Latent Epigenetic Programs in Müller Glia Contribute to Stress, Injury, and Disease Response in the Retina.

Previous studies have demonstrated the dynamic changes in chromatin structure during retinal development that correlate with changes in gene expression. However, a major limitation of those prior studies was the lack of cellular resolution. Here, we integrate single-cell (sc) RNA-seq and scATAC-seq with bulk retinal data sets to identify cell type-specific changes in the chromatin structure during development. Although most genes' promoter activity is strongly correlated with chromatin accessibility, we discovered several hundred genes that were transcriptionally silent but had accessible chromatin at their promoters. Most of those silent/accessible gene promoters were in the Müller glial cells. The Müller cells are radial glia of the retina and perform a variety of essential functions to maintain retinal homeostasis and respond to stress, injury, or disease. The silent/accessible genes in Müller glia are enriched in pathways related to inflammation, angiogenesis, and other types of cell-cell signaling and were rapidly activated when we tested 15 different physiologically relevant conditions to mimic retinal stress, injury, or disease in human and murine retinae. We refer to these as "pliancy genes" because they allow the Müller glia to rapidly change their gene expression and cellular state in response to different types of retinal insults. The Müller glial cell pliancy program is established during development, and we demonstrate that pliancy genes are necessary and sufficient for regulating inflammation in the murine retina in vivo. In zebrafish, Müller glia can de-differentiate and form retinal progenitor cells that replace lost neurons. The pro-inflammatory pliancy gene cascade is not activated in zebrafish Müller glia following injury, and we propose a model in which species-specific pliancy programs underly the differential response to retinal damage in species that can regenerate retinal neurons (zebrafish) versus those that cannot (humans and mice).

Identification of a modular super-enhancer in murine retinal development.

Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage- and cell-type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.

The myogenesis program drives clonal selection and drug resistance in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single-cell and single-nucleus RNA sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show that chemotherapy eliminates the most proliferative component with features of myoblasts within embryonal RMS; after treatment, the immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, these data serve as a proof of concept that targeting each developmental state in embryonal RMS is an effective strategy for improving outcomes by preventing disease recurrence.

Retinoblastoma from human stem cell-derived retinal organoids.

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Children with germline mutations in RB1 have a high likelihood of developing retinoblastoma and other malignancies later in life. Genetically engineered mouse models of retinoblastoma share some similarities with human retinoblastoma but there are differences in their cellular differentiation. To develop a laboratory model of human retinoblastoma formation, we make induced pluripotent stem cells (iPSCs) from 15 participants with germline RB1 mutations. Each of the stem cell lines is validated, characterized and then differentiated into retina using a 3-dimensional organoid culture system. After 45 days in culture, the retinal organoids are dissociated and injected into the vitreous of eyes of immunocompromised mice to support retinoblastoma tumor growth. Retinoblastomas formed from retinal organoids made from patient-derived iPSCs have molecular, cellular and genomic features indistinguishable from human retinoblastomas. This model of human cancer based on patient-derived iPSCs with germline cancer predisposing mutations provides valuable insights into the cellular origins of this debilitating childhood disease as well as the mechanism of tumorigenesis following RB1 gene inactivation.

Nucleome Dynamics during Retinal Development.

More than 8,000 genes are turned on or off as progenitor cells produce the 7 classes of retinal cell types during development. Thousands of enhancers are also active in the developing retinae, many having features of cell- and developmental stage-specific activity. We studied dynamic changes in the 3D chromatin landscape important for precisely orchestrated changes in gene expression during retinal development by ultra-deep in situ Hi-C analysis on murine retinae. We identified developmental-stage-specific changes in chromatin compartments and enhancer-promoter interactions. We developed a machine learning-based algorithm to map euchromatin and heterochromatin domains genome-wide and overlaid it with chromatin compartments identified by Hi-C. Single-cell ATAC-seq and RNA-seq were integrated with our Hi-C and previous ChIP-seq data to identify cell- and developmental-stage-specific super-enhancers (SEs). We identified a bipolar neuron-specific core regulatory circuit SE upstream of Vsx2, whose deletion in mice led to the loss of bipolar neurons.