September 2013
Volume 54, Issue 9
Free
Cornea  |   September 2013
Wounded Embryonic Corneas Exhibit Nonfibrotic Regeneration and Complete Innervation
Author Notes
  • Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 
  • Correspondence: Peter Lwigale, Rice University, 6100 Main Street, Houston, TX 77025; lwigale@rice.edu
Investigative Ophthalmology & Visual Science September 2013, Vol.54, 6334-6344. doi:10.1167/iovs.13-12504
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      James W. Spurlin, III, Peter Y. Lwigale; Wounded Embryonic Corneas Exhibit Nonfibrotic Regeneration and Complete Innervation. Invest. Ophthalmol. Vis. Sci. 2013;54(9):6334-6344. doi: 10.1167/iovs.13-12504.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Wound healing in adult corneas is characterized by activation of keratocytes and extracellular matrix (ECM) synthesis that results in fibrotic scar formation and loss of transparency. Since most fetal wounds heal without scaring, we investigated the regenerative potential of wounded embryonic corneas.

Methods.: On embryonic day (E) 7 chick corneas were wounded by making a linear incision traversing the epithelium and anterior stroma. Wounded corneas were collected between E7 and E18, and analyzed for apoptosis, cell proliferation, staining of ECM components, and corneal innervation.

Results.: Substantial wound retraction was observed within 16-hours postwounding (hpw) and partial re-epithelialized by 5-days postwounding (dpw). Corneal wounds were fully re-epithelialized by 11 dpw with no visible scars. There was no difference in the number of cells undergoing apoptosis between wounded and control corneas. Cell proliferation was reduced in the wounded corneas, albeit mitotic cells in the regenerating epithelium. Staining for alpha–smooth muscle actin (α-SMA), tenascin, and fibronectin was vivid but transient at the wound site. Staining for procollagen I, perlecan, and keratan sulfate proteoglycan was reduced at the wound site. Wounded corneas were fully regenerated by 11 dpw and showed similar patterns of staining for ECM components, albeit an increase in perlecan staining. Corneal innervation was inhibited during wound healing, but regenerated corneas were innervated similar to controls.

Conclusions.: These data show that minimal keratocyte activation, rapid ECM reconstruction, and proper innervation occur during nonfibrotic regeneration of the embryonic cornea.

Introduction
Damage or infection in the adult corneal stroma often results in fibrotic scarring and loss of transparency. 1,2 Tissue repair in adult corneas elicits an elaborate cascade of responses including recruitment of inflammatory cells, cytokine mediated apoptosis, activated proliferation, 3 differentiation of repair myofibroblasts, 4 and remodeling of extracellular matrix (ECM) components. 5,6 Tissue remodeling continues in the adult corneal stroma during the healing process, which leads to scar formation and vision impairment. 7,8 Despite extensive characterization of wound healing events in adult corneas and the accumulating knowledge of the regenerative potential of fetal wounds, it is not known whether wounded embryonic corneas heal scar free. 
Fetal tissues regenerate rapidly with no detectable scar formation. 9 Several mechanisms have been proposed as the basis for the scar-free regeneration of fetal wounds. The level of inflammation during the healing process is crucial in determining the magnitude of scarring. Wounded adult tissues demonstrate significant granulation and inflammation during repair, whereas fetal wounds exhibit minimal inflammation. 10,11 In addition, fetal fibroblasts simultaneously coordinate the synthesis and remodeling of ECM proteins, resulting in fiber orientations conducive to scar-free tissue regeneration. 12 Other studies suggest that the gestational period in which the wound is generated is critical to scarless regeneration of tissue. 1315  
This remarkable regenerative potential of embryonic tissues is conserved in fetal skin, heart, and lung wounds, and in bone fractures. 16 In rabbit, mouse, rat, chicken, nonhuman primates, and human embryonic tissues, fetal skin matrices possess an intrinsic potential to restore proper dermal components, resulting in repaired tissue that is indistinguishable from surrounding dermis. 12,1721 Similarly, complete fractures in mouse and lamb fetal bones regenerate rapidly and heal scar free, with minimal or no callus formation. 22,23 However, regeneration of fetal wounds is not recapitulated in all tissues since early gestational wounds made in the diaphragm or intestine form scars. 24,25 Despite the organ specificity to scar-free regeneration, this aspect of fetal wound healing has not been investigated in the embryonic cornea. 
To examine the regenerative potential of embryonic corneal wounds, we utilized a method recently developed to access chick embryos in ovo during late stages of development. 26 Corneas were wounded at E7 and analyzed at various time points during wound healing. We found that embryonic corneal wounds initially increase in size but heal without visible fibrosis. Immunohistologic analysis of wounded embryonic corneas revealed transient change in expression of ECM components, which were restored to normal levels in the healed corneas. Furthermore, we showed that Sema3A mRNA was elevated and innervation of wounded embryonic corneas was inhibited during wound healing, but healed corneas were fully innervated. These findings contribute to our understanding of the events that orchestrate scar-free regeneration of wounded embryonic corneas. 
Methods
Embryo Preparation and Wounding of the Cornea
Fertilized White Leghorn chick ( Gallus gallus domesticus ) eggs were obtained from Texas A&M Poultry Center (College Station, TX) and prepared as previously described. 26 Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Rice University. Briefly, eggs were incubated at 38°C and processed through a series of manipulations to remove the extra embryonic membranes and enable access to the right eye of E7 embryos in ovo. Corneas were wounded at E7 using a micro-dissecting knife (30° Angled Micro-Dissecting Knife; Fine Science Tools, Foster City, CA). An incision was made across the cornea with lacerations traversing the corneal epithelium, basement membrane, and anterior stroma (Figs. 1A, 1B). Ringer's solution containing penicillin (50 U/mL) and streptomycin (50 μg/mL) is added to embryos after wounding and the eggs are sealed with transparent tape and re-incubated. 
Figure 1
 
Wound healing of embryonic corneas. (A) Comparison of wounded and nonwounded stage-matched corneas between 0 and 11 dpw. Arrowheads indicate the extent of wound caused by linear incision. (B) Cross-sections through wounded corneas immunostained with laminin showing the wound at 0 dpw, wound retraction at 3 dpw, and the re-epithelialized cornea at 11 dpw. Brackets and asterisks denote wounded region. (C) Analysis of the wound size at different time points. Wound size was measured using ImageJ (National Institutes of Health, Bethesda, MD) and percent wounded area was calculated by dividing the area of the wound by the total area of the cornea. ep, epithelium; st, stroma; en, endothelium. Scale bars: 1 mm (A), 100 μm (B).
Figure 1
 
Wound healing of embryonic corneas. (A) Comparison of wounded and nonwounded stage-matched corneas between 0 and 11 dpw. Arrowheads indicate the extent of wound caused by linear incision. (B) Cross-sections through wounded corneas immunostained with laminin showing the wound at 0 dpw, wound retraction at 3 dpw, and the re-epithelialized cornea at 11 dpw. Brackets and asterisks denote wounded region. (C) Analysis of the wound size at different time points. Wound size was measured using ImageJ (National Institutes of Health, Bethesda, MD) and percent wounded area was calculated by dividing the area of the wound by the total area of the cornea. ep, epithelium; st, stroma; en, endothelium. Scale bars: 1 mm (A), 100 μm (B).
Immunohistochemistry
Embryos were collected at various time points during the wound healing process. After decapitation, eyes were collected in Ringer's solution and fixed over night in 4% paraformaldehyde at 4°C. Corneas were dissected from the eyes, infused with 5% and 15% sucrose, then embedded in gelatin containing 15% sucrose. Corneas were cryosectioned at 8 and 10 μm then prepared for immunostaining using standard protocols. The following antibodies were used diluted in antibody buffer (PBS containing 0.2% Triton X-100 and 0.2% bovine serum albumin [BSA]): mouse antikeratan sulfate proteoglycan-I22 27 (KSPG, IgG1; Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA) was diluted 1:30 to label keratocytes, mouse anti–alpha-smooth muscle actin (α-SMA; IgG2a; Sigma, St. Louis, MO) was diluted 1:400 to label myofibroblasts, rabbit anticleaved caspase 3 (Cas3a; IgG; Cell Signaling, Danvers, MA) was diluted 1:1000 to label apoptotic cells, rabbit antiphosphohistone H3 (pH3) (IgG; Cell Signaling) was diluted 1:400 to label proliferating cells, mouse antiprocollagen I (IgG1, DSHB) was diluted 1:30, mouse antifibronectin (IgG2a, DSHB), mouse antitenascin (IgG1, DSHB), mouse antilaminin (IgG1, DSHB), and mouse antiperlecan (IgG1, DSHB) were diluted 1:30 to label ECM components. The following secondary antibodies (Molecular Probes, Eugene, OR) were used at 1:200; Alexa 594 goat anti-mouse IgG2a, Alexa 488 goat anti-mouse IgG1, Alexa 488 goat anti-rabbit IgG. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to show all nuclei. Slides were cover-slipped with Perma Flour (Thermo Scientific, Rockford, IL) and fluorescent images were captured using a Zeiss Axiocam mounted on a Zeiss Axioskop 2 microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). 
Whole-Mount Immunohistochemistry
Fixed corneas were trimmed at the limbal region and immunostained as described. 28 To label corneal nerves, mouse antineuron-specific β-tubulin (TUJ1, IgG2a; Covance, Dedham, MA) was used diluted 1:500, followed by Alexa 488 goat anti-mouse (IgG2a; Molecular Probes) secondary antibody diluted at 1:200. Tissues were rinsed and mounted in PBS for imaging. Some immunostained tissues were cryosectioned at 8 to 10 μm and counterstained with DAPI. Fluorescent images of whole-mount corneas and sections were captured as described above. 
Quantitative Real-Time PCR
Tissue from wounded and nonwounded controls of similar size were dissected from identical regions of the cornea and collected in Ringers solution. Messenger RNA was isolated from wounded and nonwounded corneas with Trizol (Invitrogen), following manufacturer's protocol. Corneal tissue was pooled from two embryos at embryonic day (E) 8 (16-hours postwounding [hpw]) and single corneas were used for E10 to E14 (3–7-days postwounding [dpw]). Genomic DNA was digested using Turbo DNA-free Kit (Ambion, Austin, TX) and cDNA was generated using qScript cDNA SuperMix (Quanta Biosciences, Hercules, CA). Quantitative real-time PCR was conducted on a CFX96 Real-time instrument/C1000 Thermal Cycler (BioRad, Gaithersburg, MD) using PerfeCta SYBR Green SuperMix (Quanta Biosciences). The primers used for GAPDH, were as follows; FWD: 5′-GATTCTACACACGGACACTT-3′; REV: 5′-TCTGCCCATTTGATGTTGCT-3′. Primers used for Sema3A transcript amplification were previously described. 29 Relative transcript levels were calculated by normalizing cycle threshold (Ct) values to house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ratiometrically comparing fold changes in transcript levels between paired wounded and nonwounded controls; this was performed in triplicate for each time point. 
Cell Death and Proliferation
Cas3a staining identifies a terminal signaling component of caspase-mediated apoptosis and pH3 is a marker for chromosome condensation during mitosis. Quantification of cells undergoing apoptosis or mitotically active cells was performed by counting the number of stained cells on seven randomly selected sections from each sample. Three wounded and nonwounded corneas were analyzed for each time point. Statistical analysis was performed using Student's t-test. 
Results
Wound Healing in Embryonic Chick Corneas
To determine the regeneration potential of embryonic chick corneas, wounds were created in the right eyes at E7. At this time, the three cellular layers of the cornea are formed 30 and express characteristic markers in the epithelium, 31 stroma, 32,33 and endothelium. 31 Analysis of wounded corneas at different time points (0–11 dpw) revealed progressive retraction of the wound into an ovoid shape between 0 and 3 dpw, followed by epithelialization between 5 and 9 dpw, and complete regeneration of the epithelial layer by 11 dpw (Fig. 1A). Cross-sections through corneas collected at 0 dpw show that the wound penetrates the epithelial layer and basement membrane as confirmed by the absence of laminin staining in the wounded site (Fig. 1B). Similar absence of subepithelial laminin staining was observed in adult corneal wounds. 34 At 3 dpw, a wide region of denuded stroma was not covered by the epithelium and laminin-rich basement membrane (Fig. 1B). However, by 11 dpw, laminin staining was restored and continuous in the basement membrane of regenerated (Fig. 1B, asterisks) and surrounding epithelium. Despite the slow healing rate, the majority of the wounded embryonic corneas (n = 6/7) did not form visible fibrotic scars after complete re-epithelialization, and appeared as transparent as the nonwounded controls at 11 dpw (Fig. 1A). Due to variation in wound size and healing between embryos, we measured the size of the denuded area of each wounded cornea between 0 and 11 dpw. Our results show that on average, embryonic cornea wounds initially expand and reach a maximum increase in area at 3 dpw, then decrease in size until they are completely re-epithelialized at 11 dpw (Fig. 1C). Together, these findings indicate a unique pattern in embryonic cornea wound healing that involves initial wound retraction, followed by re-epithelialization and scar-free regeneration. 
Apoptosis and Proliferation in Embryonic Corneal Wounds
Cytokine-mediated keratocyte apoptosis and proliferation play a major role in initiating the wound healing response in adult corneas. 3 To determine if wounded embryonic corneas undergo a similar response, we examined wounded and nonwounded corneas at various stages of healing for Cas3a and pH3 staining. On average we observed one Cas3a-positive cell for every three sections (Figs. 2A–C; arrows), and there was no significant difference between wounded and nonwounded corneas throughout the healing process (Fig. 2A). In contrast, relatively more cells were undergoing cell proliferation. In nonwounded corneas at E8 there were 16.56 ± 4.7 pH3-positive cells/section, and at E10 the number increased to 20.89 ± 3.2 pH3-positive cells/section. However, the number decreased to 13.02 ± 4.3 pH3-positive cells/section by E12, and continued to decrease through E18 (Fig. 2D). Despite a similar trend in cell proliferation in wounded corneas between 16 hrpw and 5 dpw, there were significantly fewer pH3-positive cells (13.26 ± 1.7 cells/section, P = 0.022) at 3 dpw. By 11 dpw, a similar number of pH3-positive cells were observed in nonwounded and wounded corneas. Despite the low number of proliferating cells in the wounded corneas, we observed clusters of pH3-positive cells in the regenerating corneal epithelium at 5 dpw (Fig. 2E; arrowheads). This was in contrast to the punctate pH3 staining observed in the epithelial layer of control corneas (Fig. 2F). 
Figure 2
 
Quantification of cell death and proliferation during wound healing of the embryonic cornea. The number of (A) cas3A-positive cells or (D) pH3-positive cells were counted on seven random sections and averaged for three wounded and nonwounded corneas at each time point. *P < 0.05. (B, C) Cas3a staining (arrows) in representative sections of (B) 16 hrpw and (C) stage-matched control corneas. (E, F) Localization of pH3-postive cells (arrowheads) in the corneal epithelium (E) at the wound margin at 5 dpw, and (F) in stage-matched control. Brackets denote wound region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 50 μm.
Figure 2
 
Quantification of cell death and proliferation during wound healing of the embryonic cornea. The number of (A) cas3A-positive cells or (D) pH3-positive cells were counted on seven random sections and averaged for three wounded and nonwounded corneas at each time point. *P < 0.05. (B, C) Cas3a staining (arrows) in representative sections of (B) 16 hrpw and (C) stage-matched control corneas. (E, F) Localization of pH3-postive cells (arrowheads) in the corneal epithelium (E) at the wound margin at 5 dpw, and (F) in stage-matched control. Brackets denote wound region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 50 μm.
Expression of α-SMA Is Transient During Wound Healing of Embryonic Corneas
Following mechanical insult to the adult corneal stroma, keratocytes within the wound temporally transdifferentiate into contractile myofibroblasts, as denoted by the expression α-SMA. 4,35 Few α-SMA–positive cells were detected in sections of healing embryonic corneas as early as 16 hrpw (Fig. 3A, arrowhead). Endogenous staining for α-SMA was observed in the cornea endothelium until E9 and its pattern of expression was not affected by wounding (data not shown). However, the unepithelialized wound stained intensely for α-SMA at 3 dpw (Fig. 3B), but it was restricted to the superficial cells within approximately 8 to 15 μm of the stroma. Interestingly, α-SMA staining was abolished in the wound by 5 dpw (Fig. 3C), and it remained undetectable in the re-epithelialized corneas at 11 dpw (Fig. 3D). These results show that α-SMA is transiently expressed at the wound surface during regeneration of the embryonic cornea. 
Figure 3
 
Expression of α-SMA during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing: (A, B) α-SMA-positive staining in the wound at 16 hrpw (arrowhead) and 3 dpw. Note, the cornea endothelium stains positive for α-SMA at 16 hrpw. (C) Alpha–smooth muscle actin staining is not detected at 5 dpw, and (D) after re-epithelialization. Brackets and asterisks denote wounded region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 100 μm.
Figure 3
 
Expression of α-SMA during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing: (A, B) α-SMA-positive staining in the wound at 16 hrpw (arrowhead) and 3 dpw. Note, the cornea endothelium stains positive for α-SMA at 16 hrpw. (C) Alpha–smooth muscle actin staining is not detected at 5 dpw, and (D) after re-epithelialization. Brackets and asterisks denote wounded region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 100 μm.
Expression of Fibronectin and Tenascin Is Increased During Wound Healing of the Embryonic Cornea
Fibronectin and tenascin are essential for epithelial and keratocyte migration into the adult cornea wound. 36,37 To determine whether there is a correlation between the localization of fibronectin and tenascin and re-epithelialization of embryonic corneal wounds, we analyzed their expression during the healing process. At 2 dpw, fibronectin staining was elevated in the anterior stroma of the wound, whereas tenascin staining was strong beneath the epithelium at the wound margin, but low in the denuded area (Fig. 4A). Fibronectin and tenascin did not overlap in the anterior stroma of the wound. This pattern is different from the anterior expression of fibronectin and posterior expression of tenascin in the basement membranes of nonwounded corneas (Fig. 4A'). At 3 dpw, both fibronectin and tenascin were expressed vividly in the unepithelialized wound, but tenascin staining extended beneath the regenerating epithelium (Fig. 4B). At 5 dpw, fibronectin was low at the wound site although tenascin remained strong and dispersed in the anterior stroma (Fig. 4C). Fibronectin was not expressed in stage-matched controls for 3 to 5 dpw, but tenascin was expressed in the stroma and posterior basement membrane (Figs. 4B', 4C'). By 11 dpw, only tenascin was detected in the anterior basement membrane of the re-epithelialized cornea and its distribution corresponded with that of stage-matched control (Figs. 4D, 4D'). Our results show transient expression of fibronectin and tenascin at the surface of the denuded stroma, suggesting that they function in epithelial migration during wound healing of the embryonic cornea. 
Figure 4
 
Expression of fibronectin and tenascin during wound healing of the embryonic cornea. Cross-sections through embryonic corneas immunostained for fibronectin and tenascin showing: (AC) Nonoverlapping staining for fibronectin and tenascin localized in the anterior region of the wound between 2 and 5 dpw. (A'C') Staining for fibronectin and tenascin in stage-matched control corneas. (D) At 11 dpw, fibronectin is downregulated in the re-epithelialized wound and tenascin is expressed in the anterior–posterior gradient similar to stage-matched control (D'). Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Figure 4
 
Expression of fibronectin and tenascin during wound healing of the embryonic cornea. Cross-sections through embryonic corneas immunostained for fibronectin and tenascin showing: (AC) Nonoverlapping staining for fibronectin and tenascin localized in the anterior region of the wound between 2 and 5 dpw. (A'C') Staining for fibronectin and tenascin in stage-matched control corneas. (D) At 11 dpw, fibronectin is downregulated in the re-epithelialized wound and tenascin is expressed in the anterior–posterior gradient similar to stage-matched control (D'). Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Procollagen I Staining Is Reduced During Wound Healing of the Embryonic Cornea
At 2 dpw, continuous staining of procollagen I was detected in the anterior region of the wound and Bowman's membrane of the adjacent nonwounded epithelium (Figs. 5A, 5A'). Between 3 and 5 dpw, procollagen I staining was absent in the wound surface and minimal in the wounded stroma (Figs. 5B, 5B', 5C, 5C'). At 5 dpw, procollagen I was restored in the Bowman's membrane subjacent to the regenerating epithelium (Fig. 5C, arrowheads). By 11 dpw, similar anterior to posterior gradient of procollagen I staining was observed in wounded and control corneas (Figs. 5D, 5D'). 
Figure 5
 
Procollagen I expression in healing embryonic corneal wounds. Cross-sections through embryonic corneas showing expression of procollagen I in: (AD) wounded, and (A'D') stage-matched controls. Arrowheads indicate procollagen staining in regenerated epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Figure 5
 
Procollagen I expression in healing embryonic corneal wounds. Cross-sections through embryonic corneas showing expression of procollagen I in: (AD) wounded, and (A'D') stage-matched controls. Arrowheads indicate procollagen staining in regenerated epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Distribution of Proteoglycans During Wound Healing of Embryonic Corneas
In the nonwounded corneas, expression of perlecan was initially restricted to the basement membranes of the corneal epithelium and endothelium between E8 and E12 (Fig. 6A, arrowheads). By E16, perlecan was also expressed in the stroma at low levels, and at E18 it was vivid in the stroma and basement membranes (Fig. 6A). In contrast, perlecan was not detected in the wound region between 2 and 5 dpw (Fig. 6B), but it was detected at low levels in the basement membrane of the regenerating cornea epithelium at 5 dpw (Figs. 6B, 6Bi, arrowheads). At 11 dpw, perlecan staining was stronger in the stroma of the regenerated cornea (Fig. 6B) compared with stage-matched control (Fig. 6A, E18). 
Figure 6
 
Expression of perlecan in the embryonic cornea. During (A) cornea development and (B) wound healing. Arrowheads in (A) indicate localization of perlecan to the anterior and posterior basement membranes. Arrowheads in (B) indicate perlecan staining in the anterior basement membrane during re-epithelialization. Brackets and asterisks denote wounded region. Scale bars: 100 μm; inset 50 μm.
Figure 6
 
Expression of perlecan in the embryonic cornea. During (A) cornea development and (B) wound healing. Arrowheads in (A) indicate localization of perlecan to the anterior and posterior basement membranes. Arrowheads in (B) indicate perlecan staining in the anterior basement membrane during re-epithelialization. Brackets and asterisks denote wounded region. Scale bars: 100 μm; inset 50 μm.
Keratan sulfate proteoglycan (KSPG) is synthesized in the embryonic cornea stroma by E6, 32 making it an early marker for neural crest differentiation into keratocytes. Staining for KSPG was lower in the stroma of the wound compared with the surrounding nonwounded region at 2 dpw (Figs. 7A–A''). At 5 dpw, the intensity of KSPG staining increased in the stroma of the re-epithelialized wound to levels that matched the nonwounded region (Figs. 7B–B'''). By 11 dpw, KSPG staining in the wound site (Figs. 7C, 7C'') was similar to the nonwounded region and control (Fig. 7C'). Combined, these results show that corneal proteoglycans are restored to endogenous levels during regeneration of the embryonic cornea. 
Figure 7
 
Expression of KSPG during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing expression of KSPG at: (AA'') 2 dpw, (BB''') 5 dpw, and (CC') 11 dpw. Control staining is from nonwounded stage-matched cornea. Arrowheads indicate regenerating epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm (AC), 50 μm (A'C', Control).
Figure 7
 
Expression of KSPG during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing expression of KSPG at: (AA'') 2 dpw, (BB''') 5 dpw, and (CC') 11 dpw. Control staining is from nonwounded stage-matched cornea. Arrowheads indicate regenerating epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm (AC), 50 μm (A'C', Control).
Corneal Innervation Is Temporarily Inhibited During Wound Healing of the Embryonic Cornea
Presumptive corneal nerves form a pericorneal nerve ring prior to their projection into the chick cornea. 3840 Subsequently, stromal nerve bundles reach the cornea center (Fig. 8A) and project into the epithelial layer by E12. 41 Analysis of embryonic corneal wounds at 5 dpw, revealed minimal nerve projection into the re-epithelialized region (Fig. 8B, bracket). At high magnification, a region that appeared to be innervated shows a nerve bundle defasciculating into leashes that project into the distal region of the regenerating epithelium (Fig. 8B', arrow and arrowheads). Optical section of the same region shows the stromal nerve bundle projecting into the regenerating epithelium (Fig. 8B”, arrow), and the extent of epithelial innervation at the wound site (Fig. 8B”, arrowhead). Despite the lack of axon projections during wound healing, there was no defect in the pattern and level of innervation between the 11 dpw wounded corneas and stage-matched controls (Figs. 8C, 8D). 
Figure 8
 
Innervation of wounded embryonic corneas. Whole-mount immunostaining with antiβ-neurotubulin antibody (TUJ1) showing nerve projections into (A, C) nonwounded and (B, D) wounded corneas. (B'') Optical section of the wound region shown in (B'). Bracket (B) indicates regenerating epithelium. White dotted line (B, B') demarcates the wound boundary. Yellow dotted line (B'') designates epithelialstromal boundary. Arrows and arrowheads (B', B'') indicate stromal nerve and epithelial nerve leashes, respectively. (E) Quantitation of Sema3A transcripts. The values shown at each time point are the ratios of fold expression levels between wounded and nonwounded corneas.
Figure 8
 
Innervation of wounded embryonic corneas. Whole-mount immunostaining with antiβ-neurotubulin antibody (TUJ1) showing nerve projections into (A, C) nonwounded and (B, D) wounded corneas. (B'') Optical section of the wound region shown in (B'). Bracket (B) indicates regenerating epithelium. White dotted line (B, B') demarcates the wound boundary. Yellow dotted line (B'') designates epithelialstromal boundary. Arrows and arrowheads (B', B'') indicate stromal nerve and epithelial nerve leashes, respectively. (E) Quantitation of Sema3A transcripts. The values shown at each time point are the ratios of fold expression levels between wounded and nonwounded corneas.
Next, we wanted to determine whether the axon guidance molecule Sema3A, which regulates corneal innervation, 39,42 is responsible for the delayed innervation of the wounded embryonic corneas. Quantitation of mRNA levels by qRT-PCR revealed that Sema3A transcripts (Fig. 8E) increased during wound healing up to 3 dpw (1.72 ± 0.22–fold greater than paired controls). At 5 dpw, the Sema3A level decreased to 1.41 ± 0.21, and then returned to near basal level (0.95 ± 0.37) by 7 dpw. Altogether, our results demonstrate that innervation of the wounded embryonic cornea is delayed during regeneration, but it is fully recovered after wound healing. We also show that Sema3A-mediated repulsion of corneal nerves may play a role during this process. 
Discussion
Using a technique recently developed to access late-stage chick embryos in ovo, 26 we wounded embryonic corneas and investigated their regeneration potential. We report that despite the slow re-epithelialization of the embryonic corneal wound, keratocytes in the wounded stroma exhibit dynamic changes that result in nonfibrotic regeneration. This is consistent with the absence of increased apoptosis and proliferation, and the rapid upregulation of fibronectin, tenascin, and α-SMA at the wound surface, combined with the rapid turnover of perlecan, collagen I, and KSPG. Furthermore, we showed that Sema3A mRNA was elevated in wounded corneas and nerve projections were inhibited at the wound periphery, but innervation of the regenerated corneas was not affected. Combined, our results indicate that several key steps in the wound healing process of embryonic corneas differ from previously characterized cascades in fibrotic postnatal 6,43 or adult 3,3337,44,45 corneal wounds. These differences may account for the scar-free regeneration of the embryonic corneal wounds. 
In this study we show that embryonic chick corneal wounds resulting from linear incisions traversing the epithelium and anterior stroma retract until 3 dpw, after which they undergo re-epithelialization that is completed at approximately 11 dpw. A possible explanation for the initial retraction of embryonic corneal wounds is the IOP-dependent growth phase during the early stages of avian ocular development. Ocular growth in chicks is rapid and dependent on the IOP between E4 and E10, and then it slows dramatically during subsequent development when the IOP is stable. 46 Therefore, concomitant with the period of elevated IOP, corneal wounds generated at E7 increased in size until 3 dpw (E10). In addition, re-epithelialization did not occur during wound retraction, since increased proliferation of the epithelium at the leading edge was not observed until 5 dpw. This slow re-epithelialization does not occur in fetal cutaneous wounds, which heal quickly due to rapid re-epithelialization. 11,47 Also similar wounds in adult corneas close rapidly and they are fully re-epithelialized within 2 to 3 days. 48,49  
The first cellular responses detected after trauma in adult corneal wounds are the cytokine-induced apoptosis and proliferation of keratocytes, 3,50,51 triggered by the damage to the corneal epithelium and its basement membrane. Within 4-hours postinjury, a large number of keratocytes at the wounded site undergo apoptosis, thus, considered to be earliest indicator of the adult corneal wound healing response. 50 Despite the increase in wound size in embryonic corneas, there was no increased apoptosis in the epithelium and stroma during healing. Decrease in cellular density of keratocytes in the wounded corneas could be attributed to stretching of the wound as a result of IOP and eye growth. With the exception of the regenerating epithelium, cell proliferation was lower in the wounded corneas compared with stage-matched controls. Augmented cell proliferation in the corneal epithelium is probably due to the re-epithelialization process and induced by a process that does not affect the keratocytes. The absence of increased apoptosis and keratocyte proliferation suggests that the pro-inflammatory cytokines associated these cellular responses in the adult corneal wounds 50 are not active in the embryonic wound. Therefore, the absence of increased keratocyte turnover at the wound site may contribute to scar-free regeneration. 
Synthesis of contractile α-SMA fibrils and increased expression of stress response proteoglycans are associated with fetal and adult wound healing. 5255 Our results show that the expression of α-SMA is restricted to the surface of the denuded stroma and that it is transient. Comparatively, expression of α-SMA in neonatal and adult corneal wounds extends deep in the stroma and it persists for several weeks to months. 43,56,57 Expression of α-SMA at the wound surface suggests that the role of myofibroblasts in promoting wound closure is conserved in embryonic and adult corneas. In contrast to adult cornea wounds where persistent α-SMA–positive cells are associated with increased opacity, 58 its dynamic expression in the embryonic corneal wounds corresponds to wound closure, whereas its absence may contribute to the nonfibrotic regeneration. 
Another aspect of keratocyte activation is the production of stress response ECM components that increase corneal matrix structural integrity and facilitate wound closure of damaged corneas. During wound healing in the embryonic cornea, the upregulation of stress fibers such as fibronectin and tenascin, is limited to the anterior denuded stroma. The spatiotemporal expression of α-SMA and fibronectin during wound healing suggests that fibronectin is secreted by the α-SMA–positive cells at the wound surface. Previous in vitro studies have shown that myofibroblasts produce fibronectin in vitro. 59 Tenascin staining extended beneath the epithelium surrounding the wound and in the denuded area it was posterior to the region of fibronectin and α-SMA staining. This staining pattern suggests that tenascin is synthesized by the corneal epithelium and keratocytes in the wounded region. Tenascin has multiple binding sites for fibronectin, 60,61 and fibronectin is downregulated in corneal wounds of tenascin knockout mice, 36 suggesting that tenascin may act as a scaffold for fibronectin with in the wound. Both fibronectin and tenascin are upregulated in adult corneal wounds 62,63 where they mediate cell migration and survival. 37,63,64 Therefore, elevated staining of these two molecules in the embryonic cornea wounds suggests that they are involved in epithelial cell migration and wound closure. Their transient synthesis at the wound site correlates with minimal matrix deposition, which may account for the absence of fibrosis in the healed embryonic cornea. 
The ultrastructure, composition, and protein levels of endogenous ECM components are altered during fibrotic corneal wound healing. 6,7,65,66 In adult scar-forming tissues, excess deposition of collagen occurs within 24 hours of injury. 67,68 In contrast, our results show that collagen I synthesis is reduced during embryonic corneal wound healing, but it is restored in the regenerated corneas. This is similar to the rapid matrix turnover and deposition of collagen fibrils observed during scar-free regeneration of fetal skin. 14,69,70 Since collagen I is the predominant fibril collagen in the avian cornea, 71 and it is critical to the establishment of a biostable and transparent stromal matrix during development, 8,33,72 its rapid turnover during wound healing of the embryonic cornea may contribute to the absence of fibrosis. 
Perlecan is a heparan sulfate proteoglycan found in basement membranes of the cornea of both postnatal and adult corneas. 73 Our results show that expression of perlecan is initially restricted to cornea basement membranes, but it is later detected throughout the stroma of the developing avian cornea at E18. Interestingly, corneal transparency increases at this time, reaching maximum levels by E19. 74 In fibrotic corneal scars, perlecan staining persists at high levels. 6,43,75 Perlecan staining was restored to relatively low levels in the in the epithelial basement membrane, but the regenerated epithelium appeared normal. This could be due to the elevated levels of perlecan staining that persisted in the stroma, and surprisingly, did not translate into visible fibrosis. 
Staining for KSPG was also reduced during the wound healing process, but it was restored to control levels in the regenerated embryonic corneas. In adult mice, expression of KSPG is downregulated in the corneal wounds, but it returns to normal levels after 12-weeks postwound. 76 Deficiencies in corneal KSPGs, such as keratocan and lumican, result in altered corneal structure 77 and diminished corneal transparency. 78 Therefore, the rapid turnover of KSPG synthesis may contribute to the normal appearance of the healed embryonic cornea. 
In adult corneal wounds, nerves fail to regenerate and re-innervate healed tissue poorly, 79,80 which may result in diminished corneal sensitivity, 81 keratoconus, 82 and neurotrophic keratitis. 83 Our results show that despite the slow innervation of the embryonic cornea during wound healing, regenerated corneas were innervated at the same level as nonwounded controls. The slow nerve projection into the healing embryonic cornea coincides with the elevation of Sema3A mRNA. Upregulation of Sema3A in adult corneal wounds 84,85 correlates with decreased innervation, whereas application of a Sema3A inhibitor increased nerve regeneration. 86 Therefore, the transient increase of Sema3A is consistent with the normal innervation of healed embryonic corneas. 
In this study, we report our novel finding that chick embryonic corneal wounds undergo rapid regeneration with transient events that promote a wound-healing cascade, which leads to nonfibrotic restoration of the cornea. Our results show that wounding does not induce increased cell death or proliferation in embryonic corneas. One possibility is that resident keratocytes transdifferentiate and upregulate synthesis of fibronectin and tenascin, which promotes re-epithelialization. Concomitantly, keratocytes at the wound site downregulates synthesis of corneal ECM components, but promptly recovers and generates a nonfibrotic cornea. This study also presents the chick embryonic cornea as a valuable model for elucidating novel mechanisms of corneal fibrosis. Ongoing work is aimed at determining the molecular mechanisms that regulate keratocyte differentiation and nonfibrotic regeneration of the embryonic corneal wound. 
Acknowledgments
The authors thank members of the Lwigale lab for discussions of the raw data and editing the early version of the manuscript. 
Supported by National Institutes of Health Grant EY022158 (PYL) and T32 training Grant EY007102-19 (JWS III). 
Disclosure: J.W. Spurlin III, None; P.Y. Lwigale, None 
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Figure 1
 
Wound healing of embryonic corneas. (A) Comparison of wounded and nonwounded stage-matched corneas between 0 and 11 dpw. Arrowheads indicate the extent of wound caused by linear incision. (B) Cross-sections through wounded corneas immunostained with laminin showing the wound at 0 dpw, wound retraction at 3 dpw, and the re-epithelialized cornea at 11 dpw. Brackets and asterisks denote wounded region. (C) Analysis of the wound size at different time points. Wound size was measured using ImageJ (National Institutes of Health, Bethesda, MD) and percent wounded area was calculated by dividing the area of the wound by the total area of the cornea. ep, epithelium; st, stroma; en, endothelium. Scale bars: 1 mm (A), 100 μm (B).
Figure 1
 
Wound healing of embryonic corneas. (A) Comparison of wounded and nonwounded stage-matched corneas between 0 and 11 dpw. Arrowheads indicate the extent of wound caused by linear incision. (B) Cross-sections through wounded corneas immunostained with laminin showing the wound at 0 dpw, wound retraction at 3 dpw, and the re-epithelialized cornea at 11 dpw. Brackets and asterisks denote wounded region. (C) Analysis of the wound size at different time points. Wound size was measured using ImageJ (National Institutes of Health, Bethesda, MD) and percent wounded area was calculated by dividing the area of the wound by the total area of the cornea. ep, epithelium; st, stroma; en, endothelium. Scale bars: 1 mm (A), 100 μm (B).
Figure 2
 
Quantification of cell death and proliferation during wound healing of the embryonic cornea. The number of (A) cas3A-positive cells or (D) pH3-positive cells were counted on seven random sections and averaged for three wounded and nonwounded corneas at each time point. *P < 0.05. (B, C) Cas3a staining (arrows) in representative sections of (B) 16 hrpw and (C) stage-matched control corneas. (E, F) Localization of pH3-postive cells (arrowheads) in the corneal epithelium (E) at the wound margin at 5 dpw, and (F) in stage-matched control. Brackets denote wound region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 50 μm.
Figure 2
 
Quantification of cell death and proliferation during wound healing of the embryonic cornea. The number of (A) cas3A-positive cells or (D) pH3-positive cells were counted on seven random sections and averaged for three wounded and nonwounded corneas at each time point. *P < 0.05. (B, C) Cas3a staining (arrows) in representative sections of (B) 16 hrpw and (C) stage-matched control corneas. (E, F) Localization of pH3-postive cells (arrowheads) in the corneal epithelium (E) at the wound margin at 5 dpw, and (F) in stage-matched control. Brackets denote wound region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 50 μm.
Figure 3
 
Expression of α-SMA during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing: (A, B) α-SMA-positive staining in the wound at 16 hrpw (arrowhead) and 3 dpw. Note, the cornea endothelium stains positive for α-SMA at 16 hrpw. (C) Alpha–smooth muscle actin staining is not detected at 5 dpw, and (D) after re-epithelialization. Brackets and asterisks denote wounded region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 100 μm.
Figure 3
 
Expression of α-SMA during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing: (A, B) α-SMA-positive staining in the wound at 16 hrpw (arrowhead) and 3 dpw. Note, the cornea endothelium stains positive for α-SMA at 16 hrpw. (C) Alpha–smooth muscle actin staining is not detected at 5 dpw, and (D) after re-epithelialization. Brackets and asterisks denote wounded region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 100 μm.
Figure 4
 
Expression of fibronectin and tenascin during wound healing of the embryonic cornea. Cross-sections through embryonic corneas immunostained for fibronectin and tenascin showing: (AC) Nonoverlapping staining for fibronectin and tenascin localized in the anterior region of the wound between 2 and 5 dpw. (A'C') Staining for fibronectin and tenascin in stage-matched control corneas. (D) At 11 dpw, fibronectin is downregulated in the re-epithelialized wound and tenascin is expressed in the anterior–posterior gradient similar to stage-matched control (D'). Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Figure 4
 
Expression of fibronectin and tenascin during wound healing of the embryonic cornea. Cross-sections through embryonic corneas immunostained for fibronectin and tenascin showing: (AC) Nonoverlapping staining for fibronectin and tenascin localized in the anterior region of the wound between 2 and 5 dpw. (A'C') Staining for fibronectin and tenascin in stage-matched control corneas. (D) At 11 dpw, fibronectin is downregulated in the re-epithelialized wound and tenascin is expressed in the anterior–posterior gradient similar to stage-matched control (D'). Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Figure 5
 
Procollagen I expression in healing embryonic corneal wounds. Cross-sections through embryonic corneas showing expression of procollagen I in: (AD) wounded, and (A'D') stage-matched controls. Arrowheads indicate procollagen staining in regenerated epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Figure 5
 
Procollagen I expression in healing embryonic corneal wounds. Cross-sections through embryonic corneas showing expression of procollagen I in: (AD) wounded, and (A'D') stage-matched controls. Arrowheads indicate procollagen staining in regenerated epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm.
Figure 6
 
Expression of perlecan in the embryonic cornea. During (A) cornea development and (B) wound healing. Arrowheads in (A) indicate localization of perlecan to the anterior and posterior basement membranes. Arrowheads in (B) indicate perlecan staining in the anterior basement membrane during re-epithelialization. Brackets and asterisks denote wounded region. Scale bars: 100 μm; inset 50 μm.
Figure 6
 
Expression of perlecan in the embryonic cornea. During (A) cornea development and (B) wound healing. Arrowheads in (A) indicate localization of perlecan to the anterior and posterior basement membranes. Arrowheads in (B) indicate perlecan staining in the anterior basement membrane during re-epithelialization. Brackets and asterisks denote wounded region. Scale bars: 100 μm; inset 50 μm.
Figure 7
 
Expression of KSPG during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing expression of KSPG at: (AA'') 2 dpw, (BB''') 5 dpw, and (CC') 11 dpw. Control staining is from nonwounded stage-matched cornea. Arrowheads indicate regenerating epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm (AC), 50 μm (A'C', Control).
Figure 7
 
Expression of KSPG during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing expression of KSPG at: (AA'') 2 dpw, (BB''') 5 dpw, and (CC') 11 dpw. Control staining is from nonwounded stage-matched cornea. Arrowheads indicate regenerating epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm (AC), 50 μm (A'C', Control).
Figure 8
 
Innervation of wounded embryonic corneas. Whole-mount immunostaining with antiβ-neurotubulin antibody (TUJ1) showing nerve projections into (A, C) nonwounded and (B, D) wounded corneas. (B'') Optical section of the wound region shown in (B'). Bracket (B) indicates regenerating epithelium. White dotted line (B, B') demarcates the wound boundary. Yellow dotted line (B'') designates epithelialstromal boundary. Arrows and arrowheads (B', B'') indicate stromal nerve and epithelial nerve leashes, respectively. (E) Quantitation of Sema3A transcripts. The values shown at each time point are the ratios of fold expression levels between wounded and nonwounded corneas.
Figure 8
 
Innervation of wounded embryonic corneas. Whole-mount immunostaining with antiβ-neurotubulin antibody (TUJ1) showing nerve projections into (A, C) nonwounded and (B, D) wounded corneas. (B'') Optical section of the wound region shown in (B'). Bracket (B) indicates regenerating epithelium. White dotted line (B, B') demarcates the wound boundary. Yellow dotted line (B'') designates epithelialstromal boundary. Arrows and arrowheads (B', B'') indicate stromal nerve and epithelial nerve leashes, respectively. (E) Quantitation of Sema3A transcripts. The values shown at each time point are the ratios of fold expression levels between wounded and nonwounded corneas.
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