13 research outputs found
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In Situ Hybridization Assay-Based Small-Molecule Screening in Zebrafish
In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts.Stem Cell and Regenerative Biolog
Medulloblastoma therapy generates risk of a poorly-prognostic H3 wild-type subgroup of diffuse intrinsic pontine glioma: a report from the International DIPG Registry
Abstract
With improved survivorship in medulloblastoma, there has been an increasing incidence of late complications. To date, no studies have specifically addressed the risk of radiation-associated diffuse intrinsic pontine glioma (DIPG) in medulloblastoma survivors. Query of the International DIPG Registry identified six cases of DIPG with a history of medulloblastoma treated with radiotherapy. All patients underwent central radiologic review that confirmed a diagnosis of DIPG. Six additional cases were identified in reports from recent cooperative group medulloblastoma trials (total n = 12; ages 7 to 21 years). From these cases, molecular subgrouping of primary medulloblastomas with available tissue (n = 5) revealed only non-WNT, non-SHH subgroups (group 3 or 4). The estimated cumulative incidence of DIPG after post-treatment medulloblastoma ranged from 0.3–3.9%. Posterior fossa radiation exposure (including brainstem) was greater than 53.0 Gy in all cases with available details. Tumor/germline exome sequencing of three radiation-associated DIPGs revealed an H3 wild-type status and mutational signature distinct from primary DIPG with evidence of radiation-induced DNA damage. Mutations identified in the radiation-associated DIPGs had significant molecular overlap with recurrent drivers of adult glioblastoma (e.g. NRAS, EGFR, and PTEN), as opposed to epigenetic dysregulation in H3-driven primary DIPGs. Patients with radiation-associated DIPG had a significantly worse median overall survival (median 8 months; range 4–17 months) compared to patients with primary DIPG. Here, it is demonstrated that DIPG occurs as a not infrequent complication of radiation therapy in survivors of pediatric medulloblastoma and that radiation-associated DIPGs may present as a poorly-prognostic distinct molecular subgroup of H3 wild-type DIPG. Given the abysmal survival of these cases, these findings provide a compelling argument for efforts to reduce exposure of the brainstem in the treatment of medulloblastoma. Additionally, patients with radiation-associated DIPG may benefit from future therapies targeted to the molecular features of adult glioblastoma rather than primary DIPG.https://deepblue.lib.umich.edu/bitstream/2027.42/145180/1/40478_2018_Article_570.pd
Zebrafish as a model for normal and malignant hematopoiesis
Zebrafish studies in the past two decades have made major contributions to our understanding of hematopoiesis and its associated disorders. The zebrafish has proven to be a powerful organism for studies in this area owing to its amenability to large-scale genetic and chemical screening. In addition, the externally fertilized and transparent embryos allow convenient genetic manipulation and in vivo imaging of normal and aberrant hematopoiesis. This review discusses available methods for studying hematopoiesis in zebrafish, summarizes key recent advances in this area, and highlights the current and potential contributions of zebrafish to the discovery and development of drugs to treat human blood disorders
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Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4)
Recent small RNA sequencing data has uncovered 3′ end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3′ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3′ mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3′ mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3′ miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development
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Adenosine signaling promotes hematopoietic stem and progenitor cell emergence
Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates
Mutations in QARS, Encoding Glutaminyl-tRNA Synthetase, Cause Progressive Microcephaly, Cerebral-Cerebellar Atrophy, and Intractable Seizures
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Additional file 1: of Medulloblastoma therapy generates risk of a poorly-prognostic H3 wild-type subgroup of diffuse intrinsic pontine glioma: a report from the International DIPG Registry
Table S1. Treatment details for primary medulloblastoma. Table S2. Multivariate analysis of overall survival for primary and radiation-associated DIPGs. Table S3. Sequencing of radiation-associated DIPGs. Figure S1. Immunohistochemical staining for H3K27Ă‚Â M of positive and negative control pediatric high-grade gliomas. Figure S2. Diagnosis and management of case 2, which included non-standard treatment for medulloblastoma. Figure S3. Diagnosis and management of case 3, which included standard therapy for medulloblastoma. (DOCX 5366 kb