41 research outputs found
Joint interpretation of AER/FGF and ZPA/SHH over time and space underlies hairy2 expression in the chick limb
Embryo 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.FCT, Portugal [SFRH/BD/33176/2007]; Ciencia2007 Program Contract (Portuguese Government); IBB/CBME, LA; FCT, Portugal (National and FEDER COMPETE Program funds) [PTDC/SAU-OBD/099758/2008, PTDC/SAU-OBD/105111/2008]info:eu-repo/semantics/publishedVersio
Watch-ing out for chick limb development
Time control is a crucial issue during embryonic development. Nevertheless, little is known about how embryonic cells measure time. Until recently, the only molecular clock known to operate during vertebrate embryonic development was the somitogenesis clock, exclusively functioning in coordinating the precise timing of each new pair of somites formed from the presomitic mesoderm. We have recently evidenced that a similar molecular clock also underlies the timing at which autopod chondrogenic precursors are laid down to form a skeletal limb element. In addition, we herein suggest that the molecular clock is not the only parallelism that can be established between somitogenesis and limb-bud development. In an evolutionary perspective, we support the previously proposed idea that the molecular mechanisms involved in the segmentation of the body axis may have been partially reused in the mesoderm of the lateral plate, thereby allowing the emergence of paired appendages.Financial support was provided by
FCT/FEDER (POCTI/BCI/42040/2001) and by the
EU/FP6-Network of Excellence-Cells into Organs.
S.P. (SFRH/BPD/26638/2006) was supported by FCT,
Portugal
Portuguese contributions to the discovery and characterization of the embryonic molecular clock
Embryonic development is strictly regulated both in time and in space. This extraordinary control is clearly evidenced during the process of somitogenesis. In this process, pairs of somites are formed periodically, such that the time required to form a new somite pair is constant and species specific. The tight temporal control underlying somitogenesis has been shown to depend upon a molecular clock, manifested by the cyclic expression of an increasing number of genes in the unsegmented paraxial mesoderm. Portuguese researchers have been intimately connected to the achievements that have been made in this new field of research: the somitogenesis molecular clock. This article intends to report the Portuguese contributions to the discovery and characterization of the molecular clock underlying somite formation and possibly other embryonic processes. This work inspired many scientists around the world and it has been followed in Portugal by teams that keep on pursuing the characterization of the machinery of this molecular oscillator and its function in the acquisition of both temporal and positional information during development.Financial support was provided by FCT/FEDER (POCTI/BCI/42040/2001) and by the EU/FP6-Network of Excellence-Cells into Organs. S.P. (SFRH/BPD/26638/2006) was supported by FCT, Portugal
Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex
<p>Abstract</p> <p>Background</p> <p>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 <it>hairy1 </it>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 <it>hairy1 </it>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.</p> <p>Results</p> <p>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 <it>sap18 </it>and <it>sin3a </it>genes during chick embryo development, using <it>in situ </it>hybridization experiments. We found that both <it>sap18 </it>and s<it>in3a </it>expression patterns co-localize <it>in vivo </it>with <it>hairy1 </it>expression domains in chick rostral presomitic mesoderm and caudal region of somites.</p> <p>Conclusion</p> <p>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 <it>sap18 </it>and <it>sin3a </it>are not expressed in the PSM territory where <it>hairy1 </it>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.</p
Timing embryo segmentation: dynamics and regulatory mechanisms of the vertebrate segmentation clock
All 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.Fundacao paraa Ciencia e a Tecnologia, Portugal [SFRH/BD/27796/2006, SFRH/BPD/80588/2011]; Programa Operacional Regional do Norte (ON.2) [NORTE-07-0124-FEDER-000017]; Centro de Biomedicina Molecular e Estrutural, LA; Fundacao para a Ciencia e a Tecnologia (National and FEDER COMPETE Program funds) [PTDC/SAU-BID/121459/2010, PTDC/SAU-OBD/099758/2008]; [PEst-OE/EQB/LA0023/2011
Neural crest ontogeny during secondary neurulation: a gene expression pattern study in the chick embryo
In the prospective lumbo-sacral region of the chick embryo, neurulation is achieved by cavitation of the medullary cord, a process called secondary neurulation. Neural crest cells (NCC) are generated in this region and they give rise to the same types of derivatives as in more rostral parts of the trunk where neurulation occurs by dorsal fusion of the neural plate borders (primary neurulation). However, no molecular data were available concerning the different steps of their ontogeny. We thus performed a detailed expression study of molecular players likely to participate in the generation of secondary NCC in chick embryos between Hamburger and Hamilton stages 18-20 (HH18-20) at the level of somites 30 to 43. We found that specification of secondary NCC involves, as in primary neurulation, the activity of several transcription factors such as Pax3, Pax7, Snail2, FoxD3 and Sox9, which are all expressed in the dorsal secondary neural tube as soon as full cavitation is achieved. Moreover, once specification has occurred, emigration of NCC from the dorsal neuroepithelium starts facing early dissociating somites and involves a series of changes in cell shape and adhesion, as well as interactions with the extracellular matrix. Furthermore, Bmp4 and Wnt1 expression precedes the detection of migratory secondary NCC and is coincident with maturation of adjacent somites. Altogether, this first study of molecular aspects of secondary NCC ontogeny has revealed that the mechanisms of neural crest generation occurring along the trunk region of the chick embryo are generally conserved and independent of the type of neurulation involved.We are grateful to our colleagues for helpful discussions. We thank Dr Jean-Loup Duband for fibronectin and NC1 antibodies and Dr James Briscoe for Sox9 plasmid. This work has been supported by Centre National de la Recherche Scientifique (CNRS), University Paris 6 (UPMC), Fundacao para Ciencia e Tecnologia (FCT), Association Francaise contre les Myopathies (AFM). LO is a recipient of a grant from FCT (SFRH/BD/1185812003)
Comprehensive analysis of fibroblast growth factor receptor expression patterns during chick forelimb development
Specific 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.Fundacao para a Ciencia e Tecnologia [SFRH/BD/33176/2007]; Portuguese Government; Fundacao para a Ciencia e Tecnologia, Portugal [OBD/099758/2008]; EU; IBB/CBM
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.info:eu-repo/semantics/publishedVersio
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.Fundacao para a Ciencia e a Tecnologia (FCT), Portugal [PTDC/SAU-OBD/105111/2008, UMINHO/BI/7/2014, SFRH/BPD/65652/2009]; Ciencia Program Contract; Programa Operacional Regional do Norte (ON. 2) [NORTE-07-0124-FEDER-000017]; FCT (National and FEDER COMPETE Program) [PTDC/SAU-BID/121459/2010, PTDC/SAU-OBD/099758/2008]; [PEst-OE/EQB/LA0023/2011]info:eu-repo/semantics/publishedVersio
Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm
The absence of ectoderm impairs somite formation in cultured presomitic mesoderm (PSM) explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show in chick that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior PSM, which fails to form somites when cultured for 6 hours. By contrast, explants isolated with collagenase retain their fibronectin matrix and form somites under identical culture conditions. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. Furthermore, we show that pancreatin-isolated PSM explants cultured in fibronectin-supplemented medium, form significantly more somites than control explants. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, whereas all but the anterior-most region of the PSM expresses the fibronectin assembly receptor, integrin alpha5 (Itga5). We thus propose that the ectoderm-derived fibronectin is assembled by mesodermal alpha5beta1 integrin on the surface of the PSM. Finally, we demonstrate that inhibition of fibronectin fibrillogenesis in explants with ectoderm abrogates somitogenesis. We conclude that a fibronectin matrix is essential for morphological somite formation and that a major, previously unrecognised role of ectoderm in somitogenesis is the synthesis of fibronectin.Fundação para a Ciência e a Tecnologia
(FCT)/FEDER projects POCTI/BCI/40754/2001 and POCI/BIA-BCM/59201/2004
and the FP6/EU Network of Excellence ‘Cells into Organs’ of which P.R., C.L., R.P.A., G.R., I.P. and S.T. are members. L.C. was supported by the European
Social Fund contract 1/3.2/PRODEP/2001. R.P.A. was supported by FCT
(SFRH/BPD/9432/2002)