Final Report Summary - MOSIMANN ZEBRAFISH (Mechanisms of earliest hematopoietic cell fate control)
The circulatory system entails the heart, endothelium, and blood cell types that cooperate together to establish life-long physiological functions. The circulatory system arises during embryo development from an elusive territory in the developing vertebrate embryo, the so-called lateral plate mesoderm (LPM). What molecular mechanisms specify the LPM within the embryo and drive its cells into its final organ fates remains incompletely understood. We hypothesize that a dedicated molecular program in the earliest LPM cells drives their emergence and coordinated specification into heart, blood, and blood vessel lineages; we are investigating this hypothesis with latest transgenic tools and genome engineering in the zebrafish (Danio rerio). A deeper understanding of LPM formation and patterning will provide a developmental framework of how the vertebrate body plan evolved and become so successful. Mechanistic insights into fundamental processes of early LPM formation will further help in deciphering the causes and mechanisms of inherited cardiovascular disorders.
The zebrafish provides a powerful model to investigate LPM formation with rapid development, optical translucency, and potent genetic tools. In our previous and ongoing work, we have uncovered several gene-regulatory elements that are specifically active in the emerging LPM in different vertebrate species. These regulatory elements allowed us to drive fluorescent reporters to visualize the emerging LPM in previously unattainable detail and to track their long-term specification into descendant cell types.
These experiments have enabled us to establish the LPM origin of previously un-trackable cell lineages in the embryo. We in particular employed selective plane illumination microscopy (SPIM, also called lightsheet microscopy) to image the developing zebrafish embryo in toto towards understanding the architecture and dynamics of LPM formation and subsequent heart, blood, and blood vessel emergence. Towards elucidating the molecular program that drives uncommitted embryo cells into LPM and subsequently circulatory system cell lineages, we have investigated transcription factor combinations that together cooperate in the regulation of key LPM-expressed genes. These experiments hint at an evolutionarily conserved, ancient molecular program that drives LPM formation seemingly universally in all vertebrates. Our transgenic reporters have further revealed previously unattainable aspects of cardiovascular development, including the continuous formation of the heart ventricle from a bilateral progenitor cell sheath.
To rapidly reveal the contribution of individual genes and gene-regulatory elements in the genome, we have harnessed the CRISPR-Cas9 system to achieve maximized mutagenesis efficiency in zebrafish embryos. Paired with dedicated software analysis of the resulting mutagenesis on DNA sequence level, these approaches now allow us to probe the contribution of transcription factors and their bound gene-regulatory elements in early LPM cells to reveal the fundamental gene program at the base of heart, blood, and blood vessel formation during development. We have also harnessed this technology to assess the genotype-phenotype correlation of candidate genes implicated in congenital heart disease, with first candidates emerging from our screening.
Altogether, our ongoing work has revealed new details of the earliest steps of circulatory system formation in the embryo and has established powerful new genetic tools to investigate development with the zebrafish model. Our results have provided mechanistic insights into processes that are perturbed in congenital cardiovascular diseases and form a platform for genotype-phenotype analysis in vivo.
The zebrafish provides a powerful model to investigate LPM formation with rapid development, optical translucency, and potent genetic tools. In our previous and ongoing work, we have uncovered several gene-regulatory elements that are specifically active in the emerging LPM in different vertebrate species. These regulatory elements allowed us to drive fluorescent reporters to visualize the emerging LPM in previously unattainable detail and to track their long-term specification into descendant cell types.
These experiments have enabled us to establish the LPM origin of previously un-trackable cell lineages in the embryo. We in particular employed selective plane illumination microscopy (SPIM, also called lightsheet microscopy) to image the developing zebrafish embryo in toto towards understanding the architecture and dynamics of LPM formation and subsequent heart, blood, and blood vessel emergence. Towards elucidating the molecular program that drives uncommitted embryo cells into LPM and subsequently circulatory system cell lineages, we have investigated transcription factor combinations that together cooperate in the regulation of key LPM-expressed genes. These experiments hint at an evolutionarily conserved, ancient molecular program that drives LPM formation seemingly universally in all vertebrates. Our transgenic reporters have further revealed previously unattainable aspects of cardiovascular development, including the continuous formation of the heart ventricle from a bilateral progenitor cell sheath.
To rapidly reveal the contribution of individual genes and gene-regulatory elements in the genome, we have harnessed the CRISPR-Cas9 system to achieve maximized mutagenesis efficiency in zebrafish embryos. Paired with dedicated software analysis of the resulting mutagenesis on DNA sequence level, these approaches now allow us to probe the contribution of transcription factors and their bound gene-regulatory elements in early LPM cells to reveal the fundamental gene program at the base of heart, blood, and blood vessel formation during development. We have also harnessed this technology to assess the genotype-phenotype correlation of candidate genes implicated in congenital heart disease, with first candidates emerging from our screening.
Altogether, our ongoing work has revealed new details of the earliest steps of circulatory system formation in the embryo and has established powerful new genetic tools to investigate development with the zebrafish model. Our results have provided mechanistic insights into processes that are perturbed in congenital cardiovascular diseases and form a platform for genotype-phenotype analysis in vivo.