NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterning.

The NineTeen Complex (NTC), also known as pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. We show that NTC-subunit Salsa, the Drosophila ortholog of human RNA helicase Aquarius, is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken Germline depletion of Salsa and splice site mutations within gurken first intron impair both adult female fertility and oocyte dorsal-ventral patterning, due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether, our results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR.

The T-box transcription factor brachyury behaves as a tumor suppressor in gliomas.

The oncogene brachyury (TBXT) is a T-box transcription factor that is overexpressed in multiple solid tumors and is associated with tumor aggressiveness and poor patient prognosis. Gliomas comprise the most common and aggressive group of brain tumors, and at the present time the functional and clinical impact of brachyury expression has not been investigated previously in these neoplasms. Brachyury expression (mRNA and protein) was assessed in normal brain (n = 67), glioma tissues (n = 716) and cell lines (n = 42), and further in silico studies were undertaken using genomic databases totaling 3115 samples. Our glioma samples were analyzed for copy number (n = 372), promoter methylation status (n = 170), and mutation status (n = 1569 tissues and n = 52 cell lines) of the brachyury gene. The prognostic impact of brachyury expression was studied in 1524 glioma patient tumors. The functional impact of brachyury on glioma proliferation, viability, and cell death was evaluated both in vitro and in vivo. Brachyury was expressed in the normal brain, and significantly downregulated in glioma tissues. Loss of brachyury was associated with tumor aggressiveness and poor survival in glioma patients. Downregulation of brachyury was not associated with gene deletion, promoter methylation, or inactivating point mutations. Brachyury re-expression in glioma cells was found to decrease glioma tumorigenesis by induction of autophagy. These data strongly suggest that brachyury behaves as a tumor suppressor gene in gliomas by modulating autophagy. It is important to note that brachyury constitutes an independent positive biomarker of patient prognosis. Our findings indicate that the role of brachyury in tumorigenesis may be tissue-dependent and demands additional investigation to guide rational interventions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

StemMapper: a curated gene expression database for stem cell lineage analysis.

Transcriptomic data have become a fundamental resource for stem cell (SC) biologists as well as for a wider research audience studying SC-related processes such as aging, embryonic development and prevalent diseases including cancer, diabetes and neurodegenerative diseases. Access and analysis of the growing amount of freely available transcriptomics datasets for SCs, however, are not trivial tasks. Here, we present StemMapper, a manually curated gene expression database and comprehensive resource for SC research, built on integrated data for different lineages of human and mouse SCs. It is based on careful selection, standardized processing and stringent quality control of relevant transcriptomics datasets to minimize artefacts, and includes currently over 960 transcriptomes covering a broad range of SC types. Each of the integrated datasets was individually inspected and manually curated. StemMapper's user-friendly interface enables fast querying, comparison, and interactive visualization of quality-controlled SC gene expression data in a comprehensive manner. A proof-of-principle analysis discovering novel putative astrocyte/neural SC lineage markers exemplifies the utility of the integrated data resource. We believe that StemMapper can open the way for new insights and advances in SC research by greatly simplifying the access and analysis of SC transcriptomic data. StemMapper is freely accessible at http://stemmapper.sysbiolab.eu.

Mechanisms of vertebrate embryo segmentation: Common themes in trunk and limb development.

Various ultradian rhythms ensure proper temporal regulations during embryo development. The embryo molecular clock, which was first identified in the presomitic mesoderm (PSM) underlying periodic somite formation, is one among them. Somites are the earliest manifestation of the segmented vertebrate body and they are formed with strict temporal precision. The tetrapod limb is also a segmented structure and the formation of limb bone elements have also been associated with a molecular clock, operating in the distal limb mesenchyme. In both the PSM and the distal limb mesenchyme, the molecular clock (MC) is influenced by FGF, SHH and RA, which are also the key regulators of the development of these tissues. While somitogenesis has been continuously scrutinized to understand the mechanisms of the MC, the limb bud has served as an outstanding paradigm to study how a cohort of undifferentiated cells is organized into functional 3D structures. The fact that both the trunk and limb development are shaped by the MC and by common signaling molecules has prompted the exciting possibility of establishing parallelisms between somitogenesis and limb development. Systematically correlating various parameters during trunk and limb development will help us to appreciate the common principles underlying segmented structure formation and allow the rise of new questions in order to fill the gaps in our present understanding. In this review we have established the parallelisms between somitogenesis and limb development at the level of gene expression patterns and their regulation. Finally, we have also discussed the most evident new avenues this exercise could open to the scientific community.

Getting a handle on embryo limb development: Molecular interactions driving limb outgrowth and patterning.

Development of the vertebrate embryo involves multiple segmentation processes to generate a functional, articulated organism. Cell proliferation, differentiation and patterning involve spatially and temporally regulated gene expression and signal transduction mechanisms. The developing vertebrate limb is an excellent model to study such fine-tuned regulations, whereby cells proliferate and are differentially sculptured along the proximal-distal, anterior-posterior and dorsal-ventral axes to form a functional limb. Complementary experimental approaches in different organisms have enhanced our knowledge on the molecular events underlying limb development. Herein, we summarize the current knowledge of the main signaling mechanisms governing vertebrate limb initiation, outgrowth, specification of limb segments and termination.

The embryonic Brachyury transcription factor is a novel biomarker of GIST aggressiveness and poor survival.

BACKGROUND: The T-box transcription factor Brachyury was recently reported to be upregulated and associated with prognosis in solid tumors. Here, we proposed to evaluate the potential use of Brachyury protein expression as a new prognostic biomarker in gastrointestinal stromal tumors (GIST). METHODS: Brachyury protein expression was analyzed by immunohistochemistry in a cohort of 63 bona fide GIST patients. Brachyury expression profiles were correlated with patients' clinicopathological features and prognostic impact. Additionally, an in silico analysis was performed using the Oncomine database to assess Brachyury alterations at DNA and mRNA levels in GISTs. RESULTS: We found that Brachyury was overexpressed in the majority (81.0 %) of primary GISTs. We observed Brachyury staining in the nucleus alone in 4.8 % of cases, 23.8 % depicted only cytoplasm staining, and 52.4 % of cases exhibited both nucleus and cytoplasm immunostaining. The presence of Brachyury was associated with aggressive GIST clinicopathological features. Particularly, Brachyury nuclear (with or without cytoplasm) staining was associated with the presence of metastasis, while cytoplasm sublocalization alone was correlated with poor patient survival. CONCLUSIONS: Herein, we demonstrate that Brachyury is overexpressed in GISTs and is associated with worse outcome, constituting a novel prognostic biomarker and a putative target for GIST treatment.

Spatio-temporal dynamics of early somite segmentation in the chicken embryo.

During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is controlled by the somitogenesis embryo clock (EC), which was first described as gene expression oscillations of hairy1 (hes4) in the presomitic mesoderm of chick embryos with 15-20 somites. Here, the EC displays the same periodicity as somite formation, 90 min, whereas the posterior-most somites (44-52) only arise every 150 minutes, matched by a corresponding slower pace of the EC. Evidence suggests that the rostral-most somites are formed faster, however, their periodicity and the EC expression dynamics in these early stages are unknown. In this study, we used time-lapse imaging of chicken embryos from primitive streak to somitogenesis stages with high temporal resolution (3-minute intervals). We measured the length between the anterior-most and the last formed somitic clefts in each captured frame and developed a simple algorithm to automatically infer both the length and time of formation of each somite. We found that the occipital somites (up to somite 5) form at an average rate of 75 minutes, while somites 6 onwards are formed approximately every 90 minutes. We also assessed the expression dynamics of hairy1 using half-embryo explants cultured for different periods of time. This showed that EC hairy1 expression is highly dynamic prior to somitogenesis and assumes a clear oscillatory behaviour as the first somites are formed. Importantly, using ex ovo culture and live-imaging techniques, we showed that the hairy1 expression pattern recapitulates with the formation of each new pair of somites, indicating that somite segmentation is coupled with EC oscillations since the onset of somitogenesis.

Sonic hedgehog in temporal control of somite formation.

Vertebrate embryo somite formation is temporally controlled by the cyclic expression of somitogenesis clock genes in the presomitic mesoderm (PSM). The somitogenesis clock is believed to be an intrinsic property of this tissue, operating independently of embryonic midline structures and the signaling molecules produced therein, namely Sonic hedgehog (Shh). This work revisits the notochord signaling contribution to temporal control of PSM segmentation by assessing the rate and number of somites formed and somitogenesis molecular clock gene expression oscillations upon notochord ablation. The absence of the notochord causes a delay in somite formation, accompanied by an increase in the period of molecular clock oscillations. Shh is the notochord-derived signal responsible for this effect, as these alterations are recapitulated by Shh signaling inhibitors and rescued by an external Shh supply. We have characterized chick smoothened expression pattern and have found that the PSM expresses both patched1 and smoothened Shh signal transducers. Upon notochord ablation, patched1, gli1, and fgf8 are down-regulated, whereas gli2 and gli3 are overexpressed. Strikingly, notochord-deprived PSM segmentation rate recovers over time, concomitant with raldh2 overexpression. Accordingly, exogenous RA supplement rescues notochord ablation effects on somite formation. A model is presented in which Shh and RA pathways converge to inhibit PSM Gli activity, ensuring timely somite formation. Altogether, our data provide evidence that a balance between different pathways ensures the robustness of timely somite formation and that notochord-derived Shh is a component of the molecular network regulating the pace of the somitogenesis clock.

Retinoic acid signaling regulates embryonic clock hairy2 gene expression in the developing chick limb.

Embryo development proceeds under strict temporal control and an embryonic molecular clock (EC), evidenced by cyclic gene expression, is operating during somite formation and limb development, providing temporal information to precursor cells. In somite precursor cells, EC gene expression and periodicity depends on Retinoic acid (RA) signaling and this morphogen is also essential for limb initiation, outgrowth and patterning. Since the limb EC gene hairy2 is differentially expressed along the proximal-distal axis as growth proceeds, concomitant with changes in flank-derived RA activity in the mesenchyme, we have interrogated the role of RA signaling on limb hairy2 expression regulation. We describe RA as a positive regulator of limb hairy2 expression. Ectopic supplementation of RA induced hairy2 in a short time period, with simultaneous transient activation of Erk/MAPK, Akt/PI3K and Gli3 intracellular pathways. We further found that FGF8, an inducer of Erk/MAPK, Akt/PI3K pathways, was not sufficient for ectopic hairy2 induction. However, joint treatment with both RA and FGF8 induced hairy2, indicating that RA is creating a permissive condition for p-Erk/p-Akt action on hairy2, most likely by enhancing Gli3-A/Gli3-R levels. Finally, we observed an inhibitory action of BMP4 on hairy2 and propose a model whereby RA shapes limb hairy2 expression during limb development, by activating its expression and counteracting the inhibitory action of BMP4 on hairy2. Overall, our work reports a novel role for RA in the regulation of limb clock hairy2 gene expression and elucidates the temporal response of multiple intracellular pathways to RA signaling in limb development.

The vertebrate Embryo Clock: Common players dancing to a different beat.

Vertebrate embryo somitogenesis is the earliest morphological manifestation of the characteristic patterned structure of the adult axial skeleton. Pairs of somites flanking the neural tube are formed periodically during early development, and the molecular mechanisms in temporal control of this early patterning event have been thoroughly studied. The discovery of a molecular Embryo Clock (EC) underlying the periodicity of somite formation shed light on the importance of gene expression dynamics for pattern formation. The EC is now known to be present in all vertebrate organisms studied and this mechanism was also described in limb development and stem cell differentiation. An outstanding question, however, remains unanswered: what sets the different EC paces observed in different organisms and tissues? This review aims to summarize the available knowledge regarding the pace of the EC, its regulation and experimental manipulation and to expose new questions that might help shed light on what is still to unveil.

gga-miRNOME, a microRNA-sequencing dataset from chick embryonic tissues.

MicroRNAs (miRNAs) are small non-coding RNA molecules, with sizes ranging from 18 to 25 nucleotides, which are key players in gene expression regulation. These molecules play an important role in fine-tuning early vertebrate embryo development. However, there are scarce publicly available miRNA datasets from non-mammal embryos, such as the chicken (Gallus gallus), which is a classical model system to study vertebrate embryogenesis. Here, we performed microRNA-sequencing to characterize the early stages of trunk and limb development in the chick embryo. For this, we profiled three chick embryonic tissues, namely, Undetermined Presomitic Mesoderm (PSM_U), Determined Presomitic Mesoderm (PSM_D) and Forelimb Distal Cyclic Domain (DCD). We identified 926 known miRNAs, and 1,141 novel candidate miRNAs, which nearly duplicates the number of Gallus gallus entries in the miRBase database. These data will greatly benefit the avian research community, particularly by highlighting new miRNAs potentially involved in the regulation of early vertebrate embryo development, that can be prioritized for further experimental testing.

Cell-Fibronectin Interactions and Actomyosin Contractility Regulate the Segmentation Clock and Spatio-Temporal Somite Cleft Formation during Chick Embryo Somitogenesis.

Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of meso1 in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin-fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin-fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed meso1 and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of hairy1/hes4 oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin-integrin-ROCK-NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts.

Altered Cogs of the Clock: Insights into the Embryonic Etiology of Spondylocostal Dysostosis.

Spondylocostal dysostosis (SCDO) is a rare heritable congenital condition, characterized by multiple severe malformations of the vertebrae and ribs. Great advances were made in the last decades at the clinical level, by identifying the genetic mutations underlying the different forms of the disease. These were matched by extraordinary findings in the Developmental Biology field, which elucidated the cellular and molecular mechanisms involved in embryo body segmentation into the precursors of the axial skeleton. Of particular relevance was the discovery of the somitogenesis molecular clock that controls the progression of somite boundary formation over time. An overview of these concepts is presented, including the evidence obtained from animal models on the embryonic origins of the mutant-dependent disease. Evidence of an environmental contribution to the severity of the disease is discussed. Finally, a brief reference is made to emerging in vitro models of human somitogenesis which are being employed to model the molecular and cellular events occurring in SCDO. These represent great promise for understanding this and other human diseases and for the development of more efficient therapeutic approaches.

Brachyury Is Associated with Glioma Differentiation and Response to Temozolomide.

Glioblastomas (GBMs) are the most aggressive tumor type of the central nervous system, mainly due to their high invasiveness and innate resistance to radiotherapy and chemotherapy, with temozolomide (TMZ) being the current standard therapy. Recently, brachyury was described as a novel tumor suppressor gene in gliomas, and its loss was associated with increased gliomagenesis. Here, we aimed to explore the role of brachyury as a suppressor of glioma invasion, stem cell features, and resistance to TMZ. Using gene-edited glioma cells to overexpress brachyury, we found that brachyury-positive cells exhibit reduced invasive and migratory capabilities and stem cell features. Importantly, these brachyury-expressing cells have increased expression of differentiation markers, which corroborates the results from human glioma samples and in vivo tumors. Glioma cells treated with retinoic acid increased the differentiation status with concomitant increased expression of brachyury. We then selected TMZ-resistant (SNB-19) and TMZ-responsive (A172 and U373) cell lines to evaluate the role of brachyury in the response to TMZ treatment. We observed that both exogenous and endogenous brachyury activation, through overexpression and retinoic acid treatment, are associated with TMZ sensitization in glioma-resistant cell lines. In this study, we demonstrate that brachyury expression can impair aggressive glioma features associated with treatment resistance. Finally, we provide the first evidence that brachyury can be a potential therapeutic target in GBM patients who do not respond to conventional chemotherapeutic drugs.

Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex.

BACKGROUND: The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners. RESULTS: Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites. CONCLUSION: Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since sap18 and sin3a are not expressed in the PSM territory where hairy1 presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites.

rdml: A Mathematica package for parsing and importing Real-Time qPCR data.

OBJECTIVE: The purpose and objective of the research presented is to provide a package for easy importing of Real-Time PCR data markup language (RDML) data to Mathematica. RESULTS: Real-Time qPCR is the most widely used experimental method for the accurate quantification of gene expression. To enable the straightforward archiving and sharing of qPCR data and its associated experimental information, an XML-based data standard was developed-the Real-Time PCR data markup language (RDML)-devised by the RDML consortium. Here, we present rdml, a package to parse and import RDML data into Mathematica, allowing the quick loading and extraction of relevant data, thus promoting the re-analysis, meta-analysis or experimental re-validation of gene expression data deposited in RDML format.

Brachyury as a potential modulator of androgen receptor activity and a key player in therapy resistance in prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed neoplasm and the second leading cause of cancer-related deaths in men. Acquisition of resistance to conventional therapy is a major problem for PCa patient management. Several mechanisms have been described to promote therapy resistance in PCa, such as androgen receptor (AR) activation, epithelial-to-mesenchymal transition (EMT), acquisition of stem cell properties and neuroendocrine transdifferentiation (NEtD). Recently, we identified Brachyury as a new biomarker of PCa aggressiveness and poor prognosis. In the present study we aimed to assess the role of Brachyury in PCa therapy resistance. We showed that Brachyury overexpression in prostate cancer cells lines increased resistance to docetaxel and cabazitaxel drugs, whereas Brachyury abrogation induced decrease in therapy resistance. Through ChiP-qPCR assays we further demonstrated that Brachyury is a direct regulator of AR expression as well as of the biomarker AMACR and the mesenchymal markers Snail and Fibronectin. Furthermore, in vitro Brachyury was also able to increase EMT and stem properties. By in silico analysis, clinically human Brachyury-positive PCa samples were associated with biomarkers of PCa aggressiveness and therapy resistance, including PTEN loss, and expression of NEtD markers, ERG and Bcl-2. Taken together, our results indicate that Brachyury contributes to tumor chemotherapy resistance, constituting an attractive target for advanced PCa patients.

Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris.

In Saccharomyces cerevisiae the activity for the lactate-proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a V (max) of 2.1 nmol x s(-1) x mg of dry weight(-1). Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest V (max) (0.84 nmol x s(-1) x mg of dry weight(-1)) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo.

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.