Differential Developmental Deficits in Retinal Function in the Absence of either Protein Tyrosine Sulfotransferase-1 or -2

To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1−/− and Tpst2−/− mice, retinal function was compromised during early development. However, Tpst1−/− retinas became electrophysiologically normal by postnatal day 90 while Tpst2−/− mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina

Number of animals tested in Figure 3D, E & F.

<p>Number of animals tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039702#pone-0039702-g003" target="_blank">Figure 3D, E & F</a>.</p

Primary antibodies and lectins used for tissue labeling.

<p>Primary antibodies and lectins used for tissue labeling.</p

Electroretinographic responses from <i>Tpst1^−/−</i> and <i>Tpst2^−/−</i> retinas.

<p><b>A.</b> Representative waveforms recorded at different light intensities from <i>wt</i>, <i>Tpst1^−/−</i> or <i>Tpst2^−/−</i> retinas under scotopic conditions. <b>B & C,</b> in each panel, the dashed line represents the 95% confidence interval for responses obtained from 3 150-day-old <i>wt</i> littermate controls. The a- and b-waves obtained from 3 150-day-old <i>Tpst1^−/−</i> mice (squares) fell near the lower limit of this interval at lower light intensities, but were well within the range at higher light intensities. In contrast, a- and b-waves recorded from 3 123-day-old <i>Tpst2^−/−</i> mice (triangles) were reduced in amplitude and fell outside the 95% confidence interval at all light intensities tested. Development of the (<b>D</b>) scotopic a-wave, (<b>E</b>) scotopic b-wave, and (<b>F</b>) photopic b-wave responses for <i>wt</i> (dotted line), <i>Tpst1^−/−</i> (squares), and <i>Tpst2^−/−</i> (triangles) mice. Note that all responses from <i>Tpst2^−/−</i> mice were lower than those for <i>Tpst1^−/−</i> and <i>wt</i> mice at all ages. Error bars represent standard error of the mean. The differences between <i>wt</i> and <i>Tpst2^−/−</i> presented in <b>D</b> are statistically significant between P30 and P300 (P<0.05–0.001) while the differences between <i>wt</i> and <i>Tpst1^−/−</i> are only statistically significant at P30 and P60 (P<0.05–0.001). The differences between <i>wt</i> and <i>Tpst2^−/−</i> presented in <b>E</b> are statistically significant between P60 and P600 (P<0.01–0.001) while the differences between <i>wt</i> and <i>Tpst1^−/−</i> are only statistically significant at P60 (P<0.05). The differences between <i>wt</i> and <i>Tpst2^−/−</i> presented in <b>F</b> are statistically significant for all time points tested (P<0.05–0.001) while the differences between <i>wt</i> and <i>Tpst1^−/−</i> are statistically insignificant for all time points tested. Number of animals tested in <b>D, E & F</b> are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039702#pone-0039702-t003" target="_blank">Table 3</a>.</p

Knockout of TPST-1 or TPST-2 does not disrupt rod and cone-specific domains in the interphotoreceptor matrix (IPM).

<p><b>A–C.</b> In the retinas of (<b>A</b>) <i>wt</i>, (<b>B</b>) <i>Tpst1^−/−</i>, and (<b>C</b>) <i>Tpst2^−/−</i> mice, the IPM surrounding rod outer segments (<i>arrows</i>) is labeled by wheat germ agglutinin (WGA; <i>green</i>). The IPM surrounding the inner and outer segments of cones (<i>arrowheads</i>) also shows labeling by WGA. <b>D–F:</b> Peanut agglutinin (PNA) specifically labels the IPM surrounding cones (<i>arrows</i>) in the retinas of (<b>D</b>) <i>wt</i>, (<b>E</b>) <i>Tpst1^−/−</i>, and (<b>F</b>) <i>Tpst2^−/−</i> mice, but, as appropriate, does not label the IPM surrounding rods. Photoreceptor nuclei in the outer nuclear layer (ONL) are counterstained with DAPI (blue) in panels D–F. IS, inner segment layer. Scale bars  = 20 µm for all panels.</p

Effects of <i>TPST-1</i> and <i>TPST-2</i> knockout on neuronal populations, morphology and synaptic protein expression.

*<p><i>Tpst DKO</i> data from Sherry et al., 2010.</p

Immunohistochemical localization of sulfated proteins.

<p>Retinas from (<b>A</b>) wildtype (<i>wt</i>), (<b>B</b>) <i>Tpst1^−/−,</i> (<b>C</b>) <i>Tpst2^−/−</i> and (<b>D</b>) <i>Tpst</i> DKO mice were labeled using the PSG2 antibody. Labeling of <i>wt</i> retina showed signal in the sclera (SC), retinal pigment epithelium (RPE), photoreceptor outer segments (OS), inner segments (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). Arrows in panel B indicate cells in the proximal INL that retained sulfated proteins in absence of TPST-1. Retinas were from 30-day old <i>wt</i>, <i>Tpst1^−/−</i> and <i>Tpst2^−/−</i> mice while the DKO retina was from a 21-day old mouse. Scale bar  = 50 µm.</p