TIP60 up-regulates ΔNp63α to promote cellular proliferation.

An estimated 5.4 million cases of nonmelanoma skin cancer are reported in the United States at an associated cost of $4.8 billion. ΔNp63α, a proto-oncogene in the p53 family of transcription factors, is overexpressed in squamous cell carcinoma (SCC) and associated with poor prognosis and survival. ΔNp63α elicits its tumorigenic effects in part by promoting cellular proliferation and cell survival. Despite its importance in SCC, the upstream regulation of ΔNp63α is poorly understood. In this study, we identify TIP60 as a novel upstream regulator of ΔNp63α. Using a combination of overexpression, silencing, stable expression, and pharmacological approaches in multiple cell lines, we showed that TIP60 up-regulates ΔNp63α expression. Utilizing cycloheximide treatment, we showed that TIP60 catalytic activity is required for stabilization of ΔNp63α protein levels. We further showed that TIP60 coexpression inhibits ΔNp63α ubiquitination and proteasomal degradation. Stabilization of ΔNp63α protein was further associated with TIP60-mediated acetylation. Finally, we demonstrated that TIP60-mediated regulation of ΔNp63α increases cellular proliferation by promoting G2/M progression through MTS assays and flow cytometry. Taken together, our findings provide evidence that TIP60 may contribute to SCC progression by increasing ΔNp63α protein levels, thereby promoting cellular proliferation.

Myocardium and BMP signaling are required for endocardial differentiation.

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1 expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardium-depleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation.

ETS transcription factors Etv2 and Fli1b are required for tumor angiogenesis.

ETS transcription factor ETV2/Etsrp functions as a key regulator of embryonic vascular development in multiple vertebrates. However, its role in pathological vascular development has not been previously investigated. To analyze its role in tumor angiogenesis, we utilized a zebrafish xenotransplantation model. Using a photoconvertible kdrl:NLS-KikGR line, we demonstrated that all tumor vessels originate from the existing embryonic vasculature by the mechanism of angiogenesis. Xenotransplantation of mouse B16 melanoma cells resulted in a significant increase in expression of the ETS transcription factors etv2 and fli1b expression throughout the embryonic vasculature. etv2 null mutants which undergo significant recovery of embryonic angiogenesis during later developmental stages displayed a strong inhibition of tumor angiogenesis. We utilized highly specific and fully validated photoactivatable morpholinos to inhibit Etv2 function after embryonic vasculogenesis has completed. Inducible inhibition of Etv2 function resulted in a significant reduction of tumor angiogenesis and inhibition of tumor growth. Furthermore, inducible inhibition of Etv2 function in fli1b mutant embryos resulted in even stronger reduction in tumor angiogenesis and growth, demonstrating that Etv2 and Fli1b have a partially redundant requirement during tumor angiogenesis. These results demonstrate the requirement for Etv2 and Fli1b in tumor angiogenesis and suggest that inhibition of these ETS factors may present a novel strategy to inhibit tumor angiogenesis and reduce tumor growth.

ETS transcription factors in embryonic vascular development.

At least thirteen ETS-domain transcription factors are expressed during embryonic hematopoietic or vascular development and potentially function in the formation and maintenance of the embryonic vasculature or blood lineages. This review summarizes our current understanding of the specific roles played by ETS factors in vasculogenesis and angiogenesis and the implications of functional redundancies between them.

Etv2 and fli1b function together as key regulators of vasculogenesis and angiogenesis.

OBJECTIVE: The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis. APPROACH AND RESULTS: Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites. CONCLUSIONS: Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.

Muscle-derived microRNAs correlated with thigh lean mass gains during progressive resistance training in older adults.

Resistance training (RT) remains the most effective treatment for age-related declines in muscle mass. However, many older adults experience attenuated muscle hypertrophy in response to RT when compared with younger adults. This may be attributed to underlying molecular processes that are dysregulated by aging and exacerbated by improperly prescribed RT weekly volume, intensity, and/or frequency doses. MicroRNAs (miRNAs) are key epigenetic regulators that impact signaling pathways and protein expression within cells, are dynamic and responsive to exercise stimuli, and are often dysregulated in diseases. In this study, we used untargeted miRNA-seq to examine miRNA in skeletal muscle and serum-derived exosomes of older adults (n = 18, 11 M/7 F, 66 ± 1 yr) who underwent three times per wk RT for 30 wk [e.g., high intensity three times/wk (HHH, n = 9) or alternating high-low-high (HLH) intensity (n = 9)], after a standardized 4-wk washin. Within each tissue, miRNAs were clustered into modules based on pairwise correlation using weighted gene correlation network analysis (WGCNA). Modules were tested for association with the magnitude of RT-induced thigh lean mass (TLM) change [as measured by dual-energy X-ray absorptiometry (DXA)]. Although no modules were unique to training dose, we identified miRNA modules in skeletal muscle associated with TLM gains irrespective of exercise dose. Using miRNA-target interactions, we analyzed key miRNAs in significant modules for their potential regulatory involvement in biological pathways. Findings point toward potential miRNAs that may be informative biomarkers and could also be evaluated as potential therapeutic targets as an adjuvant to RT to maximize skeletal muscle mass accrual in older adults.NEW & NOTEWORTHY In this work, we identified a set of microRNAs correlated with thigh lean mass gains in a group of older adults. To our knowledge, this is the first time these microRNAs have been identified as novel predictive biomarkers correlating with lean mass gains in aging adults. As biomarkers, these may help interventionalists identify older individuals that are positively responding to an exercise intervention.

Sexually segregated housing results in improved early larval survival in zebrafish.

Large-scale aquaculture facilities require highly optimized husbandry protocols that maximize fecundity and embryo health while minimizing cost and effort. Although zebrafish are being increasingly used for preclinical drug screens, functional genomic research and toxicological and behavioral studies, many of the basic husbandry procedures that are used for these fish have not been thoroughly tested. In this study, the authors compared the breeding success of zebrafish housed in sex-separated and those housed in mixed-gender arrangements. They observed a significant increase in fecundity (egg production) between the first and the third breeding and found that egg survivorship tended to increase during successive pairings. The authors also found that zebrafish had higher fecundity, egg viability and seemed to have a higher breeding success rate when males and females were housed separately than when they were housed together.

Differential MicroRNA Signatures in the Pathogenesis of Barrett's Esophagus.

OBJECTIVES: Barrett's esophagus (BE) is the precursor lesion and a major risk factor for esophageal adenocarcinoma (EAC). Although patients with BE undergo routine endoscopic surveillance, current screening methodologies have proven ineffective at identifying individuals at risk of EAC. Since microRNAs (miRNAs) have potential diagnostic and prognostic value as disease biomarkers, we sought to identify an miRNA signature of BE and EAC. METHODS: High-throughput sequencing of miRNAs was performed on serum and tissue biopsies from 31 patients identified either as normal, gastroesophageal reflux disease (GERD), BE, BE with low-grade dysplasia (LGD), or EAC. Logistic regression modeling of miRNA profiles with Lasso regularization was used to identify discriminating miRNA. Quantitative reverse transcription polymerase chain reaction was used to validate changes in miRNA expression using 46 formalin-fixed, paraffin-embedded specimens obtained from normal, GERD, BE, BE with LGD or HGD, and EAC subjects. RESULTS: A 3-class predictive model was able to classify tissue samples into normal, GERD/BE, or LGD/EAC classes with an accuracy of 80%. Sixteen miRNAs were identified that predicted 1 of the 3 classes. Our analysis confirmed previous reports indicating that miR-29c-3p and miR-193b-5p expressions are altered in BE and EAC and identified miR-4485-5p as a novel biomarker of esophageal dysplasia. Quantitative reverse transcription polymerase chain reaction validated 11 of 16 discriminating miRNAs. DISCUSSION: Our data provide an miRNA signature of normal, precancerous, and cancerous tissue that may stratify patients at risk of progressing to EAC. We found that serum miRNAs have a limited ability to distinguish between disease states, thus limiting their potential utility in early disease detection.

microRNA involvement in the onset and progression of Barrett's esophagus: a systematic review.

Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that develops from Barrett's esophagus (BE), an intestinal metaplasia of the distal esophagus. microRNAs (miRNAs), short non-coding regulatory RNAs, are frequently dysregulated in BE and are thought to play key roles in the onset of BE and its progression to EAC. miRNAs thus have potential diagnostic and prognostic value and are increasingly being used as cancer biomarkers. This review summarizes the current literature related to miRNAs that are dysregulated in BE within the context of Hedgehog, Notch, MAPK, NF kappa-B, Wnt and epithelial-mesenchymal transition (EMT) signaling which are thought to drive BE onset and progression. This comprehensive analysis of miRNAs and their associated signaling in the regulation of BE provides an overview of vital discoveries in this field and highlights gaps in our understanding of BE pathophysiology that warrant further investigation.

Identification of novel ΔNp63α-regulated miRNAs using an optimized small RNA-Seq analysis pipeline.

Advances in high-throughput sequencing have enabled profiling of microRNAs (miRNAs), however, a consensus pipeline for sequencing of small RNAs has not been established. We built and optimized an analysis pipeline using Partek Flow, circumventing the need for analyzing data via scripting languages. Our analysis assessed the effect of alignment reference, normalization method, and statistical model choice on biological data. The pipeline was evaluated using sequencing data from HaCaT cells transfected with either a non-silencing control or siRNA against ΔNp63α, a p53 family member protein which is highly expressed in non-melanoma skin cancer and shown to regulate a number of miRNAs. We posit that 1) alignment and quantification to the miRBase reference provides the most robust quantitation of miRNAs, 2) normalizing sample reads via Trimmed Mean of M-values is the most robust method for accurate downstream analyses, and 3) use of the lognormal with shrinkage statistical model effectively identifies differentially expressed miRNAs. Using our pipeline, we identified previously unrecognized regulation of miRs-149-5p, 18a-5p, 19b-1-5p, 20a-5p, 590-5p, 744-5p and 93-5p by ΔNp63α. Regulation of these miRNAs was validated by RT-qPCR, substantiating our small RNA-Seq pipeline. Further analysis of these miRNAs may provide insight into ΔNp63α's role in cancer progression. By defining the optimal alignment reference, normalization method, and statistical model for analysis of miRNA sequencing data, we have established an analysis pipeline that may be carried out in Partek Flow or at the command line. In this manner, our pipeline circumvents some of the major hurdles encountered during small RNA-Seq analysis.

ΔNp63α and microRNAs: leveraging the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a cellular reprogramming mechanism that is an underlying cause of cancer metastasis. Recent investigations have uncovered an intricate network of regulation involving the TGFß, Wnt, and Notch signaling pathways and small regulatory RNA species called microRNAs (miRNAs). The activity of a transcription factor vital to the maintenance of epithelial stemness, ΔNp63α, has been shown to modulate the activity of these EMT pathways to either repress or promote EMT. Furthermore, ΔNp63α is a known regulator of miRNA, including those directly involved in EMT. This review discusses the evidence of ΔNp63α as a master regulator of EMT components and miRNA, highlighting the need for a deeper understanding of its role in EMT. This expanded knowledge may provide a basis for new developments in the diagnosis and treatment of metastatic cancer.

Dose-dependent effects of chemical immobilization on the heart rate of embryonic zebrafish.

The small size and optical transparency of zebrafish embryos and larvae greatly facilitate modern intravital microscopic phenotyping of these experimentally tractable laboratory animals. Neither the experimentally derived dose-response relationships for chemicals commonly used in the mounting of live fish larvae, nor their effect on the stress of the animal, are currently available in the research literature. This is particularly problematic for IACUCs attempting to maintain the highest ethical standards of animal care in the face of a recent spate in investigator-initiated requests to use embryonic zebrafish as experimental models. The authors address this issue by describing the dose-dependent efficacy of several commonly used chemical mounting treatments and their effect on one stress parameter, embryo heart rate. The results of this study empirically define, for the first time, effective, minimally stressful treatments for immobilization and in vivo visualization during early zebrafish development.

High-speed confocal imaging of zebrafish heart development.

Due to its optical clarity and rudimentary heart structure (i.e., single atrium and ventricle), the zebrafish provides an excellent model for studying the genetic, morphological, and functional basis of normal and pathophysiological heart development in vivo. Recent advances in high-speed confocal imaging have made it possible to capture 2D zebrafish heart wall motions with temporal and spatial resolutions sufficient to characterize the highly dynamic intravital flow-structure environment. We have optimized protocols for introducing fluorescent tracer particles into the zebrafish cardiovasculature, imaging intravital heart wall motion, and performing high-resolution blood flow mapping that will be broadly useful in elucidating flow-structure relationships.

An optimized method for delivering flow tracer particles to intravital fluid environments in the developing zebrafish.

Growing evidence suggests that intravital flow-structure interactions are critical morphogens for normal embryonic development and disease progression, but fluid mechanical studies aimed at investigating these interactions have been limited in their ability to visualize and quantify fluid flow. In this study, we describe a protocol for injecting small (≤1.0 µm) tracer particles into fluid beds of the larval zebrafish to facilitate microscale fluid mechanical analyses. The microinjection apparatus and associated borosilicate pipette design, typically blunt-tipped with a 2-4 micron tip O.D., yielded highly linear (r(2)=0.99) in vitro bolus ejection volumes. The physical characteristics of the tracer particles were optimized for efficient particle delivery. Seeding densities suitable for quantitative blood flow mapping (≥50 thousand tracers per fish) were routinely achieved and had no adverse effects on zebrafish physiology or long-term survivorship. The data and methods reported here will prove valuable for a broad range of in vivo imaging technologies [e.g., particle-tracking velocimetry, µ-Doppler, digital particle image velocimetry (DPIV), and 4-dimensional-DPIV] which rely on tracer particles to visualize and quantify fluid flow in the developing zebrafish.

Normal interstitial flow is critical for developmental lymphangiogenesis in the zebrafish.

BACKGROUND: The lymphatic system plays a critical role in the body's fluid and protein homeostasis, immune regulation, and dietary fat absorption. One of the major pathologies of the lymphatic system is primary lymphedema, which occurs in approximately 0.6% of live births and is caused by missing or impaired lymphatic vessels. Although there is a great need for medical intervention into diseases of the lymphatic system, very little is known about its development or how it maintains integrity over time. Recent studies have suggested that biophysical components, such as local extracellular fluid flow, may be important factors during initiation of lymphangiogenesis. We hypothesize that interstitial fluid flow functions as an important morphoregulator during developmental lymphangiogenesis. METHODS AND RESULTS: In the present study we use pharmacological agents and a mutant fish line to modulate interstitial flow. Our data confirm that a sufficient increase or decrease in interstitial flow can profoundly affect lymphatic patterning and may result in a lymphedema-like phenotype. Proper interstitial flow appears to be necessary during LEC migration for proper lymphatic development. CONCLUSIONS: These results support the contention that interstitial flow is an important morphoregulator of developmental lymphangiogenesis.