Our previous RNA-seq analysis of gene expression during pNC differentiation into corneal cells identified upregulation of Npnt transcripts during chick corneal development (Bi and Lwigale, 2019). To define the expression of Npnt mRNA and protein distribution during this process, we performed section in situ hybridization and immunohistology, respectively. Consistent with our RNA-seq data (Bi and Lwigale, 2019), we found that Npnt is undetectable in the pNC by embryonic day (E) 4 and in the presumptive corneal endothelium at E5, but it is primarily expressed in the retinal pigment epithelium (RPE) and presumptive lens fiber cells at these time points (Figure 1A and B). However, by E6, we found that in addition to the RPE and lens, Npnt is vividly expressed by the second wave of migratory pNC that eventually differentiate into the stromal keratocytes (Figure 1C, arrowhead). Expression of Npnt persisted in the corneal stroma through E12 (Figure 1—figure supplement 1A–C), but it is undetectable at E15 (data not shown).

Because Npnt is a secreted protein that plays a role in cell migration (Kuek et al., 2016; Magnussen et al., 2021; Yamada and Kamijo, 2016), we examined its localization at similar time points during cornea development. At E4, Npnt is localized in the optic cup, lens epithelium, surrounding the pNC cells adjacent to the nascent cornea (Figure 1D–F, arrow), and in the primary stroma of the cornea (Figure 1D–F, asterisk). At E5, strong Npnt staining is observed in the primary stroma (Figure 1G–I, asterisk). By E6, vivid Npnt staining was localized in the anterior region of the cornea and diffusely throughout the primary stroma (Figure 1J–L). At this time, the migratory pNC also stained positive for Npnt (Figure 1L, arrowheads). Strong expression of Npnt persists in the basement membrane of the corneal epithelium through (Figure 1—figure supplement 1C and D) and is also faintly detectable in the corneal epithelial cells by E12 (Figure 1—figure supplement 1E). Interestingly, the protein expression did not correlate with the strong mRNA expression in the stroma during later stages of cornea development. This mismatch could be due to post-transcriptional regulation that prevents protein expression. It is also possible that post-translational modification by enzymes such as matrix metalloproteinases (MMPs), which are temporally and spatially regulated in the corneal ECM during development (Huh et al., 2007), could lead to protein degradation. A previous study showed that Npnt can be modified by MMP cleavage (Toraskar et al., 2019). Given that the changes in protein localization occur after the second wave of migration, we can conclude from our results that the expression of Npnt coincides with pNC ingression into the cornea, implicating a potential role during development.

To investigate the function of Npnt during early corneal development, we generated several RCAS-GFP-Npnt-shRNA (Npnt^kd) constructs in chick DF-1 cells (Figure 2—figure supplement 1). The construct with the highest knockdown of Npnt was used for the experiments. Constructs containing only GFP (RCAS-GFP) or scrambled short hairpin (sh)RNA (RCAS-GFP-Scr-shRNA) were used as control. The viral constructs were injected to cover the entire cranial region of HH7-8 (Hamburger and Hamilton, 1951) chick embryos (Figure 2A). Using this approach, the viral constructs affect all the ocular regions where Npnt is expressed, including the neural progenitors of the optic cup and neural crest cells, and the ectodermal progenitors of the lens (Figure 1A–C). Embryos were reincubated and collected at E7, then the anterior eyes were screened for GFP as an indicator for the extent of viral infection. Only the eyes that showed robust GFP expression were evaluated for knockdown of Npnt expression (Figure 2A). Histological analysis of E7 corneas based on hematoxylin and eosin (H&E) staining revealed that knockdown of Npnt (N = 4) resulted in reduction of corneal thickness (Figure 2C) compared to control (N = 5; Figure 2B). Measurements taken in the mid-corneal regions (Figure 2D and E; arrowheads) showed significant reduction in corneal thickness in Npnt^kd corneas (Figure 2F). These data suggest that Npnt plays a critical role during early development of the cornea.

Next, we reasoned that because knockdown of Npnt causes a significant reduction in corneal thickness, one or more of its receptors should be expressed by the migratory pNC during cornea development. In this study, we focused on α8β1 because of its strong affinity for Npnt (Brandenberger et al., 2001). Although α8β1 was observed to promote spreading of trunk neural crest in vitro (Testaz et al., 1999), little is known about its expression and function in the cranial neural crest. Our analysis by in situ hybridization revealed that Itgα8 was expressed in the leading edge of the periocular mesenchyme prior to pNC migration into the primary stroma of the nascent cornea (Figure 3A, arrow). Subsequently, Itgα8 was maintained in the periocular mesenchyme but also expressed in the corneal endothelium at E5 (Figure 3B, arrows) and stroma at E6 (Figure 3C).

Given the striking expression of Itgα8 by the migratory pNC, we first assessed the potential for Itgα8-Npnt signaling in vitro using explanted periocular mesenchyme from the leading edge adjacent to the presumptive cornea (Figure 3A and D). Mesenchyme explants were cultured on slides coated with Npnt in the presence or absence of a peptide inhibitor previously shown to specifically inhibit binding of α8β1 with Npnt (Figure 3D; Sato et al., 2009). In the absence of the inhibitor, explants attached to the Npnt-coated slides and numerous migratory cells formed a halo around the explant within 12–17 hr of incubation (Figure 3E, Figure 3—video 1). In contrast, we observed that treatment with the α8β1 inhibitor significantly decreased the density of migratory cells from the explant (Figure 3F and G, Figure 3—video 2). These results indicate that α8β1 functions as a receptor for Npnt signaling in the presumptive corneal pNC, and that it is required for their migration.

Given that Itgα8-expressing pNC appear to be in direct contact with Npnt secreted into the primary stroma, we wanted to characterize the role of Itgα8 during pNC migration. For this analysis, we generated and tested several RCAS-GFP-Itgα8-shRNA constructs and validated one that showed highest knockdown of Itgα8 in DF-1 cells (Figure 2—figure supplement 1C) and in vivo (Figure 4—figure supplement 1). As a first step, we analyzed the effect of Itgα8 knockdown on the first wave of pNC migration that forms the corneal endothelium at E5. We found that at this time point relatively fewer GFP-positive cells expressing the Itgα8^kd construct appeared to migrate into the cornea (Figure 4B, arrows) compared to RCAS-GFP control, which showed robust occupation of GFP-positive cells in the corneal endothelium (Figure 4A). Despite the attenuated migration of pNC expressing the Itgα8^kd construct during the first wave, we did not observe defects in the corneal endothelium. One possibility is that pNC, which do not endogenously express Itgα8 (Figure 3A), may also contribute to the corneal endothelium, albeit at a lower level, but they are able to compensate for the Itgα8 knockdown, resulting in the formation of a normal endothelial layer. Nonetheless, histological analysis at E7 revealed reduction in corneal thickness following Itgα8 knockdown (Figure 4D) compared to control corneas (Figure 4C). Measurements and quantification of cells in the mid-corneal regions showed an overall significant reduction in corneal thickness and cell number, but no difference in cell density between Itgα8^kd and control corneas (Figure 4E). Furthermore, we performed a bromodeoxyuridine (BrdU) assay and observed no significant differences in cell proliferation between Itgα8^kd (Figure 4G) and control corneas (Figure 4F). As indicated in our analysis at E5, it is likely that non-Itgα8-expressing pNC may compensate during the second wave of pNC migration, but not to the extent that abrogates the corneal thinning defect, possibly due to the relatively large number of cells required for the formation of the stroma. These results indicate that Itgα8 is required for pNC migration into the cornea. Given that the corneal thinning defect following knockdown of Itgα8 correlates with Npnt knockdown, our results suggest that Npnt-Itgα8 signaling plays an important role during pNC migration into the cornea.

Given that knockdown of both Npnt and Itgα8 resulted in corneal thinning, combined with our observation that Npnt is expressed during the second wave of migration at E6, we investigated whether overexpression of Npnt affects cornea development. We generated and tested viral constructs containing the full-length Npnt gene (Npnt^oe) in DF1 cells (Figure 2—figure supplement 1C). Control and Npnt^oe constructs were injected in HH7-8 embryos as described (Figure 2), and the corneas were collected for analysis at E7, E9, and E15. First, we confirmed the overexpression of Npnt mRNA and protein in the corneas. At E9, expression of Npnt was localized in the corneal stroma during normal development (Figure 5A). However, Npnt^oe corneas showed robust expression of Npnt in the stroma, as well as ectopic expression in the corneal epithelium and endothelium, and the lens (Figure 5D). At this stage, strong staining for Npnt protein was only observed in the basement membrane of the corneal epithelium and low diffuse staining in the stroma of control corneas (Figure 5B). In contrast, the Npnt^oe corneas showed strong ectopic staining for Npnt in the corneal epithelium and endothelium, and a substantial increase in protein expression in the stroma compared to the control (Figure 5E).

Next, we performed histological analysis on E7, E9, and E15 corneas to determine whether there were any morphological differences between the control and Npnt^oe corneas. Our analysis did not reveal any significant differences in corneal thickness and cell number at E7 (Figure 5G and H). By contrast, cross-sections of E9 Npnt^oe corneas revealed significant thickening and increase in the cell number (Figure 5F–H) compared with control (Figure 5C, G and H). Similar increases in corneal thickness and cell number were observed at E15 (Figure 5G and H). Given that the keratocyte density is higher in the anterior stroma than the posterior stroma (Patel et al., 2001; Berlau et al., 2002), we quantified the cell densities in these regions. Overall, we observed relatively higher cell densities in the anterior stroma of E7, E9, and E15 corneas and there were no significant differences between Npnt^oe and control corneas (Figure 5I). Posterior cell density was not affected in E7 Npnt^oe corneas, but there was significant reduction at E9 and E15.

To determine whether elevated cell proliferation was a contributing factor to the increased corneal thickness in Npnt^oe, we examined corneas at E7, E9, and E15 by performing BrdU labeling and immunofluorescent detection. Overall, we observed cell proliferation in all corneal layers, and there was a decreasing trend in the percentage of labeled cells in the stroma as development progressed from E7 to E15 (Figure 5J and K). However, our results revealed no significant increase in cell proliferation at E7 and E15. Surprisingly, there was a significant reduction in cell proliferation in E9 Npnt^oe corneas despite their increase in thickness (Figure 5K). Combined, our data indicate that overexpression of Npnt increases transcript and protein expression in the cornea, which results in increased cell number in the stroma and corneal thickness. Since these increases were not correlated with increased cell proliferation, our data suggest that the changes in corneal thickness are due to augmented pNC migration into the cornea caused by excessive expression of Npnt.

Npnt mediates signal transduction in development and cancer by binding to various receptors through the EGF-like and RGD domains (Arai et al., 2017; Kahai et al., 2010; Kuek et al., 2016; Linton et al., 2007). The marked reductions in corneal thickness observed following knockdown of both Npnt and Itgα8 (Figures 2 and 3) suggest that Npnt functions via the RGD domain during pNC migration. Given that overexpression of the full-length Npnt protein caused corneal thickening, we generated an Npnt-RAE version of the protein in which the RGD domain was mutated by substitution with RAE sequence, and an Npnt-EGF version in which both the RGD and MAM domains were truncated (Figure 6A). Constructs were injected in HH7-8 embryos, and E9 corneas were collected, sectioned, and analyzed following DAPI staining. Contrary to our observations following overexpression of the full-length protein (Figure 5), neither the Npnt-RAE nor Npnt-EGF versions caused a significant change in corneal thickness compared with the control (Figure 6B and C). Interestingly, there was a significant reduction in cell count and density in Npnt-RAE corneas, although no difference in these values was observed for Npnt-EGF (Figure 6D and E). The absence of corneal thickening following overexpression of either Npnt-RAE or Npnt-EGF constructs further suggests that the RGD domain plays an essential role in this process. Our results also indicate that the EGF-like domain does not play an essential role during pNC migration into the cornea.

Previous studies have suggested that the Rho-associated kinase (ROCK) pathway is involved with integrin α8β1 modulation of actin stress fibers in vascular smooth muscles and intestinal crypt cells (Benoit et al., 2009; Zargham et al., 2007). To explore the mechanism by which Npnt-Itgα8 signaling modulates pNC migration, we performed explant culture experiments for 24 hr on Npnt substrate as control (Figure 7A) and compared them to explants cultured in the presence of Npnt combined with various inhibitors. Given that FAK play a role in integrin-mediated migration (Ilić et al., 1995; Parsons, 2003), we first wanted to verify if Npnt-Itgα8-induced migration is mediated through the modulation of focal adhesions. We compared explants cultured on Npnt with the inhibitor for α8β1 or FAK and observed that similar to the α8β1 inhibitor (Figure 7—figure supplement 1C and D), inhibition of FAK reduced pNC migration from the explant but also severely attenuated cell attachment (Figure 7—figure supplement 1E and F).

Next, to examine if Npnt-Itgα8 signaling induces assembly of the cytoskeletal machinery required for pNC migration via the Rho pathway, we treated the explants with the ROCK inhibitor Y27632. Under these conditions, the cells dissociated from the explant, attached to the Npnt substrate, and transformed into a rounded phenotype with significantly increased surface area compared to Npnt alone (Figure 7A–C). In addition, our analysis indicated that the ROCK inhibitor significantly decreased the density of migratory cells (Figure 7D). Given that the cells covered a wider area, it is possible that the decrease in cell density could be due to the increased surface area or decreased cell migration. We therefore tested for linear patterns of cell dispersion from the explant. Our results revealed that in the presence of the ROCK inhibitor cells exhibited a significantly reduced preferred orientation (90° ± 30°) compared to Npnt alone (Figure 7E). Thus, in the presence of the ROCK inhibitor, the pNC spread out on the Npnt substrate with limited directional migration. To test if loss of directionality was caused by changes to the cytoskeletal structure, we examined the cytoskeletal machinery of the cells using phalloidin staining and focal adhesions by immunostaining for pY118 paxillin, which provides a docking site for assembly of the actin fibers that is required for cell adhesion, spreading, and migration (Nakamura et al., 2000; Turner, 2000). As previously reported (Katoh et al., 2001; Masamune et al., 2003; Takamura et al., 2001), the ROCK inhibitor attenuated the formation of actin stress fibers in pNC as indicated by the substantial reduction in the intensity of phalloidin staining (Figure 7F and G). Cells in both conditions stained positive for pY118 paxillin, but the number of puncta per cell significantly increased in the presence of the ROCK inhibitor. This result indicates that disruption of Rho kinase prevents pNC from forming the actin stress fibers and that the cells adhere to the Npnt substrate using multiple foci. Collectively, these data show that Npnt/Itgα8 signaling activates FAK/paxillin for adhesion and that the Rho signaling pathway plays an important role during pNC migration on Npnt.

While Npnt is associated with kidney, teeth, and bone development (Arai et al., 2017; Kuek et al., 2016; Linton et al., 2007), only one reference was made to its expression in the mouse lens during ocular development (Brandenberger et al., 2001). First, we confirmed that Npnt is expressed in the migratory pNC and showed that it is also localized in the optic cup and lens vesicle. Our immunohistochemistry analysis revealed that Npnt protein staining consistently corresponded with the mRNA expression during pNC migration into the developing cornea. In addition, we observed that Npnt was localized in the acellular primary stroma, suggesting that prior to its expression by the migratory pNC in the cornea, the optic cup and lens vesicle are the primary sources of Npnt in the nascent corneal ECM. The link between the primary stroma and the presumptive corneal epithelium and endothelium was established in classical studies, which showed that the lens and corneal endothelium induce the corneal epithelium to synthesize collagen and glycosaminoglycans into the underlying space (Fitch et al., 1994; Hay et al., 1979; Hay and Revel, 1969; Toole and Trelstad, 1971). Our expression analyses suggest that the primary stroma sequesters Npnt initially secreted by the optic cup and the lens, and exposes it to pNC during early corneal development.

Itgα8 is expressed in the spinal cord, optic nerve, retina, urogenital and digestive systems, and in the head epidermis (Bossy et al., 1991; Ogawa et al., 2018). α8β1 is known to modulate epithelial–mesenchymal interactions during kidney and pharyngeal development (Müller et al., 1997; Talbot et al., 2016) and promote migration of mesangial and smooth muscle cells (Bieritz et al., 2003; Zargham and Thibault, 2006). We report for the first time that Itgα8 is expressed by pNC during ocular development. Since Itgα8 heterodimerizes with Itgβ1 subunit (Bossy et al., 1991), which is robustly expressed in the periocular mesenchyme and migratory pNC (Doane and Birk, 1994), we infer that α8β1 signaling functions during corneal development in response to Npnt secreted in the primary stroma. Following delamination from the neural tube, cranial neural crest migrate in response to Fn and laminin (Duband and Thiery, 1982; Newgreen and Thiery, 1980; Sternberg and Kimber, 1986) via integrin receptors such as α1β1, α4β1, α5β1, and αVβ1 (Alfandari et al., 2003; Delannet et al., 1994; Desban and Duband, 1997; Kil et al., 1996; Lallier et al., 1994). Although neural crest cells are initially highly migratory, they remain relatively stationary upon their localization in the periocular region. Given that Itgα8 expression coincides with the onset of pNC migration, our results indicate its potential role in their ingression into the developing cornea.

The three major factors that contribute to corneal thickness during development are pNC migration, cell proliferation, and synthesis of the secondary stroma by the keratocytes. We showed that disruption of Npnt expression in the anterior ocular tissues via RCAS-mediated knockdown resulted in decreased corneal thickness. In addition to functioning in epithelial–mesenchymal interactions, Npnt has been shown to promote migration in various tissues, including vascular endothelial cells during osteogenesis (Kuek et al., 2016), infiltration of immune cells into the liver (Hong et al., 2020; Inagaki et al., 2013), and cancer metastasis (Magnussen et al., 2020; Mei et al., 2020; Wang et al., 2018). Given that Npnt is localized in the primary stroma during corneal development, we hypothesized that it may play a potential role in pNC migration. In agreement with our hypothesis, our in vitro migration assays confirmed that robust pNC migration from mesenchyme explant occurred on Npnt substrate but was abrogated in the presence of the Itgα8 inhibitor. We also observed that knockdown of Itgα8 reduced pNC migration into the cornea and phenocopied the reduction of corneal thickness observed following Npnt knockdown. In addition, we found that reduction in corneal thickness was accompanied by a decrease in cell count, but cell density and proliferation were not affected, suggesting that Npnt/Itgα8 signaling mediates pNC migration during corneal development. Given that the primary function of keratocytes is to synthesize the corneal ECM comprising collagens and proteoglycans, and represents approximately 90% of the corneal thickness (Fini, 1999; Funderburgh et al., 2003; Kao, 2010), our observation that there was no change in stromal cell density raises a possibility that matrix synthesis by the pNC that differentiated into keratocytes is not a major contributing factor to the reduction in corneal thickness. However, additional studies may be required to determine how the disruption in corneal thickness observed in this study may affect the corneal ECM and transparency at later stages of development.

Our overexpression studies showed the opposite effect whereby the full-length construct caused increased corneal thickness at E9. We also found that increased corneal thickness persisted at E15 when Npnt appears to be downregulated in the cornea. Surprisingly, the E7 corneas were not affected in the overexpression studies. This could be caused by limited pNC migration potential due to their expression of Neuropilin1, which prevents them from entering the corneal environment that contains a repulsive Semaphorin3A signal (Lwigale and Bronner-Fraser, 2009). As observed in the knockdown studies, there were no differences in cell proliferation between control and Npnt^oe corneas, suggesting that there was an increase in pNC migration. The mutant Npnt-RAE construct overexpresses approximately the same-size protein as the endogenous Npnt, while maintaining its MAM domain to support ECM–ECM interactions, but it did not impact cornea thickness. This indicates that the expression of high levels of protein does not affect cornea thickness, further implicating that Npnt functions in directing cell migration specifically through its RGD sequence during early cornea development. The reduction in cell count and density could be attributed to the mutant RAE domain outcompeting the expression of the endogenous RGD by the pNC. Similarly, the truncated version of Npnt containing only the EGF-like repeats did not affect cornea thickness, further confirming that Npnt signals through the RGD domain and Itgα8 to modulate pNC migration into the cornea.

Furthermore, our in vitro culture experiments revealed that directed migration of pNC from the explant requires activation of FAK by α8β1 to promote focal adhesions. Previous studies have shown that activation of the Rho/Rock pathway via integrin signaling drives actin remodeling that is necessary for cell adhesion and migration (Clark et al., 1998; Cox et al., 2001; Price et al., 1998). Our data also revealed that in the presence of the ROCK inhibitor pNC transformed into a rounded morphology in which assembly of the actin cytoskeletal machinery was disrupted and the number of focal adhesions increased. These results indicate a drastic change in migratory behavior that was less polarized from the explant, implying a potential involvement of the Rho/ROCK pathway in the directed migration pNC into the cornea. Given that pNC migrate on both Fn and Npnt in vitro, and that both α5β1 and α8β1 activate the Rho/Rock pathway (Benoit et al., 2009; Danen et al., 2005; White et al., 2007; Zargham et al., 2007), it is possible that Rho activity increases as the cells migrate from the periocular region into the matrix of the developing cornea due to the presence of additional cues from Npnt.

Precise coordination of multiple signals from surrounding ocular tissues orchestrates the spatiotemporal migration and differentiation of multipotent pNC during the formation of the avian corneal endothelium and stromal keratocytes. Disruptions in the sequence of these early events can lead to significant defects in corneal development. Our study provides the first characterization of Npnt/Itgα8 function in pNC that contribute to the cornea. Despite being surrounded by an ECM rich in Fn, pNC remain relatively immobile in the periocular region until some become competent to respond to Npnt cues via the expression of α8β1. This transformation may be crucial for the timely induction of directional migration of pNC towards the gradient of additional cues generated by Npnt in the primary stroma (Figure 8A and B), and therefore plays a vital role in segregating the cornea progenitors from the rest of the periocular mesenchyme. In this study, we focused on the role of Npnt/Itgα8 signaling during pNC migration. Our observation that the Npnt protein subsequently localizes to the basement membrane of the epithelial layer, combined with its pleiotropic functions during development and in disease (Yamada and Kamijo, 2016), implies that Npnt may have other functions at later stages of cornea development. One possibility is that Npnt mediates epithelial–mesenchymal interactions between the cornea epithelium and stroma by signaling through the EGF-like domain to EGFRs in the epithelial cells. Future studies will expand on these findings to determine whether the corneal thinning and thickening phenotypes observed in this study affect transparency, which develops at later stages. Elucidation of the link between Npnt signaling and corneal cell differentiation may provide useful insights for future therapeutic applications for wound healing and regeneration studies.

All experiments were performed using fertilized White Leghorn chicken eggs (Gallus gallus domesticus) obtained from Texas A&M Poultry Center (College Station, TX). Eggs were incubated at 38°C in humidified conditions until the desired stages. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Rice University.

For Npnt and Itga8 knockdown and overexpression studies, eggs were incubated for approximately 24–26 hr to obtain three-somite stage or HH8 (Hamburger and Hamilton, 1951), then windowed as previously described (Spurlin and Lwigale, 2013). A few drops of Ringer’s solution containing 100 U/mL penicillin and 100 µg/mL streptomycin (PenStrep, Thermo Fisher Scientific) were added to embryos to maintain hydration. Embryos were injected with viral constructs (see below) using a Picospritzer III pneumatic microinjection system (Parker Hannifin) in the space between the vitelline membrane and cranial region, ensuring that the neural tube and adjacent ectoderm were completely covered (Figure 2A). Injected embryos were re-incubated and collected for GFP screening at the desired stages of development ranging between E5 and E15. Only the corneas showing robust GFP expression were used for subsequent analyses.

Embryos were collected at desired stages and eyes were dissected in Ringer’s saline solution. Samples collected for H&E staining were fixed overnight at 4°C in modified Carnoy’s fixative (60% ethanol, 30% formaldehyde, and 10% glacial acetic acid). Afterward, the tissues were dehydrated in ethanol series, cleared in Histosol (National Diagnostics), embedded in paraffin blocks, and sectioned at 10 µm thickness. Sections were stained with hematoxylin for 15 s, counterstained with eosin for 40 s, dehydrated in ethanol series, and mounted with Cytoseal (Thermo Fisher Scientific) for imaging. For Npnt immunohistochemistry, eyes were fixed in methanol-acetic acid (MAA) fixative (98% methanol, 2% glacial acetic acid) that was chilled on dry ice. Samples were maintained in MAA fixative at –80°C for at least 2 days, then were gradually warmed to room temperature before further dehydration in ethanol series and embedding in paraffin blocks. Samples were sectioned at 10 µm, rehydrated, then immunostained with Npnt antibody (1:100; orb221700, Biorbyt) following standard procedures. For all the other immunohistochemistry and phalloidin staining, samples were fixed in 4% paraformaldehyde (PFA) overnight at 4°C, embedded in paraffin, and sectioned as described above. Immunostaining with anti-GFP (1:500; A-6455, Invitrogen) and anti-fibronectin (1:30; B3/D6, Developmental Studies Hybridoma Bank) was used to detect protein expression. Explant cultures were immunostained with anti-phosphorylated paxillin (1:200; 44-722G, Invitrogen). Phalloidin-568 (1:200; A-12380, Invitrogen) and DAPI (Roche) were used to show total cell distribution and actin organization in whole-mount, sectioned tissue, and explant cultures. The following secondary antibodies (Invitrogen) were used at 1:200: Alexa-488 goat anti-rabbit IgG, Alexa-594 goat anti-rabbit IgG, Alexa-594 goat anti-mouse IgG2a, and Alexa-488 goat anti-mouse IgG1.

Eyes were fixed in modified Carnoy’s fixative, embedded in paraffin, and sectioned as described above. Riboprobes were generated from gene fragments using cDNA pooled from E7 anterior eyes and cloned into TOPO-PCRII (Invitrogen). Digoxigenin (DIG)-labeled riboprobes were synthesized following the manufacturer’s protocol (DIG Labeling Kit, Roche). Primers used to generate the riboprobes are listed in Supplementary file 1. Sections were hybridized with riboprobe at 52°C (Npnt) and 60°C (Itgα8) overnight. Hybridization was detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche) and color was developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma). Following color development, sections were fixed with 4% PFA, mounted in Cytoseal, and imaged using an Axiocam mounted on an AxioImager2 microscope (Carl Zeiss).

RCAS (Hughes et al., 1987) virus was used for stable and prolonged expression of shRNA constructs used for knockdown and the overexpression studies. To produce RCAS viral stocks, chick fibroblasts (DF-1 cells; lot 58217603 with no mycoplasma contamination detected, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin (referred to here as complete DMEM, Thermo Fisher Scientific). Cells were transfected at about 70% confluency with plasmid DNA containing the RCAS constructs using Lipofectamine 3000 (Invitrogen). Transfected cells were grown for 4 days to enable viral replication. Media containing viral particles were collected on subsequent days, pooled, and centrifuged at 21,000 rpm (Beckman) for 1.5 hr at 4°C to concentrate the virus. Virus pellets were resuspended in DMEM and stocks at approximately 1–7 × 10⁶ Ifu/mL were stored in –80°C until use.

shRNA target sequences used for Npnt^kd and Itgα8^kd (Supplementary file 2) were designed using the BLOCK-iT RNAi Designer tool (Thermo Fisher Scientific) and linked to their reverse complementary sequence by a loop sequence, TTCAAGAGA. A short termination sequence and restriction enzyme sites were added at each end to create fragments that were ligated into a pSLAX shuttle vector (Hughes et al., 1987) and to add a chick U6 (Cu6) promoter and a GFP reporter. The shRNA sequences were cloned into the RCAS vector (Figure 2—figure supplement 1A and B) by homologous recombination using a CloneEZ kit (GenScript) as previously described (Kwiatkowski et al., 2017; Ojeda et al., 2017). The Npnt^oe overexpression constructs were either generated by substitution of the shRNA with a full-length coding sequence of Npnt in the vector above or using a modified vector where the full-length Npnt protein was driven by a promoter within the viral long terminal repeats (LTRs) with eGFP linked by an IRES motif (Figure 2—figure supplement 1B). Constructs containing either mutated Npnt with the RGD sequence changed to RAE (PRGDVFIPRQPGVSNNLFEILEIER to PRAEVFIPRQPGVSNNLFEILAIAR) or the truncated version of Npnt containing only the N-terminal EGF-like repeats domain (Npnt-EGF) were commercially synthesized (GenScript) and cloned into the modified RCAS vector. All constructs were validated using primers for Npnt and Itga8 (Supplementary file 3).

BrdU was used to assay for cell proliferation. The BrdU solution was prepared at a final concentration of 10 µM in complete media. Eyes were collected at desired stages and injected into the anterior chamber between the cornea and lens with BrdU solution (approximately 50–100 µL depending on development stage), then cultured in BrdU solution for 2 hr at 37°C. Eyes were rinsed in phosphate-buffered saline , fixed in 4% PFA, embedded in paraffin, and sectioned as described above. BrdU-positive cells were identified by immunohistochemistry using anti-BrdU antibody (1:30; G3G4, Developmental Studies Hybridoma Bank). Sections were counterstained with DAPI.

Embryos were collected at E4 in Ringer’s solution, and the anterior eyes were dissected and digested in dispase (1.5 mg/mL, Worthington) for 10 min at 37°C. The periocular mesenchyme was isolated by physically removing the presumptive cornea epithelium, lens, and the optic cup. The mesenchyme ring was further trimmed using tungsten needles to obtain cells that are proximal to the presumptive cornea, then dissected into approximately 120 × 120 µm explants. Nunc Lab-tek II 8-well chamber slides (Sigma) were coated with 1.5 µg/cm² with poly-d-lysine (MP Biomedicals) for 1 hr at room temperature, followed by recombinant mouse or human Npnt (R&D Systems) at 1.5 µg/cm² for 2 hr at 37°C. Mesenchyme explants were transferred to the coated slides containing complete media, with and without inhibitors and incubated at 37°C in a humidified tissue culture incubator with 5% CO₂ for 12–24 hr. The α8β1 peptide inhibitor following the 23-mer sequence PRGDVFIPRQPTNDLFEIFEIER (Sato et al., 2009) was commercially generated (GenScript) and used at a 10 µM working concentration. The small-molecule FAK inhibitor PF-573228 (Sigma; Slack-Davis et al., 2007) and ROCK inhibitor Y27632 (Sigma; Uehata et al., 1997) were used at 10 µM working concentration. Explants with migratory cells were imaged with a Rebel T6s camera (Canon) mounted on an Axiovert 40C microscope (Carl Zeiss). The magnitude of cell migration from explants was calculated by measuring the density of cells in a 175 µm × 175 µm area located 270 µm from the center of the explant. The orientation of pNC migration from explants was analyzed as previously described (Babaliari et al., 2018). Briefly, confluent regions of cells proximal to the explant were segmented and linearized, then the ImageJ Directionality plugin using local gradient orientation (Liu, 1991) was applied. The cellular features oriented to 90° ± 30° were considered to be aligned perpendicular to the explant. Mean integrated peak areas, bounded by 60° and 120°, were used to compare the magnitude of perpendicular alignment in all conditions.

Mesenchyme explants were prepared and cultured as described above in media containing 0.1 µg/mL Hoechst dye solution (Thermo Fisher Scientific) used to label all nuclei. Chamber slides containing attached explants were imaged at 3 min and 27 s intervals for 17 hr using an FV1200 laser scanning microscope (Olympus) with a stage top incubator (Tokai Hit). Movies were generated using Imaris Software (Oxford Instruments).

ImageJ software was used to measure the staining intensity or tissue morphological features, such as cornea thickness and cell density. The number of cornea sections analyzed is summarized in each figure legend. All statistical analyses were conducted using GraphPad Software. Data are presented as scatterplot with mean values. Statistical significance was determined by two-tailed unpaired Student’s t-test and was used to compare the differences between means. Samples with p-values < 0.05 were considered significant.