The Interactive Fly
Genes involved in tissue and organ development
Mesoderm migration is a pivotal event in the early embryonic development of animals. One of the best-studied examples occurs during Drosophila gastrulation. Here, mesodermal cells invaginate, undergo an epithelial-to-mesenchymal transition (EMT), and spread out dorsally over the inner surface of the ectoderm. Although several genes required for spreading have been identified, the inability to visualise mesodermal cells in living embryos has hampered gathering of information about the cell rearrangements involved. Several mechanisms, such as chemotaxis towards a dorsally expressed attractant, differential affinity between mesodermal cells and the ectoderm, and convergent extension, have been proposed. This study resolved the behaviour of Drosophila mesodermal cells in live embryos using photoactivatable-GFP fused to alpha-Tubulin (PAGFP-Tub). By photoactivating presumptive mesodermal cells before gastrulation, it was possible to observe their migration over non-fluorescent ectodermal cells. The outermost (outer) cells, which are in contact with the ectoderm, migrate dorsolaterally as a group but can be overtaken by more internal (inner) cells. Using laser-photoactivation of individual cells, it was then shown that inner cells adjacent to the center of the furrow migrate dorsolaterally away from the midline to reach dorsal positions, while cells at the center of the furrow disperse randomly across the mesoderm, before intercalating with outer cells. These movements are dependent on the FGF receptor Heartless. The results indicate that chemotactic movement and differential affinity are the primary drivers of mesodermal cell spreading. These characterisations pave the way for a more detailed analysis of gene function during early mesoderm development (Murray, 2007).
Using a combination of whole mesoderm and single-cell photoactivation this study has observed the combination of cell behaviours employed by Drosophila mesodermal cells to form a monolayer, providing insights into the mechanisms responsible for this important part of gastrulation. The first observation was that outer cells moved dorsolaterally over the ectoderm. Although this is not unexpected, it nevertheless confirms a central prediction of the chemoattraction model: that cells migrate in a dorsolateral direction. Remarkably, it was then observed that inner cells are able to overtake outer cells to achieve a more dorsal position. Single-cell labelling then showed that these inner cells were likely to have originated from a position adjacent to the centre of the ventral furrow. Significantly, inner lateral (IL) cell progeny invariably move away from the midline, suggesting that they receive a directional guidance cue from the dorsal region of the ectoderm, again consistent with a chemoattraction model (Murray, 2007).
A complication in the simple chemoattraction model is that the two likely chemoattractants, Pyr and Ths, are initially expressed in quite broad lateral domains. During mesoderm migration, however, pyr expression does become restricted to the more dorsal parts of the ectoderm, whereas ths is expressed in a complementary fashion in the ventral regions of the neurogenic ectoderm. It has been suggested that the two ligands may have different binding affinities, and that the refinement of Pyr expression to more dorsal positions could guide mesodermal cells dorsally. An alternative is that those regions of the ectoderm that are not yet covered with mesodermal cells, such as the dorsal ectoderm, are highly attractive to mesodermal cells simply because the FGF ligands that they are producing are not being bound and internalised by outer cells already in contact with the ectoderm (Murray, 2007).
An alternative to chemoattraction that has been suggested is that FGFR activation is permissive rather than instructive and simply imparts a degree of motility to cells, allowing them to disperse until they are able to contact the ectoderm. This motility, combined with a steric hindrance effect, in which cells tended to move into unoccupied territory, could theoretically achieve a monolayer in the absence of directional cues. It would be expected, however, that if IL cell progeny were simply made motile and moved randomly, that cells adjacent to the midline would sometimes cross the midline to contact the ectoderm on the opposing side. This was never observed (Murray, 2007).
The movement of inner cells past the lateralmost outer cells is also consistent with the differential affinity model, according to which mesodermal cells form strong adhesions with the ectoderm. Cells not already in contact with the ectoderm would either intercalate between existing outer cells, or, as seen here, move past them. The fact that intercalation was not seen suggests either that outer cells adhere strongly to the ectoderm and do not easily move apart, or, again, that outer cells are masking FGF produced in the ectoderm. If a differential affinity model is active, the most likely candidate adhesion molecules would be integrins, which are expressed at the interface of the mesoderm and ectoderm, although there is, as yet, no published evidence for a functional role for integrins in this process (Murray, 2007).
During the initial migration of outer cells over the ectoderm it was found that cells maintained their position relative to their immediate neighbours. This result supports the argument against the convergent extension model. If convergent extension was a primary driving force behind lateral spreading, one would expect to see widespread intercalation throughout the mesoderm as inner cells pushed in between existing outer cells. This was not observed, although the possibility cannot be ruled out that some degree of intercalation does occur during this migration phase. Intercalation does, however, appear to play a part during the later stages of the formation of the monolayer, where inner medial (IM) cell progeny are seen appearing at the ectoderm. The timing of this event, at around the time of the second mitosis, suggests that the sudden lateral spreading that accompanies the second mitotic wave (50 minutes of development) may be due to the intercalation of a pool of inner cells. One possibility is that the adhesion between the mesodermal cells and the surrounding cells, both mesodermal and ectodermal, is decreased as they go through mitosis, permitting the inner cells access to their preferred position in association with the ectoderm. Thus, although a general convergent extension is not in evidence, intercalation does appear to contribute to mesoderm spreading (Murray, 2007).
On the basis of these observations, the following model of mesoderm cell behaviour following ventral furrow formation is presented. Following the breakdown of the epithelium, the first division results in a rapid spreading down onto the ectoderm, presumably due to decreased adhesion between mesodermal cells. Cells that are thereby placed in contact with the ectoderm start to polarise and proceed to migrate dorsolaterally as a group. Outer cells form a strong adhesive contact with the ectoderm, which prevents inner cells from intercalating between them and instead forces inner cells either to take up positions that outer cells vacate near the midline or move past them to more dorsal positions. Inner lateral cells receive a directional cue from the dorsal ectoderm guiding them laterally, over the outer cells. In this manner, by the time of the second mitosis the ectoderm is largely covered by mesodermal cells. Inner medial cell progeny that have failed to contact the ectoderm during the initial spreading are prevented from doing so by cells already strongly adhered to the ectoderm until the time of the second division. The second division then allows the remaining inner cells to contact the ectoderm. This intercalation produces a rapid lateral extension followed by a general retraction as the cells exit mitosis and re-establish adhesive contacts, with the ectoderm finally forming the monolayer (Murray, 2007).
The combination of behaviours observed may represent the most efficient way to rapidly spread one tissue over another. The tendency for cells to migrate dorsolaterally helps to constantly make space for those cells placed nearer the midline. If cells that contacted the ectoderm never moved away, it would mean that internal cells would have to travel further and further dorsally to find space on the ectoderm. In a similar manner, if chemotaxis towards a dorsally placed attractant was the only mechanism operating, one might expect that cells would continue moving dorsally, even if this resulted in an excess of cells in dorsal positions and a deficit closer to the midline. The tendency of mesodermal cells to develop and maintain a strong adhesive contact with the ectoderm would help ensure that all parts of the ectoderm remain covered. Finally, having a period of intercalation serves to give any remaining inner cells a chance to finally contact the ectoderm (Murray, 2007).
The resolution of mesodermal cell behaviour described in this study will make it possible analysis in greater detail of the migration defects in mutants such as htl and pebble. It will also make it possible to test whether cell rearrangements are normal in those situations in which directional information is lost, but in which spreading still occurs (e.g. rescue with activated Htl, or widespread, non-localised expression of FGF ligands). Finally, it will be of interest to determine whether the behaviors observed are typical of mesoderm migration in other systems. In mouse embryos, mesodermal cells emanating from the primitive streak migrate out over the basal surface of the primitive ectoderm to eventually form the mesodermal layer of cells. The cell rearrangements that occur during this process are not known. Photoactivatable GFP, which has provided such a versatile analysis tool here, could be applied to cultured mouse embryos to resolve these events (Murray, 2007).
The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo, these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. This paper analyzes the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. Secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. This contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization; the latter does not require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Therefore, this analysis of fog function provides a direct link from patterning to cell shape change (Dawes-Hoang, 2005).
Investigation of fog function began with an analysis of
Fog protein distribution within the cells of the ventral furrow and
posterior midgut. In both cases Fog protein was found to be present
in a characteristically punctate pattern; the protein is distributed
unevenly within the cells. The distribution of Fog is polarized with
more Fog puncta present on the apical compared with the basal side of
the cells. This punctate staining is consistent with the localization of
signaling molecules to vesicles involved in both signal production and
reception. To investigate this possibility further,
distribution of Fog was examined in embryos carrying a
temperature-sensitive mutation in the gene shibire, which encodes the
Drosophila homolog of dynamin. At the non-permissive temperature, this
mutation blocks endocytosis, and exocytosis is also compromised. When
embryos are shifted to the non-permissive temperature during early
gastrulation (earlier shifts severely disrupt the process of cellularization)
the Fog protein is already being made and some protein may already be
undergoing endocytosis. However, the localization of Fog in
these embryos is still clearly disrupted, with much less punctate staining and
a decrease in apical polarization. This suggests that the punctate staining of Fog in normal embryos may arise from localization to vesicles derived
through endocytosis, and this supports the hypothesis that fog
encodes a secreted protein. The apical polarization of Fog therefore
raises the possibility that apical secretion and reception of Fog
signal may provide a mechanism for restricting Fog function to the
apical side of the cell (Dawes-Hoang, 2005).
To understand the molecular basis of the control of the cytoskeleton by
Fog, changes in myosin II dynamics were investigated in
fog mutant embryos. Analysis of myosin dynamics is easiest in the
posterior midgut where fog is the primary pathway controlling cell
constriction and the geometry of the egg enables visualization of a myosin
lightchain-GFP fusion (sqhGFP) in time-lapse movies of living
embryos. During gastrulation myosin localizes to the apical side of cells
throughout the posterior midgut primordium of control embryos. However, in
fog mutant embryos of the same age, the apical localization of myosin
is severely disrupted and is restricted to just a few cells underlying the
pole cells. Analysis of myosin localization in fixed embryos also reveals a disruption to apical
localization, both in the posterior midgut and the ventral furrow of
fog mutants. This is consistent with previous data showing that
myosin is also disrupted in concertina (cta) mutants. In the ventral furrow only a subset of cells (39%) localizes myosin apically in fog114 mutant embryos. The fog114 allele is an RNA null. The patchiness of this defect in the ventral furrow of
fog114 mutants therefore probably reflects the redundancy
with additional pathways that control cell shape change in these cells and/or a
small maternal contribution of fog (Dawes-Hoang, 2005).
To determine whether fog is not only necessary but also sufficient
to localize myosin to the apical side of cells, a UASfog
transgene was constructed. Despite high levels of fog
expression from this transgene during cellularization, there is no apparent change in myosin localization. Myosin localizes normally to the cellularization front and the subsequent basal loss of myosin in the ventral most cells and the increased depth of cellularization in these cells that occurs in normal embryos also occur in these fog-overexpressing embryos (Dawes-Hoang, 2005).
The first effects of fog expression are seen at the onset of
gastrulation. In embryos uniformly expressing fog the apical
localization of myosin now occurs in all cells instead of being restricted to
a ventral domain. In the ventral cells of these fog-overexpressing embryos, the apical localization of myosin precedes the apical localization in the more lateral and dorsal cells and reaches a higher level. It is also a higher level than in the ventral cells of control embryos. It is unclear whether this reflects
higher levels of fog expression in the ventral cells (owing to both
endogenous and UASfog expression) or whether it reflects an earlier
or increased competence of these ventral cells to react to fog
signal. The apical localization of myosin in the lateral and dorsal cells of
fog-overexpressing embryos continues throughout gastrulation and occurs
without any concomitant reduction in levels of basally localized myosin. This raises the possibility that the apical and basal localizations of myosin may be
independently controlled (Dawes-Hoang, 2005).
Not all fog-overexpressing embryos show the same degree of ectopic
apical myosin localization in lateral and dorsal cells. Furthermore, limited
apical myosin staining is occasionally seen in control embryos. This variability was quantified over five separate experiments. During cellularization, onset of gastrulation and later gastrulation 0%, 73% and 84% of fog-overexpressing embryos show ectopic apical myosin compared with 0%, 8% and 22%
of controls respectively (Dawes-Hoang, 2005).
In wild-type embryos myosin accumulates apically in all cells after the
completion of ventral furrow invagination, at the onset of germ band extension.
Therefore, apical accumulation of myosin in dorsal and lateral cells of
apparently gastrulating embryos may occur as the result of a delay in ventral
furrow formation. To investigate this possibility, time-lapse
movies of gastrulating embryos were followed and morphology was examined in precisely timed embryo collections. In both cases, a slight delay was found in the completion of ventral furrow formation in fog-overexpressing embryos compared with controls. In equivalently aged collections, only 32% of
control embryos were undergoing ventral furrow formation compared with 45%
of fog-overexpressing embryos. This implies that
fog-overexpressing embryos take about 1.4 times longer to complete
ventral furrow formation than control embryos. However, this is considerably
less than the ~3.5 times delay that would be required to account for the
large difference seen in apical myosin localization between the
UASfog-expressing embryos and controls. Assuming that if the process
were to take twice as long in UASfog embryos this would account for
50% of the embryos showing apical myosin simply because they are in fact
older, it is estimated that the process would have to be ~3.5 times as long to
account for the actual increased numbers of embryos seen (Dawes-Hoang, 2005).
Therefore fog-overexpressing embryos show a consistent increase in
apical myosin staining in the lateral and dorsal cells of gastrulating embryos
when compared with controls, and this increase is too large to be explained by
the slight delay in gastrulation. It is concluded that fog signaling is
both necessary and sufficient to localize myosin II to the apical side of
cells (Dawes-Hoang, 2005).
It is possible that fog provides a signal to localize or transport
myosin apically, and myosin is then activated to interact and contract with
actin. An intriguing alternative, however, is that fog itself may be
activating myosin contractility, initiating an active motor-driven mechanism
of myosin localization. To help distinguish between these two possibilities,
a form of myosin was constructed that is no longer able to interact or contract
with actin and it was asked if this form of myosin was still able to localize
normally (Dawes-Hoang, 2005).
Myosin is a hexamer comprising two myosin heavy chains (MHCs), two
essential light chains and two regulatory light chains (RLCs). It is the
globular head domain of the MHC subunits that interacts directly with actin
and contains the region of ATPase activity that drives this actin-based motor.
In addition, the ATPase activity and strength of actin binding can be modified
through phosphorylation of the regulatory light chains, while the coiled-coil
tail domains of the MHCs are required for assembly of multiple myosin
molecules into organized filaments (Dawes-Hoang, 2005).
A myosin-YFP transgene (mYFP-myosin IIDN) was constructed in
which the YFP moiety has replaced the actin-binding motor head domain of the
myosin heavy chain, zipper. Based on equivalent modifications in Dictyostelium, mYFP-myosin IIDN homodimers should completely lack actin binding and contractility, and the 'single headed' wild-type myosin/mYFP-myosin IIDN heterodimers should have severely decreased actin binding and contractility. Consistent with this, it was found that YFP-containing myosin isolated from mYFP-myosin IIDN expressing Drosophila
embryos shows reduced actin binding when compared with wild-type myosin in a
standard spin down assay. However, no dominant-negative activity of this transgene during embryogenesis was detected, presumably because of the high levels of endogenous myosin (Dawes-Hoang, 2005).
To analyze the localization of this mYFP-myosin IIDN, the Gal4 system was used to express the transgene uniformly in embryos that also carry
wild-type copies of zipper. For comparison the following were examined: (1) a fully functional myosin-GFP fusion, in which GFP is fused to the myosin light chain, sqhGFP, and (2) the endogenous myosin II of wild-type embryos. No differences were found between the localization patterns of sqhGFP and
endogenous myosin, and only the endogenous myosin will be referred to (Dawes-Hoang, 2005).
When cells divide during later stages of development, the non-functional
mYFP-myosin IIDN shows a localization similar to endogenous myosin. Both localize to the contractile ring as it forms, constricts and then disappears following the completion of cell cleavage. Similarly, during cellularization,
mYFP-myosin IIDN localizes to the cellularization front in a manner similar to endogenous myosin. As reported for sqhGFP, the
mYFP-myosin IIDN tends to form aggregates in the interior of the
embryo. In time-lapse movies, the aggregates associate with the cellularization front, which 'clears' them from the outer edges of the embryos as cellularization
proceeds, but they do not fully integrate into the regular hexagonal array of
mYFP-myosin IIDN associated with the advancing furrows (Dawes-Hoang, 2005).
The first differences between functional and non-functional myosin are
observed at the onset of gastrulation. Unlike endogenous myosin, mYFP-myosin
IIDN fails to localize apically at the onset of ventral furrow
formation and throughout later stages of apical constriction and invagination. The ability of these cells to undergo normal ventral furrow formation despite a lack of apically localized mYFP-myosin IIDN presumably reflects the
activity of endogenous zipper. Both endogenous myosin and mYFP-myosin
IIDN are lost from the basal side of the invaginating ventral
furrow cells. This basal loss is slightly delayed and patchy for mYFP-myosin
IIDN, but otherwise proceeds normally (Dawes-Hoang, 2005).
The requirement for actin binding and subsequent actin-dependent
contractile activity therefore appears to distinguish two functionally
different modes of myosin localization: an actin-independent mode of
localization during cellularization and cytokinesis, and a second mode during
gastrulation where localization to the apical side of the cell is dependent
upon actin binding/contractility. It is possible that the mYFP-myosin
IIDN is defective in ways other than its ability to interact with
actin. However, equivalent constructs in Dictyostelium do not effect any other
aspects of myosin function, including RLC phosphorylation or filament assembly.
Therefore, although such secondary effects can not be entirely ruled out,
the defects seen are most likely a result of the inability to interact with
actin and at the very least distinguish two different types of myosin
localization to the apical and basal sides of the cell. They also highlight the
potential importance of actin-myosin interaction and contractility as a target
for fog signaling (Dawes-Hoang, 2005).
The components acting downstream of fog to mediate its effects on
the cytoskeleton are largely unknown. One candidate, RhoGEF2 (a guanine
nucleotide exchange factor that promotes Rho activation) has been shown to be
required for ventral furrow formation and can genetically interact with a
fog transgene. However, embryos mutant for RhoGEF2 have a much
more severe disruption of ventral furrow formation than embryos mutant for
fog and the point at which the products of these two genes interact
on a mechanistic or subcellular level is unknown. Recent studies have shown a
requirement for RhoGEF2 in controlling actin dynamics/stability
during cellularization and have also shown a disruption to myosin localization at
gastrulation. Therefore the re-localization of myosin during
cellularization and gastrulation was analyzed in RhoGEF2 mutants and
previous studies were extended by looking at a potential downstream effector of
RhoGEF2 signaling (Dawes-Hoang, 2005).
Embryos mutant for RhoGEF2 localize myosin normally to the forming
cellularization front. However, unlike fog mutants, the RhoGEF2
embryos show defects in cellularization, including an irregular, wavy
cellularization front. This implies that although RhoGEF2 function is not required
to localize myosin to the cellularization front it is required to maintain the
normal structure of the cellularization front and that the presence of myosin
is not itself sufficient to maintain a straight cellularization front. This is
consistent with previous studies of RhoGEF2 mutants and a potential
role in controlling actin but not myosin dynamics
(Dawes-Hoang, 2005 and references therein).
However, despite the defects during early cellularization, RhoGEF2
mutant embryos that reach the end of cellularization look remarkably normal. The
irregularity of the cellularization front recovers, particularly in the
ventral cells, and both the increased cell depth and basal loss of myosin
occur normally in these cells. However, in RhoGEF2 embryos, precisely
staged for the onset of gastrulation, there is an absolute failure to
re-localize myosin to the apical side of the ventral cells, despite a normal
loss of myosin from the basal side of these cells. This is consistent
with independent mechanisms controlling the basal loss and apical accumulation
of myosin during gastrulation and demonstrates an absolute requirement for
RhoGEF2 in apical myosin localization. It also confirms the previous report of
RhoGEF2 being required for apical myosin in ventral furrow cells
(Nikolaidou, 1994; Dawes-Hoang, 2005).
RhoGEF2 interacts with myosin in other systems through the Rho-kinase
family of Ser/Thr kinases that inhibit myosin phosphatase and also directly
phosphorylate myosin. Both these activities led to activation of actin binding
by myosin and increased actomyosin based contractility. Additional myosin
activators include MLCK and citron kinase but the extent to which these
different activators play specific or overlapping roles with Rho-kinase is
unclear, and the role of any of these myosin activators during
Drosophila gastrulation is not known (Dawes-Hoang, 2005).
Therefore embryos were produced mutant for Drosophila Rho-kinase
(Drok) by making germline clones of two Drok alleles, both
of which produced similar phenotypes. Myosin localizes to the cellularization
front of Drok mutant embryos but often does so unevenly and, as for
RhoGEF2, the cellularization front is 'wavy'. Unlike the
RhoGEF2 mutant embryos, the nuclei of Drok mutant embryos
have striking defects, including displacement into the interior of the embryo
leaving reduced numbers at the cortex: these remaining nuclei are often of increased size and irregular morphology. It is unclear to what extent these nuclear phenotypes may represent an earlier defect during cell-cycle/nuclear division (Dawes-Hoang, 2005).
Despite these defects, many Drok mutant embryos complete cellularization and though the increased depth of cellularization in ventral cells is difficult to discern, basal loss of myosin proceeds normally. However, Drok mutant embryos show a complete failure to localize myosin to the apical side of the ventral cells at the onset of gastrulation. At later stages of gastrulation, the outer layer of wild-type embryos consists of a single cell layered epithelium that folds in specific locations during germband extension. In Drok
mutant embryos this morphology is severely disrupted and the outer epithelium
becomes multilayered and irregular, containing large often rounded cells. Drok is therefore required to maintain epithelial integrity (Dawes-Hoang, 2005).
Both Drok and RhoGEF2 mutant embryos show defects during
cellularization and then fail to localize myosin to the apical side of ventral
cells at gastrulation. However, it is unlikely that the earlier cellularization defects are what prevent the later apical myosin localization because many other cellularization mutants, such as nullo, display severe cellularization defects but still go on to localize myosin to the apical side of ventral cells at the onset of gastrulation. The failure of Drok and RhoGEF2 mutant embryos to localize myosin apically during gastrulation therefore probably reflects a direct requirement for both these genes in the apical localization of myosin. Despite these disruptions to gastrulation, Drok embryos do still produce Fog protein that is as punctate and apically concentrated as in wild-type embryos. This is therefore consistent with a model whereby Drok driven activation of myosin contractility drives myosin apically in response to fog and RhoGEF2 signaling (Dawes-Hoang, 2005).
To examine the morphological consequences of fog-induced myosin
re-localization, scanning electron microscopy (SEM) was performed on embryos
overexpressing fog. A range of phenotypes was seen consistent with
previous reports in which fog was expressed from a heat-shock
promoter. It is difficult to predict the types of defects to expect
in fog overexpressing embryos, as ventral furrow cells already
express fog and cells outside the ventral furrow may require
additional factors for full shape changes. Furthermore, early defects may lead
to non-specific later defects by the end of gastrulation. However, apical
flattening is the very first effect seen, coincident with the
apical re-localization of myosin and this raises the issue of how these two
processes are connected (Dawes-Hoang, 2005).
This connection is likely to require adherens junctions that anchor the
actin-myosin cytoskeleton to the cell membrane and hold the cells of an
epithelium together. Previous studies have concentrated on the role of
junctions in cell polarity and maintaining integrity of epithelial sheets, or
in cell rearrangements that do not involve changes in cell shape. Much
less is understood about the role of adherens junctions in specific aspects of
cell shape change (Dawes-Hoang, 2005).
Therefore the behavior of adherens junctions was analyzed in
fog-overexpressing embryos. At the completion of cellularization the
embryo consists of a single layered epithelium with the basal junctions of
cellularization located just apical to the myosin rich cellularization front
and the newly forming adherens junctions located about 6 µm in from the
apical surface of the embryo. At the onset of ventral furrow formation, adherens junctions in the ventral most region of the embryo shift to a completely apical location as the cell surfaces flatten, whereas the junctions in more lateral cells
maintain their sub-apical position. These relative positions are maintained during the phase of apical constriction as basal junctions gradually disappear. When fog is expressed throughout the embryo the apical shift of adherens junctions occurs normally in the ventral furrow region, but under these conditions also occurs in more lateral and dorsal cells and these junctions are more tightly condensed than the equivalent junctions of control embryos. The apical
localization of myosin seen in fog-overexpressing embryos therefore
correlates with an apical shift in adherens junctions (Dawes-Hoang, 2005).
The adherens junctions are possibly being pulled into an apical position
because of forces generated by contractile myosin that has been apically
re-localized in response to fog signal. To investigate the connection
between myosin contractility and adherens junctions, myosin localization was examined in embryos that lack adherens junctions (Dawes-Hoang, 2005).
It is not possible to examine embryos totally lacking junctional components
such as Armadillo (Arm) because the maternally supplied components are required
earlier during oogenesis. To get around this problem use was made of the
effects of nullo protein. Expression of nullo during late
cellularization completely blocks the formation of apical spot junctions.
To confirm that results using this technique are due to the lack of adherens
junctions and not to additional effects of nullo expression, the analysis was
repeated with embryos made from arm043A01
germline clones. The arm043A0 allele is of the 'medium
class' of arm alleles, lacking the last few Arm repeats and the
entire C terminus. Germline clones of this class of alleles produce sufficient
levels of Arm function to enable a few eggs to complete oogenesis but
subsequent function of Arm in the embryo is severely compromised and these
embryos fail to assemble apical adherens junctions. The same results were found using both techniques (Dawes-Hoang, 2005).
In both cases, myosin localizes normally to the basal cellularization front and to the apical surface of cells in the ventral furrow. This implies that
functional Arm-containing junctions are not required for myosin to become
localized within the cell. However, subsequent events are affected. As ventral
furrow cells of wild-type embryos undergo apical constriction, myosin is seen
throughout the apical surface of cells, but in embryos
lacking junctions to tether the actin-myosin network, myosin appears to
contract into the center or side of the cell forming a tight 'ball' of
presumably contracted myosin. The most likely explanation of these results is that myosin
contractility is normal in cells lacking adherens junctions but when myosin is
no longer tethered to junctions it contracts without being able to exert force
on the plasma membrane. As a result, these cells are unable to flatten or
constrict their apical surfaces. These results suggest that apically localized
myosin is contractile and that this contractility alone is not sufficient to
result in changes in cell shape but must be tethered to the apical adherens
junctions to elicit apical flattening and constriction (Dawes-Hoang, 2005).
Adherens junctions are also known to play an important role in establishing
and maintaining apicobasal polarity in epithelial cells. However, the
results demonstrate that the polarizing signal for the apical activation of
myosin is not dependent upon any polarizing influence emanating from intact
apical adherens junctions. This is consistent with the idea that it is the Fog
protein, through its apical secretion and reception, that provides the
polarizing signal for myosin activation and that this process is independent
of intact adherens junctions (Dawes-Hoang, 2005).
Thus, this study demonstrates that fog signal is both necessary and sufficient to trigger the relocalization of myosin to the apical side of the cell. This raises the possibility that a secreted signal is used as a means of producing
a polarized response. In this case, secreting a signaling molecule on the
apical side of the cell could be used to ensure an apically localized response
to that signal. In support of this model, it was found that Fog protein is
indeed apically concentrated and therefore comprises the earliest apically
polarized component of this pathway. It will be interesting to see if the
fog independent, parallel pathway of apical myosin recruitment uses a
similar mechanism (Dawes-Hoang, 2005).
It was also demonstrated that apical myosin localization requires the ability of myosin to interact and/or contract with actin. Furthermore, it was shown that
fog signaling results in a shift of adherens junctions from their
usual apicolateral position to a more apical position and that these junctions
are necessary to translate contractile forces into physical changes in cell
shape (Dawes-Hoang, 2005).
Taken together these data suggest the following model. Expression of the
patterning gene twi in the prospective mesoderm cells results in
activation of fog transcription. The resulting Fog protein
is then secreted from the apical surface of the cells and this signal
activates fog receptors. The degree to which this activation is
paracrine versus autocrine has yet to be determined. The apically activated
receptors trigger a transduction pathway involving the G-alpha subunit,
Concertina, and the Rho activator RhoGEF2. A downstream target of this pathway
is Rho-kinase, which in turn activates the ability of myosin to interact and
contract with actin in this sub-apical region of the cell. A localized source
of activated actin-myosin contractility initiates an active motor-driven
mechanism of myosin localization that concentrates contractile myosin to the
apical side of the cell. This actin-myosin network is tethered to the cell
surface through adherens junctions. Contraction of this network therefore puts
tension on the junctions, pulling them into a completely apical location and
flattening the domed apical surface in the process. Continued contraction
exerts further tension and ultimately pulls the junctions together so much
that the entire apical cell surface constricts. Intriguingly, RhoGEF2 protein
can associate with the tips of microtubules in cultured cells. The
extent to which this may add to a polarization of the fog pathway
during gastrulation and how this ties in with the above model will therefore
be interesting avenues for further investigation. It will also be important to
examine any changes to the actin and microtubule organization of these
cells (Dawes-Hoang, 2005).
A hallmark of epithelial invagination is the constriction of cells on their apical sides. During Drosophila gastrulation, apical constrictions under the control of the transcription factor Twist lead to the invagination of the mesoderm. Twist-controlled G protein signaling is involved in mediating the invagination but is not sufficient to account for the full activity of Twist. A Twist target was identified, the transmembrane protein T48, which acts in conjunction with G protein signaling to orchestrate shape changes. Together with G protein signaling, T48 recruits adherens junctions and the cytoskeletal regulator RhoGEF2 to the sites of apical constriction, ensuring rapid and intense changes in cell shape (Kolsch, 2007).
Apical constriction of cells can contribute to the invagination of epithelia, such as during gastrulation or organogenesis, and the closure of wounds. In the Drosophila embryo, apical constrictions occur along the ventral side of the blastoderm epithelium, leading to the formation of the ventral furrow and the invagination of the mesoderm. Proteins necessary for the mechanics of these cell shape changes include the Rho guanosine 5'-triphosphate-exchange factor RhoGEF2 and a heterotrimeric G protein. Whereas RhoGEF2 is essential for furrow formation, disruption of the heterotrimeric G protein, such as by loss of its α subunit Concertina (Cta), leads to a delay but no lasting defects in mesoderm morphogenesis. These maternally supplied proteins must be activated under the control of the zygotic genome in the embryo (Kolsch, 2007).
Twist is the zygotic transcriptional activator that is essential for the cell shape changes that produce the ventral furrow. One of its targets is the transcriptional repressor Snail, which is also essential for mesodermal morphogenesis (Kolsch, 2007).
However, the cell biological events responsible for the cell shape changes must ultimately be regulated by targets that are not transcription factors. Of the known Twist targets, only one, folded gastrulation (fog), is involved in mediating shape changes. Mutants in fog, which codes for a secreted peptide, show the same defects as embryos lacking Cta. Fog is therefore thought to act in the same pathway as Cta, which is referred to as Fog/Cta signaling (Kolsch, 2007).
Fog/Cta signaling is thought to cause changes in the actin cytoskeleton in conjunction with RhoGEF2. Recruitment of myosin from basal to apical in constricting ventral cells is partly dependent on Fog/Cta and absolutely dependent on RhoGEF2. Furthermore, the mammalian homologs of RhoGEF2 and Cta interact. Finally, binding of Drosophila RhoGEF2 to microtubules by means of EB1 is disrupted by activated Cta. Given that myosin recruitment and apical constriction are reduced but not abolished in the absence of Fog/Cta, there must be other factors regulated by Twist that explain its effects on apical constriction (Kolsch, 2007).
In a screen for genes that mediate the zygotic control of gastrulation, the region uncovered by the chromosomal deficiency Df(3R)TlP was found to be necessary for the proper formation of the ventral furrow. Phenotypic analysis and molecular mapping of a set of overlapping deficiencies identified the gene T48 as being responsible for the defects seen in Df(3R)TlP. T48 is expressed in the mesoderm. It codes for a predicted protein with a signal peptide and a potential transmembrane domain. When an internally hemagglutinin-tagged T48 protein (T48HA) was expressed in embryos, it localized at the peripheries of blastoderm cells, consistent with a close association with or insertion into the plasma membrane. Optical cross-sections showed that T48HA is targeted to the apical membrane (Kolsch, 2007).
No other structural motifs are recognizable in the protein. However, the C-terminal amino acid sequence -Ile-Thr-Thr-Glu-Leu (-ITTEL) conforms to the class I consensus for peptides that interact with PDZ domains. T48 has no obvious human ortholog but shows some similarity to the intracellular part of Fras1, which also has a PDZ-binding motif. To find candidates for PDZ domains that might interact with T48, the putative PDZ-binding sequence was analyzed with an algorithm designed to determine the PDZ domains that show the optimal fit for any given peptide. Of the predicted interactors, RhoGEF2 was particularly interesting in view of its role in ventral furrow formation. Furthermore, the mammalian ortholog of RhoGEF2 has been shown to bind to Plexin-B1 by means of a PDZ-binding motif (-Val-Thr-Asp-Leu) very similar to that of T48 (Kolsch, 2007).
Whether the C terminus of T48 is indeed able to interact with RhoGEF2 was tested. A 35S-labeled C-terminal peptide of T48 preferentially coprecipitated with the PDZ domain of RhoGEF2 rather than those of other PDZ domain-containing proteins, in contrast to Crumbs, which was used as a control and which preferentially coprecipitated with PDZ domains from its physiological interaction partner Stardust, as well as Bazooka. In Schneider S2 cells, a green fluorescent protein (GFP)-tagged RhoGEF2 PDZ domain or full-length RhoGEF2 was localized in the cytoplasm or formed intracellular aggregates when expressed alone, but localized to the plasma membrane when coexpressed with T48. In both assays, the interaction required the presence of the -ITTEL motif and was not seen with other PDZ domains. Thus, T48 interacts with RhoGEF2 by means of its PDZ-binding motif and is able to enrich RhoGEF2 to the plasma membrane (Kolsch, 2007).
To understand the function of T48 during gastrulation, the subcellular localization of RhoGEF2 and its dependence on T48 were studied in the developing embryo. Before gastrulation, the apical surfaces of the blastoderm epithelium are dome shaped and the developing adherens junctions are located subapically. RhoGEF2 is associated with the basally located furrow canals, whereas Armadillo is found just below this site and at a subapical position of the lateral cell membranes (Kolsch, 2007).
After cellularization was completed, these distributions changed specifically in ventral cells. Even before morphological changes occurred, RhoGEF2 and Armadillo disappeared from the basal ends. Subsequently, Armadillo disappeared from its subapical site and accumulated apically. A weak association of RhoGEF2 with the apical plasma membrane was seen at this stage (Kolsch, 2007).
As cells begin to flatten apically, high levels of both RhoGEF2 and Armadillo accumulate apically. Although they concentrated in the same region of the cell, Armadillo was restricted to the cell junctions, whereas RhoGEF2 was often more enriched between these sites. Notably, movement of the adherens junctions occurred not only in constricting cells but also in the more lateral mesodermal cells that flattened and became stretched on their apical sides (Kolsch, 2007).
To examine whether these processes depend on T48, stage-selected T48 mutant embryos were stained. Loss of RhoGEF2 and Armadillo from the basal side was unaffected in these embryos, as was the apical concentration of Armadillo. The cells flatten apically and lengthen, but the absence of constrictions results in a thick placode rather than an indentation. Localization of RhoGEF2 to the apical membrane is slightly delayed and possibly reduced. T48 therefore contributes to but is not essential for the recruitment of RhoGEF2 to the apical membrane. This is consistent with the observation that furrow formation is not completely abolished, but only delayed or weakened. Therefore other mechanisms were examined that might participate in RhoGEF2 localization (Kolsch, 2007).
As in the case of T48, mutations in the Fog/Cta pathway delay but do not abolish apical constriction and furrow formation. It was therefore considered whether Fog/Cta signaling might cooperate with T48 to recruit RhoGEF2. In embryos lacking Cta, the recruitment of RhoGEF2 was weakened. Combining mutations in cta and T48 resulted in much more notable effects. These cta,T48 embryos failed to make a furrow; the lack of apical constrictions was mirrored by a failure to accumulate RhoGEF2 apically. Thus, T48 and Fog/Cta signaling act in parallel to concentrate RhoGEF2 apically (Kolsch, 2007).
Severe defects were also observed in the behavior of the adherens junctions in the double-mutant embryos. Armadillo staining disappeared from its tight subapical localization but did not reaccumulate apically. Thus, movement of the junctions is not simply mediated by a tensile force from the constricting actin cytoskeleton: an independent step of at least partial disassembly must occur. It is speculated that this might be controlled by Snail, which regulates the disassembly of cell junctions in vertebrates. It was found that the disassembly of Armadillo from the subapical position was indeed blocked in snail (but not in twist) mutant embryos. Thus, Snail acts in parallel to Twist to direct the disassembly of subapical junctions, a process to which currently unknown Twist targets may also contribute (Kolsch, 2007).
Having observed that T48 and Fog/Cta activation are required for the apical localization of RhoGEF2 and Armadillo, whether T48, like Fog/Cta signaling, was able to trigger their relocalization in other cells was also tested. Ubiquitous expression of T48 in the embryo led to a concentration of RhoGEF2 at the apical membranes of lateral cells. Armadillo localization in ectodermal cells was no longer restricted to a distinct subapical domain but extended to the apical end of the lateral membranes in many cells. When T48 was coexpressed with activated Cta, this effect was slightly enhanced, and some embryos showed morphological defects (Kolsch, 2007).
With T48, a missing factor has been found in the control cascade from transcriptional regulation by Twist to the cell biological mediators of furrow morphogenesis. Two Twist targets, Fog and T48, appear to act in separate pathways that converge on RhoGEF2, which integrates the signal to activate myosin and modify the actin cytoskeleton. This model shows the maternally supplied RhoGEF2 is largely attached to microtubules by means of EB1. The onset of Twist expression has two effects. Fog is synthesized, which triggers the activation of Cta. This in turn releases RhoGEF2 from the microtubules that, by analogy to its vertebrate homologs, may bind to Cta through its RGS domain, allowing some myosin activation and constriction. In parallel, T48 is synthesized and targeted to the apical membrane, where it acts to concentrate RhoGEF2 through its PDZ-binding motif. In the absence of Fog-mediated displacement of RhoGEF2 from EB1, T48 can probably still recruit sufficient freely diffusible RhoGEF2 to allow slow constriction. Only when both mechanisms fail are the downstream events of constriction and junction reassembly abolished completely (Kolsch, 2007).
The utilization of Gα12/13 proteins and a microtubule-bound RhoGEF have also been reported in vertebrate gastrulation. The absence of an obvious homolog of T48 in vertebrates might suggest that this element of the control mechanism is unique to Drosophila gastrulation. However, the PDZ-binding motif in Plexin-B1 is similar to that of T48 and acts during neuronal growth cone remodeling by recruiting PDZ-RhoGEF. Therefore, this mechanism of controlling cell shape may operate in a variety of systems (Kolsch, 2007).
References Dawes-Hoang, R. E., Parmar, K. M., Christiansen, A. E., Phelps, C. B., Brand, A. H. and Wieschaus, E. F. (2005). folded gastrulation, cell shape change and the control of myosin localization. Development 132(18): 4165-78. 16123312
Kolsch, V., Seher, T., Fernandez-Ballester, G. J., Serrano, L. and Leptin, M. (2007). Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2. Science 315(5810): 384-6. Medline abstract: 17234948
Murray, M. J. and Saint, R. (2007). Photoactivatable GFP resolves Drosophila mesoderm migration behaviour. Development 134(22): 3975-83. Medline abstract: 17942486
Genes involved in tissue development
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