11. VL Flashcards
Zebrafish is a model organism for…
…cardian regeneration
What can the cardiac BAF chromatin remodeling complex do?
Directed trans-differentiation of mouse mesoderm to heart tissue by defined factors (performed 2009)
cBAF complex:
Bafc60/Smarcd3b (Wir schließen daraus, dass Baf60c für die Koordinierung eines Genexpressionsprogramms unerlässlich ist, das die grundlegenden funktionellen Eigenschaften von Kardiomyozyten reguliert.)
Gata5
Tbx5
How to search for genes that could mediate lineage reprogramming?
singel cell reconstruction of developmental trajectories during zebrafish embryogenesis
A. Single-cell transcriptomes were collected from zebrafish embryos at 12 developmental stages (colored dots) spanning 3.3 to 12 hpf.
B. t-distributed stochastic neighbor embedding (tSNE) plot of the entire data set, colored by stage (as in A) based on transcriptional similarity. Developmental time was a strong source of variation in the data
C. tSNE plot of data from two stages . Cells from early stages formed large continuums in the plot, whereas more discrete clusters emerged at later stages
D. 1- Transition probabilities are computed from the distances between transcriptomes and used to connect cells with similar gene expression; 2- From a user-defined “root”, pseudotime is calculated as the average number of transitions required to reach each cell from the root; 3- Trajectories from user-defined “tips” back to the root are identified by simulated random walks that are biased toward transitioning to cells younger or equal in pseudotime; 4- URD reconstructs a branching tree structure by joining pairs of trajectories where they pass through the same cells; 5- The data are visualized with a force-directed layout based on cells’ visitation frequency by the random walks from each tip
E. Force-directed layout of early zebrafish embryogenesis, colored by stage (as in A)
Association of developmental trajectories with temporal gene expression patterns
2 Approaches to stimulate heart regeneration
injury: 1. cell therapy with transplanted cells
2. stimulation of endogenous regenetration
Three major cell types are activated with developmental gene expression upon injury
myocardium (gata4), epicardium (raldh2), endocardium (raldh2)
Screening for factors involved in cardiomyocyte proliferation
cmlc2
One can use tomo-seq to Identifies Genes with Spatially Restricted Expression in the Regenerating Heart. How does that technique work?
(C) The tomo-seq procedure: cryoinjured zebrafish ventricles were sectioned from apex to base. RNA from single sections was extracted, followed by reverse transcription and barcoding, after which the samples were pooled for linear amplification and sequence library preparation.
(D) Pairwise correlation between individual sections across all genes.
(E) Hierarchical clustering of Z score transformed expression profiles of all genes with expression peak
Ventricular myosin heavy chain (Vmhc) expression is detected in the developing heart of myl7:GFP transgenic embryos at 2 days post fertilization (arrow), but not in uninjured adult hearts. Vmhc is again expressed in GFP cardiomyocytes at 7 dpi
Bone Morphogenetic Proteins (BMPs) are a group of signaling molecules that belongs to the Transforming Growth Factor-β (TGF-β) superfamily of proteins.
BMP Signaling Is Required for Myocardial Regeneration
and for for Cardiomyocite dedifferentiation
BMP Signaling is Required for Injury-Induced but Not Physiological Cardiomyocyte Proliferation
Screening for active enhancers during regeneration
during regeneration:
(B) Left: fold change in enrichment of H3.3 in promoter regions for genes with increased (Up) or decreased (Down) expression during regeneration. Right: fold change of gene expression for genes either enriched with or depleted of H3.3 at their promoter regions.
(C) In situ hybridization showing transcripts from genes that increase in H3.3 occupancy during regeneration.
Nrg1 expression
Induced Nrg1 expression stimulates overt CM proliferation in the absence of injury. CM H3.3 profiling after 7 days of induced Nrg1 stimulation revealed greater overlap with the profile of regenerating CMs (89%) than that of uninjured CMs (80%)
-> last step: finding motives
