HUMAIRA Tasnuva

Intro

Role of Cilia in Heart DevelopmentTasnuva HumairaMolecular Medicine 2015/16Academic Supervisors: Dr Timothy J Chico and Dr Jarema MalickiDepartment of Infection, Immunity & Cardiovascular DiseaseUniversity of Sheffield

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Background: CiliaCilia are protuberant, thick organelles present in Eukaryotic cellsTwo types:- primary or non motile cilia- motile ciliaPrimary cilia were discovered in 18981Each mammalian cell has one primary ciliamImportant for cellular signaling and mechanosensation

1. Satir P, Christensen ST. Structure and function of mammalian cilia. Histochemistry and Cell Biology. 2008;129(6):687-693.

Cilia are protuberant, thick organelle present in Eukaryotic cellsThere are two types of cilia, primary (non motile) and motile

Primary cilia were discovered in 18981. Each mammalian cell has one primary ciliam

Primary cilia is important for cellular signaling and mechanosensation

However their involvement with diseases like congenital heart disease, polycystic kidney, genetic ciliary dysfunction augment their roles in organ development1

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Background: Primary Cilia

Some examples of ciliary protein mutationMutation in tg737 & pkd1 results in altered sheer stress response22.Nauli, S.M., Kawanabe, Y., Kaminski, J.J., Pearce, W.J., Ingber, D.E., and Zhou, J. (2008). Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1. Circulation 117, 1161-11713. Follit, J.A., Agustin, J.T.S., Xu, F., Jonassen, J.A., Samtani, R., Lo, C.W., and Pazour, G.J. (2008). The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex. Plos Genetics 44. Kinzel, D., Boldt, K., Davis, E.E., Burtscher, I., Trumbach, D., Diplas, B., Attie-Bitach, T., Wurst, W., Katsanis, N., Ueffing, M., et al. (2010). Pitchfork Regulates Primary Cilia Disassembly and Left-Right Asymmetry. Developmental Cell 19, 66-775. Clement, C.A., Kristensen, S.G., Mollgard, K., Pazour, G.J., Yoder, B.K., Larsen, L.A., and Christensen, S.T. (2009). The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation. Journal of Cell Science 122, 3070-3082Mutation in gmap210 affects ciliary transport process3Mutation in Rab8, Rab11, ArI13b affects ciliary endocytic machinery4Mutation in transcription factors Ptch-1, Gli, Smo disrupt signalling for continuous cellular turnover5Schematic representation of ciliary structure

Cilia are protuberant, thick organelle present in Eukaryotic cellsThere are two types of cilia, primary (non motile) and motile

Primary cilia were discovered in 18981. Each mammalian cell has one primary ciliam

Primary cilia is important for cellular signaling and mechanosensation

However their involvement with diseases like congenital heart disease, polycystic kidney, genetic ciliary dysfunction augment their roles in organ development1

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Background: Zebrafish in cardiac development

Animal model to observe cardiac development

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Background: Zebrafish to see effects of cilia6.. Fox, C., Manning, M.L., and Amack, J.D. (2015). Quantitative description of fluid flows produced by left-right cilia in zebrafish. Methods in cell biology 127, 175-1877. Lahvic, J.L., Ji, Y., Marin, P., Zuflacht, J.P., Springel, M.W., Wosen, J.E., Davis, L., Hutson, L.D., Amack, J.D., and Marvin, M.J. (2013). Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish. Developmental Biology 384, 166-1808. Kallakuri, S., Yu, J.X.A., Li, J.D., Li, Y.Y., Weinstein, B.M., Nicoli, S., and Sun, Z.X. (2015). Endothelial Cilia Are Essential for Developmental Vascular Integrity in Zebrafish. Journal of the American Society of Nephrology 26, 864-875Use of Zebrafish in this projectPresence of cilia in Transgenic (Tg) embryosEffects of global and/or cell specific ciliary mutation in ZebrafishAsymmetric fluid flow resulting in disruption of L-R axis of organs6Depleted hspb7 and hspb12 expression lead to cellular migration defects of the heart7Signalling pathways regulating vascular integrity8Few examples of using Zebrafish (Danio rerio) to see ciliary effects

Transparent embryosLarge number of offspring from each pairQuick developmental processEasy genetic manipulationWidely known genomeRepeated use of same embryo/fishCost effectivenessStructural similarity of ZF heart with human heartSimilar genes warking on heart in ZF and human

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Background: Zebrafish variants for the projectMutation is in ift54, which is important for encoding intraflagellar transport (IFT) protein To investigate the role of cilia in Zebrafish heart development, a number of mutant and Tg variants have been created Mutant variant ElipsaCreated to observe the effects of cilia depletion


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HypothesisGlobal and/or endothelial cell specific Elipsa mutants display defective cardiac development

AimsAim 1To image cilia in the developing heart, quantifying number and site of ciliaAim 2Examining the effects of global Elipsa mutation on Zebrafish cardiac development Aim 3To establish the efficiency and effect of a novel conditional rescue approach for Elipsa mutation in Zebrafish

Methodology: Cilia in heartOutcrossing of two transgenic lines Gene expressed in cilia, arl13b- Tg arl13b:GFP- GREEN ciliaX- Tg kdrl:RFP- RED vascular endothelial cells (EC) Gene expressed in ZF vessels, kdrlRED vascular structure with GREEN cilia


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Sample is loaded in capillary tube

Camera takes a stack of imagesOne image is taken in each plane

Observing Z-stacks seem like a movie iiiicrossing the sample

Selective Plane Illumination MicroscopyImages are collected in Z-plane, hence the name Z-stacksMethodology: Cilia in heart

- The sample is moved through the light sheet- Camera takes a stack of images, one in each plane- The laser stimulates all the fluorophores in that plane- So when all the stacks are observed at once, it is seen as a continuous movie going from one side to another- Enables us to see the whole structure

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Methodology: Cardiac Development in ift54 mutantStep 1: Incrossing ift54 mutants -/- (Mutant)Step 2: Observing HR and chambers under high power Stereomicroscopeii. +/- (Heterozygotes)iii. +/+ (Wild Type)

Observing heart rate (HR) and heart chamber parameters to see its effectsProgeny areStep 3: Genotyping the samples to confirm their genetic backgrounds

TNF RECEPTOR-ASSOCIATED FACTOR 3-INTERACTING PROTEIN 1

The TRAF3IP1 gene encodes the homolog of intraflagellar transport protein IFT54, a subunit of the IFT-B complex that mediates anterograde transport in cilia

Slide info from (7)

Change image of embryo to mine

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Methodology: Cardiac Development in ift54 mutant

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Atrial PhasesVentricular Phases

Methodology: Cardiac Development in ift54 mutant

ift54 mutant (Elipsa) Conditional RescueTerm to identify Elipsa mutant expressing mutation only in ECDefining ciliary involvement in organ developmentMethodology: Conditional rescue



Slide info from (7)


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ift54 mutant (Elipsa) Conditional RescueTerm to identify Elipsa mutant expressing mutation only in ECDefining ciliary involvement in organ development, it is considered thatMethodology: Conditional rescueIf cilia are eliminated, there are some visible defects in organ

ConcludingCILIA are necessaryMutation to eliminate cilia comes with severe pleitotropic outcomesFatality from ift54 mutation



Slide info from (7)


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Crossed with

The resultSchematic representation of the conditional rescue**Information and diagrams collected from Stone ElworthyWithout fatalityMethodology: Conditional rescueFloxing: sandwiching of a DNA sequence between two Lox-P sites Cre-Lox: A site specific recombinase technology used to carry deletion, insertion, translocation and inversions of specific DNA

Tg (ubi:loxp-ift54-loxP-myr-mcherry)

Cre-Lox: A site specific recombinase technology used to carry deletion, insertion, translocation and inversions of specific DNA

Floxing: sandwiching of a DNA sequence between two Lox-P sites

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Methodology: Conditional rescueStep 1: Screening according to phenotype

Phenotype difference starts to show by 3dpfStep 2: Observing their survival from 1dpf-5dpfStep 3: Genotyping the samples to confirm their genetic backgrounds



Slide info from (7)


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Cilia present around the heart at1dpfResults: Cilia in heart

Imaging cilia at different time points with number and quantification

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Cilia present in the heart at 2dpfResults: Cilia in heart

Z-stacks are going from the bottom of the heart to the top, crossing the chambers and atrio-ventricular valve

Imaging cilia at different time points with number and quantification

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Trend toward slightly reduced HR in Curly Vs Straight phenotypen= 15-30/groupAnalysis done in GraphPad Prism 7.0, one-way AnovaPCR and sequencing identified all curly ones as Elipsa mutant (-/-)

Results: Cardiac Development in ift54 mutant

During the atrial phases, the size of the chambers is higher in WT or +/- than the -/- n= 15-30/groupAnalysis done in GraphPad Prism 7.0Results: Cardiac Development in ift54 mutant

Analysis of the data, so far

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n= 15-30/groupAnalysis done in GraphPad Prism 7.0Results: Cardiac Development in ift54 mutant

Same phenomenon was observed in ventricular phasesTrend of slight raised chamber size in 3dpf in -/-Analysis of the data, so far

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Collected videos showed the difference in chamber sizes

WT or +/--/-

Results: Cardiac Development in ift54 mutant

Sequencing resultPhenotype73 Homozygous MutantsCurly tail96 Wild Type or HeterozygotesStraight tail

Analysis done blindedGenotyping of the imaged embryos confirmation of their genetic backgroundSnapGene software was used to analysis the sequencing resultsSimilar to the gathered data while observing themResults: Cardiac Development in ift54 mutant

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After noting the phenotype that were differentiating -/- from WT and +/- at 3dpf4/4Genotyping to confirm genetic background and to see if the conditionally rescued ift54 mutant were alive after 5dpf

Red vasculature expressing kdrl:creResults: Conditional rescue- Survival of the progeny was followed up to 5dpfSPIM imaging to see if they have RED vasculature (mutation only in EC), showing effective cre-lox with kdrl, confirming successful floxing

Green heart: Indicates a single transgene insertionRed vasculature: Indicates rescue of the curly phenotype

Tg (ubi:loxp-ift54-loxP-myr-mcherry) with floxed ift54 driven by ubiquitin promoter, provides replacement ift54 function in ZF homo mutants for elipsa

Cre recombinase mediated excision of the floxed ift54 results in the transgene irreversibly switching to eexpress membrane tethred mcherry in place of ift54.

Crossing new Tg with Tg (kdrl:cre) to specifically eliminate cilia just in endothelial cells and then analyse the vascular phenotype

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After noting the phenotype that were differentiating -/- from WT and +/- at 3dpf4/4Genotyping to confirm genetic background and to see if the conditionally rescued ift54 mutant were alive after 5dpf, awaiting sequencing result

Red vasculature expressing kdrl:creResults: Conditional rescue- Survival of the progeny was followed up to 5dpfSPIM imaging to see if they have RED vasculature (mutation only in EC), showing effective cre-lox with kdrl, confirming successful floxing

Green heart: Indicates a single transgene insertionRed vasculature: Indicates rescue of the curly phenotype

Tg (ubi:loxp-ift54-loxP-myr-mcherry) with floxed ift54 driven by ubiquitin promoter, provides replacement ift54 function in ZF homo mutants for elipsa

Cre recombinase mediated excision of the floxed ift54 results in the transgene irreversibly switching to eexpress membrane tethred mcherry in place of ift54.

Crossing new Tg with Tg (kdrl:cre) to specifically eliminate cilia just in endothelial cells and then analyse the vascular phenotype

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Conditionally rescued Elipsa embryos were observed till 5dpf ageThe survival graph shows a marginal high number of deaths between 3dpf and 4dpfAnalysis done in GraphPad Prism 7.0Results: Conditional rescue

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ConclusionConclusion and future work: Cilia in heartCilia were observed to be present in heart at 1-3dpfFuture workIncrease the sample size of imaged embryos at each time pointVascular 3D imaging to localize and quantify cilia

Conclusion and future work:Cardiac development in ift54 mutantsConclusionGlobal ift54 mutants appear to have smaller cardiac chambers than their heterozygote or WT siblingsFuture workCollected videos will be analyzed to get an idea on how the cardiac conduction travels through the chambers and if it has been affected by the mutation

Conclusion and future work: Conditional rescueConclusionSurvival data of 0-5dpf, imaging and genotype of the conditionally rescued ift54 mutants showed the rescue approach was successfulRepeat experiment, keeping offspring from each pair separate, and comparing the percentage of each genotype per parent pairFuture work

AcknowledgementsDr Timothy J Chico and Dr Jarema Malicki, for their guidance, knowledge and the wonderful opportunity to work in this fieldKaren Plant and Dr Richard Macguire, for their precious time for training, instructions and encouragementKarishma Chhabria, Stone Elworthy, Pradeep Kaul and everybody else in C10 laboratory, room C06 and aquaria staffFor being our safety net!!

My upmost gratitude to the audience for being patient as I was presentingI will be delighted to answer the questions you have!

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HUMAIRA Tasnuva

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