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- Mechanisms of vertebrate embryo segmentation: Common themes in trunk and limb developmentPublication . Sheeba, C.J.; Andrade, Raquel P.; Palmeirim, IsabelVarious 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. (C) 2016 Elsevier Ltd. All rights reserved.
- Patterning in time and space: HoxB cluster gene expression in the developing chick embryoPublication . Gouveia, Analuce; Marcelino, Hugo M.; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P.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.
- Sonic hedgehog in temporal control of somite formationPublication . Resende, Tatiana P.; Ferreira, Monica; Teillet, Marie-Aimee; Tavares, Ana Teresa; Andrade, Raquel P.; Palmeirim, IsabelVertebrate 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.
- rdml: A Mathematica package for parsing and importing Real-Time qPCR dataPublication . Magno, Ramiro; Duarte, Isabel; Andrade, Raquel P.; Palmeirim, IsabelObjective 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.
- Timing embryo segmentation: dynamics and regulatory mechanisms of the vertebrate segmentation clockPublication . Resende, Tatiana P.; Andrade, Raquel P.; Palmeirim, IsabelAll vertebrate species present a segmented body, easily observed in the vertebrate column and its associated components, which provides a high degree of motility to the adult body and efficient protection of the internal organs. The sequential formation of the segmented precursors of the vertebral column during embryonic development, the somites, is governed by an oscillating genetic network, the somitogenesis molecular clock. Herein, we provide an overview of the molecular clock operating during somite formation and its underlying molecular regulatory mechanisms. Human congenital vertebral malformations have been associated with perturbations in these oscillatory mechanisms. Thus, a better comprehension of the molecular mechanisms regulating somite formation is required in order to fully understand the origin of human skeletal malformations.
- Reprogramming iPSCs to study age-related diseases: models, therapeutics, and clinical trialsPublication . Esteves, Filipa; Brito, David; Rajado, Ana Teresa; Silva, Nádia; Apolónio, Joana; Roberto, Vania Palma; Araújo, Inês Maria; Nóbrega, Clévio; Castelo-Branco, Pedro; Bragança, José; P. Andrade, Raquel; M. Calado, Sofia; Faleiro, L; Matos, Carlos A; Marques, Nuno; Marreiros, Ana; Nzwalo, Hipólito; Pais, Sandra; Palmeirim, Isabel; S, Simão; Joaquim, Natércia; Miranda, Rui; Pêgas, António; Raposo, Daniela Marques; Sardo, AnaThe unprecedented rise in life expectancy observed in the last decades is leading to a global increase in the ageing population, and age-associated diseases became an increasing societal, economic, and medical burden. This has boosted major efforts in the scientific and medical research communities to develop and improve therapies to delay ageing and age-associated functional decline and diseases, and to expand health span. The establishment of induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells has revolutionised the modelling and understanding of human diseases. iPSCs have a major advantage relative to other human pluripotent stem cells as their obtention does not require the destruction of embryos like embryonic stem cells do, and do not have a limited proliferation or differentiation potential as adult stem cells. Besides, iPSCs can be generated from somatic cells from healthy individuals or patients, which makes iPSC technology a promising approach to model and decipher the mechanisms underlying the ageing process and age-associated diseases, study drug effects, and develop new therapeutic approaches. This review discusses the advances made in the last decade using iPSC technology to study the most common age-associated diseases, including age-related macular degeneration (AMD), neurodegenerative and cardiovascular diseases, brain stroke, cancer, diabetes, and osteoarthritis.
- Joint interpretation of AER/FGF and ZPA/SHH over time and space underlies hairy2 expression in the chick limbPublication . Sheeba, Caroline J.; Andrade, Raquel P.; Palmeirim, IsabelEmbryo development requires precise orchestration of cell proliferation and differentiation in both time and space. A molecular clock operating through gene expression oscillations was first described in the presomitic mesoderm (PSM) underlying periodic somite formation. Cycles of HES gene expression have been further identified in other progenitor cells, including the chick distal limb mesenchyme, embryonic neural progenitors and both mesenchymal and embryonic stem cells. In the limb, hairy2 is expressed in the distal mesenchyme, adjacent to the FGF source (AER) and along the ZPA-derived SHH gradient, the two major regulators of limb development. Here we report that hairy2 expression depends on joint AER/FGF and ZPA/SHH signaling. FGF plays an instructive role on hairy2, mediated by Erk and Akt pathway activation, while SHH acts by creating a permissive state defined by Gli3-A/Gli3-R>1. Moreover, we show that AER/FGF and ZPA/SHH present distinct temporal and spatial signaling properties in the distal limb mesenchyme: SHH acts at a long-term, long-range on hairy2, while FGF has a shortterm, short-range action. Our work establishes limb hairy2 expression as an output of integrated FGF and SHH signaling in time and space, providing novel clues for understanding the regulatory mechanisms underlying HES oscillations in multiple systems, including embryonic stem cell pluripotency. (C) 2012. Published by The Company of Biologists Ltd.
- Comprehensive analysis of fibroblast growth factor receptor expression patterns during chick forelimb developmentPublication . Sheeba, Caroline J.; Andrade, Raquel P.; Duprez, Delphine; Palmeirim, IsabelSpecific interactions between fibroblast growth factors (Fgf1-22) and their tyrosine kinase receptors (FgfR1-4) activate different signalling pathways that are responsible for the biological processes in which Fgf signalling is implicated during embryonic development. In the chick, several Fgf ligands (Fgf2, 4,8, 9, 10, 12, 13 and 18) and the four FgfRs (FgfR 1, 2, 3 and 4) have been reported to be expressed in the developing limb. The precise spatial and temporal expression of these transcripts is important to guide the limb bud to develop into a wing/leg. In this paper, we present a detailed and systematic analysis of the expression patterns of FgfR1, 2, 3 and 4 throughout chick wing development, by in situ hybridisation on whole mounts and sections. Moreover, we characterize for the first time the different isoforms of FGFR1-3 by analysing their differential expression in limb ectoderm and mesodermal tissues, using RT-PCR and in situ hybridisation on sections. Finally, isoform-specific sequences for FgfR1IIIb, FgfR1IIIc, FgfR3IIIb and FgfR3IIIc were determined and deposited in GenBank with the following accession numbers: GU053725, GU065444, GU053726, GU065445, respectively.
- Getting a handle on embryo limb development: Molecular interactions driving limb outgrowth and patterningPublication . Sheeba, C.J.; Andrade, Raquel P.; Palmeirim, IsabelDevelopment 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. (C) 2015 Elsevier Ltd. All rights reserved.
- Establishment of an induced pluripotent cell line (ABCRIi001-A) from an elderly female for ageing researchPublication . Esteves, Filipa; Vilhena Catarino Brito, David; Rajado, Ana Teresa; Silva, Nádia; Apolónio, Joana; Roberto, Vânia; Andrade, Raquel; Calado, Sofia; Faleiro, Maria Leonor; Albuquerque Andrade de Matos, Carlos Adriano; Marques, Nuno; Marreiros, Ana; Nzwalo, Hipólito; Pais, Sandra; Palmeirim, Isabel; Simãoa, Sónia; Joaquim, Natércia; Miranda, Rui; Pêgas, António; Raposo, Daniela Marques; Sardo, Ana; Araújo, Inês; Nóbrega, Clévio; Castelo-Branco, Pedro; Bragança, JoséHuman induced pluripotent stem cells (hiPSCs) hold promises to model and understand human diseases, including those associated with ageing. Here, we describe ABCRIi001-A, a hiPSC line generated from peripheral blood mononuclear cells (PBMCs) of a 79-year-old female enrolled in a study for development of an ageing score (ALFA Score). PBMCs were reprogrammed using three Sendai virus-based reprogramming vectors (hKOS, hc-Myc, and hKlf4). ABCRIi001-A showed normal morphology and karyotype, viral clearance, absence of genomic aberrations, and their pluripotency was confirmed by expression of pluripotency-related markers and their ability to differentiate into the three germ layers. ABCRIi001-A is valuable for ageing-related studies.