Person: Walsh, Ryan M.
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Walsh
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Ryan M.
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Walsh, Ryan M.
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Publication Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency(Public Library of Science, 2015) Li, Lin; Walsh, Ryan M.; Wagh, Vilas; James, Marianne F.; Beauchamp, Roberta L.; Chang, Yuh-Shin; Gusella, James; Hochedlinger, Konrad; Ramesh, VijayaThe multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.Publication Small molecules facilitate rapid and synchronous iPSC generation(2014) Bar-Nur, Ori; Brumbaugh, Justin; Verheul, Cassandra; Apostolou, Effie; Pruteanu-Malinici, Iulian; Walsh, Ryan M.; Ramaswamy, Sridhar; Hochedlinger, KonradThe reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2, and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is protracted and inefficient, resulting in heterogeneous cell populations. Here we show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-beta inhibitor (termed “AGi”) facilitates more synchronous and rapid iPSC formation from a variety of mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 hours of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.Publication Loss of the Histone Demethylase Phf8 Is Compatible With Development but Confers Resilience to Anxiety and Depression(2016-01-12) Walsh, Ryan M.; Eggan, Kevin; Gregory, Richard; Mostoslavsky, GustavoPhf8 is a histone demethylase associated with human developmental disorders and cancer. Early studies of Phf8 in humans indicated that inactivating mutations in the gene were linked to syndromic intellectual disability with cleft lip and palate (Siderius syndrome). However, Phf8’s functional role in regulating mammalian development and behavior has not been demonstrated. In this thesis I present my findings on a knockout mouse model of Phf8, which I have generated and characterized to interrogate Phf8’s function in these processes in an in vivo mammalian system. Unexpectedly, I did not detect any gross physiological defects nor intellectual disability, but instead report here that mice null for Phf8 are resilient to anxiety and depression. I further characterized the molecular nature of Phf8’s role regulating mammalian behavior by performing RNA-seq on regions in the brain key to mediating anxiety disorders and depression. Here I find evidence that Phf8 directly regulates multiple serotonin receptors in the prefrontal cortex, which have a long standing link to anxiety disorders and depression, suggesting a likely mechanism for resiliency. In addition to its proposed function in behavior and development, multiple studies have implicated Phf8 cancer. It has been suggested that Phf8 can behave as an oncogene, notably in the case of T-ALL, where it cooperates with the Notch pathway and is required to drive tumor proliferation in vitro and in xenograft models. Within this thesis I will also present my findings on Phf8’s function in the context of an in vivo model of T-ALL. I do not observe a requirement for Phf8 in T-ALL nor does its loss seem to at all impair the progression of T-ALL driven by a constitutively active Notch1. However, I do observe a subtle defect in T-cell development, which is likely consistent with its function in effecting Notch signaling. The data within this thesis represent the first characterization of a mammalian knockout model of Phf8 and describe a novel role for this gene in the regulation of anxiety and depression.Publication Ascorbic Acid Prevents Loss of Dlk1-Dio3 Imprinting and Facilitates Generation of All-iPS Cell Mice from Terminally Differentiated B Cells(Nature Publishing Group, 2012) Stadtfeld, Matthias; Apostolou, Effie; Chen, Taiping; Oi, Steen; Bestor, Tim; Ferrari, Francesco; Choi, Jiho; Walsh, Ryan M.; Kim, Sang Yong; Shioda, Toshi; Park, Peter; Hochedlinger, KonradThe generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor–mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs.