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).
Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. This study shows, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. These results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally (Martin, 2009).
During Drosophila gastrulation, apical constriction of ventral cells facilitates the formation of a ventral furrow and the subsequent internalization of the presumptive mesoderm. Although myosin is known to localize to the apical cortex of constricting ventral furrow cells, it is not known how myosin produces force to drive constriction. Understanding this mechanism requires a quantitative analysis of cell and cytoskeletal dynamics. Methods were developed to reveal and quantify apical cell shape with Spider-GFP, a green fluorescent protein (GFP)-tagged membrane-associated protein that outlines individual cells. Ventral cells were constricted to about 50% of their initial apical area before the onset of invagination and continued to constrict during invagination. Although the average apical area steadily decreased at a rate of about 5 microm2 min-1, individual cells showed transient pulses of rapid constriction that exceeded 10-15 microm2 min-1. During the initial 2 min of constriction, weak constriction pulses were often interrupted by periods of cell stretching. However, at 2 min, constriction pulses increased in magnitude and cell shape seemed to be stabilized between pulses, leading to net constriction. These two phases probably correspond to the 'slow/apical flattening' and 'fast/stochastic' phases that have been described previously. Overall, cells underwent an average of 3.2 ± 1.2 constriction pulses over 6 min, with an average interval of 82.8 ± 48 s between pulses (mean ± s.d., n = 40 cells, 126 pulses). Constriction pulses were mostly asynchronous between adjacent cells. As a consequence, cell apices between constrictions seemed to be pulled by their constricting neighbours. Thus, apical constriction occurs by means of pulses of rapid constriction interrupted by pauses during which cells must stabilize their constricted state before reinitiating constriction (Martin, 2009).
To determine how myosin might generate force during pulsed constrictions, myosin and cell dynamics were simultaneously imaged by using myosin regulatory light chain (spaghetti squash, or squ) fused to mCherry (Myosin-mCherry) and Spider-GFP. Discrete myosin spots and fibres present on the apical cortex formed a network that extended across the tissue. These myosin structures were dynamic, with apical myosin spots repeatedly increasing in intensity and moving together (at about 40 nm s-1) to form larger and more intense myosin structures at the medial apical cortex. This process, which is referred to as myosin coalescence, resulted in bursts of myosin accumulation that were correlated with constriction pulses. The peak rate of myosin coalescence preceded the peak constriction rate by 5-10 s, suggesting that myosin coalescence causes apical constriction. Between myosin coalescence events, myosin structures, including fibres, remained present on the cortex, possibly maintaining cortical tension between constriction pulses. Contrary to the purse-string model, no significant myosin accumulation was seen at cell-cell junctions. To confirm that constriction involved medial myosin coalescence and not contraction of a circumferential purse-string, constriction rate was correlated with myosin intensity at either the medial or junctional regions of the cell. Apical constriction was correlated more significantly with medial myosin, suggesting that, in contrast to the purse-string model, constriction is driven by contractions at the medial apical cortex (Martin, 2009).
Myosin coalescence resembled contraction of a cortical actin-myosin network. Therefore, to determine whether apical constriction is driven by pulsed contractions of the actin-myosin network, the organization of the cortical actin cytoskeleton was examined. In fibroblasts and keratocytes, actin network contraction bundles actin filaments into fibre-like structures. Consistent with this expectation was the identification of an actin filament meshwork underlying the apical cortex in which prominent actin-myosin fibres spanning the apical cortex appeared specifically in constricting cells. An actin-myosin network contraction model would predict that myosin coalescence results from myosin spots exerting traction on each other through the cortical actin network. To test whether myosin coalescence requires an intact actin network, the actin network was disrupted with cytochalasin D (CytoD). Disruption of the actin network with CytoD resulted in apical myosin spots that localized together with actin structures and appeared specifically in ventral cells. Myosin spots in CytoD-injected embryos showed more rapid movement than those in control-injected embryos, suggesting that apical myosin spots in untreated embryos are constrained by the cortical actin network. Although myosin movement was uninhibited in CytoD-treated embryos, myosin spots failed to coalesce and cells failed to constrict. Because myosin coalescence requires an intact actin network, it is proposed that pulses of myosin coalescence represent contractions of the actin-myosin network (Martin, 2009).
Because actin-myosin contractions occurred at the medial apical cortex, it was unclear how the actin-myosin network was coupled to adherens junctions. Therefore E-Cadherin-GFP and Myosin-mCherry were imaged to examine the relationship between myosin and adherens junctions. Before apical constriction, adherens junctions are present about 4 microm below the apical cortex. As apical constriction initiated, these subapical adherens junctions gradually disappeared and adherens junctions simultaneously appeared apically at the same level as myosin. This apical redistribution of adherens junctions occurred at specific sites along cell edges (midway between vertices). As apical constriction initiated, these sites bent inwards. This bending depended on the presence of an intact actin network, which is consistent with contraction of the actin-myosin network generating force to pull junctions. Indeed, myosin spots undergoing coalescence were observed to lead adherens junctions as they transiently bent inwards. Thus, pulsed contraction of the actin-myosin network at the medial cortex seems to pull the cell surface inwards at discrete adherens junction sites, resulting in apical constriction (Martin, 2009).
The transcription factors Twist and Snail regulate the apical constriction of ventral furrow cells. Snail is a transcriptional repressor whose target or targets are currently unknown, whereas Twist enhances snail expression and activates the expression of fog and t48, which are thought to activate the Rho1 GTPase and promote myosin contractility. To examine the mechanism of pulsed apical constriction further, how Twist and Snail regulate myosin dynamics was tested. In contrast to wild-type ventral cells, in which myosin was concentrated on the apical cortex, twist and snail mutants accumulated myosin predominantly at cell junctions, similarly to lateral cells. These ventral cells failed to constrict productively, which supported the cortical actin-myosin network contraction model, rather than the purse-string model, for apical constriction. twist and snail mutants differentially affected the coalescence of the minimal myosin that did localize to the apical cortex. Although myosin coalescence was inhibited in snail mutants, it still occurred in twist mutants, as did pulsed constrictions. This difference was also observed when Snail or Twist activity was knocked down by RNA-mediated interference. However, the magnitude of constriction pulses in twistRNAi embryos was greater than that of twist mutant embryos, suggesting that the low level of Twist activity present in twistRNAi embryos enhances contraction efficiency by activating the expression of snail or other transcriptional targets. Myosin coalescence was inhibited in snail twist double mutants, demonstrating that the pulsed constrictions in twist mutants required snail expression. Thus, the expression of snail, not twist, initiates the actin-myosin network contractions that power constriction pulses (Martin, 2009).
Net apical constriction was inhibited in both snailRNAi and twistRNAi embryos. It was therefore asked why the pulsed contractions that were observed in twistRNAi embryos failed to constrict cells. Using Spider-GFP to visualize cell outlines, it was found that although constriction pulses were inhibited in snailRNAi embryos, constriction pulses still occurred in twistRNAi embryos. However, the constricted state of cells in twistRNAi embryos was not stabilized between pulses, resulting in fluctuations in apical area with little net constriction. This stabilization defect was not due to lower snail activity, because these fluctuations continued when snail expression was driven independently of twist by using the P[sna] transgene. Although the frequency and magnitude of constriction pulses in such embryos were similar to those in control embryos, stretching events were significantly higher in twistRNAi; P[sna] embryos, suggesting a defect in maintaining cortical tension. This defect might result from a failure to establish a dense actin meshwork, because both twist mutants and twistRNAi embryos had a more loosely arranged apical meshwork of actin spots and fibres than constricting wild-type cells did. twist expression therefore stabilizes the constricted state of cells between pulsed contractions (Martin, 2009).
Thus, a 'ratchet' model is proposed for apical constriction, in which phases of actin-myosin network contraction and stabilization are repeated to constrict the cell apex incrementally. In contrast to the purse-string model, it was found that apical constriction is correlated with pulses of actin-myosin network contraction that occur on the apical cortex. Pulsed cortical contractions could allow dynamic rearrangements of the actin network to optimize force generation as cells change shape. Because contractions are asynchronous, cells must resist pulling forces from adjacent cells between contractions. A cortical actin-myosin meshwork seems to provide the cortical tension necessary to stabilize apical cell shape and promote net constriction. The transcription factors Snail and Twist are critical for the contraction and stabilization phases of constriction, respectively. Thus, Snail and Twist activities are temporally coordinated to drive productive apical constriction. Despite the dynamic nature of the contractions in individual cells, the behaviour of the system at the tissue level is continuous, in a similar manner to convergent extension in Xenopus. Pulsed contraction may therefore represent a conserved cellular mechanism that drives precise tissue-level behaviour (Martin, 2009).
Understanding how forces and material properties give rise to tissue shapes is a fundamental question in developmental biology. Although Drosophila gastrulation is a major system for investigating tissue morphogenesis, there does not exist a consensus mechanical model that explains all the key features of this process. One key feature of Drosophila gastrulation is its anisotropy - the mesoderm constricts much more along one axis than along the other. Previous explanations have involved graded stress, anisotropic stresses or material properties, or mechanosensitive feedback. This study shows that these mechanisms are not required to explain the anisotropy of constriction. Instead, constriction can be anisotropic if only two conditions are met: the tissue is elastic, as was demonstrated in a recent study, and the contractile domain is asymmetric. This conclusion is general and does not depend on the values of model parameters. This model can explain classical tissue grafting experiments and more recent laser ablation studies. Furthermore, this model may provide alternative explanations for experiments in other developmental systems, including C. elegans and zebrafish (Doubrovinski, 2018).
During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. This study shows that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. Mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. It is proposed that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding (Kanesaki, 2013).
The coordinated movement of cells is one of the foundations of tissue morphogenesis. The forces driving the cellular movements are generated by surface dynamics, such as rearrangements of cell adhesions and changes of the contractility of cortical acto-myosin networks. However, the surface mechanics resisting deformation forces and maintaining cortical integrity are not well understood (Kanesaki, 2013).
The shape of the cell surface can change dynamically. One notable surface feature is the bleb, a spherical protrusion of the plasma membrane observed in diverse cellular processes such as locomotion, division, and apoptosis. Formation of blebs is driven by hydrostatic pressure in the cytoplasm. According to the current model, blebbing starts with local compression of the cytoskeletal network and proceeds according to a subsequent increase of the pressure. The compression of the cytoskeleton is mediated by the contractile force of non-muscle myosin II (MyoII). Though it has been shown that various cells, such as germ line and cancer cells, utilise blebs for their motility, the role of blebs and the mechanism of blebbing in tissue morphogenesis are still largely unclear (Kanesaki, 2013).
Invagination of a cellular layer is one of the common events in tissue morphogenesis. In gastrulation in Drosophila, ventral cells of the blastoderm embryo invaginate and then differentiate to mesoderm. The process of mesoderm invagination can be grossly divided into two sequential steps: apical constriction and furrow internalisation. During apical constriction, ventral cells collectively contract their apices and consequently form a shallow furrow on the embryo. During furrow internalisation, the ventral furrow becomes deeper and the layer of cells becomes engulfed in the embryonic body. The molecular and cellular mechanisms underlying apical constriction have been studied extensively. The change of cellular shape is mediated by integrated functioning of the cortical acto-myosin network and cellular adherens junction complex. The force driving the constriction is generated by pulsed contractility of MyoII. The tensile force from individual cells is transmitted to epithelial tissue through the adherens junction, and the tissue generates feedback force that leads to anisotropic constriction of ventral cells (Kanesaki, 2013).
Heterotrimeric Gprotein (HGP) has an important role in apical constriction in Drosophila gastrulation. Signaling triggered by the extracellular ligand folded gastrulation (fog) promotes surface accumulation of MyoII in ventral cells, and the Fog signaling is mediated through an HGP α subunit encoded by concertina (cta). HGP belongs to the GTPase family, and its activity is regulated by multiple factors, including guanine nucleotide exchange factor (GEF). A previous study showed that ric-8 mutation results in a twisted germ-band due to abnormal mesoderm invagination. ric-8 was first identified as a gene responsible for synaptic transmission in Caenorhabditis elegans, and was shown to interact genetically with EGL-30 (C. elegans Gαq). Nematoda and vertebrate Ric-8 has GEF activity and positively regulates HGP signalingin vivo and in vitro. In Drosophila, Ric-8 is essential for targeting of HGPs toward the plasma membrane and participates in HGP-dependent processes such as asymmetric division of neuroblasts (Kanesaki, 2013 and references therein).
In this study, the precise role of ric-8 in mesoderm invagination was investigated. It was found that cortical stability of ventral cells is impaired in a ric-8 mutant. By a combination of genetic and pharmacological analyses, blebbing of ventral cells was found to be induced by either disruption of cortical actin or mutation of ric-8. It is suggested that HGP signaling constitutively organises cortical actin, thereby reinforcing the resistance of cells against deformation (Kanesaki, 2013).
Ventral cells intrinsically exhibit a few small blebs during mesoderm invagination. This indicates that surface contraction during apical constriction induces blebbing even in normal invagination. This study found that Ric-8 and HGP signaling are required for suppression of abnormally large blebs, and for the stabilisation of the cortex in invaginating cells. The physical mechanism underlying blebbing has been studied extensively in cultured cells. The contractile force of the acto-myosin network causes an increase of hydrostatic pressure in the cytoplasm, which leads to detachment of the plasma membrane from the cortical actin layer. The dynamics of blebs observed in ric-8 ventral cells were similar to those reported in cultured cells in terms of time and size, suggesting that the mechanisms underlying blebbing in these two systems are conserved (Kanesaki, 2013).
The average size of blebs changes as development proceeds: blebs become larger during furrow internalisation. Immuno-fluorescence imaging revealed that MyoII is abnormally accumulated beneath enlarged blebs in the ric-8 mutant. This correlation suggests that MyoII acts to induce an increase of hydrostatic pressure. Although MyoII is an indispensable factor for apical constriction, its activity can also cause malformation of the cells. How MyoII accumulates abnormally in the ric-8 mutant remains unclear. It cannot be ruled out that other processes of mesoderm invagination, such as mechanical stress from surrounding cells, also contributes to the enlargement of blebs. During apical constriction, epithelial tissue generates tension along the anterior-posterior axis, and ventral cells undergo constriction in an anisotropic manner. Similar force may also be generated at the tissue level during furrow internalisation, causing the cells there to be squeezed, and consequently increasing the intracellular pressure. Blebbing in the ric-8 mutant may be a consequence of abnormal cytoskeletal networks and physical stress acting cell to cell. In normal situations, cells would resist such physical stress and maintain the surface integrity, thereby supporting correct morphogenetic movements (Kanesaki, 2013).
This study demonstrates that HGP signaling has two functions in mesoderm invagination: induction of the apical constriction via MyoII accumulation and maintenance of the cellular surface via organisation of cortical actin. Although Fog is required for apical constriction, F-actin is organised in a Fog-independent manner, suggesting that these two functions are regulated in different ways. cta mutants and G-iα65A mutants showed similar phenotypes regarding cortical actin, suggesting that these Gα paralogs have overlapping functions. Because the Drosophila genome encodes 6 Gα subunits and 5 of them are expressed in early embryos, the contribution of G α paralogs other than Cta and G-iα65A to the suppression of blebbing cannot be rule out. The finding that ric-8, Gβ13F, and Gγ1 mutants showed blebbing, a hallmark of severely disturbed cortical actin, supports the idea that multiple HGP pathways control cortical actin redundantly. However, currently it is not known whether those signaling pathways act on the same downstream effectors. Considering that most blastoderm cells showed a dispersed signal of GFP-Moesin in the mutants, HGPs appear to be rather constitutive regulators of cortical actin organisation. Nevertheless, the abnormality of the cortex does not affect the morphology of the 'standstill' cells that do not carry out the inward movement. Thus, HGPs are required to reinforce the cortex so that the cells can endure the stress generated during tissue folding (Kanesaki, 2013).
It was previously reported that ric-8 is required for Drosophila gastrulation. This study extensively investigated mesoderm invagination and found that apical constriction is indeed compromised in the ric-8 mutant. Based on the observation of Fog-dependent MyoII accumulation, it is concluded that ric-8 is required for Fog-Cta signaling. It is unlikely that this phenotype is a secondary consequence of the disorganised F-actin in the ric-8 mutant, because actin was organised normally in the fog mutant embryo and ectopic Fog expression induced cell flattening even in late B-treated embryos. These findings instead suggested that Fog-Cta signaling and actin organisation are separate pathways and Ric-8 is involved in both pathways (Kanesaki, 2013).
Given that HGPs constitutively regulate F-actin, the signaling seems to be active in most blastoderm cells. Some unknown extracellular ligand and its receptor thus appear to be expressed to activate HGPs. It is also possible that cytoplasmic HGP regulators such as Pins, Loco, or other RGS proteins are involved in the activation. In the formation of the blood-brain barrier in Drosophila, Pins and Loco positively regulate HGP signaling. Embryos mutant for Pins also show abnormal cellular movements during mesoderm invagination. It is also intriguing to hypothesise that Ric-8 participates in the activation of HGPs through its GEF activity, which has been characterised both in vivo and in vitro. This hypothesis suggests the possibility that HGPs are endogenously activated. Future analysis of the responsible cytoplasmic regulators may clarify the mechanism of HGP regulation, and may give new insights regarding the intricate network of HGP signaling in animal development (Kanesaki, 2013).
How might HGP be functionally linked to actin polymerisation? Since G α12/13 participates in the activation of Formin family proteins in mammalian fibroblasts and a human Formin inhibits the formation of blebs in a prostate cancer cell line, a candidate factor regulating actin filaments downstream of HGP could be Diaphanous (Dia), a Drosophila Formin. Although it has been shown that organisation of actin via Dia is required for ventral furrow invagination, it is unclear whether Dia is also required for cortical stability during morphogenesis. Considering that Dia is an actin nucleator, it is speculated that Dia might act in the assembly of the actin meshwork and thereby reinforce the cortex. Indeed, it was observed that the dia mutant embryos showed cellular deformation during gastrulation, suggesting the functional relevance of the actin nucleator in the suppression of blebs. Further analysis will be required to clarify the functions of Dia (Kanesaki, 2013).
Previous studies demonstrated that ventral cells form a particular type of F-actin meshwork that makes a basic frame for apical constriction. RhoA- and Abelson-mediated signaling is required for organisation of the apical F-actin meshwork, while the Fog-Cta pathway is not. Thus, it is surprising that the mutants for HGPs, including Cta, showed a defect of cortical actin. HGP signaling may organise only a moiety of F-actin which is distinct from the one specifically accumulated at apices. HGP signaling regulates the organisation of cortical actin and mediates the establishment of the blood-brain barrier in Drosophila , suggesting that this function of HGPs is rather common in fly embryogenesis (Kanesaki, 2013).
Gastrulation of Drosophila melanogaster proceeds through sequential cell movements: ventral mesodermal (VM) cells are induced by secreted Fog protein to constrict their apical surfaces to form the ventral furrow, and subsequently lateral mesodermal (LM) cells involute toward the furrow. How these cell movements are organized remains elusive. This study observed that LM cells extend apical protrusions and then undergo accelerated involution movement, confirming that VM and LM cells display distinct cell morphologies and movements. In a mutant for the GPCR kinase Gprk2, apical constriction expands to all mesodermal cells and the involution movement is abolished. In addition, the mesodermal cells halt apical constriction prematurely in accordance with the aberrant accumulation of Myosin II. Epistasis analyses revealed that the Gprk2 mutant phenotypes are dependent on the fog gene. Overexpression of Gprk2 suppresses the effects of excess Cta, a downstream component of Fog signaling. Based on these findings, it is proposed that Gprk2 attenuates and tunes Fog-Cta signaling to prevent apical constriction in LM cells and to support appropriate apical constriction in VM cells. Thus, the two distinct cell movements in mesoderm invagination are not predetermined, but rather are organized by the adjustment of cell signaling (Fuse, 2013).
In the Gprk2 mutant embryos, cell movements triggered by Fog signaling were compromised. fog is genetically epistatic to Gprk2, indicating that Gprk2 functions by acting on Fog signaling. LM cells undergo apical constriction in the Gprk2 mutant, suggesting that Gprk2 normally inhibits Fog signaling in LM cells. Premature termination of apical constriction and abnormal accumulation of Myosin were also observed in the Gprk2 mutant, suggesting that Gprk2 adjusts Fog signaling to an appropriate level in VM cells. Thus, Gprk2 regulates Fog signalingin a cell group-dependent manner. But what are the underlying molecular mechanisms (Fuse, 2013)?
It is known that GPCR kinase phosphorylates the C-terminal region of GPCR, and regulates GPCR signaling by multiple mechanisms. The phosphorylated GPCR dissociates from the G protein and is internalized from the plasma membrane. This produces a negative-feedback loop for GPCR signaling. Theoretically, the negative-feedback loop stabilizes the signaling and generates biphasic output from fluctuating inputs: OFF for low inputs and ON for high inputs. It is speculated that Gprk2 might phosphorylate a GPCR and might generate biphasic output for Fog signaling in a spatial manner: OFF in LM cells and ON in VM cells. The Fog receptor is expected to be a GPCR, since a G protein (Cta) functions downstream of Fog. Identification of the Fog receptor would help to clarify the molecular functions of Gprk2 (Fuse, 2013).
The kinase activity of Gprk2 is essential for gastrulation. Although it is not yet known what substrates are phosphorylated by Gprk2 in this process, one might be Gprk2 itself because it was observed that Gprk2 protein was phosphorylated in S2 cultured cells and that the phosphorylation was abolished in the K338R mutant of Gprk2. Autophosphorylation of other GPCR kinases has been demonstrated previously and is thought to stimulate their binding to GPCR. Autophosphorylation of Gprk2 might play a similar role (Fuse, 2013).
In addition to its kinase activity, GPCR kinase has an RGS domain, which exhibits GAP (GTPase activating protein) activity and functions in recycling of the Gα protein. Therefore, whether Gprk2 exhibits GAP activity for Cta is an intriguing issue. Indeed, this possibility was supported by genetic data showing that Gprk2 suppresses the effect of Cta overexpression, but not that of Cta Q303L, the GTP-bound form of Cta protein. Cta Q303L might not be subject to the inhibitory effect (GAP activity) of Gprk2, although the alternative explanation has not been ruled out that the inhibition of Cta Q303L might require more Gprk2 protein than does the inhibition of wild-type Cta. Considering that GPCR kinase regulates GPCR signaling by multiple mechanisms, it is suggested that the repression of Cta activity might be one of several means by which Gprk2 regulates Fog signaling (Fuse, 2013).
Fog signaling stimulates the apical localization of Myosin, which generates a force to constrict the apical cell surface. In the wild-type embryo, Myosin protein appears and disappears at the apical surface in a dynamic pattern that they described as 'pulsed coalescence'. In the Gprk2 mutant, Myosin continued to accumulate on the entire apical surface of mesodermal cells. Similar phenomena were also observed in Cta-overexpressing ectodermal cells, and this phenotype was suppressed by simultaneous expression of Gprk2. It is suggested that Gprk2 normally attenuates Fog-Cta signaling to an appropriate level, and such refinement might contribute to controlling the dynamics of Myosin protein (Fuse, 2013).
Previous studies showed that Gprk2 acts in Hedgehog (Hh) signaling for imaginal disc patterning. In this process, Gprk2 phosphorylates a GPCR, Smoothened, and potentiates Hh signaling. Thus, Gprk2 plays roles in multiple signaling pathways in various contexts during development (Fuse, 2013).
The movements of LM cells were characterized, and were found to extended apical protrusions. Some examples have been documented of the extension of protrusions by epithelial cells, such as dorsal ectodermal cells of embryos and wing disc cells of larvae in Drosophila. However, the mechanisms that induce the protrusion and the roles of protrusion in directional cell movement are not understood. Since it was observed that apical protrusions in LM cells always pointed toward the ventral furrow and that cells close to the furrow extended longer protrusions than cells distant from it, it is speculated that the apical protrusion might be induced by the apically constricting neighbors. Indeed, in cta mutant embryos the apical protrusions did not always point toward mid-ventral, but rather frequently pointed toward the slight depressions that were formed at random positions by uncoordinated apical constriction. One possibility is that mechanical or chemical signals that emanate from apically constricting cells might induce apical protrusions in surrounding cells (Fuse, 2013).
Apical protrusions became apparent when LM cells started to accelerate toward the ventral furrow. From this observation, it is supposed that the directional protrusion might contribute to the movement of LM cells. Given that the apical protrusion of LM cells is analogous to the pseudopod of cultured cells, the apical protrusion might act as a scaffold for pulling the cell body into the furrow. The fact that the apical protrusion was also observed in some ectodermal cells, which never undergo involution movement, suggests that the apical protrusion is not sufficient to induce involution movement and that other mechanisms might regulate the cell movement in parallel (Fuse, 2013).
Drosophila mesoderm invagination is driven by sequential movements of different cells. The apical constriction of VM cells is one of the essential movements in this process. It is expected that the involution movement of LM cells might be another of the cell movements driving mesoderm invagination. The movements of different cells would probably influence each other in a complex manner. Observations of LM movements might be explained by such a coordination of cell movements. For example, the apical constriction of VM cells might stretch LM cells and thereby prevent LM cell apical constriction, as previously suggested. VM cells might then continue to move inward and pull LM cells toward the ventral furrow. In addition, ectodermal cells might generate a force to push mesodermal cells inward. These possibilities are not mutually exclusive. Further analyses are required to clarify the role of each cell movement and the effect of coordinated movements in mesoderm invagination (Fuse, 2013).
In the Gprk2 mutant, LM cells underwent apical constriction instead of involution movement. Given the inhibition of Fog signaling in the wild-type LM cells, involution movement might be a default state of mesodermal cells without Fog signaling. As noted above, in the fog and cta mutant, apical constriction occurs in some VM cells, and involution-like movement operates in an uncoordinated manner. These uncoordinated cell movements finally result in disorganized, but nearly complete, mesoderm invagination. Thus, apical constriction and involution movements seem to be alternative choices for mesodermal cells, and robust mesoderm invagination might progress via either type of cell movement. In normal Drosophila embryos, cell movements are spatially and temporally organized, and such organization might ensure the correct shape of gastrulae (Fuse, 2013).
Cell movements in gastrulation show diversity among insects. For example, mosquito embryos undergo only apical constriction and no apparent involution process. Locust embryos undergo neither apical constriction nor involution, but instead utilize the delamination of individual mesodermal cells. Compared with gastrulation in these insects, Drosophila gastrulation is a more complex process and is completed within a shorter time (15 minutes compared with hours). The highly organized cell movements in Drosophila might enable this rapid completion of gastrulation. The molecular mechanisms underlying the evolution of insect gastrulation are an intriguing issue for future studies (Fuse, 2013).
The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, this study shows in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning, and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fog's role during cellularization is widely conserved and therefore might represent the ancestral function of the pathway (Benton, 2019).
During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. This work develops a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. Novel data is introduced that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. Maintenance of this constant volume is shown to be sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. The specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, this model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that is observed in the actual embryo, with no active force generation required other than apical constriction (Polyakov, 2014).
Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much is now understood of the molecular mechanism responsible for apical constriction and of the tissue-scale integration of the ensuing in-plane deformations, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, this study used the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging leading to tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, this idea is now challenged, and it is shown that embryo-scale force balance at the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation (Fierling, 2022).
Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, an important role was identified for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. Depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. These findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding (Fuentes, 2022).
Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. This study reports the function of a common pathway comprising the heterotrimeric G proteins Gα12/13 (Concertina), Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog (Poor gastrulation, Pog & CG31660) was identified as being required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. It is proposed that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators (Kerridge, 2016).
During tissue morphogenesis, cells rearrange their contacts to invaginate, intercalate, delaminate or divide. During Drosophila gastrulation, invagination of the presumptive mesoderm in the ventral region of the embryo and of the posterior midgut requires apical cell constriction, a geometric deformation that occurs in different organisms. Elongation of the ventral–lateral ectoderm requires cell intercalation, a general topological deformation associated with junction remodelling. In the ectoderm, the so-called ‘vertical junctions’, oriented along the dorsal–ventral axis, shrink, followed by extension of new ‘horizontal’ junctions along the anterior–posterior axis. Despite differences in the cell deformations associated with intercalation and apical constriction, recent studies revealed that both processes require myosin II (MyoII) contractility. Cell shape changes rely on the pulsatile activity of MyoII in the apical–medial cortex, whereby MyoII undergoes cycles of assembly and disassembly allowing stepwise deformation1. Moreover, each step of deformation is stabilized and thereby retained, contributing to the irreversibility of tissue morphogenesis. In the mesoderm, each phase of apical area constriction mediated by MyoII pulses is followed by a phase of shape stabilization involving persistence of medial MyoII. In the ectoderm, medial–apical MyoII pulses flow anisotropically towards vertical junctions resulting in steps of shrinkage that are stabilized by a planar-polarized pool of junctional MyoII. This ratchet-like behaviour of MyoII is regulated by the Rho1–Rok pathway and requires quantitative control over MyoII activation. Low Rho1/Rok activity fails to form actomyosin networks, intermediate activation establishes MyoII pulsatility and high activation confers stability. The signalling mechanisms that cause stepwise activation of MyoII by Rho1 remain unknown. It is also unclear whether different pathways for Rho1 activation operate in the mesoderm and in the ectoderm as indeed Rho1 can be activated by numerous signalling mechanisms or whether a common pathway might exist (Kerridge, 2016).
Tissue-specific factors can result in polarized shape changes by signalling through cell surface receptors. For instance, in Drosophila ectoderm, pair rule genes encoding transcription factors control planar-polarized enrichment of MyoII through the combinatorial expression of the surface proteins Toll2, Toll6 and Toll8 in stripes. Likewise, in the mesoderm, Twist and Snail induce expression of Fog, a secreted ligand, and a G-protein-coupled receptor (GPCR) Mist (methuselah-like 1), which is reported to transduce Fog. The downstream G protein Gα12/13 (known as Concertina (Cta) in Drosophila) is required for RhoGEF2 and thereby MyoII apical recruitment. As RhoGEF2 is a known GEF for Rho1, the requirement of Gα12/13 for RhoGEF2 apical recruitment suggests that GPCRs and G-protein signalling mediate MyoII activation through the Rho1 pathway. These considerations prompted asking whether G-protein signalling directly controls the different regimes of MyoII dynamics (pulsatility and/or stability) in the mesoderm and planar polarized activation of Rho1 and MyoII in the ectoderm (Kerridge, 2016).
This study reports the function of the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and regulating MyoII dynamics both in the mesoderm and in the ectoderm. Receptor activation, through the GEF activity of the GPCR, converts Gα from an inactive GDP-bound state, in a complex with Gβγ, to an active GTP-bound state. This results in dissociation of Gβγ, enabling binding of both Gα–GTP and Gβγ to their respective effectors for signalling. This study found that Gα12/13 and the Gβ13F/Gγ1 complex constitute distinct signalling modules, which regulate MyoII dynamics medial–apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog, was found to be required for cell shape changes associated with both mesoderm invagination and ectoderm elongation. During these morphogenetic events, Smog functions with other GPCRs, Mist in the mesoderm and an as yet unknown GPCR in the ectoderm, to activate the Rho1–Rok pathway. This results in stepwise activation of Rho1 and MyoII, ensuring irreversible cell shape changes (Kerridge, 2016).
First, this study reports that Gα12/13 and Gβ13F/Gγ1 function as distinct signalling modules that control Rho1 and MyoII in different domains. Gα12/13 activates medial–apical MyoII through its effector RhoGEF2 both in the ectoderm and the mesoderm. In mammals, p115–RhoGEF interacts directly with Gα12 suggesting that this may be a conserved signalling module. In contrast, Gβ13F/Gγ1 activates MyoII both at cell junctions and in the medial–apical domain. This modularity may provide distinct regulatory mechanisms for the activation of MyoII in different subcellular compartments owing to the existence of different molecular effectors of Gα–GTP and Gβγ. Second, stepwise activation of Rho1 by multiple GPCRs and their ligands determines the emergence of a pulsatile regime medial–apically, or stable activation. In the mesoderm, Smog and Mist GPCRs, together with high expression of their ligand Fog, ensure stabilization and rapid (<5 min) accumulation of MyoII ensuring apical constriction. In the ectoderm, low Fog expression and thus lower activation of Gα12/13 and RhoGEF2 is responsible for intermediate medial–apical activation of MyoII and pulsatility. Indeed, Fog, constitutively active Gα12/13QL and RhoGEF2 overexpression all lead to stable accumulation of MyoII instead of pulsation, similar to constitutively active RhoV14 (Kerridge, 2016).
Interestingly, the same receptor Smog controls MyoII activation in different subcellular domains during intercalation and apical constriction begging the question of how activation of Gα12/13 and Gβγ is differentially achieved in the ectoderm and the mesoderm. The polarization of Smog activation is to some extent imparted by the ligand. Fog/Smog regulates medial–apical accumulation of MyoII in the two tissues: Fog induces medial Rho1 and Rok activation in the mesoderm and ectoderm and, when ectopically expressed in the ectoderm, it can increase Rho1 and Rok in the medial cortex. This argues that another mechanism results in junction-specific activation of Smog, Gβ13F/Gγ1, Rho1 and Rok in the ectoderm (Kerridge, 2016).
It is possible that an unknown ectoderm-specific ligand activates Smog specifically at junctions. Junctional localization of the Rho1 pathway by Smog may also be imparted by subcellular processing of Smog signalling, such as localization/activation of downstream effectors of Gα12/13 and Gβγ. The recently identified Toll receptors required for MyoII planar-polarized activation may bias Smog signalling although the molecular mechanisms remain unclear. This could be through localization of RhoGEFs. In the mesoderm, the transmembrane protein T48 localizes RhoGEF2 apically through binding to its PDZ domain, and is required for apical MyoII activation in parallel with Smog, Gα12/13 and Gβγ. Similarly, other GEFs may be required for junctional Rho1 activation by Smog (Kerridge, 2016).
What might be the advantage of having multiple GPCRs? Gastrulation sets the foundation for all other future processes in development and hence requires robustness. GPCRs with similar functions yet subtle differences such as ligand specificity may offer advantages compared with single ligand–receptor pairs. For instance, high cortical tension associated with mesoderm invagination may require multiple GPCRs activating parallel pathways to attain efficiency of the process. Moreover, multiple GPCRs may concede tissue-specific regulation of the common G-protein subcellular pathways. Finally, multiple GPCRs can allow stepwise activation of MyoII. Although activation by one GPCR is sufficient to induce pulsatility, more GPCRs are required to shift the actomyosin networks to more stable regimes (Kerridge, 2016).
The discovery that Smog and heterotrimeric G protein activate Rho1 and MyoII in two different morphogenetic processes provides a potentially general molecular framework for tissue mechanics. It is proposed that different developmental inputs tune a common GPCR/G-protein signalling pathway to direct specific patterns and levels of Rho1 activation. Quantitative control specifies the regime of MyoII activation through Rho1, namely pulsatility or stability of MyoII. Modular control defines the subcellular domains where MyoII accumulates (medial–apical or junctions) depending on molecular effectors. How developmental signals tune GPCR signalling will be important to decipher (Kerridge, 2016).
Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. This study measured the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. It was established that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be approximately 103-fold as viscous as water. Elasticity was shown to depend on the actin cytoskeleton, and these results are discussed as to how they relate to existing mechanical models of morphogenesis (Doubrovinski, 2017).
Sculpting organism shape requires that cells produce forces with proper directionality. Thus, it is critical to understand how cells orient the cytoskeleton to produce forces that deform tissues. During Drosophila gastrulation, actomyosin contraction in ventral cells generates a long, narrow epithelial furrow, termed the ventral furrow, in which actomyosin fibres and tension are directed along the length of the furrow. Using a combination of genetic and mechanical perturbations that alter tissue shape, this study demonstrated that geometrical and mechanical constraints act as cues to orient the cytoskeleton and tension during ventral furrow formation. An in silico model of two-dimensional actomyosin meshwork contraction was developed, demonstrating that actomyosin meshworks exhibit an inherent force orienting mechanism in response to mechanical constraints. Together, these in vivo and in silico data provide a framework for understanding how cells orient force generation, establishing a role for geometrical and mechanical patterning of force production in tissues (Chanet, 2017).
Tissue morphogenesis is strikingly robust. Yet, how tissues are sculpted under challenging conditions is unknown. This study combined network analysis, experimental perturbations, and computational modeling to determine how network connectivity between hundreds of contractile cells on the ventral side of the Drosophila embryo ensures robust tissue folding. Two network properties were identified that mechanically promote robustness. First, redundant supracellular cytoskeletal network paths ensure global connectivity, even with network degradation. By forming many more connections than are required, morphogenesis is not disrupted by local network damage, analogous to the way redundancy guarantees the large-scale function of vasculature and transportation networks. Second, directional stiffening of edges oriented orthogonal to the folding axis promotes furrow formation at lower contractility levels. Structural redundancy and directional network stiffening ensure robust tissue folding with proper orientation (Yevick, 2019).
Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, it was find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. It is proposed that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling (Guo, 2022).
Src kinases are important regulators of cell adhesion. This study has explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. During cell intercalations, Src42A was shown to be required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, Src42A was shown to act in concert with Abl kinase, which has also been implicated in cell intercalations. These data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time (Chandran, 2023).
Compartmental boundaries physically separate developing tissues into distinct regions, which is fundamental for the organisation of the body plan in both insects and vertebrates. In many examples, this physical segregation is caused by a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. This study performed an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, from the start of germband extension at gastrulation and throughout the germband extended stages (stages 6 to 11). First, it was found that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared with wild-type and ftz mutant embryos, it was shown that Tartan is required during germband extension but not beyond. Candidate genes were identified that could take over from Tartan at PSBs, and it was confirmed that at germband extended stages, actomyosin enrichment at PSBs requires Wingless signalling (Sharrock, 2022).
The mesoderm invagination of the Drosophila embryo is known as an archetypal morphogenic process. To explore the roles of the active cellular forces and the regulation of these forces, this study developed an integrated vertex model that combines the regulation of morphogen expression with cell movements and tissue mechanics. The results suggest that a successful furrow formation requires an apical tension gradient, decreased basal tension, and increased lateral tension, which corresponds to apical constriction, basal expansion, and apicobasal shortening respectively. The model also considers the mechanical feedback which leads to an ectopic twist expression with external compression as observed in experiments.The model predicts that ectopic invagination could happen if an external compressive gradient is applied (Jiang, 2023).
Shape changes of epithelia during animal development, such as convergent extension, are achieved through concerted mechanical activity of individual cells. While much is known about the corresponding large scale tissue flow and its genetic drivers, the question of cell-scale coordination remains open. It is proposed that this coordination can be understood in terms of mechanical interactions and instantaneous force balance within the tissue. Using whole embryo imaging data for Drosophila gastrulation, this study exploited the relation between balance of local cortical tension forces and cell geometry. This unveils how local positive feedback on active tension and passive global deformations account for coordinated cell rearrangements. A model was developed that bridges the cell and tissue scale dynamics and predicts the dependence of total tissue extension on initial anisotropy and hexagonal order of the cell packing. This study provides general insight into the encoding of global tissue shape in local cell-scale activity (Brauns, 2023).
Cephalic furrow formation (CFF) is a major morphogenetic movement during gastrulation in Drosophila melanogaster embryos that gives rise to a deep, transitory epithelial invagination. Recent studies have identified the individual cell shape changes that drive the
initiation and progression phases of CFF; however, the underlying mechanics are not yet well understood. During the progression phase, the furrow deepens as columnar cells from both the anterior and posterior directions fold inwards rotating by 90°. To analyze the mechanics of this process, this study has developed an advanced two-dimensional lateral vertex model that includes multinode representation of cellular membranes and allows capturing of the membrane curvature associated with pressure variation. These investigations reveal some key potential mechanical features of CFF, as follows. When cells begin to roll over the cephalic furrow cleft, they become wedge shaped as their apical cortices and overlying membranes expand, lateral cortices and overlying membranes release tension, internal pressures drop, and basal cortices and membranes contract. Then, cells reverse this process by shortening apical cortices and membranes, increasing lateral tension, and causing internal pressures to rise. Since the basal membranes expand, the cells recover their rotated columnar shape once in the furrow. Interestingly, these findings indicate that the basal membranes may be passively reactive throughout the progression phase. It was also found that the smooth rolling of cells over the cephalic furrow cleft necessitates that internalized cells provide a solid base through high levels of membrane tension and internal pressure, which allows the transmission of tensile force that pulls new cells into the furrow. These results led to the suggestion that CFF helps to establish a baseline tension across the apical surface of the embryo to facilitate cellular coordination of other morphogenetic movements via mechanical stress feedback mechanisms (Niloy, 2023).
During early development, myosin II mechanically reshapes and folds embryo tissue. A much-studied example is ventral furrow formation in Drosophila, marking the onset of gastrulation. Furrowing is driven by contraction of actomyosin networks on apical cell surfaces, but how the myosin patterning encodes tissue shape is unclear, and elastic models failed to reproduce essential features of experimental celf contraction profiles. The myosin patterning exhibits substanatial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. In this study, using biophysical modeling wviscous forces were found to offer the principle resistance to actomyosin-driven apical constriction. In consequence, tissue shape is encoded in the direction-dependent curvature of the myosin patterning which orients an anterior-posterior furrow. Tissue contraction is highly sensitive to cell-to-cell myosin fluctuations, explaining furrowing failure in genetically perturbed embryos whose fluctuations are temporally persistent. In wild-type embryos, this catastrophic outcome is averted by pulsatile myosin time-dependence, a time-averaging effect that rescues furrowing. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms (Zhu, 2023).
Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. This study examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within only 2 hr of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized 'on the go.' The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of deoxyATP (dATP) present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust, with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis (Song, 2017).
Tissue morphogenesis arises from controlled cell deformations in
response to cellular contractility. During Drosophila gastrulation, apical activation
of the actomyosin networks drives apical constriction in the
invaginating mesoderm and cell-cell intercalation in the extending
ectoderm. Myosin II (MyoII; Zipper) is
activated by cell-surface G protein-coupled receptors (GPCRs), such as
Smog and Mist, that activate G
proteins, the small GTPase Rho1, and
the kinase Rok. Quantitative
control over GPCR and Rho1 activation underlies differences in
deformation of mesoderm and ectoderm cells. The GPCR Smog activity is
concentrated on two different apical plasma membrane compartments, i.e.,
the surface and plasma membrane invaginations. Using fluorescence
correlation spectroscopy, the surface of the plasma membrane was
probed, and it was shown that Smog homo-clusters in response to its
activating ligand Fog. Endocytosis
of Smog is regulated by the kinase Gprk2 and beta-arrestin-2 that clears
active Smog from the plasma membrane. When Fog concentration is high or
endocytosis is low, Smog rearranges in homo-clusters and accumulates in
plasma membrane invaginations that are hubs for Rho1 activation. Lastly,
this study found higher Smog homo-cluster concentration and numerous
apical plasma membrane invaginations in the mesoderm compared to the
ectoderm, indicative of reduced endocytosis. Dynamic partitioning of
active Smog at the surface of the plasma membrane or plasma membrane
invaginations has a direct impact on Rho1 signaling. Plasma membrane
invaginations accumulate high Rho1-guanosine triphosphate (GTP)
suggesting they form signaling centers. Thus, Fog concentration and Smog
endocytosis form coupled regulatory processes that regulate differential
Rho1 and MyoII activation in the Drosophila embryo (Jha, 2018).
Tissue morphogenesis requires control over changes in cell shape and cell-cell contacts, which depend on the spatiotemporal regulation of actomyosin contractility. In Drosophila embryos, mesoderm invagination is driven by apical constriction, a geometric cell shape change facilitated by medial-apical Myosin II activation. In the ectoderm, tissue extension arises from cell-cell intercalation, whereby cells undergo neighbor exchange through the polarized remodeling of cell junctions. Junction remodeling is driven by medial-apical MyoII contractile pulses and MyoII planar polarized accumulation (Jha, 2018).
Actomyosin contractility is regulated by conserved signaling pathways. MyoII regulatory light chain is activated by Rho-kinase (Rok) downstream of the small GTPase Rho1, which in turn is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). This conserved pathway was shown to be under the direct control of signaling at the cell surface, such as Celsr in vertebrate neural tube formation and G protein-coupled receptors (GPCRs) in early Drosophila embryos. The GPCRs Mist (Manning, 2013) and Smog (Kerridge, 2016) transduce signals from the secreted ligand Fog in the Drosophila presumptive mesoderm (Mist and Smog) and ectoderm (Smog). Medial-apical MyoII activation progresses downstream of hetero-trimeric G proteins Gα12/13, Gβ13F, and Gγ1 in both mesoderm and ectoderm. In the mesoderm, high medial-apical MyoII activation is under a stable regime that ensures persistent apical constriction, while in the ectoderm, intermediate medial-apical MyoII activation is under a pulsatile regime that enables cell-cell intercalation. Therefore, to understand how quantitative activation of MyoII is generated and its temporal dynamics encoded, it is necessary to decipher the regulation of GPCR signaling (Jha, 2018).
Differential MyoII activation in the mesoderm and ectoderm is partly imparted by the ligand Fog, co-expression of Mist and Smog in the mesoderm, as well as by the mesoderm-specific transmembrane protein T48, which enhances apical recruitment of RhoGEF2 and, thereby, is proposed to potentiate Rho1 and MyoII activation. High Fog expression in mesoderm activates high MyoII, while in the ectoderm low Fog expression leads to low activation of MyoII. However, in general, ligand availability is one of several mechanisms impacting GPCR activation and signaling. Various cell culture studies have focused on the other modalities that regulate GPCR signaling. The major regulators of GPCR signaling are G protein-coupled receptor kinases (GRKs) that phosphorylate GPCRs and trigger signal termination, by allowing β-arrestin binding and recruitment of other adaptor proteins. In turn, β-arrestins direct activated receptors to clathrin-coated pits and remove them from the plasma membrane by endocytosis. While removal of activated GPCRs from the plasma membrane via endocytosis terminates GPCR signaling, it also reduces the number of receptors present on the surface for ligand stimulation. This effectively sets a quantitative control over GPCR signaling via endocytosis. Drosophila has only one non-visual GRK (Gprk2) and one non-visual β-arrestin-2 (kurtz). Gprk2 mutant mothers show aberrant contractility in the mesoderm lateral cells, and it was suggested that Gprk2 attenuates Fog-dependent MyoII activation in these cells. Eggs lacking Kurtz display cuticle phenotypes and suggest gastrulation defects. These data indicate that Kurtz plays a role with Gprk2 to terminate Fog signaling and could control Rho1 and MyoII via GPCR endocytosis. Its function in the mesoderm and ectoderm has not been addressed (Jha, 2018).
Conventionally, GPCR signaling from the plasma membrane is thought to occur via ligand binding and subsequent signal transduction via G proteins that relay the information to the interior of the cell. Apart from GPCR endocytosis, the localization of GPCR within the cell membrane will influence GPCR signaling. Lateral movement of GPCRs within the plasma membrane is often restricted to specific nano-domains, suggesting that selective compartmentalization is necessary for efficient signaling as it can increase GPCR localization and clustering. GPCR clustering in the form of homo- and hetero-oligomers has been reported to control both signal amplification as well as receptor recycling. Whether the main role of GPCR clustering is for chaperoning active receptors for transport or to control GPCR signaling specificity remains unclear, especially during development. To understand GPCR signaling during tissue morphogenesis, it is important to elucidate both the clustering of GPCRs at the plasma membrane and the role of endocytosis (Jha, 2018).
This study investigated the quantitative regulation of the GPCR Smog signaling by endocytosis in both the ectoderm and the mesoderm. Fog was shown to promote homo-clusters of Smog, while endocytosis rapidly removes Smog homo-clusters from the surface of the plasma membrane in the ectoderm. Dynamic partitioning of active Smog homo-clusters in two plasma membrane compartments, the surface or the plasma invaginations, was shown to directly impact Rho1 and MyoII activation. In the mesoderm, numerous apical plasma membrane invaginations and high Smog homo-clusters correlate with high Rho1 and MyoII activation compared to the ectoderm (Jha, 2018).
Epithelial cells exhibit different types of cell deformations owing to quantitative control over cell contractility that arises from contraction of the actomyosin cytoskeleton. GPCR signaling relays information conveyed by tissue-specific factors in the mesoderm and ectoderm to control this quantitative regulation during tissue morphogenesis. Rho1-dependent activation of MyoII during both apical constriction in the mesoderm and cell-cell intercalation in the ectoderm is controlled by GPCR signaling. Activation of the GPCR Smog underlies Rho1 activation in both mesoderm and ectoderm. It is believed that differential regulation of the GPCR Smog and other GPCRs underlies these tissue-specific differences in MyoII activation. This partly relies on the fact that Fog, the activating ligand, is present at higher levels in the mesoderm than in the ectoderm. This work sheds new light on this process by probing the plasma membrane organization and distribution of Smog in conditions that affect both endocytosis and production of the ligand Fog (Jha, 2018).
Probing the ectodermal cells with FCS, Smog homo-clusters on the surface of apical plasma membrane is reported and this process depends on Fog. When Fog is absent, such as in a fog-dsRNA, the brightness per Smog::GFP unit is lower, suggesting that Fog induces the formation of Smog homo-clusters. Dynamic exchange of homo-clustered Smog occurs sbetween the surface and plasma membrane invaginations. This dynamic distribution of Smog between the two plasma membrane compartments is strongly dependent upon both the rate of Smog endocytosis and Fog concentration. Increasing Fog concentration or reducing Smog endocytosis enhances the presence of Smog homo-clusters in apical plasma membrane invaginations, which results in an apparent decrease in Smog homo-clusters at the cell surface. When Fog concentration is high under conditions where Smog endocytosis is reduced, for example, when β-arrestin-2 is knocked down, Smog homo-clusters accumulate at the surface as well as in the plasma membrane invaginations. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that control the Smog cluster formation and influence the distribution of active Smog in different plasma membrane compartments. Importantly, this controls the quantitative activation of Rho1 and MyoII. Under low-endocytosis regimes (Gprk2 or β-arrestin-2 knockdowns in the ectoderm), high levels of active Rho1 accumulate in the apical plasma membrane invaginations. It is proposed that the apical plasma membrane invaginations are signaling hubs, where signaling components could concentrate, give rise to high G protein signaling (e.g., Gα12/13), and sustain high MyoII activation. The size and stability of these signaling invaginations is tuned by endocytosis, and they may provide a means to control the strength and persistence of signaling. Pulsatile active Rho1 in the ectodermal cells requires intermediate Rho1 activation. In the ectoderm, low Fog expression and rapid Smog endocytosis by Gprk2 and β-arrestin-2 lead to intermediate activation of Rho1. In turn, intermediate Rho1 activation at the apical plasma membrane creates the conditions required for self-organized actomyosin dynamics associated with pulsation (Jha, 2018).
This study also points to the possibility of tissue level regulation of endocytosis and plasma membrane compartmentalization of GPCRs. Large apical plasma membrane invaginations are observed in the mesoderm compared to the ectoderm. In the mesoderm, Smog accumulates in larger, more numerous, apical plasma membrane invaginations, and it displays larger Smog homo-clusters compared to in the ectoderm. In the mesoderm, Rho1 and MyoII activation is higher. Another GPCR, Mist produced in the mesoderm, works synergistically with Smog to boost Rho1 and MyoII activation (Manning, 2013). This is also due to the expression of another GPCR Mist in the mesoderm and to Fog being present at higher levels in the mesoderm. Ectodermal cells have similar properties of high Smog homo-clusters when Fog is overexpressed and GPCR endocytosis is slowed down. An intriguing possibility is that Smog and potentially Mist endocytosis is downregulated in the mesoderm compared to the ectoderm. Interestingly, the E3 ubiquitin ligase Neuralized (Neur), which is uniformly expressed in the embryo, is inhibited in the ectoderm by the small proteins of the Bearded (Brd) family. Brd genes are repressed by the mesoderm transcription factor Snail, so that Neur is only active in the mesoderm. In a Brd mutant, where Neur becomes active in the ectoderm, MyoII activation is increased and Neur degradation or repression in the mesoderm following Brd overexpression both reduce MyoII activation. Previous studies have shown that the E3 ubiquitin ligase targets β-arrestin-2 for ubiquitination and degradation, and, thereby, it affects endocytosis and signaling by GPCRs. It is possible that GPCR endocytosis could be reduced in the mesoderm due to increased Neur activity in this tissue. This may depend on the downregulation of several target proteins, such as β-arrestin-2 (Jha, 2018).
Selective compartmentalization of GPCR on the plasma membrane as in the case of large apical plasma membrane invaginations can increase the concentration and the probability of GPCR clustering and oligomerization. The current data suggest that the dynamic modulation of GPCR signaling can be achieved by a change in their cluster/oligomer formation. Receptor oligomerization may enlarge the signaling capacities by the recruitment of more downstream signaling components during GPCR signaling. G proteins are reported to be expressed at low concentration, and selective compartmentalization of GPCRs on the plasma membrane further increase the probability of GPCR clustering and oligomerization for efficient signaling. Investigation of G protein activation by different GPCRs in vivo will be needed to test if a similar mechanism is in place during epithelial morphogenesis (Jha, 2018).
During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. This study used optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. Precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes (Izquierdo, 2018).
The results presented in this study show that localized activation of Rho signaling at the apical surface of cells, which are otherwise not programmed to invaginate, is sufficient to cause tissue invagination and to recapitulate major cell- and tissue-level behaviors associated with endogenous invagination processes. Mechanisms other than apical constriction control a variety of different forms of invaginations during animal development. The current results do not challenge this view, rather, they argue that if considering a monolayer of epithelial cells, apical constriction is sufficient to fold it into a U-shape invagination. However, apical constriction is not sufficient to drive closure of an invagination into a tube-like structure, as seen for example during ventral furrow formation. Additional pushing forces exerted by lateral non-invaginating cells and/or loss of myosin II from the basal surface and basal relaxation might be required to complete this step (Izquierdo, 2018).
At the tissue-level, the contractile behavior of individual cells depends on the geometry of photo-activation. While a square box results in isotropic apical constrictions, a rectangular shape causes cells to constrict preferentially along the minor axis of the photo-activated area and to elongate along the major axis. This anisotropic contractile behavior resembles the one of ventral furrow cells, which are also organized in a rectangular pattern and constrict preferentially along the short axis of the tissue. Anisotropy in ventral furrow cells is not genetically determined but arises as a consequence of tissue geometrical and mechanical constraints. Consistent with these studies, the increase in the degree of anisotropic constriction as a function of the rectangularity of the photo-activated area can be explained if considering that it is mechanically less favorable to shrink cells along the major axis of a rectangle than along the short axis. Indeed, the former deformation requires the endpoints of the constricting tissue to move farther, and thus a larger deformation of neighboring tissues along that axis (Izquierdo, 2018).
The results also reveal an interesting correlation between pulsatile constrictions and tissue invagination. During endogenous morphogenetic processes, two different pulsatile behaviors have been described. One is based on cycles of myosin II accumulation at the medio-apical plane of the cell, during the contraction phase, and dissolution during the relaxation phase. This type of pulsatile behavior has been first described during dorsal closure in Drosophila and it is not linked to tissue invagination. Another type of pulsatile behavior is based on an incremental accumulation of myosin II at each contraction, which is followed by a stabilization period of cell shape without an intervening relaxation phase (ratcheted contractions). Ratcheted contractions require the radial polarization of Rho signaling components. However, in certain mutant conditions that interfere with the establishment of radial polarity, contractions become non-ratcheted with myosin II miss-localizing at three-ways junctional vertices. The optogenetic-induced pulsatile contractions described in this study display a non-ratchet behavior, and similarly to dorsal closure contractions, are characterized by myosin II medio-apical accumulation and dissolution in phase with contraction and relaxation of the apical surface. However, differently from dorsal closure, optogenetic-induced pulsatile contractions display a higher degree of synchrony with photo-activated cells constricting and relaxing in concert. This difference could be explained if considering that a light pulse provides a coherent and synchronous input, while activation of signaling in a developing tissue might be more subject to cell-to-cell variability. Lack of tissue internalization upon induction of pulsatile constrictions is likely due to the absence of a stabilization phase after constriction of the apical surface, which might result in a dissipation of the forces that are normally needed to build tension and drive invagination. Consistently, continuous administration of light induced synchronous contractile behavior and invagination, mimicking the activity of signaling molecules such as Fog whose function is to control the transition from stochastic to collective contractile behavior during ventral furrow invagination. Pulsatile behavior could be elicited either by a discontinuous administration of light, or by continuous illumination at a lower laser power, or by a single pulse at a higher laser power. These results are interpreted to suggest that pulsations can be induced by the stimulation of a Rho-dependent mechano-chemical oscillatory system up to a certain threshold, above which cells constrict without pulsing. Stimulation of Rho signaling above a certain threshold could override, for example, the activity of a RhoGAP, which is required to control the normal spatio-temporal dynamics of Rho GDP/GTP cycling. In agreement with this proposal, pulsatile constrictions during ventral furrow invagination require the activity of a specific RhoGAP. However, while ventral cells pulse with a mean period of ~80 s, optogenetic-induced pulsations display a mean period of ~150 s, a limit probably imposed by the reversion kinetics of the CRY2/CIB1 system in the dark (Izquierdo, 2018).
In conclusion, these data illustrate the utility of applying concepts of synthetic biology (e.g., precise orthogonal control over signaling pathways, guided cell behavior) to the field of tissue morphogenesis and in particular of how the nascent field of synthetic morphogenesis can help defining the minimum set of requirements sufficient to drive tissue remodeling. The data argue that while normally tissue differentiation and tissue shape are intimately linked, it is possible to direct tissue shape without interfering with complex layers of gene regulatory network and tissue differentiation programs. This might have important implications also for tissue engineering, where it might be desirable to shape any given tissue of interest without changing its fate (Izquierdo, 2018).
Epithelial folding is typically driven by localized actomyosin contractility. However, it remains unclear how epithelia deform when myosin levels are low and uniform. In the Drosophila gastrula, dorsal fold formation occurs despite a lack of localized myosin changes, while the fold-initiating cells reduce cell height following basal shifts of polarity via an unknown mechanism. This study shows that cell shortening depends on an apical microtubule network organized by the CAMSAP protein Patronin. Prior to gastrulation, microtubule forces generated by the minus-end motor dynein scaffold the apical cell cortex into a dome-like shape, while the severing enzyme Katanin facilitates network remodelling to ensure tissue-wide cell size homeostasis. During fold initiation, Patronin redistributes following basal polarity shifts in the initiating cells, apparently weakening the scaffolding forces to allow dome descent. The homeostatic network that ensures size/shape homogeneity is thus repurposed for cell shortening, linking epithelial polarity to folding via a microtubule-based mechanical mechanism (Takeda, 2018).
Epithelial folding is a fundamental morphogenetic process in which two-dimensional (2D) epithelial sheets deform into 3D, folded structures. Epithelial folds form throughout animal development, giving rise to internal tissue structures and functional organ units. Folding of an epithelial monolayer can be induced by local forces generated at the site of initiation, or resulting from global stresses that buckle the tissue by exploiting intrinsic mechanical inhomogeneities. Both types of folding have been predominantly associated with modulation of myosin-dependent contractility. However, for epithelial folding events that lack overt myosin changes, it remains unclear what active mechanism underlies tissue deformation (Takeda, 2018).
Dorsal fold formation during Drosophila gastrulation has emerged as a crucial model for alternative folding mechanisms since myosin levels are low and uniform across the tissue. Following the completion of cellularization that forms the first embryonic epithelial layer, folding begins as two stripes of initiating cells straddling across the dorsal surface at stereotypical locations become shorter than their neighbours, leading to the formation of anterior and posterior dorsal folds eventually. Prior to cell shortening, Par-1, the MARK family kinase that specifies the basal-lateral membrane, retracts its apical margin and becomes downregulated, resulting in basal repositioning of adherens junctions in the initiating cells. Following such basal polarity shifts, the apices of initiating cells shrink and descend below the embryonic surface. How basal polarity shifts result in shrinkage and descent of the apices to reduce cell height was not known. As Par-1 has recently been shown to control the localization of the microtubule minus-end protector Patronin, this study investigated the possibility that Patronin-dependent modulation of the microtubule network underlies dorsal fold initiation (Takeda, 2018).
This study has identified a non-centrosomal microtubule network that is anchored and crosslinked to the apical cortex to exert pushing forces, thereby scaffolding the spherical apical shape of the epithelial cells in the Drosophila embryo prior to gastrulation. The data suggest that this microtubule network possesses an intrinsic, feedback-dependent remodelling mechanism that helps dampen the mechanical noises arising within the tissue, thus ensuring size homogeneity, echoing previous reports on microtubule-dependent cell size homeostasis. As gastrulation commences, coupling to the basal polarity shifts repurposes this homeostatic network for cell shortening (Takeda, 2018).
The microtubule forces exerted by the apical network must exist prior to, and in the opposite direction to, the movement of dome descent, thus contrasting with myosin-dependent apical constriction whose amplitude and directionality positively correlate with cell shape changes. It is proposed that dome descent in the dorsal fold system may be induced by residual stresses that compress inward and initially counterbalance the microtubule-dependent outward pushing forces, but become dominant as the basal redistribution of Patronin weakens the outward forces. This model is mechanically similar to the shape control of red blood cells where the microtubule-based marginal band pushes the cortex to counterbalance cortical tension generated by the Spectrin/actin-based membrane skeleton. It is possible that Spectrin and/or additional microtubule/actin dual linkers, such as the spectraplakin protein Shot, underlie the hypothetical counterbalancing stresses, and it is worth noting that recent work implicated links between Spectrin/Shot and Patronin/CAMSAP. Thus, in the context of the current model, Par-1 downregulation initially causes a transient expansion of the apical volume as a result of basal shifts of junctions. The expanded apical dome probably becomes mechanically imbalanced due to basal redistribution of the Patronin-anchored microtubule network, such that the pre-existing cortical compression stresses shrink the apex to drive dome descent. When such an imbalance occurs in a localized manner, a mechanical weak point could be produced to allow for initiation of folding. Conversely, global perturbation via stiffening (overexpression of Patronin) or softening (knockdown of Dynein) of the cortices probably causes a uniform deviation of such a balanced state, which instead blocks folding (Takeda, 2018).
The Patronin/CAMSAP proteins have emerged as key regulators of the microtubule minus ends, particularly for the non-centrosomal microtubules. During early cellularization prior to its transitioning to the apical cortex, however, Patronin is localized to the centrosome and appears to be required for the anchorage of centrioles and nucleus. Similar phenotypes have also been reported for the mammalian intestinal epithelia. These data could suggest an additional function on the centrosomal microtubules, which may also account for Patronin's involvement in the apical translocation of Bazooka. Intriguingly, junctional positioning in the patronin bazooka double RNAi embryos exhibits both a wide basal spread and an apical shift, respective features observed in the patronin and bazooka single knockdowns. Thus, Patronin may be required for junctional positioning in a Bazooka-independent manner. Moreover, the double RNAi embryos do not fold, even though the junctions are differentially positioned. It may be possible that the tissue-level mechanical coupling via junctions becomes defective due to the loss of Bazooka, resulting in ineffective transmission of tensile or compressive forces necessary for folding (Takeda, 2018).
The data implicate a cell shape control function for the Patronin/CAMSAP proteins in the epithelial system. The apical microtubule network that was observed resembles a similar structure previously described in the mammalian epithelial cells and could be a common feature for some epithelial systems. Highly pliable epithelial cells owing to low actomyosin-based rigidity such as those found on the dorsal side of the Drosophila gastrula may depend on the microtubule-based forces to define their shape, contrasting with epithelial contexts where cortical actomyosin forces dominate and cell shapes themselves dictate the spatial arrangement of microtubule filaments, but not vice versa. Evidence has emerged in certain contexts wherein actomyosin and microtubule networks are both involved in cell shape changes for the initiation of folding. It will be interesting to see whether context-specific interplay between these two mechanical systems underlies the formation of distinct morphological features (Takeda, 2018).
Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, was followed in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. It is concluded that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos (Rahimi, 2019).
The early Drosophila embryo provides extreme challenges for the generation and maintenance of extracellular morphogen gradients. Most notably, the peri-vitelline fluid surrounding the embryo facilitates rapid diffusion of molecules. In addition, the alteration in the surface of the plasma membrane at every nuclear division provides an active mixing force. Thus, analysis of the early morphogen gradients operating in this environment, including ventral Spz/Toll activation and the subsequent BMP gradient patterning the dorsal aspect, should consider this highly dynamic environment. In the case of the Toll pathway, the active Spz ligand is generated by proteolytic processing within the extra-embryonic peri-vitelline fluid in a broad ventral region, defined by the activation domain of the Easter (Ea) protease. The generation of a sharp Spz activation gradient within this broad ventral domain of processing takes place by diffusion-based shuttling. Previous work demonstrated that the pro-domain of Spz plays an instructive role in delivering the active, cleaved ligand towards the ventral midline. While a variety of experiments and computational analyses indicated the utilization of a 'self-organized shuttling' mechanism in this context, it was imperative to visualize the actual dynamics of the process (Rahimi, 2019).
It was possible to infer the dynamics of the extracellular Spz gradient by following the kinetics of Dl-GFP nuclear accumulation in individual live embryos during the final syncytial nuclear division cycles and the early phase of NC 14. Nuclear levels of Dl are not a direct readout of the extracellular gradient, since accumulation of Dl in the nuclei is re-initiated at the onset of every nuclear cycle. Nevertheless, it is possible to infer key features of the extracellular Spz gradient from this dynamic behavior. Using this approach hallmarks of ligand shuttling were identified, most notably the lateral overshoot and the presence of two lateral peaks which converge to a central ventral peak. This convergence takes place within a timeframe of minutes, and repeats at every nuclear cycle. Since new protein molecules of the extracellular components are continually translated, the ongoing activity of the shuttling process is vital. Therefore, shuttling is important not only for generating the gradient, but also for maintaining it, in the face of rapid diffusion and mixing within the peri-vitelline fluid. Importantly, by ~10-15 minutes into NC 14, when the robust induction of transcription of the cardinal zygotic Dl-target genes twt and sna ensues, the nuclear gradient of Dl is sharp and a single ventral peak is resolved (Rahimi, 2019).
Having described the dynamics of Dl-nuclear entry and gradient formation, an experimental approach was used in order to examine regulatory processes affecting Toll signaling. The Wnt family ligand WntD provides an essential buffering system to variations in Toll signaling between embryos. wntD is an early zygotic gene that is expressed initially at the posterior-ventral region of the embryo, and its expression levels depend on the magnitude of Toll signaling. Although WntD is produced locally, the rapid secretion and diffusion of the protein in the peri-vitelline space generates a uniform attenuation of Toll signaling throughout the embryo surface. The activity of WntD leads in different embryos to convergence of the variable global Toll activation gradient to a similar pattern, which is dictated by the fixed final signaling level that shuts off wntD expression. This paradigm, termed 'distal pinning', is achieved in this case by an induction-contraction mechanism (Rahimi, 2019).
Secreted WntD is recruited to the plasma membrane by binding to its receptor Fz4. Epistasis assays have indicated that WntD exerts its inhibitory effect on Toll signaling by associating with the extracellular domain of Toll, thereby reducing the number of Toll receptors that are available for binding Spz. Bearing the cardinal features of shuttling in mind, this mode of inhibition implies that the effect of WntD would be global and non-autonomous, and will actually change the shape of the gradient, making it sharper. The observed dynamics of Dl-GPF in wntD mutant embryos indeed confirms this prediction. The shuttling process is driven by competition between the inhibitory Spz pro-domain and the Toll receptor for binding free, active Spz. Binding to the pro-domain is favored in the lateral part of the embryo, where its concentration is higher, while in more ventral regions binding to Toll takes over. Since WntD impinges on the extracellular properties of the Toll receptor, the active ligand is deposited in more ventral regions, where the concentration of the pro-domain is lower. Thus, WntD does not simply reduce the overall profile of Toll activation, but actually re-directs the ligand from the lateral regions to the ventral domain. Previous work has shown that accumulation of excess ligand in the peak by shuttling is an effective mechanism to buffer noise. Since activation in this region is already maximal, the excess ligand will not alter the resulting cell fates (Rahimi, 2019).
The rapid timing of processes in the early embryo and the short duration of interphases between nuclear divisions raises the question of whether it is actually possible to produce sufficient levels of WntD that will drive the morphogen profile to the desired equilibrium. When monitoring wntD transcription directly utilizing the MS2 system, most, if not all embryos were shown to express wntD indicating that Toll signaling overshoots in most embryos. Furthermore, within single embryos the number of nuclei expressing wntD was reduced between NCs 12 and 13, and completely terminated by NC 14, implying that WntD impinges on the Toll gradient and its own expression by this time. The intronless arrangement of the wntD gene and the rapid secretion of the protein, which does not require post-translational modifications (Herr et al., 2012), may facilitate the process (Rahimi, 2019).
The ventral cohort of zygotic target genes including twi and sna is induced by the Toll activation gradient, and the threshold for their induction corresponds to ~50% of maximal Dl-nuclear localization. Within the ventral domain, nuclei exhibit a similar level of sna transcription.
These genes are triggered at NC 14 after the Dl gradient is stabilized and a distinct activation peak generated (Rahimi, 2019).
Are there zygotic target genes that respond to the dynamics of Dl nuclear targeting, before it stabilizes? This appears to be the case for T48, which encodes a transmembrane protein that facilitates recruitment of RhoGEF2 and ultimately Rho and actomyosin, to mediate apical constriction of invaginating mesodermal cells. Graded distribution of myosin II was shown to be critical for proper invagination of these cells to form the ventral furrow (Rahimi, 2019).
This study provides evidence that the graded distribution of T48 mRNA results from the dynamics of Dl-nuclear re-entry at NC 14. The ventral-most cells reach the threshold of T48 induction earlier than more lateral cells, and hence will express the gene longer. Integration of the length of expression along the D-V axis then leads to a gradient of cytoplasmic T48 mRNA accumulation. This example represents a unique case, where graded morphogen activation instructs the generation of a gradient of target-gene expression. The strict dependence on the timing of transcription initiation provides another mechanism to generate differences between adjacent nuclei along the D-V axis (Rahimi, 2019).
In conclusion, this work has utilized live imaging of Toll pathway activation, to identify and characterize the hallmarks of ligand shuttling. This process is rapid and takes place continuously throughout the final nuclear division cycles, to generate and maintain a sharpactivation gradient in the diffusible environment of the peri-vitelline fluid. WntD impinges on Spz shuttling, and is responsible not only for buffering variability between embryos, but also for generating a sharp activation peak. This peak is utilized to induce a graded expression of a zygotic target gene that is essential for executing processes that drive gastrulation. Thus, diffusion-based ligand shuttling, coupled with a dynamic readout, establishes a refined pattern within the environment of early embryos (Rahimi, 2019).
During development, coordinated cell shape changes and cell divisions sculpt tissues. While these individual cell behaviors have been extensively studied, how cell shape changes and cell divisions that occur concurrently in epithelia influence tissue shape is less understood. This question was addressed in two contexts of the early Drosophila embryo: premature cell division during mesoderm invagination, and native ectodermal cell divisions with ectopic activation of apical contractility. Using quantitative live-cell imaging, it was demonstrated that mitotic entry reverses apical contractility by interfering with medioapical RhoA signaling. While premature mitotic entry inhibits mesoderm invagination, which relies on apical constriction, mitotic entry in an artificially contractile ectoderm induced ectopic tissue invaginations. Ectopic invaginations resulted from medioapical myosin loss in neighboring mitotic cells. This myosin loss enabled non-mitotic cells to apically constrict through mitotic cell stretching. Thus, the spatial pattern of mitotic entry can differentially regulate tissue shape through signal interference between apical contractility and mitosis (Ko, 2020).
During embryo development, tissues often undergo multiple concomitant changes in shape. It is unclear which signaling pathways and cellular mechanisms are responsible for multiple simultaneous tissue shape transformations. This study focussed on the process of concomitant tissue folding and extension that is key during gastrulation and neurulation. The Drosophila embryo was used as model system and focus was placed on the process of mesoderm invagination. This study shows that the prospective mesoderm simultaneously folds and extends. Mesoderm cells, under the control of anterior-posterior and dorsal-ventral gene patterning synergy, establish two sets of adherens junctions at different apical-basal positions with specialized functions: while apical junctions drive apical constriction initiating tissue bending, lateral junctions concomitantly drive polarized cell intercalation, resulting in tissue convergence-extension. Thus, epithelial cells devise multiple specialized junctional sets that drive composite morphogenetic processes under the synergistic control of apparently orthogonal signaling sources (John, 2021).
Formation of the ventral furrow in the Drosophila embryo relies on the apical constriction of cells in the ventral region to produce bending forces that drive tissue invagination. In a recent paper it was observed that apical constrictions during the initial phase of ventral furrow formation produce elongated patterns of cellular constriction chains prior to invagination; it was argued that these are indicative of tensile stress feedback. This study quantitatively analyzed the constriction patterns preceding ventral furrow formation and found that they are consistent with the predictions of an active-granular-fluid model of a monolayer of mechanically coupled stress-sensitive constricting particles. The model shows that tensile feedback causes constriction chains to develop along underlying precursor tensile stress chains that gradually strengthen with subsequent cellular constrictions. As seen in both this model and available optogenetic experiments, this mechanism allows constriction chains to penetrate or circumvent zones of reduced cell contractility, thus increasing the robustness of ventral furrow formation to spatial variation of cell contractility by rescuing cellular constrictions in the disrupted regions (Holcomb, 2021).
The intrinsic genetic program of a cell is not sufficient to explain all of the cell's activities. External mechanical stimuli are increasingly recognized as determinants of cell behavior. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic program of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells and thereby tissue folding. However, some cells do not constrict but instead stretch, even though they share the same genetic program as their constricting neighbors. This study shows that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress-strain responses do not reproduce experimental observations, but simulations in which cells behave as ductile materials with nonlinear mechanical properties do. These models show that this behavior is a general emergent property of actomyosin networks in a supracellular context, in accordance with experimental observations of actin reorganization within stretching cells (Bhide, 2021).
Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. This study reports that PtdIns(3,4,5)P(3) is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP(3), and disruption of PIP(3) reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP(3) enrichments are developmentally regulated, with mesodermal cells having high apical PIP(3) while germband cells have higher interfacial PIP(3). Finally, this study shows that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues (Miao, 2021).
Cell shaping processes use contractile force generation to drive the active contraction of specific cell surfaces that causes tissues to adopt new morphogenetic forms. This selective contraction of cell surfaces drives a diverse range of processes from tissue invagination to cell intercalation to epithelial cell extrusion and wound healing. A key discovery in the last decade of work on these processes is that they are often pulsatile in nature, with highly transient actomyosin populations that briefly apply a tensioning force to an area of the cell cortex. This has raised a central outstanding question-if actomyosin networks display such pulsed behaviors, how does processivity emerge from cyclic systems? To obtain lasting changes from such systems, there is a requirement for ratcheting mechanisms that gain irreversible changes out of periodic, contractile cycles (Miao, 2021).
Several potential molecular mechanisms of cell ratcheting have been discovered, ranging from processes that direct the turnover and reformation of elastic cortical networks to pathways that rely on direct remodeling of the plasma membrane. Previous work has shown that a membranous ratchet centered on SET-domain-binding factor (Sbf) RabGEF and Rab35 function is a critical part of the pathway used to remodel the cell surfaces during early morphogenesis in Drosophila. This membrane ratcheting module is deployable to specific cell surfaces-during cell intercalation, Sbf and Rab35 tubular invaginations of the plasma membrane are primarily found at planar polarized contracting interfaces, where they mediate the processive loss of AP (anterior-posterior) surfaces required for neighbor exchange through endocytic mechanisms. Alternatively, during the apical constrictions that drive invagination of the ventrally located mesoderm, Sbf and Rab35 are enriched at the apical surface, where they direct the ratcheted loss of apical surface areas. However, how this ratcheting activity is selectively recruited to specific cell surfaces is unknown. Although actomyosin function is required to terminate Sbf/Rab35 compartments, it is not required for Sbf and Rab35 recruitment. Thus, Sbf and Rab35 recruitment is not downstream of actomyosin function, suggesting that yet-to-be-identified mechanisms are in place to control Sbf/Rab35 compartmental formation (Miao, 2021).
One potential important cue for directing the localized activities of plasma-membrane-associated proteins are the lipid-signaling phosphoinositides (PIPs), which are often found in microdomains in the plasma membrane. PIPs have been shown to be potent regulators of both membrane trafficking and cytoskeletal networks. The various PIP phospho-species, especially PI(4,5)P2 and PI(3,4,5)P3, can regulate the activity of a number of endocytic-regulatory proteins, such as AP-2 and Dynamin. PIPs are also implicated in controlling actin assembly and plasma membrane-cytoskeletal linkage by binding directly and tightly to at least 30 regulatory proteins. PI(3,4,5)P3 in particular has been deeply implicated in the regulation of membrane trafficking processes such as regulated endocytosis and exocytosis. Upregulation of PIP3 levels induces recycling of the epidermal growth factor receptor to the cell surface, and PIP phospho-balance can regulate syncytial cleavage furrow lengths in the fly embryo. Further, recently published work suggests that PIPs may lay downstream of Toll receptor activity and Src activation in the germband epithelium. However, how PIPs regulate the dynamic cell shape changes that occur as epithelial sheets change dimensions is unclear, and the function of PIP phospho-species during Drosophila gastrulation remains to be closely examined. This study identified phosphatidylinositol phosphates as providing lipid-based membrane cues for morphogenesis and cellular gastrulation dynamics in the early Drosophila embryo. PI(3,4,5)P3 inhibition leads to a potent disruption of contractile cellular behaviors, and PIP3 function controls a 'constriction switch' that determines if epithelial cells enter into an apical constriction or cell interface contraction regime. This switch in contractile surfaces then determines which of two primary morphogenetic paradigms the developing tissue will follow-a primarily intercalatory process driven by neighbor exchange events, or the loss of apical areas that lead to furrow formation and tissue invagination (Miao, 2021).
The ability of epithelial cells to remodel specific cellular surfaces is central to determining cell dimensions as well as the neighbor community that they will interact with and adhere to. This ability to either grow or contract certain cell sides will determine overall cell shape, and the cumulative effects of these cell shape changes determines tissue behaviors and morphologies. By regulating the contraction of apical surfaces versus cell-cell interfaces, a tissue can drive events as diverse as furrow formation and cell ingression to cell intercalation and the intermixing of cells along the AP axis. However, it has been unclear whether direct cues reside within the plasma membrane that may guide and control the engagement of contractile forces. This study examined the function of plasma membrane phospholipids in recruiting Sbf-Rab35-driven ratcheting. PI(3,4,5)P3 was shown to regulates a switch in ratcheting engagement-a reduction in PIP3 levels causes a reorientation of Sbf-Rab35 compartment formation to apicomedial surfaces. This relocalization is sufficient to change the reversible oscillations in cell area that occur in the wild-type germband epithelium into a processive regime in which apical cell areas shrink and ectopic furrows are formed. Sbf, the guanine nucleotide exchange factor for Rab35, can directly bind PIP3, and PIP3 levels and sites of enrichment are differentially regulated between the germband and ingressing mesoderm to provide a differential lipid-based cue between these two tissues (Miao, 2021).
It is interesting to note that while ectopic furrows form after PIP3 disruption, these furrows are often disorganized and lack the regular appearance of the main ventral furrow that drives the ingression of mesoderm during gastrulation. In some respects, this is to be expected-whether the major portion of the embryo is transformed to attempt to contract apical surfaces, then cells will be engaged in a contractile tug-of-war against each other. This condition is likely shown by the juxtaposition of small and large cells, which is observed in both the germband as well as the ventral furrow after PIP3 disruption. The uneven contraction of apical surfaces may also reflect the different temporal dynamics of Sbf-Rab35 compartments after PIP3 disruption. The new medial compartments that form after PIP3 disruption are much more stable (indeed, lifetimes up to 10x longer in some measurements) and robust than those that form in either the germband or ventral furrow in control embryos. Previous work has shown the importance of contractile cycles to achieve a uniform overall contraction of the apical surfaces. For example, in circumstances where pulsatility, but not contractility, is compromised, contractile networks have been observed to tear and separate-this results in a similar loss of cell area uniformity as detected after PIP3 levels are downregulated. Thus, the change in Sbf-Rab35 compartment function to much more stable and longer cycles may enhance the tug-of-war element of the cell contractions previously referenced, producing 'winner' cells of much smaller apical areas and 'loser' cells that cannot shrink against the pulling forces of neighboring cells and thus possess larger apical areas. It is intriguing that the pulsatility of contraction appears to be such a fundamental element of contractile processes-pulsatility has been observed across a huge variety of contraction-driven processes ranging from wound healing to compaction of the mouse embryo to neuroblast ingression (Miao, 2021).
Another interesting aspect of this work is that both phosphatidyl inositol phosphate species and JAK/STAT-dependent signaling control where ratcheting engagement occurs. If either of these pathways is disrupted, then a medial signal dominates and Sbf-Rab35 compartment formation occurs in a central, apical location. The current model suggests that PIP3 and JAK/STAT signaling may provide a dispersal signal that guides the compartments from a single apico-central location to the cell periphery (in the case of germband epithelial cells) or to smaller, more dispersed apical locations (in cells of the ventral furrow). Based on the direct binding of PIP3 by the Sbf RabGEF in the PIP binding assay, this dispersal may be through a direct interaction. The experiments did not have the resolution to determine if small PIP3 microdomains exist in the plasma membrane, or if further systems direct the formation of smaller, compartmental assemblies. On the other hand, how does JAK/STAT direct ratcheting engagement? Previous work examining the apical constrictions driven by an absence of JAK/STAT signaling implicated a repression of WASP actin networks that, when activated, may cause the enhanced recruitment of apical myosin II populations. Interestingly, this fits with the current step detection measurements. In addition to the changes in Sbf-Rab35 localization, pulsed contractions are stronger and more sustained in JAK/STAT embryos than in PIP3-disrupted embryos. However, previous results have shown that the generation of Sbf-Rab35 compartments is independent of myosin II function. Thus, from these results, we suggest that, while PIP3 directly regulates ratcheting engagement, JAK/STAT may regulate both the underlying oscillatory machinery as well as the strength of the medial signal that directs ratcheting engagement. It is interesting to note that, through the use of automated step detection measurements based on mean squared displacements, it has been previously shown that the oscillatory machinery appears to be strengthened specifically in the ventral cells that will undergo processive apical constriction to form the ventral furrow, which suggests a commonality with the behaviors observed after JAK/STAT disruption (Miao, 2021).
Phosphatidylinositol phosphates and their function in morphogenetic processes
PIP phospho-species are attractive candidates to provide important spatial information as they are directly embedded in target membranes. PIP3 has long been implicated in distinguishing the leading edge in migrating cells and is rapidly upregulated after cells are stimulated with chemoattractant where it promotes F-actin assembly necessary for cell crawling. However, how PIP3 regulates gastrulation events has been relatively unstudied. A recent work has demonstrated the Pi3K92E can bind to active Toll receptors and Src signaling complexes, and is planar polarized at AP interfaces, in keeping with our finding of developmental enrichment of PIP3 in the germband and suggesting an intriguing connection between planar positional information and PIP function (Tamada, 2021). PIP3 is necessary for cell migration events in the mesenchymal gastrulation movements that occur in zebrafish, while data from Drosophila have shown that PIP3 levels are upregulated after wounding and help cells recognize affected surfaces. Disrupting PIP3 levels disrupted dorsal closure in the late embryo, where, once again, PIP3 is found at higher levels specifically at those surfaces that are driving tissue remodeling. This work similarly finds the PIP3 levels are developmentally patterned, where they are enriched at contractile surfaces. Other work has shown that a PIP2/PIP3 balance affects actomyosin contractility during the cellularization process that creates the early embryonic epithelium through the recruitment of an actin stabilizer, bottleneck. Sbf-Rab35 compartments have represented an interesting convergence point between pathways that directly regulate cell membrane remodeling and those that control cortical force generation. Going forward, it will be interesting to examine if this convergence includes similar higher-level regulation of the protein networks that have been implicated in migrating systems (Miao, 2021).
This work demonstrates a fundamental switch in contractile behaviors depending on the activity and localization of the PI(3,4,5)P3 lipid cue. There were several limitations to these studies: first, many of the functional disruptions relied on pharmacological or shRNA knockdown lines which often produce only hypomorphic disruptions. Phenotypes were confirmed with secondary shRNA lines that targeted different regions of the selected mRNA and yet produced similar defects; however, deeper disruption of these genes may produce more severe defects at these stages or earlier in development. Second, a PIP3 biosensor (tGPH-GFP) was used to detect PIP3 localization and levels-this is the standard in the field but represents an indirect binder of PIP3, so future probe development may allow a better resolution of PIP3 behaviors. As was mentioned, it would be very interested to be able to better address if PIP3 microdomains exist in the plasma membrane, and if these may directly trigger site-specific Sbf/Rab35 compartmental formation, but these were not resolvable with the combination of probe and microscopy elements. Finally, gastrulation in the early Drosophila embryo shows a remarkable robustness to disruption, so additional phenotypes may be concealed by compensatory mechanisms that were not detected in our analysis (Miao, 2021).
Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets, which are driven by cell divisions, cell shape changes, and cell intercalations. Each of these behaviors has been associated with distinct aspects of gastrulation and has been a subject of intense research using genetic, cell biological, and more recently, biophysical approaches. Most of these studies, however, focus either on cellular processes driving gastrulation or on large-scale tissue deformations. Recent advances in microscopy and image processing create a unique opportunity for integrating these complementary viewpoints. This study takes a step toward bridging these complementary strategies and deconstruct the early stages of gastrulation in the entire Drosophila embryo. The approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. The detected events are then mapped back onto the blastoderm shell, providing an intuitive visual means to examine complex cellular activity patterns within the context of their initial anatomic domains. By analyzing these maps, it was identified that the loss of nearly half of surface cells to invaginations is compensated primarily by transient mitotic rounding. In addition, by analyzing mapped cell intercalation events, direct quantitative relations between intercalation frequency and the rate of axis elongation were derived. This work is setting the stage for systems-level dissection of a pivotal step in animal development (Stern, 2022).
References
Benton, M. A., Frey, N., Nunes da Fonseca, R., von Levetzow, C., Stappert, D., Hakeemi, M. S., Conrads, K. H., Pechmann, M., Panfilio, K. A., Lynch, J. A. and Roth, S. (2019). Fog signaling has diverse roles in epithelial morphogenesis in insects. Elife 8. PubMed ID: 31573513
Bhide, S., Gombalova, D., Monke, G., Stegmaier, J., Zinchenko, V., Kreshuk, A., Belmonte, J. M. and Leptin, M. (2021). Mechanical competition alters the cellular interpretation of an endogenous genetic program. J Cell Biol 220(11). PubMed ID: 34449835
Brauns, F., Claussen, N. H., Wieschaus, E. F. and Shraiman, B. I. (2023). Epithelial flow by controlled transformation of internal force-balance geometry. bioRxiv. PubMed ID: 37398061
Chandran, L., Backer, W., Schleutker, R., Kong, D., Beati, S. A. H., Luschnig, S. and Muller, H. J. (2023). Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation. Development 150(2). PubMed ID: 36628974
Chanet, S., Miller, C. J., Vaishnav, E. D., Ermentrout, B., Davidson, L. A. and Martin, A. C. (2017). Actomyosin meshwork mechanosensing enables tissue shape to orient cell force. Nat Commun 8: 15014. PubMed ID: 28504247
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. PubMed ID: 16123312
Doubrovinski, K., Swan, M., Polyakov, O. and Wieschaus, E. F. (2017). Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids. Proc Natl Acad Sci U S A 114(5): 1051-1056. PubMed ID: 28096360
Doubrovinski, K., Tchoufag, J. and Mandadapu, K. (2018). A simplified mechanism for anisotropic constriction in Drosophila mesoderm. Development. PubMed ID: 30401702
Fierling, J., John, A., Delorme, B., Torzynski, A., Blanchard, G. B., Lye, C. M., Popkova, A., Malandain, G., Sanson, B., Etienne, J., Marmottant, P., Quilliet, C. and Rauzi, M. (2022). Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation. Nat Commun 13(1): 3348. PubMed ID: 35688832
Fuentes, M. A. and He, B (2022). The cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination. Development 149(6). PubMed ID: 35302584
Fuse, N., Yu, F. and Hirose, S. (2013). Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation. Development 140: 4246-4255. PubMed ID: 24026125
Guo, H., Swan, M. and He, B. (2022). Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination. Elife 11. PubMed ID: 35195065
Holcomb, M. C., Gao, G. J., Servati, M., Schneider, D., McNeely, P. K., Thomas, J. H. and Blawzdziewicz, J. (2021). Mechanical feedback and robustness of apical constrictions in Drosophila embryo ventral furrow formation. PLoS Comput Biol 17(7): e1009173. PubMed ID: 34228708
Izquierdo, E., Quinkler, T. and De Renzis, S. (2018). Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis. Nat Commun 9(1): 2366. PubMed ID: 29915285
Jha, A., van Zanten, T. S., Philippe, J. M., Mayor, S. and Lecuit, T. (2018). Quantitative control of GPCR
organization and signaling by endocytosis in epithelial morphogenesis. Curr Biol 28(10): 1570-1584 PubMed ID: 29731302
Jiang, J. and Aegerter, C. M. (2023). An integrated vertex model of the mesoderm invagination during the embryonic development of Drosophila. J Theor Biol 572: 111581. PubMed ID: 37481232
John, A. and Rauzi, M. (2021). A two-tier junctional mechanism drives simultaneous tissue folding and extension. Dev Cell. PubMed ID: 33891900
Kanesaki, T., Hirose, S., Grosshans, J. and Fuse, N. (2013). Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation. Mech Dev 130: 132-142. PubMed ID: 23085574
Kerridge, S., Munjal, A., Philippe, J. M., Jha, A., de Las Bayonas, A. G., Saurin, A. J. and Lecuit, T. (2016). Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis. Nat Cell Biol 18(3): 261-70. PubMed ID: 26780298
Ko, C. S., Kalakuntla, P. and Martin, A. C. (2020). Apical constriction reversal upon mitotic entry underlies different morphogenetic outcomes of cell division. Mol Biol Cell: mbcE19120673. PubMed ID: 32129704
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. PubMed ID: 17234948
Manning, A. J., Peters, K. A., Peifer, M. and Rogers, S. L. (2013). Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog. Sci Signal 6: ra98. PubMed ID: 24222713
Martin, A. C., Kaschube, M. and Wieschaus, E. F. (2009). Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457(7228): 495-9. PubMed ID: 19029882
Miao, H., Vanderleest, T. E., Budhathoki, R., Loerke, D. and Blankenship, J. T. (2021). A PtdIns(3,4,5)P(3) dispersal switch engages cell ratcheting at specific cell surfaces. Dev Cell 56(18): 2579-2591. PubMed ID: 34525342
Murray, M. J. and Saint, R. (2007). Photoactivatable GFP resolves Drosophila mesoderm migration behaviour. Development 134(22): 3975-83. PubMed ID: 17942486
Niloy, R. A., Holcomb, M. C., Thomas, J. H. and Blawzdziewicz, J. (2023). The mechanics of cephalic furrow formation in the Drosophila embryo. Biophys J. PubMed ID: 37571824
Polyakov, O., He, B., Swan, M., Shaevitz, J. W., Kaschube, M. and Wieschaus, E. (2014). Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation. Biophys J 107: 998-1010. PubMed ID: 25140436
Rahimi, N., Averbukh, I., Carmon, S., Schejter, E. D., Barkai, N. and Shilo, B. Z. (2019). Dynamics of Spaetzle morphogen shuttling in the Drosophila embryo shapes gastrulation patterning. Development 146(21). PubMed ID: 31719046
Rauzi, M., Krzic, U., Saunders, T. E., Krajnc, M., Ziherl, P., Hufnagel, L. and Leptin, M. (2015). Embryo-scale tissue mechanics during Drosophila gastrulation movements. Nat Commun 6: 8677. PubMed ID: 26497898
Sharrock, T. E., Evans, J., Blanchard, G. B. and Sanson, B. (2022). Different temporal requirements for tartan and wingless in the formation of contractile interfaces at compartmental boundaries. Development 149(21). PubMed ID: 36178136
Song, Y., Marmion, R. A., Park, J. O., Biswas, D., Rabinowitz, J. D. and Shvartsman, S. Y. (2017). Dynamic control of dNTP synthesis in early embryos. Dev Cell 42(3): 301-308. PubMed ID: 28735680
Stern, T., Shvartsman, S. Y. and Wieschaus, E. F. (2022). Deconstructing gastrulation at single-cell resolution. Curr Biol. PubMed ID: 35290798
Takeda, M., Sami, M. M. and Wang, Y. C. (2018). A homeostatic apical microtubule network shortens cells for epithelial folding via a basal polarity shift. Nat Cell Biol 20(1): 36-45. PubMed ID: 29203884
Tamada, M., Shi, J., Bourdot, K. S., Supriyatno, S., Palmquist, K. H., Gutierrez-Ruiz, O. L. and Zallen, J. A. (2021). Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity. Dev Cell. PubMed ID: 33932332
Yevick, H. G., Miller, P. W., Dunkel, J. and Martin, A. C. (2019). Structural redundancy in supracellular actomyosin networks enables robust tissue folding. Dev Cell. PubMed ID: 31353314
Zhu, H. and B, O. S. (2023). Actomyosin pulsing rescues embryonic tissue folding from disruption by myosin fluctuations. bioRxiv. PubMed ID: 36993262
Genes involved in tissue development
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