Organization and development of the S-cone system

National Eye Institute

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Abstract

For proper vision, it is critical to correctly specify photoreceptors. Foundational work across vertebrates has identified transcription factors that are required for the generation of photoreceptors progenitors during development. Crucial studies in mice led to the discovery of key transcription factors that control subtype-specific photoreceptor fate; NRL and its downstream effector NR2E3 are required for the specification of rods. Interestingly, mutations in NR2E3 in humans also lead to a failure in rod specification and ultimately result in impaired visual acuity, abnormal color vision, night blindness and retinal degeneration. In addition to conserved factors involved in rod specification, THRb is required for L-cone specification in mice, zebrafish, and, most likely, birds. In the absence of NRL, NR2E3 or THRb, mouse photoreceptor progenitors acquire an S-cone fate. Therefore, S-cone specification has been deemed as a passive process. Yet mounting evidence, derived mainly from work in non-mouse species, challenges this simplistic model. For example, while Nr2e3 is also required for the generation of rods in other vertebrates, Nrl is dispensable. In addition, UV- and S-cone specification in zebrafish is far from passive and requires the action of transcription factors. Moreover, genes that regulate opsin expressione.g. Rora, Rorb or Rxrg in mouse, or survival of retinal progenitorse.g. six6, six7 and gdf6a in zebrafish can also cause subtype-specific alterations in photoreceptor development. In general, these studies highlight that our understanding of the specification of photoreceptor subtypes is still incomplete and piecemeal and that many other genes are likely to be involved in this complex process. We seek to provide the resources and establish the methods required to efficiently identify additional genes involved in photoreceptor specification as well as other photoreceptor subtype-specific functions. First, we obtain a deep and high-quality transcriptomic profile (RNA-seq) of the five zebrafish photoreceptor subtypes. Second, we explore this RNA-seq dataset and identify multiple transcription factors that could potentially be involved in the specification of photoreceptor subtypes. Third, we show that a CRISPR-based F0-screening approach is a reliable platform for identifying transcription factors involved in photoreceptor specification. We initially benchmarked our method by replicating known phenotypes of Foxq2 and Nr2e3 mutants: F0 larvae that carry mutations in foxq2 lose S cones while those that carry mutations in nr2e3 lose rods. Subsequently, we explore four additional candidates (Skor1a, Sall1a, Lrrfip1a, Xbp1) and find that they are not critical to specify photoreceptor subtypes. Finally, we describe novel roles for the transcription factors Tbx2a and Tbx2b in photoreceptor specification, demonstrating that UV-cone specification requires both Tbx2a and Tbx2b, and that Tbx2a and Tbx2b, respectively, maintain the identity of L cones and S cones by repressing M-cone cell fate. In the future, our dataset and methods can be applied to further our understanding of how photoreceptors acquire their final identities and to explore other important aspects of photoreceptor biology that also differ between subtypes (phototransduction, metabolism, synaptic wiring, etc.). This knowledge can be used to inform strategies to control the photoreceptor differentiation in organoidsa potential gateway for cell-replacement therapies in retinal degenerations.