molecules in the developing ponto-cerebellar projection system. Using in situ hybridization, we detected mRNAs for BMP receptor 1A (BMPR1A), BMP receptor 1B (BMPR1B), and BMP receptor type 2 (BMPR2) in the PGN at P0, the time when pontine mossy fiber axons extend into the cerebellar cortex. By P14, detection of BMPR1B mRNA was reduced, while signals for BMPR1A and BMPR2 expression persist ( Figure 4A). Within the cerebellar cortex significant expression of several BMP ligands was observed consistent with previous reports ([ [46][47][48] and unpublished data). We focused our analysis on BMP4 as it is highly expressed in Purkinje cells and dynamically regulated during the refinement of mossy fiber connectivity ( Figure 4B). At P0, BMP4 mRNA is abundant in proliferating and premigratory granule cells of the EGL, and in scattered Purkinje cells (identified by their large diameter). BMP4 expression in Purkinje cells was reduced at P7, the time when mossy fiber-Purkinje cell synapses are most common, and expression was strongly up-regulated in Purkinje cells by postnatal day 14 ( Figure 4B). At P21, BMP4 was highly expressed in Purkinje cells. In addition a subset of large diameter cells in the IGL (presumably Golgi cells) expressed BMP4. In summary, BMP4 and its signaling receptors are appropriately positioned to regulate mossy fiber target selection during postnatal development.

Activation of the BMP Signaling Pathway in Pontine Neurons In Vivo
BMP-receptor activation results in phosphorylation of cytoplasmic SMAD proteins that translocate to the cell nucleus and activate transcription [49,50]. Classical morphogenetic functions of BMPs depend on SMAD phosphorylation but phospho-SMAD (pSMAD)-independent BMP signaling read-outs have also been described [34,[51][52][53]. Robust SMAD phosphorylation was detected when recombinant BMP4 was added to cultured pontine explants in vitro and phosphorylation was prevented by coapplication of the antagonist noggin ( Figure S5A). Quantitative evaluation of SMAD phosphorylation in PGN in vivo using Western blot and immunohistochemistry revealed a dynamic regulation, with moderate levels at P0, strongly increased levels at P14, and persistent pSMAD immune-reactivity at P21 ( Figure  S5B-D). SMAD1,5,8 protein levels were not significantly altered during this developmental time period, suggesting that regulation of SMAD signaling occurs primarily at the level of SMAD phosphorylation (unpublished data). This demonstrates a functional BMP signaling pathway in developing pontine neurons in vitro and in vivo.
Given that BMP4 was dynamically expressed in Purkinje cells we asked whether Purkinje cell-derived BMP4 was required for SMAD activation in pontine neurons. We analyzed conditional BMP4 fl/fl ::Pcp2 cre/cre knockout (BMP4 cKO) mice lacking BMP4 expression selectively in Purkinje cells. In the Pcp2 cre knock-in line, cre-mediated recombination is detected during late embryonic stages and specifically in Purkinje cells ( [54] and Figure S6). Ablation of BMP4 expression was verified by in situ hybridization ( Figure S6C). Interestingly, SMAD activation in the pontine gray nucleus was not dramatically altered in BMP4 cKO mice ( Figure  S5D,E). While we cannot completely exclude that some of the pSMAD signal is due to incomplete ablation of the BMP4 expression in Purkinje cells, these results indicate that Purkinje cell-derived BMP4 might not be essential for pSMAD activation in pontine nuclei during postnatal development. Notably, Purkinje cells express significant amounts of BMP7 and other BMP growth factors during postnatal development which might be responsible for the persistent SMAD phosphorylation in the absence of BMP4 (unpublished data).
Importantly, signaling activities have been described for specific BMP growth factors that control cytoskeletal rearrangements through pSMAD-independent pathways [34,[51][52][53]. In commissural spinal neurons such BMP signals represent extrinsic cues for the initial polarization of axons [55,56]. Therefore, we examined the possibility that target-derived BMP signaling might regulate axon development and axon-target interactions of pontine mossy fibers.

BMP4 Is a Negative Regulator of Mossy Fiber Growth
Previous work demonstrated that cerebellar explants cultured in vitro release a growth inhibiting activity for mossy fibers which is thought to resemble a target-derived stop signal for afferents [57]. Explants from the PGN exhibit robust radial axon outgrowth. However, when pontine explants are co-cultured with cerebellar tissue, axon growth on the side facing the cerebellar tissue is reduced, suggesting the presence of a growth inhibiting activity derived from cerebellar tissue ( Figure 5A). In order to assess a possible role for BMPs in this process, we applied the soluble BMP antagonist noggin to the culture medium. Noggin addition blocked cerebellar growth retardation activity in this assay ( Figure 5B,D). To directly examine whether BMP4 might exert such growthinhibiting activity, we combined pontine explants with BMP4expressing HEK293 cells in collagen gel co-cultures. Using this assay, we observed that BMP4 was sufficient to negatively regulate mossy fiber growth in vitro ( Figure 5C). Importantly, this activity could be neutralized by addition of noggin to the collagen gel matrix, indicating that the growth regulation was indeed mediated by BMP signaling ( Figure 5E). Therefore, BMP4 negatively regulates pontine mossy fiber growth in vitro.

Purkinje Cell-Derived BMP4 Is a Negative Signal for Mossy Fiber Afferent-Target Interactions
Based on the dynamic regulation of BMP4 expression in Purkinje cells and the repulsive activity of BMP4 towards mossy fiber axons in vitro, we hypothesized that BMP4 might control either initial mossy fiber-Purkinje cell interactions, the detachment of mossy fiber-Purkinje cell contacts, or both. To explore these possibilities, we further examined the BMP4 cKO mice. Given the important patterning functions of BMP signaling in early cerebellar development [46,47,58] we first asked whether the overall anatomical organization of the cerebellar cortex or specification of cerebellar cell types was perturbed in the mutant mice. No significant changes were detected in the foliation pattern, cerebellar layering, specification of the major cell types, and expression of transcriptional markers and signaling molecules ( Figure S7). In DiI labeled preparations, the number of labeled pontine mossy fiber axons or density of Purkinje cells observed in the cerebellar cortex of BMP4 cKO mice was not significantly different from control littermates or wild-type animals ( Figure S7G and unpublished data). Finally, the development of climbing fibers and formation of vGlut2-positive climbing fiber synapses on the Purkinje cell dendrites was not noticeably altered in the BMP4 cKO mice ( Figure S8).
Next, we examined whether loss of Purkinje cell-derived BMP4 resulted in defects in mossy fiber-Purkinje cell contact formation, synapse formation, and/or synapse elimination. In BMP4 cKO mice the fraction of pontine mossy fibers that penetrated into the Purkinje cell territory at postnatal day 0 was increased approximately 2-fold as compared to control ( Figure 6A,B). Moreover, the number of Purkinje cells receiving mossy fiber contacts was increased 7-fold at P0 and remained significantly increased over the following 2 wk. In our wild-type analysis (Figure 2) we identified a peak in mossy fiber elimination from the Purkinje cell territory in the second postnatal week. Mossy fiber elimination was quantitatively compared using an elimination index for the fraction of mossy fibers removed from the PCL between P7 and P14 ([MFs PCL P7-MFs PCL P14] / MFs PCL P7). In the cKO animals elimination of mossy fibers still occurred but the elimination index for control and cKO mice was reduced to about 50% of that in control animals ( Figure 6C). When normalized to the length of mossy fiber segments in the Purkinje cell territory, the density of contacts per 100 mm mossy fiber length was more than 3-fold increased at postnatal day 7 ( Figure 6C). These observations highlight an essential function for BMP4 in the control of initial mossy fiber-Purkinje cell contact formation during the first postnatal week as well as the subsequent removal of mossy fiber processes from the Purkinje cell territory.
Considering the developmental regulation of the filling fraction observed in wild-type animals ( Figure 3) we further examined mossy fiber-Purkinje cell contacts, synapses, and filling fractions at P7 using correlated light-electron microscopy. As in wild-type and control tissue, the majority of putative mossy fiber-Purkinje cell somatic contacts identified in BMP cKO mice indeed represented direct cellular appositions (38 direct contacts out of 40 potential contacts analyzed, Figure 6D,E). Some mutant contacts were characterized by unusual, irregular synapse-like profiles ( Figures 6D, S4B). However, the filling fraction was substantially reduced in the cKO as only 11% of these contacts exhibited synaptic ultrastructure ( Figure 6E). Based on the correlated light-EM analysis and the calculated filling fraction, the total density of mossy fiber-Purkinje cell synapses was not significantly changed, indicating that the excess contacts do not efficiently differentiate into synaptic structures. These experiments identify BMP4 as a retrograde signal that specifically controls mossy fiber-Purkinje cell contact formation and highlight that independent programs regulate contact versus synapse formation during postnatal development.
If loss of BMP4 from Purkinje cells results in exuberant pontine mossy fiber-Purkinje cell contacts during early postnatal development, do these aberrant interactions perturb the placement or specificity of synapses in the mature cerebellum? Using in utero electroporation, we marked pontine mossy fibers in control and BMP4 cKO animals and examined their projection pattern at P21 when cerebellar development is essentially complete (Figure 7). While in wild-type mice mossy fibers were restricted to the IGL and did not protrude into the molecular layer, we observed overshooting axons in the BMP4 cKO mice ( Figure 7A). A subset of mossy fiber axons penetrated more than 20 mm beyond the Purkinje cell somata into the molecular layer, a phenotype never observed in control cerebella ( Figure 7B). Within the molecular layer, most mossy fiber axons had a smooth appearance but some developed swellings comparable to simple mossy fiber rosettes.
Overshooting mossy fiber axons have been observed previously in mouse mutants with perturbed granule cell migration [59]. However, we did not observe ectopic granule cells in the molecular layer of BMP4 cKO mice ( Figure 7C). Instead, highresolution analysis of the overshooting mossy fiber axons revealed that some established direct contacts with the dendritic tree of Purkinje cells. Other overshooting axons formed contacts with neurogranin-positive Golgi cells ( Figure 7C). Notably, Golgi cells are one of the specific synaptic targets of ponto-cerebellar mossy fibers in the IGL. However, in wild-type mice mossy fiber synapses are excluded from the distal dendritic arbors in the IGL. Finally, we examined the position of pontine mossy fiber synapses in the IGL and observed a significant shift of mossy fiber rosettes towards the PCL (within 40 mm of the Purkinje cell somata, Figure 7). Therefore, loss of Purkinje cell-derived BMP4 results in persistent alterations in mossy fiber connectivity in the mature cerebellum.

Discussion
Cerebellar circuits and the ''crystalline'' architecture of the cerebellar cortex are a prime example of the precision of neuronal connectivity. In this study, we identified cellular and molecular mechanisms orchestrating aspects of afferent-target specificity in cerebellar networks. First, we demonstrate that synaptic specificity of pontine mossy fibers emerges in a protracted, stepwise process that encompasses extensive contacts and synapse formation with Purkinje cells. Second, we identify BMP4 as a retrograde, Purkinje cell-derived signal that negatively regulates mossy fiber-Purkinje cell contacts and synaptic specificity.

BMP Growth Factors as Retrograde Signals
BMPs are key regulators of patterning and cell fate decisions, but novel functions in neuronal wiring are emerging [49,[60][61][62]. In the vertebrate central nervous system BMPs (and the related TGFbeta growth factors) control initial axon orientation and axon regeneration [55,56,[63][64][65]. Moreover, retrograde, targetderived BMP signaling has been examined in the peripheral nervous system [66][67][68][69]. At the Drosophila neuromuscular junction a muscle-derived BMP-analogue regulates synaptic growth and homeostatic signaling [27,29,30,43,70]. Whether BMP growth factors have similar retrograde signaling activities in the central nervous system and, specifically in axon-target interactions in vertebrates, has remained unclear. In our experiments, we explored retrograde BMP signaling in the mouse pontocerebellar system and uncovered a novel function during the development of synaptic target specificity. In this system, BMP4 acts as a negative signal that limits interactions of mossy fibers with Purkinje cells, a transient target cell. The dynamic regulation of BMP4 expression in Purkinje cells mirrors the assembly of mossy fiber-Purkinje cell contacts and synapses, with a transient peak at P7 where BMP4 expression is low. Thereafter, BMP4 is strongly up-regulated and mossy fiber-Purkinje cell contacts are eliminated.

Regulation of Mossy Fiber Target Specificity by BMP4
The mossy fiber phenotypes in the BMP4 cKO mice highlight a critical function of Purkinje cell-derived BMP4 in mossy fiber-Purkinje cell interactions. In the cKO mice, there is a substantial increase in mossy fiber-Purkinje cell contacts at early postnatal stages (P0-P7). This supports an essential repulsive role for BMP4 in target recognition which limits the initial mossy fiber-Purkinje cell contacts and restricts the invading mossy fiber axons to their target territory in the IGL. The correlated light-electron microscopy analysis enabled us to dissociate changes in contact and synapse formation in the cerebellar system. Notably, while BMP4 cKO mice exhibit a 3-fold increase in mossy fiber-Purkinje cell contact density we did not detect a comparable increase in synapse density. Therefore, axon target contacts and synapse formation are controlled by different signaling systems.
The subsequent, removal of mossy fiber-Purkinje cell contacts and elimination of mossy fiber processes from the Purkinje cell territory was significantly delayed, and after completion of cerebellar development, we observed persistent overshooting mossy fiber projections in the Purkinje cell and molecular layers. Some overshooting axons retain interactions with Purkinje cells, while others form contacts on distal Golgi cell dendrites. Notably, Golgi cells are appropriate synaptic partners of mossy fibers, but in BMP4 cKO cerebella mossy fiber-Golgi cell interactions are observed ectopically in the molecular layer. These findings support an important role for Purkinje cell-derived BMP4 in eliminating mossy fiber projections from this area, in addition to its function in regulation of the early mossy fiber-Purkinje cell contacts.
Within the IGL, the placement of mossy fiber rosettes was shifted towards the Purkinje cell layer, further supporting a repulsive role for Purkinje cell-derived BMP4. However, the fact that most mossy fiber axons did not overshoot to the molecular layer indicates that there are additional signals that restrict mossy fibers to the IGL. BMP2 and 7 transcripts are up-regulated in Purkinje cells of the cKO mice (unpublished data) and may partially compensate for the loss of BMP4. Moreover, the specificity of mossy fiber connectivity is likely to emerge not only from negative, Purkinje cell-derived signals but from an interplay with positive signals derived from the appropriate target cells. Granule cells express FGF22, Wnt7a, and neuroligins which all have been demonstrated to have positive, synaptogenic activities towards mossy fiber afferents [16][17][18]. Therefore, presentation of these synaptogenic signals by mature granule cells which strongly increase in number at later postnatal stages (P7-P21) may compete with the constant number of Purkinje cells for mossy fiber contact. A prediction of this model is that direct mossy fiber-Purkinje cell synapses would persist in the absence of granule cells. This is, indeed, observed in agranular cerebella of mouse mutants or after Red arrows point at putative contacts between mossy fibers and Purkinje cell soma. More mossy fiber segments extensively invade into the Purkinje cell territory in the BMP4 cKO tissue. (B) Quantitative analysis from camera lucida drawings of DiI-Calbindin double-labeled material showing significant increases in the percentage of mossy fiber segments entering the Purkinje cell territory and in the number of Purkinje cells receiving mossy fiber contacts (n.5,000 mossy fiber segments from 220 areas of 175 mm6120 mm630 mm from 22 animals). Gray bars are control, black bars are cKO. ** p,0.01, *** p,0.001. (C) Elimination index for removal of mossy fibers from the PCL between P7 and P14 in control (gray) and BMP4 cKO mice (black bar). (D) Combinations of camera lucida drawings, schematic representations, and electron micrographs as in Figure 3. Two upper panels: At P7, many synaptic contacts in BMP cKO mice exhibit the same ultra-structural features as in the wild type. Lower left panel: Non-synaptic contacts between one mossy fiber segment and two neighboring Purkinje cells. Lower right panel: Some mutant contacts exhibit unusual ultra-structure with aggregated synaptic vesicles and ruffled plasma membranes at the contact site, characteristics never observed in wild-type or control mice. (E) Quantification of the density of EM-verified mossy fiber-Purkinje cell somatic contacts per 100 mm mossy fiber length in the PCL at P7 for BMP4 fl/fl control mice (gray) and BMP4cKO mice quantified by light microscopy (black). The filling fraction was substantially reduced in the BMP4 cKO tissue. Based on the contact density and filling fraction, an estimated synapse density was calculated which shows no significant difference in control and cKO mice. Data were collected from .3 animals per genotype, .50 areas per animal, .12 mossy fiber segments per area. *** p,0.001. doi:10.1371/journal.pbio.1001013.g006 irradiation where mossy fiber target selectivity can be examined in the absence of the appropriate synaptic targets [71]. Importantly, while most mossy fibers were appropriately restricted to the IGL, the positioning of mossy fiber rosettes within the IGL was shifted closer to the Purkinje cell layer ( Figure 7D), consistent with the loss of a negative regulator of synaptic connectivity in Purkinje cells of BMP4 cKO mice.

Transient Target Cells in the Development of Neuronal Connectivity
The finding that the development of mossy fiber target specificity involves not only extensive contact but also synapse formation with Purkinje cells argues against a model of absolute recognition specificity for unique synaptic targets. This remodeling of transient target interactions is reminiscent of interactions in the thalamo-cortical projection and for Cajal Retzius cells in the hippocampus [72][73][74]. In both cases, afferents enter the target territory before their appropriate target cells have fully differentiated and form transient synapses on a third cell type (subplate neurons and Cajal Retzius cells, respectively). This situation in the hippocampus is comparable to the transient mossy fiber-Purkinje cell synapses described in our study that are elaborated during early postnatal development when only few granule cells have descended into the forming IGL. While the initial assembly of such transient contacts is comparable, the mechanism of contact removal is fundamentally different. Elimination of transient synapses received by subplate and Cajal Retzius neurons occurs via programmed cell death of the transient target cells. By contrast, removal of mossy fiber-Purkinje cell interactions occurs independently of Purkinje cell death and requires signals for contact destabilization.

Bug or Feature?
The existence of widespread mossy fiber-Purkinje cell interactions during development poses the question of whether these synapses simply represent an imprecision in the initial transsynaptic interactions or whether transient contacts serve a purpose in the development of functional cerebellar circuits. In the hippocampus, Cajal Retzius cells appear to be required for the laminar specificity of entorhinal axon projections [74]. Similarly, in the absence of subplate neurons, thalamocortical axons do not establish appropriate synaptic connectivity [75,76]. Therefore, transient mossy fiber-Purkinje cell interactions might similarly contribute to the assembly of cerebellar circuits. The cerebellar cortex is subdivided into longitudinal bands identified by specific molecular codes of gene expression in Purkinje cells [77,78]. This code develops during the first postnatal weeks, coincident with emergence of mature cellular and sub-cellular targeting specificity of both climbing and mossy fiber afferents. Recent tracing studies indicate that there is a precise somatotopic matching of pontine and climbing fibers [79,80]. This raises the possibility that transient mossy fiber-Purkinje cell interactions might provide a mechanism to coordinate mossy fiber and climbing fiber development and, thereby, serve a functional role in the assembly of cerebellar circuits.

Mouse Strains
All animal experiments were reviewed and approved by the institutional animal care and use committee of Columbia University and the cantonal veterinary office Basel, respectively. Mice were of the NMRI (Figure 1) and C57BL/6 strains (all other experiments). PCP2 cre knock-in mice were previously described [54]. The conditional BMP4 floxed allele (BMP4 fl ) was generously provided by Dr. Brigid Hogan [81]. Htr5b-GFP mice are BAC transgenic mice generated by the GENSAT consortium [82] and were obtained from the MMRRC repository. Thy1.2-GFP (GFP-O) mice were generated by Drs. Sanes and Feng [38] and were obtained from the Jackson Laboratory. R26-lox-stop-lox-YFP were described in [83].

In Utero Electroporation of Precerebellar Neuron Precursors
Timed-pregnant mice (NMRI or C57BL6 background) were used at embryonic day 14.5 following the protocol described in [37]. After electroporation, the mice were brought to term, pups were sacrificed by transcardial perfusion with 4% paraformaldehyde in 100 mM Na-phosphate buffer (pH7.4), and tissue from successfully electroporated pups (P7, P14, P21) was processed for immunohistochemistry.
Most procedures followed standard protocols; see Text S1 for details.

Confocal Microscopy Analysis
High-resolution images of 30 to 40 mm z-stacks consisting of 0.45 mm thick optical sections were acquired using Zeiss LSM510, a Zeiss LSM5 Exciter, or a LIS-spinning disk confocal system. Direct apposition of cellular markers was identified by rotating the 3D reconstruction of the stacks using Imaris Software (Bitplane). Quantitative
Quantitative assessment of mossy fiber invasion into Purkinje cell territory was performed on camera lucida drawings of DiI and calbindin double-labeled material (50 mm coronal ''thick'' sections, 1006 objective) of P0, P7, P14, and P21 cerebellar hemispheres (crus1, crus2, and simplex lobules). All camera lucida drawings contained all of the labeled mossy fiber segments and Purkinje cell outlines drawn from a fixed area size of 175 mm horizon-tal6120 mm vertical630 mm deep (thickness of one Purkinje cell soma), encompassing the upper IGL and PCL, and spanning a stretch containing on average 40 Purkinje cells at P0 (before PC alignment occurs), and 9 Purkinje cells at P7-P21. For the sake of consistency, and since Purkinje cell density and the angle of the mossy fiber segments penetrating the PCL differs at the base, versus apex, versus sides of the folia, areas for analysis were always drawn from the sides of the folia. The percentage of mossy fiber segments invading into the PCL out of all mossy fiber segments drawn per area was scored (.20 segments per area from .25 areas obtained from 5-9 animals per time point, .3 50 mm section per animal were analyzed). For the quantification of Purkinje cells receiving putative somatic contacts from mossy fibers, contacts were judged as varicosities in the mossy fiber axon at the site apparently immediately adjacent to Purkinje cell soma. For quantification of contact density per mossy fiber length (in Figure 7D) camera lucida drawings were scanned at 600 dpi, and the length of mossy fiber segments in the PCL was measured using line tool in NeuronJ [87]. The number of putative contacts on Purkinje cell somata per mossy fiber segment was scored visually, using the criteria described above.
The filling fraction was calculated as actual synapses divided by the number of contacts (EM-verified). The elimination index for mossy fibers projecting into the Purkinje cell layer (MFs PCL ) was calculated using the data points for P7 and P14 (graph Figure 6B) as follows: [MFs PCL P7-MFs PCL P14] / MFs PCL P7.

Supporting Information
Text S1 Supplemental material and methods. Western blotting, immunohistochemistry, and in situ hybridization; functional in vitro assays; correlated light-electron microscopy analysis; supplemental references. In the photographs a single plane of focus is seen, such that labeled mossy fibers appear as discontinuous segments (demarcated by red arrowheads). Somata of Purkinje and granule cells can be identified by DIC microscopy. The two cell types can be distinguished by unique cytological profiles (soma diameter, nucleoli, density of cytoplasm and nuclei). Corresponding camera lucida drawings, reconstructing mossy fiber arbors through the Zplanes of the 50 mm thick sections, are shown to the right side of the photographs (red arrowheads demarcate the same mossy fiber segments as in the photographs). Black asterisk indicates blood vessel. Scale bar: 10 mm.  Figure 7E in BMP4 cKO mouse cerebellum (P7). Note that preand postsynaptic densities are often lacking, the membrane between the mossy fiber and Purkinje cell soma is ruffled, and synaptic vesicles are irregularly shaped. (C-D) Electron micrograph (P14) showing engulfment of DiI-labeled mossy fiber process in Purkinje cell (blue overlay, red arrows, one revealing an endocytotic figure nearby). Thin glial processes can frequently be seen separating mossy fibers from the Purkinje cell soma (yellow arrow, C). (E-F) Glial ensheathment of mossy fiber processes near Purkinje cell soma in P14 wild-type cerebellum. Glial processes (identified by the presence of glycogen particles in the cytoplasm, yellow overlay) can frequently be seen to separate mossy processes from the Purkinje cell soma. Scale bar: 500 nm; ''N'' marks granule cell nuclei (green). nuclei at P14 and P21 reveals no significant differences in BMP4 +/+ ::Pcp2 cre/cre (control) and BMP4 fl/fl ::Pcp2 cre/cre (BMP4 cKO) mice. pSMAD levels were compared to actin immunoreactivity in the same sample. pSMAD:actin ratios were arbitrarily set to ''one'' for the P14 control sample in this experiment. (TIF) Figure S6 Conditional ablation of BMP4 using Pcp2cre KI mice. BMP4 conditional KO mice were obtained by crossing a floxed BMP4 allele [81] and Pcp2 cre knock-in mice [54] which express cre recombinase from the Pcp2 locus. (A) The cell-type specificity of cre-mediated recombination in Pcp2-cre knock-in mice was confirmed using a ROSA26-lox-stop-lox-YFP reporter [83]. Recombination is observed specifically in Purkinje cells (colabeled with calbindin which labels a subset and RORalpha which labels all Purkinje cells) but not in the brain stem. Note that at P0 recombination in Purkinje cells throughout the cerebellum is detected in coronal sections. Higher magnification views (lower row) reveal that at least 50% of RORalpha positive Purkinje cells show activation of the reporter. (B) Schematic drawing of the conditional BMP4 locus. Exons 3 and 4 are flanked by loxP sites.
(C) Confirmation of BMP4 expression in Purkinje cells (P14) by in situ hybridization with a probe for the ablated exon 3. Individual Purkinje cells are marked by arrowheads. Note that the high sequence similarity between BMP4 exon 3 and other BMP family members makes detection more challenging than in the experiments using probes against the UTR used in