Faculty Bibliography

As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.

Introduction: Chlorhexidine gluconate (CHX) is widely used as a preoperative surgical skin-preparation solution and intra-wound irrigation agent, with excellent efficacy against wide variety of bacteria. The cytotoxic effect of CHX on local proliferating cells following orthopaedic procedures is largely undescribed. Our aim was to investigate the in vitro effects of CHX on primary fibroblasts, myoblasts, and osteoblasts. Methods: Cells were exposed to CHX dilutions (0%, 0.002%, 0.02%, 0.2%, and 2%) for either a 1, 2, or 3-minute duration. Cell survival was measured using a cytotoxicity assay (Cell Counting Kit-8). Cell migration was measured using a scratch assay: a "scratch" was made in a cell monolayer following CHX exposure, and time to closure of the scratch was measured. Results: All cells exposed to CHX dilutions of ≥ 0.02% for any exposure duration had cell survival rates of less than 6% relative to untreated controls (p < 0.001). Cells exposed to CHX dilution of 0.002% all had significantly lower survival rates relative to control (p < 0.01) with the exception of 1-minute exposure to fibroblasts, which showed 96.4% cell survival (p = 0.78). Scratch defect closure was seen in < 24 hours in all control conditions. However, cells exposed to CHX dilutions ≥ 0.02% had scratch defects that remained open indefinitely. Conclusions: The clinically used concentration of CHX (2%) permanently halts cell migration and significantly reduces survival of in vitro fibroblasts, myoblasts, and osteoblasts. Further in vivo studies are required to examine and optimize CHX safety and efficacy when applied near open incisions or intra-wound application.

IQGAP1 is a scaffold protein involved in the assembly of adherens junctions. Our work has recently revealed a novel role for IQGAP1 in the regulation of tight junctions (TJ) through differential recruitment of claudins to the nascent TJ. Here, we discuss the potential mechanisms of this regulation, including IQGAP1 effects on CDC42, and IQGAP1 interactions with sorting/trafficking molecules (e.g. Exo70). Given the many roles of IQGAP1 and the large number of interacting partners, we focus our discussion of these functions in the context of junction formation, trafficking, growth factor signaling and cancer. We also propose a potential role for IQGAP1 in regulating epithelial integrity and compartmentalized signaling in epithelia.

Growth factors induce and pattern sensory organs, but how their distribution is regulated by the extracellular matrix (ECM) is largely unclear. To address this question, we analyzed the diffusion behavior of Fgf10 molecules during sensory organ formation in the zebrafish posterior lateral line primordium. In this tissue, secreted Fgf10 induces organ formation at a distance from its source. We find that most Fgf10 molecules are highly diffusive and move rapidly through the ECM. We identify Anosmin1, which when mutated in humans causes Kallmann Syndrome, as an ECM protein that binds to Fgf10 and facilitates its diffusivity by increasing the pool of fast-moving Fgf10 molecules. In the absence of Anosmin1, Fgf10 levels are reduced and organ formation is impaired. Global overexpression of Anosmin1 slows the fast-moving Fgf10 molecules and results in Fgf10 dispersal. These results suggest that Anosmin1 liberates ECM-bound Fgf10 and shuttles it to increase its signaling range.

The proper accumulation and maintenance of stem cells is critical for organ development and homeostasis. The Notch signaling pathway maintains stem cells in diverse organisms and organ systems. In Caenorhabditis elegans, GLP-1/Notch activity prevents germline stem cell (GSC) differentiation. Other signaling mechanisms also influence the maintenance of GSCs, including the highly-conserved TOR substrate ribosomal protein S6 kinase. Although C. elegans bearing either a null mutation in rsks-1/S6K or a reduction-of-function (rf) mutation in glp-1/Notch produce half the normal number of adult germline progenitors, virtually all these single mutant animals are fertile. However, glp-1(rf)rsks-1(null) double mutant animals are all sterile, and in about half of their gonads, all GSCs differentiate, a distinctive phenotype associated with a significant reduction or loss of GLP-1 signaling. How rsks-1/S6K promotes GSC fate is unknown. Here, we determine that rsks-1/S6K acts germline-autonomously to maintain GSCs, and that it does not act through Cyclin-E or MAP kinase in this role. We found that interfering with translation also enhances glp-1(rf), but that regulation through rsks-1 cannot fully account for this effect. In a genome-scale RNAi screen for genes that act similarly to rsks-1/S6K, we identified 56 RNAi enhancers of glp-1/Notch sterility, many of which were previously not known to interact functionally with Notch. Further investigation revealed six candidates that, by genetic criteria, act linearly with rsks-1/S6K. These include genes encoding translation-related proteins, cacn-1/Cactin, an RNA exosome component and a Hedgehog-related ligand. We found that additional Hedgehog-related ligands may share functional relationships with glp-1/Notch and rsks-1/S6K in maintaining germline progenitors.

Neurofilament (NFL) proteins have recently been found to play unique roles in synapses. NFL is known to interact with the GluN1 subunit of N-methyl-D-aspartic acid (NMDAR) and be reduced in schizophrenia though functional consequences are unknown. Here we investigated whether the interaction of NFL with GluN1 modulates synaptic transmission and schizophrenia-associated behaviors. The interaction of NFL with GluN1 was assessed by means of molecular, pharmacological, electrophysiological, magnetic resonance spectroscopy (MRS), and schizophrenia-associated behavior analyses. NFL deficits cause an NMDAR hypofunction phenotype including abnormal hippocampal function, as seen in schizophrenia. NFL-/- deletion in mice reduces dendritic spines and GluN1 protein levels, elevates ubiquitin-dependent turnover of GluN1 and hippocampal glutamate measured by MRS, and depresses hippocampal long-term potentiation. NMDAR-related behaviors are also impaired, including pup retrieval, spatial and social memory, prepulse inhibition, night-time activity, and response to NMDAR antagonist, whereas motor deficits are minimal. Importantly, partially lowering NFL in NFL+/- mice to levels seen regionally in schizophrenia, induced similar but milder NMDAR-related synaptic and behavioral deficits. Our findings support an emerging view that central nervous system neurofilament subunits including NFL in the present report, serve distinctive, critical roles in synapses relevant to neuropsychiatric diseases.

Neurodegenerative disorders of ageing (NDAs) such as Alzheimer disease, Parkinson disease, frontotemporal dementia, Huntington disease and amyotrophic lateral sclerosis represent a major socio-economic challenge in view of their high prevalence yet poor treatment. They are often called 'proteinopathies' owing to the presence of misfolded and aggregated proteins that lose their physiological roles and acquire neurotoxic properties. One reason underlying the accumulation and spread of oligomeric forms of neurotoxic proteins is insufficient clearance by the autophagic-lysosomal network. Several other clearance pathways are also compromised in NDAs: chaperone-mediated autophagy, the ubiquitin-proteasome system, extracellular clearance by proteases and extrusion into the circulation via the blood-brain barrier and glymphatic system. This article focuses on emerging mechanisms for promoting the clearance of neurotoxic proteins, a strategy that may curtail the onset and slow the progression of NDAs.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Individuals can vary substantially in size, but the proportions of their body plans are often maintained. We generated smaller zebrafish by removing 30% of their cells at the blastula stages and found that these embryos developed into normally patterned individuals. Strikingly, the proportions of all germ layers adjusted to the new embryo size within 2 hours after cell removal. As Nodal-Lefty signalling controls germ-layer patterning, we performed a computational screen for scale-invariant models of this activator-inhibitor system. This analysis predicted that the concentration of the highly diffusive inhibitor Lefty increases in smaller embryos, leading to a decreased Nodal activity range and contracted germ-layer dimensions. In vivo studies confirmed that Lefty concentration increased in smaller embryos, and embryos with reduced Lefty levels or with diffusion-hindered Lefty failed to scale their tissue proportions. These results reveal that size-dependent inhibition of Nodal signalling allows scale-invariant patterning.