PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-9 (9)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division 
EMBO Reports  2014;15(4):383-391.
The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.
doi:10.1002/embr.201337424
PMCID: PMC3989669  PMID: 24599748
Asymmetric division; differentiation; MYC; neural progenitor; Notch
2.  Identification of a large protein network involved in epigenetic transmission in replicating DNA of embryonic stem cells 
Nucleic Acids Research  2014;42(11):6972-6986.
Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities and chromatin modifying complexes highly organized within the chromatin. Although much effort has been focused on identifying genome-binding sites, little is known on their dynamic association with chromatin across cell divisions. Here, we used a modified version of the iPOND (isolation of proteins at nascent DNA) technology to identify a large protein network enriched at nascent DNA in ESCs. This comprehensive and unbiased proteomic characterization in ESCs reveals that, in addition to the core replication machinery, proteins relevant for pluripotency of ESCs are present at DNA replication sites. In particular, we show that the chromatin remodeller HDAC1–NuRD complex is enriched at nascent DNA. Interestingly, an acute block of HDAC1 in ESCs leads to increased acetylation of histone H3 lysine 9 at nascent DNA together with a concomitant loss of methylation. Consistently, in contrast to what has been described in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs. Our results are therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication.
doi:10.1093/nar/gku374
PMCID: PMC4066787  PMID: 24852249
3.  Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart 
BMC Physiology  2012;12:3.
Background
Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.
Results
Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.
Conclusion
The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
doi:10.1186/1472-6793-12-3
PMCID: PMC3334682  PMID: 22449203
Heart; Screening; Zebrafish; Small molecule; Ex vivo; Ca2+ signaling
4.  The retinoic acid inducible Cas-family signaling protein Nedd9 regulates neural crest cell migration by modulating adhesion and actin dynamics 
Neuroscience  2009;162(4):1106-1119.
Summary
Cell migration is essential for the development of numerous structures derived from embryonic neural crest cells (NCCs), however the underlying molecular mechanisms are incompletely understood. NCCs migrate long distances in the embryo and contribute to many different cell types, including peripheral neurons, glia and pigment cells. In the present work we report expression of Nedd9, a scaffolding protein within the integrin signaling pathway, in non-lineage restricted neural crest progenitor cells. In particular, Nedd9 was found to be expressed in the dorsal neural tube at the time of neural crest delamination and in early migrating NCCs. To analyze the role of Nedd9 in neural crest development we performed loss- and gain-of-function experiments and examined the subsequent effects on delamination and migration in vitro and in vivo. Our results demonstrate that loss of Nedd9 activity in chick NCCs perturbs cell spreading and the density of focal complexes and actin filaments, properties known to depend on integrins. Moreover, a siRNA dose-dependent decrease in Nedd9 activity results in a graded reduction of NCCs migratory distance while forced overexpression increases it. Retinoic acid (RA) was found to regulate Nedd9 expression in NCCs. Our results demonstrate in vivo that Nedd9 promotes the migration of NCCs in a graded manner and suggest a role for RA in the control of Nedd9 expression levels.
doi:10.1016/j.neuroscience.2009.05.035
PMCID: PMC2797478  PMID: 19464348
HEF1; Cas-L; cytoskeleton; focal adhesion; multipotency; RA
5.  The Runx1/AML1 transcription factor selectively regulates development and survival of TrkA nociceptive sensory neurons 
Nature neuroscience  2006;9(2):180-187.
Neural crest cells (NCCs) can adopt different neuronal fates. In NCCs, neurogenin-2 promotes sensory specification but does not specify different subclasses of sensory neurons. Understanding the gene cascades that direct Trk gene activation may reveal mechanisms generating sensory diversity, because different Trks are expressed in different sensory neuron subpopulations. Here we show in chick and mouse that the Runt transcription factor Runx1 promotes axonal growth, is selectively expressed in neural crest-derived TrkA+ sensory neurons and mediates TrkA transactivation in migratory NCCs. Inhibition of Runt activity depletes TrkA expression and leads to neuronal death. Moreover, Runx1 overexpression is incompatible with multipotency in the migratory neural crest but does not induce expression of pan-neuronal genes. Instead, Runx1-induced neuronal differentiation depends on an existing neurogenin2 proneural gene program. Our data show that Runx1 directs, in a context-dependent manner, key aspects of the establishment of the TrkA+ nociceptive subclass of neurons.
doi:10.1038/nn1631
PMCID: PMC2703717  PMID: 16429136
6.  Lack of the Central Nervous System- and Neural Crest-Expressed Forkhead Gene Foxs1 Affects Motor Function and Body Weight 
Molecular and Cellular Biology  2005;25(13):5616-5625.
To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-β-galactosidase reporter gene “knock-in” (Foxs1β-gal/β-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a β-galactosidase reporter gene. Staining for β-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1β-gal/β-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1β-gal/lβ-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1β-gal/β-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.
doi:10.1128/MCB.25.13.5616-5625.2005
PMCID: PMC1157007  PMID: 15964817
7.  Whole-genome expression profiling through fragment display and combinatorial gene identification 
Nucleic Acids Research  2004;32(16):e127.
There is a growing demand for highly parallel gene expression analysis with whole genome coverage, high sensitivity and high accuracy. Open systems such as differential display are capable of analyzing most of the expressed genome but are not quantitative and generally require manual identification of differentially expressed genes by sequencing. Closed systems such as microarrays use gene-specific probes and are, therefore, limited to studying specific genes in well-characterized species. Here, we describe Tangerine, a PCR-based system that combines the scope and generality of open systems with a robust and immediate identification algorithm using publicly available sequence information. By combinatorial analysis of three independent and complete DNA indexing profiles, each displaying the complete set of expressed transcripts on capillary electrophoresis, the method allows transcripts to be simultaneously quantified and identified. The method is sensitive, accurate and reproducible, and is amenable to high-throughput automated operation.
doi:10.1093/nar/gnh126
PMCID: PMC519127  PMID: 15356287
9.  MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division 
EMBO Reports  2014;15(4):383-391.
The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.
doi:10.1002/embr.201337424
PMCID: PMC3989669  PMID: 24599748
Asymmetric division; differentiation; MYC; neural progenitor; Notch

Results 1-9 (9)