The gradual spreading of the allantoic mesothelium is the in vitro homologue of global tissue expansion during embryonic vasculogenesis: Allantoic vascular branches are subjected to tissue strain (discussed below) just as vascular sprouts in embryos are under the influence of global tissue deformations. The allantoic mesothelium is positioned between the glass substrate and the forming vascular network. As the mesothelium spreads the collective tissue motion imposes strain on the nascent vascular structures (polygons and sprouts). Similarly, during embryonic vasculogenesis in amniotes the forces of gastrulation, neurulation, and axis elongation impose long-range strain fields on the nascent vascular networks, which manifest in the correlated drift or motion of an entire vascular network (Rupp et al., 2004
, Czirok et al., 2007
). Presumably similar forces would act on angiogenic fronts during organogenesis or wound healing. In accord, allantoic vascular segments are expanded by 1) an active, sprouting process, and 2) by a passive strain generated by the gradual spreading of the explant mesothelium.
Both the passive and the autonomous components of cell motion can be estimated based on their respective statistical characteristics: Tissue motion involves deformation of a physical body, thus it is a smooth function of space and time (Czirok et al., 2004
). In contrast, correlations in cell-autonomous movements decay rapidly and are statistically distinct from those characterizing tissue motion (Sepich et al., 2005
; Zamir et al., 2005
). We present here for the first time empirical time-lapse data showing that mouse embryonic endothelial cells engage in local cell-autonomous motility within vascular sprouts — in a dynamically coherent replica of vasculogenesis.
However, the coalescence of angioblasts that normally occurs during early vasculogenesis involves VEGFR2-expressing vascular cells that do not
yet express VE-cadherin (Drake and Fleming, 2000
). In accord, rudimentary vascular structures are observed in the VE-cadherin knock out mouse (Carmeliet et al., 1999
). The mutant embryos exhibit vascular defects, but display normal angioblast differentiation. While important aspects of VE-cadherin signaling are understood (Spagnuolo et al., 2004
), the molecule’s mechanistic role in early vasculogenesis remains an area of active inquiry (Crosby et al., 2005
Previous work with VE-cadherin antibodies suggested that VE-cadherin inhibition caused the disassembly of established blood vessels. This was inferred from experiments using allantoic explant cultures fixed at two time points (Crosby et al., 2005
). Allantoides from VE-cadherin null embryos also failed to form polygonal vascular networks. Based on analysis of explants fixed after 9 and 18 hours of culture, Crosby et al (2005)
suggested that initial events of vasculogenesis including the formation of nascent endothelial tubes are not dependent on VE-cadherin activity. Our dynamic imaging studies show, however, direct unequivocal evidence that endothelial cell motion along vascular segments is reduced within hours after blocking VE-cadherin function. We also see substantially reduced expansion speeds of vascular branches in early stages of allantoic explant spreading. Interestingly, time-lapse data reveal that the early inhibitory effects do not initially
manifest themselves as a disconnected vascular network; therefore such an alteration is difficult, if not impossible to detect in specimens fixed at 9 and 18hrs. With the advantage of time-lapse imaging, however, these anomalies are readily discerned.
It is important to note that mesothelial (tissue) spreading proceeds readily in VE-cadherin inhibited cultures. However, the allantoic endothelial cells deprived of normal VE-cadherin function are limited in their ability to accommodate strain — because additional “new” endothelial cells cannot be incorporated into vascular cords
. Thus, as VE-cadherin inhibition blocks the supply of “new” additional cells advancing toward the tip, vascular branches eventually either retract or break up — resulting in the state described by Drake and colleagues (Crosby et al., 2005
Carmeliet et al (Carmeliet et al., 1999
) showed that under certain conditions loss of VE-cadherin function leads to reduced cell survival. During the investigated time frame, however, lack of VE-cadherin function does not result in detectable changes in cell viability (Carmeliet et al., 1999
). We find that CD144 treated endothelial cells continue to move for 9–18 hours, albeit at reduced velocities. Thus, inhibition of cell-autonomous motility in VE-cadherin inhibited cultures cannot be attributed to the presence of immobile dead cells (See Movies 1
). Further, dye exclusion studies showed no decrease in viability in the case of endothelial cells treated with inhibitory VE-cadherin antibodies (, Crosby et al, 2005
Based on the above results and data obtained in avian embryos (Rupp et al., 2004
), we suggest that vascular sprouting is initiated by the invasive behavior of a single “tip” cell — which engages the surrounding ECM using an integrin-dependent process. The advancing sprout, however, needs a continuous supply of endothelial cells, which may overtake, and pass by, the branch-initiating cell at the tip. Here we show that endothelial cells forming the future vascular cords are streaming along the vascular sprout, and this motion is VE-cadherin dependent. Thus, it is reasonable to postulate that VE-cadherin is required for the autonomous motility of endothelial cells that glide along vascular structures. We propose, therefore, that VE-cadherin-based motility is necessary to sustain
There are multiple ways VE-cadherin can influence cell motility. The cytoplasmic tail of VE-cadherin binds to β-catenin and consequently alters the actin cytoskeleton (Cattelino et al., 2003
; Wright et al., 2002
). Although cadherins are often thought to mediate stable cell interactions and to decrease motility, cadherin-mediated adhesion is critical for gastrulation movements of X. laevis
(Zhong et al., 1999
), or growth cone motility (Matsunaga et al., 1988
). Moreover, dynamic regulation of cell-cell adhesion is also expected to drive cell movements. For example, cadherin-mediated cell-cell contacts are hypothesized to perform an intercellular motility
) function that is analogous to the function of integrin-extracellular matrix contacts on moving cells. On the other hand, it is also known that VE-cadherin is involved in VEGF signaling. Thus, VEGF-induced mitogenic and survival signals are attenuated in the presence of blocked VE-cadherin (Carmeliet et al., 1999
Our experiments cannot directly distinguish whether VE-cadherin functions as an intercellular migration adhesion receptor, or as a VEGF co-receptor. However, the established role of integrins in VEGF-induced motility (Hood et al., 2003
) together with our failure to significantly reduce motility along vascular cords using inhibitory antibodies against α4 and α5 integrins (Perryn, 2006
) indirectly favors the interpretation that VE-cadherin functions as an intercellular motility adhesion receptor
. This assertion is most clearly supported by the data showing the prompt effect of VE-cadherin antibodies on the motility of endothelial cells along vascular segments. We did not detect reductions in endothelial cell motility when allantoides were treated with inhibitory integrin α4 and α5 antibodies (Perryn, 2006
) — although it is important to stress that our efforts to perturb integrins were neither exhaustive, nor comprehensive.
The quantitative data described above advance our understanding of how tissue dynamics impacts the construction of a new vascular pattern. In particular, the tissue drift data bring into question reports of purported vascular malformations in mutant mice where the surrounding soma is malformed. Thus if a “vascular” anomaly is observed in an embryo that is overtly malformed it is not possible to state, with certainty, whether the vascular abnormality is primary, or is due to the fact that the underlying organ or tissue deformed improperly thereby causing a secondary vascular malformation. We speculate this may be particularly troublesome when analyzing vascular amomalies in early to mid-gestation mouse embryos.
Our data define normal and perturbed parameters of murine vasculogenesis using a high-fidelity model system — cultured allantoides. Argraves and Drake (2005)
point out that the allantoic culture vasculogenesis assay is a useful and potentially important tool for understanding and quantifying endothelial cell behavior. Our empirical data show that endothelial cells differentiate from cultured allantoides and go on to participate in vascular network pattern formation; and neither CD34 antibodies nor wide-field time-lapse imaging cause detectable perturbation. This convenient culture method, when combined with our time-resolved analysis, allows computational characterization of mutant phenotypes present in the large repertoire of knockout and transgenic mice manifesting vascular defects (Argraves and Drake, 2005
). Many of these knockout and transgenic mice are embryonic lethal, therefore complicating study of late-stage embryos, fetuses or neonates. By harvesting mutant allantoides prior to lethality, the dynamics of endothelial cell behavior, and the formation of “abnormal” primary vascular networks, can be studied in the context of a precise genotype.