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

Abstract

Background: Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome.

Results: Query of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation.

Conclusions: Using genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.

Keywords: CPLANE; Eye development; IMPC; MAC spectrum; Mouse; Serine-glycine biosynthesis.

Fig. 1

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

Fig. 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 (E–H). For typically developing eye, please refer to Fig. 1

Fig. 3

Examples of embryos with homozygous null mutations of genes associated with both microphthalmia (A–G) and anophthalmia (A’–G’) at different stages of development. Typically developing mice (WT, A–D) 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’)

Fig. 4

MicroCT (μCT) images of eye abnormalities in E15.5 null (B–P, R–X), heterozygous (Q), and WT (A) embryos. Eye anomalies ranging in severity from bilateral anophthalmia (B–H), anophthalmia with or without microphthalmia (I–M), and various degrees of microphthalmia (N–X) are shown. Additional μCT data are available on the IMPC portal

Fig. 5

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

Fig. 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

Fig. 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

Fig. 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)