No co-localization of FA1/dlk1 with tyrosine hydroxylase in striatum.

<p>Representative photomicrographs of double immunofluorescence staining showing no co-localization of TH with FA1/dlk1 positive cells (arrows) in the lesioned striatum. Scale bar: 50μm.</p

Neuronal expression of FA1/dlk1 in the SNc.

<p>Double immunofluorescence staining of FA1/dlk1 and the general neuronal marker NeuN (upper row) and the astroglial marker GFAP (lower row) in the in the substantia nigra pars compacta of adult rats. Note the distinct co-localization of FA1/dlk1 with NeuN (arrows). As expected no co-localization was found for FA1/dlk1 and GFAP. Scale bar: 50 μm.</p

Co-expression of FA1/dlk1 with calbindin but not parvalbumin in the SN.

<p>Digitalized photomicrographs of FA1/dlk1-ir cells in the ventral mesencephalon of adult rats. Some calbindin (CB)-ir cells (arrowheads) showed co-localization with FA1/dlk1 (open arrowhead, upper row) in the substantia nigra pars compacta, whereas several CB-ir neurons (arrowheads, upper row) and FA1/dlk1-ir cells (arrows, upper row) did not co-localize. As expected from the distribution pattern depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116088#pone.0116088.g001" target="_blank">Fig. 1</a> no co-localization was detected for FA1/dlk1 with parvalbumin (PV) in the substantia nigra pars reticulata (lower row). Scale bar: 50μm.</p

FA1/dlk1 expression rates in the developing postnatal substantia nigra.

<p>Quantification of FA1/dlk1-ir cells co-expressing tyrosine hydroxylase (TH) (open bars) and TH-ir neurons co-expressing FA1/dlk1 (filled bars) in the substantia nigra (SN) of rats at postnatal (P) day 7, 14 and 21 (A). The percentage of TH-ir neurons co-expressing FA1/dlk1 was significantly higher at P21 as compared to P7 and P14. There was no difference in the relative co-localization observed for FA1/dlk1-ir cells expressing TH during early postnatal development. Data are expressed as mean + s.e.m. *: p<0.05 vs. percentage of TH-ir neurons co-expressing FA1/dlk1 at P21. Representative digitalized pictures of double immunofluorescence staining for TH and FA1/dlk1 in SN of P7, P14 and P21 rats (B). Scale bar: 200 μm.</p

Co-localization of striatal FA1/dlk1 with neuronal markers.

<p>Representative photomicrographs of double immunofluorescence stainings demonstrating that FA1/dlk1-ir cells located in the lesioned striatum were post-mitotic neurons co-localizing with NeuN (arrows, upper row). Moreover, most of the FA1/dlk1-ir cells co-expressed DARPP-32 (arrows, middle row). FA1/dlk1-ir cells (arrows) did not co-localize with the astroglial marker GFAP (arrowheads, lower panel). Scale bar: 50μm.</p

No co-localization of FA1/dlk1 with proliferation markers in SVZ.

<p>Representative photomicrographs of double immunofluorescence stainings of FA1/dlk1-ir cells in the striatum of adult rat brain. FA1-ir cells (arrows) did not co-localize with 5-bromo-2’-deoxyuridine (BrdU) (upper panel, arrowheads), Ki67 (middle panel, arrowheads) and doublecortin (DCX) (lower panel, arrowheads) in the subventricular zone and striatum 4 weeks after an unilateral intrastriatal 6-OHDA lesion. Scale bar: 50μm.</p

Co-expression of FA1/dlk1 with tyrosine hydroxylase and calretinin.

<p>Double immunofluorescence staining for FA1/dlk1 (FA1), tyrosine hydroxylase (TH) and calretinin (CR) in the ventral mesencephalon of adult rats. Note that nearly all FA1/dlk1-ir cells (arrows) co-localize with TH (open arrowheads), while only a very small number of TH-ir neurons did not co-express FA1/dlk1 (yellow arrowhead) (A). Triple immunofluorescence staining for FA1/dlk1, TH and CR (lower row) demonstrated co-localization of FA1/dlk1-ir cells (arrows) with both markers, TH and CR. Scale bars: 100μm (upper row in A), 50μm (lower row in A, B).</p

Reduced FA1/dlk1-ir cell densities in the 6-OHDA-lesioned rat brain.

<p>Representative photomicrographs demonstrating the loss of FA1/dlk1-ir fibers in the unilateral 6-OHDA-lesioned striatum (*) versus the unlesioned contralateral side (A). Enlarged photomicrographs showing the effect of the lesion on tyrosine hydroxylase (TH)-ir (B) and FA1/dlk1-ir (C) cells in the substantia nigra. Scale bars: 1mm (A); 500μm (B, C).</p

Higher FA1/dlk1-ir cell densities in the lesioned striatum.

<p>Quantitative analysis of FA1/dlk1-ir cell densities assessed in the dorsal striatum one month (A) and one week (B) after the unilateral striatal 6-OHDA lesions. Data are expressed as mean + s.e.m. and are given as percentage of corresponding controls. *: p<0.05 vs. corresponding control. Enlarged photomicrographs showing the effect of an unilateral 6-OHDA lesion on FA1/dlk1-ir cells in the striatum at the one-month time point (C, D). Rather few FA1/dlk1-ir cells (arrowheads) were observed in the unlesioned striatum (C) whereas higher densities of FA1/dlk1-ir neurons were present in the dopamine-depleted contralateral striatum (D). Scale bars: 200μm (overview); 50μm (inserts).</p

Expression pattern of FA1/dlk1 in the midbrain.

<p>Representative photomicrograph showing distinct FA1/dlk1 expression in the rat ventral mesencephalon, the lateral periaqueductal gray and Edinger-Westphal nucleus (A). Schematic drawing illustrating the distribution and density of FA1/dlk1-ir cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116088#sec002" target="_blank">Materials and methods</a>) (B). Photomicrographs at high magnification showing morphology of FA1/dlk1-ir cells in the substantia nigra pars lateralis (1), substantia nigra pars compacta (2), ventral tegmental area (3) and in the rostral area of Edinger-Westphal nucleus (4). Scale bars: 500μm (A, B); 100μm (1–4). Abbreviations: DpMe, deep mesencephalic nucleus; EW, Edinger-Westphal nucleus; LPAG, lateral periaqueductal gray; MGD, medial geniculate nucleus, dorsal part; MGV, medial geniculate nucleus, ventral part; MT, medial terminal nucleus of the accessory optic tract; PAG, periaqueductal gray; PBP, parabrachial pigmented nucleus; RPC, red nucleus parvicellular part; SNc, substantia nigra pars compacta; SNl, substantia nigra pars lateralis; SNr, substantia nigra pars reticularis; VTA, ventral tegmental area.</p