Chee et al. BMC Biology (2023) 21:22 BMC Biology https://doi.org/10.1186/s12915-022-01475-0 RESEARCH ARTICLE Open Access Genome‑wide screening reveals the genetic basis of mammalian embryonic eye development Justine M. Chee1†, Louise Lanoue2†, Dave Clary2, Kendall Higgins3, Lynette Bower2, Ann Flenniken4,5, Ruolin Guo4,6, David J. Adams7, Fatima Bosch8, Robert E. Braun9, Steve D. M. Brown10, H.‑J. Genie Chin11, Mary E. Dickinson12, Chih‑Wei Hsu12, Michael Dobbie13, Xiang Gao14, Sanjeev Galande15, Anne Grobler16, Jason D. Heaney12, Yann Herault17, Martin Hrabe de Angelis18, Fabio Mammano19, Lauryl M. J. Nutter4,6, Helen Parkinson20, Chuan Qin21, Toshi Shiroishi22, Radislav Sedlacek23, J‑K Seong24, Ying Xu25, The International Mouse Phenotyping Consortium, Brian Brooks26, Colin McKerlie6,27, K. C. Kent Lloyd2,28, Henrik Westerberg10 and Ala Moshiri29,30* 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 malforma‑ tions is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular devel‑ opmental 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. Addi‑ tionally, 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 These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice.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 † Justine M. Chee and Louise Lanoue are co-first authors and contributed equally to this work. *Correspondence: Ala Moshiri amoshiri@ucdavis.edu Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chee et al. BMC Biology (2023) 21:22 Page 2 of 15 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 MAC spectrum, Eye development, Mouse, IMPC, Serine-glycine biosynthesis, CPLANE Background initially form, but eventually degenerate [10]. Coloboma The molecular mechanisms of microphthalmia, ano- is a closure defect within the optic fissure, typically caus- phthalmia, and coloboma (MAC) spectrum disease are ing a “keyhole” shape within the iris, inferior lens irregu- not fully defined. This study searched 8267 single gene larity, and an inferonasal defect in the retina and choroid knockout mouse lines produced and phenotyped by the layers of the eye, though patients may only exhibit the International Mouse Phenotyping Consortium [1–4], to posterior findings. These eye malformations play a role identify the novel genes and pathways required for mam- in childhood visual impairment, with microphthalmia malian eye development and MAC spectrum disease. being present in up to 11% of blind children [11–13]. The Eye development begins shortly after gastrulation with prevalence of microphthalmia is approximated as one in the specification of a single eye field in the anterior neu- 7000 live human births, one in 30,000 live births for ano- ral plate by a set of highly conserved eye field transcrip- phthalmia, and one in 5000 live births for coloboma [14]. tion factors [5, 6]. During neurulation, two lateral optic The predominant cause of early eye malformations is her- pits form as the lateral walls of the diencephalon pro- itable in nature, and up to 10–15% of siblings of individu- trude outward, forming the left and right optic vesicles, als with microphthalmia and/or anophthalmia have a risk and can be visually detected in mice at ~E8.0 [7]. Anteri- of being affected [9]. In severe cases of bilateral micro- orly, these optic vesicles come in contact with the surface phthalmia or anophthalmia, about 80% of cases show ectoderm to form the optic placode at ~E9.0, which will loss-of-function mutations in OTX2 or SOX2 [14]. The develop into the lens and a portion of the cornea. Poste- genetic abnormalities within the other 20% of patients riorly, the neural ectoderm invaginates to form the optic with MAC spectrum disease are mostly unknown. In cup and the optic stalk which eventually form the neural addition, the pathways involved in embryogenesis of the retina, the retinal pigmented epithelium (RPE), and the eye are also incompletely understood. Therefore, through optic nerve. A furrow forms between the optic stalk and systematic forward screening of the mammalian genome, the optic cup, forming the choroidal fissure, which even- this study provides additional insight into the genetic tually fuses together to form the eye [8]. MAC spectrum basis of the morphogenetic pathways leading to MAC eye defects can occur at any stage of ocular embryogen- spectrum diseases, which may ultimately contribute to esis, with microphthalmia and anophthalmia usually better detection, diagnosis, and therapy. occurring at 4 to 6 weeks of human gestation [9]. While most of these cases occur in isolation within a continuum Results of phenotypic severity, one-third of cases present as part Query of the IMPC phenotype database (August 2022/ of a multi-systemic syndrome [10]. IMPC data release 17) identified 74 knockout mouse Clinically in humans, microphthalmia is defined as lines with significantly higher incidence of eye anomalies having an axial length of less than 10 mm (more than two compared to WT (wild-type) controls, suggesting these standard deviations below 21 mm) and a corneal diam- genes are implicated in embryonic eye development (see eter of less than 10 mm. Anophthalmia is characterized Additional file 1: Table S1 for a complete gene list). Genes as having no ocular tissue within the orbit. There are resulting in eye anomalies were noted at different stages three different categories of anophthalmia: primary ano- of development: E9.5 (n = 8), E12.5 (n = 14), E15.5 (n phthalmia caused by a failure of the optic pit to develop = 37), and E18.5 (n = 15), typically associated with the into the optic vesicle, secondary anophthalmia caused by windows of lethality of the HOM mutants. However, for a developmental failure within the anterior neural tube, 11 of the genes, MAC phenotypes were noted at more and degenerative anophthalmia, where the optic vesicles than one developmental age. As these genes were embryo Chee et al. BMC Biology (2023) 21:22 Page 3 of 15 lethal, the eye phenotypes were present predominantly in WT mice (Figs. 4 and 5). These images show that the HOM embryos (> 90%). For 27 of these 74 genes, ocular MAC spectrum ranged in severity from mild microph- anomalies were noted in HET adult mice during stand- thalmia with small discernible eyes, severe microph- ardized examination of the anterior and posterior seg- thalmia cases where there is no visible eye, but ocular ments of the eyes performed at 15 weeks of age as part of remnants are found within the orbital sockets, to anoph- the IMPC adult phenotyping pipeline. thalmia, with embryos having no visible eyes along with A search of the IMPC database for anophthalmia an empty space within the orbit. Histology examination revealed 24 knockout lines with documented evidence of a subset of these lines at E15.5 confirmed these pheno- of absent eyes in embryos: Atp13a1, Cep135, Dzip1l, types (Fig. 6). Elavl1, Ercc4, Faf2, Focad, Fuz, Gabpa, Ggnbp2, Hesx1, To verify the embryonic expression of the targeted Ino80c, Lrrc8a, Rab34, Rbm45, Rexo1, Slc25a1, Slc36a1, genes in ocular tissues, we examined HET E12.5 embryo Snx3, Tbc1d32, Tctn3, Ubn2, Uggt1, and Zfp503. Repre- staining for β-galactosidase activity, taking advantage of sentative examples are shown in Fig. 1. A similar search the inserted LacZ reporter cassette in the targeted allele. of DR17 using the term microphthalmia resulted in 22 Heterozygous embryos were chosen rather than HOM genes significant for the small eyes phenotype: Aldh1a3, in order to approximate a normal gene expression pat- Aff4, Bmp4, Cdk4, Cox6b1, Cxcr4, Dync1li1, Eef1d, Fgd1, tern and normal gross embryonic morphology (Fig. 7). Gne, Grh12, Mab21l2, Maf, Med13l, Mllt10, Mthfd2, We identified positive staining within the eyes in every Pex6, Phgdh, Ssr1, Stim1, Tdo2, and Vps26c (Fig. 2). Sev- knockout line for which corresponding LacZ-stained eral genes (n = 16) resulted in embryos expressing both E12.5 embryos were available (Fig. 7B–K), in contrast to microphthalmia and anophthalmia, including Acvr2a, LacZ stained WT embryos (Fig. 7A); magnification of Anapc15, Ankrd52, Axin2, Bmi1, Dnmt3b, Inpp5e, the eye (insets) shows positive staining within the ocular Mmachc, Mtf1, Pax6, Psph, Pygo2, Rgl1, Snx3, Tmem209, tissues. The inset photos are modified to enhance con- and Togaram1 (Fig. 3). trast to better visualize the ocular LacZ staining pattern. Facilities at some IMPC centers were equipped with The positive confirmation of ocular gene expression in μCT capability and were thus able to document oph- the presumably normal eye of all available heterozygous thalmic abnormalities with high resolution in E15.5 and embryos lends credibility to the mechanistic requirement E18.5 mutants and contrast these findings to littermate for these genes in early eye development. 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 Chee et al. BMC Biology (2023) 21:22 Page 4 of 15 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’) Chee et al. BMC Biology (2023) 21:22 Page 5 of 15 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 We performed a literature search to determine the models in peer reviewed publications, of which 15 degree to which these 74 genes had established roles in reported eye abnormalities (with 9 publications report- eye development and MAC spectrum diseases. Of the ing a MAC spectrum phenotype), and 12 KO (knock- 74 MAC genes identified in mouse embryos among the out) models with no eye phenotypes recorded. Taken 8267 single gene mutant lines produced and phenotyped together, the 47 unpublished knockout lines and the 12 by the IMPC to date, 27 lines had published knockout published knockouts with unreported eye anomalies Chee et al. BMC Biology (2023) 21:22 Page 6 of 15 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 comprise a total of 59 genes related to early eye forma- Nineteen of the 59 genes with unpublished mouse tion which were previously unrecognized as being associ- findings had a reported eye phenotype in humans either ated with eye development (Additional file 1: Table S2). from existing literature and/or from syndromes indexed Of the 15 genes with published eye anomaly reports, the in OMIM. As such, our study provided a mouse corre- results from the IMPC screen for MAC phenotype con- late for a holoprosencephaly-suspected gene from a case firmed these previously published mouse models with report, ACVR2 [20], with cyclopia (Fig. 8B) observed in MAC spectrum, such as Bcl11b−/− with gross morphol- Acvr2a−/− E15.5 embryos confirming the phenotype. ogy findings of abnormal eyelid fusion in E18.5 embryos Similarly, the microphthalmia phenotype in Aff4−/− (Fig. 8A), as reported in the extant KO mouse model E15.5 embryos is consistent with the higher incidence of [15]; Pax6−/− and Inpp5e−/− with anophthalmia [16, 17]; eye anomalies and cataracts in patients with CHOPS syn- Axin2−/− with coloboma, microphthalmia, and abnor- drome many of whom also exhibit heterozygous muta- mal eyelid fusion [18]; and Psph−/− showing strong cor- tions of the AFF4 gene [21]. relation with the phenotypes (abnormal eye muscle, optic Importantly, these 40 genes (Acvr2a, Alg10b, Anapc15, stalk, optic cup, lens morphology, absent lens) reported Ankrd52, Atp13a1, Cox6b1, Cxcr4, Dzip1l, Eef1d, Ercc4, in a Psph KO mouse model cataloged by the Deciphering Faf2, Focad, Gabpa, Ggnbp2, Gne, Ino80c, Laptm4b, the Mechanisms of Developmental Disorders (DMDD) Med1, Med13l, Mllt10, Mtf1, Mthfd2, Pcnp, Phgdh, Pigq, program [19]. Rab34, Rbm45, Rexo1, Rgl1,Slc25a1, Slc36a1, Ssr1, Stl4, Chee et al. BMC Biology (2023) 21:22 Page 7 of 15 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) Stim1, Tmem209, Ube2f, Ubn2, Uggt1, Vps26c, Zfp503) were in both groups. Within these STRING interactions, represent genes that contribute to early eye development the serine-glycine biosynthesis pathway and members that were previously unrecognized. of ciliogenesis and planar polarity effectors (CPLANE) In order to identify the novel pathways that may be are encircled; genes associated with the signaling path- implicated in early eye formation, we utilized the Panther ways regulating pluripotency of stem cells are identified tool [22] within Gene Ontology [23, 24] to determine by stars (Additional file 1: Fig. S5). In order to incorpo- which pathways are important for early eye develop- rate both our findings from Gene Ontology regarding the ment in the 74 IMPC knockout lines. This tool revealed serine-glycine metabolism and signaling pathways regu- a number of pathways that are known to be important in lating pluripotency of stem cells and our findings from early eye development based on the analysis of the 114 STRING regarding the specific genes involved within gold standard MAC spectrum genes (Additional file 1: each pathway, we utilized the Kyoto Encyclopedia of Fig. S1). There were several pathways important in early Genes and Genomes (KEGG) database [25] within the eye formation in our 74 IMPC knockout lines that were Database for Annotation, Visualization, and Integrated not implicated in the established gold standard genes. Discovery (DAVID) program to map out the pathways One of these was the serine-glycine biosynthesis pathway (Additional file 1: Figs. S2 and S3) [22, 26]. (Additional file 1: Fig. S2). There were also a number of We used CiliaCarta, a publicly available bioinformatic evolutionarily conserved pathways common to both our analysis tool [27], to investigate the proportion of genes IMPC knockout lines and the gold standard list including that have an established functional role in the primary the Hedgehog, WNT, and the TGFβ signaling pathways cilium. There were 12 of the 114 of gold standard genes (Additional file 1: Fig. S3). associated with MAC spectrum disease implicated in cili- We used the STRING biological database software to ary function. A similar analysis of our list of IMPC mouse predict protein-protein interactions within the 74 genes genes identified eight (Cep135, Dzip1l, Fuz, Inpp5e, Pex6, from the IMPC. Among the analysis of these 74 genes, Sufu, Tctn3, and Togaram1) of the 74 genes that had cili- STRING produced a network of predicted interactions ary function. These twelve human and eight mouse genes between many of the gene products. The 114 genes from and their roles in ciliopathies are summarized in Addi- the gold standard MAC spectrum disease gene list were tional file 1: Table S3. analyzed similarly for predicted interaction networks. When comparing the 74 genes generated from the IMPC Discussion database and the 114 genes from the gold standard list, The IMPC phenotyping pipeline consists of a stand- eight genes (ALDH1A3, BMP4, MAB21L2, MAF, ME13L, ardized set of minimally invasive phenotype tests for PAX6, SNX3, and TBC1D32) overlapped between the two early adult mice covering 11 organ systems from 4 to 16 groups (Additional file 1: Fig. S4). Cytoscape was used to weeks of age. Mice are then sacrificed, and samples are merge the two networks together to produce a broader collected for a standardized set of terminal tests that picture of interactions between eye development pro- include gross pathology, clinical chemistry, immunophe- teins. There were a total of 180 genes within the merged notyping, and histopathology [28]. Approximately 30% of network: 74 genes from IMPC mouse knockout models, knockout lines are homozygous lethal at various stages 114 human MAC spectrum genes, and eight of which of embryonic development [3]. A standardized embryo Chee et al. BMC Biology (2023) 21:22 Page 8 of 15 phenotyping pipeline is used by IMPC centers to identify females). The sexual dimorphism in this background rate the window of lethality, characterize embryo phenotypes of microphthalmia/anophthalmia warrants further study. using gross morphological examination, and identify Although the rd8 mutation present in C57BL/6N mice and quantify abnormalities using 3D imaging. A subset may contribute to the MAC phenotype in these 74 lines, of lines is also analyzed for gene expression pattern with the fact that the eye defects were observed in HOM/ whole mount β-galactosidase (LacZ) staining. Phenotyp- HEMI embryos (34 of 50 genes) but not in the HET and ing tests are done using standardized protocols published WT embryos, that many of the lines (Axin2, Bmi1, Fgd1, on the IMPC website [29]. Data and images collected Ggnbp2, Maf, Med13L,Pax6, Rgl1,Top2b), showed a gene during phenotyping are transferred to the IMPC Data dosage effect with increasing severity or expression with Coordination Center (DCC), quality-controlled, and ana- increasing mutated alleles, and that all eye phenotypes lyzed using OpenStats [30]. After curation by the DCC, are significantly different from WT of the same sex and the data is uploaded to the IMPC website for the bio- background point to a direct association between these medical community to freely access. The IMPC database genes and the MAC spectrum. Analyses of these 74 genes is dynamic with additional data releases several times revealed the importance of the serine-glycine pathway, each year. For example, at the time we initiated this study, the ciliogenesis and planar polarity effectors (CPLANE) the IMPC Data release 11.0 had generated 6900 unique complex, and the signaling pathways for progenitor cell gene knockout mouse lines and completed phenotyp- maintenance to be three critical mechanisms required for ing on 6440 of these lines. Our query of DR11 resulted eye formation. in 63 genes with MAC phenotypes. However, because it included lines with incomplete phenotyping, an up- Serine‑glycine pathway within the folate cycle to-date query (DR17) resulted in the loss of genes hav- Merging the 74 IMPC genes and the 114 MAC spec- ing a significant eye phenotype (n = 27) and a discovery trum genes using STRING revealed a cluster of inter- of new genes with MAC (n = 38) for revised total of 74 related genes that are involved in serine-glycine genes with MAC phenotypes. biosynthesis within the folate cycle. While folate defi- Thanks to the effort of the IMPC to produce and ana- ciency is associated with eye defects, and components lyze sex balanced cohorts of mutant mice for each gene of the serine-glycine pathway have been known to in the mouse genome, forward genetic approaches can be cause congenital eye disease [31–34], the serine-glycine applied to better understand eye development in mam- pathway has never previously been associated with the mals. In this study, we identified 74 genes associated with MAC spectrum disorder. Our query also uncovered a developmental eye defect of the MAC spectrum, in three novel genes (Mthfd2, Phgdh, Slc25a1) not previ- which 59 of these were previously unrecognized in mice ously recognized as an underlying fundamental mecha- and 40 genes novel to humans, providing new avenues of nistic factor in MAC spectrum disease and associated investigation on the molecular mechanisms regulating with this pathway (Additional file 1: Fig. S2). The serine eye development. Among the 15 genes with published glycine biosynthesis pathway is important in convert- eye phenotypes, 9 of the lines from the IMPC program ing glucose into serine and glycine to meet the cellular had eye phenotypes similar to that of published mouse requirements for serine [35]. Glucose is imported into models (Axin2, Adh1a3, Bcl11b, Fuz, Inpp5e, Mab21l2, cells and then converted into an intermediate, phos- Maf, Pax6, Pygo2); the remaining 5 lines resulted in phohydroxy pyruvate, through catalysis by phospho- embryonic eye phenotypes (Acvr2a, Bmi1, Dnmt3b, Dyn- glycerate dehydrogenase (PHGDH). This intermediate c1li1, Hesx1) more severe than the phenotypes of extant is then converted into P-serine by phosphoserine ami- models which consisted of septo-optic dysplasia (Hesx1), notransferase (PSAT1), then converted into serine by abnormal retinal vascularization (Acvr2a), and retinal phosphoserine phosphatase (PSPH) and further into and/or photoreceptor degeneration (Bmi1, Dnmt3b, Dyn- glycine by serine hydroxymethyl transferase (SHMT) c1li1). There was one exception; Bmp4 HOM embryos [36]. Mutations PHGDH, PSPH, and PSAT1 in the exhibited a less severe eye phenotype than expected from serine-glycine pathway have been associated with Neu- published model. The fact that the HOM lines generated Laxova syndrome, which is characterized by a spec- by the IMPC were embryonic lethal at E15.5 or E18.5 trum of phenotypes that vary in expression but usually compared to previous models which survive beyond manifests by perinatal lethality, microcephaly, skeletal, birth may explain why the IMPC mutants had a more and skin anomalies [37]. Eye and brain defects includ- severe eye phenotype, consistent with more severe lethal- ing shortened or absent eyelids, proptosis, microph- ity. Wild-type mice of a similar C57BL/6N background thalmia, microcephaly, lissencephaly, and agenesis of showed a rate of microphthalmia in E15.5 embryos of the corpus callosum, and neural tube defects have been 8.7% (80% females) and anophthalmia in 0.7% (88% reported [38, 39]. Chee et al. BMC Biology (2023) 21:22 Page 9 of 15 We hypothesize that mutations of genes within the ser- disruption of organogenesis during development includ- ine-glycine pathway contribute to defects in eye develop- ing the eyes. Examination of μCT images of E15.5 HOM ment, as glycine and serine are important sources of 1C Fuz mutant embryos uncovered the extent of embryonic units to the folate one-carbon metabolism (FOCM) via defects resulting from impaired primary cilia formation, the cleavage of glycine by glycine decarboxylase (GLDC) and likely subsequent impaired sonic hedgehog (SHH) [40]. Interestingly, Gldc null embryos exhibited high inci- signaling, and planar cell polarity (PCP) regulation; these dence of neural tube defects (57%) and microphthalmia include absent lungs and pituitary, agenesis of the tongue, or anophthalmia (30%) [40]. Moreover, serine and ser- hypoplastic mandible, cleft palate, Meckel’s cartilage ine-derived metabolites such as sphingolipids have been malformation, dextrocardia and heart anomalies, absent shown to support critical functions in maintaining retina or polycystic kidneys, and neural tube defects (data not integrity [41]. We postulate that by having defects within shown). serine-glycine biosynthesis, there will be decreased con- version of serine to glycine, which leads to decreased Signaling pathways regulating pluripotency of stem cells transfer of one-carbon units to the mitochondrial FOCM. STRING analysis also revealed a cluster of genes that are This in turn can lead to neural tube and subsequent (sec- involved in three critical signaling pathways that regulate ondary) embryonic eye defects on the MAC spectrum stem cell differentiation and support critical processes during embryogenesis. (cell fate determination, patterning, and polarity) during embryogenesis, including novel genes. Studies show that Ciliogenesis and planar polarity effector (CPLANE) complex early eye specification, optic cup differentiation, and eye Our IMPC query for MAC phenotype-associated genes morphogenesis are regulated by the integrated cross-talk included several members of the CPLANE complex between these signaling pathways and transcription fac- including Gabpa and Dzip1l, which were not previously tors [49–52]. Knockouts of genes involved in the SHH, associated with eye defects. Primary cilia are important the transforming growth factor beta (TGFβ)/bone mor- components of the retinal photoreceptors, the sensory phogenetic proteins (BMP), and the Wingless-related layer of the retina. The outer segment of the photore- integration site (WNT) signaling pathways have been ceptors is a highly modified primary cilium which has associated with embryonic eye defects [53–55]. Unex- evolved to play a crucial role in vision [42]. Changes in pectedly, our query of the IMPC database for embryonic primary cilium morphology or structure have been asso- eye defects revealed genes associated with the TGFβ ciated with retinal diseases such as macular diseases, (Bmp4 and Acvr2a), WNT (Axin2 and Pygo2), and SHH retinitis pigmentosa, Leber congenital amaurosis, cone (Fux, Sufu, Gabpa, Cplane1, Tctn2, and Tbc1d32) sign- dystrophies, and other photoreceptor diseases [43, 44]. aling pathways and importantly included novel genes In addition to the CPLANE genes, our search uncov- (Avcr2a and Gabpa). Additional work is required to ered a number of additional ciliopathy genes associated uncover the precise role of these novel genes in these with multi-systemic, brain, kidney, and eye ciliopathies critical embryonic signaling pathways. (Cep135, Innp5e, Pex6, Togaram1, see Additional file 1: Table S3). The members of the CPLANE complex, ACVR2A and its relationship to SHH and cyclopia by contributing to the formation of primary cilia, are Cyclopia occurs due to optic vesicle fusion, creating important regulators of the hedgehog and WNT signal- a singular eye and causing further neural abnormali- ing pathways [45]. Restricted spatial distribution of SHH ties that are incompatible with life [10]. This is the most is essential for the specification of eye progenitor cells extreme manifestation of holoprosencephaly (HPE), a [46], as SHH-mediated induction of embryonic tissues condition that results from abnormal forebrain forma- including eye formation is dependent on the establish- tion and closure during early embryonic development ment of SHH morphogenic gradients [45]. The associa- [56]. The etiology of HPE is complex and includes genetic tion between aberrant primary cilium formation, SHH mutations (several key loci have been identified, [56]) expression, and eye defects have been demonstrated for and teratogenic insults such as exposure to the naturally SUFU, Fuz, JBTS17/Cplane1, TBCD132, and TCTN3. In occurring alkaloid cyclopamine [57], retinoic acid [58], or addition, while Gabpa and Dzip1l have been previously cholesterol deficiency [59]. These teratogens were shown associated with ectopic SHH expression [47, 48], our data to abrogate the hedgehog signaling pathway [60], specifi- now confirmed a role for these two genes in cilia-SHH cally SHH, which is an important morphogen that regu- interactions and to the pathogenic mechanisms underly- lates CNS patterning and eye morphogenesis [61]. SHH ing eye defects. Specific genes of the CPLANE complex signaling within the neuroepithelium modulates the for- can affect the planar cell polarity (PCP) cascade with mation of the optic cup, optic stalk, and the retinal pig- ensuing ciliopathy-mediated defects resulting in severe mented epithelium alongside the dorso-ventral axes. Shh Chee et al. BMC Biology (2023) 21:22 Page 10 of 15 knockout mice show embryonic lethality by E9.5, with a the CPLANE complex in eye development. Additionally, phenotype showing cyclopia [62, 63]. Activin receptor there a number of genes for which our bioinformatics type 2A (ACVR2A) is a transmembrane receptor that analysis did not reveal protein interactions or networks binds to activin A. Acvr2a is largely expressed within the such as Alg10b, Atp13a1, Eef1d, Errc4, Focad, Laptm4b, head of zebrafish and plays a role in anterior-posterior Rexo1, Rgl1, Ubn2, and Vps26c. Their precise contribu- patterning. When depleting acvr2a in zebrafish, these tion to early mammalian eye development remains to be lineages exhibited ocular phenotypes such as swollen uncovered. Gene Ontology and analysis of protein-pro- eyes that were mediolaterally displaced suggesting a role tein interactions between the mouse genes in this study in eye field patterning [64]. In humans, one study showed and established human MAC genes revealed the contri- that one patient out of a cohort of 136 individuals with bution of previously unrecognized processes such as the signs of HPE had a deletion within ACVR2A, which led serine-glycine biosynthesis pathway for eye development. them to consider it an “HPE candidate” gene [20]. Within In individuals with deficiencies of enzymes involved in our STRING analysis (Additional file 1: Fig. S5), SHH serine-glycine biosynthesis, these mouse models provide is within the same network as ACVR2A, which signi- an opportunity to investigate potential therapeutic ave- fies that the sonic hedgehog and activin pathways may nues such as folate, formate, and/or B6 supplementation interact, but the relationship between ACVR2A, hedge- to reduce the occurrence of eye maldevelopment. hog signaling and midline defects such as cyclopia war- This study has several limitations. The mouse lines rant further study. A hypothesized model of cross-talk presented here in some cases coincide with extraocular between the SHH, TGFβ, and WNT pathways involving developmental anomalies such as exencephaly and crani- Acvr2a is illustrated in Additional file 1: Fig. S3. ofacial defects, suggesting they may be examples of sec- ondary anophthalmia. All mice were generated on the Conclusion C57BL/6N background which carries the rd8 mutation in Overall, phenotype screening of embryonic eyes in sin- Crb1 and can have several ocular consequences [65–67]. gle gene knockout mouse lines have revealed 40 novel Therefore, it is possible that some of the observed phe- genes that are associated with abnormalities in early eye notypes are digenic phenomena involving the targeted development. LacZ expression patterns within the het- deleted gene and Crb1. The candidate MAC genes in erozygous murine embryos further support these find- this report require further validation to confirm clini- ings by showing gene expression within the eye. Many of cal relevance. In spite of the limitations of this approach, these genes are linked to a number of conserved signal- forward genetic screening of IMPC knockout mice is a ing pathways (SHH, WNT, TGFβ), CPLANE complex, useful strategy to understand the genetic mechanisms as well as to the serine and folate metabolic pathways, of mammalian eye development, advance leading edge which influence cellular proliferation (nucleotide syn- therapeutics, and provide disease models in which to thesis) and methylation reactions (epigenetics). All of the test them. Single gene knockout mouse technology has above are likely to influence multiple cellular functions the capability to improve our understanding of human and morphogenic processes during embryonic develop- MAC spectrum disease, an important cause of child- ment. Consequently, the majority of mutant embryos hood blindness. We encourage physicians to validate the exhibited morphogenic anomalies in addition to the candidate genes discovered in IMPC mouse mutants in MAC spectrum phenotypes (Additional file 1: Table S1); unsolved cases of MAC spectrum disease. brain malformations (exencephaly and HPE) were often associated with anophthalmia and craniofacial defects Methods were often observed in mutants with microphthalmia. Bioinformatics Similarly, as described for Fuz mutants, embryos with We queried the IMPC database (data release 17, 8267 mutation of CPLANE genes exhibited a pleiotropy of KO mouse lines) for phenotypes associated with sig- defects. In one unique case, a gene (Acvr2a) was associ- nificant eye defects in E9.5, E12.5, E15.5, and E18.5 ated with cyclopia in addition to MAC anomalies con- mouse embryos using mammalian phenotype annota- firming human findings in holoprosencephaly cases. This tion terms (microphthalmia, anophthalmia, coloboma, generates hypotheses about possible cross-talk between abnormal eye morphology, abnormal optic vesicle). TGFβ and hedgehog signaling pathways in splitting the A significant eye phenotype was defined as 2 or more embryonic eye field at a very early stage of development. homozygous embryos or 4 or more heterozygous These findings highlight the requirement of SHH, WNT, embryos exhibiting an eye defect. Lines with significant and TGFβ signaling pathways regulating the pluripo- phenotypes were analyzed for statistical significance tency of stem cells for ocular morphogenesis. Similarly, using the OpenStats tool kit, available as an R pack- we confirmed the importance of ciliopathy genes and age [68]. Mutant genotypes with a P-value less than Chee et al. BMC Biology (2023) 21:22 Page 11 of 15 0.0001 were considered a significant difference from Embryo phenotyping controls at the IMPC level; we identified 57 genes that Embryos (E9.5, E12.5, E15.5, and E18.5) were recovered met this statistical threshold. Additionally, we included from time-mated heterozygous crosses. Fertilization lines with a genotype effect with P-values greater than (0 h) was set as the midpoint of the dark cycle prior to 0.0001 and less than 0.05; there were 17 genes that the detection the copulation plugs. Embryos were dis- met these criteria. In total, we identified 74 genes with sected free from their placenta and membranes in phos- embryo mutants having one or more significant eye phate-buffered saline (PBS minus ­Ca++/Mg++), and the phenotype, the vast majority being microphthalmia and yolk sacs were collected for genotyping. Eye develop- anophthalmia. ment and phenotype were assessed through gross mor- Using the search engines www.​pubmed.​gov and www.​ phological examination of individual embryos under a google.​com/​schol​ar, the set of 74 genes was queried phase microscope. Eye anomalies were noted using stage within existing literature to identify previously pub- appropriate gross morphology annotation terms and lished mouse knockout models with eye abnormalities. photo-documented when possible. For the eye morphol- To determine if each gene had been associated with eye ogy assessment, the examiner was masked to the geno- disease in humans, search terms related to the eyes and type of the embryos since PCR-based genotyping of the its associated structures, such as eye, anophthalmia, corresponding yolk sacs took place subsequent to the microphthalmia, coloboma, or retina were used. For a embryo phenotyping process. E15.5 and E18.5 embryos given gene, if there was not an extant knockout model, were transferred to warm heparinized (1 U/ml) PBS and or the published knockout reported no ocular phenotype euthanized by exsanguination. Individual embryos were in mouse embryos, and no ocular phenotypes associated submerged in fixative (4% paraformaldehyde/PBS) and with it in humans, it was designated as a “novel MAC rocked at 4 °C overnight or up to 7 days depending on gene.” The final literature search looked for review arti- the age/size of the embryo and then stored at 4 °C in PBS cles and case reports that documented genes in humans containing 0.02% sodium azide until processed for μCT related to MAC spectrum disorder, so that a gold stand- or histology. A minimum of 28 live embryos were col- ard list of published human genes (n = 114) could be cre- lected at each time point including at least eight homozy- ated for comparison to the 74 IMPC mouse genes. After gotes for each knockout mouse line. converting the 74 mouse gene symbols to the human orthologues, these two groups of genes were analyzed LacZ staining using the STRING [69] biological database software to E12.5 mouse embryos were stained for β-galactosidase predict protein-protein interactions within and between activity using X-gal [71]. Embryos were dissected free of the groups, and molecular functions were generated extraembryonic membranes in warm PBS, transferred using the Panther tool [22] within Gene Ontology [22, 24, to 12-well plates, and fixed individually in a 10% buff- 69]. The relationships between our genes within STRING ered formalin solution containing glutaraldehyde (8%), are characterized with a medium level of confidence EGTA (0.5M), and IGEPAL (10%) for 60 min at room (0.40–0.69), high confidence (0.70–0.89), or the highest temperature with gentle rocking. The yolk sacs were col- confidence (0.90–1.0), with most gene relationships fall- lected for genotyping at the time of dissection. Following ing within the medium level of confidence [70]. several washes to remove the fixative, the embryos were Animals immersed in the X-gal solution and stained for ~ 18 h at All institutions involved in this study operated under room temperature, protected from light with moderate the regulation and accreditation from their national ani- agitation. Embryos were postfixed in 10% buffered for- mal welfare committee [Institutional Animal Care and malin, cleared with sequential solutions of glycerol and Use Committee (IACUC) or Animal Care Committee potassium hydroxide (1%) and stored in 70% glycerol (ACC)]. This ensured that each institution would oper- until image acquisition and evaluation of staining pattern. ate under standardized, ethical procedures that mini- mized pain and suffering for all mice included in this Micro‑computed tomography (μCT) imaging work. The IMPC utilizes a systematic approach towards For μCT, individual E15.5 and E18.5 embryos were phenotyping mice that are either homozygous (HOM) for incubated in a hydrogel stabilizing solution (4% PFA, knockout of a single-gene or heterozygous (HET) for the 4% acrylamide, 0.05% bis-acrylamide, 0.25% VA044 Ini- gene if the homozygous mice are subviable or lethal. For tiator, 0.05% saponin in PBS) for 3 days at 4 °C to pre- X-linked genes, the knockout male mice are designated serve tissue integrity. Thereafter, the vials containing the hemizygous (HEMI). The strategies utilized for gene embryos were placed in a desiccation chamber and satu- knockout can be accessed on http://​www.​mouse​pheno​ rated with nitrogen gas to replace the air and embryos type.​org/​about-​ikmc/​targe​ting-​strat​egies. were incubated in a 37 °C water bath. Finally, embryos Chee et al. BMC Biology (2023) 21:22 Page 12 of 15 were removed from the encasing hydrogel, swiped clean, Supplementary Information and immersed in a Lugol solution [0.7% iodine solu- The online version contains supplementary material available at https://​doi.​ tion (0.1N)] for at least 24 h at room temperature while org/​10.​1186/​s12915-​022-​01475-0. rocking and then embedded in 1% agarose and oriented Additional file 1: Table S1. Knockout mice exhibiting phenotypic embry‑ for μCT imaging. Embryos were imaged using a high- onic eye defects. The embryonic eye defects were divided into four cat‑ resolution MicroXCT-200 specimen CT scanner (Carl egories: Abnormal eyelid fusion, abnormal optic vesicle formation, micro‑ Zeiss X-ray Microscopy). The CT scanner has a vari- phthalmia, and anophthalmia. If a knockout strain of the gene has already been created, a PMID is listed, along with a description of the ocular able X-ray source capable of a voltage range of 20–90 defect, age, and zygosity of the mice upon diagnosis if pertinent. If there is kV with 1–8 W of power. Embryos were placed on the an ocular defect present within humans (Human Eye PMID), or if there is a scanner’s sample stage, which has a submicron level of weak, potential correlation between the gene and an ocular defect in any species besides mice and humans (Associated Phenotypes), the PMID is position adjustments. Scan parameters were adjusted listed as well. Any systemic co-phenotypes (numerical code listed below) for each embryonic age, based on the manufacturers associated with either homozygous or heterozygous mice are listed, along recommended guidelines. A total of 1600 image projec- with the description of the heterozygous eye phenotype. From the IMPC database, if a photo of ocular defects was present in heterozygous adult tions were obtained over a 360° rotation. The camera pix- mice, the type of photo taken was described in the spreadsheet. Addition‑ els were binned by two to increase signal to noise in the ally, from the same database, the phenotypic embryonic eye defects image and the source-detector configuration resulted in were labelled for each knockout strain, along with the age the mice were diagnosed with the defect (E9.5, E12.5, E15.5, E18.5), their homozygous a voxel size of 11.4791 μm. Images were reconstructed viability, the zygosity of the mice with the phenotypic eye defect, and with a smoothing factor of 0.7 and a beam hardening of a ratio of mutant KO to normal KO mice by sex. In conclusion, the IMPC 0.2 into 16-bit values with common global minimum and yielded 74 genes that created knockout strains with an embryonic eye defect, 27 of which had already had previously published mouse knock‑ maximum values for proper histogram matching. outs, but only15 of these knockouts exhibiting an eye abnormality. There are a total of 59 genes that are associated with embryonic eye defects in Histology mice of which 19 genes had a previously reported human eye phenotype. Therefore, there are a total of 40 genes not previously associated with eye Embryos were dehydrated in a graded series of alcohol defects; these are bolded and highlighted in red in the first column of the washes, cleared in xylene, embedded in paraffin, and cut in table. 1 = Endocrine/exocrine 11 = Respiratory 2 = Taste/olfaction 12 = serial frontal sections (5 μm) using a microtome (Thermo Cardiovascular 3 = Ear, hearing, vestibular 13 = Behavior, Neurologic 4 = Craniofacial 14 = Metabolism/homeostasis 5 = Muscle phenotype 15 Scientific Micron HM355S, Leica Biosystems, Deer Park = Growth and Body Size 6 = Immune/Hematopoietic 16 = Reproduc‑ IL) and collected on slides for hematoxylin and eosin stain- tive System 7 = Skeletal Phenotype 17 = Embryonic 8 = Limbs, digits, ing using a standard protocol. Slides were scanned using a tail 18 = Mortality, aging 9 = Integumentary/Pigmentation 19 = Eyes 10 = Digestive 20 = Urinary. Table S2. The Gold standard list of 114 genes ScanScope XT Slide Scanner and viewed with the Image was created from a list of previously published genes that contribute to Scope software, version 10.2.2.2319 (Aperio). MAC spectrum disorders in humans. The 74 IMPC list of genes is listed beside it, and the genes present on both the gold standard list and IMPC list are highlighted in yellow. References from which the gold standard Abbreviations list was curated from are listed on the column to the furthest right (PMID ACC​ Animal Care Committee or DOI). Fig. S1. Pie graphs demonstrating and comparing the molecular BMP Bone morphogenetic proteins pathways of both the 74 IMPC genes and the 114 gold standard genes, CPLANE Ciliogenesis and planar polarity effectors using the Panther function on Gene Ontology. Pathways with the red DAVID Database for Annotation, Visualization and Integrated Discovery box are only implicated from the mouse data from our genes. The genes DCC Data Coordination Center implicated within the pathways with the red box are labeled next to DMDD Deciphering the Mechanisms of Developmental Disorders the pathway. Fig. S2. Serine-Glycine biosynthesis pathway derived from FOCM Folate one-carbon metabolism the KEGG pathway on DAVID, based on pathways and gene interactions GLDC Glycine decarboxylase predicted by gene ontology and STRING analysis. IMPC genes with MAC HEMI Hemizygous phenotypes (blue) are highlighted. Fig. S3. Interrelationships between HET Heterozygous pathways involved in stem cell maintenance and proliferation derived HOM Homozygous from the KEGG pathway on DAVID. Established MAC genes (pink) and HPE Holoprosencephaly IMPC genes with MAC phenotypes (blue) are highlighted. Red indicates IACUC​ Institutional Animal Care and Use Committee a gene in both groups, and SOX2 is highlighted (pink) as an established IMPC International Mouse Phenotyping Consortium critical MAC spectrum disease gene. Fig. S4. Analysis of protein-protein KEGG Kyoto Encyclopedia of Genes and Genomes interactions between the IMPC genes and the Gold Standard genes within KO Knockout a merged network. Cytoscape was the software platform used to merge MAC Microphthalmia, anophthalmia, and coloboma the two networks together, and the protein interactions between the PBS Phosphate-buffered saline two groups of genes were analyzed using the STRING biological database PCP Planar cell polarity software program. The arrows point towards genes that are initially not PHGDH Phosphoglycerate dehydrogenase incorporated into STRING clusters until merged together within the PSAT1 Phosphoserine aminotransferase Cytoscape platform. Fig. S5. Analysis of protein-protein interactions in the PSPH Phosphoserine phosphatase 74 IMPC genes (left) and the 114 Gold Standard genes (pink, right) using SHMT Serine hydroxymethyl transferase STRING biological database software. Among the IMPC genes, strongly TGFβ Transforming growth factor beta significant (P<0.0001, green) and conventionally significant (P<0.05, yel‑ WNT Wingless-related integration site low) genes are shown. Red genes are those that also appear on the list of WT Wild-type gold standard MAC genes. Genes with stars (n=6) are members of signal‑ μCT Micro-computed tomography ing pathways regulating pluripotency of stem cells. Clusters encircled Chee et al. BMC Biology (2023) 21:22 Page 13 of 15 Declarations represent the serine-glycine pathway (red box) and Cplane1 complex (blue box). Table S3. Genes implicated in ciliopathies from both the IMPC Ethics approval and consent to participate and Gold Standard list of genes. The genes on column A represent IMPC All institutions involved in this study operated under the regulation and genes that are implicated in ciliopathies, while the genes in column F accreditation from their national animal welfare committee [Institutional represent Gold Standard genes that are implicated in ciliopathies. Animal Care and Use Committee (IACUC) or Animal Care Committee (ACC)]. Consent for publication Acknowledgements Not applicable. The following authors are IMPC members that contributed to this study: Cheryl Ackert-Bicknell, David J Adams, Douglas Adams, Anne-Tounsia Adoum, Competing interests Juan A Aguilar-Pimentel, Uchechukwu Akoma, Dalila Ali-Hadji, Oana V Amarie, The authors declare that they have no competing interests. Philippe André, Aurelie Auburtin, Chaouki Bam’Hamed, Johannes Beckers, Joachim Beig, Zorana Berberovic, Alexandr Bezginov, Marie-Christine Birling, Author details Katharina Boroviak, Joanna Bottomley, Antje Bürger, Dirk H Busch, Natalie 1 Oakland University William Beaumont School of Medicine, Rochester, MI, C Butterfield, Pilar Cacheiro, Julia Calzada-Wack, Emma L Cambridge, Susan USA. 2 Mouse Biology Program, University of California Davis, Davis, CA, USA. Camilleri, Marie-France Champy, Heather Cater, Philippe Charles, Elissa J 3 University of Miami: Miller School of Medicine, Miami, FL, USA. 4 The Centre Chesler, Yi-Li Cho, Audrey E. Christiansen, Valentina Cipriani, Nicola Cockle, for Phenogenomics, Toronto, ON, Canada. 5 Lunenfeld-Tanenbaum Research Gemma Codner, Amie Creighton, Maribelle Cruz, Katharine F Curry, Abigail Institute, Sinai Health, Toronto, ON, Canada. 6 The Hospital for Sick Children, D’Souza, Ozge Danisment, Daniel Delbarre, Hannah F Dewhurst, Brendan Toronto, ON, Canada. 7 The Wellcome Trust Sanger Institute, Wellcome Doe, Alex Dorr, Florian Giesert, Graham Duddy, Kyle Duffin, Amal El Amri, Genome Campus, Hinxton, Cambridge, UK. 8 Centre of Animal Biotechnology Hillary Elrick, Mohammad Eskandarian, Martin Fray, Anthony Frost, Helmut and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona, Barcelona, Fuchs, Valerie Gailus-Durner, Karen K Gampe, Milan Ganguly, David Gan‑ Spain. 9 The Jackson Laboratory, Bar Harbor, ME, USA. 10 Medical Research non, Lillian Garrett, Marina Gertsenstein, Diane Gleeson, Leslie Goodwin, Council Harwell Institute, Mammalian Genetics Unit and Mary Lyon Centre, Jochen Graw, Kristin Grimsrud, Hamed Haselimashhadi, Liane Hobson, Harwell Campus, Oxfordshire, UK. 11 National Laboratory Animal Center, Andreas Hörlein, Sabine M Hölter, Seung-Hyun Hong, Neil Horner, Amanda National Applied Research Laboratories (NARLabs), Taipei City, Taiwan. G. Trainor, Ziyue Huang, Coleen Kane, Yulia Katsman, Lance C. Keith, Lois 12 Department of Molecular and Human Genetics, Baylor College of Medicine, Kelsey, Janet Kenyon, Ruairidh King, Piia Keskivali-Bond, Andrea Kirton, Tanja Houston, TX, USA. 13 Phenomics Australia, The John Curtin School of Medi‑ Klein-Rodewald, Thomas Klopstock, Davide Komla-Ebri, Tomasz Konopka, cal Research, Canberra, Australia. 14 Nanjing Biomedical Research Institute, Ralf Kühn, Fiona Kussy, David Lafont, Qing Lan, Denise G Lanza, Valerie Laurin, Nanjing University, Nanjing, China. 15 Indian Institutes of Science Education Elise Le Marchand, Sophie Leblanc, Victoria D Leitch, Chris Lelliott, Christoph and Research, Pune, India. 16 Faculty of Health Sciences, PCDDP North-West Lengger, Lauri Lintott, John G Logan, Isabel Lorenzo, Ann-Marie Mallon, Naila University, Potchefstroom, South Africa. 17 Institut de Génétique et de Biologie S Mannan, Susan Marschall, Melissa L. McElwee, Matthew Mckay, Robbie SB Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France. 18 Ger‑ McLaren-Jones, Jeremy Mason, Terrence F Meehan, David Miller, Michayla man Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum Moore, Violeta Munoz-Fuentes, Stephen A Murray, Dong Nguyen-Bresinsky, München, Neuherberg, Germany. 19 Monterotondo Mouse Clinic, Italian Oskar Oritz, Panos Pandis, Helen Parkinson, Alexandru Parlog, Amit Patel, Guil‑ National Research Council (CNR), Monterotondo Scalo, Italy. 20 European Bioin‑ laume Pavlovic, Monica Pereira, Kevin Peterson, Vivek Philip, Andrea S Pollard, formatics Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, UK. Jan Prochazka, Dawei Qu, Ayexa Ramirez, Sean Rangarajan, Tara L. Rasmus‑ 21 National Laboratory Animal Center, National Applied Research Laboratories, sen, Birgit Rathkolb, Mike Relac, Kyle Roberton, Willson Roper, Stéphane Beijing, China. 22 RIKEN BioResource Center, Tsukuba, Japan. 23 Czech Center Rousseau, David W Rowe, Jan Rozman, Jennifer Ryan, Edward J Ryder, Luis for Phenogenomics, Institute of Molecular Genetics of the Czech Academy Santos, Adrián Sanz-Moreno, Joel Schick, Zachary Seavey, John R Seavitt, of Sciences, Vestec, Czech Republic. 24 Research Institute for Veterinary Science, Claudia Seisenberger, Mohammed Selloum, Xueyuan Shang, Dong-Guk Shin, College of Veterinary Medicine, Seoul National University, Seoul, South Korea. Michelle Simon, Gillian Sleep, Damian Smedley, Tania Sorg, Penny C Sparkes, 25 CAM‑SU Genomic Resource Center, Soochow University, Suzhou, China. Nadine Spielmann, Ralph Steinkamp, Michelle Stewart, Claudia Stoeger, Ewan 26 Ophthalmic Genetics and Visual Function Branch, National Eye Institute, Straiton, Karen L Svenson, Holly Swash, Lydia Teboul, Sandra Tondat, Irina NIH, Bethesda, MD 20892, USA. 27 Department of Laboratory Medicine & Treise, Catherine Tudor, Rachel Urban, Valerie E Vancollie, Laurent Vasseur, Igor Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada. Vukobradovic, Hannah Wardle-Jones, Jonathan Warren, Marie Wattenhofer- 28 Department of Surgery, School of Medicine, University of California Davis, Donze, Sara E Wells, Jacqueline K White, Jean-Paul Wiegand, Amelia Willett, Sacramento, CA, USA. 29 Department of Ophthalmology & Vision Science, Catherine Witmeyer, Eckhard Wolf, Leeyean Wong, Joshua Wood, Wolfgang School of Medicine, University of California Davis, Sacramento, CA, USA. 30 UC Wurst, Catherine Xu, and Annemarie Zimprich. 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