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Elucidation of the chondrogenic program using a combination of biology and technology Garcha, Kamal 2008

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Elucidation of the Chondrogenic Program Using aCombination of Biology and TechnologybyKamal GarchaHBSc., The University of Western Ontario, 2000MSc., The University of Western Ontario, 2003A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Anatomy & Cell Biology)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)October, 2008© Kamal Garcha, 2008General AbstractSkeletogenesis is associated with the aggregation of mesenchymal cells intocondensations that prefigure the mature skeleton under the influence of numeroussignaling molecules. In the developing murine limb, Fibroblast growth factors (FGFs)(principally 4 and 8) are expressed in the apical ectodermal ridge (AER) and signalto the underlying distal mesenchymal cells. In this manner, FGFs are thought toinfluence the proliferation of chondroprogenitors, thereby modulating the growth ofpre-cartilage condensations. To study these effects the chondrogenic program canbe effectively modeled in vitro using high-density cultures of primary cells isolatedfrom the murine limb bud (El 1.5). Treatment of distal mesenchymal cultures withFGF4 leads to a transient increase in proliferation and expansion ofprechondrogenic condensations. Using transcriptional profiling with DNAmicroarrays we observed that consistent with the changes observed in cellproliferation, FGF4 treatment resulted in an 18 fold increase in the expression ofCdkn2b — a cyclin dependent kinase inhibitor. SiRNA knockdown of Cdkn2b resultedin sustained mesenchymal proliferation. Further, we determined that FGF4 regulatesCdkn2b through a MEKI/ERK-dependent pathway. Additionally, we show that FGF4promotes cell survival through the regulation of NFiB via upregulation of its activatorRIPK4.To enhance our studies of chondrogenesis, a high throughput reporter genebased assay was developed. Using this technology we performed a chemicalgenetic screen of -1500 chemical compounds to assess their ability to regulatereporter gene activity. Of these compounds, 28 yielded a >2.5 fold increase in11S0X516/9 reporter gene activity. Secondary histological screens confirmedincreased cartilage formation in response to treatment of primary limb cultures withseveral of these newly identified chemical enhancers of chondrogenesis. Throughbioinformatic analysis of microarray data in comparison withthe known targets of thescreened chemical compounds, we have shown that inhibition of potassium channelKCND2 (Kv4.2) promotes chondrogenesis. Additionally, it was found thatprochondrogenic bone morphogenetic protein 4 (BMP4) downregulated theexpression of Kcnd2. These data have revealed an unanticipatedrole of potassiumchannels during chondrogenesis. Thus by using a combined approach of microarrayanalysis and chemical genetics we have further characterized the intricacies of thechondrogenic program.111Table of ContentsGeneral Abstract.iiTable of Contents ivListof Tables ixList of Figures xList of Abbreviations XiiCo-Authorship Statement xvChapter 1 11.1 General Introduction 11.1.1 Significance 11.1.2 Background 41.2 Overview of Vertebrate Limb Development 51.2.1 Limb Bud Initiation and Early Outgrowth 51.2.3 Models of Limb Patterning 71.2.4 Emergence of a Chondrogenic Anlagen 81.2.5 Signaling Networks Involved in Limb Development 81.2.6 FGFs and Their Associated Receptors 101.2.7 Mutations in FGFs and FGFRs 141.2.8 Regulation of Cell Proliferation by the CDKN2IINK Family 151.2.9 Mitogen Activated Protein Kinase (MAPK) Signaling 171.2.10 Role of Nuclear Factor Kappa B in Limb Development 18iv1.2.11 Regulation of NF-KB .191.3 Transcriptional Regulation of Chondrogenesis 201.3.1 Importance of SOX9 201.4 Methodology and Strategies 221.4.1 Primary Cell Culture Model for Studying Chondrogenesis 221.4.2 Assessing the Status of Chondroblast Differentiation 241.4.3 Review of Transfection Technology 251.4.4 Design and Application of New Technology 271.4.5 Stabilization of Transfection Complexes 281.4.6 Enhancement of Experimental Throughput 291.4.7 Small-Molecule Screening 291.5 Overview of Thesis 301.57 Topics Addressed 311.6 Figures 321.7 References 44Chapter 2 62FGF Signals in the Embryonic Limb Regulate Cell Proliferation and SurvivalThrough Cdkn2b and the NF-icB Signaling Pathway 622.2 Introduction 642.3 Results 662.4 Discussion 73V2.4.1 FGFs and Cell Proliferation .742.4.2 FGFs, NF-icB and Cell Survival 762.4.3 FGF Signaling and Chondrogenesis 782.5.1 Reagents 792.5.2 Plasmid Constructs 802.5.3 Establishment and Transfection of Primary Limb MesenchymalCultures 802.5.4 Assessment of PLM Cell Proliferation 822.5.5 Rhodamine - Peanut Agglutinin (PNA) Labeling 832.5.6 Culture of PLM cells in the Absence of Serum Factors 832.5.7 Limb Bud Organ Culture and Bead Implantation 832.5.8 Transcriptional Profiling with Microarrays: Experimental Design andAnalysis 842.5.9 Quantitative Real-Time PCR 852.5.10 Statistical analysis 862.6 Acknowledgements 862.7 Figures 872.8 References 104Chapter3 110Chemical Genetics Reveals a Novel Role for Potassium Channels inChondrogenesis 1103.1 Abstract 111vi3.2 Introduction .1123.3 Results and Discussion 1143.4 Methods and Materials 1223.4.1 Reagents 1223.4.2 Small Molecule Libraries 1223.4.3 Establishment and Transfection of Primary Limb MesenchymalCultures 1223.4.4 RNA Collection From E11.5 Mouse Limbs 1253.4.5 Quantitative Real-Time PCR 1253.4.6 Microscopy and Image Acquisition 1253.4.7 siRNA Knockdown in PLM Cultures 1263.4.8 Statistical Analysis 1263.5 Acknowledgements 1263.6 Figures 1283.7 References 139Chapter 4 1414.1 Discussion 1414.2 Context-Dependent Gene Regulation 1424.3 Identifying Transcription Factors 1434.4 Implications and Applications of Serum-Free Culture Studies 1454.5 High-Throughput Design to Aid in the Discovery of Chondrogenicvii4.5 High-Throughput Design to Aid in the Discovery of ChondrogenicMechanisms 1474.6 Small-Molecule Screening to Identify Chondrogenic Modulators 1474.7 Systems Biology as a Predictive Tool 1494.8 Regulation and Potential Role of Kv4.2 Channels in Chondrogenesis 1504.9 Impact of Research 1524.10 Future Directions 1544.11 Concluding Remarks 1564.12 Figures 1584.13 References 162viiiList of Tables2.1 qRT-PCR Analysis of Selected Genes in E11.5 DM Cultures 103ixList of Figures1.1 Overview of Mammalian Development 331.2 Three Perspectives on Proximal-to-Distal Patterning inDeveloping Limb Buds 351 .3 The Developmental Progression of Undifferentiated LimbMesenchyme to Cartilaginous Skeletal Precursors in the Limb 371 .4 FGF Receptors and FGF Signal Transduction 391.5 Rel/NF-kB Signal Transduction 411.6 Schematic Representation of the ModifiedEffecteneTMTransfection Strategy for Enhanced Transfection and Storage ofTransfection-Ready DNA Complexes Facilitating Large-ScaleTransfections 432.1 FGF4 Regulates Expression of the Chondroblastic Phenotype andProliferation of Limb Mesenchymal Cells 892.2 Identification of FGF4-Regulated Genes in Limb-Derived DistalMesenchymal Cells 912.3 Cyclin Dependent Kinase Inhibitor 2b is Up-Regulated by FGF4in vitro and in Organ Culture 932.4 FGF4 Regulation of Cdkn2b Does Not Involve TGFbI Signaling 952.5 FGF4 Regulates Cell proliferation Through Cdkn2b and ThisInvolves the MEKI/ERK Signaling Pathway 972.6 FGF4 Activates the NF-kB Signaling Pathway in Part ThroughMEKI/ERK-Mediated Upregulation of Ripk4 99x2.7 The MEKI/ERK and NF-icB Signaling Pathways are BothRequired for FGF4-Mediated Cell Survival and CartilageFormation 1023.1 Disaccharides Such as Sucrose, and to a Greater ExtentTrehalose, Increase Transfection Efficiency ofEffecteneTMandFacilitate the Storage of Transfection-Ready DNA 1293.2 Optimization and Validation of 384-well Format for ChondrogenicAssays in PLM Cultures 1313.3 Identification of Novel Chondrogenic Modulators Using a ChemicalBiology Strategy 1333.4 The Prochondrogenic Activity of Butamben is Associated With itsPotassium Channel Blocking Capability 1363.6 BMP4 Regulates Chondrogenesis Through Regulation of Kcnd2Expression and Consequently Potassium Channel Activity 1384.1 Schematic Summary of FGF4 Mediated Signal Transduction inPLM Cultures as Evidenced in Chapter 2 1594.2 Overview of the Strategies Developed to Efficiently Assess theRole of Genes and Compounds 161xiList of AbbreviationsAER apical ectodermal ridgeAgcl aggrecanBAB butambenBMP bone morphogenetic proteinCDKN cyclin dependant protein kinase inhibitorcDNA complementary deoxyribonucleic acidcol collagencMEK constitutively active MEKCspg4 chondroitin sulfate proteoglycan 4DMSO dimethyl sulfoxideDM distal mesenchymedn dominant negativeDNA deoxyribonucleic acidE embryonic dayEGFP enhanced green fluorescent proteinEomes EomesoderminERK extracetlular signal related kinaseES cell embryonic stem cellFBS fetal bovine serumFGF fibroblast growth factorFGFR FGF receptorxiiGDF growth/differentiation factorHMG high mobility groupIDR interdigital region1KB inhibitory kappa BIKK 1KB kinaseKCND2 potassium voltage -gated channel, Shal-related subfamily, member 2Scal stem cell antigen 1MAPK mitogen-activated protein kinaseMEK MAPK kinasemRNA messenger ribonucleic acidN-cad N-cadherinNCAM neural cell adhesion moleculeNF-KB nuclear factor kappa BOgn osteoglycinPBS phosphate buffered salinePCR polymerase chain reactionPD proximo-distalPKC protein kinase CPLM primary limb mesenchymePM proximal mesenchymePNA peanut agglutininPZ progress zoneqRT-PCR quantitative real-time PCRxliiRA retinoic acidRARE retinoic acid response elementRipk4 receptor-interacting serine- threonine kinase 4RNA ribonucleic acidrRNA ribosomal RNAsIRNA short interfering RNAShh sonic hedgehogSmad homology to Sma and MAD (mothers against decapentaplegic)Sox SRY-like HMG boxSRY sex-determining region of Y chromosomeTGFf Transforming growth factor f3Wnt winglessZic zinc finger protein of the cerebellumZPA zone of polarizing activityxivCo-Authorship StatementChapter 2: FGF Signals in the Embryonic Limb Regulate Cell Proliferation andSurvival Through Cdkn2b and the NF-icB Signaling PathwayAll of the following were carried out by Kamal Garcha and T. Michael Underhill:identification and design of research program, performing the research, dataanalyses and manuscript preparation.A former post-doctoral fellow, Lisa M. Hoffman, isolated RNA used in microarrayexperiments described in Chapter 2.Chapter 3: Chemical Genetics Reveals a Novel Role for Potassium Channels inChondrogenesisAll of the following were carried out by Kamal Garcha and T. Michael Underhill:identification and design of research program, performing the research, dataanalyses and manuscript preparation.xvChapter 11.1 General IntroductionIn the early mammalian embryo, coordinated signaling events lead towidespread changes in gene expression resulting in tissue morphogenesis. Thetemporal and spatial regulation of developmental programs is tightly regulated byvarious molecular mediators such that structures arise in a precise order. During limbdevelopment, an interplay of molecular signaling between the overlying ectoderm andthe underlying mesenchyme gives rise to specialized tissues that direct thedevelopment of skeletal elements. Of particular interest is a specialized structure atthe most distal edge of the developing limb comprised of a pseudostratified layerofepithelium —the apical ectodermal ridge (AER) that governs the outgrowth andpatterning of the limb. This thesis examines the role of an AER expressed signalingmolecule -fibroblast growth factor 4 (FGF4) in the developing limb, with a particularfocus on FGF4 mediated mesenchymal cell proliferation and survival. Additionally, achemical biology approach was employed to identify other pathways that regulatemesenchymal cell function and differentiation.11.1 SignificanceCartilage functions as a template for endochondral bone formation. Thedifferentiation and growth of cartilaginous tissues are processes vital to properskeletal development and function, and in children cartilage within the growth platealso allows for longitudinal growth of bones. As such, developmental deformitiesarise from aberrant growth and differentiation of the chondrogenic anlagen duringskeletogenesis. Cartilage has limited reparative ability, such that small chondrogenic1lesions in humans are a contributing factor to the development of osteoarthritis. Inadults, osteoarthritis presents a major medical, social and economic burden onsociety, as the current incidence in North America is —10% within the generalpopulation ( is known about cartilage repair; however, reparative processes in othertissues involve at least in part, the recapitulation of developmental programs utilizedin their initial formation. Adult tissue regeneration resembles embryonic development,specifically, progenitor cells are recruited and induced to differentiate and giverise tonew tissues that have the form and function of the original tissue. Tobetterunderstand the molecular mechanisms involved in chondrogenesis, embryoniccartilage formation can be effectively modeled in vitro using mesenchymalcellsisolated from the embryonic mouse limb. Our studies have involved signalingfactorsessential in limb induction with a particular focus on the transcriptional regulationofcartilage specific genes during the initial specification of the limb skeletaltemplate.Understanding the molecular programs operating in embryonic chondrogenesisshould provide therapeutic clues for enhancing adult cartilage repair.Bioinformatic approaches are commonly used to examine changesin genetranscription in response to various stimuli. The aim of these approachesis to identifythose genes and gene families that are downstream targets of the stimuli.Therefore,to further study the regulation of chondrogenesis, we performed microarrayexperiments on samples collected from primary limb mesenchymal (PLM)culturestreated with previously identified factors essential to limb formation. Validationofthese data and the characterization of the role of several of these genes, is the focus2of chapter 2. Briefly, the role of FGF4 in the developing limb was examinedat thelevel of transcription by examining previously generated microarray data.Previousobservations indicated that treatment of PLM cultures with exogenous FGF4inducedtransient PLM proliferation, and thereby yielded an increase inprecartilaginouscondensations. However, the mechanism responsible for the attenuationof FGF4induced cellular proliferation had not been identified. We therefore hypothesizedthata cell cycle inhibitor was likely responsible for attenuating FGF4 mediatedproliferation. In Chapter 2 we provide evidence for the attenuation ofFGF4 inducedproliferation by one of its downstream targets - cyclin dependent kinaseinhibitor 2b(Cdkn2b); a cell cycle inhibitor. Additionally Chapter 2 describes a new in vitroculturemodel using serum-free conditions to ascertain the role of developmentalfactors onchondrogenesis. Specifically we show that FGF4 promotes mesenchymalcellproliferation and survival.The emergence of genome-wide approaches within the last fewyears hasrevolutionized our understanding of the molecular mechanismsunderlying variousbiological processes. These new approaches enabled unbiased strategiesto identifygene(s) that may have important roles in specific embryonic developmentalprograms. The importance of candidate genes can be evaluated empiricallybyoverexpression or by gene silencing. To this end, Chapter3 describes a noveltransfection strategy that makes it possible to perform large scale genescreens byeliminating the constraints of time associated with thepreparation of DNAtransfection mixtures. As an extension of this methodology, a chemical-genetic3screen was also carried out to identify small molecules that modulatechondrogenesis. These screens yielded several novel stimulators of chondrogenesis.By comparing the known targets of the stimulatory small-molecules with dataobtained by transcriptional profiling, we identify the importance of potassium channelKCND2 in the chondrogenic program. This technology has facilitated theimplementation of large scale gene and chemical biology screens to delineate themolecular programs operating in chondrogenesis.1.1.2 BackgroundThe vertebrate limb is complex, being comprised of several tissues with anasymmetrical arrangement of components. During embryogenesis, the majority ofthe skeletal bones are formed via endochondral ossification, whereby the futurebones are first formed as hyaline cartilage scaffolds. Specifically, mesenchymalprogenitor cells differentiate into chondrocytes giving rise to a cartilage template thatis subsequently replaced by bone in a precise arrangement. The limbs develop frompaired primordial buds that appear on the lateral surface of the embryo at specificregions referred to as limb fields along the anterior-posterior body axis. Limbformation is initiated by the selective expansion of mesenchymal cells within thelateral plate mesoderm (Olsen et al., 2000). Subsequently, through ill-definedmechanisms, a subset of mesenchymal cells is fated to become chondrocytes.These cells first become evident within precartilaginous condensations, the earliestmorphologically apparent event in skeletogenesis (Karsenty and Wagner, 2002;Mariani and Martin, 2003). During the condensation stage, numerous processesincluding mesenchymal cell recruitment, migration, and aggregation of progenitors4(Hall and Miyake, 2000; Tuan, 2004) coordinate to establish a high density ofmesenchymal cells within the developing limb. Importantly, mutants defective inmesenchymal condensation as the result of a failure to meet the required high celldensity, present with limb skeletal anomalies (Hall and Miyake, 1992; Mundlos andOlsen, 1997). Within the condensed mesenchyme, cells differentiate and establish acartilaginous matrix that will support further skeletogenesis.In an attempt to better define the molecular programs regulating the formationof the appendicular skeleton, our laboratory is characterizing the role of factorsessential for chondrogenesis in the developing limb.1.2 Overview of Vertebrate Limb Development1.2.1 Limb Bud Initiation and Early OutgrowthLimb development begins when mesenchyme cells proliferate from thesomatic layer of the limb field lateral plate mesoderm (limb skeletal precursors), andfrom the somites (limb muscle precursors). These cells accumulate under theepidermal tissue to create a rounded bulge called a limb bud. Signals from thelateral plate mesoderm (LPM) are essential for limb bud formationand it is theseLPM cells that will become the limb mesenchyme (Tickle and Munsterberg,2001).The subsequent patterning of limb mesenchyme however, is dueto interactionsbetween the mesenchyme and the overlying ectoderm (Capdevila and lzpisuaBelmonte, 2001)(Fig. 1.IA).It is proposed that as mesenchymal cells enter the limb region they secretefactors that, through reciprocal signaling with the overlying ectoderm, induce theformation of a pseudostratified, columnar epithelium termed the apical ectodermal5ridge (AER). In the limb field, the presence of the T-box transcription factors TBX4(hindlimb) and TBX5 (forelimb) precedes the signaling interaction between membersof the FGF and Wingless (WNT) families (Agarwal et al., 2003). In Tbx4’ and Tbx5’animals there is a failure of limb initiation, and FGF and WNT signaling. In theprevailing view of limb initiation, the role of the TBX transcription factors is tostimulate FgflO expression, whereas the proposed role of WNT3 is to maintain highlevels of FGF1O throughout limb initiation (Agarwal et al., 2003; Yang, 2003).Reciprocally, FGFIO is believed to maintain Wnt3 expression during the formation ofthe AER (Barrow et al., 2003b). The AER runs along the distal margin of the limband becomes a major signaling centre for the developing limb. The roles of the AERinclude maintaining the underlying mesenchyme in a plastic, proliferative phase thatenables proximal-distal outgrowth of the limb.The importance of the AER in limb outgrowth is emphasized in experimentsinvolving its removal at successive stages of development. When the AER isremoved at an early limb bud stage, the most proximal skeletal segment (stylopod:upper arm, thigh) forms, but middle (zeugopod: forearm, lower leg) and distal(autopod: wrist and hand, ankle and foot) segments are absent. When it is removedat a slightly later stage, only the autopod is missing (Rowe and FaIIon, 1982;Saunders, 1998; Summerbell, 1974). Experiments showing that distal skeletalelements could be rescued by applying beads soaked in recombinant FGF protein tothe tip of AER-denuded chick limb bud suggested that members of the FGF family ofsecreted proteins are the AER-derived signals required for limb development (Fallonet al., 1994; Niswander et al., 1993). Four of the 22 known Fgf genes, Fgf4, Fgf8,6Fgf9 and Fgfl7, display AER-specificexpression domains within the mouselimb bud(Itoh and Ornitz, 2004; Martin, 1998;Sun et al., 2000; Yu and Omitz,2008b) alongwith their respective receptors.1.2.3 Models of Limb PatterningThe prevailing model of limb patterninghas been the subject of somedebate(Dudley et al., 2002; Marianiet al., 2008; Mariani and Martin, 2003;Saunders, 2002;Tabin and Wolpert, 2007). Thereason for this controversy is relatedto the belief thatcell fate is specified by the amount oftime spent in proximityto the AER. Previouslyit has been shown that removalof the AER at successivestages results in the lossof increasingly distal limb elements(Summerbell et al., 1973). Morerecent data hassuggested that this is merely aconsequence of excessive cell death arisingfrom theremoval of the AER (Dudleyet al., 2002; Niswander and Martin,1993a; Saunders,2002) (Fig. 1 .2A). Confounding the understandingof limb patterning is more recentmolecular data related to geneexpression in the limb. To addto the possibilities, anew hybrid model is emergingbased on knockout studies involvingFGFs in the AER(Mariani et al., 2008). This newmodel, termed the “two-signaldynamic specificationmodel”, proposes thatthe AER produces distal signals, whereastissues near thebody wall produce opposing proximalsignals. Accordingly,a loss of a distal signalpermits the proximal signalto extend more distally than normal,specifying moredistal cells to give rise to structuresnormally found in the proximallimb (Fig. 1 .2B).Regardless of the proposedmodel, the absolute necessityof the AER in outgrowthand patterning of the developinglimb is undisputed.71.2.4 Emergence of a Chondrogenic AnlagenThe aggregation of mesenchymal cells is an important transient eventduringchondrogenesis. Patterning signals are expressed and regulateskeletogenesisthrough the establishment of pre-cartilage condensations. Thecells within thecondensations have altered mitotic activity, and increased cell-cell signalingandinteraction due to increased cell density. The condensed mesenchymedemarcatesthe regions in which a cartilage anlagen will form and eventually be replacedbybone. It is at this stage that condensed mesenchymal cells differentiatetochondrocytes, and begin to secrete an extracellular matrix (ECM) rich in aggrecan,collagen type II, IX, Xl, fibronectin, hyaluronan as wellas the link protein andtenascin (DeLise et al., 2000; Goldring et al., 2006; Kulyket al., 1991; Stirpe et al.,1990; Swiderski and Solursh, 1992), whereas during differentiationthe expression ofcollagen type I is downregulated. These condensed cells become encasedby theirsecreted ECM and following further maturation and hypertrophy, they producecollagen type X and downregulate the expression of collagentype II, and supportcalcification. The calcified cartilage is subsequently invaded byblood vesselscarrying osteoblasts, marking the initial stages of the template’s mineralizationtobone.1.2.5 Signaling Networks Involved in Limb DevelopmentOf the essential factors required for the coordination of signaling eventsculminating in the developed limb, ectodermal WNTs withinthe AER are among theearliest signals required to induce FGFs such as FGF1O and FGF8(Ohuchi et al.,1997). These signaling pathways act in positive feedback loops (Niswander,2003)8and promote proximal-distal growth. In thissequence of events as previouslyoutlined, TBX transcription factors (TBX4/5) inthe lateral plate mesoderm inducedownstream factors FGFIO and WNT3. WNT3acts via 3-catenin to increase FGF8in the ectoderm. FGF8 and WNT3 maintainFGFIO expression in the mesenchymeand vice versa (Agarwal et al., 2003; Barrowet al., 2003b; Tickle and Munsterberg,2001). In this manner, reciprocal FGFsignals regulate the outgrowth of the limb bud(Tickle, 2002; Yu and Ornitz, 2008a).Additionally, WNT7A produced by the dorsalectoderm signals through its downstream targetgene Lmxl in the underlying dorsalmesenchyme, and plays a criticalrole in dorsal-ventral patterning. Wnt7a isexpressed early during limb bud developmentand maintain Sonic hedgehog (Shh)expression (Shum et at., 2003; Tickle, 2003)which is required for regulating anterior-posterior limb patterning, and is necessary to maintainFgf4 expression in the AER(Laufer et al., 1994; Niswanderet al., 1994). Further adding to the complexity ofthissignaling network, the ventral ectoderm inducesthe expression of the transcriptionfactor Engrailed (EN-I), which playsa role in dorsal-ventral patterning (Fig. 1.3). Thehomeobox (Hox) transcription factorsencoded by the HoxA and HoxD gene clustersare also critical for the early eventsof limb patterning in the undifferentiatedmesenchyme, and are required for the expressionof Fgf8 and Shh (Kmita et al.,2005; Tarchini and Duboule, 2006;Tarchini et at., 2006). Taken together,the limb ispatterned through the coordinated actionof multiple signals, including the FGFs,WNTs, Hedgehogs (HH5), across allthree limb axes.As limb patterning and outgrowth progress,multiple bone morphogeneticproteins (BMP5), as their namesuggests, begin to influence bone morphogenesisby9initiating chondrogenesis. BMPs act at multiple stages to regulate the skeletogenicprogram. This process is highly dependent upon the temporal and spatial expressionof BMP receptors and BMP antagonists, such as NOGGIN and CHORDIN(Niswander, 2002; Pizette and Niswander, 2000; Tickle, 2003). In vitro and in vivostudies have also shown that BMP signaling is required both for the formation ofprecartilaginous condensations and for the differentiation of precursors intochondrocytes (Yoon and Lyons, 2004; Yoon et al., 2005). During limb development,FGFs provide a proliferative signal, and delay cellular differentiation, whereas BMPsplay a role in mesenchymal cell specification/determination and promotedifferentiation (Yoon et aL, 2006). In general, it is thought that FGF signaling andBMP signaling function antagonistically to each other during the processes of limboutgrowth (Minina et al., 2002; Niswander and Martin, 1993a; Yoon et al., 2006).1.2.6 FGFs and Their Associated ReceptorsFGFs are involved in the earliest stages of limb development and in theformation of skeletal elements within the limb. Specifically, these factors signal fromthe AER to the underlying mesenchyme to promote cellular proliferation and survival.This mesenchymal region has been termed the progress zone (PZ), consisting ofundifferentiated mesenchymal cells subjacent to the AER. FGF5 within the AERsignal to the underlying mesenchyme and maintain a population of progenitor cellsrequired for the outward growth of the limb and the formation of skeletal elements (Liet al., 2005; Shum et al., 2003).The expression patterns and function of FGFs in early limb development havebeen identified as key components of AER signaling to the underlying mesenchyme10and have been reviewed extensively (Mariani et al., 2008; Martin, 1998; Niswanderet al., 1993; Sun et al., 2000; Sun et al., 2002b). Briefly, FGF ligands signal throughbinding and activation of one of four high-affinity FGF receptors (FGFR5) whichrepresent a subclass of the tyrosine kinase receptor family (Itoh and Ornitz, 2004;Thisse and Thisse, 2005) (Fig. 1.4). FGF receptors contain, in their full-length form,a hydrophobic leader sequence, three immunoglobulin-like (IgI, II, and Ill) domains,an acidic box, a transmembrane domain, and a divided tyrosine kinase domain (Li etal., 2005). As a result of alternative splicing, numerous functional isoforms can alsobe generated. Individual FGFR proteins bind multiple FGFs but also display aunique pattern of affinities for the different ligands (De Moerlooze and Dickson,1997). However, as ligand binding is also greatly influenced by the distribution ofheparan sulfate proteoglycans (HSPGs) at the cell surface and in the extracellularmatrix (ECM), it is unknown to what extent these in vitro assays reflect the ligandbinding specificity of different FGFR proteins in vivo. Once released from cells, FGFsbind avidly to HSPGs such as the syndecans, glypican, and perlecan on the cellsurface and in the extracellular matrix (ECM), which is thought to limit their diffusionfrom the source of production (Martin, 1998).FGF5 elicit their effects on cells by forming a complex that includes the ligand,a high affinity tyrosine kinase receptor, and a heparan-sulfate proteoglycan (Ornitzand Itoh, 2001; Thisse and Thisse, 2005; Yu and Ornitz, 2008a). Specifically, uponthe binding of the Iigand, FGF receptors dimerize and autophosphorylate severalintracellular tyrosine residues that serve as docking sites for Src Homology 2 domain(SH2) containing polypeptides such as phospholipase C (Marie et al., 2005; Thisse11and Thisse, 2005). The phosphotyrosinebinding domain (PTB) of the adaptorprotein FGFR substrate 2 (FRS2)binds to the FGFR in a phosphotyrosineindependent manner and is tyrosine-phosphorylatedupon activation of FGFRs(Kouhara et al., 1997). Once FRS2 is tyrosine-phosphorylated,it binds the SH2domain containing adaptor protein GRB2as well as the protein-tyrosinephosphatase SHP2 (Kouhara et al., 1997; Mohammadiet al., 1991; Xu et al., 1998).GRB2 then recruits the guanine nucleotide-releasingfactor SOS to the plasmamembrane where it subsequently leads to theactivation of the RAS small guanosinetriphosphatases (GTPases). RASsignaling activates the extracellular signal-regulated kinase (ERK) mitogen activatedprotein kinase (MAPK) pathway (Corsonet al., 2003; Roberts and Der, 2007; Xuet al., 1998).In the developing limb bud, the epithelialsplice form of FGF receptor 2(Fgfr2b) is expressed in the ectoderm,while the mesenchymal splice forms of FGFreceptor 1 (Fgfrlc) and FGF receptor 2 (Fgfr2c)are expressed in the nascent limbmesenchyme (Niswander and Martin,1993a; Orr-Urtreger et al., 1991). An earlystep in the initiation of limb bud formation involves,as previously mentioned,signaling from mesenchymally expressed FGFIOto FGFR2B which results in theformation of the apical ectodermal ridge. FGF8is subsequently expressed in theapical ectodermal ridge and is thoughtto signal back to FGFRIC and FGFR2C inlimb mesoderm (Naski et al., 1996; Ornitzand Marie, 2002a; Siliang Zhang, 2006).This pattern of reciprocal signalingis one of several essential events requiredforoutgrowth and patterning of the limb.12Mesenchymal condensation is the first morphologicevent leading to boneformation (Hall, 1987; Hall and Miyake, 1992).Fgfr2 expression is first observed inthe mesenchyme as mesenchymal cellsbegin to coalesce in the central core of thedeveloping limb (Shum et al., 2003). Atthe condensation stage of limb development,Fgfrl expression persists in limb mesenchymeand in mesenchymal cells at theperiphery of the condensation, whereas Fgfr2expression can be observed in themorphologically distinct mesenchymal condensationsbut not in the surroundingloose connective tissue (Ornitz andMarie, 2002a). These expression profiles appearto be evolutionarily conserved andhave been observed in chicken, mouse, andhuman limb development (Delezoideet al., 1998; Orr-Urtreger et al., 1991; Petersetal., 1992; Szebenyi et al., 1995).At the initial onset of chondrogenesis Fgfr3expression is first observed in the chondrocyteswithin the center of the condensedmesenchyme (Peters et al., 1993). Importantly,although the FGFRs are dynamicallyexpressed in the developing limb, the mechanismsregulating their expression are ill-defined.FGF activity in the early limb is required for proliferationof the distal-tip cellsand prevention of apoptosis (Dudleyet al., 2002; Sun et al., 2002b). At later stages,FGF signaling plays an integral role in chondrocyteproliferation and differentiation(Ornitz and Marie, 2002a). Consistent withtheir expression, FGFs have been foundto play multiple roles in limb and skeletal developmentwhile aberrant FGF signalingleads to a spectrum of limb malformations includingsyndactyly, truncations anddwarfism (Ornitz and Marie, 2002a).These studies have suggested multiple rolesfor13FGF signaling in accordance with the differential expression of Fgfrs in thechondrogenic program.1.2.7 Mutations in FGFs and FGFRsTargeted deletion of members of the FGF family and FGFRs in the mousereveal that FGF signaling is essential for cell proliferation and survival in thepreimplantation mouse embryo (Feldman et al., 1995), as well as for cell migrationduring gastrulation (Itoh and Ornitz, 2004; Zhang et al., 2004). Conditional knockoutsenabled the analysis of later stages of embryogenesis and determined that FGFsignaling plays a role in development of the limb buds, brain, and lung in addition tonumerous other tissues and organs (Itoh and Ornitz, 2004).The requirement for proper FGF signaling in skeletal development is evidentin congenital anomalies that arise from mutations of the receptors affecting theextracellular, transmembrane, or intracellular domains. A severe condition known asAchondrodysplasia (ACH), characterized by reduced growth of the long bones withproximal segments more greatly affected than distal, arises from autosomaldominant mutations of FGFR3. Typically, the mutations result in the substitution ofamino acids (glycine to arginine) in the FGFR3 transmembrane domain, making thereceptor constitutively active (Naski et al., 1996).Conditional knockout studies to determine the function of FGFs in the AERhave revealed that only FGF8 removal significantly impacts limb development(Lewandoski et al., 2000; Moon and Capecchi, 2000) resulting in an absence ofsome skeletal elements. Although FGF4 mutants have normal limbs, compoundmutations of both FGF4 and FGF8 result in mesenchymal cell death in the limb bud14and a failure of the limb structures to form (Boulet et aL, 2004). In FGF8 mutantsalone, FGF4 is believed to partially compensate for the lack of FGF8 and isupregulated in the AER (Lewandoski et aL, 2000; Moon and Capecchi, 2000). Thecurrent view of the AER FGFs is that they exhibit overlapping functions, and it is thetotal amount of FGFs produced from the AER during limb development that isessential for skeletogenesis (Delgado et al., 2008). However some FGFs, such asFGF8, figure more prominently in this process.1.2.8 Regulation of Cell Proliferation by the CDKN2IINK FamilyIn the developing limb, rapid cellular proliferation results in the outgrowth ofthe future appendicular structures. Proliferation is tightly regulated which iscoordinated through the actions of protein effectors composed of cyclins and cyclindependent kinases (CDKs). Of the many types of kinases, the progression of cellsthrough the G1-S transition requires the activity of cyclin D-CDK4/6 and cyclin E/ACDK2 complexes (Niswander et al., 1994; Reynisdottir et al., 1995a). The activity ofthese kinases is differentially regulated by the cyclin dependent kinase inhibitors(CDIs), also known as the CDKN2 family (or INK4 family), and the CIP/KIP familiesof cell cycle inhibitors. The CIP/KIP membersp2lciP2l27KiPland p572associatewith and inactivate cyclin E-CDK2 and cyclin A-CDK2 complexes, whereas theirassociation with cyclin D-CDK4 or cyclin D-CDK6 have been shown to bestimulatory (Niswander et al., 1994; Reynisdottir et al., 1995a; Wolfraim et al., 2004).By competitive interaction, binding of CIP/KIP proteins with cyclin D-CDK4/6 kinasesprevents their interaction with cyclin E/A-CDK2. This facilitates the role of thesekinases in completing the G1 phase and their role in the DNA synthesis phase of the15cell cycle. The members of the CDKN2 family havea different mode of action.CDKN2A, CDKN2B, CDKN2C, and CDKN2D inhibit the catalyticbinding betweenCDK4 and CDK6 kinases and the regulatorydomains on the cyclin D subunits(Niswander et al., 1994; Queue et al., 1995; Reynisdottiret al., 1995a; Takeuchi etal., 1995).The role of the CDKN2 proteins in the regulationof cellular proliferation hasbeen well studied in several cell types. Collectively the studies showan increase inCDKN2 proteins during the G1 phase, resulting ina decrease in cell proliferation,followed by the differentiation of some cell lineages. Initially, interest inthe CDKN2family resulted from genetic linkage studies which showed thatan inheritedpredisposition to melanoma insome families could be traced to a putative tumorsuppressor on chromosome 9, specifically 9p21(Quelle et al., 1995; Takeuchi et al.,1995). Through positional cloning, the Cdkn2a(Ink4a) gene was identified along witha closely related Cdkn2b (Ink4b) gene. CDKN2A and CDKN2B havesimilar modesof action and serve as tumor suppressors byacting as inhibitors of the CDKs thatcontrol cellular proliferation by regulating theprogression of the cell cycle from theG1 phase. Though the role of these genes is pivotalin determining the state of thecell, it has been difficult to detect Cdkn2a or Cdkn2b expressionduringembryogenesis (Gil and Peters, 2006). Giventhe nature of the developing limb, inwhich there is an acute phase of regulated growth,which is regulated at least in partby FGFs, we sought to characterize the function of FGFs in regulatingmesenchymalcell proliferation.161.2.9 Mitogen Activated Protein Kinase (MAPK) SignalingMAPK cascades are essential signaling pathways involved in the regulation ofcell proliferation, survival and differentiation (Corson et al., 2003; Roberts and Der,2007), that function downstream of cell surface receptors such as the FGFRs. Thesesignaling networks are comprised of three protein kinases that act as a signalingrelay controlled in large part by protein phosphorylation. These kinases are: MAPKkinase kinase (MAPKKK), MAPK kinase (MAPKK), and a MAPK (Corson et al.,2003; Johnson and Lapadat, 2002). Through the action of RAS, its downstreameffector RAF serine/threonine kinase is activated and plays a key role in regulatingMAPK signaling. In an evolutionarily conserved pathway, RAF activates theMAPKJERK kinase (MEK)1/2 dual specificity protein kinases, which then activateERK1I2 (Roberts and Der, 2007). Activated ERKs then translocate to the nucleus,where they phosphorylate and regulate various transcription factors leading tochanges in gene expression.The MEK/ERK pathway is one of the major downstream pathways of FGFreceptors (Corson et al., 2003; Murakami et al., 2000). Consistent with this, in theembryonic limb, the FGFRs and phospho-ERK show colocalized expression, andinhibition of FGFR results in the a loss of phospho-ERK signaling (Corson et al.,2003). Further, overexpression of constitutively active phospho-MEKI in limbmesenchyme, inhibits chondrogenic differentiation, whereas pharmacologicalinhibition of phospho-MEKI promotes chondrogenesis (Bobick and Kulyk, 2004; Ohet al., 2000). These data are consistent with the assertion that the role of FGFs is to17promote appendicular growth by negatively regulating chondrogenesis via MEK/ERKsignaling.1.2.10 Role of Nuclear Factor Kappa B in Limb DevelopmentThe AER FGFs stimulate mesenchymal proliferation to permit appositionalgrowth of the limb, and also promote mesenchymal cell survival, requiringtheactivation of specific transcription factors. Originally identified as a nuclear factorthatbinds the kappa (K) light chain enhancer in B-cells (Sen and Baltimore, 1986),NF-KBis now recognized as an important transcription factor involvedin many cellularprocesses including cell survival/death. The generalized name -NF-KBcan representthe NE-KB and Rel protein superfamily, subfamily (p100, p105, and Relish), orthemajor heterodimer in most cells, p50-ReIA (Gilmore, 2006). Duringlimbmorphogenesis, it has been established that NE-KB is vital for the formationof theapical ectodermal ridge, and its transcription is regulated in part by AERderivedsignals such as the FGEs (Bushdid et al., 1998). As mentioned earlier, the AERisrequired to maintain the proliferative, undifferentiated state of the leadingedge of thedeveloping limb, whereas the underlying mesenchyme (the progresszone, PZ), isrequired to maintain the AER. Studies performed on chick embryos haveshown thatNE-KB transcriptionally regulates the expression of factors necessaryfor thepatterning of the limb, as well as limb mesenchymal cell survival (Bushdidet al.,1998; Kanegae et al., 1998). The establishment of a feedback loop betweensignalsfrom the AER and the signals from the PZ is required for limb outgrowth.Disruptionof the NE-KB signaling pathway results in the loss of proper geneactivationresulting in limb truncation (Bushdid et al., 1998; Kanegae et al., 1998; Perkinset al.,181997), highlighting the importance of NF-KB as a component of the networkoperating in epithelial-mesenchymal cell communication.1.2.11 Regulation of NF-icBThe complexity of NF-KB regulation is highlighted by the observations thatmore than 200 physiological stimuli have been shown to activate these transcriptionfactors (Tergaonkar, 2006). NF-KB is initially located in the cytoplasm typically as aheterodimeric protein consisting of 50 and 65 kilodalton subunits (p50 and RelA) ofthe Re! family. NF-KB is often found in an inactive complex associated with inhibitory1KB proteins (Fig. 1.5). Under the appropriate stimulus orstimuli, 1KB is releasedfrom NF-KB, subsequently NE-KB translocates to the nucleus to initiate transcriptionof a variety of genes including those involved in the cell cycle/survival (Fig. 1.5).Spatiotemporal expression of two NE-KB members - c-Re! and Re/A, in the distalmesenchyme implies a role for NE-KB signaling in the regulation of limbdevelopment. In support of this observation, c-Re! expression is diminished followingAER ablation, and is subsequently rescued by the addition of FGF4 (Bushdidet al.,1998). Experiments involving 1KB mutants decreased the amount of nucleartranslocated NE-KB available for DNA binding, and have shown the decreasedexpression of critical genes in the PZ. Experiments with these mutants have alsoindirectly shown the decreased expression of genes in the AER and zone ofpolarizing activity (ZPA) (Kanegae et al., 1998). Overall, the intrinsic functionof NFKB during limb development requires the proper functioning of all components of thepathway.19Due to the numerous pathways NE-KB can influence and its implicatedrole innumerous biological processes and diseases, regulationof this diverse transcriptionfactor has been studied quite extensively. The 1KB sequesteringof NE-KB in thecytoplasm represents a molecular mechanism of inhibiting NE-KBsignaling.Conversely, activators of NE-KB facilitate its translocationto the nucleus to promotetranscription of target genes. The receptor interacting proteins (RIPs)are importantregulators of cell proliferation and differentiation (Adamset al., 2007). One suchactivator of NE-KB is receptor-interacting serine-threonine kinase 4- RIPK4 (alsoknown as DIK, RIP4 and PKK). Overexpression studies have shownthat RIPK4increases NF-KB activity, whereas kinase inactive versions of RIPK4have adominant negative effect on NE-KB induction (Meylanet al., 2002b). Experimentsinvolving NE-KB loss-of-function have suggestedthat its role is to mediatemesenchymal maintenance of the AER as the mutant phenotype isa consequenceof improper epithelium-mesenchyme communication resulting insevere limbtruncation and defects (Bushdid et al., 1998). Interestingly, animals deficientin Ripk4have shortened limbs with syndactyly —a condition in whichthe fusion of two or moredigits occurs involving the soft tissue, bones, or both (Holland etal., 2002).1.3 Transcriptional Regulation of Chondrogenesis1.3.1 Importance of SOX9Appendicular skeletal development initiates shortly after the outgrowthof thelimb bud with the formation of a histologically identifiablemesenchymalcondensation, marked by the restricted expression of the nuclear transcriptionfactorSox9 (Fig. 1.1 B). This transcription factor is required for the expression ofthe type II20collagen gene (CoI2al) (Kosher et al., 1986; Nah et al., 1988; Shum et al., 2003;Wright et al., 1995) and is essential for chondrogenesis. Sox9 belongs to the SRY(sex-determining region on the Y chromosome) family and contains an HMG (highmobility group) box DNA binding domain (Lefebvre et al., 1997; Ng et al., 1997;Wright et al., 1995). Mutations of Sox9 have shown it to be a master regulator ofchondrogenesis (Healy et al., 1996; Wright et al., 1995). Sox9’ mouse embryos dieperinatally and exhibit severe hypoplasia of the cartilage that is associated withlower levels of cartilage matrix genes (Bi et al., 2001). Similarly, Sox9haploinsufficiency in humans causes campomelic dysplasia, characterized by severeskeletal dysmorphology (Wunderle et al., 1998). Further analyseswith conditionalinactivation of Sox9 at varying times during mouse limb development have revealedthat it is required for mesenchymal condensation and subsequent chondroblastdifferentiation (Akiyama et al., 2002; Akiyama et al., 2004).Two other members of the SOX family of transcription factors —SOX5 andSOX6 function cooperatively with SOX9 to regulate CoI2al expression (Lefebvre etal., 2001; Smits et al., 2001). It is proposed that the ability of SOX5 andSOX6 toform homo and hetero dimers facilitates their binding to 2 HMG domainssimultaneously (Ikeda et al., 2005). SOX5/6/9 are commonly referredto as the SOXtrio, as they are co-expressed in chondroprogenitors and chondrocytes (Akiyamaetal., 2002; Lefebvre et al., 1998; Smits et al., 2001). SOX9 functionsat multiple pointsduring chondrogenesis and is involved in the commitmentof undifferentiatedmesenchymal cells into the chondrogenic lineage, and is required for mesenchymalcondensation and the expression of Sox5 and Sox6. Embryoniclimbs in which Sox921was inactivated prior to mesenchymalcondensation showed no expression of Sox5and Sox6, whereas inactivation of Sox9 afterthe condensation stage resulted inembryos with skeletal defects characteristicof Sox516 double null mutants (Akiyamaet al., 2002). Thus, S0X516 appear tobe required to direct overt chondroblastdifferentiation, and the accompanying increasein expression of genes encodingextracellular matrix proteins (Ikedaet al., 2005). Proper SOX function duringchondrogenesis is absolutely required for normallimb development.1.4 Methodology and Strategies1.4.1 Primary Cell Culture Model for StudyingChondrogenesisThe basic parameters to study chondrogenesisin vitro were developed over30 years ago by Ahrens and colleagues (Ahrenset al., 1977). The chondrogenicprogram can be effectively modeled in vitrousing high-density micromass culturesofprimary cells isolated from the vertebratelimb bud (murine embryonic age day 11.5).Under these conditions, prechondrogeniccells begin to condense within —24 hrs andform cartilage nodules within —72 hrs. Further,these cells exhibit robust phenotypicresponses to signaling moleculessuch as BMPs, FGFs and WNTs. Althoughchondrogenesis can be studied using establishedchondrogenic cell lines, thesecultures require —10 days before the initialcartilage nodules become apparent,unless otherwise treated with chondrogenicfactors (Denker et al., 1999). An obviousadvantage of the primary cell micromasstechnique is the reduced time-framerequired to elaborate the chondrogenic program,without the additional concerns oflong-term cultures (eg. contamination).Additionally, primary cultures allow the ability22to study intrinsic genetic and cell cycle events in the developing mesenchyme thatmay be absent or misrepresented in established cells lines (ie. cell cycle inhibitors).Although the traditional micromass technique has withstood the test of time,there are several shortcomings which include the absolute requirement of a highdensity of cells per micromass. This poses a particular problem in experiments inwhich cells are harvested from the distal mesenchyme yielding few cells. These cellsprovide a more homogeneous population, suitable for genetic analysis, and allow thestudy of the molecular events subjacent to the AER. Routinely, per dissected litter ofmurine E11.5 embryos, -100 p.L of distal limb mesenchymal cells are harvested (ata final density of 2.0 x i07 cells/mi), and are plated as 10 jiL micromass cultures.This limited abundance of cells curtails large-scale experiments in the 24-wellformat, as the requirement of experimental controls/plate consumes one-sixth of theavailable cells since the majority of our experiments are performed in quadruplicate.The traditional 24-well based approach has severely hampered our throughput. Tocircumvent such limitations, and further delineate the chondrogenic program a novel384-well based culture method has been developed. This more efficient methodreplaces the traditional 24-well based assays and allows the amount of primary cellsand reagents consumed per experiment to be greatly reduced. The 384-well systemprovides a reliable means of assessing the role of factors on chondrogenesis whileincreasing the scale of throughput 16-fold.At the onset of the research for this thesis, there was a necessity to developtechnology that would enable high throughput screening of single genes and multiplegene crosses in PLM cultures. This need was brought about by the abundance of23transcriptional data obtained through bioinformatic data mining of previouslygenerated microarrays. Logistically, it became apparent that transfections on a largescale would require the development of an enhanced transfection strategy toperform functional assays.1.4.2 Assessing the Status of Chondroblast DifferentiationThe effects of signaling molecules (BMP5, FGFs, etc.) on SOX5/619 activity,and consequently CoI2al expression, have been reported by our laboratory andothers (Hoffman et al., 2006; Murakami et al., 2000; Semba et al., 2000; Weston etal., 2000; Zehentner et al., 1999; Zeng et al., 2002). To provide a read-out on thestatus of chondroblast differentiation, we have employed a SOX5/6/9-responsivereporter construct consisting of a 48 bp enhancer element derived from the firstintron of CoI2al (Lefebvre et al., 1997; Ng et al., 1997; Zhao et aL, 1997). Thiselement has been reiterated 4 times, and placed upstream of a minimal CoI2alpromoter and luciferase gene (Figure 1.IC). This 48 bp region is necessary forSOX5/6 and 9 binding, and thereby transcription of CoI2al (Lefebvre et al., 1997).Previously, using this reporter, we have assessed the action of numerous factorswhich regulate chondrogenesis such as BMPs and retinoids (Hoffman et al., 2006;Weston et al., 2002). These effects have been verified by alcian blue staining(staining glycosaminoglycans within cartilage nodules), which shows intensecartilage nodule staining in response to BMP4 treatment. Studies using this reporterin combination with other genes in co-transfection based assays have providedimportant information on the molecular networks operating in chondrogenesis.241.4.3 Review of Transfection TechnologyThe ability to introduce nucleic acids into cells has enabled the advancementof our knowledge of genetic regulation and protein function within eukaryoticcells,tissues, and organisms. The transfer of recombinant genes intoa variety ofeukaryotic cultured cells, commonly known as transfection, is an extensivelyusedapproach in assessing gene function. Under specific conditions, eukaryoticcells cantake up exogenous DNA, and a portion of this DNA becomes translocatedto thenucleus. These events have been exploited to obtain both transientand stableexpression of various genes. Factors that influence the entry of DNAinto the cellinclude the barrier of the cell membrane to the chargeof the DNA, as well as its sizeand conformation. Once inside the cell, the intact DNA must avoidenzymaticcleavage, in order to enter the nucleus where it can be transcribed.These factorshamper transfection efficiency. To circumvent these concerns, a widerange ofmethods have been developed to facilitate transfection, with the aim ofefficientlydelivering DNA into target cells and protecting it from nucleasedegradation.The use of viruses (retroviruses or adenoviruses)is the most efficient for celltransduction, but presents several disadvantages relatedto immunogenicity, DNAsize restriction, and large-scale production constraints (Chen andOkayama, 1988;Feigner et al., 1987; FeIgner and Ringold, 1989; Manninoand Gould-Fogerite,1988). Synthetic means of delivering DNA to the nucleus have beendeveloped toovercome these limitations and some of the primaryadvantages of this approach arethat the composition of these synthetic reagents is known, they can formcomplexeswith DNA that are either slightly toxic or completely non-toxic to cells, theyhave few25size limitations, and they areeasy to prepare in large quantities (Gaoet al., 2007).Synthetic transfection reagents inducecellular uptake of DNA by forming complexeswith nucleic acids resulting ina positively charged complex. These complexesbindto the negatively charged cell membranethrough ionic interaction and enter the cellthrough endocytosis, without significantcytotoxicity. Cationic lipids and polymersareprominent synthetic reagents. Thecombination of negatively charged DNAwithpositively charged lipids produces condensedparticles known as lipoplexes (Feigneret at., 1987; FeIgner and Ringold, 1989;Gao et at., 2007; Pedroso de Lima etal.,2001). Using positively chargedpolymers to compact DNA (calledpolyplexes),FeIgner et at. (1987) showedan ability to transfect cells with nucleic acids.Theoriginal technology has sincebeen modified and the number of synthetic reagents,along with the variety of commerciallyavailable forms, have greatly increased.Asthe demand for rapid, high efficiency transfectionshas increased, a number of otherproducts (non-liposomal lipids, synthetic polymers,etc.) have been developed thatmediate the transport ofgenes into cells.A problem associated with the majorityof non-viral gene-delivery agentsistheir relatively low transfection efficiency(Nikcevic et al., 2003) whichcan beinfluenced by a number of factors.Parameters that are taken into considerationwhen optimizing transfection efficiencyinclude cell type or cell lineto be used,culture conditions, and transfection reagent (Arnoldet al., 2004). These concernsare based on observations that certaincell types are intrinsically easierto transfectthan others. Although the exact reasonfor these differences is unknown, importantfactors influencing the successor failure of transfection include the qualityof the26transferred DNA as well as its size, configuration, quantity, and the mitotic state ofthe cells to be transfected. Traditionally, primary cells have been difficult to transfectand various strategies have been devised to overcome these limitations.1.4.4 Design and Application of New TechnologyIn order to study the regulation of genes involved in chondrogenicdifferentiation, it was essential for us to transiently introduce recombinant plasmidDNA into primary limb mesenchymal (PLM) cells isolated from the mouse atembryonic age 11.5 with high efficiency. Transient transfections are easy to prepare,and as a consequence facilitate high-throughput approaches. Earlier work from ourlab involved transfections using Fugene6TM (Roche), a cationic lipid based reagent,which yielded sufficient transfection efficiency. However, these transfectionsrequired preparation in the absence of serum for maximal activity. It is presumedthat a polyvalent negatively charged serum component inhibits the formation oftransfection complexes. Ideally, transfection in the presence of serum yields bettercell growth, function and viability, and reduced cytotoxic effects associated withtransfection reagents (Arnold et aL, 2004). These inherent issues of transfection,coupled with the value of the primary cells to be transfected prompted us to find abetter solution and thereby circumvent some of the present limitations oftransfection. To this end, we began experimenting with EffecteneTM (Qiagen) whichis also a cationic lipid based transfection reagent. UsingEffecteneTM,transfectionscan be prepared in the presence of serum, with the additional benefit of being ableto optimize several components of the transfection reagent, as they are notpremixed by the manufacturer. Chapter 3 will describe the optimized transfection27parameters forEffecteneTMwith PLM cell cultures. We describe a means ofincreasing the transfection efficiency of this transfection reagentby usingdisaccharides (Fig. I .6B).1.4.5 Stabilization of Transfection ComplexesPreservation of biological materials such as proteins, enzymes, membranesand mammalian cells has been a source of great interest over thepast 2 decades(Beattie et al., 1997; Crowe et al., 1998; Leslie et al.,1995; Powers et al., 1986;Sowemimo-Coker et al., 1993). In nature, stabilization inresponse to stresses suchas desiccation or freezing is a common practice in many plants andanimals. Theseorganisms accumulate large amounts of sugars in response to physiologicalstress(Sun et al., 2002a). In particular, disaccharides such as sucrose and trehaloseplay akey role in the desiccation and preservation process.Two different hypotheses have been postulatedby which sugars protectbiological materials in the desiccated state, namely the glass formationhypothesis,and the water-replacement hypothesis. In the glass formation hypothesis,theformation of stable glasses reduces molecularmobility and enables long-termstorage (Leslie et al., 1994; Sun et al., 1996). The water-replacementhypothesissuggests that replacement of water molecules by sugar molecules andthe directinteraction of sugars with polar residues (throughhydrogen bonding during thedesiccation process) allows biological structures to maintain their conformationalstructure in the dried state (Crowe et al., 1996; Sowemimo-Cokeret al., 1993; Sun etal., 1996) (Fig. 1.6A). Given that disaccharides can impart stabilityto biological28components, such as lipids, we evaluated their ability to stabilize lipid-based DNAtra nsfections.1.4.6 Enhancement of Experimental ThroughputThe development of the 384-well based PLM transfection strategy hasprovided the means for enhancing our experimental throughput as the DNAtransfection mixtures can be prepared well in advance of their use, therebyeliminating the constraints of time imposed by conventional transfection strategies.Additionally, by adapting our 24-well based experiments to the new 384- welltechnology we have increased the scale of our experiments by 16-fold, in a highlyreproducible fashion. We have demonstrated the merits of this technology bycomparative studies of a small-scale gene-screen between conventional 24 wellbased experiments and 384 well based experiments using the aforementionedSOX5/6/9-luciferase reporter gene as a readout.1.4.7 Small-Molecule ScreeningA relatively new and powerful approach to defining the mechanismsunderlying biological processes is the use of small-molecule chemical compoundlibraries. Small-molecules are defined as carbon-based compounds with a molecularweight under 500 (Kawasumi and Nghiem, 2007). Using a phenotype-basedapproach for screening small-molecules, compounds that produce a phenotype ofinterest can be identified and subsequently the target of the compound can provideinformation on the underlying biological process(es).As an extension of the transfection technology, we performed a smallmolecule screen of a library of compounds with known biological activity. The goal of29this endeavor was to create a technologyto enable IdentifIcatIon of both pro- andanti-chondrogenlc factors to aid In our understandingof the chondrogenlc program.The data gathered from thIs study has provenbeneficial In further characterizing thebasic mechanisms regulating cartIlageformatIon. We have also identified numerousnew pro-chondrogenic compoundsthat will hopefully lead to the developmentof newtherapeutics to treat the debilitating conditionsassocIated with dIseased anddamaged cartilage.1.5 OvervIew of ThesisThe progression of limb development Involvesan Interplay of signalIngnetworks that when InvestIgated,provide as many answers as theydo newquestions. With an observed phenotypeand worldng hypothesis, we show InChapter 2 a systematic approach to IdentIfyingand characterizing genes of InterestIn the developing limb. Consequently,we also clarify the role of FGF4Inmesenchymal survival and providenew methodology to examIne therole ofdevelopmental factors In Isolationor limited combination.Chapter 3 provIdes a technologIcalapproach aImed at Increasing throughputto enable large-scale analysisof gene function in chondrogenesis. We provideanew technology to Increasethroughput and to combIne chemical bIology withgene-based approaches. To thIs end,we used this technology to Implementa chemIcalblology screen. The InformatIonfrom thIs screen combIned with edstlngmicroarraydata sets has provided new insIghts Intothe potentIal role of potassium channels Inchondrogenesls. The overall goalhas been to further define the chondrogenlc30program using an integrated approach of hypothesis-based and discovery-drivenresearch.1.57 Topics AddressedChapter 2: FGF Signals in the Embryonic Limb Regulate Cell ProliferationandSurvival Through Cdkn2b and the NF-icB Signaling PathwayResearch outlined herein is directed towards defining the molecularnetworksregulated by fibroblast growth factor 4 in the limb mesenchyme.Objectives:i To investigate the mechanisms underlying FGF4action in cell proliferation andsurvival.ii To determine the role of FGF4 in chondrogenesis.Chapter 3: Chemical Genetics Strategy Leads to the Identificationof NovelPathways Important in ChondrogenesisObjectives:i To develop and implement a chemical biology screen foridentification ofchondrogenic modulators.ii To gain new insights into molecular programsthat regulate chondrogenesis.311.6 FiguresFigure 1.1. Overview of Murine Limb Skeletal Development. A, Duringendochondral ossification a cartilage template is established and subsequentlyremodeled to bone. This process is initiated through the action of multiple signals,culminating in the generation of cartilaginous anlagens that prefigure the skeleton.B, Type I collagen (Coil) expression is observed throughout the early limb and inprecartilaginous condensations. SOX9 an HMG box-containing transcription factor,plays a central role in regulating chondrocyle commitment and differentiation. Type IIcollagen (Coi2al) is abundantly expressed in chondroblasts and its initial expressionis dependent upon SOX5/619. Analysis of the CoI2al promoter has revealed a 48bp sequence essential for S0X51619 binding. C, Use of reporter genes (luciterase orfluorescent protein) containing multiple copies of the 48 bp sequence enablemonitoring of S0X51619 activity in PM cultures which provides a reliable readout onthe status of chondroblast differentiation and cartilage formation.32AS)day 10.5day 11.5)iiiieru\t%itb2.5- -p——•d(I.I 14.5B condensation stagecommitment-. I--mesenchymal cell chondroprogenitorchondroblastCoilSox9Coi2C___ILLucfferas. EYFP33Figure 1.2. Three Perspectives on Proximal-to-Distal Patterning in DevelopingLimb Buds. A, The “progress zone” model, predicts that the fate of proximal(prospective upper arm; blue) elements is specified prior to the fate of more distalelements, as the limb grows. Changes in cell fate occur in the progress zone,adjacent to the AER. In the “prespecification” model, proximal-to-distal fates arebelieved to be prespecified early in development and the observed temporal eventsin skeletal development result from the selective expansion of these prespecifieddomains, along the acquisition of definitive cell fate. Following removal of the AERthe “progress zone” model predicts that the specification clock is arrested and (inthis case) distal specification never occurs. In the “prespecification” model removalof the AER prevents the expansion of prespecified distal domains because of celldeath. In both cases, the same results are expected. (adapted from Duboule, 2002)B, The “two-signal dynamic specification” model for limb proximal-distal patterningpredicts that proximal domains of the embryonic limb containing proximal signals(blue) are specified by opposing distal signals (red) released from the AER. Thismodel predicts that in AER mutants, or following surgical removal of the AER, thedistal signal is reduced in proportion to the proximal signal. The proximal signal isnot restricted and interacts with cells that in wild-type limbs would only receive distalsignals. Due to decreased AER signaling, the limbs are smaller and distal structuresare missing as the result of cell death in the distal region. (adapted from Mariani etal., 2008)V 341 •..••••••....APreSpeCiatbohlModelProgressZoneBo-signalDynamic SpecificationModefDistalAdaptedfrom Duboule, 2002proximal•••..•••••.•••..•.zzAdapted from Mariani et al., 2008•• •..••••••35Figure 1.3. The Developmental Progression of Undifferentiated LimbMesenchyme to Cartilaginous Skeletal Precursors in the Limb. A, Limboutgrowth begins as a protrusion of undifferentiated mesenchyme covered byectoderm expressing FGF1O and WNT3 (purple) (embryonic age (E) -9.O). Thedistal edge of the ectoderm thickens to form the apical ectodermal ridge (AER)comprised of pseudostratified columnar epithelium which initiates the expression ofFGF8, BMP2, BMP4, and MSX2 (blue). B, During early appendicular growth, themesenchymal core expresses numerous Hox genes (gray). The cells in the posteriordomain of the proximal region of the ZPA secrete SHH and BMP4 (pink), while theanterior cells of the proximal region secrete only BMP4 (brown). The ectodermsecretes factors such as FGF4 in the posterior of the AER, WNT7A in the dorsalregions, and EN1 in the ventral segments (—E1O.O). C, The mesenchyme condensesand the cells (red) secrete a variety of signaling factors such as GDF5, BMP2,BMP4, BMP7, and have elevated levels of SOX9 (-E1 1.5). D, Cellular differentiationoccurs in a proximal to distal sequence such that the humerus differentiates prior tothe radius and ulna, followed by the digits. Differentiating chondrocytes (green)secrete NOGGIN, and proliferating chondrocytes (blue) initiate IHH expression. Theinterdigital mesenchyme (orange) undergoes apoptosis, initiated by a combination ofsignals such as BMP2, BMP4, and MSX2. The cartilaginous templates aresegmented into individual skeletal elements via joint formation (yellow) regulated byfactors such as GDF5, WNTI4, and CHORDIN (—E14.O). These elements aresubsequently mineralized to form skeletal structures. (adapted from Shum et al.,2003)36DA B C(-E9.O) (—E1O.O) (-€11.5)•Intermediate Mesoderm: FGFIO,WNT3S Apical Ectodermal Ridge:FGF8, BMP2, BMP4, MSX2, WNT3Ventral Ectoderm: EN-IDorsal Ectoderm :WNT7APosterior Ridge Ectoderm:FGF4Entire Limb Bud: Hox genes• Zone of Polarizing Activity:SHH, BMP4• Anterior Mesoderm: BMP4•Cell Condensation andPerichondrium:GDF5, BMP2, BMP4, BMP7,SOX9Developing Joints:GDF5, WNTI4,CHORDINSInterdigital MesenchymalCells: BMP2,BMP4, MSX2• Chondrocytes: NOGGIN•Proliferating Chondrocytes:IHHAdapted from Shum et aL, 2003(-€14.0)norAntDorsaçVentralPosterior37Figure 1.4. FGF Receptors and FGF Signal Transduction. FGFRs are modularproteins comprising 3 immunoglobulin domains (IgI, Igil and Iglil). IgI and IgIl areseparated by an acidic box (AD). IgIl contains a heparin binding domain (HBD). TheIgill domain is followed by a unique transmembrane (TM), a juxtamembrane (JM)and a kinase domain (KD) interrupted by an interkinase domain (IKD). FGF ligandslinked to heparin sulfate proteoglycan (HSPG) bind to Igil and Iglil of FGFR. Thiscauses the dimerization and subsequent transactivation by phosphorylation ofspecific tyrosine residues. The two main transduction pathways involvephospholipase C-y (PLOy) and RAS/MAP kinase. The SH2 domain of the PLOyinteracts with the phosphorylated Y766 of the activated receptor. The activated PLOyhydrolyzes the phosphatidyl-inositol-4, 5-diphosphate (P1 P2) to inositol-1 ,4, 5-triphophate (1P3) and diacylglycerol (DAG). 1P3 releases Ca2 while DAG activatesprotein kinase C-S (PKCS). Activated PKCS activates RAE by phosphorylating itsS338 and stimulates the downstream pathway in a RAS independent manner. Themain pathway involves the interaction of the docking protein FRS2cL with the amino-acid residues 407— 433. FRS2cL is activated by phosphorylation on multiple tyrosineresidues and then interacts with and activates GRB2 linked to SOS, a nucleotideexchange factor involved in the activation of RAS. Once activated, RAS activatesRAF which stimulates MEK which in turn phosphorylates MAP kinase ERK whichtranslocates to the nucleus and phosphorylates specific transcription factors (TF).These TFs induce the expression of specific EGF target genes. (Adapted fromThisse and Thisse, 2005)38NtExtracellular SpaceTM t TMHBDAdapted from Thisse and Thisse,2005Intracellular SpaceJMKDSH2PLCyIKDPIP2KDNucleus39Figure 1.5. ReIINF-KB Signal Transduction. In the classical pathway, varioussignals converge on activation of the 1KB kinase (IKK) complex. IKK thenphosphorylates 1KB at 2 N-terminal serines, which signals it for ubiquitination andproteolysis. Freed NE-KB (p50-ReIA, in this case) enters the nucleus and activatesgene expression. One NE-KB target gene encodes 1KB. The newly synthesized 1KBcan enter the nucleus, remove NE-KB from DNA, and export NE-KB back to itsresting state in the cytoplasm. Thick lines indicate the activating pathway; thin linesindicate the inactivating pathway.40STIMULIExtracellular SpaceCytoplasm1KB Kinase/ProteasomeAdapted from Gilmore, 200641Figure 1.6. Schematic Representation of the ModifiedEffecteneTMTransfectionStrategy for Enhanced Transfection and Storage of Transfection-Ready DNAComplexes Facilitating Large-Scale Transfections. A, One of the proposedbiological roles of disaccharides such as trehalose, is to prevent the formation ofcrystal lattices which are known to fracture lipid micelles and lipid membranes underconditions of stress attributed to desiccation as well as freezing. Trehalose additionresults in the formation of a glass like state and accommodates the fluidity of thelipid micelles and membranes. 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Shhestablishes an Nkx3.2iSox9 autoregulatory loop that Is maIntained by BMPsignals to Induce somitic chondrogenesis. Genes & Development 16:1990-2005.Zhang, V., Y. Lin, C. Bowles, and F. Wang. 2004. Direct cell cycle regulation by thefibroblast growth factor receptor (FGFR) kinase through phosphorylatlondependent release of Cksl from FGFR substrate 2. Journal of BiologicalChemlsby. 279:55348-54.Zhao, Q., H. Eberspaecher, V. Lefebvre, and B. De Crombrugghe. 1997. Parallelexpression of Sox9 and Col2al in cells undergoing chondrogenesis.Developmental Dynamics. 209:377-86.61Chapter 2:FGF Signals in the Embryonic Limb Regulate Cell Proliferation andSurvival Through Cdkn2b and the NF-icB Signaling PathwayA version of this chapter will be submitted for publication. Garcha, K., Hoffman,L.M., and T. M. Underhill. FGF Signals in the Embryonic Limb Regulate CellProliferation and Survival Through Cdkn2b and the NE-KB Signaling Pathway.622.1 AbstractGenetic studies have defined critical roles for the fibroblast growth factor(FGF) family in multiple aspects of limb ontogenesis. However, the molecular basisof these activities is poorly defined. Here we demonstrate that FGF4 regulates cellproliferation in the limb mesenchyme through induction of Cdkn2b via a TGF31-independent MEK1/ERK-dependent pathway. FGF4 also expandsthechondroprogenitor population and this is associated with increased expression ofprogenitor cell markers, Ly6a and Z1c2, 3 and 5. In the limb bud FGF5 are requiredfor cell survival and we show that this involves FGF-mediated activation of NF-icBsignaling through MEKI-dependent localized upregulation of the NE-KB activator,Ripk4. Further, in chondrogenic-deficient serum-free cultures of limb mesenchymalcells, FGF4 alone is sufficient to promote expression of Sox6, Sox9 andachondrocytic fate. Collectively, these findings define an important linkage betweenthe EGF and NE-KB signaling pathways, and establish a novel role for FGEs inchondrogenesis.632.2 IntroductionAppendicular skeletogenesis involves the coordinated action of multiplesignaling pathways that influence mesenchymal cell survival, proliferation,specification and differentiation. The fibroblast growth factor (FGF) family figuresprominently in many of these processes. Several Fgfs are expressed in thedeveloping limb, including Fgf4 and 8 which are localized to the apical ectodermalridge (AER), and whose encoded products signal to the underlying mesenchyme.Extirpation of the ridge leads to extensive mesenchymal cell death and limbtruncations, which can be rescued by the addition of FGF2 or 4 (Fallon et al., 1994;Niswander et al., 1993).The FGF5 signal through their cognate receptors, the FGF receptors(FGFR5), which belong to the tyrosine receptor kinase superfamily. Four Fgfrs havebeen identified in addition to several splice variants. Within the skeletogenicprogram they are sequentially expressed (reviewed in Ornitz and Marie, 2002), withFgfrl appearing in the limb mesenchyme, followed by Fgfr2 in precartilaginouscondensations and Fgfr3 in chondrocytes. Deletion of Fgfrl in the mesenchyme ofthe early limb bud though leads to appreciable cell death in the mesenchyme andloss of distal skeletal elements (Li et al., 2005). Similarly, conditional knockout ofFgfr2 in the AER leads to extensive cell death within the limb mesenchyme and lossof digits (Yu and Ornitz, 2008). Further, deletion of both Fgfrl and 2 in the limbmesenchyme leads to severe skeletal hypoplasia, reduced cell proliferation andincreased cell death (Yu and Ornitz, 2008). Embryos conditionally deleted for Fgf4in the AER do not present with a limb phenotype, whereas Fgf8 AER-specific64knockouts present with skeletal malformations that overlap with the defectsobserved in the Fgfr knockouts (Barrow et al., 2003; Boulet et al., 2004; Lewandoskiet al., 2000; Sun et al., 2002; Trowbridge et al., 2006). Fgf8 and Fgf4/8 doubleknockouts exhibit increased mesenchymal cell death. Comparison of the phenotypesobserved in the ligand versus receptor knockouts also suggests that FGFs functiondirectly in chondrogenesis (Yu and Ornitz, 2008). These studies have defined criticalroles for FGF signaling in regulating mesenchymal cell survival and proliferation, andestablishment of the chondrogenic anlagen.Secreted FGFs signal through the FGFRs to affect the activity of severalpotential downstream pathways that include the mitogen-activated protein kinase(MAPK), phosphatidylinositol-3’ kinase, STAT and Src tyrosine kinase signalingpathways (reviewed in Eswarakumar et al., 2005). The MAPK pathway, inparticular, the MEK1/ERK is an important downstream effector of FGF signaling inthe developing limb, as activated ERK is observed in a proximal-distal gradient in thelimb mesenchyme (Corson et al., 2003). Furthermore, evidence indicates that inchondrocytes, FGFs induce growth arrest via activation of the ERKI/2 pathway(Krejci et al., 2008; Raucci et al., 2004).In the developing limb, signals from the apical ectodermal ridge (AER) playimportant roles in regulating cell proliferation, survival and chondrogenesis ofunderlying mesenchymal cells. Studies in chick and in mouse have shown thatFGF signals from the ectoderm have critical roles in the regulation of theseprocesses although the mechanisms and targets of FGF signaling in themesenchyme are poorly defined. In this study, we provide mechanistic insights into65FGF action in cell survival and proliferation, and demonstrate that FGF alonestimulates chondrogenesis under conditions not conducive for cartilage formation.2.3 ResultsFGF signaling plays a fundamental role in regulating multiple aspects ofmesenchymal cell behavior during limb outgrowth and associated skeletogenesis.To better understand FGF action(s), we have isolated distal mesenchymal (DM)cells subjacent to the AER and assessed their responsiveness to FGF4 and 8.When plated in high density or micromass culture conditions, these cells closelyrecapitulate the chondrogenic events observed in vivo (Fig. 2.IA). Indeed, analysisof CoI2al expression in these cultures reveals that the cells begin condensing within24 h of plating (weak CoI2al expression), and form cartilage nodules shortlythereafter, as evidenced by the intense expression of CoI2al. In comparison,treatment of cultures with 20 ng/mI FGF4 delays expression of the chondroblasticphenotype, as observed with alcian blue staining, CoI2al expression and aSOX5/619 responsive reporter gene (Fig. 2.1A, C). SOX9 is both necessary andsufficient for chondroblast differentiation, and this reporter has been found to providean accurate read-out on the status of chondroblast differentiation (Hoffman et al.,2006; Muramatsu et al., 2007; Weston et al., 2002). In organ culture of limb budsdissected from El 1.5 Co12-EGFP transgenic mouse embryos (Grant et al., 2000),implantation of an FGF4-soaked bead similarly reduces transgene expression (Fig.2.IB). Cartilage formation is also initially delayed in limb mesenchyme culturesestablished from Co12-EGFP mice, although analysis at later time points (Day 7)demonstrates an increase in the size and number of cartilage nodules. Consistent66with this observation, FGF4 treatment leads to an appreciable increase in the size ofprecartilaginous condensations in comparison to control cultures, as determined byrhodamine-PNA staining and CoI2al expression (Fig. 2.IA). Further, 12 h treatmentwith FGF4 significantly stimulates BrdU incorporation that is indicative of increasedcell proliferation. This effect is transient, however, since cell proliferation in FGFtreated cultures returns to control levels by 24 h (Fig. 2.1D). Collectively, thesefindings indicate that FGF4, both in vitro and in organ culture, influences expressionof the chondroblastic phenotype and regulates proliferation of primary limbmesenchyme (PLM) cells. To better understand the molecular basis of theseactions, transcriptional profiling was subsequently employed.Cultures derived from distal mesenchyme were established and treated with20 ng/ml of FGF4, RNA collected and transcript abundance measured usingAffymetrix U74 V2 arrays A and B. Two hundred and forty five genes were found tobe induced at least 3-fold at 24 and 72 h post treatment; of particular interest,several of the Zic gene family members known to be associated with variousprogenitor cell populations were found to be markedly induced by FGF4 treatment(Fig. 2.2, Table I) (Aruga et al., 2002). Similarly, Ly6a (Scal), a cell surface markeroften associated with “stem” or progenitor populations was also significantly induced(Fig. 2.2, Table I) (Holmes and Stanford, 2007). Consistent with an FGF4-induceddelay in differentiation, several cartilage-associated genes were down-regulated, aswere several transcription factors including Eomes, Foxp2 and Sim2 (Fig. 2.2, TableI). Interestingly, the gene that exhibited the greatest increase(—18 fold) inexpression within 24 h of treatment was Cdkn2b, a cyclin-dependent kinase inhibitor67that negatively regulates cell proliferation (Gil and Peters, 2006). While Cdkn2a andCdkn2b are co-regulated, Cdkn2a expression only increased 3 fold (Fig. 2.3C),and other detected Cdkns showed little change in expression. In a variety of celltypes, including chondrocytes, FGF signaling has paradoxically been shown toreduce cell proliferation or induce growth arrest (Dailey et al., 2005). Thus, stronginduction of Cdkn2b by FGF4 is consistent with our observation that FGF4 induces atransient increase in cell proliferation.The ability of FGF4 to stimulate Cdkn2b at the transcript and protein level wasvalidated using qRT-PCR and western blotting (Fig. 2.3B). Further analysis ofCdkn2b expression at 75’ intervals over a 24 hour time period, reveals that FGF4induces Cdkn2b expression almost immediately, with Cdkn2b expression beingelevated 10 fold, relative to untreated controls within 2.5 h of addition. Further, inlimb organ culture, implantation of FGF4-soaked beads leads to a 5-fold increasein Cdkn2b expression (Fig. 2.3D). Several previous reports have shown thatCdkn2b expression is stimulated by TGF31 (Reynisdottir et al., 1995); indeed,further analysis of our microarray data reveals that FGF4 also induces Tgf/31, asfurther confirmed with qRT-PCR in treated PLM cultures and in organ culture (Fig.2.4A). Thus, we hypothesized that FGF5 regulate Cdkn2b expression throughTGFI3I.To confirm the activity of the Tgf/31 used in this study, we evaluated it onthe SOX9 reporter gene; consistent with previously described pro-chondrogenicactivity ofTGFI31(Chimal-Monroy et al., 2003), we demonstrate that treatment ofPLM cultures with TGFf31 increases reporter gene activity 3-fold. Furthermore,this stimulatory effect can be effectively abrogated in the presence of theTGFI3type68I receptor antagonist SB 431542 (Fig. 2.4B). Unexpectedly, treatment of DMcellswith TGF1 has no significant effect on Cdkn2b transcriptabundance or on theactivity of a reporter gene that encompasses the TGFI3I-responsiveregion within theCdkn2b promoter (Li et al., 1995) (Fig. 2.4C, D). In contrast, FGF4 stimulatesCdkn2b promoter activity. Together, these results indicate that FGF4 operatesthrough a TGFI3I-independent pathway to regulate Cdkn2b expression.FGFs function through several signaling pathways to affect cell behavior(Dailey et al., 2005; Eswarakumar et al., 2005). Severalreports have illustrated animportant role for the MEK/ERK signaling pathway in chondrogenesisand in FGFaction, and as such, we sought to address the role of the MEK/ERK pathway inFGFregulation of Cdkn2b. As shown above, FGF4 treatmentstimulates Cdkn2bexpression 8-fold, but not in the presence of an exogenous MAP2KI (MEK1)inhibitor, U0126, to a final concentration of 10 jiM (Fig. 2.5A). FGF4-mediatedstimulation of a Cdkn2b promoter-based reporter was similarly abrogatedby theaddition of U0126 (Fig. 2.5B). Cultures transfected with a minimal(-35) Cdkn2bpromoter construct displayed at least 20 fold less activity(data not shown).Consistent with the MEK1/ERK pathway playing a central role in FGF-mediatedregulation of Cdkn2b, overexpression of a constitutively activeversion of Map2kl inthe presence or absence of FGF4 stimulates Cdkn2b promoteractivity (Fig. 2.5C).Finally, the importance of Cdkn2b in FGF4-mediatedcessation in DM cellproliferation was evaluated through knockdown of Cdkn2b.In control transfectedcultures, the addition of FGF4 does not increase cell proliferationat 24 h, lendingfurther support to our findings illustrated in Fig.I D. In contrast, knockdown of69Cdkn2b leads to a similar increase in cell proliferation at both 12 h and 24 h.Cumulatively, these results show that FGF5 regulate Cdkn2b expression and cellproliferation through the MEKI /ERK pathway.Removal of the AER or deletion of Fgf8 from the AER are both associatedwith severe limb truncations and increased cell death in the limb mesenchyme,effects shown to be exacerbated with deletion of Fgf4. Interestingly, inhibition of NFKB signaling in the mesenchyme phenocopies many of the limb defects in the Fgf4/8compound knockouts (Bushdid et al., 1998; Kanegae et al., 1998). NE-KB istypically retained in the cytoplasm in a complex associated with IKK proteins. Uponactivation, however, NF-KB translocates to the nucleus where it activates targetgene expression (Hayden and Ghosh, 2008). Spatiotemporal expression of twodownstream targets of NF-KB, c-ReI and ReIA, in the distal mesenchyme furtherindicates a role for NF-KB signaling in the regulation of limb development (Bushdidet al., 1998; Kanegae et al., 1998). In addition, c-ReI expression is diminishedfollowing AER ablation, and subsequently rescued by the addition of EGF4 (Bushdidet al., 1998). To determine if EGF5 influence NE-KB activity, we examined NE-KB-responsive reporter gene activity in DM cells in the presence of EGE4. The additionof EGE4 to DM cells led to 4 fold increase in reporter gene activity (Fig. 2.6A).Both basal NE-KB activity and EGE4-induced activity were reduced upon cotransfection of IiB or a dominant-negative version of 1KB, IicB-2N (Fig. 2.6A). Toidentify potential mechanisms underlying FGE4 induction of NE-KB activity, themicroarray data set was queried for modulators of NE-KB. These analyses led to theidentification of receptor-interacting serine-threonine kinase 4 (Ripk4), a kinase that70has been shown to activate NE-KB (Fig. 2.2, cluster 3) (Meylan et al., 2002).Induction of Ripk4 expression in DM cultures was confirmed with qRT-PCR;interestingly, Ripk4 expression in the DM quickly declines following establishment ofcultures whereas its expression is maintained in the presence of FGF4 (Fig. 2.6B).This is consistent with the microarray profile in which Ripk4 expression declines incontrol cultures after 24h, but is maintained in FGF4-treated cultures (Fig. 2.2,cluster 3). Furthermore, Ripk4 transcripts are more abundant in the distal limb,consistent with the source of ectodermal FGFs (Fig. 2.6C). In accordance withprevious reports, heterologous expression of Ripk4 in PLM cells induces NE-KBactivity and this is further increased by FGF4 addition (Fig. 2.6D). Interestingly,expression of Ripk4 also enhances SOX9 reporter gene expression, indicating thatRipk4 activity may promote chondrogenesis (Fig. 2.6E). Similar to that observed forCdkn2b, FGF4 also regulates Ripk4 expression through the MEK1/ERK pathway, asthe MEKI inhibitor, U0126, completely inhibits FGF4-mediated induction of Ripk4(Fig. 2.6F), and overexpression of a constitutively active MEKI increases Ripk4expression (Fig. 2.6G). Further, U0126 significantly decreases NE-KB activity in DMcells, an effect that can be abrogated by expression of Ripk4 (Fig. 2.6H). Together,these results demonstrate that FGFs activate NE-KB signaling, at least in part,through up-regulation of Ripk4.Numerous studies have suggested an important role for EGEs in regulatingcell survival in the mesenchyme. To test this and the potential role of MEKI/ERKand NE-KB signaling in the regulation of these activities, we developed a low-densityserum free culture system for DM cells. When cells were seeded at sub-confluency71in 6-well dishes, and cultured in the absence of FGF4 or 8 (data not shown), noviable cells could be detected after 5 days of culture (Fig. 2.7A, B). In contrast, theaddition of 50 ng/ml FGF4 maintained cell viability such that alcian blue-positivecartilage nodules were detectable at — 10 days (Fig. 2.7A, B). Hitherto, cartilagenodules failed to form in sub-confluent limb mesenchyme cultures either in thepresence or absence of serum and/or other factors. Consistent with thisobservation, by day 6 of culture, precartilaginous condensations are observed, andstaining with PNA-rhodamine reveals a “cobblestone” distribution pattern withinprecartilaginous condensations (Fig. 2.7B).As shown previously, FGFs activate the MEKI pathway, and as revealedherein, also activates the NE-KB signaling pathways. To evaluate the importance ofboth MEKI and NF-KB signaling in cell survival, serum-free DM cultures wereincubated in the presence of either U0126 or the NE-KB inhibitor, Bay 11-7082, atconcentrations shown to have no deleterious effect on DM cells maintained in high-density culture conditions with serum (data not shown) (Fig. 2.7A). Relative tocontrol cultures, the addition of either inhibitor greatly decreases cell number, withthe Bay inhibitor having the greatest negative impact. Further, both inhibitors alsosubstantially reduce cell survival in FGE4-treated cultures (Fig. 2.7A). Bonemorphogenetic proteins (BMPs) are abundantly expressed in the distal limb andhave been shown to antagonize FGF function (Niswander and Martin, 1993b). Inaccordance with these earlier reports, we also note that the addition of BMP4compromises cell survival either alone or in EGF4-treated cultures (Fig. 2.7A). Incontrast, addition of the BMP antagonist NOGGIN leads to a small increase in cell72number in early cultures. Despite this, no viable cells were detected by day 6 ofculture. Surprisingly, as seen with BMPs, albeit to a lesser extent, NOGGIN alsocompromises cell viability in FGF4-treated cultures, indicating that BMP signaling incombination with FGFs are needed for mesenchymal cell survival. Collectively,these results demonstrate a fundamental role for FGF5, in conjunction with both theMEKI and NE-KB signaling pathways, in regulating cell viability in limbmesenchymal cells.To further evaluate the ability of FGE4 to stimulate chondrogenesis, theexpression of Sox6 and Sox9 were followed over time following addition of FGF4(Fig. 2.7C). Both genes are dynamically expressed, their expression peakingaround day 4 of culture just prior to overt cell condensation, then subsequentlydecreasing before a second significant increase to almost peak levels by day 10,coincident with the appearance of alcian blue-positive, CoI2al-expressing cartilagenodules. Sox6 expression is up-regulated during chondroblast differentiation (Smitset al., 2001), thus the early decline in Sox6 expression likely reflects“dedifferentiation” of chondrogenic cells, with subsequent increases indicative ofchondroblast differentiation; from day 6 to 10, Sox6 increases 18-fold. Theseresults clearly show that addition of FGF4 alone to serum-free cultures of DM issufficient to promote expression of the chondroblastic phenotype.2.4 DiscussionFGFs play critical roles at a number of steps within the skeletogenic program.Early in limb development, FGF signaling is required for outgrowth and patterning ofthe limb. Perturbation of EGE signaling is associated with severe limb truncations73and increased mesenchymal cell death. In this regard, AER-derived FGFs signal tothe underlying mesenchyme to maintain a population of skeletogenic progenitors bymaintaining viability, promoting expansion and preventing differentiation. To betterunderstand the molecular basis of FGF action, we have used cultures derived fromthe DM of the developing limb, a region underlying the AER. These cells exhibit aspectrum of responses to FGFs that are consistent with its activities in vivo.Microarray analysis coupled with subsequent functional analysis has providedunprecedented insights into FGF action in the limb. More specifically, we haveidentified critical linkages between FGF signaling and the NE-KB pathway, as well asFGF-regulated cell proliferation via Cdkn2b and the MEK1 signaling pathway.241 FGFs and Cell ProliferationFGFs can both stimulate and inhibit cell proliferation. FGF-mediated growtharrest has been well described in chondrocytes, and animals containing anactivating mutation of FGFR3 exhibit reduced chondrocyte proliferation whereasinactivation mutations lead to increased chondrocyte proliferation. Interestingly, cellproliferation is increased in the absence of Ffgrl in the limb mesenchyme of theearly developing limb (Li et al., 2005). Thus, FGFs appear to play paradoxical rolesin the regulation of cell proliferation. PLM cells also exhibit varied growth responsesto FGF4 and 8. For example, FGF4 or 8 induces a transient increase in cellproliferation that is quickly accompanied by a return to baseline proliferationrates.Herein Cdkn2b has been identified as mediating the growth suppressing activities ofFGFs in mesenchymal cells. As has been demonstrated recently in chondrocytes,FGF-induced growth arrest requires the MEK1/ERK pathway, and herein this74pathway has also been shown to be necessary for FGF induction of Cdkn2b (Krejciet al., 2008; Raucci et al., 2004). Other studies have reported an important role forthe Rb proteins p107 and p130 in FGF induced growth arrest in chondrocytes(Dailey et al., 2003; Raucci et al., 2004). FGF treatment was associated withdephosphorylation of p107 and p130 within 9-18h. Increased expression of CDKNscauses hypophosphorylation of Rb pocket proteins (Ashizawa, et al., 2001). Thus,these observations are also congruent with a role for FGF4-mediated growth arrestthrough Cdkn2b.TGFI31has been previously reported to induce Cdkn2b expression through amechanism involving SMAD-mediated downregulation of Myc and formation of aSMAD-activator complex on the Cdkn2b promoter (Seoane et at., 2001; Staller et al.,2001). Microarray analysis indicates that the various components (Myc, Mizi,Smad2, 3) of these complexes appear to be expressed (detected by DNAmicroarray) in DM cells, however, TGFf3I addition did not influence Cdkn2bexpression. Further, DM cells exhibit appropriate responses to TGFf3I, suggestingthatTGFI3Iregulation of Cdkn2b expression is cell-context dependent.Previous studies have shown that the cyclin-dependent kinase inhibitorsCdkn2a and Cdkn2b are regulated by the MEK1/ERK pathway (Gil and Peters,2006; Malumbres et al., 2000), however, the linkage between FGF signaling andCdkn2b expression has not been established. Cdkn2b and the closely linkedCdkn2a are both up-regulated by activation of MEKI/ERK; in accordance with thesereports, microarray analysis conducted in our study reveals that Cdkn2a expressionis also induced by FGF4(—3-fold). Thus, it seems plausible to suggest that75upregulation of Cdkn2a may also contribute to FGF4-mediated cessation of cellproliferation, however, knockdown of Cdkn2b restores growth in FGF4 treatedcultures at 24 h, indicating that if Cdkn2a is involved its role is likely minor.Moreover, our findings also explain how an absence of FGF signaling may lead toincreased cell proliferation as observed in Fgfr knockout animals. Furthermore,activation of FGF signaling or constitutively active FGFRs are associated withreduced chondrocyte proliferation; this has been recently reported to involve theMEKI/ERK pathway, which is congruent with an involvement of Cdkn2b. In thismanner, FGF-mediated induction of Cdkn2b provides a critical feedback loopensuring that cell proliferation is tightly regulated even in the continued presence ofa growth-promoting factor. Collectively, our findings demonstrate that FGFs alsonegatively impact cell proliferation through regulation of Cdkn2b, thereby providing aplausible mechanism for the contradictory mitogenic activities of FGFs.2.4.2 FGFs, NF-icB and Cell SurvivalInhibition NF-icB activity in the limb mesenchyme leads to severe skeletalreductions or truncations, and increased cell death, perhaps as a directconsequence of perturbation of NF-KB activity in the limb mesenchyme (Bushdid etal., 1998; Kanegae et al., 1998). REL is normally sequestered in a complexcontaining 1KB within the cytoplasm, however with the appropriate signal, 1KB isdegraded and thereby allowing NF-KB to enter the nucleus to regulate geneexpression. The NE-KB subunit, ReI is abundantly expressed in the progresszoneof the developing limb (Kanegae et al., 1998), but it is currently unclear as to howNE-KB signaling is activated in this region. Herein, we have demonstrated that the76NE-KB pathway is activated in distal mesenchymal cells in response to FGF4 (and 8,data not shown). Activation of NE-KB by FGF4 requires the MEK1/ERK signalingpathway, and is associated with increased expression of the NE-KB activating kinaseRipk4. RIPK4 is a member of a family of Ser/Thr kinases that influences signaltransduction pathways and leads to the activation of NE-KB. Ripk4 is expressed inthe distal limb, is activated in the limb mesenchyme by EGE4, and overexpression ofRipk4 activates NF-KB activity. In serum-free medium (SFM), limb mesenchymalcells exhibit limited survival, however this can be rescued by the addition of FGF4(and 8, data not shown). Inhibition of either the MEK1/ERK signaling pathway or theNE-KB signaling pathway in SEM conditions leads to increased cell death, indicatingthat these pathways are important in regulating mesenchymal cell survival.Interestingly, animals deficient in Ripk4 present with severe skin anomaliesassociated with abnormal keratinocyte differentiation, and limb defects characterizedby shortened limbs and syndactyly (Holland et al., 2002), the latter of which alsoappear in various FGER mutants (Ornitz, 2005).Previous studies have demonstrated an antagonistic action between FGEsand BMPs in limb outgrowth (Niswander and Martin, 1993a). While these actionsare largely recapitulated in cultures of limb mesenchyme, cell survival does appearto require some level of BMP signaling. Indeed, NOGGIN reduces cell survival inEGF4-treated cultures, albeit not to the same extent as BMP4. In more proximalderived mesenchymal cultures, BMP4 was found to support cell survival, but notchondrogenesis (data not shown).772.4.3 FGF Signaling and ChondrogenesisDeletion of ectodermally-expressed FGF4 and 8 in the developing limb leadsto a spectrum of limb defects, characterized by a progressive loss of more distalelements. From these studies it has been proposed that the roleof FGFs in limbdevelopment is to control the expansion of skeletal progenitor populations throughthe regulation of cell survival, proliferation and commitment (Sunet al., 2002; Yu andOrnitz, 2008). Together, these activities ensure that there aresufficient cells for thedevelopment of the various skeletal elements. Herein, we have clearlydemonstratedthat FGF5 influence cell proliferation and cell survival, and also the expansion ofchondroprogenitors mainly through the formation of precartilaginous condensations.Indeed, treatment of limb mesenchyme cultures with exogenous FGF4 (or 8, datanot shown) leads to a marked increase in the size of precartilaginous condensations.More significantly, addition of FGF4 alone in sub-confluent SFM cultures is sufficientto promote cell survival, cell proliferation, and the formation of pre-cartilaginouscondensations. Consistent with this, the chondroblastic marker Sox6 is markedly up-regulated during the culture period and cartilage nodules appearby day 10.Furthermore, with respect to the expansion of chondroprogenitors, FGF4 up-regulates the expression of several markers associated with progenitor populations,including the Zics and Ly6a (Scal). Microarray analysis revealsthat expression ofLy6a, Z1c2, 3 and 5 increases with FGF-4 treatment(—6, 7, 3 and 5-fold,respectively), as validated using qRT-PCR. Interestingly, these Zics in addition toLy6a are all expressed in the distal limb (Ma et al., 2002), with the Zic transcriptsbeing detected in the mesenchyme underlying the AER, and Zic2being expressed78also within pre-cartilaginous condensations, but not in chondroblasts(Nagai et al.,1997). All of these genes co-cluster with Ripk4, are enriched in the distallimb andsimilar to Ripk4 exhibit diminishing expression following culturethat can bemaintained with FGF4. Further evidence consistent with a role in regulatingexpansion of chondroprogenitors, FGF4 delays expression of the chondroblasticphenotype, as shown by reduced CoI2al expression (Fig. IA, and data notshown)and decreased S0X51619 reporter gene activity. In aggregate, this studyprovidesmechanistic insights into FGF regulation of cell proliferation andsurvival, andevidence supporting a direct role for FGFs in the early chondrogenic program.2.5 Methods and Materials2.5.1 ReagentsFGF4, BMP4, Noggin, andTGFI31(R&D Systems) were purchased from R&DSystems. FGF4 was resuspended in primary culture medium for all experimentswiththe exception of the SFM experiments in which stock solutions were preparedinSFM. FGF4 was added to media at a concentration of 20 ng/ml, with the exceptionof SFM experiments in which FGF4 was added at aconcentration of 50 ng/ml.Noggin was resuspended in sterile PBS containing 0.1% BSA, andadded to mediaat a concentration of 200 ng/ml. BMP4 and TGF-pl were resuspendedin areconstitution buffer which consisted of 4 mM hydrochloric acid containing0.1%bovine serum albumin and were added to media at a concentration of 20 and10ng/ml respectively. BAY 11-7082 (Sigma), RBI (SB 431542) (Sigma),and U0126(Promega) were dissolved in DMSO (BDH) and added to a final concentrationof5.tM, 1iM, and 10pM, respectively.792.5.2 Plasmid ConstructsThe reporter gene plasmid containing SOX9 binding sites (pGL3) waspreviously described (Weston et al., 2002), whereas the NF-KB responsiveluciferase reporter was purchased from Stratagene. The Cdkn2b-35- and Cdkn2b-463-luciferase reporter plasmids were provided by X-F. Wang (Duke University,Durham, North Carolina). Constitutively active Meki (cMEKI) was obtained fromClontech. Dr. J. Tschopp (University of Lausanne, Epilanges, Switzerland) providedus with the Ripk4 plasmid. Ixba and Iith-2N (Algarte et al., 1999) plasmids wereobtained from Dr. S. Bernier (The University of Western Ontario, London, Ontario).As a control vector, pcDNA3.I+ (Invitrogen) was used.2.53 Establishment and Transfection of Primary Limb Mesenchymal CulturesPrimary limb mesenchymal (PLM) cultures were established from CD-Imurine embryonic limbs (E1l.5) as previously described (Hoffman et al., 2006;Weston and Underhill, 2000). Briefly, the distal tips (subridge regions extending—0.3mm from the distal apex of the limb to the proximal cut edge) of these limbswere dissected and separated from the proximal regions (described in Gay andKosher, 1984). The ectoderm was enzymatically removed from these two limbregions by dispase treatment and separated by filtration through a 40 1.iM cellstrainer (BD Biosciences) giving rise to a single cell suspension. PLM cells werepelleted by centrifugation at 200 X g and resuspended to producea stock cellsuspension at a concentration of 2 x I ü cells/mI. For microarray analyses, 12-15 10jil aliquots of this suspension were plated into a 35 mm tissue culture dish (Nunc)and allowed to adhere for 1 h. After this period, 2 ml of culture medium consisting of8060% Ham’s F12 nutrient mix/40 % Dulbecco’s modified Eagle’s medium (DMEM)and supplemented with 10 % FBS (Qualified, lnvitrogen) wereadded to each wellwith or without 20 ng/ml FGF4 (R&D Systems); this time was considered T=0.Cultures were maintained for a period of up to 3 d; to minimize handling, culturemedia was replaced on alternate days. Alcian blue staining of PLM cultures wascarried out as previously described (Hoffman et al., 2006).For transfection, stock piasmid DNA5 were diluted to a concentration of Img/mi. For co-transfections, a ratio of 3:1 — gene of interest to reporter gene wasused. Luciferase reporter genes consist of a gene of interest promoter/enhancerdriven firefly (Photinus pyralis) luciferase, and a constitutively active control Renilla(Renhla reniformis) luciferase to normalize for transfection efficiency. The ratio offirefly luciferase reporter to Renilla luciferase used was 20:1. A stock of this reportergene mix was used at I mg/mi. PLM cultures were transfected usingEffecteneTM(Qiagen). Seven and a half microlitres of Effectene-complexed DNA was added to35 of the PLM cell stock. Of this mixture, 10 il spots of PLM cells were dispensedin the centre of each well. Plates were incubated under standard tissue cultureconditions for 45 minutes at which time 0.5 ml of primary culture medium was addedper well and the plates were incubated for an additional 30 minutes. Medium wasreplaced with I ml of fresh primary culture medium supplemented with factors orvehicle. At 16 h post-plating, culture medium was replaced with freshmedium. At 24h post-treatment, cells were washed with PBS and lysed in 100ilof passive lysisbuffer (Promega) for 20 mm., and dual luciferase measured as previously described.81Cdkn2b knockdown was performed using siRNAs purchased from Dharmacon.SiRNAs were transfected into PLM cells using LipofectamineTM RNAiMAX(Invitrogen). For BrdU incorporation assays, cells were transfected in suspensionwith siCdkn2b and 10 p1 micromass cultures were established as outlined above. Forexperiments involving the collection of RNA, 12-15 5iRNA-transfected cultures wereplated per well of a 6-well plate (Nunc), and 2 ml of media were added one hourpost-plating. RNA was collected as described below.2.5.4 Assessment of PLM Cell ProliferationCell proliferation in PLM cultures was determined using the of 5-bromo-2’-deoxy-uridine Labeling and Detection Kit I (Roche) - an immunofluorescence assayfor the detection of 5-bromo-2’-deoxy-uridine (BrdU) incorporated into cellular DNA.PLM cells were prepared as previously outlined, and 10 il micromass cultures wereestablished on 4-well chambered glass slides (Nunc) and incubated under standardtissue culture conditions. Culture medium was added at 1 h post plating, at whichtime FGF4 (2Ong/ml) and vehicle control were added to separate wells. This wasconsidered to be time 0. After 12 h, FGF4 and vehicle control were added to theremaining wells. At 23.5 h, BrdU labeling medium (Roche) was added to each well,and the slides were incubated for 30 mm. Medium was aspirated and the slides werewashed with PBS and fixed in an ethanol fixative consisting of 30% 50 mM glycinesolution : 70 % absolute ethanol for 30 mm at -20°C. The subsequent steps to detectBrdU positive cells were performed according to the manufacturer’s instructions.BrdU positive cells were visualized using a FITC filter set on an Axiovert S100inverted fluorescence microscope.822.5.5 Rhodamine - Peanut Agglutinin (PNA) LabelingPrimary cultures were washed twice with PBS, and fixed in 4%paraformaldehyde for 30 minutes at 4°C. Fixative was aspirated, and cultures wererinsed once with PBS. Under reduced light, rhodamine-labeled PNA (Vector Labs)was diluted in PBS to a final concentration of 10 mg/mI, and added to each well suchthat the cultures were completely covered. Plates were protected from light, andstored at 4°C overnight. Rhodamine-PNA solution was aspirated and cultures werewashed twice using PBS. PNA bound cells were visualized using epi-fluorescencemicroscopy with a Texas red filter set.2.5.6 Culture of PLM cells in the Absence of Serum FactorsUsing the previously described method of preparing PLM cell stocks, a cellsuspension was prepared at a concentration of 5 x I ü cells/mI in SFM. Of this stock,1.5 ml were dispensed per well of a 6 well plate(—8 x i05 cells/well). One hourfollowing seeding, culture medium was aspirated and replaced with fresh SFMcontaining factors or vehicle controls. Cells were incubated overnight under standardtissue culture conditions. Medium was changed daily. Cells were washed once withPBS, fixed on day 11 with 95% ethanol at -20° C overnight, and subsequentlystained with Alcian blue.2.5.7 Limb Bud Organ Culture and Bead ImplantationLimb buds from —E11.5 Col2-EGFP mice (CD-I background; derived frombreeding of heterozygous transgenic males with CD-I females) (Grant et al., 2000)were collected in cold PSA. Affi—Gel blue beads (Bio-Rad Laboratories) soaked ineither vehicle or FGF4 (20ng/il) for 2 h were transferred into the interdigital region83(IDR) of the limb buds. Limb buds were cultured on Nucleopore Track-Etchmembranes (Whatman) at the air—liquid interface on top of stainless steel mesh in12-well tissue culture plates (Corning). PSA was aspirated from each well andreplaced with BGJb medium (Invitrogen) containing 10% FBS and antibiotics. Thelevel of culture medium was maintained such that it did not exceed the height of themembranes. Limbs were incubated under standard tissue culture conditions. After a24 h incubation, EGFP expression was visualized using a fluorescence dissectionmicroscope (model MZI2; Leica).For isolation of RNA from organ cultures, culture media was aspirated andwells were washed 3 times with PBS. Limbs were removed from the membranes byagitation and transferred to 15 ml polystyrene conical tubes. Nearly all PBS wasremoved and 5 ml of RNAlater (Ambion) were added to each tube. Limbs werestored at -20°C. Using Graefe knives, areas excluding the beads and those includingthe beads were dissected and transferred to individual microfuge tubes containing700 jil of RLT lysis buffer (Qiagen RNeasy kit). Dissected limb regions werehomogenized in the RLT lysis buffer by triturating, and RNA was isolated as per themanufacturer’s protocol.2.5.8 Transcriptional Profiling with Microarrays: Experimental Design andAnalysisRNA was harvested from primary cultures using RNAeasy (QIAGEN)according to the manufacturer’s instructions. For the zero time point, cells wereallowed to attach for I h and were subsequently processed for RNA isolation. Forother cultures, the media was gently aspirated, and any remaining media was84blotted from the well before the addition of the lysis reagent. After isolation, the RNAwas precipitated, and resuspended at 2 jig/mI; RNA quality was examined on aBioanalyzer 2100 (Agilent), and the expression of Sox9 and CoI2al were measuredusing real-time PCR. For each time point, a minimum of two biological replicateswere analyzed on U74V2 chips A and B. Ten jig of RNA was labeled and hybridizedto the chips using the manufacturer’s recommended protocol. Gene expressionprofiles were subsequently analyzed using MAS 5.0 (Affymetrix) and GeneTrafficUNO bioinformatics programs (Stratagene). All datasets were initially filtered toremove genes called absent by MAS 5.0, and were further filtered as indicated in thetext using GeneSpring. Hierarchical clustering was performed in GeneSpring usingthe tree function and a Pearson correlation similarity metric.2.5.9 Quantitative Real-Time PCRTo measure the abundance of various transcripts qRT-PCR was performedusing the 7500 FAST Sequence Detection System (Applied Biosystems). Theprimer/probe sets used for detection of Sox9 and CoI2al were as described inWeston et al. (2002). For detection of all other transcripts, TaqMan Gene ExpressionAssays (Applied Biosystems) were used. Total RNA was isolated from primarycultures and limb sections as described above, and an aliquot was reversetranscribed to cDNA using a High Capacity cDNA Archive kit (Applied Biosystems).Quantification was performed using --10 ng of total RNA and the expression of allgenes relative to endogenous rRNA was determined using TaqMan RibosomalControl Reagents (Applied Biosystems).852.5.10 Statistical analysisAll luciferase assays were performed in quadruplicate and repeated usingthree distinct preparations of primary cells. Real-time PCR analysis, with theexception of the 24 h time courses, was performed in duplicate and repeated aminimum of three times with independent RNA samples. Proliferation data wereanalyzed by one-way analysis of variance, followed by a Bonferroni post test formultiple comparisons using GraphPad Prism, Version 5.0 (Graph-Pad Software,Inc.). Significance is represented as follows:*P < 0.05;**P <0.01; #, P < AcknowledgementsWe would like to thank Dr. Wang (Duke University, Durham, NC) for theCdkn2b promoter constructs and Dr. Tschopp (University of Lausanne, Switzerland)for the Ripk4 cDNA and the London Regional Genomics Centre for carrying out themicroarray experiments. K. Garcha was supported by pre-doctoral award from theStem Cell Network, L.M. Hoffman was supported by a post-doctoral fellowship fromthe Canadian Arthritis Network, and this research was funded by a grant to T.M.Underhill from the Canadian Institutes of Health Research (CIHR). TMU holds anInvestigator award from The Arthritis Society.862.7 FiguresFigure 2.1: FGF4 Regulates Expression of the ChondroblasticPhenotype andProliferation of Limb Mesenchymal Cells. A, FGF4 treatment (20 ng/ml)of PLMcultures delays chondroblastic differentiation and increases the sizeof precartilaginous condensations. PNA-rhodamine stainingat day 2 (D2) of precartilaginous condensations is greatly increased in FGF4-treatedcultures incomparison to untreated controls. Analysis of CoI2al expression by whole-mountinsitu hybridization (WISH) shows that FGF4 treatment (20ng/ml) leads to weakCo/2a 1-expressing foci of larger size as comparedto control cultures. Similarly,FGF4-induced expansion of pre-cartilaginous condensationsleads to largerchondrogenic nodules in PLM cultures derived from Co12-EGFPmice. CompareEGFP-expressing cells at D7 in untreated versus treated cultures,the formercultures are nodular in nature, whereas the FGF4cultures form a sheet oftransgene-expressing cells. Further, in FGF4-treated cultures alcianblue staining ismore diffuse and widespread. B, in organ culture FGF4 delayschondroblastdifferentiation. Implantation of a bead (white arrowheads) soaked in FGF4(50 ng/jil)(F4) decreases transgene expression versus vehicle controls (V) in organculture ofE11.5 limbs derived from Co12-EGFP mice; limbs were visualized24 h postimplantation. Limbs are excised and cultured on a stainless steelmesh at theair/media interface, following I day of culture, all of the skeletalelements across theproximo-distal (PD) axis can be observed. C, treatmentof DM and to a lesser extentPM-derived PLM cultures with FGF4 (10 ng/ml)reduces SOX5/6/9 activity,suggesting that FGF4 inhibits chondroblast differentiation. D, FGF4(20 ng/ml)87transiently increases cell proliferation as revealed by a significant increase in BrdUincorporation at 12 h, but not at 24 h. Magnification bars in A from top to bottom, barrepresents, 0.25 mm, 0.3 mm, 0.25 mm and 1 mm.88A(0150C —.0V C“- COo.CtrI FGF4D2PNA-RhodamineD1D3BCDCoI2al WISHD4I-4-C.)II0D-JD7pG L3(4X48)-Luco CtrI• FGF4o Ctrl• FGF4CoI2al-EGFPDM PM200D4Alcian blue50•012h 24h89Figure 2.2: Identification of FGF4-Regulated Genes in Limb-Derived DistalMesenchymal Cells. Hierarchical clustering in GeneSpring was performed usingthe Pearson correlation similarity measure to identify FGF-regulated genesexhibiting similar patterns of expression. Two-hundred and forty-five (3-fold cut-off)genes were clustered and several patterns emerged, four of which are shown. Manyof the cartilage-expressed genes associated with chondroblast differentiation aredown-regulated in FGF4-treated cultures (cluster 1), highlighted in red. Otherclusters (2 and 4) contain genes that are up-regulated by FGF4 (highlighted in blue),whereas cluster 3 contains genes that are normally down-regulated upon culturing(highlighted in yellow), but whose expression is maintained in the presence of FGF4.Several genes connected with cell proliferation were also identified in FGF4-treatedcultures including Cdkn2b (cluster 2) and Tgf/31 (cluster 3).90IEgfrAgclAgtr2LoxAicamcmCoilla2Cspg4OgnMialFoxclHidNpr3Nt5eMatn4Foxp2Dab2Eya2S1m2Wwp2SostdclOddi- + -+ F40 24 72 Time(h)32AnxalLohllcr2aCdkn2bPcp4ilAgpt2irak3KifIaGfra2Ptger4Sdccag33lAnxa8Ptgs2Coi6alBgnBhihb2Atxnl_____________Igfbp5- + -+ F40 24 72 Time (h)Rgs6Sic 16a7TgfblTraf3ip2Csn3St6gallCalbiFabp4S100a13Catna2Fabp7SlOOalCxcI5Sic 14a1GfralSqrciJPrkg2Ly6aP1a2g7Cd109Mmp3- + -+ F40 24 72 Time(h)720 24- + - +ITime (h)FGF415. 139Gap431JunibIRipk4CcdclO9bAkrlb8- + -+ F40 24 72 Time(h)91Figure 2.3: CycIin Dependent Kinase Inhibitor 2b is Up-Regulated by FGF4 invitro and in Organ Culture. A, FGF4 induced the expression of multiple membersof the Cdkn family as determined by microarray analysis, and this is especiallyapparent for Cdkn2b, which is induced 18 fold after 24 h of FGF4 treatment. Allgenes were called present in at least 4 samples. B, FGF4 induced the expression ofCdkn2b and CDKN2b as determined by qRT-PCR and western blotting,respectively. C, time course analysis of FGF4 induction of Cdkn2b by qRT-PCRrevealed that within 2.5 h (arrowhead) Cdkn2b is induced 10-fold in comparison tountreated cultures. D, in organ cultures, beads soaked in FGF4 lead to appreciableCdkn2b expression in the regions surrounding the bead (BD), but less so inmesenchyme distal (DL) or proximal (PL) to the bead.920Control FGF4300a-i_1100010010I0.1Cclkn2bCdkn2aCdknlaCdknlcCdkn2cCdkn3CdknlbCdkn2d72 72 Time(h)+FGF424hA0UU,0)0U)a,0NE0zBC+0‘I.C40.‘CUia,C,7000000- 5000030000a,’:iiooo24h 48hControl FGF4CDKN2B — JP13-ACTIN —— It—Cdkn2bCtrI0 4 8 12 16 20 24Time (h)0 CtrI • FGF4DL BD PL93Figure 2.4: FGF4 Regulation of Cdkn2b Does Not InvolveTGFI3ISignaling. A,FGF4 (20 ng/ml) increases the expression of Tgf/31 both in PLM cultures and organculture. B,TGFI3Istimulates S0X51619 activity and this can inhibited by theTGFf3IR antagonist, SB 431542 (RBI). Further, TGF1 soaked beads stimulateectopic cartilage formation in limb bud organ culture (inset). C, in comparison toFGF4,TGFI31does not stimulate the expression of Cdkn2b as determined by qRTPCR. D, TGFf3I does not stimulate the activity of a Cdkn2b-promoter derivedreporter activity, whereas treatment with FGF4 does stimulate reporter gene activity.Note, this reporter contains the previously identified TGF1-responsive element.94ATgfblTgfbl600d. xLU4000 00-j 4- C.)d. xLU0 000140 10060 20CtrIF4BControlFGF4pGL3(4X48)-LucCtrI•FGF4C800Cdkn2b4- C) D -JDLBDPLpGL2-Cdkn2b-463-LucD500L..4- C)300D -J100•2000CtrIT131F4RBITf1CtrIT31F4RBIT131CtrIT31F4RBITj31+RBI+RBIi-RBIRI‘ICDFigure 2.5: FGF4 Regulates Cell Proliferation Through Cdkn2b and ThisInvolves the MEKIIERK Signaling Pathway. A, induction of Cdkn2b by FGF4 (20nglml) is abrogated in the presence of the MEK1 inhibitor U0126 (10 1iM). B, FGF4(20 ng/ml) induces the reporter activity of a Cdkn2b-reporter encompassing theproximal region of the promoter (-463), and this reduced to less than control levelsby U0126 (10 j.tM). C, heterologous expression of a constitutively active version ofMEKI (cMEKI) induces activity of the -463 Cdkn2b reporter. D, siRNA-mediatedknockdown of Cdkn2b in PLM cultures increases cell proliferation in comparisontosiCtrl-transfected cultures. The extent of knockdown was determined using qRTPCR and found to be 65 %.96ABpGL2-Cdkn2b-463-Luc400EJCtrICCtrIci•FGF4€300 4- C.)a. xCFGF4w200a)Da)CD1000__________________CtrIU0126CD4-—.ci4-’—C.)cia)4-oøcuC.>G)X+Iiia)Da)CDCdkn2bCtrIU0126CtrIcMeklsiCtrisiCdkn2bsiCtrisiCdkn2bCDFigure 2.6: FGF4 Activates the NF-icB Signaling Pathway in Part ThroughMEKIIERK-Mediated Upregulation of Ripk4. A, FGF4 (20 ng/ml) treatment for24h induces the activity of an NE-KB reporter gene and this can be effectivelyinhibited by co-expression of either a construct encoding 1KB or a stabilized versionof 1KB, IKB-2N. B, the expression of Ripk4, an activator of the NF-KB pathway,progressively declines following culture of PLM cells, however, Ripk4 expression ismaintained with FGF4 treatment (20 ng/ml). C, Ripk4 transcripts are more abundantin distal limb sections (#1) as compared to proximal sections (#2-5). D,overexpression of Ripk4 induces NF-KB activity in PLM cultures and this is furtherincreased in the presence of FGE (20 ng/ml). E, expression of Ripk4 stimulatesactivity of the S0X516/9 reporter gene in PLM cells, however, this is attenuated forthe most part by the addition of FGF4 (20 ng/ml). F, FGF4-mediated induction ofRipk4 expression is inhibited by U0126. G, expression of a constitutively activeMEKI in PLM cells induces the expression of Ripk4. H, NF-KB reporter activity issubstantially decreased in U0126-treated PLM cultures and this can be partiallyrescued by overexpression of Ripk4 at 24 h post transfection.98100axLU60G)020I4-C)D-JBI‘IC-)D-J400300200100ACN F-icB-LucID CtrI• FGF44-C)1200I—80x!“CtrI •FGF4 Ripk4CtrI kB IicB-2No 4 8 12 16 20 24Time (h)EpGL3(4X48)-Luc12345Section #F GRipk4Q CtrI• FGF402700I.4-,C)500aLU30001000- + -+ FGF4CtrI Ripk4HI4-C)aLiiC0700€5004-,C)D—I 3001000NF-icB-LucDCtrII• U0126j.CtrI U0126 CtrI cMeklICtrI Ripk499Figure 2.7: The MEKIIERK and NF-KB Signaling Pathways are Both Requiredfor FGF4-Mediated Cell Survival and Cartilage Formation. A, a low-densityserum free culture model was developed to assess the role of FGF4 and varioussignaling pathways in mesenchymal cell survival. In the absence of FGF4, no viablecells could be detected after 6 days of culture, and the addition of U0126 (10 jiM) orthe NE-KB inhibitor BAY 11-7082 (5 jiM) reduced cell viability to 4 and 1 days,respectively. The addition of FGF4 (50 ng/mI) markedly stimulated cell number,however, this was inhibited by the addition of either U0126 or BAY 11-7082. Theaddition of BMP4 (10 ng/ml) also negatively influenced cell viability in the presenceor absence of FGF4, whereas, NOGGIN (100 ng/ml) reduced cell viability in oldercultures(>4 days). B, FGF4 is sufficient to stimulate chondrogenesis in sub-confluent monolayer PLM cultures. After I day (Dl), there are visibly more cells inthe EGF4-treated cultures in comparison to controls. By day 10 (D10), numerouschondrogenic nodules can be visualized in the FGF4-treated cultures and these canbe stained with alcian blue. Further, several regions of PNA-rhodamine staining(arrowheads) can be visualized in the FGF4-treated cultures and at highermagnification, intensely PNA-positive cells (arrows) can be visualized overlyingweaker PNA-positive cells (bright field). These latter cells exhibit a honeycomb-likepattern of PNA binding (arrowhead), consistent with condensation formation. C, theSox genes 6 and 9 are dynamically expressed under these conditions. Sox6 and 9exhibit 2 waves of expression, the first wave appearing just before overtcondensation (D4) followed by a large increase in Sox6 from D7-10 thataccompanies chondroblast differentiation. Magnification bars, in B, top panel left to100right, the bars represent, 0.25 mm, 0.25 mm, 0.5 mm and 0.5 mm; bottom panel, leftto right, the bars represent 0.25 mm, 0.06 mm and 0.06 mm.101A1600120EILJi.?[4j6D ControlD U0126• Bay 11-7082• FGF4• U0126+FGF4• BAYII-7082+FGF4• BMP4D BMP4+FGF4• Noggin,• Noggin+FGF4Time (Days)[18 10Dl 010 DII(-)F4BCF4Alcian blueD7Co3CoCO0.xUI0C0CD04-00—0-- Sox6-A--Sox90 2 4 6 8 10Time (Days)102Table 2.1: qRT-PCR Analysis of Selected Genes in E11.5 DM Cultures24h 48hGene Ctrl FGF4 CtrI FGF4BhIhb2 1.0 4.6 5.2 4.5Dachl 1.0 1.5 1.4 2.1Eomes 1.0 0.1 0.5 0.3Foxp2 1.0 0.2 1.6 0.1Ly6a 1.0 7.9 1.0 6.7Runxl 1.0 4.6 2.0 3.8S1m2 1.0 0.1 2.0 0.5Zic2 1.0 6.6 0.2 3.9*Fold change is determined by normalization to untreated24h control culture.1032.8 ReferencesAlgarte, M., H. Nguyen, C. Heylbroeck, R. Lin, and J. Hiscott. 1999. IkappaBmediated inhibition of virus-induced beta interferon transcription. Journal ofVirology. 73:2694-702.Aruga, J., T. lnoue, J. Hoshino, and K. 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Chemical Genetics Reveals a Novel Role for Potassium Channels inChond rogenesis.1103.1 AbstractTo gain insights into the molecular programs regulating chondrogenesisachemical genetics approach was employed. Implementation ofthese strategiesinvolved the development and validation of screens in primarycultures of murinelimb mesenchymal (PLM) cells. PLM culturesclosely recapitulate in vivochondrogenesis and within 2-3 days in culture, chondrogenicprogenitorsdifferentiate into matrix-secreting chond roblasts. Chond roblastdifferentiation isassociated with increased S0X51619 activity and a SOX5/6/9-responsivereportergene was used to follow expression of the chondroblastic phenotype.Compoundlibraries representing 1,500 compounds were screenedin 384-well format, and 28compounds were found to increase reporter gene activity > 2.5 fold.In secondaryscreens, 7 of 28 active compounds stimulated cartilage formation asassessed byalcian blue staining. Interestingly, one of these compounds, butamben(butyl 4-aminobenzoate), a member of the benzocaine family of analgesics, exhibitedprochondrogenic activity. The high affinity target of this compound is thepotassiumchannel, KCND2, which through transcriptional profiling was foundto bedownregulated by bone morphogenetic protein (BMP) 4. Further,butamben couldrescue cartilage formation in the presence of the BMP antagonist,NOGGIN.Together, these results reveal an unanticipated and novel role forpotassiumchannels in chondrogenesis.1113.2 IntroductionCartilage plays a fundamental role in the development of much of theskeleton, as long bones are formed from a cartilage precursor, which is derived fromcondensed mesenchyme. Numerous factors and pathways have been shown toplay an important role in regulating the chondrogenic program. These studies havebeen greatly aided by the development of in vitro primary cultures that faithfullyrecapitulate the in vivo sequence of events. High density or micromass cultures ofprimary limb mesenchymal (PLM) cells can be established from the embryonic limbmesenchyme and within a short period these cultures give rise to bona fide cartilagenodules. Two main stages can be readily identified, precartilaginous condensationsand cartilage nodules. Initially prechondrogenic cells aggregate to formprecartilaginous condensations and subsequently cells within the centre differentiateinto chondroblasts. Critical to these steps is SOX9, a transcription factor belongingto the Sry-related high mobility group (HMG) box gene family, whose expressionpresages and regulates the expression of type II collagen (CoI2al) (Lefebvre et al.,1997; Ng et al., 1997). Sox9 is required for both commitment and differentiation ofchondroblasts. The onset of chondroblast differentiation is associated with increasedexpression of L-Sox5 and Sox6, and together with Sox9 they increase theexpression of CoI2al and other chondrogenic genes. By transfecting PLM cultureswith a firefly luciferase based reporter gene consisting of four, 48bp- fragments ofthe CoI2al enhancer, to which SOX5/619 have been shown to bind, ourgroupamong others have demonstrated a reliable means of assessing the status ofchondroblast differentiation (Hoffman et al., 2006; Lefebvre et al., 1997; Ng et al.,1121997; Weston et al., 2002; Weston et al., 2000). Using thisassay, in previousstudies we showed an inverse correlation between the activity of the retinoidsignaling pathway and chondroblast differentiation (Weston et al., 2000). Moleculesthat enhance retinoid signaling interfere with chondroblast differentiation and this isassociated with decreased S0X51619 activity. In addition to following S0X51619activity in PLM cultures, monitoring activity of the retinoid pathway with a RAresponsive reporter gene has been found to provide a useful measure of cartilageformation (Weston et aL, 2002). Recent studies have also shown that the prochondrogenic BMPs regulate cartilage formation in part by decreasing activity of theretinoid signaling pathway (Hoffman et al., 2006).Chemical genetics has proven a powerful approach to identifying newpathways and targets operating within a biological program (Yeh and Crews, 2003).Further, this strategy has enabled identification of useful molecular tools in which tomanipulate molecular and cellular processes. Herein, a chemical genetics strategywas implemented to identify novel modulators of the chondrogenic program. Highthroughput screens utilizing PLM cultures were developed and validated in 384-wellformat and small molecule libraries were screened to identify compounds thatinfluenced S0X516/9 activity; these compounds were also testedon a retinoic acidresponse element (RARE) reporter. Several small molecules with pro-chondrogenicactivity were identified and one of these, a potassium channel modulator,appears tofunction downstream of BMP signaling in chondrogenesis.1133.3 Results and DiscussionTo better understand the molecular mechanisms underlying chondrogenesiswe previously developed low throughput reporter gene-based assays to interrogategene function in this program (Hoffman et al., 2006; Weston et al., 2002). Thepremise for the assay was based on the observations that at the onset ofchondroblast differentiation CoI2al expression is greatly elevated and this ispreceded by increased expression of L-Sox5 and 6. To efficiently follow this event,a reporter gene derived from the CoI2al gene containing binding sites for SOX5/619was used and subsequently shown to provide an accurate measure of the status ofchondroblast differentiation (Hoffman et al., 2006; Weston et al., 2002). Briefly theassay involves co-transfection of PLM cultures at the time of seeding and analysis ofreporter gene typically at 48 h post-transfection. Importantly, it is during this timeframe that chondroprogenitors in the culture differentiate into chondroblasts. In itspresent incarnation, the chondrogenic assay was implemented in a 24-well format,which limited throughput as PLM cultures had to be transfected and seededmanually. Further, PLM cells have to be cultured at high-density and isolated on theday of transfection as low-density cultures or prior culturing limits their chondrogenicpotential. To enable implementation of robust large-scale screens for chondrogenicmodulators, methods were developed to increase transfection efficiency whilepermitting storage of a prepared DNA/transfection mixture.To improve PLM transfection efficiency several transfection reagents wereevaluated and Effectene was found to exhibit greater transfection activity in PLMcells than other reagents such as Fugene6 (Roche) (data not shown). To further114streamline and increase transfection efficiency and throughput, methods weredeveloped whereby DNAltransfections could be prepared en masse and stored. Forthis purpose, various disaccharide stabilizers were tested for their potential toenhance the integrity of the lipid-basedEffecteneTMtransfection reagent and therebyallow longer-term storage. Disaccharides have been used in both cryopreservationand desiccation to enhance preservation of biologics or cells (Beattie et al., 1997;Chen et al., 2000; Crowe et al., 1998; Crowe et al., 1994; Leslie et al., 1995). TheEffecteneTMtransfection methodology condenses DNA in a buffered solution (ECbuffer) and to this buffer the disaccharide sucrose or trehalose was added to a finalconcentration of 0.4M. Inclusion of either 0.4M sucrose (S) or trehalose (T) to theEffecteneTMEC buffer led to an increase in transfection efficiency of PLM cultures(Fig. 3.IA). Transfection efficiency with 0.4M sucrose supplementation wasincreased —1.7 fold, and that of 0.4M trehalose containing mixtures increased 2.7fold as determined by measuring the activity of phRL-SV4O. This plasmid expressesRenhlla luciferase under the control of a constitutive SV4O promoter/enhancer (Fig.3.IA). Importantly, the addition of either sucrose or trehalose to the transfectionmixtures does not affect culture biology, as the addition of BMP4 (20 ng/ml) led tosimilar increases in the activity of a SOX5/6/9-responsive reporter gene[pGL3(4X48)] (Fig. 3.1B). To test the ability of the disaccharides to enable short andlong-term storage ofDNAEffecteneTMtransfection mixtures, aliquots of thesetransfection formulations were stored at -20°C for a period of 1 month or 3 years.Following which time they were transfected into freshly isolated PLM cells, andcultured in the presence or absence of BMP4. Disaccharide containing preparations115retained a minimum of —80% transfection activity following 1 month of storage,however standard formulations showed a 40 fold reduction in transfectionefficiency (Fig. 3.1A). Unexpectedly, after 3 years of storage the disaccharide-containing transfection mixtures still retained robust activity that was > 15,000 foldhigher than control transfections (Fig. 3.IA). It is difficult to make a directcomparison between the 2 groups (1 month versus 3 year storage), as serum lot andtransfection lots are different. Nonetheless, in comparison to control transfections,the addition of either disaccharide (trehalose afforded a 4-fold greater protectionthan sucrose) enabled short or long-term storage of the DNAltransfection mixturewith minimal loss of activity (Fig. 3.IA). Further, as observed in the fresh and short-term transfections, BMP4 exhibited similar trends in biological activity in thedisaccharide-containing 3-year stored DNA/transfection mixtures. Together, theseresults demonstrate that supplementation of transfection mixtures with trehaloseimproves transfection efficiency in addition to permitting long-term storage withminimal loss of transfection activity.The increased transfection efficiency afforded by the inclusion of trehaloseinto the transfection reagent was key in enabling implementation of screens in 384-well plates. Previous attempts in adopting this higher-density format were met withproblems related to reduced transfection efficiency, and consequently highervariability and lower signal/noise ratios (Garcha and Underhill, unpublished).Transfection methodology and cell density was optimized for the 384-well format(Fig. 3.2A). Optimization was carried out with a series of co-transfections withknown chondrogenic-modulatory genes at various cell densities and compared to116activity in assays conducted in standard 24-well format (Fig. 3.2B). Consistent withprevious studies, modulation of the retinoid and/or TGF/BMP signaling pathwaysaffected activity of the SOX5/6/9-responsive reporter (Fig. 3.2B) (Hoffman et al.,2006; Weston et al., 2002). Inhibition of retinoid signaling (Cyp26al) or activation ofBMP signaling (BMP4, Bmprlb) was associated with increased reporter geneactivity and vice versa, and this was in accordance with the previously reportedeffects of these pathways on chondroblast differentiation and cartilage formation(Hoffman et al., 2006). Further, overexpression of Sox5, Sox6 or a gene encodingan activated form of Mkk6 (MKK6E) also increased reporter gene activity as reportedpreviously (Lefebvre et al., 1998; Weston et aI., 2002). In general, the 90,000cells/well in 384-well plates correlated well with the results from the 24-well formatand provided a reliable and robust read-out of S0X516/9 activity.The aforementioned chondrogenic assay was used to execute a forwardchemical genetics screen to identify chondrogenic-modulatory molecules, with theintention that the identity of these molecules would provide insights into themolecular programs regulating chondrogenesis. For these reasons, screens wereinitially carried out with compound libraries where some target information wasavailable, such as natural product and drug libraries. Two collections were screenedincluding, the Prestwick Chemical Library® and the Biomol Natural Products Library.The Prestwick Chemical Library® contains 1120 small molecules, 90% beingmarketed drugs and 10% bioactive alkaloids or related substances, whereascompounds included in the Biomol Natural Products Library (361 compounds)consist of highly purified natural products of known structure and pharmacological117activity. Compounds (— 15 tM) were transferred by manual pinning into 384-wellplates 16 h post-transfection and luciferase activity was measured following 24 hof subsequent incubation. Compounds that reduced Renilla luciferase activity 50%or more (with the exception of 1 compound, Cytochalasin B, see below) in bothreporter gene assays (4X48 and RARE -a retinoic acid response element reportergene—as an indicator of anti-chondrogenic activity) in comparison to controls wereeliminated from further analysis as these were deemed to have cytotoxic activities,this yielded a list of 1418 compounds out of 1481. Based on previous reporter geneanalyses with various factors, a 2.5 fold RLU cut-off was initially used for selection ofmolecules with putative anti-chondrogenic or pro-chondrogenic activity, and 65 and28 compounds were found to meet this criterion, respectively (Fig. 3.3A, B). Of thecompounds that reduced 4X48 activity, several CYP inhibitors were identifiedincluding the azole-containing compounds (i.e. ketoconazole, enilconazole, andbutoconazole nitrate) that also stimulate RARE activity in addition to severalretinoids (i.e. retinoic acid, isotretinoin and 9-cis retinoic acid). Ketoconazole haspreviously been shown to be both a potent inhibitor of chondrogenesis and to reduce4X48 activity in PLM cultures (Hoffman et al., 2006). The 28 “pro-chondrogenic”compounds were further tested for their ability to stimulate chondrogenesis inhistological assays (Fig. 3.3B). Of the 28, 7 compounds were found to stimulatealcian blue staining, while the other compounds were all found to inhibit alcian bluestaining (Fig. 3. 3C). This latter observation may be a consequence of the fact thatcultures stained for alcian blue were treated for 3 days with compound, as opposedto transfected cultures that were exposed for 1 day. These comments118notwithstanding, this screening strategy successfully identified several novelchondrogenic modulators with a hit frequency of 0.5% (7 of 1418 compounds). Theemphasis for subsequent analyses was placed on alcian blue positive hits, asnegative hits while informative could result from modulation of more genericpathways/targets.The seven validated pro-chondrogenic compounds represent chemicallydistinct structures. Of these compounds cytochalasin B exhibited the greatest prochondrogenic activity as measured by reporter gene activity (Fig. 3.3B).Cytochalasin B functions primarily as a cytoskeleton disruptor by interfering withactin polymerization (Cooper, 1987). Interestingly, cytochalasin D shares thisactivity, but in these screens cytochalasin D or E had no effect (data not shown),whereas other reports have shown cytochalasin D stimulates Sox9 expression(Woods et al., 2005). In addition to its actions on actin polymerization, cytochalasinB has been shown to have additional activities distinct from cytochalasin D, whichinclude inhibition of glucose transport (Cooper, 1987). It may be these additionalproperties which are responsible for and/or augment cytochalasin B’sprochondrogenic activities.Butamben (butyl 4-aminobenzoate) another identified pro-chondrogenic smallmolecule has at least 2 targets. Butamben belongs to the benzocaine class ofanalgesics, which are known modulators of sodium and calcium channels(Beekwilder et al., 2006). Therapeutically these compounds are used in the> 100mM range. Interestingly, of this broad class of compounds, only butamben wasfound to significantly increase 4X48 reporter gene activity (Fig. 3.4A). In addition, to119interfering with calcium channels, butamben also inhibits potassium channels inparticular, Kv4.2 (Kcnd2) (Winkelman et al., 2005). This latter property does notappear to be shared by the other structurally-related benzocaine family membersand may partly explain the unique pro-chondrogenic activity of butamben (Fig. 3.4B).The dose range whereby butamben stimulates the 4X48 reporter activity and alcianblue staining is in the low micromolar range consistent with previous studies onbutamben inhibition of potassium channels (Kv4.2 channel, butambenKD 0.06 1iM;35% inhibition) (Winkelman et al., 2005) (Fig. 3.4C, data not shown). In thedeveloping limb, Kcnd2 and Sox9 are dynamically expressed and an increase inSox9 expression in limb sections 2-3 is preceded by a decrease in Kcnd2expression (Fig. 3.4D). Further, knockdown of Kcnd2 in PLM cultures led to amodest increase in Sox9 expression and this was comparable to butambenmediated induction of Sox9 following a I day treatment (Fig. 3.4E, F). To furtherassess the involvement of potassium channels in chondrogenesis, PLM cultureswere treated with the broad-spectrum potassium channel blocker, 4-aminopyridine(4-AP) (Fig. 3.4G). Similar to butamben, 4-AP also stimulated 4X48 activity andcartilage formation as assessed by alcian blue staining (Fig. 3.4G and data notshown). In aggregate, these results suggest that butamben regulateschondrogenesis at least in part through modulation of potassium channel activity.BMP5 are potent regulators of the chondrogenic program, many of whichexhibit robust pro-chondrogenic activity. Query of microarray datasets generatedfrom BMP4-treated PLM cultures (Hoffman et al., 2006), revealed that BMP4treatment reduced the expression of Kcnd2 3 fold and this was subsequently120confirmed using qRT-PCR on PLM cultures (Fig. 3.5A, B). To test if modulation ofKcnd2 by BMP4 was involved in BMP pro-chondrogenic activity, NOGGIN rescueexperiments were performed (Fig. 3.5C, D). NOGGIN a BMP2, 4 and 7 antagonist,interferes with chondrogenesis both in PLM cultures and in vivo (Pizette andNiswander, 2000; Weston et al., 2000). Addition of NOGGIN (200 ng/ml) reduces4X48 activity and decreases cartilage nodule formation in control and BMP4-treatedcultures. The addition of butamben, partially rescues both 4X48 activity andcartilage formation in the presence of NOGGIN (Fig. 3.5C, D). Together, theseresults suggest that within the chondrogenic program, a reduction in KCND2 activitypromotes expression of the chondroblastic phenotype. Further, KCND2 appearstofunction downstream of BMP signaling within chondrogenesis. However, it is notclear if BMPs affect chondrogenesis directly through down-regulation of Kcnd2orperhaps more likely, BMPs are known stimulators of chondroblast differentiation andKcnd2 is simply down-regulated as a consequence of differentiation.Potassium channels play diverse roles in cell physiology and changes in theiractivity have been shown to regulate a number of processes including cellproliferation, differentiation, and death (Burg et al., 2008; Lang et al., 2005; Pardo,2004). Potassium channel activity impacts cell proliferation and inhibitors ofKchannels have been shown to reduce cell proliferation in a variety of cell types(Pardo, 2004), thus butamben could be enhancing chondroblast differentiationthrough interfering with cell cycle progression and proliferation inchond roprogenitors, thereby indirectly stimulating differentiation. Alternatively,potassium channels also regulate cell volume and morphology and drug-induced121changes (such as with the cytochalasins) in chondroprogenitor morphologyhavebeen shown to trigger chondrocyte differentiation (Woods et al., 2005). Butambencould be acting through either of these two mechanisms, a combinationthereof orthrough additional unknown pathways. Notwithstanding, butambenand the otherpro-chondrogenic compounds will serve as useful tools for probingthe pathwaysregulating the chondrogenic program.3.4 Methods and Materials3.4.1 ReagentsBMP4, and NOGGIN recombinant proteins were purchased fromR&DSystems. NOGGIN was resuspended in sterile PBS containing 0.1% BSA,andadded to media at a concentration of 200 ng/ml. BMP4 was resuspended inareconstitution buffer which consisted of 4 mM hydrochloric acidcontaining 0.1%bovine serum albumin and was added to media at a concentration of 20ng/ml.Butamben and 4-aminopyridine were obtained from Sigma, and stock solutionswereprepared in DMSO.3.4.2 Small Molecule LibrariesAll small molecule libraries (Prestwick Chemical Library®, BiomolNaturalProducts Library) screened were kindly provided by Dr. Michel Roberge (Universityof British Columbia) and obtained through the Canadian Chemical Biology Network.All compounds were tested at a final concentration of 15M.3.4.3 Establishment and Transfection of Primary Limb Mesenchymal CulturesPrimary limb mesenchymal (PLM) cultures were established from CD-Imurineembryonic limbs (E1l.5) as previously described (Hoffman et al., 2006;Weston et122al., 2002). Limb mesenchyme was dissociated by dispase treatment and a singlecell suspension was obtained by filtration through a 40 pM cell strainer (BDBiosciences). PLM cells were pelleted by centrifugation at 200 X g and resuspendedto produce a stock cell suspension at a concentration of 2.0 x i07 cells/mi. Cellswere used for transfection (see below) or for establishment of cultures for aician bluestaining. For the latter, 10 jil of cells were spotted into the well of a 24-well plate,allowed to adhere for 1 h, following which culture medium consisting of 60% Ham’sF12 nutrient mix/40 % Dulbecco’s modified Eagle’s medium (DMEM) andsupplemented with 10 % FBS (Qualified, invitrogen) was added to each well; thistime was considered T=0. Cultures were maintained for a period of up to 4 d; tominimize handling, culture media was replaced on alternate days.Transfection of PLM cells in 24 or 384-well format was carried out usingsimilar methodology with Effectene reagent. For stabilization purposes, sucrose ortrehalose (final concentration 0.4 M) was added to the Effectene EC buffer, andestablishment of DNNtransfection mixtures was according to the manufacturersrecommendations. Luciferase reporter genes consist of a firefly reporter gene,pGL3(4X48) or RARE, and a Ron/I/a (Renhlla reniformis) luciferase reporter phRLSV4O to normalize for transfection efficiency. Reporter plasmids containing SOX9binding sites (pGL3[4X48]) or trimerized retinoic acid response elements, RARE-luc(pWlbRARE3tkLuc), were previously described (Weston et al., 2002). A ratio of20:1 of firefly luciferase reporter to Renhla iuciferase was used in all transfections.Co-transfections with genes-of-interest were set-up in a ratio of 3:1 of expressionpiasmid to reporter genes (firefly and Renhlla). For 24-well plates, DNAltransfection123mixtures were combined with PLM cells (2 x i07 cells/mi) and 10 tl of this mixturewas spotted into the centre of the well. Plates were incubated under standard tissueculture conditions for 45 minutes at which time 1.0 ml of primary culture medium wasadded per well. Co-transfections in 384-well plates were carried out in a similarmanner, with the exception that DNA/transfection mixtures were dispensed intowells, followed by media, cells, and an additional aliquot of media as describedbelow.For compound screening, transfections in 384-well plates were prepared inadvance using trehalose containing EC buffer. Briefly, 4 pi ofEffecteneTMcomplexed DNA was dispensed per well of the 384 well plate(s). Plates werecentrifuged for 1 minute at 200 x g, sealed and stored at —20°C until needed. Fortransfection, plates were thawed and 45 tl of primary culture medium was added toeach well. PLM cells were isolated as described previously and 10 tl containing 9 xi0 cells were dispensed into each well. Subsequently, 30 j.iI of primary culturemedium was pipetted into each well. Plates were incubated at 37°C in a humidifiedatmosphere and 5% CO2 for 1 hour at which time culture medium was removed andreplaced with 100 tl of fresh medium. For both plate formats, culture medium wasreplaced 16 hours post-plating with fresh medium. At this time, compounds weremanually pinned (300 nI) into each well using a 96-well dispensing unit. At 24 hourspost-treatment, cells were lysed and luciferase activity was determined accordingtothe manufacturer’s recommendations (Promega).For alcian blue staining, culture medium was aspirated and cells werewashed once with PBS. Cultures were fixed in 95 % ethanol at -20°C overnight.124Fixative was removed by aspiration and cells were sequentially washedonce withPBS, followed by 0.2 M HCI. Cells were stained overnight with a I % Alcian Bluesolution prepared in 0.2 M HCI.3.4.4 RNA Collection From El 1.5 Mouse LimbsUsing Graefe knives, El 1.5 limb buds were serially sectioned and eachregion was transferred to individual microfuge tubes containing 700m1 ofRLT lysisbuffer (Qiagen RNeasy kit). Limb sections were homogenized in the RLT lysisbufferby repeated pipetting, and RNA was isolated as per the manufacturer’s protocol.3.4.5 Quantitative Real-Time PCRTo follow the expression of transcripts for Kcnd2, and Sox9 quantitativerealtime PCR was performed using the 7500 Fast Sequence DetectionSystem (AppliedBiosystems). The primer/probe set used for detection of Sox9 was as described inWeston et al. (2002). For detection of all other transcripts, TaqMan Gene ExpressionAssays (Applied Biosystems) were used. Total RNA was isolated from primarycultures and limb sections as described above, and an aliquot wasreversetranscribed to cDNA using a High Capacity cDNA Archive kit (Applied Biosystems).Quantification was performed using —10 ng of total RNA and the expression of allgenes relative to endogenous rRNA was determinedusing TaqMan RibosomalControl Reagents (Applied Biosystems).3.4.6 Microscopy and Image AcquisitionImages of fixed cultures (in 70% ethanol) were collected at room temperatureusing a dissection microscope (Stemi SVI I Apo, SI .6x objective;Carl ZeissMicrolmaging, Inc.). Color images were acquired witha Qlmaging Retiga 1300i (12-125bit) camera and using Openlab 4 software (Improvision). Photoshop adjustments(brightness/contrast) were applied uniformly to all images.3.4.7 siRNA Knockdown in PLM CulturesKcnd2 knockdown was performed using siRNAs purchased from Dharmacon(catalogue # L-042846-00) with the corresponding siRNA control (catalogue #D-001810-OX), and transfected into PLM cells using LipofectamineTM RNAiMAX(Invitrogen). PLM cells were transfected in suspension with siKcnd2 and 10 il PLMcultures were established as outlined above. For experiments involvingthe collectionof RNA, 5iRNA 12-15 transfected PLM cultures were plated per well of a 6-well plate(Nunc), and 2 ml of media were added one hour post-plating. RNA was collected aspreviously outlined.3.4.8 Statistical AnalysisWith the exception of the small molecule screens, all experiments wereperformed a minimum of 3 times. Luciferase assays were performed inquadruplicate using 3 distinct populations of primary cells. Quantitative real-timePCR was performed in duplicate and repeated a minimum of 3 times withindependent RNA samples. Data were analyzed by one-way analysis of variance,followed by a Bonferroni post-test for multiple comparisons using GraphPad PrismVersion 5.00 (GraphPad Software, Inc.). Significance is representedas follows:*,P<0.05; Pc0.01, and***P< AcknowledgementsWe would like to thank Dr. Michel Roberge (University of British Columbia) forkindly providing us with access to the Prestwick and Biomol compound libraries and126for encouraging us to undertake these experiments. We would also like to thank Dr.John Church (University of British Columbia) for giving advice on K channels andreagents. K. Garcha was supported by pre-doctoral award from the Stem CellNetwork, and this research was funded by a grant to T.M. Underhill from theCanadian Institutes of Health Research (CIHR). TMU holds an Investigator awardfrom The Arthritis Society.1273.6 FiguresFigure 3.1. Disaccharides Such as Sucrose, and to a Greater Extent Trehalose,Increase Transfection Efficiency ofEffecteneTMand Facilitate the Storage ofTransfection-Ready DNA. A, Comparison of transfection efficiency of controlversus disaccharide containing mixtures (S, sucrose; T, trehalose); transfectionefficiency was determined by transfection of a plasmid containing Renilla luciferase(RL) under the control of a SV4O promoter and measurement of RL-activity. PLMcultures treated with BMP4 (2onglml) or vehicle for 24 h showed no difference inreporter gene activity. The addition of 0.4M sucrose or trehalose increased Renhllareporter gene detection by approximately 1.5 and 2.3 fold, respectively in freshpreparations. Following 1 month or 3 years of storage at -20°C,DNAEffecteneTMmixtures containing 0.4M sucrose or trehalose maintained most of their transfectionactivity. Numbers over bars represent ratio of S or T over Ctrl. B, Addition ofsucrose or trehalose had a negligible effect on PLM culture biology and BMP4responsiveness. PLM cultures were co-transfected with pGL3(4X48) and phRL5V40 and treated with BMP4 (20 ng/mI). In all cases, BMP4 increased RLU activityand similar inductions were observed after freezing for I month and to a lesserextent after 3 years of storage. All experiments were performed a minimum of 3times using 3 distinct populations of cells. Error bars represent SD. Significanceversus corresponding control,P<0.001.128AI Month -20° CFresh3+ Years2O0C1>C.)-J>0-JBI4-’C-)D-JCtrI S T CtrI S TBMP4CtrI S TI Month -20° CFresh______________3+ Years -20° CpGL3(4x48)-Luciferase pGL3(4x48)-Luciferase300I4-’C.)200D-J100BMP4BMP4CtrI S T CtrI S T CtrIS T129Figure 3.2. Optimization and Validation of 384-well Format for ChondrogenicAssays in PLM Cultures. Schematic representation of the 24 and 384-welltransfection strategies. A, PLM cells are collected on the day of transfection fromseveral litters of El 1.5 mouse embryos. Previously prepared DNAltransfection (Tf)mixtures are dispensed into tubes, cells added and spotted into a 24-well plate. Fortransfections in 384-well plate, DNA/transfection mixtures are aliquoted into eachwell followed by cells. Reporter gene activity (luciferase, fluorescent protein) ismeasured 48 h post-transfection. B, Optimization of assays in a 384-well format.Cells were co-transfected with plasmids encoding known chondrogenic modulatoryproteins along with pGL3(4X48) and phRL-SV4O, and relative reporter gene activitywas compared between standard 24-well assays versus various cell densities in384-well format. Cell densities were as follows: 24-well: 165000; 384-well: 120000,90000, and 60000 cells per well. Cells were treated with BMP4 (B4) (20 ng/ml) orvehicle control. Data were normalized to vehicle treated control (Ctrl) cultures cotransfected with pCDNA3.1+ and reporter gene. The responses from the 384-wellplates for the most part paralleled that observed in 24-well format. Figure insetshows basal relative reporter gene activity under the various plating conditions.130ATransfection and AssayI...4-’C-)D-JTGFIBMP PathwayPrimary Cell Collection—2 XPLM cellsI litter (10-12—E11.5 embryos)BDNA-Tfmixture24-wellEl 165000I1Ctrl384-wellEl 120000— 90000— 60000500.0Ji- + - +- + - + - + - + - + - + - + - +B4Ctrl Rara- Rarb Cyp26al Smad6 Smad7 Bmprlb Sox5 Sox6 Mkk6EVPI6ftjRetinoid Pathway131Figure 33. Identification of Novel Chondrogenic Modulators using a ChemicalBiology Strategy. A, 1482 compounds were screened in 384-well format for theirability to modulate a chondrogenic- or retinoid-responsive reporter gene,pGL3(4X48) and RARE, respectively. Using a 2.5 fold cut-off on the chondrogenicresponsive reporter gene, 28 compounds were identified; note, cytochalasin B is notshown on this graph. B, Table listing the identity of the 28 compounds and theirrespective abilities to regulate reporter gene activity. C, Secondary screening byalcian blue staining to further test prochondrogenic activity of chemical compoundsfrom table in B. PLM cultures were plated as high density micromass cultures andtreated with “hit” compounds (—15 jiM final concentration) or DMSO vehicle 16 hourspost plating. Culture medium was replaced on day 3 and cultures were stained withalcian blue on day 4. The numbers correspond to the compound numbers (#) in B.Magnification bar, 1 mm.132ABRe porterAlcian— Re orter Alcian# Compound Name 4X48 RARE blue # Compound Name 4X48 RARE blueI Clorgyline HCI 269 173 - 15 Pinocembrin 262 84 -2 5-AMP 304 109 nlc 16 7-Hydroxyflavone 306 177 -3 Apigenin 264 285 - 17 6-Methoxyluteolin 391 156 -4 Tiabendazole 296 260 nlc 18 6,7-Dihydroxyflavone 422 253 +5 Hexetidine 275 82 - 19 Tamarixetine 395 219 -6 Acacetin 238 165 - 20 Genistin 232 118 -7 Phenazopyridine HCI 262 253 + 21 5-Methoxyflavone 300 2198 Butamben 466 135 + 22 Pratol 366 191 -9 Indoprofen 268 186 nlc 23 Rhapontin 263 176 +10 Oxybenzone 510 103 + 24 5,6-Dehydrokawain 300 153 -11 Chrysin 386 220 - 25 Methysticin 351 172 +12 Kaempferol 326 151 + 26 Harringtonine 302 2913 Cyclopiazonic acid 384 65 - 27 Yangonin 367 145 -14 Cytochalasin B 1658 27 + 28 Galangine 406 151 -3500400IC.)!.300D-jI.I.•Co12-Luciferase. RARE-Luciferase.200 -I.-I..I-lip-I— I-. 1.II.0 200II;I I I400 600 800I I I1000 1200 1400C• I 24.;II*ByCtrl11 12.16 17 185 6 •.....7.•%.13 .1419 20 2126 27 2822 • 23:;24133Figure 3.4. The Prochondrogenic Activity of Butamben is Associated With itsPotassium Channel Blocking Capability. A, With the exception of butamben,benzocaine derivatives or related compounds (15iM treatment) exhibit negligiblepro-chondrogenic activity as determined by examination of pGL3(4X48) reportergene activity in PLM cultures. B, Structures of benzocaine and the chemically-related compound, butamben. Note the similarity in their structures, however, anadditional ethyl group is present on the side-chain of butamben in comparison tobenzocaine. C, Treatment of PLM cultures with butamben leads to an increase inpGL3(4X48) reporter gene activity in a dose dependent manner. Control cultures(Ctrl) were treated with DMSO vehicle. Transfected PLM cultures were treated withvarious doses of butamben 16 h post-transfection and reporter gene activity wasmeasured 24 h later. D, Kcnd2 and Sox9 exhibit dynamic expression patterns in theEl 1.5 mouse limb bud. qRT-PCR was used to examine gene expression in limbsections (inset) from the distal (1) to proximal (5) region. Sox9 expression iselevated in more proximal sections and this is congruent with the distal-proximalgradient of increasing chondroblast differentiation. Kcnd2 is expressed to higherlevels in the distal limb and expression declines in more proximal regions. E,Treatment with butamben (5 tiM) also increases Sox9 expression as determined byqRT-PCR in PLM cultures. Cultures were treated for I or 3 days and geneexpression quantified. F, SiRNA-mediated knockdown of Kcnd2 increases Sox9expression in PLM cultures. Knockdown efficiency of<40% was achieved and thiswas accompanied by a modest 20% increase in Sox9 expression. G, Treatment ofPLM cultures with 4-aminopyridine (4-AP), a broad spectrum potassium channel134blocker, increases pGL3(4X48) reporter gene activity. An experimental plan similarto that described in C was used. Significance versus corresponding control;*,P<0.05;**,P < 0.01; P < 0.001.135Benzocaine ButambenA B300dLII200100. .eO OOHHC DB Kcnd26001.2 - —- Sox94001-***TIiiI*********0.6-2001dillill123450.2 -01j1J1203011750- . I I I1 2 3 4 5Butamben [jiM]Section #E F GHHSox9Kcnd2 Sox9 N120•’ft€100100£200*300160•siCtrI DsiKcnd2**120C)80 ** o60ñ 60180-jCLU04040401004)0202010 — 0 0- 0CtrI BAB CtrI BAB0 0.05 0.1 0.25 0.5 1Dl D34-AP [mM]136Figure 3.5. BMP4 Regulate Chondrogenesis Through Regulation of Kcnd2Expression and Consequently Potassium Channel Activity. A, Expressionprofiling of BMP4-treated PLM cultures reveals that BMP4 downregulates theexpression of Kcnd2. B, qRT-PCR confirms that BMP4 (2Ong/ml) decreases Kcnd2expression in PLM cultures in comparison to vehicle controls. Treatment withbutamben (BAB) (15 jtM) showed no appreciable effect on Kcnd2 transcriptabundance. C, The BMP antagonist NOGGIN (200ng/ml) reduced both basal andBMP4-induced pGL3(4X48) reporter activity by 5 and 16-fold, whereas NOGGINonly partially attenuated (0.3 fold) the prochondrogenic activity of BAB (15 jiM). D,Alcian blue staining of treated cultures showed that the addition of butambenpartially rescues chondrogenesis in the presence of NOGGIN. Error bars representSD. Significance is shown as,P < 0.05; P < 0.01;***,P < 0.001. Magnificationbar, 0.5 mm.137.11CtrI BMP4 BAB CtrI BMP4 BABDl D3CtrI NOGGINCtrI .BABA Blu/Z$.0.12502O0150I.0.xLiia 100C,50•0Kcnd2Ii’a)NOEI0zI I I I I0 24 24 72 72 Time (h)- + - +BMP4C D1000IC.)600D-J200***L********I- + - + -+ NOGGINCtrI BMP4 BAB1383.7 ReferencesBeattie, G.M., J.H. Crowe, A.D. Lopez, V. Cirulli, C. Ricordi, and A. Hayek. 1997.Trehalose: a cryoprotectant that enhances recovery and preserves function ofhuman pancreatic islets after long-term storage. Diabetes. 46:519-23.Chen, T., A. Fowler, and M. Toner. 2000. Literature review: supplemented phasediagram of the trehalose-water binary mixture. Cryobiology. 40:277-82.Cooper, J.A. 1987. Effects of cytochalasin and phalloidin on actin. J Cell 8101.105:1473-8.Crowe, J.H., J.F. Carpenter, and L.M. Crowe. 1998. The role of vitrification inanhydrobiosis. Annual Review of Physiology. 60:73-103.Crowe, J.H., S.B. Leslie, and L.M. Crowe. 1994. Is vitrification sufficient to preserveliposomes during freeze-drying? Cryobiology. 31:355-66.Hoffman, L.M., K. Garcha, K. Karamboulas, M.F. Cowan, L.M. Drysdale, W.A.Horton, and T.M. Underhill. 2006. BMP action in skeletogenesis involvesattenuation of retinoid signaling. Journal of Cell Biology. 174:101-13.Lefebvre, V., W. Huang, V.R. Harley, P.N. Goodfellow, and B. de Crombrugghe.1997. SOX9 is a potent activator of the chondrocyte-specific enhancer of thepro alphal(ll) collagen gene. Molecular& Cellular Biology. 17:2336-46.Lefebvre, V., P. Li, and B. de Crombrugghe. 1998. A new long form of Sox5 (LSox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperativelyactivate the type II collagen gene. EMBO Journal. 17:5718-33.139Leslie, S.B., E. Israeli, B. Lighthart, J.H. Crowe, and L.M. Crowe. 1995. Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying. Applied & Environmental Microbiology. 61:3592-7.Ng, L.J., S. Wheatley, G.E. Muscat, J. Conway-Campbell, J. Bowles, E. Wright, D.M.Bell, P.P. Tam, K.S. Cheah, and P. Koopman. 1997. SOX9 binds DNA,activates transcription, and coexpresses with type II collagen duringchondrogenesis in the mouse. Developmental Biology. 183:108-21.Pizeffe, S., and L. Niswander. 2000. BMPs are required at two steps of limbchondrogenesis: formation of prechondrogenic condensations and theirdifferentiation into chondrocytes. Developmental Biology. 219:237-49.Weston, A.D., R.A. Chandraratna, J. Torchia, and T.M. Underhill. 2002.Requirement for RAR-mediated gene repression in skeletal progenitordifferentiation. Journal of Cell Biology. 158:39-51.Weston, A.D., V. Rosen, R.A. Chandraratna, and T.M. Underhill. 2000. Regulation ofskeletal progenitor differentiation by the BMP and retinoid signaling pathways.Journal of Cell Biology. 148:679-90.Winkelman, D.L., C.L. Beck, D.L. Ypey, and M.E. O’Leary. 2005. Inhibition of the Atype K+ channels of dorsal root ganglion neurons by the long-durationanesthetic butamben. JPharmacolExp Ther. 314:1177-86.Woods, A., G. Wang, and F. Beier. 2005. RhoA/ROCK signaling regulates Sox9expression and actin organization during chondrogenesis. J Biol Chem.280:11626-34.140Chapter 44.1 DiscussionOver the past 30 years, genes and their protein products have beenscrutinized for their role in the patterning of the skeletal elements of the limb(Tickle,2003). The importance of the AER at the leading edge of the developinglimb ishighlighted by its function in promoting the proliferation and survival of the underlyingmesenchyme (Lu et al., 2006). Of the signaling networks mediated by the AER, theFGFs are crucial to the primordial mesenchyme and promote cell survivalandmesenchymal cell expansion (Dudley et al., 2002; Niswander and Martin, 1993;Sunet al., 2002a).To further our understanding of the molecular mechanisms underlying FGF4action in the limb, in vitro cultures of limb mesenchymal cells were employed.Observations that FGF4 transiently increased distal mesenchymal (DM) cellproliferation and thereby led to increased mesenchymal condensation, promptedtheinvestigation of the transient nature of this response to FGF4. By gathering unbiasedgenome-wide transcriptional data, an ‘-18-fold increase in the transcriptionalabundance of a cyclin dependent kinase inhibitor (Cdkn2b) was observed withinthefirst 24 hr following treatment. Similarly, anti-proliferative CDKN2B levels increase inresponse to FGF4 in an MEKI -dependent manner.The importance of the AER FGFs is evident in FGF8 loss-of-function mutantmice which present with limb truncations, whereby compound mutations with FGF4,further increase the severity of disrupted limb morphogenesis. These experimentsare complemented by AER removal studies (Sun et al., 2000; Sun et al., 2002b). In141both cases, an increase in mesenchymal cell death is observed resulting intruncated or absent limbs. Previous studies have shown that disruption of NFKB,which is located within the ‘progress zone’, results in limb defects consistent withFGF mutants (Kanegae et al., 1998). Chapter 2 provides evidence for FGF4regulation of NFKB through the activator RIPK4, in a MEKI- dependent manner.Further, inhibition of NFKB negatively impacts cell survival (Fig. 4.1). Collectively,these findings define an important linkage between the FGF and NE-KB signalingpathways, and establish a novel role for FGF5 in chondrogenesis.4.2 Context-Dependent Gene RegulationPrevious studies have demonstrated that transcriptional control of Cdkn2b invarious established cell lines, many of which are tumor derived, is mediated by TGF131(Li et al., 1998; Li et al., 1995). Examination of an FGF4 treated DM microarraydata set, revealed an increase in Tgf-/31 which was validated by qRT-PCR. Theseobservations provided support for previous studies and outlined a simplifiedsequence of events - induction of TGF-j31 by FGF4 led to the observed induction inCDKN2B. To validate the regulation of this gene in the context of the developinglimb mesenchyme, we obtained several luciferase based reporter genes containingfragments of the Cdkn2b promoter region. In stark contrast to the previous studies,TGF-131 failed to induce reporter gene activity in the limb mesenchyme, whereasFGE4 treatment increased reporter gene activity consistent with previous studies (Liet al., 1998; Li et al., 1995). Additionally, qRT-PCR showed that Cdkn2b transcriptabundance was unaltered by TGF-f31 treatment, and the TGF-p1 specific inhibitor(RBI) failed to block the induction of Cdkn2b by FGF4 in this context. In the142developing limb, FGF4 influences the transcription of Cdkn2b -independent of TGF3I signaling.The importance of context as it pertains to genes and their regulation, addsan additional level of complexity. Based on current knowledge, context-dependentgene regulation depends on combinatorial regulation - the principle by which genesare activated or repressed by specific combinations of transcription factors underprecise conditions (Mayo et al., 2006; Tuch et al., 2008). The plasticity of generegulatory networks is of interest because it allows the generation of novelphenotypes. For example, transcription factors that are activated by mitogenactivated protein kinase (MAPK) signaling, as have been shown to be involveddownstream of FGF signaling, regulate very different gene expression programs indifferent cell types (Zeitlinger et al., 2003). Presumably, the binding and activity ofthese transcription factors depends on other factors, some of which include thecombination of other transcription factors, signaling activities, and the cell-typespecific chromatin environment (Michelson, 2002; Tuch et al., 2008).4.3 Identifying Transcription FactorsAs a logical next step in characterizing FGF induction of Cdkn2b, it will benecessary to understand the crosstalk between DNA and transcription factor(s).Candidate transcription factors (FOXOs, HDACI, OCT-I, SMRT, SMADs, SP1, andnumerous others) (Feng et al., 2000; Hitomi et al., 2007; Katayama et al., 2008)have been proposed to regulate the transcription of Cdkn2b however pertainingtothe context-dependent nature of transcription, these candidates will need to betested empirically in PLM cells. In light of the identified MEKI/ERK-dependent143regulation of Cdkn2b, it may be of particular interest to examine transcription factorsthat have been previously identified as being similarly regulated. MEKI/ERKregulated transcription factors include: ETS, ELK-I, STAT1/3, c-MYC/n-MYC, c-EQS(Kang et al., 2005; Lee et al., 2008; Thisse and Thisse, 2005).The promoter region required for activation of transcription of Cdkn2b in PLMcultures has been identified as a 463 bp fragment encompassing the proximalpromoter upstream of the transcriptional start site. Using bioinformatic approachesputative DNA binding motifs of transcription factors that may bind this region of DNAcan be identified. By constructing nested deletions in this promoter region and thenassessing the ability of transcription factors to stimulate reporter gene activity,possible transcription factors mediating Cdkn2b expression can be identified.Previous studies in HaCaT cells, have shown that a minimal 113 bp promotersequence of the Cdkn2b promoter contains binding sites for transcription factor SP1.Eurther these studies have shown that SPI is sufficient for inducing the transcriptionof this gene via TGF-f31 (Li et al., 1998). The importance of context dependent genetranscription needs to be addressed, as in PLM culturesTGF-131 doesnot induceCdkn2b. Eurthermore, overexpression of SPI in PLM cultures does not induce theCdkn2b reporter gene -consisting of 463 bp of the promoter region which includesthe proposed SPI regulatory region (data not shown).DNA-binding proteins can be purified by DNA-affinity column chromatographyand subsequently identified by mass spectroscopy. Thereby, using nested deletionsto identify minimal promoter sequences required for Cdkn2b induction, transcriptionfactors that bind these sequences can be identified. Briefly, promoter fragments are144bound to DNA-affinity beads, incubated with cell lysates, and subsequently purifiedDNA-binding proteins are eluted and analyzed by mass spectroscopy to determinetheir identity. Subsequent protein overexpression and siRNA knockdown of geneexpression can then be used to validate the regulatory role of these transcriptionfactors.FGF4 induced transcription of Cdkn2b is regulated by MEKI/ERK signaling.By comparing known downstream transcription factors in the MEK1/ERK pathwaywith FGF4 treated PLM microarray data, it should be possible to identify likelycandidates involved in the regulation of Cdkn2b. To determine which of the identifiedtranscription factor(s) are involved, a systematic approach using overexpression (orsiRNA knockdown) of candidate transcription factors can be used to determine theeffect of FGF4-mediated induction of Cdkn2b promoter activity.Additionally, the yeast one-hybrid system can be used to identify which of theproposed transcription factors bind the promoter region of Cdkn2b. By generatingfusion proteins consisting of the transcription factors fused to transcription activationdomains (Deplancke et al., 2004), the interaction of these proteins with the Cdkn2bpromoter region can be assessed by reporter gene activity. Identificationof thetranscription factor(s) downstream of MEKI/ERK signaling that regulate Cdkn2btranscription will aid in characterizing this FGF4- induced pathway.4.4 Implications and Applications of Serum-Free Culture StudiesIn Chapter 2, we have shown that in the absence of serum factors, FGF4 cannot only induce DM cell survival, but can also support proliferation, condensationand differentiation of these cells — the hallmarks of limb development. Remarkably,145we have shown that at low-density, chondrogenic cells expand to formcondensations and eventually cartilage. A pillar of the micromass culture method, isthe requirement that limb mesenchymal cells be plated at high density, whereas atlow density they quickly lose their chondrogenic potential (Ahrens et al., 1977). Thisability of FGF4 to facilitate the recapitulation of the chondrogenic program has shedlight on the developmental role of FGF4 in the AER. Previous studies have shownthat following removal of the AER, FGF4 can functionally replace the AER and directthe formation of the limb (Sun et al., 2002b). In this instructive role within the limb,FGF4 governs the interplay between signaling networks present within the overlyingectoderm and within the mesenchyme. As suggested by the “two-signal dynamicspecification model” (Mariani et at., 2008), FGF4 may play a role in maintaining thedistal domain of the limb. Additionally, as the cells begin to condense anddifferentiate, perhaps the role of FGF4 becomes permissive rather than instructiveand simply promotes cell survival and maintenance of the chondrogenic potential.This would be consistent with our serum free studies, in which cells rapidlyproliferate under these conditions, and once a particular density is achieved (usuallyby day 6) pre-cartilage nodules form, indicative of the earliest stages ofchondrogenic differentiation.These newly developed culture conditions provide a unique environmentwithin which to study PLM cultures without the variable effects of animal serum. Thismethod provides a model system to systematically test the importance of otherfactors. Using this method it should be possible to determine the temporalsignificance of signaling events, by sequentially adding in known factors involved in146chondrogenesis. Establishment of SFM cultures (treated with FGF4) for a shortperiod (eg. 4 days) followed by supplementation with known factors expressedduring limb formation and subsequent analyses, should further facilitate delineationof the molecular mechanisms operating in the chondrogenic program.4.5 High-Throughput Design to Aid in the Discovery ofChondrogenic MechanismsTo increase our experimental throughput and to incorporate more unbiasedfunctional screens, efficient methods were developed for following differentiationevents in primary cells. We enhanced our transfection efficiency by taking advantageof the lipid stabilizing effects of disaccharides. The addition of trehalose to theproprietary formulation ofEffecteneTMprovided the most efficient transfectioncondition, but this stabilization also facilitated the storage of transfection complexes.This novel advance afforded us for the first time, the ability to exploit large-scalePLM cell transfection, and observe the effects of small-molecule compounds onreporter gene output (primarily SOX5/6/9-luciferase) (Fig. 4.2).4.6 Small-Molecule Screening to Identify Chondrogenic ModulatorsChemical genetics utilizes diverse small-molecule compounds to delineatebiological events in a manner similar to the strategies of classical genetics involvingmutagenesis — systematically altering one component of a pathway/gene andobserving the outcome (usually phenotype). As this discovery-based methodologyoften involves the screening of thousands of chemical compounds without a specifichypothesis, recently it has only been exploited by the pharmaceutical industry. Thisseemingly “kitchen sink” approach takes diverse libraries of chemical compounds147and screens them in the hopes of discovering a novel compound that affects thebiology of interest.Determining the precise mechanism of action of a small-molecule (ie. itscellular target) remains quite challenging. New bioactive small-molecules areidentified either on the basis of their ability to produce a specific phenotype (‘forwardchemical genetics’) or on their ability to interact with a specific target (‘reversechemical genetics’) (Kawasumi and Nghiem, 2007; Lokey, 2003; Mayer, 2003). Inthe case of forward chemical genetics, the small-molecule’s mechanism of action isoften unknown, whereas in the case of reverse chemical genetics, the small-molecule’s target is presumably known, but its in vivo specificity must beestablished. Cell-permeable small-molecules permit the precise timing of theirdelivery (and duration), whereas mutational studies as well as RNA interference(RNAi) do not. The importance of having temporal control facilitates the developmentof new therapeutics (Eggert et al., 2004) whereby control over drug administrationand duration of its effect can be monitored.The most widely used approach for inhibiting biological pathways on agenome-wide basis is RNAi (Eggert et al., 2004). An important feature of thismethodology is the high degree of specificity to silence the target gene,and therebypermits a systematic approach to identifying the role of each component/gene in apathway. Although the transcriptional profile of small-molecule treatment mayprovide an enormity of data, extrapolating the mechanism of the small-molecule fromthose data has been a challenge and continues to be so. New comparativeapproaches relying on databases of transcriptional profiles are likelyto facilitate this148process and make it more systematic. As such, by grouping uncharacterizedcompounds with those of known mechanism on the basis of the similarity of theirtranscriptional profiles, hypotheses can be generated and tested regarding theirmechanism of action. Similarly, data collected from genome-wide RNAi screens canbe used to support the discovery of “hits”. These experimental approaches are easilyadaptable to the PLM screening technique, and the use of fluorescent protein basedreporter genes in combination with automated microscopy platforms (ie. CellomicsKineticScan) will further accelerate advances in data collection (outlined in Fig. 4.2).4.7 Systems Biology as a Predictive ToolSystems biology represents an analytical approach to define the relationshipsamong all elements within a biological system. Systems may include simplegeneralized cellular processes involving few proteins within one cell or a greatercomplexity involving groups of cells within a tissue working in a coordinated manner(Kawasumi and Nghiem, 2007; Kitano, 2002; Siliang Zhang, 2006). Regardless ofthe application, the goal of systems biology is to establish the relationship betweenthe components that comprise the system. In other words, systems biology attemptsto explain “the whole” in terms of the summation of its “parts”.By using the integrative approach of systems biology, transcriptional profilingdata consisting of thousands of genes can be cross-compared with databasespertaining to identified small-molecule targets, and in our case reporter gene data.From the standpoint of chondrogenesis as outlined in Chapter 3, using either retinoidresponsive or 50X5/619 reporter genes, a database of small-molecule activities (prochondrogenic, anti-chondrogenic) can be easily constructed. To this end, reporter149gene data gathered in Chapter 3 has been compiled. As demonstrated,bioinformatically probing microarray datasets aided in the identification of themolecular basis of one of the many screening “hits”. The pro-chondrogenic activity ofbutamben -a potassium channel (KCND2, Kv4.2) blocker led to the examination oftranscriptional data of BMP4 treated PLM cultures. BMP4 downregulates thetranscriptional abundance of Kcnd2. Functional analyses confirmed that inhibition ofthis particular potassium channel’s (Kv4.2) function stimulated S0X51619 activity andcartilage formation. Additionally, a wide-spectrum potassium channel blocker, 4-aminopyridine, also promoted cartilage formation. The importance of inhibitingpotassium channels during chondrogenesis has never been investigated.The ultimate multidisciplinary goal of a systems biology approach in thisregard would be to construct mapped relationships between compounds and genes,thereby providing greater resolution of the molecular events that govern variouschondrogenic events (eg. proliferation, condensation, differentiation etc.).4.8 Regulation and Potential Role of Kv4.2 Channels inChondrogenesisThe Kv4.x family of potassium channels are expressed in many tissuesthroughout the body and are present in particularly high levels in the brain and heart(Birnbaum et al., 2004). The complex regulation of these channels has beeninvestigated in numerous tissues and organisms. Attempts to determine themechanism(s) regulating Kv4.2 channels have shown protein kinase C (PKC) to beinvolved in suppressing this potassium channel by phosphorylation in ventricularmyocytes (Apkon and Nerbonne, 1988), dendrites in the hippocampus (Hoffman and150Johnston, 1998), and in Xenopus oocytes (Nakamura et aL, 1997). Although the roleof potassium channels has not been characterized in the developing limb,asdemonstrated in Chapter 3, inhibition of Kv4.2 channel activity increaseschondrogenesis. Previous studies have shown that PKC stimulates chondrogenesis(Lim et al., 2003; Lim et al., 2000; Sonn and Solursh, 1993), which may in part bethrough the suppression of Kv4.2 channels as in other tissues.Inhibition of potassium channels leads to an increase in cellular volume dueto the inability of the cells to pump out potassium ions. This increase in osmoticpressure disrupts the cytoskeleton. Previous reports have shown that inhibition ofpotassium channels induces differentiation of cells due to the stresses imposed onthe cytoskeleton by increased cell volume (Goldring et al., 2006; Lang et al., 2007).Interestingly, disruption of the cytoskeleton has also been shown to inducechondrogenesis (Goldring et al., 2006; Lim et al., 2000). As chondrogenic cells beginto differentiate within the condensed mesenchyme, they undergo a state ofhypertrophy in which the cell volume increases by —20 fold (Goldring et al., 2006).Numerous studies have suggested that modulation of potassium channels in avariety of cells promotes cellular differentiation (Biella et al., 2007; Felipeet al.,2006; Iwamoto et al., 2007). Perhaps the most remarkable and convincingexperiment showing the involvement of potassium channels in limb morphogenesis,is a recent report stating that modulation of the expression of potassium channels inXenopus Iaevis embryos results in the generation of additional limbs (Ingber andLevin, 2007). Likely through indirect means, BMP4 causes the downregulation ofKcnd2 expression, and this is evident in the ability of butamben to partially rescue151the phenotype produced by the BMP-antagonist NOGGIN. In light of theseexperiments, the often overlooked role of ion signaling in the context of limbdevelopment needs to be examined to delineate a broader view of cellular eventssuch as cytoskeletal rearrangement and effects on cellular volume.4.9 Impact of ResearchClarification of the function of developmental factors involved inchondrogenesis provides a more complete understanding of the sequence of eventsbeginning with limb bud initiation and terminating with the identifiable adult limb.Through necessity, novel techniques and approaches have been developedthroughout the course of this research. These include: 1) quantitative information viaqRT-PCR of regions which surround bead implants in the developing limb to providein vivo confirmation of molecular events observed in vitro. This technique hasprovided a fast, quantitative alternative to whole mount in situ hybridization (WISH),and permits the detection of subtle changes in gene expression that may otherwisebe overlooked as background signal in a conventional WISH. 2) Establishment ofparameters for low-density serum free PLM cultures that can recapitulate thechondrogenic program under the influence of FGF4. To date, the ability to culturePLM cells under low-density, serum free conditions that permit cartilage formationhas not been reported. As such, the ability to determine the function of FGF or othersignaling molecules in isolation (and combinations) hasnot been assessed untilnow. As an extension of this method, genes of interest (GOl) or siRNAscan betransfected into PLM cells under these conditions to assess their impact onchondrogenesis. 3) Enhancement ofEffecteneTMbased transfections to improve152transfection efficiency and facilitate the storage of transfection-ready DNAcomplexes. PLM cells are challenging to transfect which is in part correlated to thedensity at which PLM cultures are established. As such, the modified transfectionreagent alleviates one constraint imposed by the PLM micromass culture method(>2 fold increase in transfection efficiency).Further, as PLM cells are laboriouslyharvested and transfected on the same day, the ability to prepare DNA transfectioncomplexes well in advance of their use, facilitates large-scale experimentation. 4)Development and application of 384-well based PLM cell transfection schema tofacilitate large-scale gene and chemical compound screens. Isolation of PLM cellsand their subsequent transfection requires an efficient strategy to extract as muchinformation from these valuable populations of cells. Similarly, empirical testing ofGOls requires an efficient means of assessing their possible chondrogenicmodulatory function. This is the only technology currently available to screen genesand small-molecule compound libraries for potential modulators of chondrogenesis.5) Identification of small-molecule compounds that stimulate S0X516/9-reporter geneactivity, in particular butamben, that may lead to novel therapeutics fordiseased/damaged cartilage. Until recently, small-molecule screening has been atool exclusively utilized by the pharmaceutical industry to identify chemicals ofinterest using established cell lines. Here we have shown that in an academicenvironment, using primary cells, “hit” compounds can be identified for their possiblerole in regulating chondrogenesis. Further, we have shown that data obtained via thechemical screen can be analyzed in a comparative manner against transcriptional153data obtained through microarray analyses, thus providing a more completeunderstanding of the mechanisms involved in the biological process.In aggregate, the findings of this research have further elucidated the role ofFGF4 in the developing limb, and provided a new means of identifying andcharacterizing both genes and small-molecule compoundsas they relate tochondrogenesis. Specifically, the requirement of FGF4 to induce mesenchymalproliferation, and survival has been shown, and albeit throughthe contribution ofdifferent genes, both events require downstream activation ofthe MEKI/ERKpathway. By developing new methodology to circumvent the current limitationsof thetransfection efficiency of PLM cells, a new high-throughput primary cell basedapproach has been devised. The data obtained have identified novel small-moleculecompounds that stimulate chondrogenesis, and provided insights into thechondrogenic regulatory function of potassium channels.4.10 Future DirectionsAlthough we have identified CDKN2B as a molecular mechanism thatattenuates FGF4-mediated proliferation, we have yet to define the transcriptionfactor(s) responsible for its regulation. As proposed above, an empirical approachwill be used to search for likely candidates. Using both open access databases andthe available literature as a guide, we can narrow the possibilities.To investigate and validate the proposed roles of various signaling moleculesduring chondrogenesis, the serum-free culture conditions willbe used to evaluatethe impact of signaling proteins. In this regard, it will be of interest to test differentmembers of the FGF family to determine if any overlap in function occurs. In addition154to treatment of SFM cultures with factors, overexpression of genes encoding themand/or other genes of interest will be used to determining their effect on PLMsurvival and differentiation. Although we can pharmacologically block NFiBsignaling and prevent mesenchymal survival, the absolute requirement of itsactivator —RIPK4, needs to be determined. To test this requirement, overexpressionof Ripk4 as well as siRNA knockdown will be evaluated for effects on cell survival.Since the overall goal of this research was to further characterize thechondrogenic program, it is fifing that we have developed a new tool to aid in thediscovery and characterization of chondrogenic networks. Using the chemical-genetics approach, additional small-molecule libraries will be screened using theaforementioned reporter genes. It may be of importance to revisit previouslyscreened libraries using additional reporter genes (eg. CDKN2B or NFKB andothers). By generating datasets for numerous reporter genes relevant inchondrogenesis, the overall understanding of the contribution of multiple pathways inthe chondrogenic program can be evaluated. To date, we have begun the compilinga database consisting of reporter gene data. Additionally, we have amassednumerous microarray datasets. The goal is to use a bioinformatic approach tointegrate these data and categorize our findings, such that initially pro- and antichondrogenic effectors can be grouped, and subsequently, subgroups will begenerated based on the underlying mechanisms of action. Additionally, using the384 well system, a large scale 5iRNA library screen will likely prove invaluable asspecific targets will be the focus of analysis and these data will also be incorporatedinto our database. With reference to Chapter 3, using this methodology both BMP4155and butamben would have been initially grouped for prochondrogenic activity, andthen subgrouped based on their ability to inhibit potassium channel Kv4.2 activity.Interestingly, this gives rise to the possibility of bioinformatically prescreening andperhaps using customized targeted small-molecule libraries prior to experimentation.Despite the apparent appeal of this approach, actual biological events my dictate therequirement of a more empirical approach, especially when the targets of the small-molecules are unknown. Similarly, although enhancing/inhibiting the activity of abiological process may yield a desired phenotype, the downstream effects need tobe addressed. As such, the downstream effects of inhibiting Kv4.2 (KCND2) activityneed to be identified.The establishment of a cross-referenced database, consisting of bothtranscriptional and reporter gene datasets will undoubtedly increase our globalunderstanding of signaling events during chondrogenesis.4.11 Concluding RemarksThe combined approach of biology and technology has arisen out ofnecessity. As the focus of chapter 2, we showed a step-wise progression from anobserved biological phenotype (FGF4 induced transient proliferation of PLM cells)which led to a hypothesis (involvement of a cell cycle inhibitor), followed by theidentification of a gene of interest (using bioinformatics). Subsequent validationstudies confirmed the transcription, translation and determined the mechanism bywhich this GOl was regulated (MEKI/ERK). The prime focus was on one gene,however consequently in this pursuit we identified numerous others. The approachof Chapter 3 was discovery-based with the hypothesis that of the chemical156compounds screened, modulators of chondrogenesis would be identified. The needexisted for a new way of approaching the dilemma of having an overabundance oftranscriptional data obtained through bioinformatic data mining of microarrays. Assuch, we developed a high-throughput transfection technology to aid incharacterizing the chondrogenic role of hundreds, if not thousands of genes eitheralone or in combination. This technology was easily adaptable to small-moleculescreening, and led us to numerous lead compounds. This technological approachidentified a particular biological event (inhibition of Kv4.2 channel function), whichhad not been previously identified as involved in cartilage formation. It is throughboth traditional and novel approaches that we have gained new insights into thebasic molecular programs regulating limb skeletogenesis.“Technology will continue to drive biology, and biology will continue to drivetechnology. The emergence of noteworthy techniques and pivotal findings requiresthat the funding and facilities to pursue imaginative ideas be available and that thosealong the whole spectrum of knowledge be encouraged to participate together. Andthose who are trained in this spirit may make the most remarkablecontributions. “(Fields, 2001)1574.12 FiguresFigure 4.1. Schematic Summary of FGF4 Mediated Signal Transduction in PLMCultures as Evidenced in Chapter 2. FGF4 bind to its receptor and inducesdownstream targets to activate the MAP kinase signaling cascade. MEK/ERKsignaling activates transcription of Cdkn2b and Ripk4, which can be blocked byU0126 - a MEK inhibitor. CDKN2B accumulation in the cytosol inhibits mesenchymalcell proliferation by binding to CDK 4 or 6 and inducing allosteric changes thatabrogate the binding of CDKs to Cyclin D. RIPK4 activates NFKB, whichsubsequently translocates to the nucleus and promotes cell survival.158Extracellular- 01 CDCytosol NucleusERK(MAPK)RAFCyclinDCDKN2BICDK4I6(MAPKKK)U0126—1MEK(MAPKK)cellproliferationRIPK4)NFKB)cellsurvival1BAYI1-7082TFI)Cdkn2bTFI)‘Ripk4Figure 4.2. Overview of the Strategies Developed to Efficiently Assess the Roleof Genes and Compounds. Primary limb mesenchymal (PLM) cells are harvestedfrom El 1.5 murine embryos. Previously prepared transfections are spotted into 384-well plates followed by primary cells and culture medium. The luciferase-basedscreen provides a high degree of sensitivity and dynamic range, thus enablingreliable detection of very small changes in reporter gene activity. Potential “hits” thatmodulate reporter gene activity at least 2.5 fold are further evaluated forchondrogenic function. The secondary screen, relies on histological staining usingalcian blue, to validate pro/anti-chondrogenic factors. The tertiary screen isperformed using the Cellomics KineticScan Reader (KSR) which provides a greateramount of biological information via a fluorescent reporter; however, sensitivity anddynamic range are reduced.160OverviewofScreeningStrategiesDevelopedtoEfficientlyAssesstheRoleofGenesIdentifiedThroughBioinformaticsCellCollectionTransfectionReactionsPrimaryScreen:Luciferase-based-mediumthroughput(100-4,000weilslday)-end-pointanalysis(fireflyandrenillaluciferases)-limitedbiologicalinformation-decentdynamicrange-highsensitivitydissociate384-wellplate(5-7plateslexpt.)(—500-700transfectionsperdayinquadruplicate)I,SSaw:y-basedV-1,000+cDNAs-freezeforstorage-reportergene(s)(lucorFP)CellomicsKineticScanReader-mediumthroughput(>3,000wells/day)-noreagentcosts-multipleend-points-high-content(i.e.cellcycle,clustering,migration,morphology,distribution,etc.)-mediumsensitivityremovebuds—E11.5mix,plateanddispensemedia- 0)ControlTreated4.13 References:Ahrens, P.B., M. Solursh, and R.S. Reiter. 1977. 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