Sox2 co-occupies distal enhancer elements with cell-type-specific POU factors to specify cell identity in embryonic stem cells and neural precursor cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections."June 2012." Cataloged from student submitted PDF version of thesis.Includes bibliographical references.Sox2 is a master regulator of two distinct cellular states, that of pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs), but what common or distinct roles Sox2 may play in these cell types not fully understood. Further, the molecular mechanisms by which Sox2 can specify two distinct cell identities are as of yet unclear. This thesis is aimed at answering these fundamental questions. In ESCs, Sox2 was associated with a subset of poised regulators of nervous system development, and upon differentiation into NPCs Sox2 selectively activates those which are important for progenitor cell state, while keeping others poised to become activated in later neural development. These data suggested that Sox2 might act as a pioneer factor for neural development throughout embryogenesis. While Sox2 is known to co-occupy target loci in ESCs with the POU factor Oct4, in NPCs Sox2 interacts with the central-nervous-system-expressed POU factors Brn1 and Brn2. By utilizing distinct composite Sox:Octamer motifs in each cell type, Sox2:POU modules control the expression of thousands of genes involved in the development of the neural lineage in a cell-type-specific manner. These data advance our understanding of the mechanism by which transcription factors control cell fate transitions, and indicate that combinatorial interactions between transcription factors may be a pervasive mechanism of transcriptional control in developmentby Michael A. Lodato.Ph.D

Evidence that APP gene copy number changes reflect recombinant vector contamination [preprint]

Mutations that occur in cells of the body, called somatic mutations, cause human diseases including cancer and some neurological disorders1. In a recent study published in Nature, Lee et al.2 (hereafter “the Lee study”) reported somatic copy number gains of the APP gene, a known risk locus of Alzheimer’s disease (AD), in the neurons of AD-patients and controls (69% vs 25% of neurons with at least one APP copy gain on average). The authors argue that the mechanism of these copy number gains was somatic integration of APP mRNA into the genome, creating what they called genomic cDNA (gencDNA). We reanalyzed the data from the Lee study, revealing evidence that APP gencDNA originates mainly from contamination by exogenous APP recombinant vectors, rather from true somatic retrotransposition of endogenous APP. Our reanalysis of two recent whole exome sequencing (WES) datasets—one by the authors of the Lee study3 and the other by Park et al.4—revealed that reads claimed to support APP gencDNA in AD samples resulted from contamination by PCR products and mRNA, respectively. Lastly, we present our own single-cell whole genome sequencing (scWGS) data that show no evidence for somatic APP retrotransposition in AD neurons or in neurons from normal individuals of various ages

Black-Hole Spin Dependence in the Light Curves of Tidal Disruption Events

A star orbiting a supermassive black hole can be tidally disrupted if the black hole's gravitational tidal field exceeds the star's self gravity at pericenter. Some of this stellar tidal debris can become gravitationally bound to the black hole, leading to a bright electromagnetic flare with bolometric luminosity proportional to the rate at which material falls back to pericenter. In the Newtonian limit, this flare will have a light curve that scales as t^-5/3 if the tidal debris has a flat distribution in binding energy. We investigate the time dependence of the black-hole mass accretion rate when tidal disruption occurs close enough the black hole that relativistic effects are significant. We find that for orbits with pericenters comparable to the radius of the marginally bound circular orbit, relativistic effects can double the peak accretion rate and halve the time it takes to reach this peak accretion rate. The accretion rate depends on both the magnitude of the black-hole spin and its orientation with respect to the stellar orbit; for orbits with a given pericenter radius in Boyer-Lindquist coordinates, a maximal black-hole spin anti-aligned with the orbital angular momentum leads to the largest peak accretion rate.Comment: 16 pages, 15 figures, 1 table, PRD published versio

Chemistry in a gravitationally unstable protoplanetary disc

Until now, axisymmetric, alpha-disc models have been adopted for calculations of the chemical composition of protoplanetary discs. While this approach is reasonable for many discs, it is not appropriate when self-gravity is important. In this case, spiral waves and shocks cause temperature and density variations that affect the chemistry. We have adopted a dynamical model of a solar-mass star surrounded by a massive (0.39 Msun), self-gravitating disc, similar to those that may be found around Class 0 and early Class I protostars, in a study of disc chemistry. We find that for each of a number of species, e.g. H2O, adsorption and desorption dominate the changes in the gas-phase fractional abundance; because the desorption rates are very sensitive to temperature, maps of the emissions from such species should reveal the locations of shocks of varying strengths. The gas-phase fractional abundances of some other species, e.g. CS, are also affected by gas-phase reactions, particularly in warm shocked regions. We conclude that the dynamics of massive discs have a strong impact on how they appear when imaged in the emission lines of various molecular species.Comment: 10 figures and 3 tables, accepted for publication in MNRA

The collapse of protoplanetary clumps formed through disc instability: 3D simulations of the pre-dissociation phase

We present 3D smoothed particle hydrodynamics simulations of the collapse of clumps formed through gravitational instability in the outer part of a protoplanetary disc. The initial conditions are taken directly from a global disc simulation, and a realistic equation of state is used to follow the clumps as they contract over several orders of magnitude in density, approaching the molecular hydrogen dissociation stage. The effects of clump rotation, asymmetries, and radiative cooling are studied. Rotation provides support against fast collapse, but non-axisymmetric modes develop and efficiently transport angular momentum outward, forming a circumplanetary disc. This transport helps the clump reach the dynamical collapse phase, resulting from molecular hydrogen dissociation, on a thousand-year timescale, which is smaller than timescales predicted by some previous spherical 1D collapse models. Extrapolation to the threshold of the runaway hydrogen dissociation indicates that the collapse timescales can be shorter than inward migration timescales, suggesting that clumps could survive tidal disruption and deliver a proto-gas giant to distances of even a few AU from the central star.Comment: Accepted for publication in MNRA

A Triple Protostar System Formed via Fragmentation of a Gravitationally Unstable Disk

Binary and multiple star systems are a frequent outcome of the star formation process, and as a result, almost half of all sun-like stars have at least one companion star. Theoretical studies indicate that there are two main pathways that can operate concurrently to form binary/multiple star systems: large scale fragmentation of turbulent gas cores and filaments or smaller scale fragmentation of a massive protostellar disk due to gravitational instability. Observational evidence for turbulent fragmentation on scales of $>$1000~AU has recently emerged. Previous evidence for disk fragmentation was limited to inferences based on the separations of more-evolved pre-main sequence and protostellar multiple systems. The triple protostar system L1448 IRS3B is an ideal candidate to search for evidence of disk fragmentation. L1448 IRS3B is in an early phase of the star formation process, likely less than 150,000 years in age, and all protostars in the system are separated by $<$200~AU. Here we report observations of dust and molecular gas emission that reveal a disk with spiral structure surrounding the three protostars. Two protostars near the center of the disk are separated by 61 AU, and a tertiary protostar is coincident with a spiral arm in the outer disk at a 183 AU separation. The inferred mass of the central pair of protostellar objects is $\sim$1 M$_{sun}$, while the disk surrounding the three protostars has a total mass of $\sim$0.30 M_{\sun}. The tertiary protostar itself has a minimum mass of $\sim$0.085 M$_{sun}$. We demonstrate that the disk around L1448 IRS3B appears susceptible to disk fragmentation at radii between 150~AU and 320~AU, overlapping with the location of the tertiary protostar. This is consistent with models for a protostellar disk that has recently undergone gravitational instability, spawning one or two companion stars.Comment: Published in Nature on Oct. 27th. 24 pages, 8 figure

A Large Double-ring Disk around the Taurus M Dwarf J04124068+2438157

Planet formation imprints signatures on the physical structures of disks. In this paper, we present high-resolution ($\sim$50 mas, 8 au) Atacama Large Millimeter/submillimeter Array (ALMA) observations of 1.3 mm dust continuum and CO line emission toward the disk around the M3.5 star 2MASS J04124068+2438157. The dust disk consists only of two narrow rings at radial distances of 0.47 and 0.78 arcsec ($\sim$70 and 116 au), with Gaussian $\sigma$ widths of 5.6 and 8.5 au, respectively. The width of the outer ring is smaller than the estimated pressure scale height by $\sim25\%$, suggesting dust trapping in a radial pressure bump. The dust disk size, set by the location of the outermost ring, is significantly larger (by $3\sigma$) than other disks with similar millimeter luminosity, which can be explained by an early formation of local pressure bump to stop radial drift of millimeter dust grains. After considering the disk's physical structure and accretion properties, we prefer planet--disk interaction over dead zone or photoevaporation models to explain the observed dust disk morphology. We carry out high-contrast imaging at $L'$ band using Keck/NIRC2 to search for potential young planets, but do not identify any source above $5\sigma$. Within the dust gap between the two rings, we reach a contrast level of $\sim$7 mag, constraining the possible planet below $\sim$2--4 $M_{\rm Jup}$. Analyses of the gap/ring properties suggest a $\sim$Saturn mass planet at $\sim$90 au is likely responsible for the formation of the outer ring, which can be potentially revealed with JWST.Comment: 15 pages, 5 figures. Accepted for publication in Ap

A conscious mouse model of gastric ileus using clinically relevant endpoints

BACKGROUND: Gastric ileus is an unsolved clinical problem and current treatment is limited to supportive measures. Models of ileus using anesthetized animals, muscle strips or isolated smooth muscle cells do not adequately reproduce the clinical situation. Thus, previous studies using these techniques have not led to a clear understanding of the pathophysiology of ileus. The feasibility of using food intake and fecal output as simple, clinically relevant endpoints for monitoring ileus in a conscious mouse model was evaluated by assessing the severity and time course of various insults known to cause ileus. METHODS: Delayed food intake and fecal output associated with ileus was monitored after intraperitoneal injection of endotoxin, laparotomy with bowel manipulation, thermal injury or cerulein induced acute pancreatitis. The correlation of decreased food intake after endotoxin injection with gastric ileus was validated by measuring gastric emptying. The effect of endotoxin on general activity level and feeding behavior was also determined. Small bowel transit was measured using a phenol red marker. RESULTS: Each insult resulted in a transient and comparable decrease in food intake and fecal output consistent with the clinical picture of ileus. The endpoints were highly sensitive to small changes in low doses of endotoxin, the extent of bowel manipulation, and cerulein dose. The delay in food intake directly correlated with delayed gastric emptying. Changes in general activity and feeding behavior were insufficient to explain decreased food intake. Intestinal transit remained unchanged at the times measured. CONCLUSION: Food intake and fecal output are sensitive markers of gastric dysfunction in four experimental models of ileus. In the mouse, delayed gastric emptying appears to be the major cause of the anorexic effect associated with ileus. Gastric dysfunction is more important than small bowel dysfunction in this model. Recovery of stomach function appears to be simultaneous to colonic recovery

Synthetic recording and in situ readout of lineage information in single cells

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here, we describe a new synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out in single cells in situ. This system, termed Memory by Engineered Mutagenesis with Optical In situ Readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by Cas9-based targeted mutagenesis, and read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). To demonstrate a proof of principle of MEMOIR, we engineered mouse embryonic stem (ES) cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of the lineage trees of cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which ES cells switch between two gene expression states. Finally, using simulations, we showed how parallel MEMOIR systems operating in the same cell can enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single cell readout across diverse biological systems

Appropriateness of treatment recommendations for PPI in hospital discharge letters

International audienc