Research

The vertebrate vasculature consists of an extensive branched network of blood vessels that supplies all tissues with essential nutrients and oxygen. As such, new blood vessel morphogenesis (or angiogenesis) is critical for the development, growth and repair of almost all organs and tissues. Moreover, imbalances in angiogenesis contribute to numerous disease states, including blindness, arthritis, ischaemic disorders and cancer. Thus, manipulation of angiogenesis has clear therapeutic potential, yet, we are just beginning to decipher the complex mechanisms underpinning new blood vessel growth.

Like any newly forming or regenerating tissue, in angiogenesis cells must precisely coordinate a diverse suite of cellular behaviours (e.g. cell migration, proliferation, polarity and differentiation) to collectively build functional organs. To do this, endothelial cells employ a core toolkit of morphogenetic mechanisms (e.g. collective cell migration, competitive cell fate decisions, tissue branching, patterning/guidance mechanisms and tubulogenesis), processes that are broadly conserved across many tissue systems. Thus, by defining the basic mechanisms of angiogenesis, we aim to both reveal fundamental principles underpinning tissue building relevant to many tissue contexts, as well as define putative therapeutic approaches to tackling pathological angiogenesis.

Of note, our recent research interests include:

 

Coordination of collective cell migration by asymmetric cell divisions:

tip stalk
(Fig. 1) Endothelial tip and stalk cells display distinct behaviours and functions: Time-lapse images of mosaic labelled (lyn-mCherry) individual tip or stalk cells and quantification of the dorsal movement of tip or stalk cell nuclei and the top, bottom and centre of migrating cells. Tip cells are highly motile. Stalk cells are less motile and undergo cell elongation (Costa et. al., 2016. Nat. Cell Biol.).

Angiogenesis is coordinated by the collective migration of specialised leading “tip” and trailing “stalk” endothelial cells (Fig. 1). The molecular determinants of tip versus stalk identity are well known, namely VEGFR and Notch signalling, respectively. However, the importance of endothelial cell division in coordinating tip-stalk specification in angiogenesis was only recently revealed by my lab. Using an interdisciplinary approach combining in silico computational modelling studies with in vivo live single-cell imaging approaches in the zebrafish model, we demonstrated that tip cell divisions are asymmetric and elegantly coordinate the collective migration of branching endothelial cells by self-generating the tip-stalk hierarchies that drive angiogenesis (Fig. 2; Costa et. al., 2016. Nat. Cell Biol.; Costa et. al., 2017. Cell Cycle). We find that asymmetric positioning of the mitotic spindle in dividing tip cells generates daughter cell size asymmetries, differential Vegfr signal levels, and establishes the post-mitotic leader-follower hierarchies that coordinate collective cell behaviour in angiogenesis. Likewise, our collaborator (Claudia linker, KCL) uncovered functional conservation of such asymmetric division in the coordination of trunk neural crest cell morphogenesis (Richardson et. al., 2016. Cell Rep.). Thus, we have defined asymmetric division as a novel mechanism for the coordination of communal cell identity decisions in collective cell migration that likely directs tissues morphogenesis in many cellular contexts. Studies probing how these asymmetric divisions are controlled, how they regulate post-mitotic cell fate and their conserved function in the control of other morphogenetic tissue systems are all active areas of research in my lab.

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(Fig. 2) Asymmetric tip cell divisions self-generate the tip-stalk heirarchy: Time-lapse images of an individual dividing lyn-mCherry and a-tubulin-GFP-expressing tip cell in a zebrafish embryo. The mitotic spindle moves ventrally to drive asymmetric division and differential daughter tip-stalk identity (Costa et. al., 2016. Nat. Cell Biol.).

 

 

Spatial control of tissue morphogenesis via mRNA compartmentalisation:

The polarised targeting of diverse mRNAs to the front of motile cells is a hallmark of cell migration, yet, precise functional roles for such targeting of mRNAs has long remained unknown. Likewise, their relevance to modulation of tissue dynamics in vivo is unclear (Herbert et. al., 2019. Essays in Biochem.). Using single-molecule analysis, endogenous gene-edited mRNAs and zebrafish in vivo live-cell imaging, my lab has recently shown that this compartmentalisation of mRNAs uniquely acts as a molecular compass that orients motile cell polarity to spatially direct tissue movement (Costa et. al., 2019. BioRxiv). Using cell fractionation to isolate motile cell protrusions and RNAseq we defined distinct clusters of mRNAs that have unique spatial distributions in migrating endothelial cells, both in vitro and in vivo. For example, the mRNA for RAB13 is universally spatially targeted to motile processes of diverse cell types (Fig. 3).

mRNA
(Fig. 3) RAB13 mRNA is targeted to endothelial cell motile processes: Endothelial cell protrusions were fractionated to identify protrusion-enriched mRNAs by RNAseq. Endogenous RAB13 mRNA is localised to motile protrusions versus the cell body-localised control mRNA, GAPDH (Costa et. al., 2019. bioRxiv).

Importantly, we find that the targeting of certain mRNAs to the front of motile cells leads to precise local control of subsequent protein expression, which drives regional remodelling of key cytoskeletal structures in cells that orient cell and tissue movement. Hence, disruption of this mRNA compartmentalisation depolarises motile cells and disrupts the guidance of newly forming blood vessels during vascular development in vivo in zebrafish embryos. What’s more, considering we have identified numerous mRNAs that exhibit this polarised subcellular localisation (as well as possessing putative motile functions), spatial compartmentalisation of numerous mRNAs likely coordinates diverse aspects of motile cell behaviour. Studies investigating the mechanisms triggering such polarised targeting of certain mRNAs (but not others), the function of compartmentalised mRNAs in directing motile cell behaviour and if this phenomenon can be hijacked to predictably direct cell and tissue movement are currently ongoing.

 

Transcriptional control of collective tissue behaviour:

To identify new determinants of the tip versus stalk cell identity that coordinates collective cell movement in angiogenesis, we have employed an integrated pharmacological and transcriptomic strategy for the detection of novel tip and stalk cell-associated genes in vivo (Herbert et. al., 2012. Current Biology). Upon incubation of zebrafish embryos with well-established pharmacological inhibitors Vegfr or Notch, both alone or in combination, we could either repress tip cell formation (Vegfr inhibition), induce ectopic tip cell formation (Notch inhibition) or recover ectopic tip cell formation (Notch + Vegfr inhibition). Subsequent FACS isolation of GFP-positive endothelial cells and multifactorial transcriptomic comparison with DMSO-treated controls identified previously unknown tip cell restricted genes that are tightly spatiote­­mporally associated with tip cell behavior in vivo.

tip cell genes
(Fig. 4) Identification of tip cell-enriched transcripts: Using inhibitors of Notch (DAPT) and VEGFRs (SU5416) we could identify transcripts associated with tip cell-specific signalling in vivo. These transcripts are often highly enriched in sprouting vessels (arrows), such as tm4sf18 and hlx1, but are excluded from the rest of the non-angiogenic vasculature that is stained with kdrl (Page et. al., 2019. Cell Rep.; Herbert et. al., 2012. Curr. Biol.).

This previous work has allowed us to define key transcriptional switches that couple selection of tip versus stalk identity to induction of cell-type-specific behaviours. For example, we have previously shown that the tip cell restricted gene hlx1 coordinates stalk versus tip cell behaviour in vivo, although the mechanisms involved remain unclear (Herbert et. al., 2012. Curr. Biol.). Moreover, we revealed that tip cell-restricted expression of the tetraspanin gene, tm4sf18, drives positive-feedback modulation of Vegfr signalling and spatiotemporally regulates tip-stalk signalling dynamics and identity decisions in vivo (Page et. al., 2019. Cell Rep.). Work defining the localisation, regulation and function of other previously unidentified tip and stalk cell specific transcripts uncovered by these studies are currently in progress.