Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1

During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity

Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1

During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity.Depto. de Bioquímica y Biología MolecularFac. de MedicinaTRUEpu

Knock-down of <i>Pax6</i> in the embryonic retina does not interfere with the generation of RGCs.

<p>Frontal cryostat sections of E19.5 retinas after electroporation at E13.5 with shRNAs constructs control and targeting <i>Pax6</i> and immunostained (red) for Pax6 and Caspase 3 (<b>a</b>) or Islet-1/2 and Brn3 (<b>b</b>) as indicated in the panels. Electroporated cells are visualized with GFP (green). The graphs show the quantification of the proportion of the double positive cells for the indicated marker. Note that shRNA efficiently target Pax6 without inducing cell death (a) or changes in expression of markers for postmitotic RGCs (b). Bar indicates 10 in (a) and 40 µm in (b). Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01. Number of axons per condition >60 (n = 3). INL, inner layer; VZ, ventricular zone.</p

Ectopic expression of <i>Pax6</i> stimulates axonal growth in cortical neurons.

<p>NSCs were nucleofected with CAG-empty vector or CAG-<i>Pax6</i> and co-electroporated with CAG-<i>GFP</i>. <b>a</b>) The graph shows the percentage of nucleofected neurons with respect to their axonal length after 9 days of differentiation. Over-expression of <i>Pax6</i> increments the axonal length compared with control neurons. <b>b</b>) The graph shows quantification of the axonal length of a cohort of BrdU positive neurons. <b>c</b>) Nucleofected neurons were identified by the specific expression of β-tubulin III and GFP. BrdU staining allowed us to compared neurons of similar birth dates. The effect in axonal growth is unrelated to an early exit from the cell cycle. Arrows point to BrdU stained nuclei. Bar indicates 50 µm. <b>d</b>) Cortical primary neurons were also transfected with CAG-empty vector or CAG<i>-Pax6</i>, and with CAG-<i>GFP</i>. Transfected primary axons were identified by GFP and MAP1b staining. Arrows indicate the distal part of the axon stained by MAP1b. Bar indicates 70 µm. <b>e</b>) The graph shows the percentage of primary neurons with respect to their axonal length. Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01.</p

Knock-down of <i>Pax6</i> blocks SFRP1 stimulated growth of retinal axons.

<p><b>a</b>) Low magnification images (a) and confocal micrographs (b) of explants from control (a) or electroporated retinal explants (b) seeded onto laminin coated coverslip and cultured in the absence or presence of recombinant SFRP1 as indicated in the panels. Explants in (a) were stained with β-tubulin III whereas those in (b) were co-electroporated with CAG-GFP and shRNA control or shRNA targeting <i>Pax6</i> and axons were visualized by GFP expression. Graph shows quantification of the proportion of total axon longer than 900 (a) or 200 (b) µm. Note that knocking-down <i>Pax6</i> inhibits the axonal response stimulated by SFRP1. Bar indicates 300 (a) and 150 µm (b). Data are expressed as the mean ± SD. (*) p<0.05 comparing stimulated populations; (**) p<0.01 and (***) p<0.001 compared with control.</p