Short Running Title: Atoh7 genetic networks and human eye diseases. Nonstandard Abbreviations: RPCs: retinal progenitor cells NCRNA: retinal non-attachment ONH: optic nerve hypoplasia ONA: optic nerve aplasia PHPV: persistent hyperplastic primary vitreous RGCs: retinal ganglion cells EFTFs: eye field transcription factors ABSTRACT: Expression of the bHLH transcription protein Atoh7 is a crucial factor conferring competence to retinal progenitor cells for the development of retinal ganglion cells. A number of studies have emerged establishing ATOH7 as a retinal disease gene. Remarkably, such studies uncovered ATOH7 variants associated with global eye defects including optic nerve hypoplasia, microphthalmia, retinal vascular disorders and glaucoma. The complex genetic networks and cellular decisions arising downstream of atoh7 expression, and how their dysregulation cause development of such disease traits remains unknown. To begin to understand such Atoh7-dependent events in vivo we performed transcriptome analysis of wild type and atoh7 mutant (lakritz) zebrafish embryos at the onset of retinal ganglion cell differentiation. We investigated in silico interplays of atoh7 and other disease-related genes and pathways. By network reconstruction analysis of differentially expressed genes we identified gene clusters enriched in retinal development, cell cycle, chromatin remodelling, stress response and Wnt pathways. By weighted gene coexpression network we identified coexpression modules affected by the mutation and enriched in retina development genes tightly connected to atoh7. We established the groundwork whereby Atoh7-linked cellular and molecular processes can be investigated in the dynamic multitissue environment of the developing normal and diseased vertebrate eye. 3 KEY WORDS: Atoh7, Ath5, retinal ganglion cells, transcriptome analysis; inherited eye diseases; human retina; zebrafish 6 anesthetized for 5-10 min in Ethyl 3-aminobenzoate methanesulfonate (MS-222) (Sigma-Aldrich, Saint Louis, MO, USA) in E3 medium. The corresponding body of each embryo was collected and used immediately to perform genotyping analysis to identify the corresponding to lakritz and wild type eyes. All embryos were collected from the same batches of fish stock to maintain a uniform genetic background. DNA extraction from zebrafish body biopsies and genotyping Genomic DNA extraction from each single body was performed in 100 μl of lysis buffer containing Proteinase K-20mg/ml (EuroClone S.p.A. Milan, Italy), 2 M Tris-HCl ph 8.0, 0.5 M EDTA ph 8.0 and 5 M NaCl, 20 % SDS in a final volume of 50 μl ultra H20. After 3 hours of incubation at 65°C, the gDNA was purified with an Ethanol precipitation step and resuspended in 50 μl of Dnase/Rnase H2O. The genotyping was performed by Restriction Fragment Length Polymorphism (RFLP) assay as previously described 6 . An ∼ 300 bp fragment of atoh7 was PCR amplified with 1 U Taq DNA polymerase (Applied Biosystems by Life Technologies, Carlsbad, CA, USA) according to manufacturer protocols in a 30μl PC...