Distinct Roles for Neuropilin1 and Neuropilin2 during Mouse Corneal Innervation

Trigeminal sensory innervation of the cornea is critical for protection and synthesis of neuropeptides required for normal vision. Little is known about axon guidance during mammalian corneal innervation. In contrast to the chick where a pericorneal nerve ring forms via Npn/Sema signaling, mouse corneal axons project directly into the presumptive cornea without initial formation of an analogous nerve ring. Here we show that during development of the mouse cornea, Npn1 is strongly expressed by the trigeminal ganglion whereas Npn2 is expressed at low levels. At the same time Sema3A and Sema3F are expressed in distinct patterns in the ocular tissues. Npn1sema−/− mutant corneas become precociously and aberrantly innervated by nerve bundles that project further into the corneal stroma. In contrast, stromal innervation was not affected in Npn2−/− mutants. The corneal epithelium was prematurely innervated in both Npn1sema−/− and Npn2−/− mutants. These defects were exacerbated in Npn1sema−/−;Npn2−/− double mutants, which in addition showed ectopic innervation of the region between the optic cup and lens vesicle. Collectively, our data show that Sema3A/Npn1 and Sema3F/Npn2 signaling play distinct roles and both are required for proper innervation of the mouse cornea

Innervation of the Mouse Cornea during Development

This article describes innervation of the mouse cornea at different time points during development and reveals a novel behavior that is different from chick

Timing of the innervation of the corneal epithelium is disrupted in <i>Npn1^Sema−/−</i> and <i>Npn2^−/−</i> mutants.

<p>Cross sections of E15.5–E16.5 (A–B) Wild type, (C–D) <i>Npn1^Sema−/−</i>, and (E–F) <i>Npn2^−/−</i> corneas immunostained with TuJ1 and counterstained with DAPI. In wild type corneas, axons projection toward the epithelium at E15.5 (A) and innervate it at E16.5 (B). The corneal epithelium is innervated by E15.5 in <i>Npn1^Sema−/−</i> (C, asterisk) and <i>Npn2^−/−</i> (E, asterisk) mutant corneas. Scale bar: 50 µm.</p

Expression of <i>Npn1</i> and <i>Npn2</i> and their ligands <i>Sema3A</i> and <i>Sema3F</i> during eye development.

<p>Levels of mRNA expression were revealed by section in situ hybridization on transverse sections through the cranial region of wild-type mice. (A) <i>Npn1</i> is expressed in the trigeminal ganglion (tg), periocular mesenchyme (pm), optic cup (oc) and hyaloid vasculature (black arrowhead) at E12.5. (C′–E′) The trigeminal ganglion remains positive for <i>Npn1</i> during corneal innervation between E14.5–E16.5. (B) <i>Sema3A</i> is expressed in the lens, pericorneal epithelium, and in the ocular and eyelid mesenchyme (m), but void from the periocular region (arrows) at E12.5. (B–E) This pattern of <i>Sema3A</i> expression is maintained at E14.5–E16.5, except in the pericorneal epithelium (white arrowheads) where it is down regulated at E16.5. (F) In contrast with <i>Npn1</i>, <i>Npn2</i> is expressed at low levels in the trigeminal ganglion but strongly expressed around the optic nerve (arrow), hyaloid vasculature, and periocular mesenchyme at E12.5. (H′–J′) Low levels of Npn2 expression are maintained in the trigeminal ganglion throughout E14.5–E16.5 with strong expression restricted to the trigeminal root (asterisks). (G) Expression of <i>Sema3F</i> is ubiquitous in the eye region at E12.5. (H–J) Between E14.5–16.5, expression of <i>Sema3F</i> is prominent in the corneal epithelium, optic cup, and eyelids. Scale bars: 200 µm.</p

Innervation of the corneal stroma is defective in <i>Npn1^Sema−/−</i> but normal in <i>Npn2^−/−</i> mutant embryos.

<p>Immunostaining for TuJ1 was performed on whole-mount corneas (A–I) and sections (J–R) at E12.5, E13.5 and E14.5. (A–C) Innervation of wild type cornea showing the spatiotemporal projection of nerve bundles into the four quadrants of the stroma and minimal contribution from the DT quadrant (arrow in C). (D) At E12.5, <i>Npn1^Sema−/−</i> eyes show several aberrant projections (asterisks) that also innervate the DT quadrant (arrow) and extend close to the corneal periphery (black arrowheads). (E) By E13.5, nerve bundles from all quadrants including the DT quadrant (arrowhead) innervate the cornea and some project across the diameter of the cornea (asterisk). (F) At E14.5, extensively bifurcated nerve bundles cover most of the cornea. (G–I) Innervation of <i>Npn2^−/−</i> eyes resembled wild type at these stages of corneal development. (J–R) Representative sections through wild type, <i>Npn1^Sema−/−</i> and <i>Npn2^−/−</i> mutant corneas showing the extent of nerve projections at each developmental stage. All sections were counterstained with DAPI. Arrowheads in J, M, and P show relative projection of nerve bundles in the periocular region at E12.5. (S) Quantification of nerves in wild type, <i>Npn1^Sema−/−</i> and <i>Npn2^−/−</i> mutant corneas was carried out by measuring the lengths of nerve bundles in the cornea at E13.5–E15.5; for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037175#s2" target="_blank">Methods</a> section. For all samples n = 8, except <i>Npn2^−/−</i> mutants where n = 6 for E14.5 and E15.5. Error bars = SEM. Scale bars: 100 µm. ***, P<0.001.</p

Loss of Npn1 signaling through Sema disrupts the pattern of innervation of the corneal quadrants.

<p>Whole mount corneas were immunostained with TuJ1 and imaged. The lengths of nerve bundles projecting into each quadrant were quantified as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037175#s2" target="_blank">Methods</a> section. For all graphs, Y-axis is average length of corneal nerves (µm) and X-axis is cornea quadrants. (A–C) Wild type, (D–F) <i>Npn1^Sema−/−</i> mutant, and (G–I) <i>Npn2^−/−</i> mutant eyes were analyzed at E13.5–15.5. ANOVA with a Tukey post test was performed on all data sets. For all samples n = 8, except <i>Npn2^−/−</i> mutants where n = 6 for E14.5 and E15.5. No bracket indicates P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001.</p

Proposed model for Sema/Npn signaling during mouse corneal innervation.

<p>(A) Schematic diagram showing the spatiotemporal expression of <i>Sema3A</i> and <i>Sema3F</i> in wild type embryos, which guide axons during stromal and epithelial innervation. Innervation of the wild type mouse corneal stroma and epithelium respectively commence at E13.5 and E16.5. (B) In <i>Npn2^−/−</i> mutant embryos, stromal innervation is similar to wild type but the epithelium is prematurely innervated by E15.5. (C) In <i>Npn1^Sema−/−</i> mutants, the stroma and epithelium are both prematurely innervated by several nerve bundles that are misguided into the cornea. (D) The corneal innervation defects shown in <i>Npn1^Sema−/−</i> and <i>Npn2^−/−</i> mutants are exacerbated in the absence of Sema/Npn signaling (<i>Npn1^Sema−/−;Npn2^−/−</i> double mutants) by E13.5. In addition, nerve bundles project into ectopic regions of the eye between the optic cup and lens vesicle.</p