Person: Hrvatin, Sinisa
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Hrvatin
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Sinisa
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Hrvatin, Sinisa
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Publication Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex(2017) Hrvatin, Sinisa; Hochbaum, Daniel; Nagy, M. Aurel; Cicconet, Marcelo; Robertson, Keiramarie; Cheadle, Lucas; Zilionis, Rapolas; Ratner, Alex; Borges-Monroy, Rebeca; Klein, Allon; Sabatini, Bernardo; Greenberg, MichaelActivity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders.Publication Single-cell transcriptomics of the developing lateral geniculate nucleus reveals insights into circuit assembly and refinement(National Academy of Sciences, 2018) Kalish, Brian; Cheadle, Lucas; Hrvatin, Sinisa; Nagy, M. Aurel; Rivera, Samuel; Crow, Megan; Gillis, Jesse; Kirchner, Rory; Greenberg, MichaelCoordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.Publication MARIS: Method for Analyzing RNA following Intracellular Sorting(Public Library of Science, 2014) Hrvatin, Sinisa; Deng, Francis; O'Donnell, Charles W.; Gifford, David K.; Melton, DouglasTranscriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.Publication Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3(Nature Publishing Group, 2012) Blum, Barak; Hrvatin, Sinisa; Schuetz, Christian; Bonal, Claire; Rezania, Alireza; Melton, DouglasInsulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro. We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.Publication Exploring the Use of Human Pluripotent Stem Cells to Create Functional Pancreatic \(\beta\) Cells(2013-03-18) Hrvatin, Sinisa; Melton, Douglas A.; Weir, Gordon; Eggan, Kevin; Greiner, Dale; Zon, LeonardDirected differentiation of human pluripotent stem cells (hPSCs) has the potential to produce human cell types that can be used for disease modeling and cell transplantation. Two key challenges in the differentiation from hPSCs to \(\beta\) cells are the specification from pancreatic progenitors to insulin-expressing \((INS^+ )\) cells and the maturation of \(INS^+\) cells into glucose responsive β cells. To address the first, two high-content chemical screens identified PKC inhibitors as inducers of \(INS^+\) cells from pancreatic progenitors. PKC inhibition generated up to tenfold more \(INS^+\) cells while PKC agonists blocked differentiation into \(INS^+\) cells. Transplantation of \(PKC\beta\) inhibitor-treated pancreatic progenitors, containing higher proportions of endocrine progenitors and endocrine cells, resulted in mature \(\beta\) cells showing higher levels of glucose-stimulated human c-peptide production in vivo. This indicates that in vitro derived \(INS^+\) cells might be competent to mature into functional \(\beta\) cells. To address the second challenge, we first studied mouse and human \(\beta\) cell maturation in vivo. Postnatal mouse \(\beta\) cell maturation was marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3. To study human \(\beta\) cell maturation, a Method for Analyzing RNA following Intracellular Sorting (MARIS) was developed and used for transcriptional profiling of sorted human fetal and adult \(\beta\) cells. Surprisingly, transcriptional differences between human fetal and adult \(\beta\) cells did not resemble differences between mouse fetal and adult \(\beta\) cells, calling into question inter-species homology at the late stages of development. A direct comparison between hPSC-derived \(INS^+\) cells, and \(\beta\) cells produced during human development is essential to validate directed differentiation and provide a roadmap for maturation of hPSC-derived \(INS^+\) cells. Genome-wide transcriptional analysis of sorted \(INS^+\) cells derived from three hPSC-lines suggest that different lines produce highly similar \(INS^+\) cells, confirming robustness of directed differentiation protocols. Furthermore, nonfunctional hPSC-derived \(INS^+\) cells resemble human fetal \(\beta\) cells, which are distinct from adult \(\beta\) cells. We therefore suggest that in vitro directed differentiation mimics normal human development and reveal differences in gene expression that may account for the functional differences between hPSC-derived \(INS^+\) cells and true \(\beta\) cells.