RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis

The RNA interference technique is a powerful tool to understand gene function. Intriguingly, RNA interference cannot only be used for cells in vitro, but also in living organisms. Here, we have adapted the method for use in the chick embryo. However, this technique is limited by the uncertainty in predicting the RNAi transfection efficiency and site in the embryo. Hence, we elaborated a modified vector system, pEGFP-shRNA, which can coexpress enhanced green fluorescent protein (EGFP) and short hairpin RNA (shRNA) simultaneously to facilitate analysis of gene silencing in chicken embryos. We tested the silencing of two highly conserved genes (cAxin2, cParaxis), which play crucial roles in chicken embryonic developmental processes. For each target gene, four to five small DNA inserts, each of them encoding one shRNA, were selected and cloned individually to the vector downstream of the Pol III promoter (either human H1 or U6 promoter), which shared with highly conserved motifs in human and chicken. The pEGFP-shRNA constructs were electroporated into the neural tube or somites. After subsequent re-incubation of 24 h, the EGFP expression, with green fluorescent signal, indicated the transfected regions in the neural tube or somites. The EGFP expressing embryos were further submitted into the process of in situ hybridization for examination of the silencing effects. The results show that the EGFP signal in transfected areas correlated with the silencing of the target genes (cAxin2, cParaxis). The cAxin2 expression was inhibited by shRNAs of either targeting the RGS domain or the DAX domain coding region. The cParaxis mRNA level in transgenic somites and the related migratory myogenic population was also reduced. The results suggest that our novel dual expression EGFP-shRNA system opens a new possibility to study gene function in a convenient and efficient way. © 2005 Elsevier Inc. All rights reserved

A novel interaction between ATOH8 and PPP3CB

ATOH8 is a bHLH transcription factor playing roles in a variety of developmental processes such as neurogenesis, differentiation of pancreatic precursor cells, development of kidney and muscle, and differentiation of endothelial cells. PPP3CB belongs to the catalytic subunit of the serine/threonine phosphatase, calcineurin, which can dephosphorylate its substrate proteins to regulate their physiological activities. In our study, we demonstrated that ATOH8 interacts with PPP3CB in vitro with different approaches. We show that the conserved catalytic domain of PPP3CB interacts with both the N-terminus and the bHLH domain of ATOH8. Although the interaction domain of PPP3CB is conserved among all isoforms of calcineurin A, ATOH8 selectively interacts with PPP3CB instead of PPP3CA, probably due to the unique proline-rich region present in the N-terminus of PPP3CB, which controls the specificity of its interaction partners. Furthermore, we show that inhibition of the interaction with calcineurin inhibitor, cyclosporin A (CsA), leads to the retention of ATOH8 to the cytoplasm, suggesting that the interaction renders nuclear localization of ATOH8 which may be critical to control its activity as transcription factor

The dermomyotome ventrolateral lip is essential for the hypaxial myotome formation

Background The myotome is the primitive skeletal muscle that forms within the embryonic metameric body wall. It can be subdivided into an epaxial and hypaxial domain. It has been shown that the formation of the epaxial myotome requires the dorsomedial lip of the dermomyotome (DML). Although the ventrolateral lip (VLL) of the dermomyotome is believed to be required for the formation of the hypaxial myotome, experimentally evidence for this statement still needs to be provided. Provision of such data would enable the resolution of a debate regarding the formation of the hypaxial dermomyotome. Two mechanisms have been proposed for this tissue. The first proposes that the intermediate dermomyotome undergoes cellular expansion thereby pushing the ventral lateral lip in a lateral direction (translocation). In contrast, the alternative view holds that the ventral lateral lip grows laterally. Results Using time lapse confocal microscopy, we observed that the GFP-labelled ventrolateral lip (VLL) of the dermomyotome grows rather than translocates in a lateral direction. The necessity of the VLL for lateral extension of the myotome was addressed by ablation studies. We found that the hypaxial myotome did not form after VLL ablation. In contrast, the removal of an intermediate portion of the dermomyotome had very little effect of the hypaxial myotome. These results demonstrate that the VLL is required for the formation of the hypaxial myotome. Conclusion Our study demonstrates that the dermomyotome ventrolateral lip is essential for the hypaxial myotome formation and supports the lip extension model. Therefore, despite being under independent signalling controls, both the dorsomedial and ventrolateral lip fulfil the same function, i.e. they extend into adjacent regions permitting the growth of the myotome

The Emergence of Embryonic Myosin Heavy Chain during Branchiomeric Muscle Development

A prerequisite for discovering the properties and therapeutic potential of branchiomeric muscles is an understanding of their fate determination, pattering and differentiation. Although the expression of differentiation markers such as myosin heavy chain (MyHC) during trunk myogenesis has been more intensively studied, little is known about its expression in the developing branchiomeric muscle anlagen. To shed light on this, we traced the onset of MyHC expression in the facial and neck muscle anlagen by using the whole-mount in situ hybridization between embryonic days E9.5 and E15.5 in the mouse. Unlike trunk muscle, the facial and neck muscle anlagen express MyHC at late stages. Within the branchiomeric muscles, our results showed variation in the emergence of MyHC expression. MyHC was first detected in the first arch-derived muscle anlagen, while its expression in the second arch-derived muscle and non-somitic neck muscle began at a later time point. Additionally, we show that non-ectomesenchymal neural crest invasion of the second branchial arch is delayed compared with that of the first brachial arch in chicken embryos. Thus, our findings reflect the timing underlying branchiomeric muscle differentiation

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

BAF(mSWI/SNF) complex regulates mediolateral cortical patterning in the developing forebrain

Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny

Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying Both Ligand and siRNA

Gastric cancer is the second leading cause of cancer-related death worldwide. RNA nanotechnology has recently emerged as an important field due to recent finding of its high thermodynamic stability, favorable and distinctive in vivo attributes. Here we reported the use of the thermostable three-way junction (3WJ) of bacteriophage phi29 motor pRNA to escort folic acid, a fluorescent image marker and BRCAA1 siRNA for targeting, imaging, delivery, gene silencing and regression of gastric cancer in animal models. In vitro assay revealed that the RNA nanoparticles specifically bind to gastric cancer cells, and knock-down the BRCAA1 gene. Apoptosis of gastric cancer cells was observed. Animal trials confirmed that these RNA nanoparticles could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues. The volume of gastric tumors noticeably decreased during the course of treatment. No damage to important organs by RNA nanoparticles was detectible. All the results indicated that this novel RNA nanotechnology can overcome conventional cancer therapeutic limitations and opens new opportunities for specific delivery of therapeutics to stomach cancer without damaging normal cells and tissues, reduce the toxicity and side effect, improve the therapeutic effect, and exhibit great potential in clinical tumor therapy

Mesenchymal stem cell-based therapies for treating well-studied neurological disorders: a systematic review

BackgroundMillions of people across the globe are affected by conditions like Amyotrophic Lateral Sclerosis (ALS), Parkinson’s Disease (PD), Multiple Sclerosis (MS), Spinal Cord Injury (SCI), and Traumatic Brain Injury (TBI), although most occurrences are common in the elderly population. This systematic review aims to highlight the safety of the procedures, their tolerability, and efficacy of the available therapies conducted over the years using mesenchymal stem cells (MSCs) in treating the neurological conditions mentioned above.MethodsPubMed was used to search for published data from clinical trials performed using mesenchymal stem cells. Studies that provided the necessary information that mentioned the efficacy and adverse effects of the treatment in patients were considered for this review.ResultsIn total, 43 manuscripts were selected after a strategic search, and these studies have been included in this systematic review. Most included studies reported the safety of the procedures used and the treatment’s good tolerability, with mild adverse events such as fever, headache, mild pain at the injection site, or nausea being common. A few studies also reported death of some patients, attributed to the progression of the disease to severe stages before the treatment. Other severe events, such as respiratory or urinary infections reported in some studies, were not related to the treatment. Different parameters were used to evaluate the efficacy of the treatment based on the clinical condition of the patient.ConclusionMesenchymal stem cells transplantation has so far proven to be safe and tolerable in select studies and patient types. This systematic review includes the results from the 43 selected studies in terms of safety and tolerability of the procedures, and several adverse events and therapeutic benefits during the follow-up period after administration of MSCs

Nano-sulforaphane attenuates PhIP-induced early abnormal embryonic neuro-development

Background: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyrimidine (PhIP), one of the most abundant heterocyclic aromatic amines (HAA) formed by cooking meat at high temperatures, may modify humans and rodents through the metabolic process prior to affecting nervous system development. in humans and rodents may be modified by metabolic processes and then affecting nervous system development. Methods: In this paper, PhIP was used to prepare a chicken embryo model with abnormal embryonic nervous system defects. Sulforaphane (SFN) is a derivative of a glucosinolate, which is abundant in cruciferous vegetables, and can pass through the placental barrier. Moreover, SFN has antioxidant and anti-apoptotic functions and is considered as a bioactive antioxidant with significant neuroprotective effects. Nano-sulforaphane (Nano-SFN, Sulforaphane nanoparticles) was prepared by self-assembly using biocompatible, biodegradable methoxy polyethylene glycol 5000-b-polyglutamic acid 10000 (mPEG5K-PGA10K) as the substrate, to explore the new application of Nano-SFN and its modified compounds as leading compounds in protecting against the abnormal development of the embryonic nervous system. Results: The results show that Nano-SFN could protect against PhIP-induced central nervous system (CNS, derived from neural tube) and peripheral nervous system (PNS, derived from neural crest cells, NCCs) defects and neural tube defects (NTDs), and increase the embryo survival rate. Conclusions: This study indicates that Nano-SFN can effectively alleviate the developmental defects of embryonic nervous system induced by PhIP in the microenvironment and has a protective effect on embryonic development. It not only helps with expanding the application of SFN and improving its medicinal value, but also provides a possibility of SFN being developed as a novel drug for neuroprotection