DOCSLIB.ORG

A Balance of Positive and Negative Regulators Determines the Pace Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Balance of Positive and Negative Regulators Determines the Pace Of 1 2 3 A balance of positive and negative regulators determines the 4 pace of the segmentation clock. 5 6 Guy Wiedermann1, Robert Bone1, Joana Clara Silva, Mia Bjorklund, Philip Murray2$, 7 J. Kim Dale$* 8 9 Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dow 10 Street, Dundee DD1 5EH, UK. 11 2Division of Mathematics, University of Dundee, Dow Street, Dundee DD1 5EH, UK 12 13 14 1These two authors contributed equally to this work 15 $Joint senior authors 16 17 Running Title: Increasing NICD stability slows the segmentation clock 18 19 *Corresponding author: j.k.dale @dundee.ac.uk 20 Tel : +44 01382 386290 21 Fax: +44 01382 385386 22 1 23 Abstract 24 Somitogenesis is regulated by a molecular oscillator which drives dynamic gene expression 25 within the PSM with a periodicity tightly coupled to somite formation. Previous mathematical 26 models that invoke the mechanism of delayed negative feedback predict the oscillation period 27 depends on the sum of delays in negative-feedback loops and the inhibitor half-lives. We 28 develop a model to explore the possibility positive feedback also plays a role. The model 29 predicts increasing the half-life of the positive regulator, Notch intracellular domain (NICD), can 30 lead to elevated NICD levels and an increase in the oscillation period. To test this, we 31 investigate a phenotype induced by various small molecule inhibitors in which the clock is 32 slowed. We observe elevated levels and prolonged half-life of NICD. Reducing NICD production 33 rescues these effects. These data provide the first indication tight control of the turnover of 34 positive as well as negative regulators of the clock determines its periodicity. 35 36 Key Words: Notch, embryo, chick, segmentation clock, oscillation, somite, negative feedback, 37 positive feedback, nonlinear dynamics 38 2 39 Introduction 40 The formation of the vertebrate skeleton and associated musculature relies on the ordered and 41 timely segmentation of mesodermal tissue during early embryonic development. Segmentation is 42 progressive and occurs in an anterior to posterior direction as the body axis elongates (reviewed 43 in Oates et.al. 2012). Mesodermal segments, or somites, ‘pinch off’ at the rostral limit of the two 44 rods of pre-somitic mesoderm (PSM) that lie on either side of the caudal neural tube, with a 45 distinct, species-specific periodicity; every 90 minutes in the chicken and every 120 minutes in the 46 mouse. This periodicity is regulated by a molecular oscillator, known as the segmentation clock, 47 which is defined by cyclic gene expression within PSM cells that has a periodicity which regulates 48 that of somite formation. These “clock” genes belong to the Notch, WNT and FGF signalling 49 pathways and are expressed in a cyclical pattern in the PSM of a wide variety of vertebrate 50 species (Krol et.al.., 2011; reviewed in Dequéant & Pourquié, 2008; Gibb et.al.. 2010; Maroto 51 et.al.., 2012). On a single cell level in the vertebrate PSM, this oscillatory gene expression is 52 believed to be established through negative feedback loops of unstable clock gene products. 53 54 Previous mathematical models of the somitogenesis clock that invoke the mechanism of delayed 55 negative feedback predict that the period can be approximated as a sum of the delays involved in 56 transcription, splicing, translation and transport and the half-lives of inhibitors of clock gene 57 expression (Lewis, 2003; Monk 2003; Ay et.al.., 2013; Hanisch et.al.., 2013). Subsequent studies 58 have confirmed that the period can be altered by genetically modifying the delays in transcription 59 of clock genes encoding negative regulators or by removing key inhibitory regulators of this 60 network (e.g. Hes7, Her6, Her7, Nrarp) (Harima, Y., et.al.. 2013; Takashima et.al.., 2011; Oates 61 et.al.., 2012; Hoyle, N.P., and Ish-Horowicz D. 2013; Schröter and Oates, 2010; Kim et.al.., 2011; 62 Herrgen et.al.., 2010). Moreover, it has been shown that tight regulation of clock gene mRNA 63 turnover and degradation is gene specific and regulated at the level of the 3’UTR (Nitanda Y 64 et.al.., 2014; Hilgers V et.al. 2005). In contrast, there has been relatively little experimental work 65 demonstrating the predicted role of protein stability of inhibitory clock gene products in 66 regulating the periodicity of clock gene oscillations (Hirata 2004). 67 68 It is notable that models that invoke delayed negative feedback as the mechanism underlying the 69 somitogenesis clock were developed primarily from zebrafish data, where the clock period is 70 relatively short and there are significantly fewer oscillating components (Krol et.al.., 2011). 71 Moreover, in zebrafish there is experimental evidence that Notch plays a key role in the 72 synchronisation of neighbouring oscillators (Delaune et.al.., 2012) but that it is not necessary for 3 73 oscillations themselves (Ozbudak and Lewis, 2008). In contrast, Notch signalling in mouse and 74 chick is thought to be essential for oscillations (Ferjentsik et.al.., 2009). One crucial factor in that 75 regard is the intracellular domain of the Notch1 receptor, (NICD), which is cleaved following 76 ligand activation, translocates to the nucleus and activates Notch target clock gene expression. 77 NICD itself is unstable and, at least in mouse, NICD production appears as pulsatile, spatio- 78 temporal waves that traverse the rostro-caudal axis of the PSM in the manner of a clock gene 79 (Huppert et.al.., 2005). Importantly, pulsatile Notch1 protein expression was recently shown to be 80 dependent on Notch signalling in the PSM of both chick and mouse (Bone et.al.., 2014); this raises 81 the possibility that, in mouse and chick at least, positive feedback, mediated by Notch and NICD, 82 plays a role in the clock mechanism. 83 84 In this study we develop a mathematical model of the clock that describes both repression and 85 NICD-mediated activation of clock gene transcription. The model predicts that increasing the half- 86 life of NICD results in elevated levels of NICD and a longer clock period. Notably, these effects are 87 not observed in the model upon increasing the half-life of the repressing clock protein. To test 88 this prediction, we use a pharmacological approach that robustly delays clock gene oscillations 89 across the chick and mouse PSM. Under these conditions, using a custom made antibody that 90 recognises endogenous chick NICD, we show that, concomitant to delaying the pace of 91 oscillations, these inhibitors increase levels of NICD and increase the half-life of NICD. In 92 agreement with model predictions, we rescue the phase patterns of clock gene expression by 93 offsetting increased NICD stability through reducing NICD production. These data therefore 94 suggest that increasing the half-life of cNICD and thus increasing its turnover time increases the 95 period of the somitogenesis clock. 96 97 4 98 Materials and Methods 99 Experimental Procedures 100 Chick culture 101 Fertilized chick (Gallus gallus) embryos from Winter Farm, Cambridge, UK, were incubated for 102 approximately 40 hours at 38°C 5% CO2 to yield embryos between Hamburger Hamilton (HH) 103 stages 10-12 as judged by somite number and morphology (Hamburger and Hamilton, 1992). 104 Explants were prepared as previously described (Palmeirim et.al.., 1997). In brief, for the half- 105 embryo assay: the caudal end of the chicken embryo is isolated to contain the most caudal somite 106 pairs and the tail tissues. The isolated tail is then bisected down the midline along the neural tube 107 to produce two identical left and right half-PSM explants which allows an internal control from 108 the same embryo with which to compare the oscillation phase of cLfng expression by in situ 109 hybridisation, following culturing of the two halves in various culture conditions. The following 110 small molecule inhibitors were typically used at the given concentrations and dissolved in 111 Dimethyl sulfoxide (DMSO) unless otherwise stated: 150 nM LY411575 unless otherwise stated 112 (generated in house, University of Dundee; Ferjentsik Z et.al., 2009; Bone et. al., 2014), 1 μM 113 MLN4924 (generated in house, University of Dundee); 20 μM Cycloheximide (Tocris Bioscience; 114 dissolved in ethanol); 100 μM XAV939 (Tocris Bioscience); 25-50 μM Cyclopamine (Calbiochem); 115 10 μM Roscovitine (Calbiochem); 10 μM 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) 116 (Sigma); 1 μM PHA-767491 (kind gift from Nathanael Gray, HARVARD.EDU). Titration assays were 117 performed to establish the lowest concentration that abolishes expression of control target 118 genes. For Fix and culture: both half-PSM explants were treated with inhibitor but while one was 119 removed from culture after 3 hours (‘fix’), the other side was cultured for an additional 45 120 minutes (‘culture’) which corresponds to half of the usual 90 minute cycle for a complete 121 oscillation of cLfng in the chicken PSM. Variations include culturing the “culture” side for “90” or 122 “120” minutes to determine the length of an oscillation. For the Western blot analysis half-PSMs 123 from 9 embryos were cultured in DMSO control vehicle and the corresponding 9 half-PSMs 124 cultured in the presence of a small molecule inhibitor. Following 3 hours of culture, both pools 125 were treated with 20 μM Cycloheximide for an additional 30 minutes before protein lysates were 126 prepared simultaneously. For the half-life assay: half-PSMs from 9 embryos were pooled and both 127 pools were cultured in each of the reagents for 3 hours and following 30 minute exposure to 128 cycloheximide, the decay of cNICD protein was monitored in the presence of these inhibitors by 129 lysing PSM explants at different time points.
Load more

Recommended publications
  • The Regulation of Lunatic Fringe During Somitogenesis
    THE REGULATION OF LUNATIC FRINGE DURING SOMITOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Emily T. Shifley ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Susan Cole, Advisor Professor Christine Beattie _________________________________ Professor Mark Seeger Advisor Graduate Program in Molecular Genetics Professor Michael Weinstein ABSTRACT Somitogenesis is the morphological hallmark of vertebrate segmentation. Somites bud from the presomitic mesoderm (PSM) in a sequential, periodic fashion and give rise to the rib cage, vertebrae, and dermis and muscles of the back. The regulation of somitogenesis is complex. In the posterior region of the PSM, a segmentation clock operates to organize cohorts of cells into presomites, while in the anterior region of the PSM the presomites are patterned into rostral and caudal compartments (R/C patterning). Both of these stages of somitogenesis are controlled, at least in part, by the Notch pathway and Lunatic fringe (Lfng), a glycosyltransferase that modifies the Notch receptor. To dissect the roles played by Lfng during somitogenesis, we created a novel allele that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng∆FCE1). Lfng∆FCE1/∆FCE1 mice have severe defects in their anterior vertebrae and rib cages, but relatively normal sacral and tail vertebrae, unlike Lfng knockouts. Segmentation clock function is differentially affected by the ∆FCE1 deletion; during anterior somitogenesis the expression patterns of many clock genes are disrupted, while during posterior somitogenesis, certain clock components have recovered. R/C patterning occurs relatively normally in Lfng∆FCE1/∆FCE1 embryos, likely contributing to the partial phenotype rescue, and confirming that Lfng ii plays separate roles in the two regions of the PSM.
    [Show full text]
  • Caspase-8, Receptor-Interacting Protein Kinase 1 (RIPK1), and RIPK3 Regulate Retinoic Acid-Induced Cell Differentiation and Necroptosis
    Cell Death & Differentiation (2020) 27:1539–1553 https://doi.org/10.1038/s41418-019-0434-2 ARTICLE Caspase-8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3 regulate retinoic acid-induced cell differentiation and necroptosis 1,2 1,3 4 3 1,2,4 Masataka Someda ● Shunsuke Kuroki ● Hitoshi Miyachi ● Makoto Tachibana ● Shin Yonehara Received: 1 July 2019 / Revised: 4 October 2019 / Accepted: 4 October 2019 / Published online: 28 October 2019 © The Author(s) 2019. This article is published with open access Abstract Among caspase family members, Caspase-8 is unique, with associated critical activities to induce and suppress death receptor-mediated apoptosis and necroptosis, respectively. Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1 or RIP1) and RIPK3 to activate mixed lineage kinase domain-like (MLKL). Disruption of Caspase-8 expression causes embryonic lethality in mice, which is rescued by depletion of either Ripk3 or Mlkl, indicating that the embryonic lethality is caused by activation of necroptosis. Here, we show that knockdown of Caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripk1 and Ripk3; however, the enhancement of RA-induced 1234567890();,: 1234567890();,: cell differentiation is independent of Mlkl and necrosome formation. RA treatment obviously enhanced the expression of RA-specific target genes having the retinoic acid response element (RARE) in their promoter regions to induce cell differentiation, and induced marked expression of RIPK1, RIPK3, and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RIPK1 and RIPK3 to translocate into the nucleus and to form a complex with RA receptor (RAR), and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3.
    [Show full text]
  • Regulation of Myogenesis by Cardiotrophin-1 and Tgfp Signalling
    Regulation of Myogenesis by Cardiotrophin-1 and TGFp signalling Tetsuaki Miyake A dissertation submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Programme in Biology York University Toronto, Ontario, Canada Jan 2010 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-64946-6 Our file Notre reference ISBN: 978-0-494-64946-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation.
    [Show full text]
  • Species-Specific Oscillation Periods of Human and Mouse Segmentation
    bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Species-specific oscillation periods of human and mouse segmentation clocks 2 are due to cell autonomous differences in biochemical reaction parameters 3 4 Mitsuhiro Matsuda1, 2, Hanako Hayashi1, Jordi Garcia-Ojalvo3, Kumiko Yoshioka- 5 Kobayashi4, Ryoichiro Kageyama4, Yoshihiro Yamanaka5, Makoto Ikeya5, Junya 6 Toguchida4, 5, Cantas Alev5, Miki Ebisuya1, 2* 7 8 1RIKEN Center for Biosystems Dynamics Research (RIKEN BDR) 9 2-2-3 Minatojima-minamimachi, Chuo-ku, 650-0047 Kobe, Japan 10 2European Molecular Biology Laboratory (EMBL) Barcelona 11 Dr. Aiguader 88, 08003 Barcelona, Spain 12 3Department of Experimental and Health Sciences, Universitat Pompeu Fabra 13 Dr. Aiguader 88, 08003 Barcelona, Spain 14 4Institute for Frontier Life and Medical Sciences, Kyoto University 15 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan 16 5Center for iPS Cell Research and Application (CiRA), Kyoto University 17 53 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan 18 19 20 *Correspondence to M Ebisuya ([email protected]) 21 1 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
    [Show full text]
  • Investigating the Roles of Hes and Sox Genes During Embryogenesis
    Investigating the roles of Hes and Sox genes during embryogenesis of the spider P. tepidariorum Christian Louis Bonatto Paese This thesis was submitted in partial fulfillment of the requirements of the award of Doctor of Philosophy June 2018 “Our greatest glory is not in never falling, but in rising every time we fall” Confucius Aquele uísque “Lembro-me daqueles dias, Em que assistíamos ao pica-pau E rachavamos o bico dando risada de algumas passadas. Lembro-me de que possuíamos aspirações e sonhos selvagens. Cada qual, à sua maneira, pôs-se a caçar seu horizonte. Sabíamos que nenhuma estrada se emenda no passado, Todas se amarram apenas no porvir, E, só com muita sorte, nos achamos novamente. Mas a memória, ah! Essa é uma cruel concubina, Pois nunca nos esquecemos daqueles dias. Eles voltam em espasmos mistos de tristeza e alegria. No saldo da vida, Parece que atingimos muito do que poderíamos, Mas sempre ficamos na dúvida De uma dose daquele uísque: Velho olho vermelho.” Pablo Jamilk Essa tese é o resultado de 3 anos que me trouxeram um incrivel auto-aprendizado. Todo esse esforço é dedicado a minha família – em especial aos meus sobrinhos Leonardo e Leticia. Table of Contents Abstract ...................................................................................................................................................... 6 List of Abbreviations .................................................................................................................................. 8 List of Figures ............................................................................................................................................
    [Show full text]
  • The Transcriptome of Utricle Hair Cell Regeneration in the Avian Inner Ear
    The Journal of Neuroscience, March 5, 2014 • 34(10):3523–3535 • 3523 Development/Plasticity/Repair The Transcriptome of Utricle Hair Cell Regeneration in the Avian Inner Ear Yuan-Chieh Ku,1 Nicole A. Renaud,1 Rose A. Veile,1 Cynthia Helms,1 Courtney C.J. Voelker,2 Mark E. Warchol,2 and Michael Lovett1 1Department of Genetics and 2Department of Otolaryngology, Washington University School of Medicine, St Louis, Missouri 63110 Sensory hair cell loss is the major cause of hearing and balance disorders. Mammals are incapable of sustained hair cell regeneration, but lower vertebrates can regenerate these mechano-electrical transducers. We present the first comprehensive transcriptome (by mRNA- Seq) of hair cell regeneration in the chick utricle. We provide pathway and pattern annotations and correlate these with the phenotypic events that occur during regeneration. These patterns are surprisingly synchronous and highly punctuated. We show how these patterns are a new resource for identifying components of the hair cell transcriptome and identify 494 new putative hair-cell-specific genes and validate three of these (of three tested) by immunohistochemical staining. We describe many surprising new components and dynamic expression patterns, particularly within NOTCH signaling. For example, we show that HES7 is specifically expressed during utricle hair cell regeneration and closely parallels the expression of HES5. Likewise, the expression of ATOH1 is closely correlated with HEYL and the HLH inhibitory transcription factors ID1, ID2, and ID4.
    [Show full text]
  • Regulation of Notch Activation by Lunatic Fringe During
    REGULATION OF NOTCH ACTIVATION BY LUNATIC FRINGE DURING SOMITOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Dustin Ray Williams Graduate Program in Molecular Genetics The Ohio State University 2014 Dissertation Committee: Susan Cole, Advisor Heithem El-Hodiri Mark Seeger Amanda Simcox Copyright by Dustin Ray Williams 2014 ABSTRACT During somitogenesis, paired somites periodically bud from the presomitic mesoderm (PSM) located at the caudal end of the embryo. These somites will give rise to the axial skeleton and musculature of the back. The regulation of this process is complex and occurs at multiple levels. In the posterior PSM, Notch activity levels oscillate as part of a clock that controls the timing of somite formation. In the anterior PSM, the Notch pathway is involved in somite patterning. In the clock, cyclic Notch activation is dependent upon periodic repression by the glycosyltransferase Lunatic fringe (LFNG). Lfng mRNA levels cycle over a two-hour period in the clock, facilitating oscillatory Notch activity. Lfng is also expressed in the anterior PSM, where it may regulate Notch activity during somite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng∆FCE) exhibit abnormal anterior development but relatively normal posterior development, suggesting distinct requirements for segmentation clock activity during the formation of the anterior skeleton compared to the posterior skeleton and tail. To further test this idea, we created an allelic series that progressively lowers Lfng levels in the PSM. We find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development.
    [Show full text]
  • EXAMINING the POST-TRANSCRIPTIONAL REGULATION of LUNATIC FRINGE (Lfng) in the MOUSE SEGMENTATION CLOCK
    EXAMINING THE POST-TRANSCRIPTIONAL REGULATION OF LUNATIC FRINGE (Lfng) IN THE MOUSE SEGMENTATION CLOCK DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kanu Wahi Graduate Program in Molecular Genetics The Ohio State University 2016 Dissertation Committee: Dr. Susan Cole, Advisor Dr. Keith Slotkin Dr. Robin Wharton Dr. Dawn Chandler Copyright by Kanu Wahi 2016 Abstract Somitogenesis is a developmental process in vertebrates involving periodic formation of somites that bud from an unsegmented region known as the pre-somitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscle in the developed organism. The process of somitogenesis is regulated by a "segmentation clock" that times somite formation and is evolutionarily conserved among vertebrates. Genes, such as Lunatic fringe (Lfng), are required for normal clock function in chickens, mice and humans and exhibit rapid cyclic expression in the PSM with a period that matches the rate of somite formation. To maintain rapid oscillations, especially in the posterior PSM, it is hypothesized that the Lfng transcript should be promptly degraded to ensure its clearance of from cells before the next round of oscillation begins. The 3’UTR of Lfng contains number of conserved sequences that could regulate Lfng expression at the post transcriptional level. In this study we explore the post- transcriptional regulation of the Lfng transcript by the Lfng 3’UTR in the context of the segmentation clock. The Lfng 3'UTR has binding sites for microRNAs (miRNAs), such as miR-125a and miR-200b that are known to affect transcript stability via the 3’UTR.
    [Show full text]
  • The Precise Timeline of Transcriptional Regulation Reveals Causation in Mouse Somitogenesis Network Bernard Fongang and Andrzej Kudlicki*
    Fongang and Kudlicki BMC Developmental Biology 2013, 13:42 http://www.biomedcentral.com/1471-213X/13/42 RESEARCH ARTICLE Open Access The precise timeline of transcriptional regulation reveals causation in mouse somitogenesis network Bernard Fongang and Andrzej Kudlicki* Abstract Background: In vertebrate development, the segmental pattern of the body axis is established as somites, masses of mesoderm distributed along the two sides of the neural tube, are formed sequentially in the anterior-posterior axis. This mechanism depends on waves of gene expression associated with the Notch, Fgf and Wnt pathways. The underlying transcriptional regulation has been studied by whole-transcriptome mRNA profiling; however, interpretation of the results is limited by poor resolution, noisy data, small sample size and by the absence of a wall clock to assign exact time for recorded points. Results: We present a method of Maximum Entropy deconvolution in both space and time and apply it to extract, from microarray timecourse data, the full spatiotemporal expression profiles of genes involved in mouse somitogenesis. For regulated genes, we have reconstructed the temporal profiles and determined the timing of expression peaks along the somite cycle to a single-minute resolution. Our results also indicate the presence of a new class of genes (including Raf1 and Hes7) with two peaks of activity in two distinct phases of the somite cycle. We demonstrate that the timeline of gene expression precisely reflects their functions in the biochemical pathways and the direction of causation in the regulatory networks. Conclusions: By applying a novel framework for data analysis, we have shown a striking correspondence between gene expression times and their interactions and regulations during somitogenesis.
    [Show full text]
  • Dynamic Expression and Essential Functions of Hes7 in Somite Segmentation
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION cells (Palmeirim et al. 1997). For the wave-like propaga- Dynamic expression and tion, chairy1 expression should oscillate synchronously essential functions of Hes7 in the PSM, but the exact mechanism for the synchro- nous oscillation remains to be determined. in somite segmentation Recent studies suggest that Notch signaling regulates 1 1 the synchronously oscillating gene expression in the Yasumasa Bessho, Ryoichi Sakata, PSM and coordinated segmentation of somites. In ze- Suguru Komatsu,2 Kohei Shiota,2 brafish, mutation for the Notch pathway is likely to de- Shuichi Yamada,1 and Ryoichiro Kageyama1,3 synchronize the oscillating gene expression (a random mixture of PSM cells that express different levels of gene 1Institute for Virus Research, Kyoto University, Kyoto expression) and this desynchronization may lead to a 606-8507, Japan; 2Department of Anatomy and loss of the coordinated somite segmentation (Holley et Developmental Biology, Kyoto University Graduate School al. 2000; Jiang et al. 2000). Similarly, in mice, Notch of Medicine, Kyoto 606-8501, Japan signaling is essential for oscillating gene expression (Jouve et al. 2000) and coordinated segmentation of The basic helix–loop–helix (bHLH) gene Hes7, a putative somites: mutations for Notch1, the ligands Delta-like 1 Notch effector, encodes a transcriptional repressor. Here, (Dll1) and Dll3, and other regulators such as Presenilin, we found that Hes7 expression oscillates in 2-h cycles in RBP-J, and lunatic fringe (Lfng) all exhibit defects of so- the presomitic mesoderm (PSM). In Hes7-null mice, mite segmentation (Swiatek et al.
    [Show full text]
  • Intronic Delay Is Essential for Oscillatory Expression in the Segmentation Clock
    Intronic delay is essential for oscillatory expression in the segmentation clock Yoshiki Takashimaa,b,c, Toshiyuki Ohtsukaa,b, Aitor Gonzáleza,b, Hitoshi Miyachia, and Ryoichiro Kageyamaa,b,1 aInstitute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan; bJapan Science and Technology Agency, Core Research for Evolutional Science and Technology, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan; and cKyoto University Graduate School of Biostudies, Kyoto 606-8502, Japan Edited by David Ish-Horowicz, Cancer Research UK, London, United Kingdom, and accepted by the Editorial Board January 14, 2011 (received for review September 27, 2010) Proper timing of gene expression is essential for many biological To determine the significance of introns in the timing of gene events, but the molecular mechanisms that control timing remain expression, we focused on the Hes7 gene in the mouse segmen- largely unclear. It has been suggested that introns contribute to tation clock. We first found that the Hes7 promoter-driven re- the timing mechanisms of gene expression, but this hypothesis has porter without any introns led to expression ∼19 min earlier than not been tested with natural genes. One of the best systems for the reporter with full introns, indicating that introns lead to about examining the significance of introns is the oscillator network in a 19-min delay in Hes7 expression. According to our previous the somite segmentation clock, because mathematical modeling mathematical model (17), if the delay is 19 min shorter, the os- predicted that oscillating expression depends on negative feedback cillatory expression of Hes7 would be abolished, although a dif- with a delayed timing.
    [Show full text]
  • Travelling Waves in Somitogenesis: Collective Cellular Properties
    bioRxiv preprint doi: https://doi.org/10.1101/297671; this version posted April 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Travelling waves in somitogenesis: collective cellular properties 2 emerge from time-delayed juxtacrine oscillation coupling 3 Tomas Tomka1, Dagmar Iber1,2,*, Marcelo Boareto1,2,* 4 Affiliations 5 1 Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, 4058 6 Basel, Switzerland 7 2 Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland. 8 *Correspondence to: [email protected], [email protected] 9 10 Key words 11 Somitogenesis, Hes/her oscillations, time delays, travelling waves, Notch signalling, mathematical 12 modelling 13 14 Abstract 15 The sculpturing of the vertebrate body plan into segments begins with the sequential formation of somites 16 in the presomitic mesoderm (PSM). The rhythmicity of this process is controlled by travelling waves of 17 gene expression. These kinetic waves emerge from coupled cellular oscillators and sweep across the 18 PSM. In zebrafish, the oscillations are driven by autorepression of her genes and are synchronized via 19 Notch signalling. Mathematical modelling has played an important role in explaining how collective 20 properties emerge from the molecular interactions. Increasingly more quantitative experimental data 21 permits the validation of those mathematical models, yet leads to increasingly more complex model 22 formulations that hamper an intuitive understanding of the underlying mechanisms. Here, we review 23 previous efforts, and design a mechanistic model of the her1 oscillator, which represents the 24 experimentally viable her7;hes6 double mutant.
    [Show full text]