Tahani BaakdhahTahani Baakdhah
Supervisor: Dr.Jens FranckSupervisor: Dr.Jens Franck
Department of BiologyDepartment of Bio...
OVERVIEW
Introduction
Gene duplication and RyR expression
Medaka fish as a model organism
Objectives
Methods
Results
Discu...
Ryanodine receptors (RyR) are large intracellular
Ca²+
release channels
Homotetramer of 2.2 million Daltons
Mammals encode three RyRs
RyR1 (skeletal) → DICR
RyR2 (cardiac) → CICR
RyR3 (ubiquitous) → CICR
Medaka RyRs
RyR1a/RyR1b
Ry...
590 Mya
440 Mya
GENEDUPLICATION
Gene Duplication
590 Mya
440 Mya
110 Mya 80 Mya
Gene Duplication
Speciation
DuplicationDuplication
Medaka RyR1Fugu RyR1
RyR1bRyR1aRyR1a RyR1b
Co-orthologues
RyR1
RyR1a RyR1b
RyR3
RyR3a RyR3b
Slow Twitch
Muscle
Fast Twitch
Muscle
? ?
Gene Subfunctionialization
1. Search for conserved noncoding sequences
(CNSs) and conserved noncoding elements
(CNEs) in RyR orthologues (zebrafish, ...
Medaka fish is native to the freshwater of Japan and
east Asia
Medaka is considered a good model for studying
gene regulat...
1. Search for conserved noncoding sequences
(CNSs) and conserved noncoding elements
(CNEs) in RyR orthologues (zebrafish, ...
Methods
Bioinformatics:
Medaka , fugu, zebrafish, human and
mouse intron sequences were obtained from
genome databases
Bio...
Hypothesis
1. Evidence of conservation between orthologues
2. Evidence of divergence between paralogues
Conserved noncodin...
Conserved Noncoding Sequences –
RyR1a Co-orthologues
Local Amino Acid Alignment –
RyR1a Orthologues
Conserved Noncoding Sequences –
RyR1b Co-orthologues
Conserved Noncoding Sequences –
RyR3a Co-orthologues
Conserved Noncoding Sequences –
RyR3b Co-orthologues
Transcription Factor Binding Sites Conserved Between Fugu
and Medaka RyR1a Co-orthologues
 Higher hits for HNF transcript...
Transcription Factor Binding Sites Conserved Between
Fugu and Medaka RyR1b Co-orthologues
 More hits for HNF transcriptio...
Transcription Factor Binding Sites Conserved Between
Fugu RyR3a and Medaka RyR3a Co-orthologues
 More hits for HNF transc...
Transcription Factor Binding Sites Conserved Between
Fugu and Medaka RyR3b Co-orthologues
 More hits for HNF transcriptio...
1. Search for conserved noncoding sequences
(CNSs) and conserved noncoding elements
(CNEs) in RyR orthologues (zebrafish, ...
Divergence Between Medaka RyR1a and
RyR1b Paralogues
Divergence Between Medaka RyR3a and
RyR3b Paralogues
Summary
A. Conserved noncoding sequences (CNSs)
1. Evidence of conservation between orthologues.
 Medaka – fugu clade hav...
B. Conserved noncoding elements (CNEs)
1. Evidence of conservation between co-
orthologues
 68% 0f CNEs disappeared in th...
1. Search for conserved noncoding sequences
(CNSs) and conserved noncoding elements
(CNEs) in RyR orthologues (zebrafish, ...
RNA Extraction and cDNA synthesis from medaka
tissues
 Developmental stages
 Dissected tissues
Primer design
 Based on ...
End Point PCR
Spatial ExpressionDevelopmental Expression
Methods and Results
Quantitative real time PCR (qRT-PCR)
 Fold expression of RyR paralogues was
calculated using 2-∆∆
CT method
 The express...
 RyR1a show early expression of the RyR1a gene starting
from stage1 to 24
 RyR1b is expressed from stage 25 onward
 sim...
RyR3a/3b first expressed in stage 25-31 (early to mid-
somite stage)
 RyR3a expression increases from stage 32 (late som...
RyR1a and RyR1b Developmental Expression
RyR3a and RyR3b Developmental Expression
RyR1a and RyR1b Expression in Medaka Tissues
Slow-twitch
muscle
Fast-twitch
muscle
RyR3a and RyR3b Expression in Medaka Tissues
Testes
Slow-twitch
muscle
Fast-twitch
muscle
 RyR1a is primarily expressed in the slow-twitch (red)
muscle which forms earlier than fast-twitch (white)
muscle during ...
Stage Fold ratio P-value
36 2.884 0.0011
39-45 3.872 0.0119
Developmental (RyR1a/b)
Developmental (RyR3a/3b)
• Fold criter...
VISTA analyses identifies conserved noncoding
elements (CNEs) in intron sequences of RyR co-
orthologues
CNEs may represen...
• Amplify all CNSs from medaka
genomic DNA to determine if
they work as enhancer
sequences
• Ligation of those regions to ...
Cis-Regulatory divergence and expression of ryanodine receptor paralogues in Medaka (Oryzias latipes)
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Cis-Regulatory divergence and expression of ryanodine receptor paralogues in Medaka (Oryzias latipes)

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Ryanodine receptors (RyRs) are large homotetrameric proteins that in mammals are encoded by three genes: RyR1 in skeletal muscle; RyR2 in cardiac and smooth muscle; and RyR3 which is expressed in a diversity of cell types. RyR channels play a central role in the excitation-contraction (EC) coupling process by mediating Ca²+ release from the sarcoplasmic reticulum (SR). RyR1 paralogues are expressed in a fiber type-specific manner in fish skeletal muscles: RyR1a in slow-twitch skeletal muscle (red muscle) and RyR1b in fast-twitch skeletal muscle (white muscle). RyR1a and RyR1b are classic examples of spatial subfunctionalization, since they share an ancestral function, yet are expressed differentially in red and white muscle fibres respectively. Gene duplication and subsequent divergence in sequence, expression and interactions are considered to be one of the major driving forces in the evolution of diversity. After the upstream promoter regions, evolutionarily conserved introns are considered the second most important sites containing gene regulatory elements that control tissue-specific expression (gene enhancers or gene silencers). Using medaka (Oryzias latipes) as a model organism, I searched the noncoding sequences in medaka RyR1 and RyR3 genes to look for conserved noncoding elements for RyR co-orthologues and paralogues. The bioinformatic analyses revealed evidence of conservation of noncoding elements for RyR co-orthologues and divergence between RyR paralogues. I also analyzed the spatial and developmental expression of the RyR paralogues (RyR1a/RyR1b; RyR3a/RyR3b) in medaka. The expression analyses revealed conserved expression patterns for the RyR co-orthologues and divergent expression of the RyR paralogues.

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Cis-Regulatory divergence and expression of ryanodine receptor paralogues in Medaka (Oryzias latipes)

  1. 1. Tahani BaakdhahTahani Baakdhah Supervisor: Dr.Jens FranckSupervisor: Dr.Jens Franck Department of BiologyDepartment of Biology University of WinnipegUniversity of Winnipeg
  2. 2. OVERVIEW Introduction Gene duplication and RyR expression Medaka fish as a model organism Objectives Methods Results Discussion Summary
  3. 3. Ryanodine receptors (RyR) are large intracellular Ca²+ release channels Homotetramer of 2.2 million Daltons
  4. 4. Mammals encode three RyRs RyR1 (skeletal) → DICR RyR2 (cardiac) → CICR RyR3 (ubiquitous) → CICR Medaka RyRs RyR1a/RyR1b RyR2 RyR3a/RyR3b
  5. 5. 590 Mya 440 Mya GENEDUPLICATION Gene Duplication 590 Mya 440 Mya 110 Mya 80 Mya
  6. 6. Gene Duplication
  7. 7. Speciation DuplicationDuplication Medaka RyR1Fugu RyR1 RyR1bRyR1aRyR1a RyR1b Co-orthologues
  8. 8. RyR1 RyR1a RyR1b RyR3 RyR3a RyR3b Slow Twitch Muscle Fast Twitch Muscle ? ? Gene Subfunctionialization
  9. 9. 1. Search for conserved noncoding sequences (CNSs) and conserved noncoding elements (CNEs) in RyR orthologues (zebrafish, medaka, fugu) 2. Investigate divergence of CNSs and CNEs in medaka RyR paralogues (RyR1a vs. RyR1b and RyR3a vs. RyR3b) 3. Determine temporal (developmental) and spatial (tissues) expression patterns for RyR1 and RyR3 in medaka Research Objectives
  10. 10. Medaka fish is native to the freshwater of Japan and east Asia Medaka is considered a good model for studying gene regulation and development Reach sexual maturity within 2 to 2.5 month Both the embryo and chorion are transparent The embryos hatch seven to ten days after fertilization The size of the Medaka genome (800 Mbp) is smaller than the zebrafish genome (1700 Mbp) Medaka as a Model Organism
  11. 11. 1. Search for conserved noncoding sequences (CNSs) and conserved noncoding elements (CNEs) in RyR orthologues (zebrafish, medaka, fugu) 2. Investigate divergence of CNSs and CNEs in medaka RyR paralogues (RyR1a vs. RyR1b and RyR3a vs. RyR3b) 3. Determine the temporal (developmental) and spatial (tissues) expression patterns for RyR1 and RyR3 in medaka Research Objectives
  12. 12. Methods Bioinformatics: Medaka , fugu, zebrafish, human and mouse intron sequences were obtained from genome databases Bioinformatics program (VISTA) was used to identify areas of sequence conservation and transcription factor binding site hits between fugu and medaka
  13. 13. Hypothesis 1. Evidence of conservation between orthologues 2. Evidence of divergence between paralogues Conserved noncoding sequences (CNSs) 50 to 200 bp Similar regions between orthologues Located within the noncoding introns Conserved noncoding elements (CNEs) 5 – 20 bp Contain transcription factors binding sites Found within CNSs
  14. 14. Conserved Noncoding Sequences – RyR1a Co-orthologues
  15. 15. Local Amino Acid Alignment – RyR1a Orthologues
  16. 16. Conserved Noncoding Sequences – RyR1b Co-orthologues
  17. 17. Conserved Noncoding Sequences – RyR3a Co-orthologues
  18. 18. Conserved Noncoding Sequences – RyR3b Co-orthologues
  19. 19. Transcription Factor Binding Sites Conserved Between Fugu and Medaka RyR1a Co-orthologues  Higher hits for HNF transcription factors (development and organogenesis) Less hits for muscle transcription factors
  20. 20. Transcription Factor Binding Sites Conserved Between Fugu and Medaka RyR1b Co-orthologues  More hits for HNF transcription factors (development and organogenesis) and muscle transcription factors (myogenesis) compared to other TFBS hits
  21. 21. Transcription Factor Binding Sites Conserved Between Fugu RyR3a and Medaka RyR3a Co-orthologues  More hits for HNF transcription factors (development and organogenesis) Less hits for EVI1 transcription factors (neurogenesis)
  22. 22. Transcription Factor Binding Sites Conserved Between Fugu and Medaka RyR3b Co-orthologues  More hits for HNF transcription factors (development and organogenesis) and EVI1 transcription factors (neurogenesis)
  23. 23. 1. Search for conserved noncoding sequences (CNSs) and conserved noncoding elements (CNEs) in RyR orthologues (zebrafish, medaka, fugu) 2. Investigate divergence of CNSs and CNEs in medaka RyR paralogues (RyR1a vs. RyR1b and RyR3a vs. RyR3b) 3. Determine temporal (developmental) and spatial (tissues) expression patterns for RyR1 and RyR3 in medaka Research Objectives
  24. 24. Divergence Between Medaka RyR1a and RyR1b Paralogues
  25. 25. Divergence Between Medaka RyR3a and RyR3b Paralogues
  26. 26. Summary A. Conserved noncoding sequences (CNSs) 1. Evidence of conservation between orthologues.  Medaka – fugu clade have more conserved noncoding regions than fugu – zebrafish  This could be explained by the closer relationship between medaka and fugu that diverged from zebrafish 110 Mya 2. Evidence of divergence between paralogues.  Lack of sequence similarity suggests rapid divergence of RyR noncoding regions followed by fixation of cis-regulatory elements
  27. 27. B. Conserved noncoding elements (CNEs) 1. Evidence of conservation between co- orthologues  68% 0f CNEs disappeared in the common ancestor before diversification of teleost fish  The remaining elements were conserved by positive selection 2. Evidence of divergence between paralogues  Fish-specific genome duplication (FSGD) in the ancestor of teleost fish is considered to be responsible for diversification of teleost fish  FSGD triggered an accelerated rate of nucleotide substitutions resulting in rapid divergence of CNEs Summary
  28. 28. 1. Search for conserved noncoding sequences (CNSs) and conserved noncoding elements (CNEs) in RyR orthologues (zebrafish, medaka, fugu) 2. Investigate divergence of CNSs and CNEs in medaka RyR paralogues (RyR1a vs. RyR1b and RyR3a vs. RyR3b) 3. Determine temporal (developmental) and spatial (tissue) expression patterns for RyR1 and RyR3 genes in medaka Research Objectives
  29. 29. RNA Extraction and cDNA synthesis from medaka tissues  Developmental stages  Dissected tissues Primer design  Based on database sequences Methods and Results
  30. 30. End Point PCR Spatial ExpressionDevelopmental Expression Methods and Results
  31. 31. Quantitative real time PCR (qRT-PCR)  Fold expression of RyR paralogues was calculated using 2-∆∆ CT method  The expression was measured relative to the average of 18S rRNA and β-actin housekeeping gene expression  Tissue with the highest Ct value was used as a calibrator Methods and Results
  32. 32.  RyR1a show early expression of the RyR1a gene starting from stage1 to 24  RyR1b is expressed from stage 25 onward  similar to zebrafish RyR1a and RyR1b temporal expression (Wu, 2011) Day 1: stage1-stage 24 ( early segmentation stage) Day 2: stage 25- stage 28 (Late segmentation stage)
  33. 33. RyR3a/3b first expressed in stage 25-31 (early to mid- somite stage)  RyR3a expression increases from stage 32 (late somite stage) onward up to adulthood RyR3b is expressed at low levels in early developmental stages and starts to increase significantly from stage 35 up to adulthood Day 2: stage 25- stage 28 (Late segmentation stage)
  34. 34. RyR1a and RyR1b Developmental Expression
  35. 35. RyR3a and RyR3b Developmental Expression
  36. 36. RyR1a and RyR1b Expression in Medaka Tissues Slow-twitch muscle Fast-twitch muscle
  37. 37. RyR3a and RyR3b Expression in Medaka Tissues Testes Slow-twitch muscle Fast-twitch muscle
  38. 38.  RyR1a is primarily expressed in the slow-twitch (red) muscle which forms earlier than fast-twitch (white) muscle during development  RyR3b is the predominant RyR3 paralogue found in brain and spinal column tissues The early expression of RyR1a, RyR1b, RyR3a and RyR3b suggest their functional significance during early stages of development Developmental Expression Spatial Expression Summary
  39. 39. Stage Fold ratio P-value 36 2.884 0.0011 39-45 3.872 0.0119 Developmental (RyR1a/b) Developmental (RyR3a/3b) • Fold criteria (fold ratio ≥ 2, P-value < 0.01) (McCarthy and Smyth, 2009) Tissue Fold ratio P-value Heart 9.79 0.01 Spinal column 16.11 0.0001 White muscle 13.457 0.0047 Ovaries 4.528 0.0013 No stages found that meet both criteria Spatial (RyR1a/b) Spatial (RyR3a/b) Tissue Fold ratio P-Value Red muscle 16.073 0.0013 Spinal column 6.357 0.0001 Paralogues Expression Ratio
  40. 40. VISTA analyses identifies conserved noncoding elements (CNEs) in intron sequences of RyR co- orthologues CNEs may represent regulatory domains conserved during the divergence of gene paralogues The temporal and spatial expression patterns show paralogue-specific expression which is consistent for both medaka and zebrafish species Conclusion
  41. 41. • Amplify all CNSs from medaka genomic DNA to determine if they work as enhancer sequences • Ligation of those regions to Tol2 transposase vector • Anti-sense oligo nucleotide that binds to mRNA and block transcription • Knocking down RyR3a/b is important to determine the physiological process that control the channel activity Future Directions

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