Robert J Johnston - US grants

Project Summary Cell fate specification is driven by lineage, signaling, and stochastic regulatory inputs. The mechanisms controlling stochastic fate specification, in which a cell randomly chooses between two or more fates, are poorly understood. Stochastic fate specification is critical for diversifying retinal neurons, olfactory sensory neurons, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms cause debilitating human disorders, including vision impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project is to determine how chromatin state and transcriptional variability control stochastic fate specification, using the random patterning of photoreceptor subtypes in the fly retina as a paradigm. The fly eye contains a random mosaic of two color-detecting R7 photoreceptor subtypes, defined by expression of Rhodopsin 4 (Rh4) or Rhodopsin 3 (Rh3). This fate decision is controlled by the transcription factor Spineless (Ss), which is expressed in a random subset of mature R7s. SsON R7s express Rh4, while SsOFF R7s express Rh3. Our data support a two-step mechanism regulating ssON/OFF expression in mature R7s. In step 1, the early enhancer drives an early pulse of ss transcription in R7 precursors that opens the chromatin at the ss locus. In step 2, the transcriptional pulse ceases and chromatin variably closes, defining the accessibility of the late enhancer. Depending on the degree of chromatin compaction, the late enhancer either turns on (open chromatin) or remains off (closed chromatin) for the lifetime of the mature R7. How regulation of transcription and chromatin compaction is integrated to turn genes randomly on or off during development is poorly understood. We will use DNA FISH, genomics, CRISPR, and lacO/LacI-based live imaging approaches to assess the role of chromatin dynamics and the temporality of regulatory inputs in the two-step mechanism (Aim 1). Identification of the source of variability driving stochastic fate specification in metazoans has not been achieved. Our data suggest that variability in transcription (initiation, elongation, frequency, and duration) in individual cells influences terminal R7 fate specification. To test this hypothesis, we will use nascent multi-color RNA FISH and MS2/MCP-based live imaging to assess transcriptional parameters and relate them to R7 subtype fates (Aim 2). Successful completion of these experiments will identify a source of variability that drives a cell fate decision and inform how molecular noise is utilized to diversify cell types during metazoan development.

Project Summary: The specification of three cone photoreceptor subtypes enables high-acuity daytime and trichromatic color vision in humans. During terminal differentiation, cone cells express opsins that define their functionalities as blue (S-opsin), green (M-opsin), or red (L-opsin) light detectors. How the three cone subtypes are generated in humans remains largely unknown. Elucidating these mechanisms will be critical for understanding and treating visual disorders including macular degeneration, cone dystrophy, retinitis pigmentosa, foveal hypoplasia, and color blindness. The goal of this project is to determine the mechanisms controlling the specification of S, M, and L cones in the human retina. Cone subtype specification occurs in a two-step process. First, a decision occurs between S and L/M cone fates. If the L/M fate is chosen, a subsequent choice is made between L and M cone fates. To study these processes, we genetically and pharmacologically manipulated human retinal organoids to test mechanisms of retinal development. We found that dynamic expression of thyroid hormone (TH)-regulating genes within the retina ensures low TH signaling early to specify S cones and high TH signaling late to produce L/M cones. Our work established human retinal organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair (Eldred et al., 2018, Science). We found that S, M, and L cones are generated in a temporal progression during development. How temporal and spatial inputs are integrated in the human retina to generate patterns of cones is poorly understood. We developed dissection, imaging, and cell identification approaches to assess the spatial distributions of cone subtypes and generate the first maps of cone subtypes across intact human retinas (Aim 1). We found that temporal regulation of TH signaling is critical for establishment and maintenance of cone subtype fates. To evaluate thyroid dysregulation and cone fate plasticity, we will define the temporal windows of TH regulation in organoids and examine cone fates in retinas from patients with late-onset thyroid dysregulation. We will determine the roles of TH-regulating genes in cone subtype specification in organoids (Aim 2). Additionally, we found that retinoic acid (RA) signaling induces S cone fate and suppresses L/M cone fate early in decision 1, and induces L cone fate and suppresses M cone fate late in decision 2. We will characterize temporal windows of RA action and assess the function and expression of RA-regulating genes in the specification of cone subtype fates in organoids (Aim 3). These experiments will delineate how regulation of TH and RA signaling specify cone subtypes in the human retina. Moreover, these experiments will provide the first direct mechanistic insight into L and M cone specification with important implications for understanding red/green color blindness and developing regenerative therapeutics for macular degeneration.