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.

The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network.

Understanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a 'master control gene' called Pax6, which is both necessary and sufficient for eye development. Yet precisely how Pax6 achieves this deeply homologous function is poorly understood. Using the chick as a model organism, we show that vertebrate Pax6 interacts with a pair of morphogen-coding genes, Tgfb2 and Fst, to form a putative Turing network, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the first organisational axis of the eye and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independently of the highly ordered tissue interactions that help to assemble the eye in vivo These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cups in vitro We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.

Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis.

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3'UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3'UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs.

Investigating chromatin accessibility during development and differentiation by ATAC-sequencing to guide the identification of cis-regulatory elements.

Mapping accessible chromatin across time scales can give insights into its dynamic nature, for example during cellular differentiation and tissue or organism development. Analysis of such data can be utilised to identify functional cis-regulatory elements (CRE) and transcription factor binding sites and, when combined with transcriptomics, can reveal gene regulatory networks (GRNs) of expressed genes. Chromatin accessibility mapping is a powerful approach and can be performed using ATAC-sequencing (ATAC-seq), whereby Tn5 transposase inserts sequencing adaptors into genomic DNA to identify differentially accessible regions of chromatin in different cell populations. It requires low sample input and can be performed and analysed relatively quickly compared with other methods. The data generated from ATAC-seq, along with other genomic approaches, can help uncover chromatin packaging and potential cis-regulatory elements that may be responsible for gene expression. Here, we describe the ATAC-seq approach and give examples from mainly vertebrate embryonic development, where such datasets have identified the highly dynamic nature of chromatin, with differing landscapes between cellular precursors for different lineages.

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.

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.

microRNAs in skeletal muscle development.

A fundamental process during both embryo development and stem cell differentiation is the control of cell lineage determination. In developing skeletal muscle, many of the diffusible signaling molecules, transcription factors and more recently non-coding RNAs that contribute to this process have been identified. This has facilitated advances in our understanding of the molecular mechanisms underlying the control of cell fate choice. Here we will review the role of non-coding RNAs, in particular microRNAs (miRNAs), in embryonic muscle development and differentiation, and in satellite cells of adult muscle, which are essential for muscle growth and regeneration. Some of these short post-transcriptional regulators of gene expression are restricted to skeletal muscle, but their expression can also be more widespread. In addition, we discuss a few examples of long non-coding RNAs, which are numerous but much less well understood.

microRNAs associated with early neural crest development in Xenopus laevis.

BACKGROUND: The neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression. RESULTS: To uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue. Ectodermal and blastula animal pole (blastula) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells. CONCLUSION: We have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm, neural or NC tissue. This has identified novel tissue specific miRNAs and highlighted differential expression of miR-427 isoforms.

Robo signaling regulates the production of cranial neural crest cells.

Slit/Robo signaling plays an important role in the guidance of developing neurons in developing embryos. However, it remains obscure whether and how Slit/Robo signaling is involved in the production of cranial neural crest cells. In this study, we examined Robo1 deficient mice to reveal developmental defects of mouse cranial frontal and parietal bones, which are derivatives of cranial neural crest cells. Therefore, we determined the production of HNK1+ cranial neural crest cells in early chick embryo development after knock-down (KD) of Robo1 expression. Detection of markers for pre-migratory and migratory neural crest cells, PAX7 and AP-2α, showed that production of both was affected by Robo1 KD. In addition, we found that the transcription factor slug is responsible for the aberrant delamination/EMT of cranial neural crest cells induced by Robo1 KD, which also led to elevated expression of E- and N-Cadherin. N-Cadherin expression was enhanced when blocking FGF signaling with dominant-negative FGFR1 in half of the neural tube. Taken together, we show that Slit/Robo signaling influences the delamination/EMT of cranial neural crest cells, which is required for cranial bone development.

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.

myomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis.

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.

Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells.

In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelial-to-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3ß) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3ß, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3ß-resistant Smad1 resulted in aberrant CPC migration trajectories. De-repression of GSK3ß by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.

Klhl31 attenuates ß-catenin dependent Wnt signaling and regulates embryo myogenesis.

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with ß-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or ß-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.

Canonical Wnt signals combined with suppressed TGFß/BMP pathways promote renewal of the native human colonic epithelium.

BACKGROUND: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. OBJECTIVE: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFß/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. DESIGN: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. RESULTS: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFß and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFß or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. CONCLUSIONS: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFß/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

Regulation of multiple target genes by miR-1 and miR-206 is pivotal for C2C12 myoblast differentiation.

MicroRNAs are short non-coding RNAs involved in post-transcriptional regulation of multiple messenger RNA targets. The miR-1/miR-206 family is expressed during skeletal muscle differentiation and is an integral component of myogenesis. To better understand miR-1/miR-206 function during myoblast differentiation we identified novel target mRNAs by microarray and characterized their function in C2C12 myoblasts. Candidate targets from the screen were experimentally validated together with target genes that were predicted by three different algorithms. Some targets characterised have a known function in skeletal muscle development and/or differentiation and include Meox2, RARB, Fzd7, MAP4K3, CLCN3 and NFAT5, others are potentially novel regulators of myogenesis, such as the chromatin remodelling factors Smarcd2 and Smarcb1 or the anti-apoptotic protein SH3BGRL3. The expression profiles of confirmed target genes were examined during C2C12 cell myogenesis. We found that inhibition of endogenous miR-1 and miR-206 by antimiRs blocked the downregulation of most targets in differentiating cells, thus indicating that microRNA activity and target interaction is required for muscle differentiation. Finally, we show that sustained expression of validated miR-1 and/or miR-206 targets resulted in increased proliferation and inhibition of C2C12 cell myogenesis. In many cases the expression of genes related to non-muscle cell fates, such as chondrogenesis, was activated. This indicates that the concerted downregulation of multiple microRNA targets is not only crucial to the skeletal muscle differentiation program but also serves to prevent alternative cell fate choices.

MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis.

Commitment of progenitors in the dermomyotome to myoblast fate is the first step in establishing the body musculature. Pax3 is a crucial transcription factor, important for skeletal muscle development and expressed in myogenic progenitors in the dermomyotome of developing somites and in migratory muscle progenitors that populate the limb buds. Down-regulation of Pax3 is essential to ignite the myogenic program, including up-regulation of myogenic regulators, Myf-5 and MyoD. MicroRNAs (miRNAs) confer robustness to developmental timing by posttranscriptional repression of genetic programs that are related to previous developmental stages or to alternative cell fates. Here we demonstrate that the muscle-specific miRNAs miR-1 and miR-206 directly target Pax3. Antagomir-mediated inhibition of miR-1/miR-206 led to delayed myogenic differentiation in developing somites, as shown by transient loss of myogenin expression. This correlated with increased Pax3 and was phenocopied using Pax3-specific target protectors. Loss of myogenin after antagomir injection was rescued by Pax3 knockdown using a splice morpholino, suggesting that miR-1/miR-206 control somite myogenesis primarily through interactions with Pax3. Our studies reveal an important role for miR-1/miR-206 in providing precision to the timing of somite myogenesis. We propose that posttranscriptional control of Pax3 downstream of miR-1/miR-206 is required to stabilize myoblast commitment and subsequent differentiation. Given that mutually exclusive expression of miRNAs and their targets is a prevailing theme in development, our findings suggest that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.

The expression and function of microRNAs in chondrogenesis and osteoarthritis.

OBJECTIVE: To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. METHODS: The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter. RESULTS: The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor ß (TGFß) ligands, and miRNA regulated TGFß signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFß signaling. CONCLUSION: MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFß signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.

Phenethyl isothiocyanate and dasatinib combination synergistically reduces hepatocellular carcinoma growth via cell cycle arrest and oxeiptosis.

Introduction: Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which is among the most lethal tumours. Combination therapy exploits multiple drugs to target key pathways synergistically to reduce tumour growth. Isothiocyanates have been shown to possess anticancer potential and to complement the anticancer activity of other compounds. This study aimed to investigate the potential of phenethyl isothiocyanate (PEITC) to synergise with dasatinib, improving its anticancer potential in HCC. Methods: MTT, 3D spheroids and clonogenic assays were used to assess the combination anti-tumour effect in vitro, whereas a murine syngeneic model was employed to evaluate the combination efficacy in vivo. DCFDA staining was employed to evaluate the production of reactive oxygen species (ROS), while flow cytometry and Western blot assays were used to elucidate the molecular mechanism of the synergistic activiy. Results: PEITC and dasatinib combination exhibited a synergistic effect in vitro and in vivo. The combination induced DNA damage and oxidative stress through the production of ROS, which led to the formation of a premature CDK1/Cyclin B1 complex associated with induction of mitotic catastrophe. Furthermore, ROS activated oxeiptosis, a caspase-independent form of programmed cell death. Conclusion: PEITC showed to enhance dasatinib action in treating HCC with increased production of ROS that induced cell cycle arrest followed by mitotic catastrophe, and to induce oxeiptosis. These results highlight the role that ITCs may have in cancer therapy as a complement of clinically approved chemotherapeutic drugs.

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. 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 focal adhesion kinase (FAK) activation. Isothiocyanates, on the other hand, are phytochemicals with broad anticancer activity and FAK inhibition capabilities. This study evaluated the synergistic effect of dasatinib and phenethyl isothiocyanate (PEITC) on HCC. The combination was tested using various assays, including MTT, adhesion, scratch, Boyden chamber, chorioallantoic membrane (CAM), and yolk sac membrane (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 the adhesion, migration, and invasion of HepG2 cells and reduced xenograft volume in the CAM assay. Additionally, the combination reduced angiogenesis in vitro, diminishing the growth of vessels in the tube formation assay. The inhibition of FAK/STAT3 signalling led to increased E-cadherin expression and reduced VEGF secretion, reducing HCC metastatic potential. Therefore, a combination 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.