Additional file 1: Figure S1. of Loss of AP-2delta reduces retinal ganglion cell numbers and axonal projections to the superior colliculus

Loss of Brn3c immunostaining in AP-2δ−/− retinal ganglion cells. Retinal tissue sections from AP-2δ+/+ and AP-2δ−/− mice at E16.5, P1, P14 and adult were co-immunostained with anti-AP-2δ and anti-Brn3c (QQ8) antibodies followed by secondary antibodies conjugated to Alexa 488 and Alexa 555, respectively. Tissue sections were counterstained with DAPI Cells immunostained with the anti-Brn3c (QQ8) antibody in AP-2δ−/− retina are indicated by arrows. Figure S2. Loss of Brn3c expression in AP-2δ−/− P1 retina. Paraffin-embedded tissue sections from P1 AP-2δ+/+ and AP-2δ−/− retina were immunostained with two anti-Brn3c antibody: mouse anti-Brn3c (Santa Cruz QQ8) and rabbit anti-Brn3c antibody (Sigma Atlas). The signal was detected using the Dako-Cytomation EnVision + anti-rabbit or anti-mouse secondary systems. Photographs were taken using a 20× lens and images merged. Figure S3. Brn3a labeling in P1 and P14 AP-2δ+/+ and AP-2δ−/− retina. Retinal tissue sections were immunostained with anti-Brn3a antibody followed by secondary antibody conjugated to Alexa 555. Tissue sections were counterstained with DAPI. Photographs were taken with a 40× lens. Figure S4. Brn3a labeling in P21 and adult AP-2δ+/+ and AP-2δ−/− retina. Retinal tissue sections were processed as described in Figure S3. Figure S5. Flat-mount of cholera toxin B (CTB)-injected retina. Retinas from AP-2δ+/+ and AP-2δ−/− mice injected with CTB were flat-mounted onto glass slides. The dorsal central regions of the retinas are shown in the upper panels and the optic discs are shown in the lower panels. Photographs were taken with a 20× lens. Scale bar = 100 μm. Figure S6. AP-2δ and AP-2β immunostaining of P1 mouse brain. Sagittal tissue sections close to the midline of wild-type P1 mouse brain were immunostained with anti-AP-2δ and anti-AP-2β antibodies. The emerging stratum zonale (SZ), stratum griseum superficiale (SGS), stratum opticum or optic layer, intermediate and deep layers of the superior colliculus (SC) are indicated. The white layer of the SC separates the SC from 2 the periaqueductal gray (PAG). The insets on the left are magnified in the diagram on the right. Both AP-2β and AP-2δ are widely expressed in the SC. Scale bars = 100 μm. Table S1. Volume of contralateral projections to the MTN using raw RGB image data and thresholded image data. Abbreviations for additional files: NBL, neuroblastic layer; INBL, inner neuroblastic layer; INL, inner nuclear layer; GCL, ganglion cell layer; ONL, outer nuclear layer. (PDF 1923 kb)