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.

1. Tahani BaakdhahTahani Baakdhah
Supervisor: Dr.Jens FranckSupervisor: Dr.Jens Franck
Department of BiologyDepartment of Biology
University of WinnipegUniversity of Winnipeg

2. OVERVIEW
Introduction
Gene duplication and RyR expression
Medaka fish as a model organism
Objectives
Methods
Results
Discussion
Summary

3. Ryanodine receptors (RyR) are large intracellular
Ca²+
release channels
Homotetramer of 2.2 million Daltons

4. Mammals encode three RyRs
RyR1 (skeletal) → DICR
RyR2 (cardiac) → CICR
RyR3 (ubiquitous) → CICR
Medaka RyRs
RyR1a/RyR1b
RyR2
RyR3a/RyR3b

5. 590 Mya
440 Mya
GENEDUPLICATION
Gene Duplication
590 Mya
440 Mya
110 Mya 80 Mya

6. Gene Duplication

7. Speciation
DuplicationDuplication
Medaka RyR1Fugu RyR1
RyR1bRyR1aRyR1a RyR1b
Co-orthologues

8. RyR1
RyR1a RyR1b
RyR3
RyR3a RyR3b
Slow Twitch
Muscle
Fast Twitch
Muscle
? ?
Gene Subfunctionialization

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. 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. 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. 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. 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. Conserved Noncoding Sequences –
RyR1a Co-orthologues

15. Local Amino Acid Alignment –
RyR1a Orthologues

16. Conserved Noncoding Sequences –
RyR1b Co-orthologues

17. Conserved Noncoding Sequences –
RyR3a Co-orthologues

18. Conserved Noncoding Sequences –
RyR3b Co-orthologues

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. 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. 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. 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. 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. Divergence Between Medaka RyR1a and
RyR1b Paralogues

25. Divergence Between Medaka RyR3a and
RyR3b Paralogues

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. 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. 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. RNA Extraction and cDNA synthesis from medaka
tissues
 Developmental stages
 Dissected tissues
Primer design
 Based on database sequences
Methods and Results

30. End Point PCR
Spatial ExpressionDevelopmental Expression
Methods and Results

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.  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. 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. RyR1a and RyR1b Developmental Expression

35. RyR3a and RyR3b Developmental Expression

36. RyR1a and RyR1b Expression in Medaka Tissues
Slow-twitch
muscle
Fast-twitch
muscle

37. RyR3a and RyR3b Expression in Medaka Tissues
Testes
Slow-twitch
muscle
Fast-twitch
muscle

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. 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. 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. • 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