Genome-wide screening reveals the genetic basis of mammalian embryonic eye development

Query of the IMPC phenotype database (August 2022/IMPC data release 17) identified 74 knockout mouse lines with significantly higher incidence of eye anomalies compared to WT (wild-type) controls, suggesting these genes are implicated in embryonic eye development (see Additional file 1: Table S1 for a complete gene list). Genes resulting in eye anomalies were noted at different stages of development: E9.5 (n = 8), E12.5 (n = 14), E15.5 (n = 37), and E18.5 (n = 15), typically associated with the windows of lethality of the HOM mutants. However, for 11 of the genes, MAC phenotypes were noted at more than one developmental age. As these genes were embryo lethal, the eye phenotypes were present predominantly in HOM embryos (> 90%). For 27 of these 74 genes, ocular anomalies were noted in HET adult mice during standardized examination of the anterior and posterior segments of the eyes performed at 15 weeks of age as part of the IMPC adult phenotyping pipeline.

A search of the IMPC database for anophthalmia revealed 24 knockout lines with documented evidence of absent eyes in embryos: Atp13a1, Cep135, Dzip1l, Elavl1, Ercc4, Faf2, Focad, Fuz, Gabpa, Ggnbp2, Hesx1, Ino80c, Lrrc8a, Rab34, Rbm45, Rexo1, Slc25a1, Slc36a1, Snx3, Tbc1d32, Tctn3, Ubn2, Uggt1, and Zfp503. Representative examples are shown in Fig. 1. A similar search of DR17 using the term microphthalmia resulted in 22 genes significant for the small eyes phenotype: Aldh1a3, Aff4, Bmp4, Cdk4, Cox6b1, Cxcr4, Dync1li1, Eef1d, Fgd1, Gne, Grh12, Mab21l2, Maf, Med13l, Mllt10, Mthfd2, Pex6, Phgdh, Ssr1, Stim1, Tdo2, and Vps26c (Fig. 2). Several genes (n = 16) resulted in embryos expressing both microphthalmia and anophthalmia, including Acvr2a, Anapc15, Ankrd52, Axin2, Bmi1, Dnmt3b, Inpp5e, Mmachc, Mtf1, Pax6, Psph, Pygo2, Rgl1, Snx3, Tmem209, and Togaram1 (Fig. 3).

Fig. 1figure 1

Examples of wild-type (WT) control (AD) and mutant embryos (EH) with homozygous null mutations of genes associated with anophthalmia at different stages of development. Typically developing mice (WT, AD) on C57BL/6N background are shown for comparison

Fig. 2figure 2

Examples of embryos with homozygous null mutations of genes associated with abnormal development of optic vesicle at E9.5 (arrows, A, B), microphthalmia and coloboma at E12.5 (C, D), and E15.5 (EH). For typically developing eye, please refer to Fig. 1

Fig. 3figure 3

Examples of embryos with homozygous null mutations of genes associated with both microphthalmia (AG) and anophthalmia (A’G’) at different stages of development. Typically developing mice (WT, AD) on C57BL/6N background are shown in Fig. 1. Note the presence of additional anomalies such as short or absent mandible (C’, D, D’, E, E’, F, F’), oral cleft (D, E, E’, G, G’), exencephaly (G, G’), and moderate to severe edema (D, D’, E’)

Facilities at some IMPC centers were equipped with μCT capability and were thus able to document ophthalmic abnormalities with high resolution in E15.5 and E18.5 mutants and contrast these findings to littermate WT mice (Figs. 4 and 5). These images show that the MAC spectrum ranged in severity from mild microphthalmia with small discernible eyes, severe microphthalmia cases where there is no visible eye, but ocular remnants are found within the orbital sockets, to anophthalmia, with embryos having no visible eyes along with an empty space within the orbit. Histology examination of a subset of these lines at E15.5 confirmed these phenotypes (Fig. 6).

Fig. 4figure 4

MicroCT (μCT) images of eye abnormalities in E15.5 null (BP, RX), heterozygous (Q), and WT (A) embryos. Eye anomalies ranging in severity from bilateral anophthalmia (BH), anophthalmia with or without microphthalmia (IM), and various degrees of microphthalmia (NX) are shown. Additional μCT data are available on the IMPC portal

Fig. 5figure 5

MicroCT images of eye abnormalities in E18.5 null (BJ) and WT (A) embryos. Eye anomalies ranging in severity from bilateral anophthalmia (B, C), anophthalmia (D, E), and various degrees of microphthalmia (FJ) are shown. Additional μCT data available on the IMPC portal

Fig. 6figure 6

Coronal sections of E15.5 WT (A), homozygous (B, C), and heterozygous (D) E15.5 embryos stained with hematoxylin and eosin showing examples of MAC phenotypes. Arrow (D) indicates the presence of ocular remnant

To verify the embryonic expression of the targeted genes in ocular tissues, we examined HET E12.5 embryo staining for β-galactosidase activity, taking advantage of the inserted LacZ reporter cassette in the targeted allele. Heterozygous embryos were chosen rather than HOM in order to approximate a normal gene expression pattern and normal gross embryonic morphology (Fig. 7). We identified positive staining within the eyes in every knockout line for which corresponding LacZ-stained E12.5 embryos were available (Fig. 7B–K), in contrast to LacZ stained WT embryos (Fig. 7A); magnification of the eye (insets) shows positive staining within the ocular tissues. The inset photos are modified to enhance contrast to better visualize the ocular LacZ staining pattern. The positive confirmation of ocular gene expression in the presumably normal eye of all available heterozygous embryos lends credibility to the mechanistic requirement for these genes in early eye development.

Fig. 7figure 7

Examples of whole embryo (E12.5) LacZ histochemistry within heterozygous C57BL/6N embryos (A-K). Heterozygous embryos were chosen since most appear phenotypically normal. Positive LacZ is taken as a surrogate of endogenous gene expression. Magnification of the eye (inset) shows the positive staining in the ocular tissues in each case

We performed a literature search to determine the degree to which these 74 genes had established roles in eye development and MAC spectrum diseases. Of the 74 MAC genes identified in mouse embryos among the 8267 single gene mutant lines produced and phenotyped by the IMPC to date, 27 lines had published knockout models in peer reviewed publications, of which 15 reported eye abnormalities (with 9 publications reporting a MAC spectrum phenotype), and 12 KO (knockout) models with no eye phenotypes recorded. Taken together, the 47 unpublished knockout lines and the 12 published knockouts with unreported eye anomalies comprise a total of 59 genes related to early eye formation which were previously unrecognized as being associated with eye development (Additional file 1: Table S2). Of the 15 genes with published eye anomaly reports, the results from the IMPC screen for MAC phenotype confirmed these previously published mouse models with MAC spectrum, such as Bcl11b−/− with gross morphology findings of abnormal eyelid fusion in E18.5 embryos (Fig. 8A), as reported in the extant KO mouse model [15]; Pax6−/− and Inpp5e−/− with anophthalmia [16, 17]; Axin2−/− with coloboma, microphthalmia, and abnormal eyelid fusion [18]; and Psph−/− showing strong correlation with the phenotypes (abnormal eye muscle, optic stalk, optic cup, lens morphology, absent lens) reported in a Psph KO mouse model cataloged by the Deciphering the Mechanisms of Developmental Disorders (DMDD) program [19].

Fig. 8figure 8

Examples of eye malformations in knockout embryonic mice different from microphthalmia and/or anophthalmia. E18.5 Bc1llb null mutant (A’) has abnormal eyelid fusion compared to wild-type C57BL/6N (A). E15.5 Acvr2a null mutant exhibits cyclopia (B’) compared to wild-type C57BL/6N mice (B)

Nineteen of the 59 genes with unpublished mouse findings had a reported eye phenotype in humans either from existing literature and/or from syndromes indexed in OMIM. As such, our study provided a mouse correlate for a holoprosencephaly-suspected gene from a case report, ACVR2 [20], with cyclopia (Fig. 8B) observed in Acvr2a−/− E15.5 embryos confirming the phenotype. Similarly, the microphthalmia phenotype in Aff4−/− E15.5 embryos is consistent with the higher incidence of eye anomalies and cataracts in patients with CHOPS syndrome many of whom also exhibit heterozygous mutations of the AFF4 gene [21].

Importantly, these 40 genes (Acvr2a, Alg10b, Anapc15, Ankrd52, Atp13a1, Cox6b1, Cxcr4, Dzip1l, Eef1d, Ercc4, Faf2, Focad, Gabpa, Ggnbp2, Gne, Ino80c, Laptm4b, Med1, Med13l, Mllt10, Mtf1, Mthfd2, Pcnp, Phgdh, Pigq, Rab34, Rbm45, Rexo1, Rgl1,Slc25a1, Slc36a1, Ssr1, Stl4, Stim1, Tmem209, Ube2f, Ubn2, Uggt1, Vps26c, Zfp503) represent genes that contribute to early eye development that were previously unrecognized.

In order to identify the novel pathways that may be implicated in early eye formation, we utilized the Panther tool [22] within Gene Ontology [23, 24] to determine which pathways are important for early eye development in the 74 IMPC knockout lines. This tool revealed a number of pathways that are known to be important in early eye development based on the analysis of the 114 gold standard MAC spectrum genes (Additional file 1: Fig. S1). There were several pathways important in early eye formation in our 74 IMPC knockout lines that were not implicated in the established gold standard genes. One of these was the serine-glycine biosynthesis pathway (Additional file 1: Fig. S2). There were also a number of evolutionarily conserved pathways common to both our IMPC knockout lines and the gold standard list including the Hedgehog, WNT, and the TGFβ signaling pathways (Additional file 1: Fig. S3).

We used the STRING biological database software to predict protein-protein interactions within the 74 genes from the IMPC. Among the analysis of these 74 genes, STRING produced a network of predicted interactions between many of the gene products. The 114 genes from the gold standard MAC spectrum disease gene list were analyzed similarly for predicted interaction networks. When comparing the 74 genes generated from the IMPC database and the 114 genes from the gold standard list, eight genes (ALDH1A3, BMP4, MAB21L2, MAF, ME13L, PAX6, SNX3, and TBC1D32) overlapped between the two groups (Additional file 1: Fig. S4). Cytoscape was used to merge the two networks together to produce a broader picture of interactions between eye development proteins. There were a total of 180 genes within the merged network: 74 genes from IMPC mouse knockout models, 114 human MAC spectrum genes, and eight of which were in both groups. Within these STRING interactions, the serine-glycine biosynthesis pathway and members of ciliogenesis and planar polarity effectors (CPLANE) are encircled; genes associated with the signaling pathways regulating pluripotency of stem cells are identified by stars (Additional file 1: Fig. S5). In order to incorporate both our findings from Gene Ontology regarding the serine-glycine metabolism and signaling pathways regulating pluripotency of stem cells and our findings from STRING regarding the specific genes involved within each pathway, we utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [25] within the Database for Annotation, Visualization, and Integrated Discovery (DAVID) program to map out the pathways (Additional file 1: Figs. S2 and S3) [22, 26].

We used CiliaCarta, a publicly available bioinformatic analysis tool [27], to investigate the proportion of genes that have an established functional role in the primary cilium. There were 12 of the 114 of gold standard genes associated with MAC spectrum disease implicated in ciliary function. A similar analysis of our list of IMPC mouse genes identified eight (Cep135, Dzip1l, Fuz, Inpp5e, Pex6, Sufu, Tctn3, and Togaram1) of the 74 genes that had ciliary function. These twelve human and eight mouse genes and their roles in ciliopathies are summarized in Additional file 1: Table S3.

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