Biology Reference
In-Depth Information
hollows out by apoptosis.
67
Epithelial growth and branching is controlled by a number of
mesenchyme-derived growth factors including FGF10, without which salivary gland
morphogenesis fails;
68
signalling by the sonic hedgehog, EGF, TGF, TNF and IGF pathways
is also probably involved.
67
Rudiments of these glands can be removed from embryos and
grown in organ culture, where they can be observed and manipulated easily. Grown in
normal media, the epithelial ampullae develop clefts and divide into separate growth fronts,
each of which develops an ampulla of its own so that branching continues (
Figure 20.11
). This
branching growth will take place when the epithelium is surrounded by mesenchyme as it
would be
in vivo
, and will also take place if it is cultured on its own as long as it is provided
with appropriate mesenchyme-derived factors and an artificial matrix.
69
This is an important
observation, because it indicates that the mechanism for branching morphogenesis lies
within the epithelium itself rather than the surrounding cells.
The epithelium grows by cell proliferation as it branches but proliferation is not required
for the development of clefts: clefts form normally even if rudiments are X-rayed or treated
with aphidicollin to reduce greatly both DNA synthesis and mitotic division.
70
This empha-
sizes that clefts are not simply places at which epithelial advance is blocked, but they rather
push into the epithelium, invading its space. The clefts, once they have formed, are associated
with collagen fibres up to 2.5
m in diameter which appear and thicken as the clefts deepen.
71
These contain several types of collagen but collagen III is particularly strong.
72
Collagen
fibres can bear considerable tension, so that their presence in the cleft may be responsible
for holding back the epithelium in the cleft while the tissue each side is free to advance.
This idea is supported by the observation that treating salivary glands with exogenous colla-
genase eliminates clefting and branching while, conversely, treating them with inhibitors of
their endogenous collagenases enhances clefting.
73,74
The organization of type I and type III collagens into the type of fibrils found in clefting
does not occur by simple self-assembly, at least in
in vivo
conditions, and instead requires
a scaffold of the extracellular matrix protein fibronectin.
75
Inhibiting the function of fibronec-
tins using antibodies, or inhibiting fibronectin synthesis using RNA interference, blocks the
formation of collagen fibrils and therefore blocks the clefting of salivary gland epithelium.
54
The fibronectin scaffold is itself placed and arranged by cell-substrate adhesion complexes
containing integrins such as
m
1, and inhibition of these integrins also blocks branching.
54
This means that, although collagen fibres control epithelial shape, the epithelial cells are still
ultimately in control of their morphogenesis since they determine where the collagen fibres
will form. Examination of the expression pattern of fibronectin mRNA supports this idea: it is
seen only in cells that are about to form or are actually forming a cleft.
54
The epithelium of the ampulla is unusually loosely connected compared with that of
the shafts and of epithelia in general, and their cells showunusually lowexpression of compon-
ents of cell-cell junctions such as E-cadherin, desmoplakins and ZO1.
54,55
This is particularly
true in the clefts themselves, and it may be another consequence of fibronectin; addition of
fibronectin aggregates to human salivary gland epithelial cells growing in culture causes
them to down-regulate their cell-cell junctions locally, where they make contact with the fibro-
nectin. The relaxing of cell-cell adhesion may help the cells, which have not yet formed a true,
sealed, lumen-containing epithelium, to 'flow' easily past the cleft point. Actinmicrofilaments
are also required for salivary gland morphogenesis. Treatment of cultured rudiments with
cytochalasin blocks morphogenesis and clefting, although clefting begins again when the
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