Cardiac injections of AntagomiRs as a novel tool for knockdown of miRNAs during heart development

Background: Studying microRNA networks during heart development is essential to obtain a better understanding of developmental defects and diseases associated with the heart and to identify novel opportunities for therapeutics. Here we highlight the advantages of chicken embryos as a vertebrate model for studying intermediate processes of heart development. Avians develop a four-chambered heart closely resembling human anatomy and they develop ex utero, which makes them easily accessible. Furthermore, embryos are available all year with a steady supply. Results: In this report we established a novel method for the knockdown of microRNA function by microinjecting AntagomiRs into the chicken heart in ovo. Our approach enables the targeted delivery of antagomirs into a locally restricted area and is not impacted by circulation. After further embryo development the successful miRNA knockdown was confirmed. Loss of function phenotypes can be evaluated rapidly, compared to more time-consuming genetic ablation experiments. The local application avoids potential systemic effects of microRNA knockdown, therefore allowing the assessment of impacts on heart development only. The method can be adjusted for different stages of chicken embryos (HH13-HH18) as well as for knockdown or targeted overexpression of coding genes. Conclusion: In conclusion our method allows targeted and locally restricted delivery of Antagomirs to the heart leading to successful knockdown of microRNA function. This method enables rapid phenotypic assessment, for example by gene expression analysis of multiple cardiac genes

csal1 Is Controlled by a Combination of FGF and Wnt Signals in Developing Limb Buds

While some of the signaling molecules that govern establishment of the limb axis have been characterized, little is known about the downstream effector genes that interpret these signals. In Drosophila, the spalt gene is involved in cell fate determination and pattern formation in different tissues. We have cloned a chick homologue of Drosophila spalt, which we have termed csal1, and this study focuses on the regulation of csal1 expression in the limb bud. csal1 is expressed in limb buds from HH 17 to 26, in both the apical ectodermal ridge and the distal mesenchyme. Signals from the apical ridge are essential for csal1 expression, while the dorsal ectoderm is required for csal1 expression at a distance from the ridge. Our data indicate that both FGF and Wnt signals are required for the regulation of csal1 expression in the limb. Mutations in the human homologue of csal1, termed Hsal1/SALL1, result in a condition known as Townes–Brocks syndrome (TBS), which is characterized by preaxial polydactyly. The developmental expression of csal1 together with the digit phenotype in TBS patients suggests that csal1 may play a role in some aspects of distal patterning

Combination of phenethyl isothiocyanate and dasatinib inhibits hepatocellular carcinoma metastatic potential through FAK/STAT3/Cadherin signalling and reduction of VEGF secretion

Cancerous cells are characterised by their ability to invade, metastasise and induce angiogenesis. To achieve this, tumour cells use various molecules that can be targeted to reverse these processes. Dasatinib, a potent Src inhibitor, has shown promising results in treating hepatocellular carcinoma (HCC) in vitro and in vivo. However, its effectiveness is limited by FAK activation. Isothiocyanates (ITCs), on the other hand, are phytochemicals with broad anticancer activity and FAK inhibition capabilities. This study evaluated the synergistic effects of dasatinib and phenethyl isothiocyanate (PEITC) on HCC. The combination was tested using various assays, including MTT, adhesion, scratch wound healing, Boyden chamber, chorioallantoic membrane (CAM), and yolk sac mem-brane (YSM) assays, to evaluate the effect of the drug combination on HCC metastatic potential and angiogenesis in vitro and in vivo. The results showed that the combination inhibited adhesion, migration, and invasion of HepG2 cells and reduced xenograft volume in the CAM assay. Addi-tionally, the combination reduced angiogenesis in vitro, reducing the growth of new vessels in the tube formation assay. The inhibition of FAK/STAT3 signalling led to increased E-cadherin ex-pression and reduced VEGF secretion, thus reducing HCC metastatic potential. Therefore, com-bination of PEITC and dasatinib could be a potential therapeutic strategy for the treatment of HCC

A transcriptional and regulatory map of mouse somite maturation

The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression and open chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals

A database of microRNA expression patterns in Xenopus laevis

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase

Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior–posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a ‘gate-keeper’ for the correct temporal and spatial development of the neural crest

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle

Somite development and regionalisation of the vertebral axial skeleton

A critical stage in the development of all vertebrate embryos is the generation of the body plan and its subsequent patterning and regionalisation along the main anterior-posterior axis. This includes the formation of the vertebral axial skeleton. Its organisation begins during early embryonic development with the periodic formation of paired blocks of mesoderm tissue called somites. Here, we review axial patterning of somites, with a focus on studies using amniote model systems – avian and mouse. We summarise the molecular and cellular mechanisms that generate paraxial mesoderm and review how the different anatomical regions of the vertebral column acquire their specific identity and thus shape the body plan. We also discuss the generation of organoids and embryo-like structures from embryonic stem cells, which provide insights regarding axis formation and promise to be useful for disease modelling