The vertebrate vasculature displays a highly reproducible and pervasive anatomy, required for carrying its multiple vital functions. Consequently, defective vessel growth contributes to the pathogenesis of multiple human diseases.
To understand the genetic pathways and cellular strategies used by developing vessels to acquire their architecture, we are using genetic approaches and imaging tools to study vascular development in zebrafish. We are focusing on answering the following questions:
- Which signaling pathways shape the anatomical pattern of the vasculature?
- What are the molecular mechanisms by which these pathways regulate the motility, shape and cell cycle of endothelial cells?
We hope that the answers to these questions will allow us to contribute to the development of therapies aimed at the regulation of blood vessel growth, like anti-cancer treatments and ischemic tissue re-vascularization.
The transparent and externally developing zebrafish embryo is the only genetic system in which blood vessel development can be visualized in vivo and in real time. In addition, animals with defective vessels survive for long periods of time due to passive oxygen diffusion, providing the opportunity to study both early and late embryonic stages of vascular patterning. In our studies, we employ transgenic animals carrying vascular fluorescent reporters and high-resolution imaging methods, such as confocal microscopy and microangiography to study gene-specific loss of function phenotypes generated by mutagenesis or morpholino injection. Want to watch an example of this powerful combination? See the development of the zebrafish trunk vasculature (formation of the intersomitic vessels) in a normal embryo (WT movie) and in an animal lacking plxnD1 activity (plxnD1 movie).
Click here to view the WT movie (8.6 MB)
Confocal time-lapse movies of the development of the intersomitic vessels in TG(fli1-EGFP)y1 embryos (Lateral views, from 20 to 32 hours post fertilization. Dorsal is to the top and anterior is to the left). Note that in wild type embryos (WT movie) the intersomitic vessels sprout at regular intervals and display thin and dynamic filopodia-like projections, which are absent from the Dorsal Aorta. The path followed by the intersomitic vessels prefigures their final shape. By contrast, in animals lacking the function of the endothelial-specific receptor plxnD1 (plxnD1 MOVIE) the intersomitic sprouts grow at irregular intervals and form an aberrant interconnected vascular network due to the formation of ectopic interconnections.
What mechanisms shape the architecture of the vasculature?
Semaphorin-PlexinD1 (Sema-PlxnD1) signaling
We have shown that somite to endothelial Sema-PlxnD1 paracrine signaling patterns a subset of the evolutionarily conserved trunk vasculature (See Figure 1), demonstrating the existence of genetically encoded programs guiding the formation of the vascular tree. This finding indicates that common cues and mechanisms shape the evolutionarily conserved anatomy of the nervous and vascular systems. However, the molecules that mediate endothelial cell repulsion downstream of Sema-PlxnD1 signaling have not been defined. We have recently identified a group of genes likely involved in this process and are currently characterizing their function.
Figure 1. A model for how repelling Sema3a signals guide patterning of the PlxnD1 expressing intersomitic sprouts. In the zebrafish trunk (a), the overlapping expression patterns of the two highly related Semaphorins sema3a1 and sema3a2 (b) define territories not permissive for the growth of plxnD1 expressing intersomitic sprouts (c). When plxnD1 activity is reduced, as in out of bounds mutants or plxnD1 morphants, the intersomitic sprouts fail to sense the sSema3a repelling cues and make pathfinding mistakes. Modified from Dev Cell. 2004 Jul; 7(1):117-23.
Lack of plxnD1 activity induces defects only in some vessels, suggesting that additional signaling cascades are required to pattern the vasculature. To uncover them, we are mapping and characterizing mutants with abnormal vessels isolated in an F3 genetic screen.
Studying vascular patterning: New tools
Our studies, as well as the findings from others suggest that a dynamic molecular landscape of different spatially restricted cues guides the formation of the vascular tree. To aid us in understanding this process, we require both misexpression tools and tissue-specific reporters. We are developing these technologies to complement our Semaphorin-PlexinD1 signaling and mutant characterization studies.