An unbiased AAV-STARR-seq screen revealing the enhancer activity map of genomic regions in the mouse brain in vivo.

Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.

The distribution of the preferred directions of the ON-OFF direction selective ganglion cells in the rabbit retina requires refinement after eye opening.

The ON-OFF direction selective ganglion cells (DSGCs) in the mammalian retina respond differentially for an object moving in different directions. DSGCs can be further segregated into four functional subtypes, namely those responsible for the detection of motion in the superior, inferior, anterior, and posterior directions of the visual field. Although it has been known that the basic neural circuit of direction selectivity is established at around the time of eye opening, it is less known if the four DSGC subtypes can be unambiguously distinguished at this time and whether their preferred directions are aligned with four canonical axes at this developmental stage. By examining the preferred directions of DSGCs in P10-12 rabbit retinas and characterizing their distribution pattern, we have shown that the preferred directions of DSGCs at around the time of eye opening are not distinctly segregated but rather are diffusely distributed along the four canonical axes. Similar results were found in the mouse retina by reanalyzing previously published data. Furthermore, taking into account the fact that the direction tuning strength of DSGCs at P10-12 is weaker than that in adults, this was found not to be correlated with their preferred directions, which suggests that the maturations of direction selectivity and preferred direction are independent processes. In addition, we also found that the subtypes of DSGCs, which do not display tracer coupling pattern in the adult, show extensive coupling at P10-12. Taken together, the present study supports that the significant refinement after eye opening is required for the development of the four functional DSGC subtypes in the rabbit retina.

Effect of visual experience on the maturation of ON-OFF direction selective ganglion cells in the rabbit retina.

Activity-dependent neural plasticity is well known in the development of the visual cortical circuitry. However, the role of neural plasticity in the developing retina is less well understood. In the light of recent findings that light deprivation alters the development of synaptic pathway in the mouse and turtle retinas, we studied whether visual experience is required for the maturation of the ON-OFF direction selective ganglion cells (DSGCs) in the rabbit retina. The DSGCs of rabbits raised under a normal light-dark cycle and in the constant darkness were recorded extracellularly at various postnatal stages. Receptive field properties, such as direction selectivity, velocity tuning, classical center-surround interaction and motion-induced surround inhibition were examined. Recorded cells were subsequently injected with Neurobiotin in order to characterize their morphological features and tracer coupling patterns. Our results revealed that visual experience is not critical for the maturation of the classical receptive field properties of the DSGCs, such as direction selectivity and velocity tuning. However, the dark-reared rabbits showed altered surround inhibition, which is mediated by the amacrine cells of the inner retina. In addition, the DSGCs of both normal- and dark-reared rabbits showed similar dendritic features and tracer coupling patterns. Taken together, this study indicates that visual experience plays a less significant role on the DS circuitry maturation in the retina than in the cortex.