1. Role of Cilia in Heart
Development
Tasnuva Humaira
Molecular Medicine 2015/16
Academic Supervisors: Dr Timothy J Chico and Dr Jarema Malicki
Department of Infection, Immunity & Cardiovascular Disease
University of Sheffield
2. Background: Cilia
Cilia are protuberant, thick organelles present in Eukaryotic cells
Two types:
- primary or non motile cilia
- motile cilia
Primary cilia were discovered in 18981
Each mammalian cell has one primary ciliam
Important 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.
3. Background: Primary Cilia
Some examples of ciliary protein mutation
Mutation in tg737 & pkd1 results in altered
sheer stress response2
2.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, 1161-1171
3. 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 4
4. 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, 66-77
5. 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,
3070-3082
Mutation in gmap210 affects ciliary transport
process3
Mutation in Rab8, Rab11, ArI13b affects
ciliary endocytic machinery4
Mutation in transcription factors Ptch-1, Gli,
Smo disrupt signalling for continuous cellular
turnover5Schematic representation of ciliary structure
5. Background: Zebrafish to see effects of cilia
6.. 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, 175-187
7. 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, 166-180
8. 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, 864-875
Use of Zebrafish in this project
• Presence of cilia in Transgenic (Tg) embryos
• Effects of global and/or cell specific ciliary mutation in Zebrafish
> Asymmetric fluid flow resulting in disruption of L-R axis of organs6
> Depleted hspb7 and hspb12 expression lead to cellular migration defects of
the heart7
> Signalling pathways regulating vascular integrity8
Few examples of using Zebrafish (Danio rerio) to see ciliary effects
6. Background: Zebrafish variants for the project
• Mutation 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 Elipsa
Created to observe the effects of cilia depletion
8. Aims
Aim 1
To image cilia in the developing heart, quantifying number and site of cilia
Aim 2
Examining the effects of global Elipsa mutation on Zebrafish cardiac
development
Aim 3
To establish the efficiency and effect of a novel conditional rescue
approach for Elipsa mutation in Zebrafish
9. Methodology: Cilia in heart
Outcrossing of two transgenic lines
> Gene expressed in cilia, arl13b
- Tg arl13b:GFP
- GREEN cilia
X
- Tg kdrl:RFP
- RED vascular endothelial cells (EC)
> Gene expressed in ZF vessels, kdrl
RED vascular structure with GREEN cilia
10. Sample is loaded in capillary tube
Camera takes a stack of images
One image is taken in each plane
Observing Z-stacks seem like a movie
iiiicrossing the sample
Selective Plane Illumination Microscopy
Images are collected in Z-plane,
hence the name Z-stacks
Methodology: Cilia in heart
11. Methodology: Cardiac Development in ift54 mutant
Step 1: Incrossing ift54 mutants
i. -/- (Mutant)
Step 2: Observing HR and chambers under high power Stereomicroscope
ii. +/- (Heterozygotes)
iii. +/+ (Wild Type)
Observing heart rate (HR) and heart chamber parameters to see its effects
> Progeny are
Step 3: Genotyping the samples to confirm their genetic backgrounds
14. ift54 mutant (Elipsa) Conditional Rescue
Term to identify Elipsa mutant expressing mutation only in EC
Defining ciliary involvement in organ development
Methodology: Conditional rescue
15. ift54 mutant (Elipsa) Conditional Rescue
Term to identify Elipsa mutant expressing mutation only in EC
Defining ciliary involvement in organ development, it is considered that
Methodology: Conditional rescue
Concluding
Mutation to eliminate cilia comes with severe pleitotropic outcomes
Fatality from ift54 mutation
16. Crossed with
The result
Schematic representation of the conditional rescue*
*Information and diagrams collected from Stone Elworthy
Without
fatality
Methodology: Conditional rescue
Floxing: “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
17. Methodology: Conditional rescue
Step 1: Screening according to phenotype
Phenotype difference starts to show by 3dpf
Step 2: Observing their survival from 1dpf-5dpf
Step 3: Genotyping the samples to confirm their genetic backgrounds
19. Cilia present in the heart at 2dpf
Results: Cilia in heart
Z-stacks are going from the bottom of
the heart to the top, crossing the
chambers and atrio-ventricular valve
20. Trend toward slightly reduced HR in
Curly Vs Straight phenotype
n= 15-30/group
Analysis done in GraphPad Prism 7.0, one-way Anova
PCR and sequencing identified all
curly ones as Elipsa mutant (-/-)
1
d
p
f
W
T
o
r
+
/-
1
d
p
f-/-2
d
p
f
W
T
o
r
+
/-
2
d
p
f-/-3
d
p
f
W
T
o
r
+
/-
3
d
p
f-/-4
d
p
f
W
T
o
r
+
/-
4
d
p
f-/-5
d
p
f
W
T
o
r
+
/-
5
d
p
f-/-
0
5 0
1 0 0
1 5 0
ift5 4 m u ta n ts : H e a rt ra te
Beats/min
Results: Cardiac Development in ift54 mutant
21. During the atrial phases, the size of the chambers is higher in WT or +/-
than the -/-
n= 15-30/group
Analysis done in GraphPad Prism 7.0
Results: Cardiac Development in ift54 mutant
1 2 3 4 5
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
A tria l S y s to le in ift5 4 m u ta n ts
A g e o f e m b ry o s (d p f)
Chambersize(Numberofpixels)
W T o r + /-
-/-
1 2 3 4 5
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
A tria l D ia s to le in ift5 4 m u ta n ts
A g e o f e m b ry o s (d p f)
Chambersize(Numberofpixels)
W T o r + /-
-/-
Analysis of the data, so far
22. n= 15-30/group
Analysis done in GraphPad Prism 7.0
Results: Cardiac Development in ift54 mutant
1 2 3 4 5
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
V e n tric u la r S y s to le in ift5 4 m u ta n ts
A g e o f e m b ry o s (d p f)
Chambersize(Numberofpixels)
W T o r + /-
-/-
1 2 3 4 5
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
5 0 0 0 0
V e n tric u la r D ia s to le in ift5 4 m u ta n ts
A g e o f e m b ry o s (d p f)
Chambersize(Numberofpixels)
W T o r + /-
-/-
Same phenomenon was observed in ventricular phases
Trend of slight raised chamber size in 3dpf in -/-
Analysis of the data, so far
23. Collected videos showed the difference in chamber sizes
WT or +/- -/-
Results: Cardiac Development in ift54 mutant
24. Sequencing result Phenotype
73 Homozygous Mutants Curly tail
96 Wild Type or
Heterozygotes
Straight tail
Analysis done blinded
Genotyping of the imaged embryos confirmation of their genetic background
SnapGene software was used to analysis the sequencing results
Similar to the gathered
data while observing them
Results: Cardiac Development in ift54 mutant
25. After noting the phenotype that were differentiating -/- from WT and +/- at 3dpf
4/4
Genotyping to confirm genetic background and to see if the conditionally
rescued ift54 mutant were alive after 5dpf
Red vasculature expressing kdrl:cre
Results: Conditional rescue
- Survival of the progeny was followed up to 5dpf
SPIM imaging to see if they have RED vasculature (mutation only in EC),
showing effective cre-lox with kdrl, confirming successful floxing
26. After noting the phenotype that were differentiating -/- from WT and +/- at 3dpf
4/4
Genotyping to confirm genetic background and to see if the conditionally
rescued ift54 mutant were alive after 5dpf, awaiting sequencing result
Red vasculature expressing kdrl:cre
Results: Conditional rescue
- Survival of the progeny was followed up to 5dpf
SPIM imaging to see if they have RED vasculature (mutation only in EC),
showing effective cre-lox with kdrl, confirming successful floxing
27. Conditionally rescued Elipsa embryos were observed till 5dpf age
The survival graph shows a marginal high number of deaths between 3dpf and 4dpf
Analysis done in GraphPad Prism 7.0
Results: Conditional rescue
1 2 3 4 5
0
5 0
1 0 0
S u rv iv a l p ro p o rtio n s : ift5 4 m u ta n ts
A g e o f e m b ry o s (d p f)
Percentsurvival
S tra ig h t ta il
C urly tail
28. Conclusion
Conclusion and future work: Cilia in heart
> Cilia were observed to be present in heart at 1-3dpf
Future work
> Increase the sample size of imaged embryos at each time point
> Vascular 3D imaging to localize and quantify cilia
29. Conclusion and future work:
Cardiac development in ift54 mutants
Conclusion
> Global ift54 mutants appear to have smaller cardiac chambers
than their heterozygote or WT siblings
Future work
> Collected 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
30. Conclusion and future work: Conditional rescue
Conclusion
> Survival data of 0-5dpf, imaging and genotype of the conditionally
rescued ift54 mutants showed the rescue approach was successful
> Repeat experiment, keeping offspring from each pair separate, and
comparing the percentage of each genotype per parent pair
Future work
31. Acknowledgements
Dr Timothy J Chico and Dr Jarema Malicki, for their guidance,
knowledge and the wonderful opportunity to work in this field
Karen Plant and Dr Richard Macguire, for their precious time for
training, instructions and encouragement
Karishma Chhabria, Stone Elworthy, Pradeep Kaul and everybody else
in C10 laboratory, room C06 and aquaria staff
For being our safety net!!
32. My upmost gratitude to the audience for being patient as I was presenting
I will be delighted to answer the questions you have!
Editor's Notes
Cilia are protuberant, thick organelle present in Eukaryotic cells
There 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
Cilia are protuberant, thick organelle present in Eukaryotic cells
There 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
Transparent embryos
Large number of offspring from each pair
Quick developmental process
Easy genetic manipulation
Widely known genome
Repeated use of same embryo/fish
Cost effectiveness
Structural similarity of ZF heart with human heart
Similar genes warking on heart in ZF and human
Transparent embryos
Large number of offspring from each pair
Quick developmental process
Easy genetic manipulation
Widely known genome
Repeated use of same embryo/fish
Cost effectiveness
Structural similarity of ZF heart with human heart
Similar genes warking on heart in ZF and human
Transparent embryos
Large number of offspring from each pair
Quick developmental process
Easy genetic manipulation
Widely known genome
Repeated use of same embryo/fish
Cost effectiveness
Structural similarity of ZF heart with human heart
Similar genes warking on heart in ZF and human
- 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
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
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
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
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
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
Imaging cilia at different time points with number and quantification
Imaging cilia at different time points with number and quantification
Green heart: Indicates a single transgene insertion
Red 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
Green heart: Indicates a single transgene insertion
Red 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