Loss-of-function mutations in FREM2 disrupt eye morphogenesis

Experimental Eye Research 181 (2019) 302–312

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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer

Loss-of-function mutations in FREM2 disrupt eye morphogenesis a,1

a,1

a

a,b

a,b

T a

Xiayin Zhang , Dongni Wang , Meimei Dongye , Yi Zhu , Chuan Chen , Ruixin Wang , Erping Longa, Zhenzhen Liua, Xiaohang Wua, Duoru Lina, Jingjing Chena, Zhuoling Lina, Jinghui Wanga, Wangting Lia, Yang Lic, Dongmei Lic, Haotian Lina,∗ a

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA c Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology and Visual Science, Beijing, 100730, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Isolated cryptophthalmos FREM2 mutation Development of eyelids Ocular abnormalities Genotype–phenotype correlation

Cryptophthalmos is a rare congenital disorder characterized by ocular dysplasia with eyelid malformation. Complete cryptophthalmos is characterized by the presence of continuous skin from the forehead over the eyes and onto the cheek, along with complete fusion of the eyelids. In the present study, we characterized the clinical manifestations of three patients with isolated bilateral cryptophthalmos. These patients shared the same c.6499C > T missense mutation in the FRAS1-related extracellular matrix protein 2 (FREM2) gene, while each individual presented an additional nonsense mutation in the same gene (Patient #1, c.2206C > T; Patient #2, c.5309G > A; and Patient #3, c.4063C > T). Then, we used CRISPR/Cas9 to generate mice carrying Frem2R725X/R2156W compound heterozygous mutations, and showed that these mice recapitulated the human isolated cryptophthalmos phenotype. We detected FREM2 expression in the outer plexiform layer of the retina for the first time in the cryptophthalmic eyes, and the levels were comparable to the wild-type mice. Moreover, a set of different expressed genes that may contribute secondarily to the phenotypes were identified by performing RNA sequencing (RNA-seq) of the fetal Frem2 mutant mice. Our findings extend the spectrum of FREM2 mutations, and provide insights into opportunities for the prenatal diagnosis of isolated cryptophthalmos. Furthermore, our work highlights the importance of the FREM2 protein during the development of eyelids and the anterior segment of the eyeballs, establishes a suitable animal model for studying epithelial reopening during eyelid development and serves as a valuable reference for further mechanistic studies of the pathogenesis of isolated cryptophthalmos.

1. Introduction Cryptophthalmos ([MIM: 123570]) is a rare congenital ocular dysplasia, the prevalence of which has generally been estimated to be 3 per 100,000 individuals in the population and was not varying significantly between different ethnicities (Morrison, 2002; Shaw et al., 2005; Leck, 1994; Källén et al., 1996). It is primarily reported as a component of Fraser syndrome (FS [MIM: 219000]), an autosomal recessive disorder accompanied by syndactyly and anomalies of the respiratory and urogenital tracts (Tessier et al., 2016). Three categories of FS have been identified: complete (total), incomplete (partial), and abortive or congenital symblepharon (Subramanian et al., 2013). Complete (total)

cryptophthalmos is characterized by the presence of continuous skin from the forehead over the eyes and onto the cheek, along with complete fusion of the eyelids (François, 1969). Only 15 cases of complete isolated bilateral cryptophthalmos have been documented (Egier et al., 2005), and these patients have an almost normal life expectancy without cognitive impairments (Smyth and Scambler, 2005). Since postnatal surgical management is technically challenging, a precise prenatal diagnosis is strategically important (Raymond et al., 2010). The vertebrate eye is a complex structure and its development involves highly organized and complex cascades of multiple transcription factors and signals (Chaerkady et al., 2013). In vertebrates, the development of the eyelids originates from the surface ectoderm and the

Abbreviations: FREM2, FRAS1-related extracellular matrix protein 2; FS, Fraser syndrome; CSPG, Chondroitin sulfate proteoglycan; MRI, Magnetic resonance imaging; CT, Computed tomography; H&E, Hematoxylin-eosin; PCR, Polymerase chain reaction; WT, Wild-type; OPL, Outer plexiform layer; PolyPhen, Polymorphism phenotyping; SIFT, Sorting intolerant from tolerant; RNA-seq, RNA Sequencing; DEGs, Differentially expressed genes ∗ Corresponding author. Xian Lie South Road 54#, Guangzhou, 510060, China. E-mail address: [email protected] (H. Lin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.exer.2019.02.013 Received 23 October 2018; Received in revised form 30 January 2019; Accepted 17 February 2019 Available online 22 February 2019 0014-4835/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Probands showing bilateral complete cryptophthalmos. (A–C) Probands from families 1–3 showing bilateral complete cryptophthalmos. (D) Ocular ultrasonography revealed bilateral gourd-shaped cysts (white arrow) without a clear internal structure. (E and F) The brain MRI from Patient #1 showed normal morphologic brain development. The black arrow denotes the developing visual cortex, the white arrow denotes extraocular muscles in the narrow orbital fat gap, and the white arrowhead denotes optic nerve enlargement in the expanded orbits.

due to the lack of suitable animal models. In this study, we present clinical findings from three patients from three unrelated consanguineous families with isolated bilateral complete cryptophthalmos at birth, and all patients shared a novel missense mutation, c.6499C > T (p.R2167W, dbSNP: rs114837786) in exon 9 of FREM2. To the best of our knowledge, this study is the first to report a loss-of-function mutation in individuals with isolated bilateral complete cryptophthalmos. Moreover, we established a mouse model that recapitulates the human complete cryptophthalmos phenotype by CRISPR-Cas9. Ocular pathological phenotypes similar to those of our patients were identified in the Frem2 mutant mice. In addition, we observed FREM2 expression in the eyes of adult mice for the first time. RNA Sequencing (RNA-seq) data identified a set of differentially expressed genes that may contribute secondarily to the phenotypes. The findings identify new targets for the clinical intervention of isolated bilateral cryptophthalmos and underscore the importance of FREM2 during the development of eyelids and the anterior segment of the eyeballs.

secondary mesenchyme (Barishak, 1992), while the development of the eyeballs requires crosstalk between the optic vesicle originating from invagination of the diencephalon, the surface ectoderm of the head, and the ocular mesenchyme near their point of contact (Zieske, 2004). Eyelid development has been divided into several distinct phases, namely eyelid formation, fusion, development, separation, and maturation (Barishak, 1992). After embryonic eyelid fusion, the cornea matures and the ocular auxiliary tissues start to form (Ohuchi, 2012). The morphogenesis of the eye, particularly the eyelid, is a dynamic process involving intraepithelial and epithelial-mesenchymal interactions between the epidermis and dermis (Barishak, 1992). FRAS1-related extracellular matrix protein 2 (FREM2) is a member of the Fras1/Frem protein family that directly interacts with constituents of connective tissue through their chondroitin sulfate proteoglycan motifs, contributing to epithelial–mesenchymal coupling (Pavlakis et al., 2011). The FREM2 protein comprises a signal peptide on the N-terminus, a region homologous to the chondroitin sulfate proteoglycan (CSPG) motifs of the NG2 protein, 5 Calx-β motifs (Calxβ), and a transmembrane region (Fig. 2B). Previous studies of patients with FS and mouse bleb mutants have revealed that FREM2 is produced by epidermal cells and deposited at the basement membrane zone (Kiyozumi et al., 2006). The FRAS1/FREM protein complex is localized in most epithelial basement membranes, including basement membranes of the epidermis, periophthalmic region, lung, neural tube and kidney (Kiyozumi et al., 2006). Studies in animals and humans have provided converging evidence that mutations in the FREM2 gene result in transient embryonic epidermal blistering, leading to the development of cryptophthalmos, syndactyly, and urogenital malformations (Jadeja et al., 2005; van Haelst et al., 2008). However, loss-of-function variants in FREM2 were only identified in patients with FS and different classes of mouse bleb mutants, but not in patients with isolated bilateral cryptophthalmos. Moreover, the regulatory role of FREM2 in epithelial reopening during eyelid development has been poorly characterized

2. Materials and methods 2.1. Patients This study was approved by the ethics committee of Zhongshan Ophthalmic Center, and adhered to the tenets of the Declaration of Helsinki. All family members provided informed consent before participation. Three unrelated patients from the southern area of China were recruited from 2012 to 2015. No teratogenic exposure was documented during pregnancy. A B-scan ultrasound (CineScan A/B, Quantel Medical) of all patients’ eyes was also performed. Ultrasound examinations of the heart, brain and kidney were performed on Patient #1 using M5 (Mindray). Patient #1 underwent a magnetic resonance imaging (MRI) examination using a whole-body 3.0 T scanner (Signal 303

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Fig. 2. Bilateral cryptophthalmos in infants is caused by mutations in FREM2. (A) Pedigrees and sequence chromatograms of FREM2 cDNA from independent families 1–3. The shared missense mutation, c.6499C > T (p.R2167W), was identified in exon 9 of the FREM2 gene. (B) The position of the mutations relative to the protein domains of FREM2. The structure of the FREM2 is shown schematically. (C) Results of the analysis of the evolutionary conservation of amino acids affected by the c.6499C > T (p.R2167W) missense mutation in FREM2. The mutant alleles are highlighted. Here, DNA refers to the coding sequence.

an intrafamilial cosegregation analysis and to determine the novelty of the variants. Validation of the identified c.6499C > T mutation in the FREM2 gene was performed using polymerase chain reaction (PCR) amplification with specific primer (FREM2-Forward, 5′-CCAAACAGGC AGAGCAAAAT-3′ and FREM2-Reverse, 5′-CAGTGTTTGGGCGTTCT TCA-3′). We performed homology modeling with SWISS-MODEL and determined the 3D protein structure using PyMOL software. Finally, we verified the novel variants by conducting an allele-specific PCR analysis in 100 healthy controls and family members. We have submitted the new variants to the Locus Specific Mutation Databases-Zhejiang University Center for Genetic and Genomic Medicine (LOVD 2.X) (http://www.genomed.org/lovd2/variants.php? action=search_unique&select_db=FREM2).

Excite). Other characteristics are shown in Table S1. 2.2. Whole exome sequencing and variant detection Four milliliters of venous blood were collected from the patients and their family members. Genomic DNA was extracted from peripheral blood using a QIAmp DNA Mini Blood Kit (Qiagen). The exome DNA was enriched using the SureSelect Human All Exon Kit (Agilent Technologies) and 100-bp paired-end sequencing was performed using the HiSeq2000 platform (Illumina). Bioinformatics were subsequently analyzed as described in our previous studies (Lin et al., 2018; Wu et al., 2017). The candidate variations were filtered against the National Center for Biotechnology Information. Variants shared by the patients but no other members of their families were selected. Polymorphism phenotyping (PolyPhen) and sorting intolerant from tolerant (SIFT) were used to predict the potential impacts of these variants. The Human Gene Mutation database was used to screen mutations reported in the published studies. In addition, HomoloGene was used to assess whether the mutated sites were conserved across different species. Sanger sequencing was used for

2.3. Animals All animals used in this study were Mus musculus, adult C57BL/6J mice (2–7 months old). All Frem2 mutant mice and WT C57BL/6J mice were handled in accordance with policies and procedures recommended by the Institutional Animal Care and Use Committee of Sun 304

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length, lens thickness, the boundaries between the lens and retina and the thickness of total retina were measured using AxioVision software (Carl Zeiss Inc.). The retinal thickness was measured at 200 μm intervals superior and inferior to the edge of the optic nerve head along the vertical meridian. All growth indices were measured three times, and the averages of all measurements were calculated.

Yat-sen University, and all procedures adhered to the ARVO Statement for the Use and Care of Animals in Ophthalmic and Vision Research. The mice were maintained on a 12-h: 12-h light-dark cycle with unlimited access to food and water. Frem2R725X/R2156W mice on a C57BL/6 background were obtained by hybridization using mouse strains carrying two different point mutations. Mice carrying the c.2173C > T (R725X) or c.6466A > T (R2156W) point mutation in the murine Frem2 gene were designed by Shanghai Model Organisms Center, Inc. (Shanghai, China) and generated using CRISPR/Cas9 technology. The sequence 5′-TCACTGTGTAC TGTAACTCT-3′ was selected as the Cas9-targeted single guide RNA (sgRNA), transcribed in vitro using the MEGAshortscriptKit (ThermoFisher, USA) and subsequently purified using the MEGAclearTM Kit to generate Frem2R725X heterozygous mice. The transcribed Cas9 mRNA and sgRNA and a 120-base pair single-stranded oligodeoxynucleotide (ssODN) were coinjected into zygotes of C57BL/ 6J mice. The genotypes of the F0 mice were validated using PCR and sequencing with the following primer pair: Frem2-F1, 5′-GCGGCTGGC CAGGTTGTGTA-3′ and Frem2-R1, 5′- GATGTGGTGGCCCTTCTCCT. CA-3'. The same procedure was applied as mentioned above to generate Frem2R2156W heterozygous mice, 5′-CTTCTGTGAGATGTTAC ACG-3′ was selected as the sgRNA. After coinjection, the genotypes of F0 mice were validated using PCR and sequencing with the primer pair: Frem2-F2, 5′-GAACATCTGAAAGCGGGTGACG-3′ and Frem2-R2, 5′-ATGTTGGACGCTTGTTGGAAAATC-3'. All the F0 mice carrying the expected point mutation were backcrossed four generations onto the C57BL/6J background. DNA extracted from tail tissues of these mice using QIAamp DNA Mini Kit (QIAGEN), amplified by PCR and confirmed by sequencing for genotyping (Fig. 4H and I). In vivo computed tomography (CT) and MRI were performed using a micro-CT system (LaTheta LCT-200) and a 7-T MRI system (Pharmascan 70/16, Bruker Biospin) (Tkatchenko et al., 2010). Prior to the examinations, the mice were anesthetized with 1% isoflurane. Gadopentetate dimeglumine (Magnevist, Berlex Laboratories) was used as the contrast agent.

2.5. Immunohistochemistry Paraffin sections were deparaffinized in xylene, rehydrated and incubated with 0.3% H2O2 for 30 min at room temperature. Antigen retrieval was performed in 0.01 M citrate buffer, pH 6, in a pressure cooker for 45 s. Then the sections were incubated with blocking solution (5% goat serum, 3% BSA, and 0.1% Triton X-100 in PBS) for 1 h at room temperature. After an overnight incubation with primary antibodies against FREM2 (sc-376555, F-1, mouse, Santa Cruz Biotechnology) at 4 °C (Takahashi et al., 2016), the sections were incubated with a peroxidase-conjugated goat anti-mouse IgG (Molecular Probes) secondary antibody at room temperature for 30 min. Portions of the sections were stained with DAPI to visualize the nuclei. Specimens were mounted using PermaFluor (Thermo) and visualized with an LSM510 laser confocal microscope (Zeiss). Specificity of the FREM2 antibody was recognized by western blots in Fig. S1. 2.6. Immunofluorescence staining Paraffin sections were deparaffinized, rehydrated and rinsed with PBS. After 3 washes with PBS, epitope unmasking was applied to reveal the antigenic determinants. Sections were washed with PBST 3 times, blocked with 5% goat serum/0.1% Triton X-100 for 1 h, and then incubated with primary antibodies against FREM2, E-cadherin (24E10, rabbit, Cell Signaling Technology) and collagen VII (PL0302263, rabbit, PLLABS) overnight. A secondary antibody conjugated with rhodamine (TRITC) or fluorescein isothiocyanate (FITC) was applied at a 1:1000 dilution for 2 h. After counterstaining with DAPI, sections were mounted and observed using a fluorescent microscope. The immunostaining intensity was measured using Image J software.

2.4. Sample preparation and histological analysis The mouse eyes and embryos were fixed with 4% paraformaldehyde, and subsequently embedded in paraffin for at least 24 h. Tissues were sectioned in a vertical pupillary optic nerve plane and stained with hematoxylin-eosin (H&E). Growth indices, including the axial

2.7. RNA-seq analysis and real-time PCR analysis We compared whole-embryonic transcriptome changes between Frem2R725X/R2156W mice and their wild-type (WT) littermates. Total Fig. 3. Superposition of modeled FREM2 protein structures. 3D models showing parts of superimposed FREM2 structures, the native (green indicates a the portion of the Nterminus of the mutant protein, and pink indicates a portion of the C-terminus of the mutant protein) and the mutant forms (red). (A) p.R2167W; (B) p.R736X; (C) p.W1770X; (D) p.R1355X. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 4. Phenotype and ocular examinations of Frem2R725X/R2156W mice. (A) The observed dysplastic eyes exhibited complete cryptophthalmos, without normal eyelids or auxiliary ocular tissues. (B) Images of the relatively normal eyes of the mice with unilateral cryptophthalmos. (C) The eye opened at 4 months of age, with a structurally disordered anterior segment. The black arrowhead denotes corneal neovascularization, the white arrowhead denotes complete iris synechia and the dotted circle shows corneal leukoma. (D) Four of seven of the animals also exhibited syndactyly. (E and F) MRI and CT scans of eyes from Frem2R725X/R2156W mice with unilateral cryptophthalmos. White arrowheads in E and F indicate cryptophthalmic eyes. (G) Separation of the cyst from the subcutaneous tissue, revealing gourd-shaped cysts in the eye socket. (H) The chromatogram and verification of the targeted Frem2 regions from Frem2R2156W heterozygous mice, (I) The chromatogram and verification of the targeted Frem2 regions from Frem2R725X heterozygous mice.

to the reference genome using HISAT2 v2.1.0. Read count for each gene were determined using HTSeq v0.6.0, and Fragments Per Kilobase Millon Mapped Reads were then calculated to estimate the expression levels. DESeq2 v1.6.3 analyzed differentially expressed genes (DEGs) and estimated the expression level of each gene per sample using a linear regression analysis; then, the P-value was calculated with the

RNA was extracted with TRIzol reagent (Invitrogen). The RNA-seq libraries were generated using Illumina TruSeq RNA Sample Preparation Kits. RNA-seq was performed using an Illumina HiSeq 2000 platform. The obtained sequence reads were trimmed and mapped to the mouse reference genome Mus_musculus GRCm38.89. Bowtie2 v2.2.3 software was used to build the genome index, and clean data were then aligned 306

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Wald test and corrected using the BH method. Genes with q ≤ 0.05 and |log2_ratio|≥1 were identified as DEGs. The Integrative Genomics Viewer was used to view the mapping results in heatmaps and scatter plots. GO enrichment analyses of the DEGs were performed using DAVID. GO terms with a Bonferroni corrected P-value of < 0.05 were considered significantly enriched functional annotations. Real-time quantitative PCR was performed on a 7900HT Real-time PCR system (Applied Biosystems) using real-time primers and TaqMan probes from Applied Biosystems. The first strand was reverse-transcribed using the Omniscript Reverse Transcription Kit (Qiagen) and random primers. Expression was normalized to Gapdh.

mutation in exon 9 of the FREM2 gene was identified in the three patients with the same causative haplotype. Other mutations were identified as follows: Patient #1, c.2206C > T (p.R736X) in exon 1; Patient #2, c.5309G > A (p.W1770X) in exon 3; and Patient #3, c.4063C > T (p.R1355X) in exon 1 (Fig. 2B). These nonsense mutations resulted in a truncated FREM2 protein which normally spans 3169 amino acids. No mutations were identified in FRAS1 and GRIP1 which have been reported in patients with FS (McGregor et al., 2003; Vogel et al., 2012) (Table S3), or genes related to ocular morphogenesis, such as SOX2, OTX2 and PAX6 (Hever et al., 2006; Chen et al., 2010; Lin et al., 2012). FREM2 is well conserved across species (Fig. 2C). The novel c.6499C > T (p.R2167W) missense mutation was predicted to affect function, with a score of 1.00 from PolyPhen and 0.01 from SIFT. The 3D structural homology modeling revealed that the c.6499C > T (p.R2167W) missense mutation resulted in a conformational change in the FREM2, in which a beta sheet became a random coil (Fig. 3).

2.8. Statistical analysis Statistical comparisons between two groups were evaluated using Student's t-test (SPSS ver. 19.0). Differences were considered significant at P < 0.05.

3.3. Phenotypes of Frem2 mutant mice generated using CRISPR-Cas9 technology

3. Results 3.1. Clinical manifestations of three patients with bilateral cryptophthalmos

Next, we established a mouse model with the compound heterozygous mutations c.2173C > T (p.R725X) and c.6466A > T (p.R2156W) using CRISPR-Cas9 technology; these mutations corresponded to the c.2206C > T (p.R736X) and c.6499C > T (p.R2167W) mutations in Patient #1, respectively. The targeted Frem2 regions were sequenced and the chromatograms from the targeted alleles are shown (Fig. 4H and I). All eleven Frem2R725X/R2156W mice displayed bilateral or unilateral congenital complete cryptophthalmos (Fig. 4A and B), and the dysplastic eyes did not have normal eyelids or ocular auxiliary tissues. In addition, the cryptophthalmic eyes were gourd-shaped cysts that were unable to easily be distinguished from normal eyes using CT or MRI (Fig. 4E and F). After separation from the subcutaneous tissue, a whole gourd-shaped cyst within the eye socket was identified (Fig. 4G). All the dysplastic eyes exhibited structural malformation in the anterior segment, including corneal neovascularization, corneal leukoma, and iris synechia, using slit lamp microscopy (Fig. 4C). Additionally, two Frem2R725X/R2156W mice born with bilateral cryptophthalmos opened one eye at an age of 4 months. Eight of eleven mice exhibited a single kidney and 4/11 mice exhibited syndactyly (Fig. 4D). One of the two Frem2R2156W/R2156W mice displayed bilateral congenital complete cryptophthalmos, and the other displayed unilateral cryptophthalmos. Both exhibited a single kidney and normal limbs. Quantification of the phenotypic penetrance in different backgrounds and phenotypes of Frem2R725X/R2156W mice are shown in Table 1a, b. H&E staining showed a clear internal structure with a disorganized or absent cornea, complete iris synechia, iris hypoplasia, a lack of the ciliary body and a lack of the pupil, indicating severe ocular developmental defects (Fig. 5A–D). Compared to the WT eyes, the dysplastic

We report three patients who all presented bilateral complete cryptophthalmos. A gross examination of the periorbital region revealed continuous, intact skin covering the upper half of the face without the normal development of eyelids, eyelashes, or eyebrows (Fig. 1A–C, consent to publish was obtained). The patients did not exhibit syndactyly, microtia, or ambiguous genitalia. The physical examination and ultrasound did not reveal any abnormalities of the heart, brain or urogenital system. Ocular B-scan ultrasound revealed bilateral gourd-shaped cysts without clear internal structures (Fig. 1D, Patient #1). MRI showed a normal brain morphology, extraocular muscles in a narrow orbital fat gap, and optic nerve enlargement in the expanded orbits (Fig. 1E, F, Patient #1). At 4 months of age, Patient #1 underwent exploratory surgery of the right orbit. No anatomical eyelid structure was identified. A transparent anterior cyst was detected underneath the subcutaneous tissue. The iris and suspensory ligament of the lens were absent. A lens-like transparent spherical body was observed in the posterior cyst. The vascular and pigment tissues in the posterior cyst were not able to be obviously identified as retinal and choroid tissues. All three patients achieved normal developmental milestones with no cognitive impairments. 3.2. Whole exome sequencing and analysis Genetic analyses revealed compound heterozygous mutations in the FREM2 gene in all three patients, and their parents were all heterozygous carriers (Fig. 2A). A shared c.6499C > T (p.R2167W) missense

Table 1a Quantification of the phenotypic penetrance in mice with different genetic backgrounds. Genes

Frem2

Sex

R725X/R2156W

Frem2R2156W heterozygous Frem2R725X heterozygous Frem2

R2156W

homozygous

Frem2R725X homozygous WT

M: 6 F: 5 M: 12 F: 8 M: 11 F: 8 M: 2 F: 0 M: 1 F: 2 M: 10 F: 10

Weight (g)

Phenotype

M: 25.8 F: 21.6 M: 25.1 F: 20.8 M: 27.3 F: 20.6 M: 24.6 F: 0 M: 25.8 F: 21.2 M: 25.3 F: 21.9

307

Cryptophthalmos

Kidney

Limbs

Unilateral: 7 Bilateral: 4 Unilateral: 1 Bilateral: 0 Unilateral: 2 Bilateral: 0 Unilateral: 1 Bilateral: 1 Unilateral: 2 Bilateral: 1 Unilateral: 0 Bilateral: 1

Unilateral: 8 Normal: 3 Unilateral: 9 Normal: 11 Unilateral: 7 Normal: 12 Unilateral: 2 Normal: 0 Unilateral: 3 Normal: 0 Unilateral: 7 Normal: 13

Syndactyly: Normal: 7 Syndactyly: Normal: 19 Syndactyly: Normal: 19 Syndactyly: Normal: 2 Syndactyly: Normal: 2 Syndactyly: Normal: 20

4 1 0 0 1 0

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Table 1b Phenotypes of Frem2R725X/R2156W mice on a C57BL/6 background. ID 1 2 3 4 5 6 7 8 9 10 11

Genes R725X/R2156W

Frem2 Frem2R725X/R2156W Frem2R725X/R2156W Frem2R725X/R2156W Frem2R725X/R2156W Frem2R725X/R2156W Frem2ΔR725X/R2156W Frem2R725X/R2156W Frem2R725X/R2156W Frem2R725X/R2156W Frem2R725X/R2156W

Sex

Age (w)

Weight (g)

Congenital cryptophthalmos

Kidney

Limbs

Notes

M M F M M M F F F F M

28 25 27.6 14.6 14.6 18 18 18 10 10 10

28.5 27.7 23.1 22.6 25.7 25.5 21.3 22.6 21.9 19.1 24.6

Unilateral Unilateral Unilateral Unilateral Unilateral Bilateral Bilateral Unilateral Unilateral Bilateral Bilateral

Unilateral Unilateral Unilateral Unilateral Bilateral Unilateral Unilateral Unilateral Unilateral Bilateral Bilateral

Normal Normal Normal Syndactyly Syndactyly Normal Syndactyly Normal Syndactyly Normal Normal

The same parents

Consangui-neous parents

Opened one eye after birth

Opened one eye after birth

Fig. 5. Ocular abnormalities in the Frem2R725X/R2156W mice. Histological sections of the eyes from adult WT (A) and Frem2R725X/R2156W mice (B–D) focusing on the anterior structures. In (B) and (C), defects in the cornea, iris, and ciliary body are evident in the cryptophthalmic eye. The black arrowheads denote the disappearance and disorganization of the corneal structure, the white arrowhead denotes complete iris synechia, the dotted circle indicates a region of iris hypoplasia, the arrows show the absence of a ciliary body and the dotted line denotes the boundary between the lens and retina. Most notably, the pupil is absent in the cryptophthalmic eyes. (D) Morphology of the relatively normal eyes of mice with unilateral cryptophthalmos. (E) Images of H&E staining of transverse sections from an E13.5 Frem2R725X/R2156W mouse embryo through the head, the black arrow points to the poorly developed eye. (F–I) Comparison of the characteristics of adult WT and Frem2R725X/R2156W mouse eyes. (F) Axial length (cryptophthalmic eyes vs WT eyes: 2777.6 μm vs 3284.9 μm, P < 0.01). (G) Lens thickness (cryptophthalmic eyes vs WT eyes: 1556.8 μm vs 2020.5 μm, P < 0.05). (H) The boundary between the lens and retina (cryptophthalmic eyes vs WT eyes: 200.68 μm vs 373.93 μm, P < 0.001). (I) The thickness of total retina (cryptophthalmic eyes vs WT eyes: 317.97 μm vs 269.50 μm, P < 0.01). The values represent the averages ± SD from at least three experiments. (*P < 0.05, **P < 0.01, and ***P < 0.001). (Scale bars in all panels = 200 μm). 308

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eyes exhibited a reduced axial length (2777.6 μm vs 3284.9 μm, P < 0.01) and reduced lens thickness (1556.8 μm vs 2020.5 μm, P < 0.05). The boundaries between the lens and retina were narrower in the eyes of the mutant mice than in the WT eyes (200.68 μm vs 373.93 μm, P < 0.001). Retinas were layered normally, but were thicker in the Frem2R725X/R2156W mice (317.97 μm vs 269.50 μm, P < 0.01). In mice with unilateral cryptophthalmos, the boundary between the lens and retina, and the retina thickness of the relatively normal eyes were comparable to the WT eyes, while the axial length and lens thickness were reduced (Fig. 5F–I). H&E staining of transverse sections from an E13.5 Frem2R725X/R2156W mouse embryo showed dysplastic eye structures in one eye before eyelid opening (Fig. 5E). The growth indices including axial length, lens thickness, the boundaries between the lens and retina and the thickness of total retina were measured three times. The averages of all measurements were calculated.

3.5. Identification of DEGs in fetal mice Eyelid opening is a complicated process requiring tight regulation by two parallel pathways: EGFR and BMP-Smad signaling (Jin et al., 2008; Dong et al., 2017; Chaerkady et al., 2013). We performed RNAseq with RNA isolated from the whole embryos of E13.5 mice and found that the levels of 18 protein-coding genes participating in the EGFR signaling pathway and 43 genes including Bmp3, Bmp6, Bmp8a participating in the BMP-Smad signaling pathway were significantly up- or down-regulated in Frem2R725X/R2156W mice compared with the WT mice. Most importantly, we observed decreased expression of genes contributing to extracellular matrix organization and cell-matrix adhesion, including Ecm2, Col6a1, Col5a3, Colla1 and Lama5. Decreased expression of cytoskeleton-related genes were also identified, including Krtl1, Krtl3, Lor and members of the Sprr family, which are involved in the process of cornification during the formation of the epidermal barrier (Tong et al., 2006; de Koning et al., 2012; Rubinstein et al., 2016). The expression of Cryba and Cryge, which are the main structural proteins of the vertebrate ocular lens and are required for lens transparency, were significantly reduced in Frem2R725X/R2156W mice (Zhao et al., 2015). Overall, we identified 661 and 1465 genes that were significantly up- or down-regulated, respectively (Fig. 7B and Dataset S1). The volcano plot shows these DEGs (Fig. 7A). In addition, the heat map presents the top 50 DEGs sorted by P-values (Fig. 7C). The lower expression of Ecm2, Col6a1, Lor, Sprr1a, Cryge, and the 3 genes Cblc, Esp8, Agr2 participating in the EGFR signaling pathway, were verified by performing real-time quantitative PCR in the eyes and whole embryos of Frem2R725X/R2156W mice (Fig. 7D). Overall, the enriched GO terms in cellular components mainly focused on the extracellular region (Fig. 7E), and the enriched GO terms in biological processes were mainly related to organ development, particularly the anatomical structure morphogenesis (Fig. 7F).

3.4. FREM2 expression in the retinas of cryptophthalmic eyes Immunohistochemical and immunofluorescence staining of the retina revealed FREM2 expression in the outer plexiform layer (OPL), which contains retinal neurons such as horizontal and Muller glia cells in adult mice (Fig. 6A–F). The thickness and intensity of FREM2 immunostaining in the OPL were not significantly different between the cryptophthalmic eyes and the WT eyes (10.1 μm vs 9.6 μm, P > 0.1, 0.0567/pixel vs 0.0563/pixel, P > 0.1, respectively). In addition, Ecadherin and collagen VII (Fig. 6C–F), which are critical for cell adhesion in the process of closure of the mouse optic fissure during development, were expressed under the outer limiting membrane in the retinal pigment epithelium, consistent with previously described (Chen et al., 2012; Stern and Temple, 2015; Palma-Nicolás and López-Colomé, 2013).

Fig. 6. FREM2 expression in the retinas of cryptophthalmic eyes. (A–B) Images of immunohistochemical staining for FREM2 in the retina. (C–F) Images of immunofluorescence staining for FREM2 (red) in the retina, sections were counterstained with antibodies against E-cadherin/collagen VII (green) and DAPI (blue). Dotted boxes show FREM2 (red) expression in the OPL of the retina in adult Frem2ΔR725X/R2156W and WT mice. (Scale bars in all panels = 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 309

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Fig. 7. Gene-expression profiles from fetal Frem2ΔR725X/R2156W mice and WT mice. (A) Volcano plot showing the top 50 DEGs. (B) 2D scatter plot showing DEGs. Genes highlighted in blue, orange, and green indicate DEGs with |log (FC)| > 1, |log (FC)| > 2, |log (FC)| > 3, respectively. (C) Heat map of the top 50 DEGs. (D) The lower expression of Ecm2, Col6a1, Lor, Sprr1a, Cryge, Cblc, Esp8, Agr2 were verified in embryonic eyes and whole embryos of Frem2R725X/R2156W mice using real-time quantitative PCR (*P < 0.05, **P < 0.01). (E and F) Enriched GO terms in cellular components and biological processes. FC, fold change. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

are associated with differences in the age at eyelid opening, and contribute to the differences in phenotype (Nosten and Roubertoux, 1988). In conclusion, the effects on eyelids phenotypes are specific to particular genotypes and uterine environments. Our study facilitates an improved understanding of eye development across vertebrate species. In addition, the results of our study should be cautiously interpreted within the context of mouse strains, as the ocular phenotypes of mice on different genetic backgrounds may be multifarious (Doetschman, 2009; Keane et al., 2011). Previous studies have reported striking defects in eyelid formation associated with ocular anomalies in Egr1−/− mice on the BALB/c background but not the C57BL/6J background (Oh et al., 2017). According to Mao et al. the capacity for genetic modification between 129B6F1 and CASTB6F1 mouse strains differs for anterior segment dysgenesis and progression to glaucoma (Mao et al., 2015). Stronger inflammatory responses in the ocular surface were observed in C57BL/6J mice following regular benzalkonium chloride administration, compared with BALB/c mice (Yang et al., 2017). Additional experiments are required to confirm whether FREM2 is a strain-specific heredity gene. Experiments designed to clarify the chronology of the pathogenesis of cryptophthalmos is also essential for identifying when defects in eyelid formation and/or opening occur. In summary, our findings extend the range of FREM2 mutations, establish a mouse model that recapitulates the human complete cryptophthalmos phenotype, and highlight the importance of FREM2 during the development of eyelids and the anterior segment of the eyeballs. Current data provide insights that will support further mechanistic studies on the pathogenesis of isolated cryptophthalmos.

Based on accumulating evidence, the FRAS1/FREM family encodes structurally similar proteins of the extracellular matrix that function in the embryonic dermal epidermal adhesion (Kiyozumi et al., 2006; Pavlakis et al., 2011). The c.6499C > T missense mutation (p.R2167W) identified in our patients was predicted to result in impaired function of the FREM2 as well as disassembly of the whole protein family. Consistent with this prediction, the ocular adnexal agenesis, microphthalmia, corneopalpebral adhesions, and iris coloboma appeared in the Frem2 mutant mice, which might be explained by abnormal mesenchyme-epithelium interactions during eyelid development (Tawfik et al., 2016; Ohuchi, 2012). Additionally, the increased thickness of the total retina of Frem2R725X/R2156W mice may be a compensatory response to the visual deprivation caused by anterior segment developmental defects, as suggested in our previous report (Long et al., 2017). In the present study, we performed immunohistochemical and immunofluorescence staining of the retina of adult mice to investigate FREM2 expression. To our knowledge, this study is the first to report the localization of FREM2 expression in the OPL of the retina which contains retinal neurons such as horizontal and Muller glia cells.The morphology of the retina was almost normal and did not show abnormal expression of FREM2 using the commercially available antibody. In addition, E-cadherin and collagen VII, which are required for cell adhesion in the process of closure of the mouse optic fissure during development, were expressed normally. An experiment on retinal function is still needed to assess genetic effects of the mutant Frem2 gene on retinal development. If the retinal function of the patients with isolated bilateral cryptophthalmos is normal, replacement surgery of the anterior segment could be performed to achieve the desired therapeutic effect. However, if the retina function is abnormal, the therapeutic effect of the operation would be relatively poor. Moreover, RNA-seq data identified DEGs in fetal Frem2R725X/R2156W mice including Ecm2, Col6a1, Lor, Sprr1a, Cryge, and the 3 genes Cblc, Esp8, Agr2 participating in the EGFR signaling pathway. While the compound heterozygous mutation in the Frem2 gene is believed to directly cause the ocular abnormalities, the different expressions of these genes may secondarily contribute to the phenotypes. In general, humans and mice share common features during eyelid morphogenesis (Teraishi and Yoshioka, 2001; Findlater et al., 1993). However, the phenotypes exhibited by the Frem2 mutant mice and patients in this study differed. While the patients all presented bilateral complete cryptophthalmos, the Frem2R725X/R2156W mice displayed bilateral or unilateral cryptophthalmos with eyelids that opened later in a few cases. Furthermore, 8/11 mice exhibited a single kidney and 4/11 mice exhibited syndactyly, similar to the clinical diagnosis of FS in humans. The differences may be attributed to the following explanations. First, although the physiology and short gestational period of mice make them ideal models of human diseases (Bradley, 2002), differences exist in gene expression between humans and mice in the early embryonic stage (Madissoon et al., 2014). Second, inbreeding is a common phenomenon in the generation of Frem2 mutant mice. However, the three patients described in this study had a sporadic disease and nonconsanguineous parents. Third, the eyelids are completely open in utero in humans but postnatally in mice (Findlater et al., 1993). A previous study revealed an interactive maternal environmental action on genotype expression in the early stages of mouse eyelid development (Nosten and Roubertoux, 1988). Differences in the uterine environment

Acknowledgments We thank Prof. Jinxin Bei (Sun Yat-sen University Cancer Center) for assisting with whole exome sequencing and Peikuan Cong (Hangzhou Beingen Biotechnology Co.Ltd) for providing technical assistance with variant detection and homology modeling of the protein structure.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exer.2019.02.013.

Funding This study was funded by the National Natural Science Foundation of China (81770967 and 81822010). The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. Prof. Haotian Lin was supported by grants from the Guangdong Provincial Natural Science Foundation for Distinguished Young Scholars of China (2014A030306030) and Guangdong Province Universities and Colleges Youth Pearl River Scholar Funded Scheme (2016).

Declarations of interests The authors declare no competing interests. 311

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Websites DAVID, http://david.abcc.ncifcrf.gov/ DbSNP, http://www.ncbi.nlm.nih.gov/SNP/ Homologene, https://www.ncbi.nlm.nih.gov/homologene. Human Gene Mutation Database, http://www.hgmd.org/ ImageJ Fiji, http://fiji.sc/Fiji. Integrative Genomics Viewer, http://software.broadinstitute.org/ software/igv/ Locus Specific Mutation Databases-Zhejiang University Center for Genetic and Genomic Medicine (LOVD2.X),http://www.genomed.org/ lovd2/variants.php?action=search_unique&select_db = FREM2. OMIM, http://www.omim.org/ PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/ SIFT, http://sift.bii.a-star.edu.sg/ SWISS-MODEL, http://swissmodel.expasy.org/ Authors’ contributions H.T.L., D.N.W. and X.Y.Z. designed the study. X.Y.Z., D.N.W., M.M.D.Y., W.R.X., E.P.L., Z.Z.L., X.H.W., D.R.L., J.J.C., Z.L.L., J.H.W., and W.T.L. performed the experiments. X.Y.Z. analyzed the data. H.T.L. and X.Y.Z. co-wrote the manuscript, Y.Z., C.C. Y.L. and D.M.L. critically revised the manuscript, and all authors discussed the results and commented on the paper. References Barishak, Y.R., 1992. Embryology of the eye and its adnexae. Dev. Ophthalmol. 24, 1–142. Bradley, A., 2002. Mining the mouse genome. Nature 420, 512–514. Chaerkady, R., Shao, H., Scott, S.G., Pandey, A., Jun, A.S., Chakravarti, S., 2013. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration. J. Proteomics 87, 122–131. Chen, M., Chen, Q., Sun, X., Shen, W., Liu, B., Zhong, X., Leng, Y., Li, C., Zhang, W., Chai, F., et al., 2010. Generation of retinal ganglion-like cells from reprogrammed mouse fibroblasts. Investig. Ophthalmol. Vis. Sci. 51, 5970–5978. Chen, S., Lewis, B., Moran, A., Xie, T., 2012. Cadherin-mediated cell adhesion is critical for the closing of the mouse optic fissure. PLoS One 7 e51705. de Koning, H.D., van den Bogaard, E.H., Bergboer, J.G., Kamsteeg, M., van VlijmenWillems, I.M., Hitomi, K., Henry, J., Simon, M., Takashita, N., Ishida-Yamamoto, A., et al., 2012. Expression profile of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins during skin barrier repair. Br. J. Dermatol. 166, 1245–1254. Doetschman, T., 2009. Influence of genetic background on genetically engineered mouse phenotypes. Methods Mol. Biol. 530, 423–433. Dong, F., Call, M., Xia, Y., Kao, W.W., 2017. Role of EGF receptor signaling on morphogenesis of eyelid and meibomian glands. Exp. Eye Res. 163, 58–63. Egier, D., Orton, R., Allen, L., Siu, V.M., 2005. Bilateral complete isolated cryptophthalmos: a case report. Ophthalmic Genet. 26, 185–189. Findlater, G.S., McDougall, R.D., Kaufman, M.H., 1993. Eyelid development, fusion and subsequent reopening in the mouse. J. Anat. 183 (Pt 1), 121–129. François, J., 1969. Malformative syndrome with cryptophthalmos. Acta Genet. Med. Gemellol. 18, 18–50. Hever, A.M., Williamson, K.A., van Heyningen, V., 2006. Developmental malformations of the eye: the role of PAX6, SOX2 and OTX2. Clin. Genet. 69, 459–470. Jadeja, S., Smyth, I., Pitera, J.E., Taylor, M.S., van Haelst, M., Bentley, E., McGregor, L., Hopkins, J., Chalepakis, G., Philip, N., et al., 2005. Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs. Nat. Genet. 37, 520–525. Jin, C., Yin, F., Lin, M., Li, H., Wang, Z., Weng, J., Liu, M., Da, D.X., Qu, J., Tu, L., 2008. GPR48 regulates epithelial cell proliferation and migration by activating EGFR during eyelid development. Investig. Ophthalmol. Vis. Sci. 49, 4245–4253. Källén, B., Robert, E., Harris, J., 1996. The descriptive epidemiology of anophthalmia and microphthalmia. Int. J. Epidemiol. 25 (5), 1009–1016. Keane, T.M., Goodstadt, L., Danecek, P., White, M.A., Wong, K., Yalcin, B., Heger, A., Agam, A., Slater, G., Goodson, M., et al., 2011. Mouse genomic variation and its effect on phenotypes and gene regulation. Nature 477, 289–294. Kiyozumi, D., Sugimoto, N., Sekiguchi, K., 2006. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects. Proc. Natl. Acad. Sci. U.S.A. 103, 11981–11986. Leck, I., 1994. Clusters of anophthalmia. Br. Med. J. 308 (6922), 205–206. Lin, Y., Gao, H., Chen, C., Zhu, Y., Li, T., Liu, B., Ma, C., Jiang, H., Li, Y., Huang, Y., et al.,

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