Co-injection of two AAV1-CAG viruses results in optimal Purkinje neuron expression of two different genes.

<p>A. Schematic representation of the two AAV transfer constructs used for co-injection. AAV1-CAG-GFP and AAV1-CAG-FGF14B-IRES-tdTomato viruses were mixed together at a ratio of 1∶5 prior to injecting into <i>Fgf14^−/−</i> cerebellum. CAG, chicken β-actin promoter with CMV enhancer; in, SV40 intron; IRES, internal ribosome entry site; pA, polyadenylation site; ITR, inverted terminal repeat. B. Confocal images of sagittal sections from an <i>Fgf14^−/−</i> cerebellum co-injected with AAV1-CAG-GFP and AAV1-CAG-FGF14B-IRES-tdTomato and immunostained for FGF14 (red). GFP expression is readily visible in Purkinje neuron somata and dendrites, and FGF14 is properly localized to the AIS. While some Purkinje neurons express FGF14 but not GFP, all GFP expressing Purkinje neurons express FGF14. Scale bars: 25 µm (B, top); 5 µm (B, bottom).</p

AAV1 delivery of FGF14B-IRES-tdTomato and FGF14B-P2A-GFP results in efficient FGF14 expression but failure of reporter gene fluorescence.

<p>A. Schematic representation of CAG-FGF14B-IRES-tdTomato AAV transfer construct. CAG, chicken β-actin promoter with CMV enhancer; in, SV40 intron; IRES, internal ribosome entry site; pA, polyadenylation site; ITR, inverted terminal repeat. B. CAG-FGF14B-IRES-tdTomato transfected into CHL1610 cells produces a diffuse cytoplasmic tdTomato expression pattern. C. Confocal image from an <i>Fgf14^−/−</i> mouse injected with CAG-FGF14B-IRES-tdTomato and immunostained for FGF14. Viral delivered FGF14 is properly localized at the Purkinje neuron AIS but tdTomato expression is not visible. D. Schematic representation of CAG-FGF14B-P2A-GFP AAV transfer construct. The arrow represents approximate location where ribosomal skipping should occur to generate two independent polypeptides. E. GFP expression in CHL1610 cells transfected with CAG-FGF14B-GFP (top) or CAG-FGF14B-P2A-GFP (bottom). FGF14B-GFP fusion protein is expressed in punctate foci surrounding the nucleus whereas FGF14B-P2A-GFP is expressed as a diffuse cytoplasmic protein, suggesting cleavage of GFP from FGF14B. F. Western blot analysis of P2A cleavage efficiency in CHL cells. CHL1610 cells were transfected with either CAG-FGF14B-GFP or CAG-FGF14B-P2A-GFP and processed for western blot 24 h after transfection. Immunoblotting for both FGF14 and GFP revealed a ∼50kDa band in CAG-FGF14B-GFP transfected cells, which is consistent with the expected size of the fusion protein. Immunoblotting for FGF14 and GFP in CAG-FGF14B-P2A-GFP transfected cells revealed ∼25kDa bands for FGF14 and GFP and no detectable ∼50kDa band, indicating efficient cleavage of the P2A peptide. G. Confocal image of <i>Fgf14^−/−</i> cerebellum injected with CAG-FGF14B-P2A-GFP and immunostained for FGF14 (red) and AnkyrinG (AnkG, blue). No GFP fluorescence is visible but viral delivered FGF14 is properly expressed at the AIS of Purkinje neurons where it colocalizes with AnkyrinG. H. Confocal image of <i>Fgf14^−/−</i> cerebellum injected with CAG-FGF14B-P2A-GFP and immunostained for FGF14 (red) and GFP (blue). Immunostaining reveals that GFP is expressed and colocalizes with FGF14 in the Purkinje neuron AIS, suggesting that P2A cleavage did not occur <i>in vivo</i>. Scale bars: 20 µm (B, E); 10 µm (C, G, H).</p

MND-tdTomato expression following injection with pulled glass pipette.

<p>Confocal images of sagittal cerebellar slices from wild type mice injected with MND-tdTomato lentivirus. Injections were performed using pulled glass pipettes and a picospritzer (see methods) to determine if injection technique affected cellular transduction pattern. Dotted lines in A and B represent the border between the Purkinje layer and granule layer. In C, dotted lines are drawn to separate the Purkinje layer from the molecular layer and granule layer. A. Widespread tdTomato expression in cells in the granule layer and processes in the molecular layer. B. A few small tdTomato expressing cells are visible in the Purkinje layer, but the majority of the tdTomato expressing cell bodies are located in the white matter (wm). C. High magnification view of tdTomato expressing cells with somata in the Purkinje layer show that their processes are relatively straight and unbranched, characteristic of Bergmann glia. m, molecular layer; p, Purkinje layer; g, granule layer. Scale bars: 100 µm (A, B); 25 µm (C).</p

Expression of reporter genes in cerebellar cells transduced with lentiviral vectors under various promoters.

<p>Representative confocal images of sagittal cerebellar sections from mice 7–14 days following intracerebellar injection of lentiviral vectors with indicated promoters. Dotted lines demarcate the border between cerebellar cortex layers. In low magnification images (B, D, F, G, and J), the line is drawn between Purkinje and granule layers. In high magnification images (A, C, E, H, and I), two lines are drawn to separate the Purkinje layer from the molecular layer and granule layer. A. Widespread GFP expression in a cerebellar lobe injected with MND-GFP. B. Single confocal section of MND-GFP transduced cerebellum at higher magnification showing absence of GFP expression in Purkinje neuron somata (asterisks). C. Low magnification of cerebellum injected with MSCV-GFP. D. High magnification of cerebellum injected with MSCV-GFP demonstrating GFP expression in small cell bodies in the Purkinje layer with radial processes extending to the pial surface, characteristic of Bergmann glia. E. Venus expression in a cerebellar lobe injected with UBC-Venus. F, G. High magnification of UBC-Venus infected cerebellum shows venus expression in multiple small cells in the granule layer (F) and a single Purkinje neuron (G). H, I. GFP expression in cerebellar lobes of two animals injected with PGK-GFP. Several GFP-expressing Purkinje neurons are visible in H, whereas most GFP-expressing cells in I are in the white matter, with a single GFP-positive Purkinje neuron. J. High magnification view of GFP-positive Purkinje neurons from H. Abbreviations: m = molecular layer; p = Purkinje layer; g = granule layer; wm = white matter. Scale bars, 25 µm (B, D, F, G, J), 100 µm (A, C, E, H, I).</p

Purkinje neuron transduction by lentiviruses and AAV.

a<p>-,≤1 Purkinje neurons transduced per 20x field; +, ≤5 Purkinje neurons transduced per 20x field; ++, >5 Purkinje neurons transduced per 20x field.</p>b<p>Cells transduced based on cell morphology and cerebellar region. GCL, granule cell layer; BG, Bergmann glia; WM, white matter; ML, molecular layer.</p

AIS expression of virally transduced FGF14 protein in <i>Fgf14^−/−</i> Purkinje neurons.

a<p>Average of three 60x fields near injection site.</p>b<p>Average of nine 60x fields near injection site.</p>c<p>Immunostaining (number of AIS stained/60x field).</p

Patterns of cellular transduction by Lenti-MND-tdTomato.

<p>A. Montage of low magnification confocal images of sagittal sections of wild type mouse cerebellum injected with MND-tdTomato (red) and immunostained for calbindin (green), a marker for Purkinje neurons. A region of tdTomato expressing cells in the Purkinje and/or molecular layer is visible near the injection site, but the vast majority of tdTomato-expressing cells are in the white matter (wm). Arrowhead indicates approximate location of injection, where some damage to brain parenchyma can be seen. A’. A higher magnification image from an adjacent slide illustrating the lack of co-localization of tdTomato-expressing processes and calbindin positive Purkinje neuron dendrites. B–E. To examine transduction patterns in more detail, MND-tdTomato was injected into L7/pcp2-GFP mouse cerebellum, and sagittal sections were examined at higher magnification. L7/pcp2-GFP mice express GFP under control of the Purkinje cell specific promoter L7/pcp2. B, GFP expression is visible in Purkinje neuron somata, dendrites, and axons (ax), which project into the white matter tract (left panel and green, right panel). tdTomato-expressing somata are located in the white matter tracts and extend short processes (center panel and red, right panel). C. Purkinje neuron axons (left panel and green, right panel) and processes expressing tdTomato (center panel and red, right panel) do not overlap. D-E. High magnification images of the Purkinje layer of L7/pcp2-GFP cerebellum injected with MND-tdTomato. D. GFP-expressing Purkinje neuron somata with characteristic highly branched dendrites (left and green, right panel). Cells expressing tdTomato are located in the Purkinje layer but somata are smaller and processes are straight and unbranched (center). tdTomato (red) expression pattern does not colocalize with GFP (green) expressing Purkinje neuron somata or dendrites (right panel). The shape and location of tdTomato expressing cells is consistent with Bergmann glia. E. Coexpression of GFP (left) and tdTomato (center) in three Purkinje neuron somata (arrowheads, and yellow, right panel). Scale bars: 100 µm (A, B), 50 µm (A’), 25 µm (D, E).</p

FGF20 initiates lateral compartment differentiation before E14.5.

<p>(A–H) Immunostaining of Myo7a in <i>Fgf20^βGal/+</i> and <i>Fgf20^βGal/βGal</i> cochlear explants treated with or without FGF9 and cultured for 5 d (schematic). Treatment of <i>Fgf20^βGal/+</i> explants with FGF9, either at E14.5 (B) or E15.5 (F), did not have any effect on hair cell number compared to untreated explants (A,E). Treatment of <i>Fgf20^βGal/βGal</i> explants with FGF9 at E14.5 resulted in increased numbers of hair cells and decreased gaps (arrows) between hair cell clusters (D) compared to untreated explants (C). Treatment of <i>Fgf20^βGal/+</i> or <i>Fgf20^βGal/βGal</i> explants with FGF9 at E15.5 did not affect hair cell number or the formation of gaps (arrows) lacking sensory cells (G, H). (I) Quantitation of the number of hair cells. The number of OHCs and total hair cells were rescued by treatment with FGF9 at E14.5 but not at E15.5. * <i>p</i><0.05. (J–M) Immunostaining for Prox1 in <i>Fgf20^βGal/+</i> and <i>Fgf20^βGal/βGal</i> explants treated with or without FGF9 at E14.5 and cultured for 5 d (schematic). Treatment of <i>Fgf20^βGal/+</i> explants with FGF9 did not affect supporting cell number (K) compared to untreated explants (J). Treatment of <i>Fgf20^βGal/βGal</i> explants with FGF9 at E14.5 resulted in increased numbers of supporting cells and decreased gaps between sensory cell clusters (M) compared to untreated explants (L). (N) Quantitation of numbers of supporting cells in explants. The number of supporting cells was partially rescued in <i>Fgf20^βGal/βGal</i> explants by treatment with FGF9 at E14.5.</p

Schematic model of sensory cell development in the organ of Corti.

<p>Diagram showing that FGF20 specifically functions to initiate lateral compartment development. The differential activity of FGF20 suggests that there may be separate progenitor cells for the medial and lateral cochlear compartments.</p

<i>Fgf20</i> is necessary for formation of the lateral compartment of the organ of Corti.

<p>Phalloidin staining of P0 and P7 cochleae showing hair cells. In <i>Fgf20^βGal/+</i> cochlea, (A) there is one row of inner hair cells (IHC) and three rows of outer hair cells (OHC) throughout the cochlea. At P0, apical hair cells are not fully differentiated; by P7, apical differentiation is complete. In <i>Fgf20^βGal/βGal</i> cochlea (B), the cochlear base (base) has one row of inner hair cells and two rows of outer hair cells. The mid-cochlea (middle) forms patches of hair cells with two rows of IHCs and three rows of OHCs separated by gaps that lack hair cells. The apex (apex) contains hair cells that are much less differentiated and the distal apex (end of apex) contains no identifiable hair cells. At P7, the hair cells in the distal apex of <i>Fgf20^βGal/βGal</i> and <i>Fgf20^βGal/+</i> cochlea are comparable. (C) Diagram showing the cochlear regions examined in (A, B); b, base; m, middle; a, apex, a′, end of apex. (D) Quantification of hair cell numbers at P4. Total numbers of IHCs and OHCs were counted from <i>Fgf20^βGal/+</i> (<i>n</i> = 3) and <i>Fgf20^βGal/βGal</i> (<i>n</i> = 3) cochlea. The numbers of IHCs were comparable, while the numbers of OHCs were decreased by 70% in <i>Fgf20^βGal/βGal</i> compared to <i>Fgf20^βGal/+</i> cochlea. Total numbers of hair cells were decreased by 50% in <i>Fgf20^βGal/βGal</i> cochlea. (E) Length of cochlea at P4. <i>Fgf20^βGal/βGal</i> cochlea length (<i>n</i> = 3) was decreased by 10% compared to <i>Fgf20^βGal/+</i> (<i>n</i> = 3). (F, G) Quantification of supporting cell numbers at P0. (F) Number of supporting cells, identified by staining for Prox1 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001231#pbio.1001231.s003" target="_blank">Figure S3G,H</a>), and hair cells, were counted from base, middle, and apex regions of the OC and normalized to 100 µm from <i>Fgf20^βGal/+</i> (<i>n</i> = 6) and <i>Fgf20^βGal/βGal</i> (<i>n</i> = 8) mice. IHC numbers were slightly increased while OHC numbers were decreased by 57% in <i>Fgf20^βGal/βGal</i> compared to <i>Fgf20^βGal/+</i> cochlea. Numbers of Deiters' cells (DC) and outer pillar cells (OPC) were decreased by 52%, whereas numbers of inner pillar cells (IPC) were only slightly decreased in <i>Fgf20^βGal/βGal</i> compared to <i>Fgf20^βGal/+</i> cochlea. (G) Ratio of hair cells and supporting cells in P0 cochlea. The ratio of outer compartment cells (OHC, OPC, DC) to inner compartment cells (IHC, IPC) was decreased in <i>Fgf20^βGal/βGal</i> compared to <i>Fgf20^βGal/+</i> cochlea. Small changes in the ratio of outer compartment supporting cells (DC+OPC) to OHCs and inner compartment supporting cells (IPC) to IHCs were observed. * <i>p</i><0.01.</p