Lateral LMC axons are misguided in absence of En1.

<p><b>(a)</b> Retrograde tracing from dorsal forelimb mesenchyme labels motor neurons in the LMCl (Lim1, blue) and showed no significant increase of aberrantly projecting LMCm neurons (Isl1/2-positive, green) in <i>En1</i>^<i>-/-</i> mutant embryos when compared to wildtype littermates. <b>(b)</b> Retrograde tracing from ventral forelimb mesenchyme labels motor neurons in the LMCm (Isl1/2, blue) and revealed a significantly increased number of Lim1-positive LMCl motor neurons that aberrantly project to the ventral limb in <i>En1</i>^<i>-/-</i> mutant embryos (Lim1-positive, green and Rhodamine-positive, red). <b>(c)</b> Staining for neurofilament in E12.5 murine embryos shows bifurcation of limb innervating axons into a dorsal (yellow dashed line) and a ventral (cyan dashed line) branch. In <i>En1</i>^<i>-/-</i> mutant embryos, a significantly higher proportion of EphA4-positive axons is observed in the ventral branch, while in wildtypes EphA4 is largely restricted to the dorsally projecting branch. Scale bar in c equals 50μm for all panels.</p

Engrailed controls the dorsal-ventral choice of lateral LMC axons <i>in vivo</i>.

<p><b>(a)</b> An ectopic source of either En1, En2, or En1-SR was established by injecting the proteins into the dorsal mesenchyme of the developing chick wing. <b>(b)</b> Immunohistochemical detection of neurofilament (red) and EphA4 (green) visualizes the dorsal-ventral branching point and EphA4-positive LMCl axons. After injection of En1 into the dorsal wing bud more EphA4-positive axons are present in the ventral branch compared to mock (PBS) injection (arrows). <b>(c)</b> Quantification of axonal EphA4 signal results in a higher average EphA4 intensity in the ventral branch after En1 injection when compared to embryos where PBS was injected EphA4 signal was not altered significantly if En2 (28.75% ± 0.48 SEM, n = 4) or En1-SR (29.75% ± 1.25 SEM) were injected into the dorsal wing mesenchyme. <b>(d)</b> The expression patterns of the dorsal marker <i>Lmx1b</i> were not altered by injection of ectopic En1 into the dorsal wing. Scale bar in d equals 100μm for b and 250μm for d.</p

Strategies for inducing expression of scFv in PV cells or in CSF.

<p><b>A)</b> Co-staining for Myc and PV in layers II-IV of the binocular zone of the visual cortex (V1b) of P30 <i>PV</i>::<i>Cre</i>, <i>PV</i>::<i>Cre;scFvPax6</i>^<i>tg/o</i> and <i>PV</i>::<i>Cre;scFvOtx2</i>^<i>tg/o</i> mice (scale bar: 100μm). The enlarged view shows the immunofluorescence overlap. <b>B)</b> Western blot of cerebrospinal fluid (CSF) protein extracts from <i>scFvOtx2</i>^<i>tg/o</i> and <i>scFvPax6</i>^<i>tg/o</i> adult mice injected (+Cre) or not (ctrl) with Cre-TAT protein in lateral ventricles. Anti-Otx2 and anti-Pax6 scFvs (revealed with anti-Myc antibody) are secreted in the CSF upon choroid plexus recombination.</p

Anti-Otx2 scFv in adult CSF restores ocular dominance plasticity.

<p><b>A)</b><i>scFvOtx2</i>^<i>tg/o</i> adult (P90) mice were injected with NaCl or Cre-TAT and, after 15 d, the left eye was sutured for 4 d. Intrinsic optical imaging was then performed in the right visual cortex to measure visual acuity for each eye. <b>B)</b> Intrinsic signals recorded for the ipsilateral eye stimulation in <i>scFvOtx2</i>^<i>tg/o</i> adult mice injected with NaCl (top) or Cre-TAT (bottom). Ten spatial frequencies (SFs) ranging from 0.03 to 1 cycle/degree were tested. The binocular zone is delineated in white (A, Anterior; M, Medial; Scale bar, 1 mm). <b>C)</b> Estimation of the ipsilateral eye visual acuity for the two mice shown in (B). Each point corresponds to the average magnitude in the binocular zone. For each animal, data were normalized by the maximal amplitude among SFs and visual acuity was defined as the intersection of a fitted line segment with the X-axis. Representative stimuli are shown below. <b>D)</b> Visual acuity of the ipsilateral eye in adult <i>scFvOtx2</i>^<i>tg/o</i> injected with NaCl or Cre-TAT. <b>E)</b> Same as (D) for the contralateral eye. <b>F)</b> Ocular dominance index at different SF thresholds in adult <i>scFvOtx2</i>^<i>tg/o</i> mice injected with NaCl or Cre-TAT. Positive values indicate a preference for the contralateral eye. (t-tests; *p<0.05; error bars indicate SEM)</p

Anti-Otx2 scFv in adult CSF activates plasticity genes.

<p><b>A)</b> Immunostaining for Otx2, PV, WFA, CR and GABA in V1b layers II-IV of adult <i>scFvPax6</i>^<i>tg/o</i> and <i>scFvOtx2</i>^<i>tg/o</i> mice, 15 days after intracerebroventricular (icv) injection of Cre-TAT protein (scale bar: 100μm). <b>B)</b> Quantification of number of Otx2-, PV-, WFA, CR- and GABA-positive cells in V1b layers II-IV of adult <i>scFvOtx2</i>^<i>tg/o</i> and <i>scFvPax6</i>^<i>tg/o</i> mice, 15 days after icv injection of Cre-TAT (in percentage of <i>scFvPax6</i>^<i>tg/o</i> mice, dashed line indicates <i>scFvPax6</i>^<i>tg/o</i> levels). Absolute numbers are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006035#pgen.1006035.s002" target="_blank">S2 Fig</a>. <b>C)</b> Quantification of <i>Arc</i>, <i>Fos</i>, <i>Nr4a1</i>, <i>Egr4</i> and <i>HPRT</i> mRNA content (relative to <i>GAPDH</i>) in V1 layer IV extracts from adult <i>scFvOtx2</i>^<i>tg/o</i> and <i>scFvPax6</i>^<i>tg/o</i> mice, 15 d after icv injection of Cre-TAT. (t-tests; 3–12 mice per group; *p<0.05, **p<0.01, ***p<0.001; error bars indicate SEM)</p