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multicellular cysts (
Figure 20.3
). When the medium is enriched with certain ramogens
)
such
as HGF that would normally be synthesized by the mesenchyme that surrounds branching
epithelia in the embryo, processes sprout from the cysts and will extend to make tubules.
5
e
7
Many authors consider this to be a good model for branching
in vivo.
8
e
11
Given that HGF is clearly important to the choice between cystic growth and branching
growth, careful study of the effects of HGF on cell behaviour seems a promisingway to discover
the morphogenetic basis of branching. Long before these culture models for branching were
developed, HGF was known to exert profound effects on MDCK cells in two-dimensional
cell culture. Normally, these epithelial cells adhere to one another strongly. If a culture plate is
seededwith a lowdensity of MDCK cells, each forms a colony that coheres strongly (supported
by E-cadherin-mediated adhesions, stress fibres and low motility), to create smooth-edged
cellular islands in a sea of empty culture plate. When HGF is added, the cells let go of each
other, become strongly motile and scatter. Indeed, the alternative name for HGF is 'Scatter
Factor'.
12
e
14
Assuming for a moment that the underlying effects of HGF onMDCK cells will be the same
whether these cells are growing on glass or in gels, could reduced adhesion and increased
motility explain branching? On its own, with no added layer of control, the answer would
appear to be no: all of the cells in a cyst would experience HGF so all would be expected to
reduce their mutual adhesions and all would be expected to become motile and to invade
the gel. In other words, we would expect once again to see scatter, but this time to see it in three
dimensions. If, however, only some cells within a cyst were responsive to HGF and others
were not, then branching could result as the responsive cells forged ahead, trying to scatter,
while the unresponsive ones clung to them and became drawn out into tubules. With this
as a model, the problem becomes one of explaining the differential sensitivity of cells neces-
sary for the model to work. The fact that the cysts in most three-dimensional culture systems
are clonal rules out any odd artefacts of variability in badly maintained cell lines
d
as does the
statistical regularity of the branching response: whatever causes differential sensitivity, it must
achieve it in intrinsically equally capable cells. What is more, it must be able to break the
symmetry of a pre-HGF round cyst and be capable of altering cells' sensitivity dynamically,
to allow branches to branch again.
One intriguing hypothesis that would satisfy all of these requirements is that the motility
response to HGF is controlled by geometry, in a way that creates positive feedback. Recently,
an undergraduate project student in my laboratory grewMDCK cells on patterned substrates
that confined them to precisely printed islands of adhesive surface in much less adhesive
surroundings. The islands, hundreds on each culture plate, were shaped so that they
included positive and negative curves of differing radii. For each zone of radius, scoring
of the percentage of cells in the zone that produced lamellipodia revealed a clear relationship
between curvature and probability of lamellipodium production, a proxy for motility: cells
bent around a convex radius were significantly more likely to make lamellipodia than unbent
cells, while those on a concave curve were less likely (
Figure 20.4
a,b). If this tendency,
measured in a two-dimensional system, is reflected in the three-dimensional gel cultures,
it would mean that any small protuberance formed by random cell jostling at the surface
)
Ramogens
molecules that promote branching, from the Latin
Ramus
meaning 'branch', and Greek
Genesis
meaning 'creation'.
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