BmpRE adult heart

Research projects

We use a number of approaches to carry out our research, including creation of numerous mutant and transgenic lines to examine gene function and image cells as they build the heart in real time. In collaboration with Dr. Michael Wilson at SickKids, we use sophisticated genomics approaches to discover genetic elements key to cardiac development and disease. By defining what is conserved between zebrafish and human, a gap that spans over 400 million years of evolution, we are starting to define the critical drivers of heart development and disease. Combined with the explosion of human genome sequences that are available, we aim to pinpoint the key genetic events that lead to cardiovascular disease, and that can be exploited for therapies. Further, we have found that the embryonic heart development program is re-deployed during adult zebrafish cardiac regeneration. We are therefore studying the ability of key developmental genes to drive a regenerative response. 

  

In the long term, this knowledge will be used to devise therapies based on activating regenerative pathways to heal the damaged heart, and using zebrafish embryos to screen for potential drugs to alleviate the burden of congenital heart defects and other cardiovascular diseases, including cerebral cavernous malformations (CCMs). Discovering how zebrafish cardiac progenitors are made and develop will therefore drive future efforts to repair what is at present irreparable heart disease. 

CURRENT PROJECTS
Trainees working on this project:

Dr. Xuefei Yuan, Mengyi Song, Nathan Stutt, Palak Desai

We have a major interest in the earliest events of heart development that regulate how cardiac progenitors, the building blocks of the heart, are first specified and diversified. Our early work with the grinch mutant has shown that function of the Apelin receptor GPCR (Aplnr) is essential for cardiac development, with aplnr/a/b mutants being heartless. This appears to be due to defects in the cardiac lineage at the earliest events of development, as the gastrulation is being initiated. To address this, we have collaborated with the lab of Dr. Michael Wilson at SickKids to undertake single cell RNAseq and ATACseq analysis of the initial stages of cardiac development in both wild type and heartless (aplnra/b and gata5/6 loss-of-function) models. This work will lead to the key gene regulatory networks that govern the earliest events of cardiac lineage specification. 

Patterning of the cardiopharyngeal mesoderm at 13 hours post-fertilization: gata5:GFP-positive cardiac mesoderm is in close proximity to irx1b and cyp26c1 expressing pharyngeal mesoderm.

Patterning of the cardiopharyngeal mesoderm at 13 hours post-fertilization: gata5:GFP-positive cardiac mesoderm is in close proximity to irx1b and cyp26c1 expressing pharyngeal mesoderm. 

Lineage progression of gata5:GFP-positive mesendoderm from 6 to 13 hours post-fertilization (hpf), assembled from single cell RNA-seq data at 6, 8, 10 and 13 hpf data.

Lineage progression of gata5:GFP-positive mesendoderm from 6 to 13 hours post-fertilization (hpf), assembled from single cell RNA-seq data at 6, 8, 10 and 13 hpf data. 

Trainees working on this project:

Meaghan Leslie, Casey Carlisle

Subsequent to cardiac lineage commitment, cardiac progenitor cells must be diversified to form the various components of the heart. Our lab was amongst the first to demonstrate that zebrafish heart development occurs via two waves of addition of cells from the so-called first and second heart fields (FHF and SHF), as is true in mammals. Subsequently, these cells differentiate to form the structures of the heart. We are utilizing a comparative genomics approach to identify genes and gene regulatory elements (enhancers) conserved in cardiac expression/activity from zebrafish to human. These conserved elements are being examined for activity in transgenic and mutant models we are generating. Long-term goals include creating zebrafish models of congenital heart disease for functional and small molecule therapeutic screens, annotating non-coding variants associated with human disease, and identifying genes and enhancers that may play roles in cardiac regeneration. 

A 48 hours post-fertilization heart, with cardiomyocyte nuclei marked with a myl7:nlsmCherry transgene, and cell membranes marked with Zn-8 immunostaining.

A 48 hours post-fertilization heart, with cardiomyocyte nuclei marked with a myl7:nlsmCherry transgene, and cell membranes marked with Zn-8 immunostaining.

A 48 hours-post-fertilization zebrafish heart, with cardiomyocytes marked with a myl7:EGFP transgene, nuclei stained with DAPI, and actin marked with Phalloidin.

A 48 hours-post-fertilization zebrafish heart, with cardiomyocytes marked with a myl7:EGFP transgene, nuclei stained with DAPI, and actin marked with Phalloidin. 

Trainees working on this project: 

Chris Onderisin, Casey Carlisle, Harsha Murthy

Unlike the case in mammals, the adult zebrafish heart has a remarkable regenerative capacity. We have generated a number of transgenic and mutant lines based on enhancers we have found to be active during cardiac development and conserved in function between zebrafish and humans. Intriguingly, we have identified several that are activated upon cardiac injury, and are currently examining their function in this process. Combined with our work in cardiac development, we aim to identify key molecular events that can be used to drive cardiac repair in the human heart. 

Activity of aCNE1, a conserved enhancer active during heart development, in cardiomyocytes contributing to cardiac regeneration in an adult zebrafish heart. A heart 7 days post-resection is shown.

Activity of aCNE1, a conserved enhancer active during heart development, in cardiomyocytes contributing to cardiac regeneration in an adult zebrafish heart. A heart 7 days post-resection is shown.

Effects of deletion of the conserved cardiac enhancer CNE1 on cardiac regeneration in zebrafish. Mutant hearts fail to fully remodel scar tissue, and do not properly replace lost myocardial tissue.

Effects of deletion of the conserved cardiac enhancer CNE1 on cardiac regeneration in zebrafish. Mutant hearts fail to fully remodel scar tissue, and do not properly replace lost myocardial tissue.

Trainees working on this project:

Dr. Tvisha Misra, Shimon Rosenthal

Cerebral Cavernous Malformations (CCMs) arise from defects in vascular stability in venous elements of the brain vasculature. Mutation of three genes, CCM1-3, have been shown to be causative of CCMs in familial cases. We have generated zebrafish ccm3 mutants, and are undertaking a detailed analysis of the molecular and cellular events that lead to CCM initiation and progression. We carry out this work with colleagues who use C. elegans genetics to define novel CCM pathway genes (Dr. Brent Derry) and proximity labelling based mass spectrometry to define in vivo partners of CCM3 (Dr. Anne-Claude Gingras). In collaboration with the Gingras lab, we are developing methods for BioID approaches in the zebrafish embryo. 

Image of the cerebral vasculature in a 48 hour post-fertilization transgenic kdrl:EGFP zebrafish embryo.

Image of the cerebral vasculature in a 48 hour post-fertilization transgenic kdrl:EGFP zebrafish embryo. 

Mutation of ccm3a/b (right) results in dilation and mispatterning of the cerebral vasculature at 52 hours post-fertilization.

Mutation of ccm3a/b (right) results in dilation and mispatterning of the cerebral vasculature at 52 h post-fertilization.   

Flattened selective plane illumination (SPIM) view of the zebrafish cerebral vasculature at 48 hours post-fertilization in a double fli1a:nlsEGFP; kdll:memb-mCherry transgenic embryo.

Flattened selective plane illumination (SPIM) view of the zebrafish cerebral vasculature at 48 hours post-fertilization in a double fli1a:nlsEGFP; kdll:memb-mCherry transgenic embryo.

Early Cardiac Lineages
Trainees working on this project:

Dr. Xuefei Yuan, Mengyi Song, Nathan Stutt, Palak Desai

We have a major interest in the earliest events of heart development that regulate how cardiac progenitors, the building blocks of the heart, are first specified and diversified. Our early work with the grinch mutant has shown that function of the Apelin receptor GPCR (Aplnr) is essential for cardiac development, with aplnr/a/b mutants being heartless. This appears to be due to defects in the cardiac lineage at the earliest events of development, as the gastrulation is being initiated. To address this, we have collaborated with the lab of Dr. Michael Wilson at SickKids to undertake single cell RNAseq and ATACseq analysis of the initial stages of cardiac development in both wild type and heartless (aplnra/b and gata5/6 loss-of-function) models. This work will lead to the key gene regulatory networks that govern the earliest events of cardiac lineage specification. 

Patterning of the cardiopharyngeal mesoderm at 13 hours post-fertilization: gata5:GFP-positive cardiac mesoderm is in close proximity to irx1b and cyp26c1 expressing pharyngeal mesoderm.

Patterning of the cardiopharyngeal mesoderm at 13 hours post-fertilization: gata5:GFP-positive cardiac mesoderm is in close proximity to irx1b and cyp26c1 expressing pharyngeal mesoderm. 

Lineage progression of gata5:GFP-positive mesendoderm from 6 to 13 hours post-fertilization (hpf), assembled from single cell RNA-seq data at 6, 8, 10 and 13 hpf data.

Lineage progression of gata5:GFP-positive mesendoderm from 6 to 13 hours post-fertilization (hpf), assembled from single cell RNA-seq data at 6, 8, 10 and 13 hpf data. 

Cardiac Development
Trainees working on this project:

Meaghan Leslie, Casey Carlisle

Subsequent to cardiac lineage commitment, cardiac progenitor cells must be diversified to form the various components of the heart. Our lab was amongst the first to demonstrate that zebrafish heart development occurs via two waves of addition of cells from the so-called first and second heart fields (FHF and SHF), as is true in mammals. Subsequently, these cells differentiate to form the structures of the heart. We are utilizing a comparative genomics approach to identify genes and gene regulatory elements (enhancers) conserved in cardiac expression/activity from zebrafish to human. These conserved elements are being examined for activity in transgenic and mutant models we are generating. Long-term goals include creating zebrafish models of congenital heart disease for functional and small molecule therapeutic screens, annotating non-coding variants associated with human disease, and identifying genes and enhancers that may play roles in cardiac regeneration. 

A 48 hours post-fertilization heart, with cardiomyocyte nuclei marked with a myl7:nlsmCherry transgene, and cell membranes marked with Zn-8 immunostaining.

A 48 hours post-fertilization heart, with cardiomyocyte nuclei marked with a myl7:nlsmCherry transgene, and cell membranes marked with Zn-8 immunostaining.

A 48 hours-post-fertilization zebrafish heart, with cardiomyocytes marked with a myl7:EGFP transgene, nuclei stained with DAPI, and actin marked with Phalloidin.

A 48 hours-post-fertilization zebrafish heart, with cardiomyocytes marked with a myl7:EGFP transgene, nuclei stained with DAPI, and actin marked with Phalloidin. 

Cardiac Regeneration
Trainees working on this project: 

Chris Onderisin, Casey Carlisle, Harsha Murthy

Unlike the case in mammals, the adult zebrafish heart has a remarkable regenerative capacity. We have generated a number of transgenic and mutant lines based on enhancers we have found to be active during cardiac development and conserved in function between zebrafish and humans. Intriguingly, we have identified several that are activated upon cardiac injury, and are currently examining their function in this process. Combined with our work in cardiac development, we aim to identify key molecular events that can be used to drive cardiac repair in the human heart. 

Activity of aCNE1, a conserved enhancer active during heart development, in cardiomyocytes contributing to cardiac regeneration in an adult zebrafish heart. A heart 7 days post-resection is shown.

Activity of aCNE1, a conserved enhancer active during heart development, in cardiomyocytes contributing to cardiac regeneration in an adult zebrafish heart. A heart 7 days post-resection is shown.

Effects of deletion of the conserved cardiac enhancer CNE1 on cardiac regeneration in zebrafish. Mutant hearts fail to fully remodel scar tissue, and do not properly replace lost myocardial tissue.

Effects of deletion of the conserved cardiac enhancer CNE1 on cardiac regeneration in zebrafish. Mutant hearts fail to fully remodel scar tissue, and do not properly replace lost myocardial tissue.

Cerebral Cavernous Malformation
Trainees working on this project:

Dr. Tvisha Misra, Shimon Rosenthal

Cerebral Cavernous Malformations (CCMs) arise from defects in vascular stability in venous elements of the brain vasculature. Mutation of three genes, CCM1-3, have been shown to be causative of CCMs in familial cases. We have generated zebrafish ccm3 mutants, and are undertaking a detailed analysis of the molecular and cellular events that lead to CCM initiation and progression. We carry out this work with colleagues who use C. elegans genetics to define novel CCM pathway genes (Dr. Brent Derry) and proximity labelling based mass spectrometry to define in vivo partners of CCM3 (Dr. Anne-Claude Gingras). In collaboration with the Gingras lab, we are developing methods for BioID approaches in the zebrafish embryo. 

Image of the cerebral vasculature in a 48 hour post-fertilization transgenic kdrl:EGFP zebrafish embryo.

Image of the cerebral vasculature in a 48 hour post-fertilization transgenic kdrl:EGFP zebrafish embryo. 

Mutation of ccm3a/b (right) results in dilation and mispatterning of the cerebral vasculature at 52 hours post-fertilization.

Mutation of ccm3a/b (right) results in dilation and mispatterning of the cerebral vasculature at 52 h post-fertilization.   

Flattened selective plane illumination (SPIM) view of the zebrafish cerebral vasculature at 48 hours post-fertilization in a double fli1a:nlsEGFP; kdll:memb-mCherry transgenic embryo.

Flattened selective plane illumination (SPIM) view of the zebrafish cerebral vasculature at 48 hours post-fertilization in a double fli1a:nlsEGFP; kdll:memb-mCherry transgenic embryo.

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Ontario Institute for Cancer Research website
SickKids Foundation website