November 2010
Volume 51, Issue 11
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Cornea  |   November 2010
Twist2: Role in Corneal Stromal Keratocyte Proliferation and Corneal Thickness
Author Affiliations & Notes
  • Linda Weaving
    From the Eye Genetics Research Group, Embryology, Children's Medical Research Institute, The Children's Hospital at Westmead, Save Sight Institute, Sydney, NSW, Australia;
    Sydney Medical School, University of Sydney, Sydney, NSW Australia;
  • Marija Mihelec
    From the Eye Genetics Research Group, Embryology, Children's Medical Research Institute, The Children's Hospital at Westmead, Save Sight Institute, Sydney, NSW, Australia;
    Sydney Medical School, University of Sydney, Sydney, NSW Australia;
  • Rebecca Storen
    From the Eye Genetics Research Group, Embryology, Children's Medical Research Institute, The Children's Hospital at Westmead, Save Sight Institute, Sydney, NSW, Australia;
    Sydney Medical School, University of Sydney, Sydney, NSW Australia;
  • Drazen Sosic
    Department of Molecular Biology, University of Texas, Southwestern Medical Centre, Dallas, Texas.
  • John R. Grigg
    From the Eye Genetics Research Group, Embryology, Children's Medical Research Institute, The Children's Hospital at Westmead, Save Sight Institute, Sydney, NSW, Australia;
    Sydney Medical School, University of Sydney, Sydney, NSW Australia;
  • Patrick P. L. Tam
    From the Eye Genetics Research Group, Embryology, Children's Medical Research Institute, The Children's Hospital at Westmead, Save Sight Institute, Sydney, NSW, Australia;
    Sydney Medical School, University of Sydney, Sydney, NSW Australia;
  • Robyn V. Jamieson
    From the Eye Genetics Research Group, Embryology, Children's Medical Research Institute, The Children's Hospital at Westmead, Save Sight Institute, Sydney, NSW, Australia;
    Sydney Medical School, University of Sydney, Sydney, NSW Australia;
  • Corresponding author: Robyn V. Jamieson, Children's Medical Research Institute, and The Children's Hospital at Westmead, Hawkesbury Road, Westmead, Sydney NSW 2145, Australia; rjamieson@cmri.usyd.edu.au
  • Footnotes
    3  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science November 2010, Vol.51, 5561-5570. doi:10.1167/iovs.09-5123
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      Linda Weaving, Marija Mihelec, Rebecca Storen, Drazen Sosic, John R. Grigg, Patrick P. L. Tam, Robyn V. Jamieson; Twist2: Role in Corneal Stromal Keratocyte Proliferation and Corneal Thickness. Invest. Ophthalmol. Vis. Sci. 2010;51(11):5561-5570. doi: 10.1167/iovs.09-5123.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: Twist2 is a member of a family of bHLH transcription factors critical for normal mesenchymal proliferation and differentiation. In this study, the authors analyzed the role of Twist2 in the eye and cornea through examination of a Twist2 loss-of-function mouse mutant.

Methods.: Twist2 expression during eye development in the mouse was investigated using RT-PCR and mRNA slide in situ hybridization. Lineage tracing was performed using Cre reporter mice. Morphometric analyses were performed, and cell proliferation and cell death were investigated by immunohistochemistry using Ki67 and cleaved caspase 3 antibodies, respectively.

Results.: In the mouse, Twist2 is expressed first in the periocular mesenchyme and subsequently in the corneal stroma and endothelium of the developing eye. Loss of Twist2 function leads to corneal thinning and a reduced population of stromal keratocytes. The reduction in the stromal cell population can be traced back to embryonic stages during which the proliferation of stromal progenitor cells is impaired and to the reduced number of proliferating cells in the corneal limbus postnatally. Adult Twist2-null mice display enophthalmia and blepharophimosis. Corneal thinning in mutant mice is not accompanied by glaucoma, an association reported in human patients.

Conclusions.: Twist2 is required for normal corneal keratocyte proliferation and eyelid morphogenesis in the mouse. Loss of Twist2 function leads to corneal thinning because of the reduction in stromal keratocyte proliferation.

The integrity of the cornea is essential for the normal refraction of light and the barrier functions of the eye. The corneal stroma constitutes the bulk of the cornea and provides transparency and strength because of its highly organized extracellular matrix, which is secreted by the stromal keratocytes. Keratocyte dysfunction underpins a number of human eye conditions: overexpansion of the keratocyte population results in corneal thickening and dystrophy 1 ; loss of cells is linked to keratoconus causing severe distortion of vision 2,3 ; and corneal thinning is associated with glaucoma, in which there is retinal ganglion cell loss leading to progressive blindness. 4 7 Precise control of keratocyte proliferation is also a critical factor in the recovery of function after corneal wounding and laser-assisted in situ keratomileusis (LASIK) refractive surgery. 8 10 Hence, corneal thickness and control of keratocyte proliferation and differentiation are critical for corneal function and normal vision. 
In the mouse, the stromal keratocytes of the cornea are derived dually from neural crest cells and the cranial mesoderm. 11 Precursors of the keratocytes populate the region between the lens placode and the presumptive corneal epithelium at embryonic day (E) 11 to E12. They continue to proliferate rapidly until E16.5, when the endothelial and stromal layers become distinct. The endothelial cells are then arrested in G1 through contact inhibition, 12 15 whereas most stromal cells exit the cell cycle but remain in G0 and become quiescent. 16 Some stromal cells continue to proliferate during the postnatal period, but this ceases by days 12 to 14 after birth, at which time the cornea reaches maturity and the eyelids open. 17  
Many members of the Twist family are known to regulate the proliferation and differentiation of mesenchymal cells. 18 26 In conjunction with the bHLH protein E12, Twist2 (also known as Dermo-1) inhibits myogenic differentiation by repressing the myogenic factors MyoD and Mef2 20,27 and osteogenic differentiation by repressing Runx2. 21,22 Enforced expression of Twist2 sustains the proliferation of bone marrow-derived mesenchymal stromal/stem cells. 28 Loss of Twist2 function in mice leads to runting, cachexia (adipose and glycogen deficiency), and thin skin with sparse hair. 25 These phenotypes may be partially accounted for by dysregulation of the proinflammatory cytokines TNF-α and IL-1β, which elicit NF-κB-mediated apoptosis. 25 Together, these findings point to a critical role of Twist2 in mediating cellular proliferation, inhibition of apoptosis, and regulation of the timing of differentiation in mesenchymal tissues. 
In the present study, we examined the role of Twist2 in the development of the corneal stroma. Our results reveal that Twist2 is specifically required for regulating corneal keratocyte proliferation and differentiation. 
Materials and Methods
Animals and Genotyping
Twist2cre mice were maintained by intercrossing heterozygotes on a mixed 129 × C57BL/6J background. In a previous study, Twist2cre/cre mice on an inbred 129 background 25 did not survive beyond 15 days of age. On the mixed background, although severe to mild runting resulted in perinatal death in some animals, 12% of Twist2cre/cre mice survived to adulthood and up to two years in some cases, allowing an analysis of eye phenotypes. PCR genotyping was performed using two sets of primers: one for amplifying the wild-type Twist2 allele from exon 1 (5′-AACTTCCTCTCCCGGAGACC-3′ and 5′-GTCTATGTACCTGGCGGCCAGCTTG-3′) and the other for amplifying the Cre transgene that replaced exon 1 in the mutant allele (5′-CTGACCGTACACCAAAATTTGCCTG-3′ and 5′-GATAATCGCGAACATCTTCAGGTTC-3′). Cre reporter mice (pCAGG- loxP-flanked bgeo/3xpA-hPLAP = Z/AP), 29 were maintained on a closed-bred ARC background. For in vivo fate mapping, Twist2 cre/+ were mated with Z/AP mice to generate the Twist2 cre/+ ;Z/AP embryos for mapping the fates of Twist2-positive cells and their derivatives. 
Age categories for adult Twist2cre mice were defined as middle-aged for animals 6 to 9 months of age and elderly for animals 18 months to 2 years of age. 
Animal experiments were approved by the Children's Medical Research Institute/Children's Hospital at Westmead Animal Care and Ethics Committee and conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Tissue Collection and Dissection
Whole embryos from pregnant mice at specific gestational ages, whole heads of newborn (postnatal day [P]0-P2) mice, and enucleated eyes of P5 and P10 and adult mice were collected. In most instances, one eye was collected for histology/immunostaining and the other was dissected for qRT-PCR into retina, lens, cornea, and iris + anterior chamber angle. 
Histology, Morphometric Analysis, and Immunohistochemistry
Embryos, whole heads, and eyes were embedded in embedding medium (Medite Tissuewax; HD Scientific, Wetherill Park, NSW, Australia) and were sectioned at 10 μm for histology and 4 μm for immunostaining. Histology sections were stained with hematoxylin and eosin or periodic acid Schiff and were photographed with an imaging microscope (Axio Imager.A1; Zeiss, North Ryde, NSW, Australia) and digital camera (SPOT; Diagnostic Instruments, Perth, WA). Morphometric measurements and cell counts were performed using the software measurement tool of the digital camera (SPOT; Diagnostic Instruments) on four to five consecutive sections from each specimen. For central corneal measurements, sections were selected that contained the widest pupil opening in pups and adults. For embryos in which the iris was not yet developed, the plane with the widest distance between the two edges of the optic cup was taken as indicating the central position. Total numbers of cells were calculated by counting the number of cells in four or five serial sections (at 40×) and taking the mean. For cell density measurements, the SPOT area tool was used to define the morphologic area for examination, and these areas were analyzed in the number of sections required so that the same total area in wild-type and mutant animals was examined for positive cells (see Fig. 5C for one example). Morphometric analysis of retinal parameters (retinal thickness, optic nerve width) and ganglion cell numbers were performed on three to six serial sections that passed through the optic nerve. Ganglion cell counts were performed by scoring all cells in the ganglion layer (excluding blood vessels) immediately adjacent to the optic nerve on 40× images. 
For immunostaining, samples were subjected to antigen retrieval and 3 × 10-minute washes in 0.01% NaBH4. 30 Staining was performed 31 using rabbit polyclonal anti-Ki67 (#16667; Abcam, Sapphire Bioscience, Redfern, NSW, Australia; 1:100) and rabbit polyclonal anti-cleaved caspase 3 32 (Asp175, #9661; Cell Signaling Technology, Danvers, MA; 1:100), followed by Alexa-Fluor 594 conjugated goat anti-rabbit secondary antibodies (Invitrogen, Mulgrave, VIC, Australia; 1:1000). Sections were counterstained with DAPI and mounted in DABCO solution (Sigma, Castle Hill, NSW, Australia). Cell counts for caspase and Ki67 staining were performed by visual scoring of stained cells. Ki67 was expressed in the actively proliferating retinal progenitor cells and lens epithelium, and these served as internal positive controls. Sections used as negative controls were of two types, those that had the primary antibody omitted and those that had the secondary antibody omitted. Photographs were taken with a fluorescence microscope (BX51; Olympus, Tokyo, Japan). 
Alkaline phosphatase staining was performed on Twist2 cre/+; ZAP/+ embryos and adult eyes using staining solution (NBT/BCIP or BM Purple; Roche, DeeWhy, NSW, Australia) as a substrate. 
In Situ Hybridization
A plasmid containing Twist2 cDNA sequences was cut with ApaI and NotI (Roche) restriction enzymes to produce sense and antisense probes from the T3 and T7 sites, respectively. Antisense and sense riboprobes for Twist2 RNA were synthesized using RNA polymerase kits (Ampliscribe; Epicenter Technologies, Astral Scientific, Gymea, NSW, Australia) and tagged (Digoxigenin-11 UTP; Roche). Hybridization was performed on cryosections affixed in a hybridization chamber gasket (Secure-Seal; Molecular Probes, Invitrogen) for 16 hours at 65°C. Substrate (BM Purple AP; Roche) was used to detect the hybridization signal. 
Reverse Transcription PCR
RNA was purified using a micro kit (RNeasy; Qiagen, Doncaster, VIC, Australia) or a reagent (Trizol; Invitrogen) according to the manufacturer's instructions. Standards were amplified using each set of primers, and products were purified using a gel extraction kit (Qiaquick; Qiagen). Purified samples were quantified using a spectrophotometer (Nanodrop; Biolabs, ThermoFisher Scientific, Scoresby, VIC, Australia), diluted to 1010 molecules/μL in MQ water and stored in aliquots at −80°C until analysis. 
qRT-PCR was performed (Sybr Green chemistry and Amplitaq Gold; Perkin Elmer, Glen Waverley, VIC, Australia) on a real-time machine (Rotorgene; Corbett Lifescience, Sydney, NSW, Australia). A number of primers for murine genes were designed and used for qRT-PCR. Primers used for investigation of cytokine levels were as follows: TNF-α, 5′-GGGGCCACCACGCTCTTCTGT-3′ and 5′-CTCCACTTGGTGGTTTGCTACGACG-3′; IL-1β, 5′-GGCAGCTACCTGTGTCTTTCCCGT-3′ and 5′-CACCAGCAGGTTATCATCATCATCCC-3′. Primers used for investigation of keratocan expression in corneal stroma were as follows: keratocan, 5′-CCGTCGAGGGGTTTTGATGTG-3′ and 5′-TTGTGGTCCGTGAATGAAGGCA-3′. Primers used for markers of ganglion cells and glaucoma were as follows: Thy-1, 5′-GCCCTATATCAAGGTCCTTACCCTAG-3′ and 5′-GTTCTGAACCAGCAGGCTTATG-3′; Nefl, 5′-CAACAAACTGGAGAATGAGC-3′ and 5′-CTGGTGAAACTGAGCCTGGT-3′; Sele, 5′-ATAATTCCTCCTGCTCCTTTGGCTG-3′ and 5′-GAACATTTCCTGATACCGTGGGCA-3′. Primers used for a photoreceptor marker were as follows: rhodopsin, 5′-TTCGGAGGATTCACCACCACC-3′ and 5′-CAGGACCACCAGGGACCACAG-3′. Samples were normalized against β-actin (primers, 5′-AGCACCCTGTGCTGCTCA-3′ and 5′-GTACGACCAGAGGCATACA-3′). 
Reactions for each sample were performed in triplicate in 100-well gene discs (Corbett Lifescience). A standard sample (1010 molecules/μL) was serially diluted to 109 molecules/μL to 102 molecules/μL to generate the standard curve, which was also run in triplicate during each qRT-PCR reaction. Positive controls (either cDNA from eye tissues of C57Bl/6 mice or placenta cDNA) and negative controls (no RT and no cDNA) were used. Data were analyzed using appropriate software (Rotorgene [Corbett Lifescience] and Excel [Microsoft, Redmond, WA]). 
Statistical Analysis
ANOVA was used for multiple comparisons and the two-tail unequal variance t-test for two-sample comparisons. 33 Linear regression was used to examine the relationship between increasing age and corneal thickness. All statistical tests were performed using Excel. P < 0.05 was considered statistically significant. 
Results
Twist2 Expressed Periocular Mesenchyme, Corneal Stroma, and Adult Cornea
Twist2 transcripts were detected by RT-PCR in many wild-type embryonic tissues (Fig. 1A). Of particular interest to our study was its expression in embryonic/fetal eye and in early postnatal and adult cornea. Twist2 expression was revealed by in situ hybridization (Fig. 1B) in the periocular mesenchyme (E10.5), the presumptive corneal stroma and endothelium but not the corneal epithelium (E12.5-E14.5), and in corneal endothelium, stroma, and limbal regions (E16.5). Twist2 was also expressed strongly in the dermis of the developing eyelids (E12.5, E14.5, E16.5) and, as in the cornea, was excluded from the ectodermally derived epidermis (Fig. 1B). Twist2 expression was undetectable in the wild-type adult cornea by in situ hybridization (data not shown) but was faintly detectable by RT-PCR (Fig. 1A). By tracking the distribution of cells expressing the Twist2-Cre activated Z/AP reporter, descendants of cells that had previously expressed Twist2 were found to populate specifically the corneal stroma and endothelium and the dermis of the eyelid in embryonic and adult eyes (Figs. 1C, 1D). 
Figure 1.
 
Expression of Twist2 in the eye. (A) Twist2 expression as determined by RT-PCR in various embryonic tissues and in the corneas of newborn and adult wild-type mice. (B) Twist2 expression revealed by in situ hybridization in wild-type embryos. At E10.5, expression could be detected in the developing corneal stroma (arrowheads) and nearby periocular mesenchyme (arrows). At E12.5, Twist2 was strongly expressed in the corneal stroma (arrowhead, inset), periocular mesenchyme, and dermis of the developing eyelids. Expression was absent from the corneal epithelium (arrow, inset). A similar pattern was observed at E14.5. By E16.5, Twist2 expression was not detected in the periocular mesenchyme (arrows) but was present in the eyelid dermis (arrowhead) and corneal stromal cells (arrowhead, inset). (C) In vivo fate mapping using Twist2cre/+;Z/AP/+ reporter mice. AP staining could be detected in cells that expressed Twist2 during development. Cells that expressed Twist2 were found in the periocular mesenchyme (arrowheads) and the developing corneal stroma (arrows) from E10.5 to E14.5. By E16.5, the Twist2-expressing cells were found in the corneal stroma (arrow) and the dermis of the overlying eyelid but no longer in the periocular mesenchyme (arrowheads). (D) AP staining of adult Twist2 cre/+;Z/AP/+ eyes confirmed that Twist2 expressing cells contributed to the corneal stroma and endothelium (arrow) but not the corneal epithelium (arrowhead). c, cornea; r, retina; lp, lens pit. Scale bar, 100 μm.
Figure 1.
 
Expression of Twist2 in the eye. (A) Twist2 expression as determined by RT-PCR in various embryonic tissues and in the corneas of newborn and adult wild-type mice. (B) Twist2 expression revealed by in situ hybridization in wild-type embryos. At E10.5, expression could be detected in the developing corneal stroma (arrowheads) and nearby periocular mesenchyme (arrows). At E12.5, Twist2 was strongly expressed in the corneal stroma (arrowhead, inset), periocular mesenchyme, and dermis of the developing eyelids. Expression was absent from the corneal epithelium (arrow, inset). A similar pattern was observed at E14.5. By E16.5, Twist2 expression was not detected in the periocular mesenchyme (arrows) but was present in the eyelid dermis (arrowhead) and corneal stromal cells (arrowhead, inset). (C) In vivo fate mapping using Twist2cre/+;Z/AP/+ reporter mice. AP staining could be detected in cells that expressed Twist2 during development. Cells that expressed Twist2 were found in the periocular mesenchyme (arrowheads) and the developing corneal stroma (arrows) from E10.5 to E14.5. By E16.5, the Twist2-expressing cells were found in the corneal stroma (arrow) and the dermis of the overlying eyelid but no longer in the periocular mesenchyme (arrowheads). (D) AP staining of adult Twist2 cre/+;Z/AP/+ eyes confirmed that Twist2 expressing cells contributed to the corneal stroma and endothelium (arrow) but not the corneal epithelium (arrowhead). c, cornea; r, retina; lp, lens pit. Scale bar, 100 μm.
Adult Twist2cre/cre Mice: Enophthalmia and Blepharophimosis
Perinatal lethality caused by runting and cachexia was observed among Twist2cre/cre mutants; however, approximately 12% survived into adulthood. Surviving adult Twist2cre/cre animals were similar to their littermates in size but generally had sparse facial fur and smaller-appearing eyes (Figs. 2A, 2B). Measurements of epicanthal length confirmed that the palpebral fissures were significantly smaller in Twist2cre/cre mice (a condition known as blepharophimosis), but the eyeballs were of normal size (Fig. 2C). Twist2cre/cre mice also displayed enophthalmia (sunken eyes; Fig. 2B). Initial investigations of skull X-rays of enophthalmic adult Twist2cre/cre animals did not identify any obvious differences in the bony orbit. Histologic examination of the eyes of P0, P1, and adult Twist2cre/cre mice showed that the enophthalmia was not caused by the agenesis of the Harderian gland. 34 It remains possible that the enophthalmia is caused by the loss of intraorbital adipose tissue (which could not be assessed in our histologic preparation that removed this tissue) because Twist2cre/cre mice have been shown previously to be deficient in adipogenesis. 22,23,25,35  
Figure 2.
 
Enophthalmia and blepharophimosis in Twist2cre/cre animals. (A, B) Twist2cre/cre (−/−) adult male (left) and normal heterozygous littermate (right). Enlarged views of the eyes are shown on the right. (B, C) Note the enophthalmic (sunken) eyes. Measurement of epicanthal length in adult Twist2 +/+ and Twist2cre/cre mice. Epicanthal length of adult Twist2cre mice was measured using calipers (inset). Twist2cre/cre animals had significantly smaller eye apertures (two-sample t-test assuming unequal variance: n = 21, Twist2 +/+; n = 8, Twist2cre/cre ; P = 0.00013). In contrast, measurements of eyeball length and width did not detect any significant difference (n = 6, Twist2 +/+; n = 4, Twist2cre/cre ; eyeball length, P = 0.713581; eyeball width, P = 0.710512). This suggests that loss of Twist2 function leads to blepharophimosis and enophthalmia rather than true microphthalmia (small eyes). Asterisk: statistically significant result.
Figure 2.
 
Enophthalmia and blepharophimosis in Twist2cre/cre animals. (A, B) Twist2cre/cre (−/−) adult male (left) and normal heterozygous littermate (right). Enlarged views of the eyes are shown on the right. (B, C) Note the enophthalmic (sunken) eyes. Measurement of epicanthal length in adult Twist2 +/+ and Twist2cre/cre mice. Epicanthal length of adult Twist2cre mice was measured using calipers (inset). Twist2cre/cre animals had significantly smaller eye apertures (two-sample t-test assuming unequal variance: n = 21, Twist2 +/+; n = 8, Twist2cre/cre ; P = 0.00013). In contrast, measurements of eyeball length and width did not detect any significant difference (n = 6, Twist2 +/+; n = 4, Twist2cre/cre ; eyeball length, P = 0.713581; eyeball width, P = 0.710512). This suggests that loss of Twist2 function leads to blepharophimosis and enophthalmia rather than true microphthalmia (small eyes). Asterisk: statistically significant result.
Early Eyelid Opening in Some Twist2cre/cre Mice
In mice, the eyelids normally open at P12 to P14. 36 In approximately one-third of postnatal Twist2cre/cre mice, eyelid opening occurred early, at P8 to P11. The remaining homozygous mutants opened their eyes according to the normal schedule. Twist2cre/cre pups frequently had thinner eyelid tissue (Figs. 3A, 3C), consistent with the dermal atrophy previously noted in these mice. 25 We could not detect any apoptosis in the eyelids of P2 mice (38 sections from four Twist2 +/+, 22 sections from three Twist2cre/cre ; data not shown). However, there was a noticeable decrease in proliferation as demonstrated by Ki67 staining (Fig. 3A). 
Figure 3.
 
Corneal thinning in Twist2cre/cre mice. (A) Immunostaining of P2 limbal sections for Ki67+ (red) proliferating cells. Sections are counterstained with DAPI (green). Twist2cre/cre pups have markedly thinner eyelid tissue (see also C) and fewer hair follicles (arrows). The hair follicles of Twist2cre/cre pups appeared to have reduced expression of Ki67 (arrows). Strong staining for Ki67 could be detected in the actively proliferating retinal progenitor cells, which serve as an internal positive control. C, ciliary body; con, conjunctiva. (B) Comparison of corneal thickness in adult Twist2 +/+ (+/+) and Twist2cre/cre (−/−) mice. Twist2cre/cre mice have visibly thinner corneal stroma (indicated by the parentheses). (C) Twist2cre/cre P2 pups have visible thinning of the cornea. The dermis of the overlying eyelids is also thin with reduced numbers of hair follicles. (D) PAS staining of adult Twist2 +/+ and Twist2cre/cre cornea. Arrow: Bowman's membrane. Scale bar, 100 μm, unless otherwise specified.
Figure 3.
 
Corneal thinning in Twist2cre/cre mice. (A) Immunostaining of P2 limbal sections for Ki67+ (red) proliferating cells. Sections are counterstained with DAPI (green). Twist2cre/cre pups have markedly thinner eyelid tissue (see also C) and fewer hair follicles (arrows). The hair follicles of Twist2cre/cre pups appeared to have reduced expression of Ki67 (arrows). Strong staining for Ki67 could be detected in the actively proliferating retinal progenitor cells, which serve as an internal positive control. C, ciliary body; con, conjunctiva. (B) Comparison of corneal thickness in adult Twist2 +/+ (+/+) and Twist2cre/cre (−/−) mice. Twist2cre/cre mice have visibly thinner corneal stroma (indicated by the parentheses). (C) Twist2cre/cre P2 pups have visible thinning of the cornea. The dermis of the overlying eyelids is also thin with reduced numbers of hair follicles. (D) PAS staining of adult Twist2 +/+ and Twist2cre/cre cornea. Arrow: Bowman's membrane. Scale bar, 100 μm, unless otherwise specified.
Twist2cre/cre pups with premature eyelid opening were prone to eye inflammation and infection. The inflammation may be exacerbated by elevated corneal and serum levels of proinflammatory cytokines TNF-α and IL-1β caused by loss of Twist2 function. 25 Our assay of cytokines including TNF-α and IL-1β in the cornea of postnatal mice showed higher expression in some individual Twist2cre/cre pups (for TNF-α, 2 of 5 P1, 1 of 5 P5, and 3 of 5 P10 Twist2cre/cre animals had increased levels); however, mean levels did not differ between wild-type and mutant animals. P10 mutant pups with high cytokine levels had open or partially open eyelids, raising the possibility of secondary induction of cytokines as a result of corneal injury or infection. To avoid the confounding effects of infection and inflammation on the corneal phenotype, mice with premature eyelid opening were excluded from further analyses. 
Corneal Thinning in Twist2cre/cre Mice
The corneas of adult Twist2cre/cre mice had normal gross morphology and remained transparent. However, histologic examination revealed significant corneal thinning in Twist2cre/cre adult and neonatal eyes (Figs. 3B, 3C). The mean central corneal thickness of adult Twist2cre/cre and Twist2 cre/+ mice was 61% and 81% that of the Twist2 +/+ cornea, respectively (Fig. 4A). PAS staining of Bowman's membrane was patchy in Twist2cre/cre adult mice, suggestive of breakages (Fig. 3D). This, combined with the central corneal thinning, may be indicative of keratoconus. Patients with keratoconus reportedly have corneal epithelial defects, although the presentation is variable. 3,37,38 However, there was no statistically significant difference in the thickness or the population of epithelial cells between Twist2 +/+ and Twist2cre/cre mice (Fig. 4A). The thinning was due primarily to the reduced thickness of the corneal stroma (Fig. 4A). 
Figure 4.
 
Corneal thinning in Twist2cre/cre mice because of a reduction in the thickness of the corneal stroma. (A) Adult Twist2cre/cre (−/−) mice have significantly reduced corneal thickness compared with Twist2 +/+ (+/+) animals (two-sample test assuming unequal variance; n = 7 per genotype, P = 0.000127) because of a reduction in stromal thickness (n = 7 per genotype, P = 0.000131) rather than epithelial thickness (n = 7 per genotype, P = 0.224433). Adult Twist2cre/cre mice have significantly fewer corneal stromal cells (n = 3 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.001653), whereas average stromal cell densities (15.7 cells/cm2 for Twist2 +/+, 18.4 cells/cm2 for Twist2cre/cre ; n = 6 each; P = 0.337201) and epithelial cell numbers (n = 7 for Twist2 +/+, n = 7 for Twist2cre/cre ; P = 0.252962) were not significantly different. (B) Keratocan expression, as determined by qRT-PCR, was unchanged in the corneas of adult Twist2cre/cre mice (n = 5 Twist2 +/+, n = 4 Twist2cre/cre , P = 0.445). (C) Corneal thickness plotted against age for Twist2cre/cre and Twist2 +/+ pups. No difference could be detected at P0. Corneal thinning in Twist2cre/cre animals became apparent by P2 but did not reach statistical significance until adulthood. P0: n = 4 Twist2 +/+, n = 5 Twist2cre/cre ; P1: n = 7 Twist2 +/+, n = 6 Twist2cre/cre ; P2: n = 4 Twist2 +/+, n = 3 Twist2cre/cre ; P5: n = 1 Twist2 +/+, n = 3 Twist2cre/cre ; P10: n = 4 Twist2 +/+, n = 2 Twist2cre/cre. Asterisk: statistically significant differences.
Figure 4.
 
Corneal thinning in Twist2cre/cre mice because of a reduction in the thickness of the corneal stroma. (A) Adult Twist2cre/cre (−/−) mice have significantly reduced corneal thickness compared with Twist2 +/+ (+/+) animals (two-sample test assuming unequal variance; n = 7 per genotype, P = 0.000127) because of a reduction in stromal thickness (n = 7 per genotype, P = 0.000131) rather than epithelial thickness (n = 7 per genotype, P = 0.224433). Adult Twist2cre/cre mice have significantly fewer corneal stromal cells (n = 3 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.001653), whereas average stromal cell densities (15.7 cells/cm2 for Twist2 +/+, 18.4 cells/cm2 for Twist2cre/cre ; n = 6 each; P = 0.337201) and epithelial cell numbers (n = 7 for Twist2 +/+, n = 7 for Twist2cre/cre ; P = 0.252962) were not significantly different. (B) Keratocan expression, as determined by qRT-PCR, was unchanged in the corneas of adult Twist2cre/cre mice (n = 5 Twist2 +/+, n = 4 Twist2cre/cre , P = 0.445). (C) Corneal thickness plotted against age for Twist2cre/cre and Twist2 +/+ pups. No difference could be detected at P0. Corneal thinning in Twist2cre/cre animals became apparent by P2 but did not reach statistical significance until adulthood. P0: n = 4 Twist2 +/+, n = 5 Twist2cre/cre ; P1: n = 7 Twist2 +/+, n = 6 Twist2cre/cre ; P2: n = 4 Twist2 +/+, n = 3 Twist2cre/cre ; P5: n = 1 Twist2 +/+, n = 3 Twist2cre/cre ; P10: n = 4 Twist2 +/+, n = 2 Twist2cre/cre. Asterisk: statistically significant differences.
The corneal stroma is composed of keratocytes and the extracellular matrix they produce. There was a significant reduction in the total number of stromal cells in the Twist2cre/cre mice (Fig. 4A), but cell density was not significantly different between Twist2 +/+ and Twist2cre/cre animals (Fig. 4A). Expression of keratocan, a major component of the corneal extracellular matrix, also did not differ between Twist2 +/+ and Twist2cre/cre mice (Fig. 4B). These results suggest that the corneal thinning in the mutant mice resulted from a paucity of keratocytes in the corneal stroma rather than a deficiency of extracellular matrix. 
Morphometric analysis revealed that the thickness of the cornea in Twist2cre/cre pups was similar to that of their Twist2 +/+ counterparts until P2 (Figs. 3C, 4C). After this time, the cornea rapidly thickened in the Twist2 +/+ mice, but the growth of the cornea in the Twist2cre/cre pups became stagnant (Fig. 4C). After P2, the Twist2cre/cre cornea was consistently thinner than the wild-type cornea (Figs. 3B, 3D, 4C). Corneal thickness did not change significantly with age in either wild-type or homozygous mutant adult Twist2 mice over an age range of 6 weeks to 10 months (regression analysis: r 2 = 0.065579, P = 0.579371705, n = 7 for Twist2 +/+; r 2 = 0.003302, P = 0.902602512, n = 7 for Twist2cre/cre ). 
Corneal Thinning in Mutant Pups Caused by Decreased Stromal Cell Proliferation
A previous study of Twist2cre/cre mice indicated that apoptosis may contribute to dermal thinning and growth retardation. 25 To assess cell death in the corneal stroma, we examined the expression of cleaved caspase 3 32 in P2, P10, and adult mice (P2: both eyes from four Twist2 +/+ and three Twist2cre/cre ; P10: both eyes from one Twist2 +/+, one Twist2 +/−, and one Twist2cre/cre ; middle-aged: one eye from each of three Twist2 +/+, two Twist2cre/cre ; elderly: one eye from each of two Twist2 +/+, two Twist2 +/−, and two Twist2cre/cre ). Apoptosis was rarely found in the Twist2 +/+ cornea. Similarly, apoptosis was rarely found in mutant corneas, with no significant increase in apoptotic cells observed in the Twist2cre/cre corneas of P2, P10, or adult mice (data not shown). Examination of E14.5 embryos also showed no sign of elevated apoptosis in the corneal primordium of mutant embryos (both eyes from four Twist2 +/+and five Twist2cre/cre ). Cellular loss by apoptosis was therefore unlikely to be the primary cause of the corneal thinning. 
To determine whether there was decreased cell proliferation in Twist2cre/cre mice, we examined the prevalence of Ki67+ cells in the corneal stroma. 39 Ki67 specifically marks cells that are within all stages of the active cell cycle but does not label cells in G0. 40 Ki67+ cells were sparse in the corneal stroma of Twist2+/+ and Twist2cre/cre mice at P2, P10, and adult ages (animal numbers as for apoptosis analysis). In contrast, many corneal endothelial cells stained positively for Ki67, consistent with the proposal that these cells are arrested in G1. 17  
Corneal epithelial stem cells are reputed to reside in the limbal region of the cornea, at the border between the cornea and the sclera. 41 44 The limbal population may also contain stem cells for the stromal keratocytes. 45 Limbal sections encompassing the outer edge of the iris (before the appearance of the lens) and the ciliary body were, therefore, examined at P2, P10, and adult ages in Twist2 +/+ and Twist2cre/cre animals (animal numbers as for apoptosis analysis). In adult mice, proliferating cells were present in the basal epithelium of the limbal region, presumed to be the progenitor cells for the constantly renewing corneal epithelium (data not shown). 
A significant reduction in Ki67+ (proliferating) cells was found in the central stromal corneal region in the Twist2cre/cre E14.5 embryos (Figs. 5A, 5B). There was also a trend toward reduced proliferation in the peripheral stromal cornea, but this did not reach statistical significance. Stromal cell density and the total number of stromal cells in the corneas of these mutant embryos were similar to those in the Twist2 +/+ embryos (n = 4 Twist2 +/+, n = 5 Twist2cre/cre ; cell density central, P = 0.543589; peripheral, 0.6966; total stromal cell number central, P = 0.098251; peripheral, P = 0.270678). No significant differences were observed in the periocular mesenchyme (Fig. 5B; n = 3 per genotype; mesenchyme cell density, P = 0.251876). More Ki76+ cells were detected in the peripheral than in the central stromal cornea in both mutant and wild-type embryos, but this difference was more marked in the mutant embryos (Twist2 +/+, 1.5:1, with 30% of peripheral and 21% of central cells Ki67+; Twist2cre/cre , 2.4:1, with 26% peripheral and 11% central). This suggests that the proliferation of presumptive stromal cells decreased as the cells moved toward the central cornea, and this decrease was greater for the Twist2cre/cre cells. In the Twist2 +/+ P2 pups, a small number of Ki67+ stromal cells were found in the corneal limbal region, and there were even fewer positive cells in the Twist2cre/cre limbus (Fig. 5C). In contrast, Ki67+ proliferating cells were rare in the adult limbal stroma. Together these findings indicate that there was reduced proliferation of the corneal stromal cells in the Twist2cre/cre embryos, which might have led to a reduced pool of keratocyte progenitors in the postnatal cornea. 
Figure 5.
 
Reduced corneal stromal keratocyte proliferation in Twist2cre/cre . (A) Immunostaining for Ki67 (red) in E14.5 embryonic eyes to detect proliferating cells. Sections were counterstained with DAPI (green). Enlargements of the central cornea are shown on the right. Examples of the area measurement tool used to calculate cell density are shown in the enlargements from the central cornea. Ki67 was expressed in the actively proliferating retinal progenitor cells and lens epithelium, and these served as internal positive controls. (B) Graphs of stromal cell counts from Ki67-stained Twist2cre/cre (−/−) E14.5 embryos. The proportion of cells expressing Ki67 (proliferating) in the central cornea of Twist2cre/cre E14.5 embryos was significantly reduced (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.00097). There was also a trend toward reduced expression of Ki67 in the peripheral cornea, but this did not reach statistical significance (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.091157). No significant difference in the proportion of Ki67+ cells could be detected in the periocular mesenchyme at E14.5 (n = 3 Twist2 +/+, n = 3 Twist2cre/cre , P = 0.164655). Graphs show mean ± SD. (C) Immunostaining for Ki67 (red) in P2 corneal limbus. Quantification of Ki67+ staining cells is shown in the graph. Numbers of Ki67+ cells appeared to be reduced in Twist2cre/cre P2 mice (n = 3 Twist2 +/+, n = 3 Twist2cre/cre ), but this could not be analyzed statistically because of the low numbers of positively staining cells. Scale bar, 100 μm. Asterisk: statistically significant results.
Figure 5.
 
Reduced corneal stromal keratocyte proliferation in Twist2cre/cre . (A) Immunostaining for Ki67 (red) in E14.5 embryonic eyes to detect proliferating cells. Sections were counterstained with DAPI (green). Enlargements of the central cornea are shown on the right. Examples of the area measurement tool used to calculate cell density are shown in the enlargements from the central cornea. Ki67 was expressed in the actively proliferating retinal progenitor cells and lens epithelium, and these served as internal positive controls. (B) Graphs of stromal cell counts from Ki67-stained Twist2cre/cre (−/−) E14.5 embryos. The proportion of cells expressing Ki67 (proliferating) in the central cornea of Twist2cre/cre E14.5 embryos was significantly reduced (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.00097). There was also a trend toward reduced expression of Ki67 in the peripheral cornea, but this did not reach statistical significance (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.091157). No significant difference in the proportion of Ki67+ cells could be detected in the periocular mesenchyme at E14.5 (n = 3 Twist2 +/+, n = 3 Twist2cre/cre , P = 0.164655). Graphs show mean ± SD. (C) Immunostaining for Ki67 (red) in P2 corneal limbus. Quantification of Ki67+ staining cells is shown in the graph. Numbers of Ki67+ cells appeared to be reduced in Twist2cre/cre P2 mice (n = 3 Twist2 +/+, n = 3 Twist2cre/cre ), but this could not be analyzed statistically because of the low numbers of positively staining cells. Scale bar, 100 μm. Asterisk: statistically significant results.
Twist2cre/cre Mice Do Not Develop Glaucoma
Central corneal thinning has been associated with and is a predictor of the development of glaucoma. 4,6,7,46 48 We therefore tested whether the central corneal thinning observed in the Twist2cre/cre mice was associated with the development of glaucoma. 
In mouse models of glaucoma and ganglion cell damage, ganglion cell death is preceded by a reduction in the expression of the two ganglion cell markers, Thy-1 and Nefl. 49 51 However, in Twist2cre/cre mice, the expression of Thy-1 (single-factor ANOVA; P5: n = 5 per genotype, P = 0.420954; P10: n = 5 Twist2 +/+, n = 4 Twist2 +/−, n = 3 Twist2 −/−, P = 0.975147; elderly: n = 10 Twist2 +/+, n = 12 Twist2 +/−, n = 3 Twist2 −/−, P = 0.51983) and Nefl (single-factor ANOVA; P5: n = 5 per genotype, P = 0.108283) was not different from that for Twist2 +/+ mice. Expression of rhodopsin, a photoreceptor cell marker, was also unchanged between wild-type and mutant animals at P5 (t-test with unequal variance; n = 5 per genotype, P = 0.849533), P10 (single-factor ANOVA; n = 5 Twist2 +/+, n = 5 Twist2 +/−, n = 3 Twist2 −/−, P = 0.486532), and elderly mice (single-factor ANOVA; n = 10 Twist2 +/+, n = 5 Twist2 +/−, n = 3 Twist2 −/−, P = 0.790197). 
Cells grown from human glaucomatous iridocorneal angle samples are reported to have increased expression of ELAM-1 (Sele), which has been suggested as a potential marker for the development of glaucoma. 52 We therefore examined Sele expression in iridocorneal angle samples from our Twist2 mice. However, we did not find any change in the expression of Sele among elderly Twist2 animals (ANOVA; n = 9 Twist2 +/+, n = 9 Twist2 +/−, n = 3 Twist2 −/−, P = 0.932666). 
A hallmark of glaucoma is the death of retinal ganglion cells, which we investigated by cleaved caspase3 immunostaining. 53,54 Apoptotic cells were rare in the retinas of 6- to 9-month-old (middle-aged) animals, and no ganglion cell death could be detected. Apoptotic cells could be detected in the retinas of elderly Twist2 +/+ and Twist2cre/cre mice, but there was no increase in the number of apoptotic ganglion cells in mutant animals. Morphometric analyses also found no difference between Twist2 +/+ and Twist2cre/cre adult mice in retinal thickness, optic nerve width, or ganglion cell number (two sample t-test assuming unequal variance; n = 7 per genotype, P = 0.900535, P = 0.074344, and P = 0.117874, respectively). These results suggest that corneal thinning in Twist2cre/cre mutant mice is not associated with the development of glaucoma. 
Discussion
The present study is the first evaluation of the role of Twist2 in eye morphogenesis. Our results show that Twist2 is expressed in the developing eye and that loss of its function perturbs eye development, resulting in corneal thinning, early eyelid opening, enophthalmia, and blepharophimosis. Twist2 activity is present in the embryonic corneal stromal/endothelial cells and the periocular mesenchyme from which they may arise, 11,17 and in vivo tracing of cell fates demonstrated that the stromal and endothelial cells in the cornea are descendants of Twist2-expressing cells. The corneal thinning caused by loss of Twist2 function likely resulted from reduced proliferation of the stromal progenitor cells. 
Central corneal thinning is reported as a strong predictive factor for the development and progression of glaucoma in epidemiologic studies. 5,6,46,47,55 It has been postulated that genes that contribute to corneal thinning are functionally common with those causing glaucoma. Our study in middle-aged and elderly Twist2cre/cre animals did not reveal any manifestations of glaucoma, such as reduction of expression of ganglion cell markers or ganglion cell death. This indicates that Twist2-mediated activity in corneal stromal proliferation is not associated with glaucomatous pathology in this genetic mouse model. 
The most obvious eye phenotype in the Twist2 mutant mice is corneal thinning with no associated anterior segment anomalies. This points to a specific effect on corneal development, in contrast to other mutants in which there is a more global impact on anterior segment development. 56 60 The corneal thinning in the Twist2 mutants is presaged by decreased cell proliferation at E14.5 and P2. In mesenchymal cells, bone, and muscle, Twist2 functions to maintain progenitor cell phenotypes and proliferative potential, and reduced activity promotes cell differentiation. 20,22,27,28 Our results indicate that Twist2 may play a similar role in keratocyte development in the cornea. Periocular mesenchyme cells destined to become corneal stromal or endothelial cells express Twist2 before and during their migration to the cornea. When these cells reach their final position, they cease to express Twist2 and lose Ki67 expression, suggesting that they are exiting the cell cycle. This process may be expedited in the Twist2cre/cre embryos, with a more rapid reduction in the population of Ki67+ cells in the central cornea compared to the peripheral cornea. These results indicate that loss of Twist2 leads to early cessation of the proliferation of corneal stromal progenitors, resulting in fewer mature stromal keratocytes for the production of the corneal matrix leading to the ensuing corneal thinning. 
Control of keratocyte proliferation is critical in disorders of corneal thinning and in the recovery of function after corneal wounding and LASIK refractive surgery. This study reveals Twist2 and its downstream targets as factors for investigation in the control of these processes. 
Footnotes
 Supported by the Ophthalmic Research Institute of Australia, the Rebecca Cooper Medical Research Foundation, and a CJ Martin Postdoctoral Fellowship (LW) and a Senior Principal Research Fellowship (PPLT) from the National Health and Medical Research Council of Australia.
Footnotes
 Disclosure: L. Weaving, None; M. Mihelec, None; R. Storen, None; D. Sosic, None; J.R. Grigg, None; P.P.L. Tam, None; R.V. Jamieson, None
The authors thank Craig Smith (Washington University, St. Louis, MO) for providing the Twist2Cre mice, Eric Olson for the Twist2 riboprobe plasmid, and Irma Villaflor, Mehtap Baserdem, and the staff of the Bioservices Unit (CMRI) for assistance with animal care. 
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Figure 1.
 
Expression of Twist2 in the eye. (A) Twist2 expression as determined by RT-PCR in various embryonic tissues and in the corneas of newborn and adult wild-type mice. (B) Twist2 expression revealed by in situ hybridization in wild-type embryos. At E10.5, expression could be detected in the developing corneal stroma (arrowheads) and nearby periocular mesenchyme (arrows). At E12.5, Twist2 was strongly expressed in the corneal stroma (arrowhead, inset), periocular mesenchyme, and dermis of the developing eyelids. Expression was absent from the corneal epithelium (arrow, inset). A similar pattern was observed at E14.5. By E16.5, Twist2 expression was not detected in the periocular mesenchyme (arrows) but was present in the eyelid dermis (arrowhead) and corneal stromal cells (arrowhead, inset). (C) In vivo fate mapping using Twist2cre/+;Z/AP/+ reporter mice. AP staining could be detected in cells that expressed Twist2 during development. Cells that expressed Twist2 were found in the periocular mesenchyme (arrowheads) and the developing corneal stroma (arrows) from E10.5 to E14.5. By E16.5, the Twist2-expressing cells were found in the corneal stroma (arrow) and the dermis of the overlying eyelid but no longer in the periocular mesenchyme (arrowheads). (D) AP staining of adult Twist2 cre/+;Z/AP/+ eyes confirmed that Twist2 expressing cells contributed to the corneal stroma and endothelium (arrow) but not the corneal epithelium (arrowhead). c, cornea; r, retina; lp, lens pit. Scale bar, 100 μm.
Figure 1.
 
Expression of Twist2 in the eye. (A) Twist2 expression as determined by RT-PCR in various embryonic tissues and in the corneas of newborn and adult wild-type mice. (B) Twist2 expression revealed by in situ hybridization in wild-type embryos. At E10.5, expression could be detected in the developing corneal stroma (arrowheads) and nearby periocular mesenchyme (arrows). At E12.5, Twist2 was strongly expressed in the corneal stroma (arrowhead, inset), periocular mesenchyme, and dermis of the developing eyelids. Expression was absent from the corneal epithelium (arrow, inset). A similar pattern was observed at E14.5. By E16.5, Twist2 expression was not detected in the periocular mesenchyme (arrows) but was present in the eyelid dermis (arrowhead) and corneal stromal cells (arrowhead, inset). (C) In vivo fate mapping using Twist2cre/+;Z/AP/+ reporter mice. AP staining could be detected in cells that expressed Twist2 during development. Cells that expressed Twist2 were found in the periocular mesenchyme (arrowheads) and the developing corneal stroma (arrows) from E10.5 to E14.5. By E16.5, the Twist2-expressing cells were found in the corneal stroma (arrow) and the dermis of the overlying eyelid but no longer in the periocular mesenchyme (arrowheads). (D) AP staining of adult Twist2 cre/+;Z/AP/+ eyes confirmed that Twist2 expressing cells contributed to the corneal stroma and endothelium (arrow) but not the corneal epithelium (arrowhead). c, cornea; r, retina; lp, lens pit. Scale bar, 100 μm.
Figure 2.
 
Enophthalmia and blepharophimosis in Twist2cre/cre animals. (A, B) Twist2cre/cre (−/−) adult male (left) and normal heterozygous littermate (right). Enlarged views of the eyes are shown on the right. (B, C) Note the enophthalmic (sunken) eyes. Measurement of epicanthal length in adult Twist2 +/+ and Twist2cre/cre mice. Epicanthal length of adult Twist2cre mice was measured using calipers (inset). Twist2cre/cre animals had significantly smaller eye apertures (two-sample t-test assuming unequal variance: n = 21, Twist2 +/+; n = 8, Twist2cre/cre ; P = 0.00013). In contrast, measurements of eyeball length and width did not detect any significant difference (n = 6, Twist2 +/+; n = 4, Twist2cre/cre ; eyeball length, P = 0.713581; eyeball width, P = 0.710512). This suggests that loss of Twist2 function leads to blepharophimosis and enophthalmia rather than true microphthalmia (small eyes). Asterisk: statistically significant result.
Figure 2.
 
Enophthalmia and blepharophimosis in Twist2cre/cre animals. (A, B) Twist2cre/cre (−/−) adult male (left) and normal heterozygous littermate (right). Enlarged views of the eyes are shown on the right. (B, C) Note the enophthalmic (sunken) eyes. Measurement of epicanthal length in adult Twist2 +/+ and Twist2cre/cre mice. Epicanthal length of adult Twist2cre mice was measured using calipers (inset). Twist2cre/cre animals had significantly smaller eye apertures (two-sample t-test assuming unequal variance: n = 21, Twist2 +/+; n = 8, Twist2cre/cre ; P = 0.00013). In contrast, measurements of eyeball length and width did not detect any significant difference (n = 6, Twist2 +/+; n = 4, Twist2cre/cre ; eyeball length, P = 0.713581; eyeball width, P = 0.710512). This suggests that loss of Twist2 function leads to blepharophimosis and enophthalmia rather than true microphthalmia (small eyes). Asterisk: statistically significant result.
Figure 3.
 
Corneal thinning in Twist2cre/cre mice. (A) Immunostaining of P2 limbal sections for Ki67+ (red) proliferating cells. Sections are counterstained with DAPI (green). Twist2cre/cre pups have markedly thinner eyelid tissue (see also C) and fewer hair follicles (arrows). The hair follicles of Twist2cre/cre pups appeared to have reduced expression of Ki67 (arrows). Strong staining for Ki67 could be detected in the actively proliferating retinal progenitor cells, which serve as an internal positive control. C, ciliary body; con, conjunctiva. (B) Comparison of corneal thickness in adult Twist2 +/+ (+/+) and Twist2cre/cre (−/−) mice. Twist2cre/cre mice have visibly thinner corneal stroma (indicated by the parentheses). (C) Twist2cre/cre P2 pups have visible thinning of the cornea. The dermis of the overlying eyelids is also thin with reduced numbers of hair follicles. (D) PAS staining of adult Twist2 +/+ and Twist2cre/cre cornea. Arrow: Bowman's membrane. Scale bar, 100 μm, unless otherwise specified.
Figure 3.
 
Corneal thinning in Twist2cre/cre mice. (A) Immunostaining of P2 limbal sections for Ki67+ (red) proliferating cells. Sections are counterstained with DAPI (green). Twist2cre/cre pups have markedly thinner eyelid tissue (see also C) and fewer hair follicles (arrows). The hair follicles of Twist2cre/cre pups appeared to have reduced expression of Ki67 (arrows). Strong staining for Ki67 could be detected in the actively proliferating retinal progenitor cells, which serve as an internal positive control. C, ciliary body; con, conjunctiva. (B) Comparison of corneal thickness in adult Twist2 +/+ (+/+) and Twist2cre/cre (−/−) mice. Twist2cre/cre mice have visibly thinner corneal stroma (indicated by the parentheses). (C) Twist2cre/cre P2 pups have visible thinning of the cornea. The dermis of the overlying eyelids is also thin with reduced numbers of hair follicles. (D) PAS staining of adult Twist2 +/+ and Twist2cre/cre cornea. Arrow: Bowman's membrane. Scale bar, 100 μm, unless otherwise specified.
Figure 4.
 
Corneal thinning in Twist2cre/cre mice because of a reduction in the thickness of the corneal stroma. (A) Adult Twist2cre/cre (−/−) mice have significantly reduced corneal thickness compared with Twist2 +/+ (+/+) animals (two-sample test assuming unequal variance; n = 7 per genotype, P = 0.000127) because of a reduction in stromal thickness (n = 7 per genotype, P = 0.000131) rather than epithelial thickness (n = 7 per genotype, P = 0.224433). Adult Twist2cre/cre mice have significantly fewer corneal stromal cells (n = 3 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.001653), whereas average stromal cell densities (15.7 cells/cm2 for Twist2 +/+, 18.4 cells/cm2 for Twist2cre/cre ; n = 6 each; P = 0.337201) and epithelial cell numbers (n = 7 for Twist2 +/+, n = 7 for Twist2cre/cre ; P = 0.252962) were not significantly different. (B) Keratocan expression, as determined by qRT-PCR, was unchanged in the corneas of adult Twist2cre/cre mice (n = 5 Twist2 +/+, n = 4 Twist2cre/cre , P = 0.445). (C) Corneal thickness plotted against age for Twist2cre/cre and Twist2 +/+ pups. No difference could be detected at P0. Corneal thinning in Twist2cre/cre animals became apparent by P2 but did not reach statistical significance until adulthood. P0: n = 4 Twist2 +/+, n = 5 Twist2cre/cre ; P1: n = 7 Twist2 +/+, n = 6 Twist2cre/cre ; P2: n = 4 Twist2 +/+, n = 3 Twist2cre/cre ; P5: n = 1 Twist2 +/+, n = 3 Twist2cre/cre ; P10: n = 4 Twist2 +/+, n = 2 Twist2cre/cre. Asterisk: statistically significant differences.
Figure 4.
 
Corneal thinning in Twist2cre/cre mice because of a reduction in the thickness of the corneal stroma. (A) Adult Twist2cre/cre (−/−) mice have significantly reduced corneal thickness compared with Twist2 +/+ (+/+) animals (two-sample test assuming unequal variance; n = 7 per genotype, P = 0.000127) because of a reduction in stromal thickness (n = 7 per genotype, P = 0.000131) rather than epithelial thickness (n = 7 per genotype, P = 0.224433). Adult Twist2cre/cre mice have significantly fewer corneal stromal cells (n = 3 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.001653), whereas average stromal cell densities (15.7 cells/cm2 for Twist2 +/+, 18.4 cells/cm2 for Twist2cre/cre ; n = 6 each; P = 0.337201) and epithelial cell numbers (n = 7 for Twist2 +/+, n = 7 for Twist2cre/cre ; P = 0.252962) were not significantly different. (B) Keratocan expression, as determined by qRT-PCR, was unchanged in the corneas of adult Twist2cre/cre mice (n = 5 Twist2 +/+, n = 4 Twist2cre/cre , P = 0.445). (C) Corneal thickness plotted against age for Twist2cre/cre and Twist2 +/+ pups. No difference could be detected at P0. Corneal thinning in Twist2cre/cre animals became apparent by P2 but did not reach statistical significance until adulthood. P0: n = 4 Twist2 +/+, n = 5 Twist2cre/cre ; P1: n = 7 Twist2 +/+, n = 6 Twist2cre/cre ; P2: n = 4 Twist2 +/+, n = 3 Twist2cre/cre ; P5: n = 1 Twist2 +/+, n = 3 Twist2cre/cre ; P10: n = 4 Twist2 +/+, n = 2 Twist2cre/cre. Asterisk: statistically significant differences.
Figure 5.
 
Reduced corneal stromal keratocyte proliferation in Twist2cre/cre . (A) Immunostaining for Ki67 (red) in E14.5 embryonic eyes to detect proliferating cells. Sections were counterstained with DAPI (green). Enlargements of the central cornea are shown on the right. Examples of the area measurement tool used to calculate cell density are shown in the enlargements from the central cornea. Ki67 was expressed in the actively proliferating retinal progenitor cells and lens epithelium, and these served as internal positive controls. (B) Graphs of stromal cell counts from Ki67-stained Twist2cre/cre (−/−) E14.5 embryos. The proportion of cells expressing Ki67 (proliferating) in the central cornea of Twist2cre/cre E14.5 embryos was significantly reduced (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.00097). There was also a trend toward reduced expression of Ki67 in the peripheral cornea, but this did not reach statistical significance (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.091157). No significant difference in the proportion of Ki67+ cells could be detected in the periocular mesenchyme at E14.5 (n = 3 Twist2 +/+, n = 3 Twist2cre/cre , P = 0.164655). Graphs show mean ± SD. (C) Immunostaining for Ki67 (red) in P2 corneal limbus. Quantification of Ki67+ staining cells is shown in the graph. Numbers of Ki67+ cells appeared to be reduced in Twist2cre/cre P2 mice (n = 3 Twist2 +/+, n = 3 Twist2cre/cre ), but this could not be analyzed statistically because of the low numbers of positively staining cells. Scale bar, 100 μm. Asterisk: statistically significant results.
Figure 5.
 
Reduced corneal stromal keratocyte proliferation in Twist2cre/cre . (A) Immunostaining for Ki67 (red) in E14.5 embryonic eyes to detect proliferating cells. Sections were counterstained with DAPI (green). Enlargements of the central cornea are shown on the right. Examples of the area measurement tool used to calculate cell density are shown in the enlargements from the central cornea. Ki67 was expressed in the actively proliferating retinal progenitor cells and lens epithelium, and these served as internal positive controls. (B) Graphs of stromal cell counts from Ki67-stained Twist2cre/cre (−/−) E14.5 embryos. The proportion of cells expressing Ki67 (proliferating) in the central cornea of Twist2cre/cre E14.5 embryos was significantly reduced (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.00097). There was also a trend toward reduced expression of Ki67 in the peripheral cornea, but this did not reach statistical significance (n = 4 Twist2 +/+, n = 5 Twist2cre/cre , P = 0.091157). No significant difference in the proportion of Ki67+ cells could be detected in the periocular mesenchyme at E14.5 (n = 3 Twist2 +/+, n = 3 Twist2cre/cre , P = 0.164655). Graphs show mean ± SD. (C) Immunostaining for Ki67 (red) in P2 corneal limbus. Quantification of Ki67+ staining cells is shown in the graph. Numbers of Ki67+ cells appeared to be reduced in Twist2cre/cre P2 mice (n = 3 Twist2 +/+, n = 3 Twist2cre/cre ), but this could not be analyzed statistically because of the low numbers of positively staining cells. Scale bar, 100 μm. Asterisk: statistically significant results.
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