The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs¹. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates². By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.

The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs (Baden et al., 2020). One might expect that retinal cell types evolved to accommodate these varied needs, but this has not been systematically studied. Here, we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a teleost fish, a bird, a reptile and a lamprey. Molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells [RGCs] and Müller glia) is striking, with transcriptomic differences across species correlated with evolutionary distance. Major subclasses are also conserved, whereas variation among types within classes or subclasses is more pronounced. However, an integrative analysis revealed that numerous types are shared across species based on conserved gene expression programs that likely trace back to the common ancestor of jawed vertebrates. The degree of variation among types increases from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified mammalian orthologs of midget RGCs, which comprise >80% of RGCs in the human retina, subserve high-acuity vision, and were believed to be primate-specific (Berson, 2008); in contrast, the mouse orthologs comprise <2% of mouse RGCs. Projections both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.

Cancer drug resistance is the leading cause of cancer related deaths. The development of drug resistance can be partially contributed to tumor heterogeneity and epigenetic plasticity. However, the detailed molecular mechanism underlying epigenetic modulated drug resistance remains elusive. In this work, we systematically analyzed epigenetic changes in tamoxifen (Tam) responsive and resistant breast cancer cell line MCF7, and adopted a data-driven approach to identify key epigenetic features distinguishing between these two cell types. Significantly, we revealed that DNA methylation and H3K9me3 marks that constitute the heterochromatin are distinctively different between Tam-resistant and -responsive cells. We then performed time-lapse imaging of 5mC and H3K9me3 features using engineered probes. After Tam treatment, we observed a slow transition of MCF7 cells from a drug-responsive to -resistant population based on DNA methylation features. A similar trend was not observed using H3K9me3 probes. Collectively, our results suggest that DNA methylation changes partake in the establishment of Tam-resistant breast cancer cell lines. Instead of global changes in the DNA methylation level, the distribution of DNA methylation features inside the nucleus can be one of the drivers that facilitates the establishment of a drug resistant phenotype in MCF7.

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

Amacrine cells (ACs) comprise a heterogeneous class of inhibitory neurons in the vertebrate retina, exhibiting morphological and functional complexity rivaling that of cortical interneurons. Here, we integrate single-cell and single-nucleus transcriptomic atlases from 24 vertebrate species to reconstruct the evolutionary origins of this extreme diversity. We identify 42 orthologous AC types (oACs), most of which exhibit a one-to-one correspondence across amniotes and, in many cases, across vertebrates. While core molecular identities are conserved, AC types vary in abundance and gene expression across species, likely reflecting adaptations to distinct visual ecologies. AC diversity scales with that of retinal ganglion cells (RGCs), indicative of co-evolution. Finally, we suggest that ACs arose from an AC-RGC hybrid precursor, with glycinergic ACs diverging early in vertebrate evolution, followed by a bifurcation between RGCs and GABAergic ACs. Together, these findings establish a unified evolutionary framework for understanding the diversity, development, and function of a class of inhibitory neurons across vertebrates.