Znf469 Plays a Critical Role in Regulating Synthesis of ECM: A Zebrafish Model of Brittle Cornea Syndrome

Abstract

Purpose: Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder characterized by extreme thinning and fragility of the cornea, and mutations in ZNF469 cause BCS-1. We aimed to establish a znf469 mutant zebrafish line to explore its roles and possible pathogenic mechanism in cornea development and disorder.

Methods: znf469^4del/4del mutant zebrafish was generated using the CRISPR/Cas9 technology. Transmission electron microscopy (TEM) was performed to examine the phenotype of the cornea in different developmental stages. RNA sequencing and quantitative real-time polymerase chain reaction were used to reveal the molecular mechanism.

Results: Macroscopically, homozygous znf469 mutant zebrafish larvae exhibited a curved body from 72 hours postfertilization, similar to kyphoscoliosis, and a noninflated swimbladder at 7 days postfertilization (dpf). TEM revealed an extreme reduction of corneal stroma thickness in homozygous znf469 mutant zebrafish in both the central and peripheral cornea from the early development stage. RNA-sequencing analysis demonstrated that the znf469 mutation leads to the decreased synthesis of various extracellular matrix (ECM) components, such as collagens and proteoglycans, but increased synthesis of 26S proteasome family members.

Conclusions: The results of our work indicate that znf469 is a critical gene that, as a widely considered transcription factor, may regulate the synthesis and degradation of a large number of ECM components that play an important role in corneal development.

Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder characterized by extreme thinning and fragility of the cornea, with keratoconus (KC) and blue sclera as major ocular manifestations. Other ocular signs include keratoglobus, high myopia, irregular corneal astigmatism, and corneal rupture.¹ Disorder of collagen-rich extracellular matrix (ECM) synthesis and homeostasis is the major cause of BCS. BCS is genetically heterogeneous and caused by biallelic mutations in either ZNF469 (BCS-1, OMIM 22920) or PRDM5 (BCS-2, OMIM 614170).² 

ZNF469 belongs to the Cys2–His2 (C2H2) zinc finger family, which is considered to encode a class of transcription factors.³ Genome-wide association studies have repeatedly indicated that ZNF469 is strongly associated with central corneal thickness.^4–10 Mounting evidence has shown that ZNF469 heterozygous missense mutations are associated with a keratoconic phenotype,^11–13 which indicates autosomal dominant inheritance. Conversely, several groups found no association between mutations in ZNF469 and KC,^14,15 suggesting a complicated pathogenic mechanism of ZNF469 mutations. Furthermore, ZNF469 is reported to be a pathogenic gene of systemic connective tissue disease in humans.¹⁶ Current evidence suggests that ZNF469 may play a role in collagen biosynthesis and assembly¹⁷ and regulation of ECM homeostasis.¹⁶ 

A recent study in a mouse model reported that homozygous Zfp469 mutant mice weigh less but are viable and fertile. Depletion of Zfp469 in mice induces decreased expression of ECM components, especially Col1a1 and Col1a2, but not Col5a1. The abnormal collagen type I/collagen type V ratio may result in collagen fibrils with smaller diameter and increased fibril density. As a result, the corneal stroma thickness of homozygous mutants is reduced by approximately 50% after conversion according to the thickness ratio of the epithelium and stroma.¹⁸ In this study, we generated a znf469 mutant zebrafish line to elucidate its roles in zebrafish corneal development and applied RNA-sequencing (RNA-seq) to elucidate the possible pathogenesis of BCS induced by znf469 mutations. 

Materials and Methods

Patient

In our previous research, seven novel mutations in ZNF469 were identified and predicted to have a pathogenic role in Chinese patients with KC.¹² Here, we evaluated a patient with severe ocular signs. The patient was a 30-year-old man. He underwent a corneal topography that used an Oculus Pentacam 1.21r41 (Oculus Optikgeräte GmbH, Wetzlar, Germany). Genomic DNA extracted from a blood sample using a Simgen Blood DNA mini kit (Simgen, Hangzhou, China) was applied to screen the mutations with next-generation sequencing technology, which is based on targeted sequence capturing technology with the SureSelect Target Enrichment Kit (Agilent Technologies, Santa Clara, CA, USA) and Illumina sequencing technology using a HiSeq sequencer (Illumina, San Diego, CA, USA). To avoid false-positive results and to ascertain the significance of the mutations in ZNF469, mutations with a minor allele frequency < 0.1% (according to data from the May 2012 release of the 1000 Genomes Project and the Single Nucleotide Polymorphism database) and absent from the results of the whole-exome sequencing data acquired from 220 Han Chinese individuals without ocular abnormalities (from a commercial database provided by the Genesky Bio-Tech company) were subsequently confirmed using Sanger sequencing technology. Written informed consent was obtained. 

Zebrafish Husbandry

All experiments, including the generation of mutants, were conducted on AB strain zebrafish. The zebrafish were maintained at 27.5°C ± 1°C with a 14-hour light/10-hour dark photocycle. Embryos were collected and maintained in E3 medium in a 28.5°C incubator. Animal care and experimental procedures were approved by the Ethics Committee on Animal Research of Zhejiang University. All procedures conformed to the protocols of the Zhejiang University Animal Care and Use Committee. 

Generation of znf469 Loss-of-Function Zebrafish Line and Genotyping

Clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) technology was applied to generate znf469 mutants following previously described protocols.¹⁹ The specific guide RNA (gRNA) sequence targeting the c.495-514 region in the single coding exon of the znf469 gene (Fig. 2B), 5′-GAATACTTGGCACATGGAAG-3′, was designed by a website (http://crispor.tefor.net) and synthesized using the MEGAscript T7 kit (AM1334; Thermo Fisher Scientific, Waltham, MA, USA). The mixtures (approximately 4 nL), including 800 ng/µL NLS-Cas9-NLS protein (Z03389; GenScript, Piscataway, NJ, USA) and 200 ng/µL single-guide RNA (sgRNA) were coinjected into one-cell zygote embryos. The founders (F0) were raised and outcrossed with wild-type (WT) fish to obtain F1 fish. The adult F1 fish were genotyped by Sanger sequencing (Fig. 2C). A 4-base pair (bp) deletion strain was detected and outcrossed with WT until F3. The mutation introduced a recognition site of NspI. The 4-bp deletion strain was genotyped by PCR and digestion with restriction enzyme NspI (R0602S; New England Biolabs, Ipswich, MA, USA) (Fig. 2E). The genotyping PCR primers were as follows: 

• F: 5′-CAAACCAACGCTCAAATCCCAC-3′
• R: 5′-CCTCTCTGAAGAGTGCCATGTGT-3′

Figure 1.

 

Clinical ocular features of the ZNF469 mutant patient. (A) Pentacam refractive maps and corneal thickness map of the right eye (OD). Thinnest corneal thickness = 435 µm, K_max = 64.9 D. (B) Pentacam refractive maps and corneal thickness map of the left eye (OS). Thinnest corneal thickness = 406 µm, K_max = 70.4 D. Pupil center is indicated by a plus sign (+), the pachy apex is marked with a filled circle (•), and the thinnest location is indicated by an open circle (○). Three black dotted lines in the corneal thickness map indicate the distribution of healthy people, and the red line indicates the fitting curve of the patient.

Transmission Electron Microscopy

Zebrafish larvae at the desired developmental stage were fixed overnight at 4°C in 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.1-M PBS. After three 15-minute washes in PBS (0.1-M, pH 7.0), larvae were postfixed with 1% SPI-Chem osmium tetroxide (02595-BA; Structure Probe, Inc., West Chester, PA, USA) for 1 hour and then washed of osmium using PBS three times, 15 minutes each. Next, the larvae were dehydrated by a graded series of ethanol (30%, 50%, 70%, 80%) and acetone (90%, 95%) for 15 minutes each. Finally, the sections were dehydrated twice for 20 minutes in absolute acetone. The larvae were placed in a 1:1 mixture of absolute acetone and the final Spurr resin (02680-AB; Structure Probe, Inc.) mixture for 1 hour at room temperature. They were then transferred to a 1:3 mixture of absolute acetone and the final resin mixture for 3 hours and to the final Spurr resin mixture overnight. Larvae were placed in an Eppendorf tube containing Spurr resin, heated at 70°C for more than 9 hours, and then sectioned in a Leica EM UC7 ultratome (Leica, Wetzlar, Germany). Every effort was made to achieve sagittal sections at the equator of the eyes. Sections were with by uranyl acetate and alkaline lead citrate for 5 to 10 minutes and observed with a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). 

RNA-Seq and Bioinformatics Analysis

Total RNA was isolated from a pool of 50 homozygous mutant or WT larvae at 7 days postfertilization (dpf). RNA-seq and analysis were performed at RiboBio (Guangzhou, China). The quality of the RNA libraries was assessed using the Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA), and libraries were sequenced by the Illumina platform with paired-end 150 bp. Clean reads were aligned to the zebrafish reference genome sequence (GRCz11) using HISAT2 2.1.0 with default parameters. HTSeq 0.12.4 was subsequently employed to convert aligned short reads into read counts for each gene model. Differential expression was assessed by DESeq2 using read counts as input. The Benjamini–Hochberg multiple test correction method was enabled. Differentially expressed genes (DEGs) were chosen according to the criteria of |log2FoldChange| > 1 and adjusted P (Q) < 0.05. The clusterProfiler 3.12.0 package in R/Bioconductor (R Foundation for Statistical Computing, Vienna, Austria)/KOBAS 3.0 was used to identify and visualize the GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by all DEGs. 

Quantitative Real-Time PCR Analysis

Total RNA was isolated from a pool of five homozygous mutants and heterozygous or WT larvae at 7 dpf using TRIzol (15596026; Thermo Fisher Scientific). RNA samples were reverse transcribed using the PrimeScript RT reagent kit (RR047A; Takara Bio, Shiga, Japan). Two-step quantitative real-time PCR (qRT-PCR) was then performed using PowerUp SYBR Green Master Mix (A25742; Thermo Fisher Scientific) on a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Data are represented as the mean ± SD of three independent biological replicates. The actb1 gene that encodes β-actin1 was used as an internal control. Data were analyzed using the 2^−ΔΔCT method for relative quantitation. All of the primers are listed in Supplementary Table S1. 

Data Analysis

The measurements of the thickness of corneal layers (total, epithelium, stroma, and endothelium) were performed on five transmission electron microscopy (TEM) digital micrographs taken of both the central and peripheral cornea using ImageJ 1.53 (National Institutes of Health, Bethesda, MD, USA). Quantitative data are shown as the means ± SD. Unpaired Student's t-test was used to compare the means from two different groups, and one-way ANOVA with multiple comparisons was used to compare the means from three or more groups using Prism 9 9.1.1 (GraphPad, San Diego, CA, USA). P < 0.05 was considered significant. 

Results

Keratoconus and Global Corneal Thinning in a Patient With a ZNF469 Mutation

The index patient described here presented with corneal stromal thinning, Vogt's striae, Fleischer ring, and Munson's sign in both eyes under the slit-lamp microscope, and his corneal topography analysis revealed reduced corneal thickness and increased corneal central curvature, and the thinnest point of the cornea had moved downward (Fig. 1), which met the diagnostic criteria for KC. Genomic DNA sequencing revealed that the patient carried a heterozygous missense variant in ZNF469 (c.3749C>T), leading to the amino acid substitution P1250L. Unknown significance was assigned to the ZNF469 (c.3749C>T:p.P1250L) variant according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology 2015 classification guidelines. Silico analysis with the Sorting Intolerant Form Tolerant (SIFT) program predicted the variant as a damaging mutation. Previous studies suggest that biallelic mutations in ZNF469 cause BCS, whereas heterozygous carriers of the pathogenic variant tend to show mild or no corneal thinning.^14,17 Considering that patients with BCS typically present with an autosomal-recessive generalized connective tissue disorder (such as the abnormalities in corneal, skin, auditory, and musculoskeletal tissue), the index patient did not meet the diagnostic criteria for BCS because he did not show any other clinical manifestations except KC and did not fit the normal recessive inheritance for the disease. In a word, this case supports the observation that heterozygous carriers of ZNF469 mutations lead to KC. 

Figure 2.

 

Generation of znf469 mutant zebrafish. (A) Sequence alignment of the seven C2H2 zinc finger protein domains in zebrafish Znf469 protein and human ZNF469 protein. The identical residues are colored in red. Residues shown in the black box are the paired cysteines and histidines, which bind the zinc ion. (B) Schematic diagram of the zebrafish znf469 gene CDS region. The single-guide RNA (sgRNA) targeting site is marked in blue, and the protospacer adjacent motif (PAM) sequence CGG is marked in red. (C) Sanger sequencing results revealed a 4-bp deletion (c.496-499del) in the znf469 homozygous mutant zebrafish. (D) Schematic representation of the protein structure of Znf469 and the position of the seven predicted C2H2 zinc finger domains. The 4-bp deletion caused a frameshift; the 166th and subsequent amino acids began to change (marked with red segment) and then generated a premature stop codon, leading to early termination of protein translation and a truncated Znf469 protein with 208 amino acids in znf469^4del/4del zebrafish. (E) The mutant zebrafish were genotyped with PCR, followed by NspI digestion. The undigested WT PCR product was loaded as a control. The mutant allele could be digested into two fragments for the addition of the NspI digestion site, but the WT allele could not be digested. (F) qRT-PCR analysis revealed no significant difference in the level of znf469 mRNA in the WT, znf469^4del/+, and znf469^4del/4del larvae at 7 dpf. Three biological replicates were performed, and the data are shown as the mean ± SD.

Generation of znf469 Mutant Zebrafish and Identification of Macroscopic Phenotype

The seven predicted C2H2 zinc finger domains of the Znf469 protein (XP_021326388.1) showed extremely high conservation compared to the human ZNF469 protein (NP_001354553.1) (Fig. 2A). Zebrafish znf469 contains a single coding exon with a coding sequence (CDS) of 11,253 bp. The CRISPR target site was designed in the CDS c.495-514 region (Fig. 2B). Sanger sequencing results revealed a 4-bp deletion in the homozygous znf469 mutant zebrafish (c.496-499del, p.Phe166Metfs*43), which introduced a recognition site of the restriction enzyme NspI. We refer to the znf469 mutant line as znf469^4del/4del (Fig. 2C). The 4-bp deletion is expected to cause a frameshift and generate a premature stop codon, leading to early termination of protein translation and a truncated Znf469 protein with 208 amino acids leading to the loss of all of the zinc finger domains in znf469^4del/4del zebrafish (Fig. 2D). PCR products amplified from extracted genomic DNA followed by NspI digestion were carried out to distinguish the homozygous mutants (Fig. 2E). No significant difference was observed for the expression levels of znf469 mRNA among heterozygous and homozygous mutation carriers and WT larvae, indicating that the mutant transcripts did not undergo nonsense-mediated decay (Fig. 2F). 

The larvae of znf469^4del/4del zebrafish had slightly curved bodies compared to WT larvae at 72 hours postfertilization (hpf), whereas the znf469^4del/+ zebrafish seemed to be unaffected (Fig. 3A). The homozygous mutation of znf469 compromised viability from 7 dpf on due to the curved body and the absence of an inflated swimbladder (Fig. 3B, marked with red *). Homozygous mutant larvae sank to the bottom and were unable to actively swim, and they eventually died at 7 to 10 dpf. The larvae of znf469^4del/+ zebrafish showed no apparent macroscopic abnormalities. Znf469^4del/+ adult zebrafish were viable and fertile. 

Figure 3.

 

Macroscopic phenotype of the znf469 mutant zebrafish. (A) The larvae of znf469^4del/4del zebrafish had slightly curved bodies compared to the WT larvae at 72 hpf. The larvae of znf469^4del/+ zebrafish showed no phenotypic abnormalities. (B) The larvae of znf469^4del/4del zebrafish lacked an inflated swimbladder (indicated by a red asterisk, *) and had a curved body compared to WT larvae at 7 dpf. The larvae of znf469^4del/+ zebrafish showed no phenotypic abnormalities.

znf469^4del/4del Mutation Results in an Extreme Reduction of Primary Stroma in Whole Cornea

The adult zebrafish cornea has five layers similar to humans: epithelium, Bowman's layer, stroma, Desçemet's membrane and endothelium. The development of zebrafish cornea occurs in several stages.^20–22 The corneal epithelial cells separate from the lens epithelial cells at 24 hpf. The stroma starts to accumulate on the internal surface of the corneal epithelium at 30 hpf. By 48 hpf, endothelial cells form a monolayer, separated from the epithelial cells by a robust stroma. At 72 hpf, the cornea contains two layers of epithelial cells: the well-organized primary stroma and a single cell layer of the endothelium. From 14 to 28 dpf, the stroma increases rapidly, and keratocytes appear in the stroma (Fig. 4A). To investigate the roles of znf469 in corneal development, we analyzed the phenotypes of WT, znf469^4del/+, and znf469^4del/4del zebrafish larvae at 48 hpf, 72 hpf, and 7 dpf using TEM. 

Figure 4.

 

Similar ultrastructure of cornea in WT, znf469^4del/+, and znf469^4del/4del zebrafish larvae at 48 hpf. (A) Schematic diagram of zebrafish corneal development. To adulthood, the epithelium increases to four to six layers, the stroma is thickened, the density of keratocytes is increased, Desçemet's membrane is visible, and the endothelium is also a monolayer. The TEM ultrastructure of the cornea is shown in WT (B, B’, C), znf469^4del/+ (D, D’, E), and znf469^4del/4del (F, F’, G) zebrafish larvae at 48 hpf. Each cornea contains a bilayer epithelium, a primary stroma, and a monolayer endothelium. Collagen fibers are visible but not well organized. B, D, and F show the cornea at low magnification. B’, D’, and F’ are the enlarged images of the primary stroma showed in the red boxes in B, D, and F, respectively; C, E, and G show the intercellular junctions between the superficial layer of the epithelium. Mp, microplicae; Ce, corneal epithelium; N, nucleus; St, stroma; En, endothelium; Le, lens epithelium; CF, collagen fiber; Tj, tight junction; BL, Bowman's layer; K, keratocyte; DM, Desçemet's membrane. Scale bars: 2 µm (B, D, F); 500 nm (B’, D’, F’); 200 nm (C, E, G).

For WT larvae at 48 hpf, the corneal epithelium had two cell layers, and the superficies formed numerous microplicae (Figs. 4B, 4C). The acellular stromal rudiment was still loose and not well organized, and some collagen fibers can be seen in the enlarged photograph (Figs. 4B, 4B’). Endothelial cells had completed their migration from the periphery to form a monolayer of endothelium (Figs. 4B, 4B’). Tight junctions were observed between the adjacent cells of the superficial layer of the epithelium (Fig. 4C). For znf469^4del/+ (Figs. 4D, 4D’, 4E) and znf469^4del/4del (Figs. 4F, 4F’, 4G) larvae at 48 hpf, the shape and structure of the cornea were similar to those of the WT larvae, suggesting that the migration and organization of the corneal epithelium and endothelium were not affected. 

For WT larvae at 72 hpf, there were two layers of epithelial cells and one layer of endothelial cells in both the central cornea (Fig. 5A) and the peripheral cornea (Fig. 5G). From 48 hpf to 72 hpf, one major change was that the primary corneal stroma developed rapidly. At 72 hpf, the thickness of the stroma increased significantly, and the collagen fibers had formed a well-organized, orthogonal, and multilayer structure (Figs. 5A, 5A’, 5G, 5G’). The other major change was that some important structures began to appear, such as the desmosomes between the adjacent cells of the two epithelial layers: the fibril-like structure in the superficial epithelium (Figs. 5B, 5G) and Bowman's layer (Figs. 5A’, 5G’). For znf469^4del/4del zebrafish at 72 hpf, the thickness of the stroma was extremely reduced compared to that of WT zebrafish at 72 hpf, and collagen fibers were barely observed and disorganized in both the central and the peripheral cornea (Figs. 5E, 5E’, 5I, 5I’). The other shapes and structures of the cornea were similar to those of the WT larvae, with a fibril-like structure, and the desmosomes (Figs. 5F, 5I). For znf469^4del/+ zebrafish at 72 hpf, no difference was observed in the corneal structure or the thickness of each layer compared to WT zebrafish (Figs. 5C, 5C’, 5D, 5H, 5H’).We measured the thickness of the total cornea and each layer of the cornea and found that, as the TEM photographs reveal, the stromal thickness of znf469^4del/4del mutant zebrafish was extremely reduced compared to that of WT zebrafish (central: 0.1942 ± 0.02607 µm vs. 0.5628 ± 0.08867 µm, P < 0.0001; peripheral: 0.2448 ± 0.0513 µm vs. 0.6668 ± 0.1200 µm, P < 0.0001). In addition, we found that the central corneal thickness of znf469^4del/4del zebrafish was thinner than that of WT zebrafish (5.215 ± 0.2018 µm vs. 5.769 ± 0.4282 µm; P = 0.0309) (Fig. 5J). For the other ocular tissues, such as retina and lens, it seems that the mutation of znf469 did not affect their morphogenesis during zebrafish development (Supplementary Fig. S1). 

Figure 5.

 

znf469 homozygous mutation in zebrafish results in a significant reduction of corneal stroma thickness at 72 hpf. The TEM ultrastructure of the cornea is shown in WT (A, A’, B, G, G’), znf469^4del/+ (C, C’, D, H, H’), and znf469^4del/4del (E, E’, F, I, I’) zebrafish larvae. The stroma components decreased significantly and the collagen fibers were few and disorderly arranged in znf469^4del/4del mutant zebrafish at 72 hpf compared to WT zebrafish. A, C, and E show the central cornea at low magnification. G, H, and I show the peripheral cornea at low magnification. A’, C’, E’, G’, H’, and I’ are the enlarged images of the primary stroma shown in the red boxes in A, C, E, G, H, and I, respectively. B, D, and F show that Fibril-like materials and cell junctions (desmosomes) can be observed in znf469^4del/+ and znf469^4del/4del zebrafish at 72 hpf, with no significant difference compared to WT zebrafish. J shows the thickness of each layer in the total, central, and peripheral cornea in WT, znf469^4del/+, and znf469^4del/4del zebrafish at 72 hpf. The thickness of the central and peripheral corneal stroma is significantly reduced in znf469^4del/4del mutant zebrafish (n = 5 for each genotype; data are shown as the mean ± SD; ^*P < 0.05, ^****P < 0.0001). Mp, microplicae; Ce, corneal epithelium; N, nucleus; St, stroma; En, endothelium; Le, lens epithelium; R, retina; BL, Bowman's layer; CF, collagen fiber; Fm, fibril-like material; Ds, desmosome. Scale bars: 1 µm (A, C, E, G, H, I); 200 nm (A’, C’, E’, G’, H’, I’); 500 nm (B, D, F).

The WT cornea at 7 dpf was similar to that observed at 72 hpf which contained two layers of epithelial cells: a stromal layer and a monolayer of endothelium. However, the epithelial cells become flattened, and the thickness of the epithelium was reduced by approximately a half comparing to that at 72 hpf (Figs. 6A, 6G). Although the stromal layer continued to grow slightly, the thickness of the stroma changed very little between 72 hpf and 7 dpf. However, the stroma was arranged in a more orderly manner (Figs. 6A, 6A’, 6G, 6G’). For znf469^4del/4del zebrafish at 7 dpf, the thickness of the stroma was also extremely reduced compared to that of WT zebrafish at 7 dpf; only a few disorganized collagen fibers were observed below Bowman's layer in the central and peripheral cornea (Figs. 6E, 6E’, 6I, 6I’, 6J). Otherwise, the shape and cellular structure of the cornea were similar to those of the WT cornea, and the desmosomes still existed (Figs. 6F, 6I). For znf469^4del/+ zebrafish at 7 dpf, no significant difference was observed in the corneal structure or the thickness of each layer compared to WT zebrafish (Figs. 6C, 6C’, 6D, 6H, 6H’, 6J). Expectedly, the corneal stroma of znf469^4del/+ adult zebrafish seemed to be unaffected compared to that of WT zebrafish (Supplementary Fig. S2). Unfortunately, due the lethality of znf469^4del/4del mutation in zebrafish, we could not investigate the phenotype in elder or adult homozygous mutant zebrafish. The above results suggest that znf469 is essential for the formation of the primary stroma during corneal development in zebrafish. 

Figure 6.

 

znf469 homozygous mutation in zebrafish results in a significant reduction of corneal stroma thickness at 7 dpf. TEM ultrastructure of the cornea is shown in WT (A, A’, B, G, G’), znf469^4del/+ (C, C’, D, H, H’), and znf469^4del/4del (E, E’, F, I, I’) zebrafish larvae at 7 dpf. The stroma components decreased significantly, and only a few orderly arranged collagen fiber lamellae can be seen in znf469^4del/4del mutant zebrafish at 7 dpf. A, C, and E show the central cornea at low magnification. G, H, and I show the peripheral cornea at low magnification. A’, C’, E’, G’, H’, and I’ are the enlarged images of the primary stroma shown in the red boxes in A, C, E, G, H, and I, respectively; B, D, and F show that fibril-like materials and cell junctions (desmosomes) could be observed in znf469^4del/+ and znf469^4del/4del zebrafish at 72 hpf, with no significant difference compared to WT zebrafish. J shows the thickness of each layer of the total, central, and peripheral cornea in WT, znf469^4del/+, and znf469^4del/4del zebrafish at 7 dpf. The thickness of the central and peripheral stroma is significantly reduced in the znf469^4del/4del mutant (n = 5 for each genotype; data are shown as the mean ± SD; ^*P < 0.05, ^****P < 0.0001). Ce, corneal epithelium; N, nucleus; St, stroma; En, endothelium; Le, lens epithelium; R, retina; BL, Bowman's layer; CF, collagen fiber; Fm, fibril-like material; Ds, desmosome. Scale bars: 500 nm (A, C, E, G, H, I); 200 nm (A’, C’, E’, G’, H’, I’, B, D).

znf469 Homozygous Mutation Leads to Downregulated Expression of Multiple Extracellular Matrix Genes

To identify the potential transcripts and pathways that znf469 regulates, and considering that ZNF469 is a pathogenic gene of systemic connective tissue disease,¹⁶ we extracted the RNA from the whole larvae of WT and homozygous mutant zebrafish at 7 dpf to apply a global transcriptome analysis using RNA-seq (National Center for Biotechnology Information Gene Expression Omnibus database, data deposit GSE225418). A total of 1227 genes were differentially expressed between the WT and homozygous mutant at the thresholds of |log2FoldChange| > 1 and Q < 0.05, of which 528 genes were upregulated and 699 genes were downregulated (Fig. 7A). All DEGs are listed in Supplementary Table S2. By function, we selected the candidate ECM-related genes that may be responsible for the corneal phenotype from the DEGs and divided them into three categories: ECM component, ECM regulator, and ECM degradation genes (Fig. 7B). As in the ECM component and regulator genes, a large number of genes encoding important corneal stroma proteins were remarkably downregulated, including most of collagen (e.g., col1a1a, col1a1b, col1a2, col2a1a, col2a1b, col5a1, col5a3b), integrin (e.g., itgb1b.2), laminin (lama1, lamb1b, and lamb4), proteoglycan (dcn, lum, kera, and gpc2), binding protein (tgfbi and hapln1a), elastin (elnb and emilin3), and keratin (krt5, krt93, and krtt1c19e) genes. In addition, the genes encoding the 26S proteasome were almost all upregulated, including, psma1, psma3–psma5, and psma6a; psmb1–psmb7; psmc1a, psmc1b, and psmc2–psmc6; psmd1, psmd3, psmd4a, psmd4b, psmd6–psmd8, psmd11a, psmd11b, and psmd12–psmd14; psme3; and adrm1, which may be related to the accelerated degradation of ECM. KEGG analysis of the DEGs revealed that the top three pathways affected by znf469 mutation were the proteasome pathway, the ECM-receptor interaction pathway, and the cell cycle pathway (Fig. 7C). Interestingly, 35 genes enriched in the proteasome pathway were all upregulated, and 19 and 23 genes enriched in the ECM-receptor pathway and cell cycle pathway, respectively, were all downregulated. The identified genes by RNA-seq were partially confirmed by qRT-PCR experiments (Fig. 7D). In summary, our data indicate that, widely considered as a transcription factor, znf469 might target a great number of ECM-related genes, including virtually all collagen and proteasome genes, to regulate the synthesis and homeostasis of ECM, thus affecting development of the corneal stroma. 

Figure 7.

 

RNA-seq analysis of transcriptomic changes in znf469 mutant larvae at 7 dpf. (A) Volcano plots depict the fold changes (log2 fold changes, x-axis) of gene expression in the two samples and the statistical significance (–log10 Q value, y-axis). |log2FoldChange)| > 1 and Q < 0.05 were considered significant. Red and green dots represent the significantly upregulated genes and downregulated genes, respectively. (B) ECM-related genes were selected from the DEGs and divided into three categories: ECM component, ECM regulator, and ECM degradation genes. (C) KEGG bubble graph of the top 15 terms related to the znf469 mutation in zebrafish. The size and color of the point indicate the gene number and P value, respectively. We mainly focused on the proteasome and ECM-receptor interaction pathways. (D) mRNA expression level analysis of ECM components and regulators identified by RNA sequencing using qRT-PCR (col1a1a, col1a1b, col1a2, col2a1a, col2a1b, col5a1, itgb1b.2, dcn, lum, kera, psma1, psmb1, psmc1a, psmd1) at 7 dpf. Three biological replicates were performed, and the data are shown as the mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Discussion

In this study, we demonstrated the ocular features of a patient with ZNF469 mutation and then we generated a znf469^4del/4del zebrafish model of BCS using CRISPR/Cas9 technology. The model reproduced the phenotypic ocular features caused by ZNF469 mutations. Deletion of znf469 led to a severe reduction in the stroma in the total cornea in zebrafish larvae from the early developmental stage (3 dpf), which is similar to the situation in which keratoconus in BCS usually appears during adolescence, even 6-week-old patients.²³ RNA-seq analysis revealed that the synthesis and degradation of a large number of ECM components, such as collagens and proteoglycans, as well as the 26S proteasome family members, are affected. 

There are both positive and negative reports on whether ZNF469 heterozygous missense mutations are related to KC.^11–15 These conflicts may be due to the difficulty of assessing the impact of missense mutations on such a poorly characterized protein in silico and the different regions and races. The patient reported in this study developed KC in BCS. In the zebrafish model, znf469^4del/+ zebrafish did not display an apparent phenotype in cornea or other tissues, which is consistent with the observation in mice.¹⁸ This observation suggests that znf469 may have varied roles in different species or that the heterozygous missense mutations in humans bring about gain-of-function or dominant-negative effects.¹⁴ Different from in mice and humans, depletion of Znf469 is lethal and induces more severe disorder in corneal stroma in zebrafish, suggesting that it may play more important roles in zebrafish. We observed only the sharp decrease in the thickness of the corneal stroma, but not in total corneal thickness. It should be because the stroma accounts for 90% of the human cornea (approximately 500 µm),²⁴ but in zebrafish at 3 to 7 dpf and adults, the percentages are only 10% to 15% and 30% to 40%, respectively,^20,25 and the depletion of Znf469 did not affect the structure of the corneal epithelia and endothelia. Unfortunately, the lethality of znf469^4del/4del in zebrafish larvae prevented us from exploring the longer course of the disease in this model. Notably, the cellular origin and composition of the corneal stroma under study here are those of the primary stroma, not the same as the thin stroma apparent in BCS, although it may be a precursor to it. 

At the molecular level, our data suggest that many more downstream candidates than current known may be affected by depletion of Znf469. Collagen I is the major component of the corneal stroma in vertebrates and is encoded by COL1A1 and COL1A2.²⁶ Consistent with in mice, reduced col1a1a was observed in znf469^4del/4del zebrafish mutants. We also observed a noninflated swimbladder in the mutants, resembling the col1a1a^−/− zebrafish model.²⁷ Primary keratocyte cultures taken from the Zfp469^BCS/BCS mouse indicate the significantly decreased synthesis of Col1a1 and Col1a2 but not Col5a1.¹⁸ In zebrafish, it seems that depletion of Znf469 affects the expression of most of collagen, such as col1a1a, col1a1b, col1a2, col2a1a, col2a1b, col5a1, and col5a3b. PRDM5 (PR/SET domain 5) is considered to act on a common pathway regulating ECM gene expression.^1,28 As a transcriptional regulator, Prdm5 targets all mouse collagen genes and some other proteoglycan genes. Moreover, Prdm5 sustains the transcription of collagen I genes by binding RNA polymerase II,²⁹ which closely resembles our results. We infer that znf469 may comparably target a large number of ECM component genes and regulates their transcription. 

An additional interesting result is the overall upregulation of proteasome family members and other regulators in znf469^4del/4del mutant zebrafish. Supporting this observation, a proteomic study of KC indicated that ubiquitin proteasomal degradation pathway members were elevated.³⁰ A patient with ZNF469 mutation showed increased urinary pyridinoline (PYR) and deoxypyridinoline (DPYR) concentrations and an elevated DPYR/PYR ratio,³¹ suggesting complicated downstream targets of ZNF469. Although we cannot exclude the possibility that the proteasome genes were upregulated due to the misfolding of the truncated Znf469 protein, we infer that ZNF469 may regulate the transcription of 26S proteasome family members, thereby promoting the degradation of ECM. Together with the reduced synthesis of ECM components, ZNF469 mutations ultimately result in all of the phenotypes. Further experiments such as chromatin immunoprecipitation followed by sequencing should be performed to clarify its roles as a considered transcription factor and the downstream candidates of Znf469. Furthermore, the RNA-seq data reported in this study were obtained from whole embryos; tissue-specific sample preparations for RNA-seq would be beneficial for obtaining insight into BCS. 

In summary, this study suggests that znf469 is a critical gene that may regulate the synthesis and degradation of a large number of ECM components and is closely related to the formation of BCS. 

Acknowledgments

The authors thank the Bio-Ultrastructure Analysis Lab of the Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University, for TEM assistance. 

Supported by grants from the Program of National Natural Science Foundation of China (82171033 and 81970781 to XS) and the Natural Science Foundation of Zhejiang Province (LD21H120001 to XS). 

Disclosure: J. Bao, None; X. Yu, None; X. Ping, None; X. Shentu, None; J. Zou, None