The Interactive Fly
Maternally transcribed genes
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Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).
To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).
This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).
Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P2, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).
The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).
The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).
A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).
A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).
In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).
De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. This study identified the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, it was determined that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression (Su, 2013).
Cellularization in the Drosophila embryo involves de novo generation of 6000 columnar epithelial cells, which are generated by the ingression of plasma membrane furrows (cleavage furrows) that enclose each nucleus. At the tip of ingressing cleavage furrows, CFT-tubules form. This study demonstrated the existence of three populations of CFT-tubules, which can de defined by different septin family members. The different populations of CFT-tubules are differentially regulated, and their presence or absence correlates with changes in cleavage furrow ingression kinetics (Su, 2013).
Septins were identified as additional factors localizing to the CFT-tubules. Of interest, not all septins localize to the same CFT-tubules or the same domain within a single CFT-tubule. This suggests that although the CFT-tubules are formed by an endocytic pathway (Sokac, 2008), the tubules are not homogeneous. Instead, tubules can contain different domains that may have different functions. Three distinct types of tubules were identified: those that contain only amphiphysin and the septins Sep1 and Sep2, those that contain only the septins Peanut, Sep4, and Sep5, and those that possess heterogeneous subdomains each defined by a distinct composition of these various components. Of importance, localization studies suggest that distinct septin complexes localize to different structures. Because Peanut, Sep4, and Sep5 do not colocalize with Sep1 and Sep2 on CFT-tubules, it is predicted that Peanut, Sep4, and Sep5 form a novel septin complex. This new septin complex may resemble the previously isolated complex of Peanut, Sep1, and Sep2, as Sep2 is most closely related to Sep5 (72% identity) and Sep1 is most closely related to Sep4 (47% identity). It was not possible to isolate individual septin complexes by immunoprecipitation, as all septins coimmunoprecipitated. This finding is consistent with studies in mammalian cells and reflects either the heterogeneous nature of septin complexes within the entire embryo or that, in part, partial septin filaments were being immunoprecipitated. Unexpectedly, Peanut did not colocalize with Sep1 and Sep2 on CFT-tubules. This observation raises the possibility that Sep1 and Sep2 alone form a complex. Septin filaments in yeast and mammalian systems are generated from octamers containing two copies of four different septins arranged in an inverted repeat; however, this may not be true for all systems. In the case of Drosophila a hexamer of Peanut, Sep1, and Sep2 has been isolated, and in Caenorhabditis elegans there are only two septin genes (Su, 2013).
Septins have predominantly been implicated in modulating events at the plasma membrane in conjunction with the actin cytoskeleton. In mammalian cells, septins have also been linked to potential roles in membrane trafficking, especially in the exocytic pathway, possibly by regulating vesicle fusion. It seems unlikely that the septins on CFT-tubules are regulating exocytosis, as all evidence suggests that exocytosis occurs at distinct apical sites in the syncytial embryo). In contrast, one study suggests a role for septins in the endocytic pathway by regulating recruitment of the coat protein complex AP-3 to lysosomal membranes (Baust, 2008). The precise roles for septins in this process are unclear. In CFT-tubules, it is possible that septins exert an effect directly on the membrane. Septins can tubulate membranes containing phosphatidylinositol (4,5)-bisphosphate, a lipid that has a key role in cytokinesis. However, the current data demonstrate that CFT-tubule formation is dependent on amphiphysin. Septins have been proposed to stabilize membranes. Therefore septins could stabilize the CFT-tubules once formed. Indeed, reduced recruitment of septins to cleavage furrows destabilizes the entire cleavage cleavage furrow. Furthermore, embryos depleted of Peanut form unstable yolk channels at the end of cellularization, further supporting the model that septins can stabilize membrane structures to which they localize. These findings also suggest that mutations that deplete septins will not allow examination of the role of septins in CFT-tubule organization and function (Su, 2013).
This study found that CFT-tubule formation requires the BAR domain of amphiphysin. The N-BAR subfamily, to which amphiphysin belongs, can bind to membranes and promote their curvature. Amphiphysin is also involved in t-tubule formation in Drosophila indirect flight muscles and mouse heart muscle. These findings suggest a conserved role for amphiphysin in promoting tubule formation and organization (Su, 2013).
Loss of amphiphysin and the prevention of CFT-tubule formation did not inhibit furrow ingression, suggesting that amphiphysin is not required for remodeling of the membrane to drive furrow ingression. Instead, loss of amphiphysin increased the rate of furrow ingression. Because amphiphysin localizes to the tip of the furrow, it is possible that amphiphysin acts as a negative regulator of furrow ingression. Alternatively, by preventing CFT-tubule formation, amphiphysin may render more plasma membrane accessible for furrow ingression, and therefore the rate of furrow ingression increases. Consistent with this model, when CFT-tubules become longer upon disruption of dynamin, the rate of cleavage furrow ingression is reduced. One potential consequence of inhibiting endocytosis at the furrow tip would be to reduce the amount of membrane available for the expansion of the plasma membrane and the ingression of the furrow. In such a scenario membrane derived from endocytosis at the tip of the furrow would be recycled back to the plasma membrane through the exocytic pathway, thereby providing sufficient membrane for the expansion and ingression of the furrow. This reduced availability of membrane could manifest itself as a reduced rate of furrow ingression seen in shibirets embryos at the nonpermissive temperature, where CFT-tubules elongate due to a failure to pinch off. The additional membrane may be especially important for the rapid increase in furrow ingression that is seen once the furrow has ingressed ∼10 μm, a depth of ingression where CFT-tubules normally become shorter and disappear (Su, 2013).
Changes in tubule parameters correlate with changes in cleavage furrow ingression kinetics, especially in the fast phase of ingression; longer, more persistent tubules correlate with slower ingression kinetics, and the absence of tubules correlates with faster ingression kinetics. If the fast phase of cleavage furrow ingression were dependent upon new membrane being inserted into the plasma membrane, then restricting membrane insertion would suppress the fast phase. If membrane was recycled by endocytosis at the cleavage furrow tips through an endocytic compartment back to the plasma membrane, then changes in CFT-tubule parameters might be expected to affect cleavage furrow ingression kinetics (Su, 2013).
In the models outlined in this study CFT-tubules would function to buffer the amount of available membrane that is accessible for efficient cleavage furrow ingression. However, no comparable measurements have been made with respect to t-tubules in muscles. Therefore it remains unclear whether the tubules in these different systems have a common function, whether they are examples of specialized endocytosis, or whether the creation of extra membrane surface area facilitates specialized functions in these different systems (Su, 2013).
Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. This problem was addressed by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. The cleavage of the Drosophila melanogaster embryo was used as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings (Mavrakis, 2014).
These findings demonstrate that septins are required during cellularization for generating curved and tightly packed actin filament networks at the tips of cellularization furrows termed furrow canals (FCs). Given that the F-actin bundling and bending activity is conserved for fly and human septins, it is predicted that septins contribute to the assembly, stabilization and contractility of cytokinetic rings. Septin depletion in dividing cultured cells, where anillin is localized to the cleavage furrow, leads to furrow instability, suggesting that the septin F-actin bundling activity could be required for proper cortical tension at the equator or/and the poles, which is in turn critical for the positioning of the cytokinetic ring46. Defective formation or stabilization of curved F-actin could further contribute to furrow shape instability. Decreased cytokinetic ring contractility was also recently confirmed in dividing Drosophila septin mutant epithelia, where anillin localizes to the cleavage furrow (Mavrakis, 2014).
Septins could also potentially contribute to the shape of FCs in actin-independent ways. Recent studies provide compelling evidence that septins regulate the mechanical properties of the cortex in non-dividing mammalian cells. Septins were also proposed to provide the cortical rigidity and membrane curvature necessary for rice blast fungal infection. It will be important to investigate whether septins bind membranes at the FC, and how this might synergize with actin crosslinking for cortex organization (Mavrakis, 2014).
The current findings indicate that the F-actin bending activity of septins requires septin hexamers and not septin filaments, although it cannot be excluded that they both function together in vivo. Septin post-translational modifications might also promote septin filament ring formation, which might in turn organize F-actin in curved bundles. Actin filaments could also potentially act in septin filament nucleation, even under conditions where septins alone do not form filaments. It will be important to compare the actin remodelling activity of septin hexamers and octamers, given that human septins (unlike Drosophila septins form octamers with hSep9 at the ends (Mavrakis, 2014).
A striking feature of septins is that they bend actin into rings and other highly curved geometries. Actin circularization is energetically unfavourable owing to the large bending rigidity of actin filaments. Thus far, stable actin circles have been reported only under strong adhesion mediated by divalent cations or positively charged lipid monolayers. The adhesion energy mediated by septins seems sufficiently high to overcome the large bending energy associated with such a highly curved geometry. In cells, septin-mediated actin curving may act in synergy with myosin-induced actin filament buckling (Mavrakis, 2014).
Septins could conceivably crosslink F-actin into loose contractile networks in processes that do not involve contractile F-actin rings, depending on the local septin and actin filament concentration and turnover, and septin post-translational modifications. Although no direct actin-septin interaction is known in interphase cells, their interplay has been reported in non-dividing cells, despite anillin's nuclear confinement. This study suggests that septins, alone or together with myosin-II, could contribute to these biological processes through their F-actin bundling activity (Mavrakis, 2014).
Mammalian septins are known to interact with exocyst components and SNAREs and to regulate membrane fusion. Membrane growth during cellularization relies on vesicular trafficking, and the exocyst is necessary for this process. As membrane growth is delayed in septin mutants, it will be important to investigate whether septins mediate trafficking events that act together with actin remodelling at the FC to drive membrane growth (Mavrakis, 2014).
Actin cytoskeletal networks push and pull the plasma membrane (PM) to control cell structure and behavior. Endocytosis also regulates the PM and can be promoted or inhibited by cytoskeletal networks. However, endocytic regulation of the general membrane cytoskeleton is undocumented. This study provides evidence for endocytic inhibition of actomyosin networks. Specifically, it was found that Steppke, a cytohesin Arf-guanine nucleotide exchange factor (GEF), controls initial PM furrow ingression during the syncytial nuclear divisions and cellularization of the Drosophila embryo. Acting at the tips of ingressing furrows, Steppke promotes local endocytic events through its Arf-GEF activity and in cooperation with the AP-2 clathrin adaptor complex. These Steppke activities appear to reduce local Rho1 protein levels and ultimately restrain actomyosin networks. Without Steppke, Rho1 pathways linked to actin polymerization and myosin activation abnormally expand the membrane cytoskeleton into taut sheets emanating perpendicularly from the furrow tips. These expansions lead to premature cellularization and abnormal expulsions of nuclei from the forming blastoderm. Finally, consistent with earlier reports, it was also found that actomyosin activity can act reciprocally to inhibit the endocytosis at furrow tips. It is proposed that Steppke-dependent endocytosis keeps the cytoskeleton in check as early PM furrows form. Specifically, a cytohesin Arf-GEF-Arf G protein-AP-2 endocytic axis appears to antagonize Rho1 cytoskeletal pathways to restrain the membrane cytoskeleton. However, as furrows lengthen during cellularization, the cytoskeleton gains strength, blocks the endocytic inhibition, and finally closes off the base of each cell to form the blastoderm (Lee, 2013).
Coupling actomyosin networks to the plasma membrane (PM)
is essential for cells to migrate, interact, change shape,
and divide. As examples, actin networks form and function
at the leading edge of migratory cells, at cell-substrate
adhesion complexes, and at cell-cell adhesion complexes in
multicellular tissues. To assemble these complexes, receptors
can physically engage the actin cytoskeleton and also induce
cytoskeletal assembly via Rho- family guanosine triphosphate
(GTP)ases and phosphoinositide signaling. Inversely,
endocytosis can remove receptors from the PM promoting
the turnover of adhesion and signaling complexes.
More generally, the close links of both actin networks and
endocytic machinery with the PM suggest possible crosstalk
between these subsystems. Indeed, endocytic signaling
nucleates local actin networks to help drive membrane invagination
and scission (Mooren, 2012; Anitei, 2012). In contrast, more widespread
membrane cytoskeleton activity can create tension that
inhibits membrane invagination. Conceivably, endocytosis
could also inhibit the membrane cytoskeleton, but such
activity is undocumented (Lee, 2013).
The syncytial Drosophila embryo is a well-established
model for studying actomyosin networks and membrane trafficking
during PM furrow ingression. In the early syncytial
embryo, nuclei divide synchronously just beneath the PM.
At each division cycle, the activities of Rho-family GTPases, the Arp2/3 complex, and the formin Diaphanous
(Dia) organize actomyosin-based PM ingressions
(pseudocleavage furrows) that surround each nucleus to prevent
nuclear collision and loss. Once ~6,000 nuclei form,
similar mechanisms induce a final round of PM ingressions.
These furrows persist and elongate through membrane trafficking
to apical and lateral sites, and with support of
actomyosin networks at their basal tips (the furrow canals). This massive PM growth cellularizes the first embryonic
epithelium, a process completed with constriction of
actomyosin rings formed at the base of each cell.
Recently, endocytic events were detected at the tips of
pseudocleavage furrows and early cellularization furrow canals
by the presence of Amphiphysin (Amph)-positive tubules
and the internalization of labeled PM (Sokac, 2008). These events have
provided a model for studying how the actin cytoskeleton
can both promote and inhibit endocytosis (Sokac, 2008; Yan, 2013). However,
the role of this endocytosis is unclear, and paradoxically, it
would appear to counteract membrane growth. This study examined
how Arf G protein (Arf) activation might be involved. In other
contexts, Arfs promote endocytosis by recruiting coat proteins,
activating lipid signaling, and triggering actin polymerization. Like other G proteins, Arfs are activated
by guanine nucleotide exchange factors (GEFs). Cytohesins
are a major class of PM Arf-GEFs (Donaldson, 2011), and roles for cytohesin
Arf-GEFs have been documented at migratory leading edges,
focal adhesions, and adherens junctions in mammalian cell
culture (Santy, 2005; Torii, 2010; Ikenouchi, 2010). Drosophila contains one cytohesin, called
Steppke (Step). Step is known to function in postembryonic
insulin and EGF signaling, which mammalian
cytohesins do as well, but its contributions to
the Drosophila embryo and to other cellular processes are
unknown. This study shows that Step promotes endocytosis
at pseudocleavage furrows and furrow canals to restrain actomyosin
networks at these sites (Lee, 2013).
These data provide the first description of cytohesin function in
a developing embryo. Drosophila Step promotes
a subset of endocytic events at the tips of ingressing PM
furrows during embryo cellularization. Endocytosis has been
documented previously at these sites (Sokac, 2008), but its role has
been unclear. By manipulating a conserved upstream activator
of endocytosis, this study has identified an important role of endocytosis
in controlling the membrane cytoskeleton. The data argue
that Step acts at furrow tips to induce local Arf-dependent
endocytosis, which in turn antagonizes Rho1-dependent
actomyosin network assembly at these sites.
It was also found that the cytoskeleton can inhibit endocytosis at
the furrow tips, as has been previously shown in this system
(Sokac, 2008; Yan, 2013) and in other contexts. An overall
model is proposed in which this reciprocal relationship is one-sided at specific
developmental stages. At newly forming PM furrows, Step
dominates, promoting endocytosis that keeps cytoskeleton
activity in check for proper pseudocleavage and cellularization
furrow architecture and growth. During later cellularization,
the cytoskeleton dominates. Zygotic expression of actin regulators
such as Nullo normally increases actomyosin activity
as cellularization proceeds and appears to work in
conjunction with Dia to block endocytic events at the furrow
tips (Sokac, 2008; Yan, 2013)]. By counteracting the inhibitory endocytosis,
cytoskeletal activity would elevate further but at these later
stages is locally restrained by a distinct mechanism requiring
Bottleneck (Schejter, 1993). To form the blastoderm, this second restraint
mechanism is removed, and contractile rings close off the
base of each cell. In the absence of the initial step-mediated
restraint mechanism, it is proposed that the cytoskeleton abnormally
dominates the relationship at all early PM furrows.
Without Step-based endocytic inhibition, it is speculated that
actomyosin networks abnormally expand and inhibit other
endocytic events leading to coexpansion of cytoskeletal polymers
and PM from the furrow tips (Lee, 2013).
An important element of the model is the local induction
of endocytic events. The data localize Step to the tips of
ingressing PM furrows, and both the loss and overexpression
of Step alter membrane organization specifically at these
sites. This localized Step-regulated activity occurs in a dynamic
global membrane trafficking system within each forming
cell. During the peripheral nuclear divisions, each nucleus
acquires its own endoplasmic reticulum and Golgi apparatus
that function with recycling endosomes to direct exocytosis
to growing PM furrows at cellularization. Simultaneously,
endocytic events occur over the apical PM and at
the furrow tips, with endocytosed material recycled
to the growing furrows. Thus, the overall membrane system
is in continual flux, and coordination by local regulation
would be expected. The data identify a polarized endocytic
activator required for the process. Step Arf-GEF activity is
critical for restraining the membrane cytoskeleton at furrow
tips, and a subset of AP-2 activities is involved as well (Lee, 2013).
How could endocytosis and actomyosin networks impact
each other at the tips of PM furrows or elsewhere? This question
can be considered from several levels of organization.
First, a simple and direct connection could be endocytic
removal of one or more PM actomyosin regulators. This work
identifies Rho1 or an upstream regulator as a candidate.
Intriguingly, membrane trafficking has been previously
linked to the Rho1 pathway in this context. Specifically, recycling
endosomes have been implicated in the trafficking of
RhoGEF2 to the PM (Cao, 2008). It was hypothesized that RhoGEF2
might also be a target of Step for its removal from the PM
but no difference was found in RhoGEF2 levels at furrow canals
in step loss-of-function embryos. Thus, Rho1 may be a more
specific target of Step, although a direct connection to the
Rho1 pathway remains to be determined. Of note, a number
of septins have also recently been observed on the Amph-positive tubules (Lee, 2013).
Second, interplay between different pools of actin is
possible. For example, actin contributes to the invagination
and scission of endocytic vesicles, and thus, endocytic
actin and other PM actin networks could compete for regulators
or components. Additionally, there could be signaling
crosstalk between regulators of the different networks. For
example, Arf signaling often elicits local Rac or Cdc42 activity, and this might trigger crosstalk affecting Rho activity. Interestingly, overexpression of Cdc42-interacting protein
4 (Cip4) appears to antagonize Dia at furrow canals,
although Cip4 mutants have no cellularization phenotype on
their own. Significantly, however, this study found that Step
acts with AP-2 to control the membrane cytoskeleton. This
Step-AP-2 cooperation suggests that clathrin-coated pits are
involved in the antagonism, although it does not exclude the
possibility of separate cytoskeletal crosstalk (Lee, 2013).
Third, larger scale interactions should be considered. Endocytosis
could remove membrane in bulk that would otherwise
support the membrane cytoskeleton, although observations
of residual furrow canal endocytic activity with step
loss of function suggest a more specific mechanism. Inversely,
the membrane cytoskeleton could block endocytosis by
elevating PM tensio or possibly by sterically blocking
endocytic machinery from accessing the PM (Lee, 2013).
Endocytic-cytoskeletal crosstalk is relevant to many cellular
processes. For example, receptor endocytosis occurs in proximity
to actomyosin networks in various contexts, including
migratory leading edges, focal adhesions, and adherens junctions. However, these endocytic events and actomyosin
networks have mainly been studied independently,
and thus their functional integration is not understood. This study highlights the possibility that endocytic activity at such assemblies could simultaneously remove receptors and
antagonize local cytoskeletal networks, with both effects promoting
complex turnover and cellular dynamics (Lee, 2013).
Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. This study shows that mutations in drop out (dop; CG6498) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions
in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. This study shows that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein (Hain, 2014).
This study is the first mutational analysis of a MAST kinase in any organism and demonstrates that the MAST kinase Dop plays an important role in plasma membrane cortex compartmentalisation during the generation of epithelial polarity in the fly. The results reported in this study demonstrate a requirement of Dop in the establishment of the furrow canal and the bAJ
at the cycle 14 transition. The defect in bAJ formation is likely to be a consequence of a failure in the initial specification
of the incipient furrows. It is proposed that Dop acts upstream in furrow canal formation by controlling the formation of F-actin-rich foci, which initiate the assembly of a specific furrow membrane cortex (Hain, 2014).
In mid-cellularisation stages, dop mutant phenotypes are reminiscent of embryos lacking the early zygotic gene bottleneck (bnk). In bnk mutants the initial formation of the cleavage furrows is normal, but then furrows close prematurely. Although it cannot be excluded that bnk might play a role in later defects associated with dop mutations, the primary defect in dop mutants concerned the lack of regular F-actin-rich furrows during the onset of cellularisation. Another early zygotic gene, nullo, is required for the proper recruitment of F-actin during furrow canal formation. Nullo and the actin regulator RhoGEF2 have been proposed to act in parallel pathways controlling processes that are distinct but both essential for F-actin network formation during the establishment of the furrow canal. Since early F-actin rearrangements are largely normal in nullo and RhoGEF2 single mutants, it is proposed that Dop is essential for the initial early focussing of F-actin, whereas Nullo and RhoGEF2 are required to elaborate and maintain F-actin levels to stabilise the furrows. The actin regulator enabled (ena) has been shown to act downstream of Abelson tyrosine kinase (Abl) in the redistribution of F-actin from the plasma membrane cortex into the furrows in both syncytial stages and cellularisation. Although ena would provide a good candidate for acting downstream of dop in the redistribution of F-actin, ena is already required for syncytial cleavages and the F-actin phenotypes in Abl mutants are much more severe than those that were found for dop mutants (Hain, 2014).
The similarity of syncytial cleavage furrows and the cleavage furrows at cellularisation raises the question of how they differ from each other. The molecular basis of the hexagonal pattern of the F-actin-rich cell cortex at the cleavage furrow relies upon the recycling endosome components Rab11 and Nuclear fallout (Nuf) and the actin polymerisation factors Dia and Scar/Arp2/3. In contrast to dop mutants, nuf, dia or Scar mutants indicate that these genes are required also for the dynamic redistribution of F-actin during syncytial development. Since Dop is a maternally supplied protein, its activity might be regulated by events triggered during the cycle 13-14 transition. The major difference between the furrows in syncytial stages and cellularisation is that metaphase furrows are formed during M phase, whereas cellularisation furrows are formed during G2 phase. Since Dop is a maternally supplied gene product, one would have to implicate regulation of Dop by zygotic factors to explain its phenotype at the cycle 13-14 transition. An alternative possibility is that Dop is regulated by phosphorylation or other post-translational modification through the cell cycle machinery and that, in the absence of Cdk1-dependent phosphorylation, its phosphorylation state is changed. This study provided evidence that Dop is indeed differentially post-translationally modified during syncytial versus cellular blastoderm stages. It is proposed that such cell cycle-dependent regulation of Dop may be crucial in transforming syncytial cleavages into persistent cellularisation furrows. Furthermore, the data suggest that this transition could require Dop-dependent regulation of dynein-associated microtubule transport (Hain, 2014).
The mechanisms for the initial localisation of Baz and E-cadherin are still unclear but, interestingly, dop is required for the localisation of both proteins. At the cycle 14 transition, E-cadherin and Arm puncta are associated with apical membrane projections and the homophilic association of these cadherin puncta is strengthened by membrane flow and is dependent on actin. Baz function allows these puncta to become tightened into sAJs. Thus, Dop might affect the stabilisation of the weakly interacting puncta either through cortical actin organisation or membrane flow. In addition to this early requirement for Baz localisation, Dop is also involved in clearing Baz from the basal cytoplasm during late cellularisation. The mechanism that eventually clears Baz from the basal cytoplasm depends on dynein-based transport. Therefore, Dop is required for dynein-based transport of different cargoes during cellularisation: lipid droplets, mRNA particles, Golgi and Baz. It is proposed that the main function of Dop in cellularisation is in regulating dynein-mediated transport of important cargos along microtubules (Hain, 2014).
This study presents the first evidence for regulation of dynein-mediated transport by a MAST family kinase. Dop is shown to controls phospho-Dic levels in a direct or indirect manner. The data are consistent with a model in which the initiation of furrow formation involves dynein-dependent transport that is controlled by Dop. In support of a role in membrane formation, this study found defects in the distribution of the recycling endosome and Golgi compartments in dop mutants. Interference with Rab11 function causes similar defects in Slam distribution as those shown by dop mutants. Therefore, Dop might control the transport of endomembrane compartments, which drive membrane growth. In addition, F-actin redistribution plays a major role in membrane cortical compartmentalisation in the initial stages of cellularisation. The focussing of F-actin to incipient furrows might involve a dynein-dependent shift of actin regulators or existing actin filaments to the furrow. An attractive hypothesis is that the translocation of F-actin and/or its regulators is coupled to an endomembrane compartment that is transported via microtubules towards the incipient furrow canals. Future studies should aim to determine which dynein cargos contribute to furrow formation and how Dop regulates Dic phosphorylation at the molecular level (Hain, 2014).
One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminates in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events have remained unclear. This study shows that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. CRISPR/Cas9 technology was used to N-terminally tag Rab8, which is then used to address both endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, while GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through exocyst tethering complex interactions (Mavor, 2016).
Cells store membrane in surface reservoirs of pits and protrusions. These membrane reservoirs facilitate cell shape change and buffer mechanical stress, but how reservoir dynamics are regulated is not known. During cellularization, the first cytokinesis in Drosophila embryos, a reservoir of microvilli unfolds to fuel cleavage furrow ingression. This study found that regulated exocytosis adds membrane to the reservoir before and during unfolding. Dynamic F-actin deforms exocytosed membrane into microvilli. Single microvilli extend and retract in ~20 s, while the overall reservoir is depleted in sync with furrow ingression over 60-70 min. Using pharmacological and genetic perturbations, this study shows that exocytosis promotes microvillar F-actin assembly, while furrow ingression controls microvillar F-actin disassembly. Thus, reservoir F-actin and, consequently, reservoir dynamics are regulated by membrane supply from exocytosis and membrane demand from furrow ingression (Figard, 2016).
Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. This study shows that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. This paper delineates a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. This unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation (Hampoelz, 2016).
Biosynthetic traffic from the Golgi drives plasma membrane growth. For Drosophila embryo cleavage, this growth is rapid, but regulated, for cycles of furrow ingression and regression. The highly conserved small G protein Arf1 organizes Golgi trafficking. Arf1 is activated by guanine nucleotide exchange factors, but essential roles for Arf1 GTPase activating proteins (GAPs) are less clear. This study reports that the conserved Arf GAP Asap is required for cleavage furrow ingression in the early embryo. Since Asap can affect multiple sub-cellular processes, genetic approaches were used to dissect the primary effect of Asap. The data argue against cytoskeletal or endocytic involvement, and reveal a common role for Asap and Arf1 in Golgi organization. Although Asap lacked Golgi enrichment, it was necessary and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site was required for Golgi organization and output. Notably, Asap re-localized to the nuclear region at metaphase, a shift that coincided with subtle Golgi re-organization preceding cleavage furrow regression. It is concluded that Asap is essential for Arf1 to function at the Golgi for cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle (Rodrigues, 2016).
In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. This study analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. The occurrence of a high mutation rate in Xenopus is reported; it was shown to be dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, a homology-directed repair contribution of Rad18 was observed in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early period Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation (Lo Furno, 2022).
Organ and tissue formation are complex three-dimensional processes involving cell division, growth, migration, and rearrangement, all of which occur within physically constrained regions. However, analyzing such processes in three dimensions in vivo is challenging. This study focused on the process of cellularization in the anterior pole of the early Drosophila embryo to explore how cells compete for space under geometric constraints. Using microfluidics combined with fluorescence microscopy, quantitative information was extracted on the three-dimensional epithelial cell morphology. A cellular membrane rearrangement was observed in which cells exchange neighbors along the apical-basal axis. Such apical-to-basal neighbor exchanges were observed more frequently in the anterior pole than in the embryo trunk. Furthermore, cells within the anterior pole skewed toward the trunk along their long axis relative to the embryo surface, with maximum skew on the ventral side. A vertex model was constructed for cells in a curved environment. The observed cellular skew was reproduced in both wild-type embryos and embryos with distorted morphology. Further, such modeling showed that cell rearrangements were more likely in ellipsoidal, compared with cylindrical, geometry. Overall, it was demonstrated that geometric constraints can influence three-dimensional cell morphology and packing within epithelial tissues (Rupprecht, 2017).
Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila. Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. This study reports a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. It is concluded that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division (Sherlekar, 2016).
Cleavage furrow formation during cell division requires a highly conserved set of cytoskeletal and membrane-trafficking proteins. Their positioning and initiation involves microtubules and the centralspindlin complex. Rho-GTPase-activating protein (RacGAP50C) of this complex positions Rho-GTP exchange factor (RhoGEF) Pebble at contractile rings, and another RhoGEF2 functions in pseudocleavage furrows to activate Rho1 for furrow initiation. Rho1 recruits formins that assemble an actin scaffold for contractile-ring formation and/or furrow initiation. Formin activity also depends on the presence of a scaffold protein, Anillin, at the contractile ring. RacGAP50C also accumulates Anillin at the furrow, which is responsible for both septin and myosin II association at the contractile ring. Cytokinesis failure increases in Caenorhabditis elegans when embryos are depleted of both Rho kinase and Anillin/septins, implying that they work together for robust furrow formation (Sherlekar, 2016).
The cell division cycle is accompanied by drastic changes in cell shape that necessitate dynamic interplay between the membrane and actin cytoskeleton. In the Drosophila syncytial embryo, nuclear division cycles 10-13 are rapid and involve dynamic pseudocleavage furrow ingression and retraction between adjacent dividing nuclei. These furrows serve to prevent spindle cross-talk across compartments during metaphase of each cycle and organize the embryo into discrete polarized functional units. Furrow positioning and initiation at this stage requires RhoGEF2 for recruiting Rho1 and the formin Diaphanous (Dia). Microtubules are required for furrow positioning, while furrow ingression involves dynamic growth of actin filaments through Profilin and the action of anticapping proteins (like Ena/VASP). The syncytial cycles are followed by massive elongation of furrows to form individual cells in a process called cellularization, during which membrane extension is fueled by flattening of apical microvilli and Rab11-mediated endocytosis and driven by an actomyosin contractile ring that, apart from actin and myosin II, also comprises Anillin, septins, RhoGEF2, and Dia. Although contractile rings first form only during cellularization in early developing Drosophila embryos, the syncytial pseudocleavage furrows contain most of the proteins present in the contractile ring such as Rho1, RhoGEF2, Dia, Anillin, and septins (Sherlekar, 2016).
F-BAR domain-containing proteins link membrane and cytoskeleton in various processes, including endocytosis, cell shape and polarity, cell motility, and cytokinesis. The yeast orthologues of F-BAR protein Cip4 are known to recruit formins and influence their nucleation and elongation activities. In addition, Hof1 (Cip4 in Saccharomyces cerevisiae) coiled-coil domain binds Septin (Cdc10) and localizes it to the bud neck. Drosophila Cip4, however, is not essential for formin Dia recruitment to cellularization furrows, and its loss does not result in a defect in cellularization but its overexpression shows dia loss-of-function phenotypes. The F-BAR domain protein, Syndapin/Pacsin (Synd), initially identified as a binding partner for Dynamin and neuronal Wiscott-Aldrich syndrome protein (N-Wasp) via its SH3 domain, participates in endocytosis and actin remodeling. Mammalian Synd1 binds to the actin nucleator Cordon bleu (Cobl)and mediates its interaction with Arp2/3 to affect actin nucleation during neuromorphogenesis. Synd, unlike other F-BAR proteins, directly binds and stabilizes F-actin and, unlike any N- or F-BAR protein, can generate a range of membrane curvatures much greater than its own intrinsic curvature. Drosophila Synd promotes expansion of the subsynaptic reticulum, which also requires actin-remodeling. Drosophila Synd also binds to Anillin via its myosin-binding domain in vitro, localizes at the cytokinetic furrow (earlier than Drosophila Cip4) in D.Mel-2 cells, and is important for cytokinesis during male meiosis in primary spermatocytes. Together these studies suggest a role for Synd in coordinated membrane and actin remodeling during cleavage furrow formation. However, no analysis of its recruitment dynamics or functional analysis in organization of actin or actin-remodeling proteins with respect to furrow initiation or extension machinery has been carried out so far. This study reports that Synd is important for syncytial Drosophila pseudocleavage furrow extension; septin Peanut (Pnut) recruitment; and distinct Dia, Anillin, and actin localization. Most significantly, Synd can recruit actin remodeling proteins, organize actin, and result in furrow extension during pseudocleavage furrow formation in rhogef2-depleted embryos (Sherlekar, 2016).
Syndapins belong to the family of highly conserved F-BAR-domain containing proteins with diverse roles in membrane tubulation, Clathrin-mediated and bulk endocytosis, and actin remodeling and cytokinesis. Synd is thus poised to play a role in processes like furrow formation, which needs orchestrated remodeling of both the membrane and the cytoskeleton. Furrow elongation in syncytial Drosophila embryos is an excellent model system to study the role of proteins that drive its formation. Previous studies show that furrow formation involves membrane addition by trafficking and membrane extension by remodeling of the actin meshwork. This study has conclusively demonstrated that Synd functions to promote furrow formation by organization and elongation of F-actin structures. Synd is essential for recruitment and distribution of Pnut and Dia on the membrane. In turn, Pnut and Dia also affect Synd distribution on the membrane. RhoGEF2/Dia and Anillin/Pnut have been previously shown to regulate F-actin architecture at cleavage and cellularization furrows, and loss of Synd in synd mutants therefore affects actin both directly and through its influence on Pnut and Dia localization. As with other actin-regulated processes, even though a linear pathway of association/regulation of these actin-remodeling proteins to the furrow membrane is unlikely, the data imply that Synd is a key component in the RhoGEF2-Dia-Anillin/Pnut pathway during actin-driven furrow elongation. Synd2 can bind and inhibit Rac1 via its SH3 domain, thus reducing Arp2/3 activity, and may therefore be able to potentiate Dia activity by increasing RhoA levels. Such a mechanism can explain increased Dia function when Synd is overexpressed in RhoGEF2 knockdown embryos, which, along with recruitment of Pnut to the membrane, can help organize actin and elongate cleavage furrows (Sherlekar, 2016).
Actin stabilization into continuous structures reversed the furrow length defect in synd mutant embryos. Jasplakinolide (Jasp) blocks actin turnover at the contractile ring and affects cleavage furrow invagination while preserving furrow integrity, and hence showed fewer punctae in synd and rhogef2 mutant embryos. CytoD, on the other hand, allows actin polymerization, and as a result, synd and rhogef2 mutant embryos treated with CytoD displayed more organized actin structures and elongated furrows. This provides mechanistic insight into how Synd functions in regulating actin polymerization and may be further investigated through kinetic studies of actin polymerization (Sherlekar, 2016).
Overexpression of Synd and not Pnut in the rhogef2RNAi-containing embryos partially reversed pseudocleavage furrow recruitment and morphology defects seen in rhogef2RNAi and increased the furrow length compared with wild type. Synd activity is thus needed at the pseudocleavage furrow for extension, and some as yet uncharacterized proteins play a role in furrow limitation. It is interesting to compare the functions of F-BAR domain proteins, Synd with Cip4 in furrow elongation and Dia recruitment. Cip4 antagonizes Dia function, and its overexpression has dia loss-of-function phenotypes like missing furrows. It is possible that opposing activities of F-BAR proteins Synd and Cip4 with respect to Dia are in a balance, and future experiments can test whether this function plays a role in limiting the growth of pseudocleavage furrows (Sherlekar, 2016).
Because Synd's SH3 domain interacts with Dynamin, and Dynamin has a role in endocytosis and furrow extension in syncytial divisions and cellularization, it remained to be investigated whether Clathrin-dependent endocytosis defects in synd mutants also affect furrow elongation. This study shows that synd mutant embryos have defects in cleavage furrow-tubule length and Rab5 vesicle numbers. Decrease in Rab5 vesicle numbers is also seen in rhogef2 mutant embryos. However, Synd-GFP overexpression in rhogef2 mutant embryos is able to reverse the furrow-extension defect without rescuing the Rab5 endocytic vesicle defect. Taken together, these data show that Synd has a role in endocytosis, but the reversal of furrow phenotypes in rhogef2 mutant embryos is due to the ability of Synd to recruit and organize actin and proteins of the actin-remodeling machinery such as Dia and Pnut (Sherlekar, 2016).
This analysis of membrane architecture and pseudocleavage furrow length in rhogef2, pnut, and synd mutants found that shorter furrows in each of these mutants were also loose/unstable and had slow lateral movement during the nuclear cycle. Septins brace the plasma membrane against aberrant cell-shape deformation and are able to tubulate phosphatidylinositol-4,5-bisphosphate liposome membranes when treated with a brain extract. It is probable that Septin-mediated membrane tubulation activity and cell-shape effects are dependent on the presence of F-BAR proteins like Synd. Sept7 mutants in Xenopus show unstable and undulating membranes during gastrulation. This substantiates Synd’s role in maintenance of membrane integrity and shape by affecting actin organization and Pnut recruitment (Sherlekar, 2016).
Overall mutant and epistatic analyses presented in this study find a significant role for the F-BAR domain protein Synd in mediating pseudocleavage furrow extension. This study favors a model in which Synd, along with Anillin and RhoGEF2, provide a platform for recruitment of Dia and Pnut to allow persistent and stable growth of actin to promote furrow elongation. Further experiments combining protein interactions and deduction of the biophysical nature of Synd-Pnut-actin association with the plasma membrane will elucidate the molecular mechanism that makes Synd an important component of pseudocleavage furrow-extension or contractile-ring assembly at large (Sherlekar, 2016).
Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. This study showed that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients (Lattao, 2021).
Autosomal recessive Chediak-Higashi syndrome (CHS) results from a mutation in the lysosomal trafficking regulator (LYST) or CHS1 gene and leads to partial albinism, neurological abnormalities, and recurrent bacterial infections. CHS cells have giant lysosome-related organelles (LROs), compartments that, in addition to lysosomal proteins, contain cell-type-specific proteins. LROs include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and pigment granules of Drosophila. Whether the giant LROs of CHS form through the excessive fusion of LROs or by inhibition of their fission is unclear (Lattao, 2021).
The compromised immune system in CHS is associated with enlarged LROs in natural-killer (NK) cells. NK cells normally become polarized with centrosomes close to their contact site with antigen-presenting cells, the immunological synapse (IS). Despite the formation of a mature IS in CHS NK cells, centrosomes do not correctly polarize and the enlarged LROs neither converge at the centrosome nor translocate to the synapse. Such findings could reflect defective microtubule (MT) organization by the centrosomes in CHS cells, and while some groups describe CHS centrosomes to nucleate fewer MTs, others report normal MT numbers, lengths, and distributions. Thus, the consequence of mutation in LYST for centrosome and MT function is unclear (Lattao, 2021).
Drosophila's LYST counterpart is encoded by mauve (mv) (CG42863). mv mutants show a characteristic eye color due to larger pigment granules, defective cellular immunity through large phagosomes, and enlarged starvation-induced autophagosomes, indicating several types of LRO are affected. The embryo's LROs are the yolk granules (YGs), which provide nutrition and energy during early development. YGs are produced and stored in the egg chamber when the yolk proteins (YPs) of follicle cells are internalized by clathrin-mediated endocytosis and trafficked through the endocytic pathway of the growing oocyte. YGs are present at the periphery of the egg until the early nuclear division cycles of the syncytial embryo, when they translocate to the interior as nuclei migrate to the embryo's cortex in nuclear division cycles 8 and 9. Nurse cells of the egg chamber also supply eggs with endoplasmic-reticulum-derived lipid droplets (LDs), which store maternally provided proteins and neutral lipids for energy and membrane biosynthesis (Lattao, 2021).
This study reveals Mauve's role in regulating LRO/YGs and MT nucleation from centrosomes through the maternal effect lethal (MEL) phenotypes of two new mutant alleles of mauve, mvrosario (mvros) and mv3. Embryos derived from mutant mv females have enlarged YGs that fuse with LDs, and this can be reverted by reducing Rab5 activity. mv-derived embryos also show compromised MT nucleation leading to defects in the embryo's mitotic cycles and cytoskeletal organization. Moreover, a requirement for Mauve in regulating MTs through the TACC/Msps pathway suggests a role for endosomal trafficking in the recruitment or maintenance of pericentriolar material (PCM) components at centrosomes (Lattao, 2021).
Previous studies of Drosophila mv mutants suggested a role for Mauve in suppressing the homotypic fusion of LROs (Rahman, 2012). This study has extended those observations by showing that Mauve also regulates heterotypic fusion between LROs and LDs and by showing that Mauve interacts with molecules that regulate the behavior of interphase and mitotic MTs. This study also shows that dominant-negative Rab5 not only rescues the LRO enlargement defect in mv-derived embryos but also ameliorates recruitment of Msps and PCM at centrosomes. The participation of LDs in LRO fusion that this study now describes could have been previously overlooked because of the lower numbers of LDs in other tissues compared with those in embryos or through specific differences in the mutant alleles under study (Lattao, 2021).
The finding that high levels of Mauve did not induce the formation of smaller sized vesicles together with live imaging of excessive fusion events of autofluorescent vesicles during oogenesis in mv mutant females are consistent with a role for Mauve as a negative regulator of vesicle fusion. The behavior of LDs and the incorporation of their content into the dramatically enlarged YGs of mv-derived embryos is also consistent with this model (Lattao, 2021).
Several lines of evidence support a role for Drosophila Mauve protein in regulating MT nucleation. First, this study found an enrichment of Mv-mCherry around the spindle and centrosomes during mitosis. Second, Mauve co-purifies with γ-tubulin and Msps. Third, the rosario phenotype of mauve-derived embryos is enhanced by mutations in d-tacc or msps, suggesting co-involvement of Mauve and the D-TACC:Msps complex in establishing and/or maintaining the MT-mediated organization of the syncytium that ensures dividing nuclei are at the cortex and endoreduplicating yolk nuclei in the interior. Fourth, embryos derived from mv mutant mothers have reduced amounts of both Msps and γ-tubulin at centrosomes, in accord with the diminished MT nucleating capacity of these centrosomes. Fifth, in line with the reduced amounts of MT nucleating molecules at centrosomes, the regrowth of de-polymerized MTs from centrosomes is compromised in mv-derived embryos (Lattao, 2021).
Mauve's co-purification with Msps, but not its D-TACC partner protein, is another indicator that Msps can exist independently of D-TACC. Indeed, Msps is present in several separate pools: independent of D-TACC at the centrosome; in complex with the D-TACC: Clathrin complex on the spindle; with the MT minus-end protein Patronin to assemble perinuclear non-centrosomal MTOCs (ncMTOCs); with the Augmin complex at kinetochores; and in complex with endosomal proteins such as Mauve. It is speculated that mutations affecting the constitution of Msps complexes at any one of these sites can affect another (Lattao, 2021).
The finding of defects in mitotic MT nucleation by centrosomes in mv-derived embryos suggests that there might be similar requirements at later developmental stages that may have been overlooked because flies can progress through most of the development without functional centrosomes (Lattao, 2021).
The increased NUF seen in mv-derived embryos is likely to be a secondary consequence of disruption to either or both membrane trafficking and mitosis. NUF was first described for the mutant of the nuf gene encoding an ADP ribosylation factor effector that associates with Rab11. Nuf protein is required to organize recycling endosomes in the coordinated processes of membrane trafficking and actin remodeling and embryos deficient for Rab11 also show a strong NUF phenotype. Together this suggests the possibility that NUF in mv mutants could result from the accumulation of endosomal components in the enlarged YGs, which would diminish numbers of recycling endosomes and their associated Rab11-Nuf complex. NUF can also occur as a Chk2 protein kinase-mediated response to DNA damage (DSBs), activated by DNA lesions at mitotic onset. However, this study found no evidence for DNA damage marked by the accumulation of phosphorylated γ-H2Av at DSBs. Finally, NUF also occurs in response to a wide range of primary or secondary mitotic defects. Indeed, failure of the sequestration of histone H2Av to LDs results in embryos that display mitotic defects, nuclear fallout, and reduced viability (Lattao, 2021).
Dominant-negative Rab5 suppresses enlarged YG formation and the mitotic defects of mv-derived embryos in accord with known roles of Rab5 at the early endosome and growing indications of a requirement for Rab5 in mitosis. Rab5 also mediates transient interactions between LDs and early endosomes that enable the transport of lipids between the two without resulting in their fusion. The possibility that Msps transiently localizes to LROs in wild-type embryos cannot be reuled out because LD-YG associations were observed in wild-type embryos and Msps is a component of LDs. The incorporation of Msps and LD markers into the enlarged YGs in mv-derived embryos is also rescued by a dominant-negative form of Rab5 and reciprocally, levels of Msps at centrosomes are restored. This suggests that mutation in mauve leads to mislocalization of Msps around YGs at the expense of its localization at the centrosome and so its availability for mitosis. Suppression of these mv phenotypes by dominant-negative Rab5 could therefore either reflect a passive restoration of the balance of Msps between YGs and spindle poles once YG fusion is prevented or a more active role of Rab5 in organizing the spindle poles (Lattao, 2021).
These findings add to a small but growing body of evidence for the roles of endocytic membrane trafficking in regulating centrosomal function. There are no reports of a membrane-independent role of Rab5, although other groups have reported examples of trafficking proteins involved in MT nucleation in a membrane-independent manner, such as ALIX, a PCM component in human and fly cells, whose recruitment depends on Cnn/Cep215 and D-Spd2/Cep192. The late endosome marker Rab11 also appears to be a part of a dynein-dependent retrograde transport pathway bringing MT nucleating factors and spindle pole proteins to mitotic spindle poles. It is not clear whether Rab5-associated structures mature to Rab11-associated structures in mitosis as they do in interphase but it seems that the two vesicle types might have overlapping functions at centrosomes in mitosis. It will be of future interest to put these current findings into context with these earlier demonstrations of roles of Rab5- and Rab11-containing endosomes in spindle function (Lattao, 2021).
The dynamic relationship between endosomal trafficking and recruitment of MT nucleating molecules onto centrosomes may all have relevance for the role of LYST at the IS and how this is affected in CHS. Thus, it is conceivable that there may be a convergence of the two functions of the LYST protein in lymphocytes, both in regulating the size of LROs and in facilitating the correct positing of centrosomes and membraneous structures. Further studies will be required to clarify the precise roles of LYST in regulating vesicle trafficking and MT nucleation in this particular cell type (Lattao, 2021).
Although the results strongly indicate Mauve to act as a negative regulator of vesicle fusion, this study did not directly assess the fusion ability of LROs. In part, this was limited by the autofluorescent nature of YGs and LDs that restricted the extent to which fluorescently tagged proteins could be used to visualize membrane components of these bodies in dynamic studies. Future work should aim to complement these findings in cell culture and in cell-free systems to determine whether the involvement of both LROs and LDs is widespread. In a similar vein, it will be important to assess whether the roles of LYST proteins in regulating MT dynamics are conserved as implied by these findings. This would require carrying out studies of MT dynamics in other cell types, particularly in mammalian cells (Lattao, 2021).
Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Using quantification of nuclei position and division orientation together with embryo explants, this study shows that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled deduction of the nature of the mechanical interaction between pairs of nuclei. This was used to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which was confirmed in vivo with laser ablation. Overall, this study shows that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity (de-Carvalho, 2022).
The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. This study shows that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. It is concluded that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, this study illuminates an essential process of embryonic multicellularity (Deshpande, 2021).
The collective behavior of the nuclear array in Drosophila embryos during nuclear cycle (NC) 11 to NC14 is crucial in controlling cell size, establishing developmental patterns, and coordinating morphogenesis. After live imaging on Drosophila embryos with light sheet microscopy, the nuclear trajectory, speed, and internuclear distance were extract with an automatic nuclear tracing method. The nuclear speed showed a period of standing waves along the anterior-posterior (AP) axis after each metaphase as the nuclei collectively migrate towards the embryo poles and partially move back. And the maximum nuclear speed dampens by 28%-45% in the second half of the standing wave. Moreover, the nuclear density is 22-42% lower in the pole region than the middle of the embryo during the interphase of NC12-NC14. To find mechanical rules controlling the collective motion and packing patterns of the nuclear array, a deep neural network (DNN) was used to learn the underlying force field from data. The learned spatiotemporal attractive force field was applied in the simulations with a particle-based model. The simulations recapitulated nearly all the observed characteristic collective behaviors of nuclear arrays in Drosophila embryos (Wu, 2021).
Syncytial cells contain multiple nuclei and have local distribution and function of cellular components despite being synthesized in a common cytoplasm. The syncytial Drosophila blastoderm embryo shows reduced spread of organelle and plasma membrane-associated proteins between adjacent nucleo-cytoplasmic domains. Anchoring to the cytoarchitecture within a nucleo-cytoplasmic domain is likely to decrease the spread of molecules; however, its role in restricting this spread has not been assessed. In order to analyze the cellular mechanisms that regulate the rate of spread of plasma membrane-associated molecules in the syncytial Drosophila embryos, a pleckstrin homology (PH) domain was expressed in a localized manner at the anterior of the embryo by tagging it with the bicoid mRNA localization signal. Anteriorly expressed PH domain forms an exponential gradient in the anteroposterior axis with a longer length scale compared with Bicoid. Using a combination of experiments and theoretical modeling, it was found that the characteristic distribution and length scale emerge due to plasma membrane sequestration and restriction within an energid. Loss of plasma membrane remodeling to form pseudocleavage furrows shows an enhanced spread of PH domain but not Bicoid. Modeling analysis suggests that the enhanced spread of the PH domain occurs due to the increased spread of the cytoplasmic population of the PH domain in pseudocleavage furrow mutants. This analysis of cytoarchitecture interaction in regulating plasma membrane protein distribution and constraining its spread has implications on the mechanisms of spread of various molecules, such as morphogens in syncytial cells (Thukral, 2022).
The boundaries of topologically associating domains (TADs) are delimited by insulators and/or active promoters; however, how they are initially established during embryogenesis remains unclear. This was examined during the first hours of Drosophila embryogenesis. DNA-FISH confirms that intra-TAD pairwise proximity is established during zygotic genome activation (ZGA) but with extensive cell-to-cell heterogeneity. Most newly formed boundaries are occupied by combinations of CTCF, BEAF-32, and/or CP190. Depleting each insulator individually from chromatin revealed that TADs can still establish, although with lower insulation, with a subset of boundaries (~10%) being more dependent on specific insulators. Some weakened boundaries have aberrant gene expression due to unconstrained enhancer activity. However, the majority of misexpressed genes have no obvious direct relationship to changes in domain-boundary insulation. Deletion of an active promoter (thereby blocking transcription) at one boundary had a greater impact than deleting the insulator-bound region itself. This suggests that cross-talk between insulators and active promoters and/or transcription might reinforce domain boundary insulation during embryogenesis (Cavalheiro, 2023).
Early embryogenesis is characterized by rapid and synchronous cleavage divisions, which are often controlled by wave-like patterns of Cdk1 activity. Two mechanisms have been proposed for mitotic waves: sweep and trigger waves. The two mechanisms give rise to different wave speeds, dependencies on physical and molecular parameters, and spatial profiles of Cdk1 activity: upward sweeping gradients versus traveling wavefronts. Both mechanisms hinge on the transient bistability governing the cell cycle and are differentiated by the speed of the cell-cycle progression: sweep and trigger waves arise for rapid and slow drives, respectively. This study, using quantitative imaging of Cdk1 activity and theory, illustrates that sweep waves are the dominant mechanism in Drosophila embryos and test two fundamental predictions on the transition from sweep to trigger waves. Sweep waves can be turned into trigger waves if the cell cycle is slowed down genetically or if significant delays in the cell-cycle progression are introduced across the embryo by altering nuclear density. Genetic experiments demonstrate that Polo kinase is a major rate-limiting regulator of the blastoderm divisions, and genetic perturbations reducing its activity can induce the transition from sweep to trigger waves. Furthermore, it was shown that changes in temperature cause an essentially uniform slowdown of interphase and mitosis. That results in sweep waves being observed across a wide temperature range despite the cell-cycle durations being significantly different. Collectively, the combination of theory and experiments elucidates the nature of mitotic waves in Drosophila embryogenesis, their control mechanisms, and their mutual transitions (Hayden, 2022).
In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10-13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. While substantial work has led to a better understanding of the core network components that are responsible for the formation of these furrows, less is known about the regulation that controls cytoskeletal and trafficking function. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. Quantitative analysis was performed of the effects of Sponge disruption on cortical cap (actin structures at the apical surface) growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. Membrane trafficking was found to be relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but Sponge was found to act as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, it is also suggested that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, the phasic requirements were demonstrated for branched F-actin and linear F-actin networks in potentiating furrow ingression. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows (Henry, 2022).
This work demonstrates the F-actin networks that support the phased ingression of syncytial furrows, while also revealing that Sponge is critically required for the recruitment of several Arp2/3 regulators to the apical and furrow-supporting cortex in the early Drosophila blastoderm. Sponge knockdown causes the mislocalization as well as altered cortical levels of Arp2/3 subunits and Arp regulators such as Scar, Coronin, Pod1, and Cortactin (see Proposed model of Sponge activity in syncytial embryos). Sponge regulation of F-actin is essential for the transition of these regulators from caps onto apical regions of the growing furrows, as Cortactin, DPod1, Coronin, and Scar are absent on ingressing furrows in Sponge knockdown embryos. This leads to inadequate branched actin network function resulting in short, broad furrows that extend no longer than 2.1 μm in length through cycle 13, and small residual cap-like structures, as small as 33% the wild-type cap area. Based on these data, a mechanism is proposed in which Sponge regulates and recruits Scar, Coronin, and DPod1 to the apical caps, while antagonizing Cortactin localization. These proteins in turn modulate filamentous actin and activate Arp2/3 activity and/or stability. As the new apical actin cap forms and expands, Arp2/3 and its regulators remain associated with the branched Actin network and are present in the ring-like structure at the transition point from cap to furrow. This appears necessary for proper linear F-actin nucleation and polymerization by Diaphanous which enables the building of sufficiently extended furrows in each syncytial cycle (Henry, 2022).
Successful cell division relies on an ingressing plasma membrane furrow physically separating neighboring nuclei. In the early Drosophila blastoderm syncytium, as successive cell cycles progress and nuclei become more densely packed, the risk of chromosomal missegregation or mitotic collapse rises if furrows do not adequately segregate neighboring nuclei or provide appropriate anchoring points for spindles. Previous work has shown that furrow length is negatively correlated with mitotic defects. In Sponge embryos, where furrows do not ingress past ~2 μm, severe missegregation in the syncytial stages causes embryos to fail to survive past cellularization (cycle 14). In control embryos, segregation defects are avoided by building furrows in two phases, Ingression I and Ingression II, with a Stabilization period juxtaposed in between. Sponge knockdown does not affect the biphasic nature of furrow ingression, as each cycle maintains two separate ingression periods as well as a measurable stabilization phase that is not significantly different in duration than in control embryos. However, the rate of ingression that Sponge furrows reach in any given ingression phase is slower than in control embryos. This reveals that Sponge does not affect select portions of furrow ingression, but is acting on all phases that promote invagination of the plasma membrane. It also remains a possibility that defects in actin and/or early ingression events may indirectly affect the degree to which later phases such as Ingression II proceeds, though Sponge protein, and the actin and/or nucleator regulatory proteins (ANRP) proteins it regulates, are present at caps and furrows throughout the syncytial cycle. Whether direct or indirect, further evidence of the requirement for Sponge during both early and late furrow phases is observed in measurements of the width of syncytial furrows. As furrows transition from Ingression I to Ingression II, furrows change in morphology from a broad and diffuse appearance to very sharply delineated furrows. In Sponge embryos, furrows possess a broader morphology throughout cycles 10-13, but still partially sharpen as later cycles transition into Ingression II phasic behaviors. Together, these data show that furrow ingression after Sponge disruption occurs in a predictable phasic pattern but that the formation, efficiency, and organization of the furrow is compromised, suggesting that Sponge acts as a master regulator of the furrow ingression process. As the results suggest that Sponge primarily impacts cortical F-actin cap components, this further indicates the importance of the actin cortex in directing proper furrow ingression dynamics (Henry, 2022).
F-actin levels are dramatically reduced both on apical caps and on furrows in Sponge embryos. As these regions have different contributions from branched and unbranched actin populations (branched is more predominant in cap regions while linear is more strongly present at the furrow) this raises the possibility that Sponge could regulate both pathways of actin polymerization. Reducing linear F-actin populations through dia knockdown results in a shortened furrow phenotype reminiscent of what is seen in Sponge. However, several characteristics of dia furrows indicate they may be controlled by a separate mechanism than those in Sponge disrupted embryos. First, furrows are able to reach a significantly greater maximum depth of 3.5 μm, which also reduces the occurrence of mitotic defects. To achieve this greater length, dia furrows ingress at maximal rates closer to those in control during the early phases of furrow formation. In the Ingression I phases of cycles 12 and 13, Sponge furrows show reduced ingression rates, while dia maximal rates are slightly higher than or equal to control furrows, showing no significant difference. It is only in Ingression II phases that dia maximum ingression rates fall behind control. Similarly, dia furrow morphologies are thinner and sharper than those in Sponge throughout cycles 10-13, and are not significantly different from control furrows during Ingression I phases. It is only in Ingression II phases that dia furrows are significantly wider than control, although they do still transition to a sharper morphology than in respective Ingression I phases. In contrast to Sponge, which is needed for both Ingression I and Ingression II, Dia appears more important for Ingression II phases that are responsible for the bulk of a given cycles maximum furrow length. However, it should be noted that both shRNA-driven disruptions and a Dia genetic allele that was used previously, although similar in phenotype, may only be partial disruptions of function, and thus additional defects could be observed with amorphic loss-of-function approaches (Henry, 2022).
When branched F-actin networks are reduced through Many factors are involved in activating, stabilizing, and otherwise regulating Arp2/3 activity during the syncytial stage of embryogenesis, including Scar, Coronin, DPod1, and Cortactin. Each of these proteins can be found on the apical caps where Arp2/3 mediated branched F-actin is prominent (Xie, 2021). These regulators are all disrupted to varying degrees on caps when Sponge is knocked down. Scar, Coronin, and DPod1 are each reduced after Sponge disruption, with remaining protein mislocalized as random puncta throughout the cytoplasm. Within these cap-like structures, Scar is the most severely diminished; from cycle 10-13, Scar intensity is on average 70% reduced from control embryos. Coronin is the next most severely affected factor with Coronin levels at residual caps being 67% lower in intensity than on control caps. The Coronin-family member DPod1 also shows a 49% reduction over cycles 10-13. DPod1 has been shown to have the strongest impact on overall F-actin and Arp2/3 intensities at the cap, and thus its reduction is likely a major contributing factor to the observed loss of F-actin intensities in Sponge embryos, with changes in Coronin and Scar function also contributing to changes in cap growth rates and sizes. One regulator that does not appear to require Sponge activity for localization to the apical cap, however, is Cortactin. While Cortactin is only present on the small residual structures in Sponge embryos, it is present on these structures in significantly increased intensity levels, suggesting that Sponge and Cortactin may possess an antagonistic relationship. Prior work has demonstrated an inhibitory function of Coronin on Cortactin function, so it may be that Sponge regulation of Coronin in turn affects Cortactin levels at the cortical cap in an opposing fashion. Alternatively, it may be that Sponge affects Cortactin levels through its regulation of small GTPase activity or through its other scaffolding domains (Henry, 2022).
In order to understand morphogenesis, it is necessary to know the material properties or forces shaping the living tissue. In spite of this need, very few in vivo measurements are currently available. Using the cellularization stage Drosophila embryo as a model, this study describes a novel cantilever-based technique which allows for the simultaneous quantification of applied force and tissue displacement in a living embryo. By analyzing data from a series of experiments in which embryonic epithelium is subjected to developmentally relevant perturbations, it is concluded that the response to applied force is adiabatic and is dominated by elastic forces and geometric constraints, or system size effects. Crucially, computational modeling of the experimental data indicated that the apical surface of the epithelium must be softer than the basal surface, a result which was confirmed experimentally. Further, the combination of experimental data and comprehensive computational model was used to estimate the elastic modulus of the apical surface and set a lower bound on the elastic modulus of the basal surface. More generally, these investigations revealed important general features that should be more widely addressed when quantitatively modeling tissue mechanics in any system. Specifically, different compartments of the same cell can have very different mechanical properties; when they do, they can contribute differently to different mechanical stimuli and cannot be merely averaged together. Additionally, tissue geometry can play a substantial role in mechanical response, and cannot be neglected (Cheikh, 2023).
To support tissue and organ development, cells transition between epithelial and mesenchymal states. This study investigated how mesoderm cells change state in Drosophila embryos and whether fibroblast growth factor (FGF) signaling plays a role. During gastrulation, presumptive mesoderm cells invaginate, undergo an epithelial-to-mesenchymal state transition (EMT) and migrate upon the ectoderm. The data show that EMT is a prolonged process in which adherens junctions progressively decrease in number throughout the mesoderm cells' migration. FGF influences adherens junction number and promotes mesoderm cell division, which is proposed to decrease cell-cell attachments to support slow EMT while retaining collective cell movement. It was also found that, at the completion of migration, cells form a monolayer and undergo a reverse mesenchymal-to-epithelial transition (MET). FGF activity leads to accumulation of beta-integrin Myospheroid basally and cell polarity factor Bazooka apically within mesoderm cells, thereby reestablishing apicobasal cell polarity in an epithelialized state in which cells express both E-Cadherin and N-Cadherin. In summary, FGF plays a dynamic role in supporting mesoderm cell development to ensure collective mesoderm cell movements as well as proper differentiation of mesoderm cell types (Sun, 2018).
Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. This study analyzed the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. Early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which this study shows to regulate actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis (Krueger, 2019).
Embryonic development starts with cleavages, a rapid sequence of reductive divisions that result in an exponential increase of cell number without changing the overall size of the embryo. In Drosophila, the final four rounds of cleavages occur at the surface of the embryo and give rise to approximately 6000 nuclei under a common plasma membrane. This study used live imaging to study the dynamics of this process and to characterize the emergent nuclear packing in this system. The characteristic length scale of the internuclear interaction was shown to scale with the density, which allows the densifying embryo to sustain the level of structural order at progressively smaller length scales. This is different from nonliving materials, which typically undergo disorder-order transition upon compression. To explain this dynamics, a particle-based model was used that accounts for density-dependent nuclear interactions and synchronous divisions. The pair statistics of the disordered packings observed in embryos was reproduced, and the scaling relation between the characteristic length scale and the density both in real and reciprocal space was recovered. This result reveals how the embryo can robustly preserve the nuclear-packing structure while being densified. In addition to providing quantitative description of self-similar dynamics of nuclear packings, this model generates dynamic meshes for the computational analysis of pattern formation and tissue morphogenesis (Dutta, 2019).
Branched actin networks driven by Arp2/3 collaborate with actomyosin filaments in processes such as cell migration. The syncytial Drosophila blastoderm embryo also shows expansion of apical caps by Arp2/3 driven actin polymerization in interphase and buckling at contact edges by MyosinII to form furrows in metaphase. The role of Syndapin (Synd), an F-BAR domain containing protein in apical cap remodelling prior to furrow extension. synd depletion showed larger apical caps. STED super-resolution and TIRF microscopy showed long apical actin protrusions in caps in interphase and short protrusions in metaphase in control embryos. synd depletion led to sustained long protrusions even in metaphase. Loss of Arp2/3 function in synd mutants partly reverted defects in apical cap expansion and protrusion remodelling. MyosinII levels were decreased in synd mutants and MyosinII mutant embryos have been previously reported to have expanded caps. It is proposed that Syndapin function limits branching activity during cap expansion and affects MyosinII distribution in order to shift actin remodeling from apical cap expansion to favor lateral furrow extension (Sherlekar, 2020).
The cell number of the early Drosophila embryo is determined
by exactly 13 rounds of synchronous nuclear
divisions, allowing cellularization and formation of the
embryonic epithelium. The pause in G2 in cycle 14 is
controlled by multiple pathways, such as activation of DNA
repair checkpoint, progression through S phase, and inhibitory
phosphorylation of Cdk1, involving the genes grapes,
mei41, and wee1. In addition, degradation of maternal
RNAs and zygotic gene expression are involved.
The zinc finger Vielfaltig (Vfl) controls expression of many
early zygotic genes, including the mitotic inhibitor
fruhstart. The functional relationship of these pathways
and the mechanism for triggering the cell-cycle pause
have remained unclear. This study shows that a novel single-nucleotide
mutation in the 3' UTR of the RNA polymerase RNPII215 gene leads
to a reduced number of nuclear divisions that is accompanied
by premature transcription of early zygotic genes and
cellularization. The reduced number of nuclear divisions in
mutant embryos depends on the transcription factor Vfl
and on zygotic gene expression, but not on grapes, the
mitotic inhibitor Fruhstart, and the nucleocytoplasmic ratio.
It is proposed that activation of zygotic gene expression is the
trigger that determines the timely and concerted cell-cycle pause and cellularization (Sung, 2012).
Embryos from germline clones of the lethal mutation X161 (in
the following, designated as mutant embryos) showed
a reduced cell number but otherwise developed apparently
normally until at least gastrulation stage. Cell specification along the anterior-posterior
and dorsoventral axes proceeded as in wild-type, as
demonstrated by the seven stripes of eve expression, mesoderm
invagination, and cephalic furrow formation. The
reduced cell number can be due to a lower number of nuclear
divisions prior to cellularization or to loss of nuclei in the
blastoderm. To distinguish these possibilities,
time-lapse recordings were performed of mutant embryos in comparision to
wild-type. To measure the cell-cycle
length, the nuclei in these embryos were fluorescently labeled.
Three types of embryos were observed: (1) with 13 nuclear divisions
with an extended interphase 13 (28 min versus 21 min in
wild-type), (2) with 12 nuclear divisions, and (3) with partly 12
and partly 13 nuclear divisions with an extended interphase
13. Because a severe nuclear fallout
phenotype was not observed, it is concluded that the reduced cell number in gastrulating
embryos is due to the reduced number of nuclear divisions.
Consistent with these observations, the number of
centromeres and centrosomes was normal in mutant embryos (Sung, 2012).
In wild-type embryos, interphase 14 is different from the preceeding
interphases, in that the plasma membrane invaginates
to enclose the individual nuclei into cells. In X161 embryos with
patches in nuclear density, furrow markers showed more
advanced furrows in the part with a lower number of divisions,
indicating a premature onset of cellularization.
Furthermore, in time-lapse recordings, the
speed of membrane invagination was measured, with no obvious difference
found between X161 and wild-type embryos. Additionally,
cellularization was investigated by live imaging with
moesin-GFP labeling F-actin. Clear accumulation
of F-actin at the furrow canals was observed in wild-type
embryos after about 20 min in interphase 14, but not in interphase
13. In X161 embryos with 12 nuclear divisions, a comparable reorganization was observed
already in interphase 13 after about 25 min. This analysis shows that both the cell-cycle
pause and cellularization are initiated in X161 embryos
earlier than in wild-type embryos (Sung, 2012).
To identify the mutated gene in X161, the
lethality and blastoderm phenotype was mapped. The X161
gene was separated from associated mutations on the chromosome
by meiotic recombination and mapped to a region
of four genes by complementation analysis with duplications
and deficiencies. Sequencing of the mapped region and
complementation tests with two independent RPII215 loss-of-function alleles, RPII215(1) and RPII215[G0040],
and a transgene comprising the RPII215 locus revealed the
large subunit of the RNA polymerase II as the mutated gene.
A single point mutation was identified in the 3' UTR of RPII215
about 40 nt downstream of the stop codon. This region in
the 3' UTR is not conserved and does not show any obvious motifs (Sung, 2012).
To test whether the mutation in the noncoding region affects
transcript or protein expression, mRNA levels were quantified by
reverse transcription and quantitative PCR and protein levels
by whole-mount staining and immunoblotting with extracts
of manually staged embryos. mRNA levels were found to be the same in wild-type and X161.
In contrast, immunohistology and immunoblotting revealed
reduced RPII215 protein levels. In summary, the analysis shows that the X161 point mutation
within the 3' UTR affects mainly RPII215 protein levels.
The precocious onset of cellularization raised the hypothesis
that the timing of zygotic gene expression may be affected
in the X161 embryos. To establish the expression profiles of
selected maternal and zygotic genes, nCounter
NanoString technology was used with embryos staged by the
nuclear division cycle. Embryos
expressing histone 2Av-RFP were manually selected 3 min
after anaphase of the previous mitosis or at midcellularization (Sung, 2012).
Expression of ribosomal proteins was analyzed.
They did not change much and were not different in wild-type
and mutant embryos, confirming the robustness of the
method. Zygotic genes, whose expression strongly increases
during the syncytial cycles, showed an earlier upregulation in
X161 than in wild-type embryos. Comparing the
profiles by plotting the ratio of the expression levels, a clear difference was revealed in cycle 12, with a factor of up to ten, indicating that zygotic genes are precociously expressed
in X161 embryos. The premature expression of early
zygotic genes was confirmed by whole-mount in situ hybridization
for slam and frs mRNA (Sung, 2012).
Next, expression profiles were analyzed of RNAs subject to
RNA degradation. Transcripts representative for
the two classes of degradation were selected, depending on zygotic gene
expression, and on egg activation. Degradation of string, twine, and smaug transcripts
in interphase 14 depends of zygotic gene expression. In
X161 mutants, the mRNA of these three genes was degraded
already in cycle 13, slightly sooner than in wild-type.
The profiles of string and twine RNA were confirmed by RNA
in situ hybridization. Consistent with the precocious RNA degradation in X161,
Twine and String protein levels decreased already in interphase 13 of
X161 embryos. Finally, the profile was analyzed of
mRNAs whose degradation depends on egg activation. No consistent pattern or clear difference was detected between the profiles of wild-type and X161
mutants. The data show that zygotic gene expression starts
earlier in X161 than in wild-type and that degradation of
mRNAs follows zygotic gene expression (Sung, 2012).
The cell cycle may be paused prematurely by altered levels
of maternal factors, such as CyclinB, grapes, and twine, or by
precociously expressed zygotic genes, such as frs and trbl. To distinguish these two options, mutant embryos with suppressed zygotic gene expression were analyzed. Embryos injected with the RNA polymerase II inhibitor α-amanitin develop until mitosis 13 but then fail to cellularize
and may undergo an additional nuclear division, depending
on injection conditions. Using this assay,
whether zygotic genes are required for the reduced number
of nuclear divisions was tested in X161 mutants. If the precocious cell-cycle
pause were due, for example, to reduced levels of
CyclinB mRNA, α-amanitin injection should not change the
reduced number of divisions. All injected
mutant embryos passed through at least 13 nuclear divisions,
similar to injected wild-type embryos, whereas injection of
water resulted in a mixed phenotype of 12 and 13 nuclear
divisions, comparable to uninjected X161 embryos. This experiment demonstrates that the reduced
division number in X161 embryos requires zygotic gene expression (Sung, 2012).
The expression of many early zygotic genes is controlled by
the zinc-finger protein Vfl (also called Zelda). Tests
were performed to see whether the precocious cell-cycle pause in X161 mutants is
mediated by vfl-dependent genes. Analysis of X161 vfl
double-mutant embryos revealed that, in contrast to X161
mutants, the cell cycle undergoes at least 13 divisions. Activation of zygotic gene
expression was further analyzed by staining for Vfl and activated RPII21.
Staining of both in presyncytial stages of X161
mutants was detected already in cycle 5. No specific staining for the activated
RPII215 was detected in X161 vfl double-mutant
embryos, and no difference in Vfl staining in syncytial embryos
was detected in wild-type and X161 embryos.
These findings show that the genes relevant for the precocious
cell-cycle pause in X161 mutants are vfl target genes.
A zygotic gene involved in cell-cycle control is frs, which is
sufficient to induce a pause of the cell cycle. Analysis
of X161 frs double-mutant embryos showed, however, that the
number of nuclear divisions was not changed as compared to
X161 single mutants. This indicates that frs is not the
only cell-cycle inhibitor expressed in the early embryo.
Proteins mediating the DNA repair checkpoint, such as
Grapes/Chk1, are required for the cell-cycle pause.
Passing normally through the nuclear division cycles, the cell
cycle shows striking abnormalities in nuclear envelope formation
and chromosome condensation in interphase 14 in
embryos from grapes females. Tests were performed to see whether the timing
of the transition in cell-cycle behavior in grapes embryos
depends on the onset of zygotic transcription by analyzing X161 grapes double-mutant embryos. Some of the X161 grapes double mutants were found to show the defects in nuclear envelope formation
and chromatin condensation already in interphase 13, indicating that
the requirement of grapes for chromatin structure shifted from interphase 14 to
13. These data suggest that the activation of grapes and the DNA checkpoint
depends on the onset of zygotic gene expression (Sung, 2012).
A factor controlling the number of nuclear divisions is the
ploidy of the embryo, given that haploid embryos undergo 14
instead of 13 nuclear divisions prior to cellularization.
Based on this and on related observations, it has been
proposed that the nucleocytoplasmic (N/C) ratio controls the
trigger for MBT. To address the functional relationship of
X161 and the N/C ratio, haploid X161 embryos were analyzed. A mixture was observed in the number
of nuclear divisions between 12 and 14 in fixed embryos. Embryos were even observed containing three patches with nuclear densities corresponding to 12, 13, and 14 nuclear divisions. About half of the embryos underwent 12 nuclear divisions, similar to X161 embryos. These data
suggest that ploidy acts independently of general onset of zygotic
transcription, which is consistent with the observation
that only a subset of zygotic genes are expressed with a delay
in haploid embryos. Consistent with this report, cellularization
starts for a first time temporarily in interphase 14 in
haploid embryos and for a second time in interphase 15. These
observations suggest that the N/C ratio in Drosophila specifically
affects cell-cycle regulators such as frs, for example,
but not general zygotic genome activation and onset of cellularization (Sung, 2012).
In summary, the data support the model that activation of
the zygotic genome controls the timing of the MBT. First, onset
of MBT is sensitive to changes in RNA polymerase II activity.
Second, the changes in zygotic gene expression in X161
embryos occur earlier than the changes in zygotic RNA degradation,
Cdc25 protein destabilization, or activation of grapes.
Third, the X161 mutant phenotype depends on zygotic transcription
and on the transcription factor Vfl, showing that the
precocious cell-cycle pause and onset of cellularization
cannot be due to changes in maternal factors, such as higher
expression of CyclinB. Although the altered levels of RNA polymerase
II in X161 mutants probably affect expression of many
genes during oogenesis, these changes seem not to matter in
functional terms, given the overall normal morphology and
specific mutant phenotype. It is conceivable that transcriptional
repressors are expressed or translated in eggs in lower
levels. In the embryo, such lower levels of repressors would
allow the trigger for onset of zygotic gene expression to reach
the threshold earlier than in wild-type embryos. The first signs
of zygotic transcription are detected already during the presyncytial
stages, before nuclear cycle 8/9. This may be the
time when the trigger for MBT is activated (Sung, 2012).
The beautiful mitotic waves that characterize nuclear divisions in the early Drosophila embryo have been the subject of intense research to identify the elements that control mitosis. Calcium waves in phase with mitotic waves suggest that calcium signals control this synchronized pattern of nuclear divisions. However, protein targets that would translate these signals into mitotic control have not been described. This study investigated the role of the calcium-dependent protease Calpain A in mitosis. Impaired Calpain A function was shown to result in loss of mitotic synchrony and ultimately halted embryonic development. The presence of defective microtubules and chromosomal architecture at the mitotic spindle during metaphase and anaphase and perturbed levels of Cyclin B indicate that Calpain A is required for the metaphase-to-anaphase transition. The results suggest that Calpain A functions as part of a timing module in mitosis, at the interface between calcium signals and mitotic cycles of the Drosophila embryo (Vieira, 2016).
Transcription factors and microRNAs (miRNAs) are two important classes of trans-regulators in differential gene expression. Transcription factors occupy cis-regulatory motifs in DNA to activate or repress gene transcription, whereas miRNAs specifically pair with seed sites in target mRNAs to trigger mRNA decay or inhibit translation. Dynamic spatiotemporal expression patterns of transcription factors and miRNAs during development point to their stage- and tissue-specific functions. Recent studies have focused on miRNA functions during development; however, much remains to explore regarding how the expression of miRNAs is initiated and how dynamic miRNA expression patterns are achieved by transcriptional regulatory networks at different developmental stages. This study has focused on the identification, regulation and function of miRNAs during the earliest stage of Drosophila development, when the maternal-to-zygotic transition (MZT) takes place. Eleven miRNA clusters comprise the first set of miRNAs activated in the blastoderm embryo. The transcriptional activator Zelda is required for their proper activation and regulation, and Zelda binding observed in genome-wide binding profiles is predictive of enhancer activity. In addition, other blastoderm transcription factors, comprising both activators and repressors, the activities of which are potentiated and coordinated by Zelda, contribute to the accurate temporal and spatial expression of these miRNAs, which are known to function in diverse developmental processes. Although previous genetic studies showed no early phenotypes upon loss of individual miRNAs, this analysis of the mir-1; miR-9a double mutant revealed defects in gastrulation, demonstrating the importance of co-activation of miRNAs by Zelda during the MZT (Fu, 2014).
Similar to protein-coding genes, miRNA genes are regulated by sophisticated spatial and temporal signals to ensure their proper production in specific cell types. The muscle-specific transcription factors Twist (Twi) and Mef2 are key activators of mir-1 in Drosophila. Genomic studies have also identified regulators of miRNAs, such as Dorsal (Dl), c-Myc (Diminutive -- FlyBase) and Ecdysone. However, for many miRNAs, particularly those differentially expressed across developmental stages, the regulatory networks that control their transcription remain unknown. This study examined the gene network that regulates miRNA functions during the maternal-to-zygotic transition (MZT), a time when developmental control is transferred from maternal products preloaded into the egg to the embryo's own genome, which in Drosophila is activated ~1 hour after fertilization. During the MZT, thousands of maternal RNAs are degraded and hundreds of newly synthesized RNAs appear; thus, the MZT represents a major reprogramming event of the early transcriptome. Previous studies have reported that the zinc-finger transcription factor Zelda (Vielfaltig -- FlyBase) plays a key role during the MZT in Drosophila, collectively activating batteries of genes involved in early developmental processes, such as sex determination, cellularization and axis patterning. Interestingly, Zelda also activates the miR-309 cluster of eight miRNAs, which is involved in the clearance of many maternally loaded mRNAs. Since Zelda plays such an extensive role in zygotic genome activation, possibly as a pioneer factor to prime genes for transcriptional activation, this study investigated the possibility that Zelda activates the miRNAs expressed during the MZT (Fu, 2014).
This study identified a group of miRNAs (11 clusters) that are zygotically expressed in cellular blastoderm embryos; Zelda was shown to regulate all 11. The enhancers of several miRNAs were localized by virtue of Zelda ChIP binding; Zelda binding sites, also known as CAGGTAG sites or TAGteam sites, in these enhancers were shown to be essential for proper activation. It was further shown that anteroposterior (AP) and dorsoventral (DV) patterning factors work together with Zelda to ensure timely and robust transcriptional activation of these miRNAs, contributing to their accurate spatial expression patterns. The reduced and disrupted miRNA expression seen in Zelda mutants affects their downstream functions in maternal mRNA degradation, cell death gene repression and Hox gene regulation. Ventral midline defects were observed during gastrulation in mir-1; miR-9a double mutants, that were not seen in either single mutant, suggesting that the coordinated activation of miRNAs by Zelda is crucial for their combinatorial function. This analysis offers a systems-level view and understanding of the early gene network. Zelda sits as a major hub in the network, globally activating both protein-coding and non-coding genes, thereby orchestrating the early developmental processes (Fu, 2014).
This study identified the set of miRNAs expressed in 2- to 3-h Drosophila embryos, a time when the MZT is well underway and the fate map of the embryo is being established. These early expressed miRNAs are regulated globally by Zelda, both directly via binding to cis-regulatory enhancers and indirectly by affecting the expression of additional transcriptional regulators. Together with previously published data on specific miRNAs, it was possible to integrate the early miRNAs, their upstream regulators and downstream targets into the early gene network (Fu, 2014).
Using expression profiling data from 2- to 3-h wild-type and zelda mutant embryos, blastoderm-specific pri-miRNA transcription units, which included seven intergenic and four intronic miRNAs (clusters) were identified. Since the expression levels of all 11 miRNAs were affected in Zelda mutants, it was possible to better distinguish blastoderm-specific isoforms, particularly in the case of intronic miRNAs, such asmir-11, which resides in an intron of E2f . Moreover, maternal E2f expression could be differentiated from zygotic expression by observing the intronic signal, which was clearly downregulated in zelda mutants (Fu, 2014).
The early miRNAs exhibit strikingly different expression patterns, and it is noteworthy that, similar to the protein-coding targets of Zelda, two different strategies are used to regulate these miRNAs. Some miRNAs, such as miR-9a, were completely abolished in Zelda mutants, indicating that Zelda is their sole activator, whereas others, such as mir-1, were affected temporally and/or spatially, indicating that Zelda works together with other factors to establish robust and precise domains of expression. For example, mir-1 downregulation in Zelda mutants is likely to be due to the cumulative effect of loss of direct inputs from Zelda and the delayed expression of twi that occurs in Zelda mutants. Thus, the effect onmir-1 is the result of a breakdown in the Zelda-Twi-mir-1 feedforward loop (Fu, 2014).
Cis-regulatory modules/enhancers of miRNAs have been predicted based on the presence of transcription factor binding or specific chromatin marks, and verified in only some cases. For example, two regions upstream of mir-1 that bind Twi/Dl were shown to drive a mir-1-like expression pattern. It was reasoned that it could be possible to locate enhancers of all early miRNAs by simply looking for Zelda-bound regions upstream of the pri-miRNA transcription units, especially since Zelda is a global activator during the MZT. This approach worked well; eight of nine enhancers recapitulated endogenous-like expression. Mutation of Zelda binding sites in enhancers further demonstrated direct Zelda input. As proof of principle, enhancers of two genes, miR-9a and mir-1, were analyzed and it was shown that mutation of the Zelda binding sites had the same effect as eliminating Zelda in trans. These results indicate that Zelda directly regulates the early expressed miRNAs, often in conjunction with other transcription factors, many of which are also regulated by Zelda. Zelda is a major hub in the early network, establishing multiple feedforward loops and closely linking the transcription factors and miRNAs expressed in this stage (Fu, 2014).
The MZT is a key event during the development of an organism, whereby the transcriptome is reprogrammed in the first few hours of development. This requires the clearance of previous information (maternal mRNA degradation) and the initiation of a new program (zygotic genome activation). The maternal mRNA degradation machinery comprises both maternally derived and zygotically derived pathways. In Drosophila, Smaug (Smg), a maternally loaded RNA-binding protein, is central to the mRNA clearance pathway. By recruiting the CCR4-NOT deadenylation complex, Smg destabilizes two-thirds of the maternal mRNAs that undergo degradation (i.e. that are unstable) upon egg activation. By contrast, miR-309 is a key component of the zygotically derived pathway to clear mRNA. When analyzing the maternal RNAs upregulated in zelda mutants, it was noted in this study that 81% of them (434) depend on zygotic degradation pathways; 125 of the 434 genes are also upregulated in miR-309 mutants, indicating that Zelda, by activating miR-309, is involved in maternal RNA degradation. Therefore, Zelda plays important roles in both of the hallmark events of the MZT. Interestingly, the miR-309 targets account for only ~30% of the unstable maternal RNAs upregulated in Zelda mutants, and another 14% are putative targets of the other early miRNAs, indicating that Zelda might activate additional zygotic pathways to mediate maternal mRNA degradation (Fu, 2014).
Several miRNAs, in addition to miR-309, have been shown to target specific mRNAs in the early embryo; for example, miR-iab-4 and miR-iab-4as target Hox genes. However, although each of the miRNAs is predicted to target hundreds of genes, in many cases the individual miRNA loss-of-function phenotypes are relatively mild. There are several explanations for this phenomenon: (1) the miRNA does not function at the time that it is expressed, but might function later; (2) miRNAs 'fine-tune' the expression levels of their target genes, which might not be reflected in obvious phenotypes when they are mutated; and (3) miRNA functions are redundant, such that knockdown of one miRNA may be compensated by another (Fu, 2014).
To better address the functions of miRNAs, investigators have used several genetic approaches: gain-of-function assays, using sensitized genetic backgrounds, and assaying double mutants. For example, the miR-6; mir-11 double mutant exhibits increased apoptosis, leading to lower survival rates compared with either of the single miRNA mutants. Using a similar approach, this study observed fully penetrant gastrulation defects in mir-1; miR-9a double mutants. Neither single mutant is embryonic lethal, nor shows any sign of ventral furrow defects; however, mir-1 mutants are larval lethal and display muscle defects, while miR-9a mutants show wing margin defects in adulthood. Importantly, the double-mutant phenotype is the earliest phenotype seen for any known miRNA, or combination of miRNAs, thus far tested. These results support the idea that co-activation of miRNAs by Zelda is required for normal development (Fu, 2014).
The mir-1; miR-9a double-mutant phenotype resembles, to some extent, the ventral furrow defects observed in RhoGEF2 loss-of-function mutant. Rho signaling is involved in the cell shape changes associated with ventral furrow invagination, and loss of Rho signaling results in very disorganized invagination. Curiously, mir-1 and miR-9a are both predicted to target RhoGAP68F, a negative regulator of Rho signaling. RhoGAP68F is maternally loaded and cleared during the MZT. It is possible that the gastrulation phenotype of the mir-1; miR-9a double mutant is caused in part by excess activity of RhoGAP68F. Although no obvious upregulation of RhoGAP68F transcripts were seen in mir-1; miR-9a mutant embryos by in situ hybridization, it is possible that subtle upregulation of RhoGAP68F, combined with effects on other predicted targets, all contribute to the gastrulation defects observed in the double mutant (Fu, 2014).
The co-activation of groups of miRNAs by master regulators such as Zelda may be crucial for miRNA activity during development, as revealed by the severe gastrulation phenotype of the mir-1; miR-9a double mutant. Such coordinated activation of miRNAs might also occur at later stages in development in tissues in which Zelda is expressed, such as the central nervous system. In the future, various combinations of mutations in miRNA genes that are co-regulated by Zelda, or other key factors, might unveil additional functions of miRNAs across development stages (Fu, 2014).
The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, this study refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. 523 proteins were determined to be high-confidence RBPs, half of which have not been previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development (Sysoev, 2015). The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. This study found that Death-associated protein kinase related (Drak) is necessary for most of the phosphorylation of myosin regulatory light chain during cellularization. Drak was shown to be required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak also is involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, these observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization (Chougule, 2016).
Tight regulation of actomyosin is likely critical for many cellular processes, but how this is accomplished is as yet poorly understood. A key input to the regulation of myosin II is through phosphorylation of the Serine-19, or the Serine-19 and Threonine-18 residues of MRLC (Spaghetti squash). The variety of MRLC kinases might allow different specific aspects of actomyosin dynamics, such as localization, organization and contraction to be regulated independently. Such a system would provide greater flexibility and control than either a single kinase, or multiple kinases acting in concert, regulating all of these functions. drak was found to be required for the organization of myosin II into contractile rings, but is not required for localization of myosin to the cellularization front. Since the majority of Sqh phosphorylation during cellularization is dependent on drak activity, Drak either regulates most aspects of myosin II dynamics during cellularization, or Drak-regulated myosin II organization is required for further function of myosin II, such as contraction (Chougule, 2016).
Myosin II is somewhat less disorganized and Sqh phosphorylation is slightly increased during late cellularization in drak mutants, suggesting that phosphorylation of myosin II by other kinases occurs during late cellularization. Thus other kinases might act synergistically with Drak to regulate actomyosin organization during late cellularization. For example, Drak function has been shown to be partially redundant with Rok function during later development. An alternative possibility is that other kinases that do not normally function in myosin II organization in the microfilament rings might phosphorylate Sqh to some degree and lead to some organization of myosin II in the absence of Drak activity (Chougule, 2016).
Myosin II has been implicated in actin bundling and F-actin organization in some contexts. Since F-actin appears to
be organized normally within drak mutant microfilament rings during early cellularization, it is concluded that myosin II does not play a role in initially organizing F-actin within the microfilament rings during cellularization. F-actin is somewhat disorganized during late cellularization in drakdel mutant embryos, but not as severely as myosin II, nor does the pattern of F-actin distribution fit the pattern of myosin II distribution in drakdel mutant embryos. These observations suggest that F-actin disorganization is an indirect consequence of Drak regulation of myosin II activity, and that F-actin disorganization might be due to actomyosin contraction defects or furrow canal structural defects (Chougule, 2016).
Anillin is required for the organization of actomyosin contractile rings during cellularization and cytokinesis. scraps (scra, anillin) mutant embryos have a myosin II organization defect somewhat similar to that of drak mutant embryos: myosin II is found in discrete bars in the actomyosin network. Despite this similarity, myosin II defects differ between scra and drak mutant embryos. Myosin II becomes more disorganized during late cellularization in scra mutant embryos. Myosin II becomes slightly better organized during late cellularization in drak mutant embryos. This organizational difference is likely caused by actomyosin contraction during microfilament ring constriction occurring in a highly disorganized cytoskeleton in scra mutant embryos, and occurring in a disorganized cytoskeleton that has slightly improved during constriction in drak mutant embryos. Anillin only interacts with myosin II when MRLC is phosphorylated. Together with these results, this suggests that Drak phosphorylation of Sqh might be necessary for Anillin-mediated myosin II organization within the contractile ring (Chougule, 2016).
Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 leads to the
unfolding of inactive myosin II hexamers into an open conformation that allows assembly of bipolar myosin II filaments and their association with F-actin to form actomyosin filaments. This is likely how Drak organizes myosin II. Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 also leads to the activation of the Mg2+-ATPase activity of myosin II that slides actin filaments past each other, causing actomyosin contraction. Three aspects of the drak mutant phenotype support the requirement for Drak in actomyosin contraction: wavy cellularization fronts caused by non-uniform furrow canal depths, abnormal microfilament ring shapes, and failure of microfilament rings to constrict during late cellularization. These are the same defects that suggest an actomyosin contraction defect in src64 mutant embryos. However, src64 mutant embryos do not show myosin II organization defects. Because effective actomyosin contraction likely requires properly organized actomyosin filaments within the contractile ring apparatus, it is unclear whether Drak directly regulates actomyosin contraction or whether Drak only enables actomyosin contraction through proper organization of myosin II within the microfilament rings. One possibility is that phosphorylation of Sqh by Drak both organizes actomyosin filaments into a contractile ring apparatus and directs actomyosin contraction. An alternative possibility is that Drak is directly responsible for organizing actomyosin filaments into a contractile ring by phosphorylating Sqh, but Drak is not directly involved in its contraction and different kinases that phosphorylate Sqh regulate actomyosin contraction. Thus, Drak could be an early regulator of myosin II activity
during cellularization, such that further phosphorylation of Sqh and myosin II-driven contraction is dependent on Drak-mediated organization of myosin II. At some level the regulation of actomyosin contraction diverges from the regulation of actomyosin filament organization: Src64 is required for contraction, but has no role in myosin II organization (Chougule, 2016).
Rescue of myosin II organization, actomyosin contraction and F-actin distribution defects in drak mutant embryos by the mono-phosphorylated SqhE21 phosphomimetic suggests that Drak-mediated mono-phosphorylation of Sqh at Serine-21 is sufficient for regulation of actomyosin dynamics during cellularization. Although the diphosphorylated SqhE20E21 phosphomimetic also rescues myosin II organization and actomyosin contraction defects, it does not rescue F-actin distribution defects in drak mutant embryos. These results are consistent with Drak primarily phosphorylating Sqh at Serine-21, and are consistent with reports that DAPK family members phosphorylate MRLC mainly at Serine-19 (Chougule, 2016).
The normal teardrop shape of the furrow canals in early cellularization is likely caused by actomyosin contraction in the microfilament rings. In drak mutant embryos, unexpanded early cellularization furrow canals and failure of many late cellularization furrow canals to expand further suggest that Drak is required for proper furrow canal structure. Some of the furrow canal structural defects in drak mutant embryos are similar to those of nullo mutant embryos: collapsed furrow canals and blebbing. However, nullo mutant embryos, as well as RhoGEF2 or dia mutant embryos, have other, more severe furrow canal defects: missing or regressing furrow canals and compromised lateral membrane-furrow canal compartment boundaries. Furthermore, cytochalasin treatment causes similar defects, suggesting that reduced F-actin levels in the furrow canals are responsible for these defects. Thus Nullo, RhoGEF2 and Dia regulate F-actin and its levels in furrow canals. These observations suggest that Drak regulates myosin II and thereby regulates actomyosin organization and contraction, and that these are necessary for structural integrity and expansion of the furrow canals, but not for their continued existence (Chougule, 2016).
The furrow canals of drak mutant embryos during late cellularization show extensive blebbing into the lumens. This is consistent with a defect in furrow canal membrane or cortex integrity. Blebs can be formed by local rupture of the cortical cytoskeleton or detachment of the plasma membrane from the cortical actomyosin cytoskeleton. Actomyosin contraction has been implicated in bleb formation. Therefore, it is proposed that blebbing in furrow canals is caused by aberrant localized actomyosin contraction during late cellularization in the disorganized actomyosin cytoskeleton of drak mutant embryos. Contraction is presumably driven by phosphorylation of Sqh by kinases other than Drak. Since actomyosin contraction occurs in a disorganized actomyosin cytoskeleton, it does not lead to uniform constriction of the microfilament rings, but instead leads to localized contraction that produces cytoplasmic blebs. However, other causes for furrow canal defects are possible. Plasma membrane attachment sites might not form or function properly in the disorganized furrow canal cytoskeleton in drak mutant embryos. The disorganized cytoskeleton might inhibit vesicle trafficking. Vesicle trafficking itself might be
defective: mammalian DAPKs have been shown to be involved in membrane trafficking and in phosphorylation of syntaxin A1.
Vesiculated lateral plasma membrane in drak mutant embryos during late cellularization suggests that the plasma membrane breaks down. Intriguingly, scra mutant embryos have lines of vesicles where the closely apposed lateral plasma membranes would have been. However in scra mutant embryos, vesiculation is observed during early cellularization, but to a lesser extent than during late cellularization. drak mutant embryos do not show lateral plasma membrane vesiculation defects until late cellularization. drak mutant defects in both the furrow canal membrane and the lateral plasma membrane might reflect a general defect in membrane integrity. It will be interesting to investigate the potential role of myosin II organization in furrow canal structure and plasma membrane integrity (Chougule, 2016).
Metazoan embryos undergo a maternal-to-zygotic
transition (MZT) during which maternal gene products are eliminated
and the zygotic genome becomes transcriptionally active. During this
process RNA-binding proteins (RBPs) and the microRNA-induced silencing
complex (miRISC) target maternal mRNAs for degradation. In Drosophila,
the Smaug (SMG), Brain
tumor (BRAT) and Pumilio (PUM)
RBPs bind to and direct the degradation of largely distinct subsets of
maternal mRNAs. SMG has also been shown to be required for zygotic
synthesis of mRNAs and several members of the miR-309
family of microRNAs (miRNAs) during the MZT. This study carried out global
analysis of small RNAs both in wild type and in smg mutants. It
was found that 85% all miRNA species encoded by the genome are present
during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting
RNAs (piRNAs) or small
interfering RNAs (siRNAs), zygotic production of more than 70
species of miRNAs fails or is delayed in smg mutants. SMG is
also required for the synthesis and stability of a key miRISC component, Argonaute
1 (AGO1), but plays no role in accumulation of the Argonaute-family
proteins associated with piRNAs or siRNAs. In smg mutants,
maternal mRNAs that are predicted targets of the SMG-dependent zygotic
miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these
miRNAs but not with SMG itself. The study hypothesizes that SMG controls
the MZT, not only through direct targeting of a subset of maternal mRNAs
for degradation but, indirectly, through production and function of miRNAs
and miRISC, which act together with BRAT and/or PUM to control clearance
of a distinct subset of maternal mRNAs (Luo, 2016).
To identify small RNA species expressed during the Drosophila MZT and to assess the role of SMG in their regulation 18 small-RNA libraries were produced and sequenced: nine libraries from eggs or embryos produced by wild-type females and nine from smg-mutant females. The 18 libraries comprised three biological replicates each from the two genotypes and three time-points: (1) 0-to-2 hour old unfertilized eggs, in which zygotic transcription does not occur and thus only maternally encoded products are present; (2) 0-to-2 hour old embryos, the stage prior to large-scale zygotic genome activation; and (3) 2-to-4 hour old embryos, the stage after to large-scale zygotic genome activation. After pre-alignment processing, a total of ~144 million high quality small-RNA reads was obtained and 110 million of these perfectly matched the annotated Drosophila genome (Luo, 2016).
Loss of SMG had no significant effect on piRNAs and siRNAs, or on the Argonaute proteins associated with those small RNAs: Piwi, Aubergine (AUB), AGO3, and AGO2, respectively. In contrast, loss of SMG resulted in a dramatic, global reduction in miRNA populations during the MZT as well as reduced levels of AGO1, the miRISC-associated Argonaute protein in Drosophila (Luo, 2016).
A pre-miRNA can generate three types of mature miRNA: (1) a canonical miRNA, which has a perfect match to the annotated mature miRNA; (2) a non-canonical miRNA, which shows a perfect match to the annotated mature miRNA but with additional nucleotides at the 5'- or 3'- end that match the adjacent primary miRNA sequence, and (3) a miRNA with non-templated terminal nucleotide additions (an NTA-miRNA), which has nucleotides at its 3'-end that do not match the primary miRNA sequence (Luo, 2016).
In these libraries a total of 364 distinct miRNA species were identified that mapped to miRBase, comprising 85% (364/426) of all annotated mature miRNA species in Drosophila. Thus, the vast majority of all miRNA species encoded by the Drosophila genome are expressed during the MZT. Overall, in wild type, an average of 75% of all identified miRNAs fell into the canonical category. The remaining miRNAs were either non-canonical (10%) or NTA-miRNAs (15%) (Luo, 2016).
To validate these sequencing results, those mature miRNA species identified in the data that perfectly matched the Drosophila genome sequence (i.e., canonical and non-canonical) were compared with a previously published miRNA dataset from 0 to 6 hour old embryos. To avoid differences caused by miRBase version, data sets from previous study were remapped to miRBase Version 19 and f99% of their published miRNA species were found to be on the miRNA list (176/178 mature miRNA species comprising 161 canonical miRNA s and 94 non-canonical miRNA s) . There were an additional 181 mature miRNA species in the library that had not been identified as expressed in early embryos in the earlier study (Luo, 2016).
As a second validation, the list of maternally expressed miRNA species (those present in the 0-to-2 hour wild-type unfertilized egg samples) were compared with the most recently published list of maternal miRNAs, which had been defined in the same manner. 99% of the 86 published maternal miRNA species were on this study's maternal miRNA list (85/86). An additional 144 maternal miRNA species in the library were identified that had not been observed in the previous study. Identification of a large number of additional miRNA species in unfertilized eggs and early embryos can be attributed to the depth of coverage of the current study. The current dataset, therefore, provides the most complete portrait to date of the miRNAs present during the Drosophila MZT (Luo, 2016).
Next, global changes in miRNA species during the MZT were analyzed in wild-type embryos. A dramatic increase was observed in the proportion of miRNAs relative to other small RNAs that was due to an increase in absolute miRNA amount rather than a decrease in the amount of other types of small RNAs. In wild-type 0-to-2 hour unfertilized eggs, the proportion of the small RNA libraries comprised of canonical and non-canonical miRNAs was 12.8%. These represent maternally loaded miRNAs since unfertilized eggs do not undergo zygotic genome activation. The proportion of small RNAs represented by miRNAs increased dramatically during the MZT, reaching 50.7% in 2-to-4 hour embryos. The other abundant classes of small RNAs underwent either no change or relatively minor changes over the same time course. It is concluded that there is a large amount of zygotic miRNA synthesis during the MZT in wild-type embryos (Luo, 2016).
For more detailed analysis of the canonical, non-canonical and NTA isoforms focus was placed on 154 miRNA species that possessed an average of > 10 reads per million (RPM) for all three isoform types in one or more of the six sample sets. A focus was placed on changes in wild type. Among all miRNAs, in wild type the proportion of canonical isoforms increased over the time-course from 69% to 83%, the proportion of non-canonical miRNAs remained constant (from 9% to 10%) , and the proportion of the NTA-miRNAs decreased (from 22% to 7%). These results derive from the fact that, during the MZT, the vast majority of newly synthesized miRNAs were canonical, undergoing a more than seven-fold increase from 103,105 to 744,043 RPM; that non-canonical miRNAs underwent a comparable, nearly seven-fold, increase from 13,902 to 92,199; whereas NTA-miRNAs underwent a less than two-fold increase, from 32,840 to 63,847, thus decreasing in relative proportion (Luo, 2016).
Whereas the proportion of the small-RNA population that was comprised of miRNAs increased fourfold over the wild-type time-course, concomitant with increases in overall miRNA abundance, there was no such increase in the smg mutant embryos: 21.9% of the small RNAs were miRNAs in 0-to-2 hour unfertilized smg mutant eggs (mean RPM = 203,415) and 20.5% (mean RPM = 196,110) were miRNAs in 2-to-4 hour smg mutant embryos (Luo, 2016).
This difference between wild type and smg mutants could have resulted from the absence of a small number of extremely highly expressed miRNA species in the mutant. Alternatively, it may have been a consequence of a widespread reduction in the levels of all or most zygotically synthesized miRNAs in smg mutants. To assess the cause of this difference, canonical miRNA reads were graphed in scatter plots. These showed that a large number of miRNA species had significantly reduced expression levels in 0-to-2 and in 2-to-4 hour smg-mutant embryos relative to wild type. Most of the down-regulated miRNA species exhibited a more than four-fold reduction in abundance relative to wild type. Furthermore, this reduction occurred for miRNA species expressed over a wide range of abundances in wild type (Luo, 2016).
Box plots were then used to analyze the canonical, non-canonical and NTA isoforms of the 154 miRNA species identified in the previous section. These showed that, in wild type, the median abundance of canonical, non-canonical and 3' NTA miRNAs increased significantly in 0-to-2 and in 2-to-4 hour embryos relative to 0-to-2 hour unfertilized eggs. In contrast, there was no significant increase in the median abundance of any of the three isoforms of miRNAs in the smg-mutant embryos. Also for all three isoform types, when each time point was compared between wild type and smg mutant, there was no difference between wild type and mutant in 0-to-2 hour unfertilized eggs but there was a highly significant difference between the two genotypes at both of the embryo time-points. Whereas the abundance of miRNAs differed between wild-type and mutant embryos, there was no difference in length or first-nucleotide distribution of canonical miRNAs, nor in the non-templated terminal nucleotides added to NTA-miRNAs (Luo, 2016).
As described above, during the wild-type MZT canonical miRNAs comprised the major isoform that was present (69% to 83% of miRNAs). It was next asked whether miRNA species could be categorized into different classes based on their expression profiles during the wild-type MZT. 131 canonical miRNA species that had > 10 mean RPM in at least one of the six datasets were analyzed. Hierarchical clustering of their log 2 RPM values identified five distinct categories of canonical miRNA species during the MZT. The effects of smg mutations on each of these classes were analyzed (Luo, 2016).
The data are consistent with a model in which SMG degrades its direct targets without the assistance of miRNAs whereas a large fraction of the indirectly affected maternal mRNAs in smg mutants fails to be degraded by virtue of being targets of zygotically produced miRNA species that are either absent or present at significantly reduced levels in smg mutants. Thus, SMG is required both for early, maternally encoded decay and for late, zygotically encoded decay. In the former case SMG is a key specificity component that directly binds to maternal mRNAs; in the latter case SMG is required for the production of the miRNAs (and AGO1 protein) that are responsible for the clearance of an additional subset of maternal mRNAs (Luo, 2016).
In Drosophila, the stability of miRNAs is enhanced by AGO1 and vice versa. Since miRNA levels are dramatically reduced in smg mutants, Ago1 mRNA and AGO1 protein levels were assessed during the MZT both in wild type and in smg mutants. In wild type, AGO1 levels were low in unfertilized eggs and 0-to-2 hour embryos but then increased substantially in 2-to-4 hour embryos. These western blot data are consistent with an earlier, proteomic, study that reported a more than three-fold increase in AGO1 in embryos between 0-to-1.5 hours and 3-to-4.5 hours. In contrast to AGO1 protein, it was found using RT-qPCR that Ago1 mRNA levels remained constant during the MZT. Taken together with a previous report that Ago1 mRNA is maternally loaded, the increase in AGO1 protein levels in the embryo is, therefore, most likely to derive from translation of maternal Ago1 mRNA rather than from newly transcribed Ago1 mRNA (Luo, 2016).
Next, AGO1, AGO2, AGO3, AUB and Piwi protein levels were analyzed in eggs and embryos from mothers carrying either of two smg mutant alleles: smg1 and smg47. The smg mutations had no effect on the expression profiles of AGO2, AGO3, AUB or Piwi. In contrast, in smg-mutant embryos, the amount of AGO1 protein at both 0-to-2 and 2-to-4 hours was reduced relative to wild type and this defect was rescued in embryos that expressed full-length, wild-type SMG from a transgene driven by endogenous smg regulatory sequences. The reduction of AGO1 protein levels in smg mutants was not a secondary consequence of reduced Ago1 mRNA levels since Ago1 mRNA levels in both the smg-mutant and the rescued-smg-mutant embryos were very similar to wild type (Luo, 2016).
A plausible explanation for the decrease in AGO1 levels in smg mutants is the reduced levels of miRNAs, which would then result in less incorporation of newly synthesized AGO1 into functional miRISC and consequent failure to stabilize the AGO1 protein. To assess this possibility, a time-course in wild-type unfertilized eggs was analyzed in which zygotic genome activation and, therefore, zygotic miRNA synthesis, does not occur. It was found that AGO 1 levels were reduced in 2-to-4 hour wild-type unfertilized eggs compared with wild-type embryos of the same age. This result is consistent with a requirement for zygotic miRNAs in the stabilization of AGO1 protein (Luo, 2016).
Next, wild-type unfertilized egg and smg-mutant unfertilized egg time-courses were compared, and AGO1 levels were found to be further reduced in the smg mutant relative to wild type. This suggests that SMG protein has an additional function in the increase in AGO1 protein levels that is independent of SMG's role in zygotic miRNA production (since these are produced in neither wild-type nor smg-mutant unfertilized eggs) (Luo, 2016).
To assess whether this additional function derives from SMG's role as a post-transcriptional regulator of mRNA, smg1 mutants were rescued either with a wild-type SMG transgene driven by the Gal4:UAS system (SMGWT) or a GAL4:UAS-driven transgene encoding a version of SMG with a single amino-acid change that abrogates RNA-binding (SMGRBD) and, therefore, is unable to carry out post-transcriptional regulation of maternal mRNAs. It was found that, whereas AGO1 was detectable in both unfertilized eggs and embryos from SMGWT-rescued mothers, AGO1 was undetectable in unfertilized eggs from SMGRBD-rescued mothers and was barely detectable in embryos from these mothers. Thus, SMG's RNA-binding ability is essential for its non-miRNA-mediated role in regulation of AGO1 levels during the MZT (Luo, 2016).
Since the abundance of SMGWT and SMGRBD proteins is very similar, the preceding result excludes the possibility that it is physical interaction between SMG and AGO1 that stabilizes the AGO1 protein. It was previously shown that the Ago1 mRNA is not bound by SMG. Thus, SMG must regulate one or more other mRNAs whose protein products, in turn, affect the synthesis and/or stability of AGO1 protein. It is known that turnover of AGO1 protein requires Ubiquitin-activating enzyme 1 (UBA1) and is carried out by the proteasome . It was previously shown that the Uba1 mRNA is degraded during the MZT in a SMG-dependent manner and that both the stability and translation of mRNAs encoding 19S proteasome regulatory subunits are up-regulated in smg-mutant embryos. It is speculated that increases in UBA1 and proteasome subunit levels in smg mutants contribute to a higher rate of AGO1 turnover and, thus, lower AGO1 abundance than in wild type (Luo, 2016).
AGO1 physically associates with BRAT. It is not known whether AGO1 interacts with PUM but it has been reported that, in mammals and C. elegans , Argonaute-family proteins interact with PUM/PUF-family proteins. Recent studies identified direct target mRNAs of the BRAT and PUM RBPs in early Drosophila embryos and showed through analysis of brat mutants that, during the MZT, BRAT directs late (i.e., after zygotic genome activation) decay of a subset of maternal mRNAs. These data permitted asking whether the maternal mRNAs that are predicted to be indirectly regulated by SMG via its role in miRISC production might be co-regulated by BRAT and/or PUM (Luo, 2016).
A highly significant overlap was found between the predicted miRNA-dependent indirect targets of SMG and both BRAT-and PUM-bound mRNAs in early embryos. This suggests that BRAT and PUM might function together with miRISC during the MZT to direct decay of maternal mRNAs (Luo, 2016).
Given that BRAT and PUM bind to largely non-overlapping sets of mRNAs during the MZT, there are three types of hypothetical BRAT-PUM-miRISC-containing complexes: one with both BRAT and PUM, one with BRAT only, one with PUM only. To assess this possibility for a specific set of zygotically produced miRNAs, the lists of mRNAs stabilized in 2-to-3 hour old embryos from miR-309 deletion mutants were compared to the lists of BRAT and PUM direct-target mRNAs. There was no significant overlap of PUM-bound mRNAs with those up-regulated in miR-309 mutants. However, there was a highly significant overlap of mRNAs up-regulated in miR-309-mutant embryos with BRAT-bound mRNAs. These results lead to the hypothesis that BRAT (but not PUM) co-regulates clearance of miR-309-family miRNA target maternal mRNAs during the MZT (Luo, 2016).
Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. This study presents an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. The deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and the X-chromosomal contribution was tracked to furrow lengthening to the nullo gene product. Checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, the turnover of maternal gene products was examined, and this was found to be a minor contributor to the developmental regulation of furrow morphologies. These results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis (Xie, 2018).
Many metabolic enzymes are evolutionary highly conserved and serve a central function for catabolism and anabolism of cells. The serine hydroxymethyl transferase (SHMT) catalysing the conversion of serine and glycine and vice versa feeds into the tetrahydrofolate mediated C1 metabolism. This study identified a Drosophila mutation in SHMT (CG3011) in a screen for blastoderm mutants. Embryos from SHMT mutant germline clones specifically arrest the cell cycle in interphase 13 at the time of the mid blastula transition (MBT) and prior to cellularisation. The phenotype is due to a loss of enzymatic activity as it cannot be rescued by an allele with a point mutation in the catalytic center but by an allele based on the SHMT coding sequence from E. coli. Onset of zygotic gene expression and degradation of maternal RNAs in SHMT mutant embryos are largely similar to wild type embryos. The specific timing of the defects in SHMT mutants indicates that at least one of the SHMT-dependent metabolites becomes limiting in interphase 13, if it is not produced by the embryo. These data suggest that mutant eggs contain maternally provided and SHMT-dependent metabolites in amounts which suffice for early development until interphase 13 (Winkler, 2017).
The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? It is proposed that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, it was demonstrated that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. This work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription (Djabrayan, 2019).
In most metazoans, early embryonic development is characterized by rapid division cycles that pause before gastrulation at the midblastula transition (MBT). These cleavage divisions are accompanied by cytoskeletal rearrangements that ensure proper nuclear positioning. However, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium. Nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows. In shackleton (shkl) mutants, this axial spreading is impaired. This study shows that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but is required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity, suggesting that a major target of cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis that were exploited to study cell-cycle control as a function of the N/C ratio. The N/C ratio is sensed collectively in neighborhoods of about 100 μm, and such collective sensing is required for a precise MBT, in which all the nuclei in the embryo pause their division cycle. Moreover, it was found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to Cdk1 dynamics. Collectively, this study revealed a new pathway controlling nuclear positioning and provides a dissection of how nuclear cycles respond to the N/C ratio (Hayden, 2022).
The tight control of the cell cycle and nuclear (cell) positioning and number is a ubiquitous feature of metazoan development and is crucial to the proper development of early embryos. This work has taken advantage of shkl mutants that have defects in nuclear spreading to identify a novel pathway involved in the control of cortical contractility and gain insights into how nuclei respond to changes in the N/C ratio. Through DNA sequencing and complementation tests, this study has identified shkl mutants as mutations of the ubiquitin ligase Cul-5. In the early embryo, Cul-5 does not regulate the cell-cycle oscillator but is required for Rho and myosin activities. Cul-5 restricts the levels of active Src kinase, which is a known regulator of the actomyosin cytoskeleton. Indeed, it was found that the cullin-5 phenotype could be largely rescued through a genetic reduction in Src activity and recapitulated through Src overexpression, indicating that a main function of Cul-5 is to downregulate Src activity. These results implicate the Cul-5/Src axis as a crucial pathway involved in the control of cortical contractility in early Drosophila embryos (Hayden, 2022).
In the early embryo, nuclei regulate their own positioning through PP1 activity that spreads from the nuclei to the cortex. This localized PP1 activity drives activation of Rho and myosin II accumulation in turn. The current results argue that Cul-5 and Src act in a pathway downstream or parallel to the cell cycle to regulate Rho activity. The molecular mechanisms by which Cul-5 and Src control Rho remain to be elucidated, as is the possible connection between the cell-cycle oscillator and Cul-5/Src activities. Since Src has been shown to regulate Rho GTPases in several contexts, these mechanisms are natural candidates for the regulation of cortical actomyosin regulation via the Cul-5/Src pathway (Hayden, 2022).
Control of the MBT by the N/C ratio is important in several species, including Drosophila and Xenopus but likely excluding zebrafish. This density of DNA (as well as nuclear size) can directly or indirectly impact multiple aspects of the MBT, namely zygotic gene expression and cell-cycle control. Previous experiments with embryos irradiated to generate different nuclear densities across the AP axis argued that nuclear cycles and zygotic activation of a large set of genes respond to the local N/C ratio. This study has exploited the changes in nuclear positioning in shkl embryos to generate a continuous range of nuclear densities. This property has lead insights into how the decision of nuclei to pause their cell cycles at the MBT is affected by the N/C ratio. The threshold for nuclear division was found to be about 70% of the density at nuclear cycle 14, which confirms previous results. This value-about halfway between the density at cycles 13 and 14-likely contributes to the robustness of the MBT. However, it is not sufficient for the robustness of the MBT. To ensure reliable lengthening of cycle 14 in all nuclei, the sensing of the N/C ratio must be averaged over hundreds of nuclei. Consistently, the results suggest that nuclei sense the local N/C ratio in neighborhoods of ~100 &mi;m. This length essentially coincides with the correlation length of the Cdk1 activity field, which is established via reaction-diffusion mechanisms. Additionally, it was found that a model based on uniform sensing of the N/C ratio fails to predict the behavior of a large fraction of nuclei. However, a model assuming a slightly higher N/C ratio threshold in the posterior is highly predictable and mainly misses the behavior of nuclei at the interface between the region of extra division and that of normal division. Thus, it is proposed that the N/C ratio is the major regulator of the cell cycle at the MBT and that no mechanism other than a slight spatial modulation of the N/C threshold is needed to account for nuclear behaviors. This spatial modulation likely reflects the fact that the rate of Cdk1 activation is also slightly graded across the AP axis. The Cdk1 activation gradient is dependent on the DNA replication checkpoint, which argues that the gradient might be controlled by an asymmetric distribution of factors controlling DNA replication and/or Chk1 activity. Alternatively, the DNA replication checkpoint and Cdk1 activity might be influenced by factors controlling AP patterning and expressed in gradients across the embryos. In the future, it will be interesting to understand the mechanisms and possible functional significance of this gradient (Hayden, 2022).
The precise coordination of biochemical and mechanical signals is a ubiquitous feature of embryonic development. In early Drosophila embryogenesis, it is necessary for the uniform positioning of nuclei and timing of the MBT. This work has identified a new pathway wherein Cul-5 regulates cortical contractility by restricting Src activity. The results investigating embryos with patchy divisions indicate that nuclei sense the N/C ratio in neighborhoods of ~100 μm and pause the cell cycle when the local density exceeds a threshold around 70% of the normal density at the MBT. Moreover, the threshold required to arrest the cell cycle is slightly graded across the AP axis and is coupled to the spatiotemporal dynamics of Cdk1. Quantitatively measuring biochemical and physical dynamics during specific morphogenic events will undoubtedly continue to reveal new insights into the mechanisms and regulations of these pathways (Hayden, 2022).
As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However it remains unclear how regions of activity are established during early embryogenesis, and if they are the product of spatially restricted or ubiquitous processes. To shed light on this question, chromatin accessibility across the anterior-posterior axis (A-P) of early Drosophila melanogaster embryos was probed by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. Genome-wide chromatin accessibility is highly similar between the two halves, with regions that manifest significant accessibility in one half of the embryo almost always accessible in the other half, even for promoters that are active in exclusively one half of the embryo. These data support previous studies that show that chromatin accessibility is not a direct result of activity, and point to a role for ubiquitous factors or processes in establishing chromatin accessibility at promoters in the early embryo. However, in concordance with similar works, this study found that at enhancers active exclusively in one half of the embryo, a significant skew was found towards greater accessibility in the region of their activity, highlighting the role of patterning factors such as Bicoid in this process (Haines, 2018).
Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. This study reports on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. The results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant (Pérez-Montero, 2013) to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process (Satovic, 2018).
This study reports that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming. On one hand, rapid incorporation was observed of the Drosophila germline specific linker histone dBigH1 into the somatic nuclei. NT experiments performed in Xenopus and mammals showed that incorporation of the oocyte specific linker histone variants B4 and H1oo into the donor nuclei is an early event in nuclear reprogramming. B4 binding precedes loading of oocyte RNApol II and expression of a dominant negative B4 form significantly inhibits transcription of many reprogrammed genes. Along the same lines, expression of H1oo in mouse ESCs impairs differentiation although it does not improve iPSC formation. How oocyte specific H1s might contribute to nuclear reprogramming remains not well understood. Oocyte specific H1s are less positively charged than their somatic counterparts and, therefore, their interaction with DNA is weaker and condense chromatin less than somatic H1s, rendering it more accessible to chromatin modifiers, remodelers and transcription factors. In this regard, Xenopus B4 is more mobile than somatic H18 and B4-containing chromatin is more accessible to remodeling factors39. B4 binds pervasively across chromatin of the donor nuclei and, concomitantly, somatic H1s are released, suggesting competition of somatic H1s by the oocyte specific variants. However, this competition does not appear to play an important role in reprogramming since overexpression of somatic H1s does not interfere with B4 binding and subsequent activation of pluripotency genes. Moreover, in mouse fibroblasts, binding of H1oo is detected 10' after NT, while release of somatic H1s occurs later at 30' after NT20. Similarly, somatic H1s replacement can last hours in NT experiments with bovine cells. Finally, the results indicate that, upon incubation in DREX, dBigH1 binds along chromatin without affecting somatic dH1 occupancy. In fact, dH1 occupancy is significantly reduced only at short incubation times when dBigH1 binding is very low (Satovic, 2018).
The results also show that DREX induces changes in the epigenetic landscape of chromatin, which are in agreement with the global epigenetic remodeling of chromatin observed during reprogramming of somatic cells to iPSCs. In particular, increased global H3Ac was observed that is maintained throughout the incubation time course. Increased histone acetylation is observed in fully reprogrammed iPSCs40 and ESC chromatin is hyperacetylated compared to differentiated cells. It was also observed that H3K4me3 levels increased more intensively at promoters of developmentally regulated genes that are silent in S2 cells but highly expressed in early embryogenesis, suggesting their reactivation. Interestingly, pluripotency-related and developmentally regulated genes are known to acquire H3K4me3 at promoters during nuclear reprogramming. Finally, though not statistically significant, global levels of H3K4me3 and the chromatin bound promoter-proximal active RNApol IIoser form tend to increase at short incubation times. In this regard, NT experiments in Xenopus showed loading of oocyte basal transcription factors and RNApol II leading to genome-wide transcriptional reprogramming and selective activation of pluripotency genes. Notably, these results showed that increased histone acetylation induced by DREX does not require binding of dBigH1, suggesting that, at least in part, the epigenetic changes occurring during reprogramming do not depend only on the activities of the oocyte specific H1s (Satovic, 2018).
Incubation in DREX also induces profound changes in chromatin/nuclear organization. On one hand, at short incubation times, DREX induces heterochromatin reorganization since HP1a/H3K9me3 foci disassemble. A decrease in the number of HP1a foci has also been reported during reprogramming to iPSC. In this regard, chromatin of pluripotent cells is largely decondensed and heterochromatin is organized in larger and fewer domains that become smaller, more abundant and hypercondensed as cells differentiate. Interestingly, incubation in DREX did not decrease H3K9me2 occupancy at multiple heterochromatic elements, suggesting that DREX affects condensation but not the actual heterochromatin content of somatic nuclei. Oocyte specific H1s might be one of the factors contributing to heterochromatin decondensation since, in humans, H1oo is required for decondensation of sperm chromatin. At long incubation times, HP1a foci reform and extrude from nuclei. Interestingly, extrusion of heterochromatic sequences was also reported in somatic plant cells undergoing meiosis. Finally, it was also observed that DREX induces disassembly of nuclear lamin, a nuclear envelope component of differentiated cells that is absent in ESCs. Similar results were reported earlier using a Drosophila oocyte cell-free extract. Nuclear lamin disassembly is considered a marker of reprogrammed cells, since it is detected at the nuclear envelope in partial iPSCs, but not in fully reprogrammed iPSCs40. Interestingly, nuclear lamin disassembly strongly correlates with heterochromatin reorganization, which might account for the heterochromatin extrusion observed after long-term exposure to DREX (Satovic, 2018).
In summary, these results show that DREX induces several changes associates with gain of pluripotency, such as binding of the germline specific linker histone dBigH1, epigenetic remodeling, heterochromatin reorganization and nuclear lamin disassembly. However, it is highly unlikely that DREX induces full reprogramming of somatic nuclei. Nevertheless, the use of DREX offers the possibility of applying the powerful genetics techniques developed in Drosophila to the analysis of factors and mechanisms involved in chromatin remodeling during this essential process (Satovic, 2018).
Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. This study shows that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, this study shows that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w(m4h) reporter. It is proposed that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1 (Orkenby, 2023).
The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. This study investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization (Zhang, 2018).
The metabolic and redox state changes during the transition from an arrested oocyte to a totipotent embryo remain uncharacterized. This study applied state-of-the-art, integrated methodologies to dissect these changes in Drosophila. Early embryos were shown to have a more oxidized state than mature oocytes. Specific alterations were identified in reactive cysteines at a proteome-wide scale as a result of this metabolic and developmental transition. Consistent with a requirement for redox change, a role was demonstrated for the ovary-specific thioredoxin Deadhead (DHD). dhd-mutant oocytes are prematurely oxidized and exhibit meiotic defects. Epistatic analyses with redox regulators link dhd function to the distinctive redox-state balance set at the oocyte-to-embryo transition. Crucially, global thiol-redox profiling identified proteins whose cysteines became differentially modified in the absence of DHD. These potential DHD substrates were validated by recovering DHD-interaction partners using multiple approaches. One such target, NO66, is a conserved protein that genetically interacts with DHD, revealing parallel functions. As redox changes also have been observed in mammalian oocytes, a link between developmental control of this cell-cycle transition and regulation by metabolic cues is hypothesized. This link likely operates both by general redox state and by changes in the redox state of specific proteins. The redox proteome defined here is a valuable resource for future investigation of the mechanisms of redox-modulated control at the oocyte-to-embryo transition (Petrova, 2018).
Although life is driven by reduction-oxidation (redox) reactions, remarkably little is known about how metabolic state interfaces with normal development. Reactive oxygen species (ROS) were first described as a byproduct of metabolism and a hallmark of disease and aging. Exciting new research, however, implicated ROS more directly in cell signaling and regulation. In a manner analogous to posttranslational modifications, ROS can alter the oxidation status of cysteine residues and thus affect protein stability, activity, and localization or protein-protein interactions. In this respect, how ROS link to the spatiotemporal regulation of development via downstream targets has not been explored thoroughly (Petrova, 2018).
The redox state in the cell exists in a dynamic balance between ROS production and removal. The source of ROS is primarily oxidative phosphorylation in mitochondria. The antioxidants catalase and superoxide dismutase (SOD) neutralize ROS in a direct enzymatic reaction. Further major ROS scavenger systems are glutathione (GSH), which exists as GSH in its reduced state and as glutathione disulfide (GSSG) in its oxidized state, and thioredoxins. GSH and thioredoxin utilize reducing power provided by oxidative metabolism, tied to redox couples such as NADPH/NADP+, to counteract oxidation via ROS. Under the redox-optimized ROS balance hypothesis, energy metabolism, antioxidants, and redox couples are in equilibrium to allow physiological ROS signaling under varying conditions. Ultimately, the redox balance sets the oxidation level of downstream targets via an interplay of scavenger systems with downstream substrates such as protein and lipids (Petrova, 2018).
A key developmental event is the oocyte-to-embryo transition, when at fertilization the highly specialized oocyte becomes a totipotent embryo. The oocyte-to-embryo transition, occurring in the absence of transcription, relies on posttranscriptional and posttranslational control. In this respect, could redox modulation of protein function bring a highly dynamic, additional level of regulation? Indeed, Dumollard (2008) showed that metabolic activity and thus the redox potential varies during mouse oogenesis and fertilization and is critical for early development. In addition, several aspects of oogenesis and early embryogenesis were blocked if proper redox balance was altered. Although these studies suggest a direct role for ROS, no comprehensive analysis has been carried out, and the downstream targets have not been investigated rigorously (Petrova, 2018).
Intriguingly, Drosophila expresses an oocyte-specific thioredoxin, Deadhead (DHD). DHD is required for early embryogenesis and timely protamine-to-histone exchange in the male pronucleus in fertilized eggs. DHD redox activity is essential for its function. A further exciting observation was that a ubiquitous thioredoxin, Trx-2, did not recognize protamines as substrates. This indicates that DHD has at least one specific target in early development. Because the protamine exchange defect did not fully account for the developmental block in the mutant, more roles and thus additional specific substrates are postulated for the functions DHD likely controls through redox regulation in the oocyte-to-embryo transition in Drosophila (Petrova, 2018).
This study describes an integrated approach to understand the role of redox in oogenesis and early embryogenesis in Drosophila. Proper redox balance is shown to be essential for meiosis completion, fertilization, and early embryogenesis. Use was made of recent technological breakthroughs enabling quantitative examination of the redox Cys-proteome. The data corroborate the emerging view that redox systems selectively maintain a nonequilibrium redox dynamic in distinct sets of the redox Cys-proteome. It is proposed that the developmentally regulated thioredoxin DHD controls a specific set of substrates by interpreting global changes at the level of energy metabolism. These findings provide a paradigm for understanding redox-sensitive targets and pathways in other organisms or systems where cell-state transitions are accompanied by global metabolic remodeling (Petrova, 2018).
Although redox state changes have been described to occur in oogenesis and embryogenesis in diverse systems, no comprehensive analysis of the regulators and targets of redox has been carried out. This work demonstrates that global redox changes occur at the oocyte-to-embryo transition in Drosophila. Redox pair measurements by LC-MS or in vivo H2O2 sensor imaging showed that embryos are more oxidized. The developmentally regulated thioredoxin DHD contributes to these changes so that the GSH/GSSG and NADH/NAD+ ratios and H2O2 levels in oocytes partially depend on its proper function. DHD works in conjunction with other redox proteins to ensure the fidelity of meiosis completion, fertilization, and early embryogenesis. DHD likely exerts its function via specific substrates, and thie study found that DHD interacts with other proteins in addition to the previously described protamines. Importantly, global changes in redox state were linked to corresponding changes in the redox-thiol proteome, and a set of specific DHD-dependent redox targets were documented. One such substrate, NO66, contributes to DHD function during early embryogenesis. This study leads to the hypothesis that metabolic changes occurring in the context of a developmental transition can be utilized to modify the function of specific downstream proteins and protein networks via dedicated redox systems (Petrova, 2018).
These work extends previous studies describing metabolic changes in Drosophila during oogenesis and embryogenesis. The metabolome changes as the Drosophila fertilized oocyte is activated to proceed through embryonic development. Within a very short time frame, 0-1 h after egg-laying, there are metabolic changes that affect, among others, the TCA cycle, amino acid metabolism, the pentose phosphate pathway, and purine metabolism. Although the mechanism by which metabolic changes and redox interact in early embryogenesis in Drosophila is yet to be determined, it is possible that this could occur via modulation of mitochondrial functions. Recently, mitochondria were shown to exist in a quiescent state in Drosophila oocytes and to reactivate in embryos. It will be interesting to investigate in the future how mitochondrial function, calcium signaling, and ROS generation and signaling are coupled at the oocyte-to-embryo transition (Petrova, 2018).
The oocyte-specific thioredoxin DHD is a key developmentally regulated node in the redox control of the oocyte-to-embryo transition in Drosophila. DHD protein levels are regulated, increasing markedly during oocyte maturation and sharply decreasing at egg activation. Interestingly, sperm-specific thioredoxins have been described in mammals as well as in Drosophila. Developmental control of thioredoxins could thus be a broader strategy to modulate cell-state transitions in other systems (Petrova, 2018).
A key question that this study poses is whether DHD exerts its essential roles at the oocyte-to-embryo transition via modulation of global redox state and/or via specific downstream targets. This study found that DHD is partially required for redox state in late oocytes. The effect on the GSH/GSSG ratio could be via thioredoxin's direct contribution in Drosophila to the GSH cycle, as no GSH reductase has been found. Interestingly, genetic analysis of interaction between dhd and trx-2 revealed an overlap of function, such that the proteins could cooperate in setting the proper GSH/GSSG ratio in late oocytes and early embryos. The genetic interactions between dhd and other genes involved in redox control argue for a global role for general redox in the oocyte-to-embryo transition. This conclusion is supported further by the observation that modulation of the redox state balance by the addition of DTT affected progression through meiosis in the in vitro system (Petrova, 2018).
In addition to redox control at a global level, this study showed both by physical interactions and reactive-thiol mass spectroscopy profiling that specific proteins are DHD targets. Along with TrxR1, a well-known DHD partner, NO66 was one of the strongest interactors. NO66 was identified by thiol-redox proteomics analysis as having a cysteine residue (C239) whose reactivity diminished, indicating thiol-oxidation, in dhdJ5/dhdP8-mutant embryos. NO66 is a JmjC domain-containing histone demethylase, shown recently to act as a suppressor of variegation and to localize to the nucleolus in flies. A link to redox control is that JmjC domain-containing proteins have been described as requiring ascorbate, a reducing agent, for optimal activity in vitro. The colocalization between DHD and NO66 in the nucleolus in S2 cells is consistent with a functional relationship. Importantly, the genetic interaction between no66 and dhd further supported this notion. DHD interacts with and/or modulates ribosomal and RNA-binding proteins and thus could control aspects of nucleolar or ribosomal function. Interestingly, the human NO66 has been described as having ribosome oxygenase activity (44). In the future, it will be important to mutate redox-sensitive cysteine residues on potential DHD targets and examine functions in vivo (Petrova, 2018).
Intriguingly, the mitochondrial NADH dehydrogenase ND-75 is among the top changed reactive cysteine-containing proteins in dhd mutants. Although the same residue (C193) is not present in the wild-type reactive Cys-proteome, the conserved cysteine Cys712 changes reactivity between oocytes and embryos. As ND-75, along with other complex I subunits, leaks into the cytoplasm in late-stage oocytes, an exciting possibility is that DHD-dependent global redox changes occur via modulation of mitochondrial metabolism. Furthermore, in an interaction partner analysis, the presence of SOD1, a thioredoxin partner previously described in plants, was noted. These observations suggest that the effect of DHD on global H2O2 as well as on NADH/NAD+ levels could be via modulation of the function of downstream targets. Thus, global and specific redox roles are likely interwoven. Overall, a model is proposed whereby DHD affects specific targets in a developmental context, thus 'translating' the metabolic and redox changes to control important regulators of the oocyte-to-embryo transition (Petrova, 2018).
Thiol-redox proteomic studies have been widely successful in understanding thiol-redox dynamics in various systems but have not been broadly applied in a developmental context. In this regard, model organisms offer the advantage of applying integrated approaches. For example, one study monitored thiol-redox changes as a function of life span in Caenorhabditis elegans. Another showed the dramatic effect of fasting on the Drosophila reactive-thiol proteome. This study demonstrates the use of thiol-redox proteomics to investigate the oocyte-to-embryo transition in Drosophila. In addition to identifying specific targets of DHD, 500 cysteines were found to become differentially reactive at egg activation. This dataset will be a valuable resource for the community. The results provide evidence that a defined set of regulators along with general redox state control the oocyte-to-embryo transition. It is concluded that remodeling of the reactive thiol proteome should be considered a fundamental part of this critical developmental window (Petrova, 2018).
The earliest stages of animal development are controlled by maternally deposited mRNA transcripts and proteins. Once the zygote is able to transcribe its own genome, maternal transcripts are degraded, in a tightly regulated process known as the maternal to zygotic transition (MZT). While this process has been well-studied within model species, there is little knowledge of how the pools of maternal and zygotic transcripts evolve. To characterize the evolutionary dynamics and functional constraints on early embryonic expression, a transcriptomic dataset was created for 14 Drosophila species spanning over 50 million years of evolution, at developmental stages before and after the MZT, and the results were compared with a previously published Aedes aegypti developmental time course. Deep conservation was found over 250 million years of a core set of genes transcribed only by the zygote. This select group is highly enriched in transcription factors that play critical roles in early development. However, a surprisingly high level of change was also identified in the transcripts represented at both stages over the phylogeny. While mRNA levels of genes with maternally deposited transcripts are more highly conserved than zygotic genes, those maternal transcripts that are completely degraded at the MZT vary dramatically between species. It was also shown that hundreds of genes have different isoform usage between the maternal and zygotic genomes. This work suggests that maternal transcript deposition and early zygotic transcription are remarkably dynamic over evolutionary time, despite the widespread conservation of early developmental processes (Atallah, 2018).
During development, cell-generated forces induce tissue-scale deformations to shape the organism. The pattern and extent of these deformations depend not solely on the temporal and spatial profile of the generated force fields but also on the mechanical properties of the tissues that the forces act on. It is thus conceivable that, much like the cell-generated forces, the mechanical properties of tissues are modulated during development in order to drive morphogenesis toward specific developmental endpoints. Although many approaches have recently emerged to assess effective mechanical parameters of tissues, they could not quantitatively relate spatially localized force induction to tissue-scale deformations in vivo. This study presents a method that overcomes this limitation. The approach is based on the application of controlled forces on a single microparticle embedded in an individual cell of an embryo. Combining measurements of bead displacement with the analysis of induced deformation fields in a continuum mechanics framework, material properties were quantified of the tissue, and their changes over time were followed. In particular, a rapid change was uncovered in tissue response occurring during Drosophila cellularization, resulting from a softening of the blastoderm and an increase of external friction. The microtubule cytoskeleton is a major contributor to epithelial mechanics at this stage. Developmentally controlled modulations was identified in perivitelline spacing that can account for the changes in friction. Overall, this method allows for the measurement of key mechanical parameters governing tissue-scale deformations and flows occurring during morphogenesis (D'Angelo, 2019).
Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) has been thought to occur in two phases, starting with a minor wave, in which a small number of genes become expressed, and progressing to the major wave, in which many more genes are activated. However, technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. This study developed an approach to isolate transcribed mRNAs and applied it over the course of Drosophila early genome activation. The results increase by 10-fold the genes reported to be activated during what has been thought of as the minor wave and shows that early genome activation is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes transcribed in the early and middle part of ZGA are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts (Kwasnieski, 2019).
The early embryos of many animals including flies, fish, and frogs have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. This study examine the effects of under and overexpression of maternal histones in Drosophila embryogenesis. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation, and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda and lack of SIN3A and associated histone deacetylases. These findings uncover a critical regulatory role for histone concentrations in ZGA of Drosophila (Wilky, 2019).
To understand the effects of histone concentration on the MBT maternally supplied histones were reduced by downregulating the gene encoding a crucial histone regulator, Stem-Loop Binding Protein (Slbp) via maternally driven RNAi. Under these conditions, histone H2B was reduced by ~50% and H3 by ~60% at the MBT. Approximately 50% of embryos laid by Slbp RNAi mothers (henceforth Slbp embryos) that form a successful blastoderm do not undergo the final division and attempt gastrulation in NC13. Another ~30% exhibit an intermediate phenotype of partial arrest, with only part of the embryo entering NC14. A minority of Slbp embryos begin gastrulation with all nuclei in NC14. NC12 duration was predictive of NC13 arrest, with NC12 being an average of ~5min longer in Slbp embryos that went on to arrest compared with those that did not arrest (Wilky, 2019).
Cellularization was first detected in wild-type (WT) embryos ~20 min into NC14. Partially arrested Slbp embryos also began cellularization ~20 min into NC14, with nuclei that arrested in NC13 waiting until the remainder of the embryo had entered NC14 to cellularize. Fully arrested embryos began cellularization ~20min into NC13, initiating cellularization one cycle early and ~20min earlier in overall developmental time than WT. Despite their reduced cell number, these embryos form mitotic domains and gastrulate without obvious defects, however they die before hatching (Wilky, 2019).
To examine the effects of increased histone concentration on developmental timing cell cycle progression was monitored in embryos from abnormal oocyte (abo) mutant mothers (henceforth abo embryos). abo is a histone locus-specific transcription factor, the knockdown of which increases the production of replication-coupled histones, particularly H2A and H2B (Berloco, 2001). abo increased H2B by ~90%, whereas total (combined replication-coupled and replication-independent) H3 was not affected in NC14 embryos. Approximately 60% of abo embryos displayed fertilization defects or catastrophic early nuclear divisions. Of abo embryos that formed a functioning blastoderm, ~6% underwent a complete extra nuclear division before gastrulating in NC15, whereas ~4% underwent a partial extra nuclear division. Embryos from abo mothers that completed total extra divisions had faster NC14s in which they did not cellularize and spent 40-60 min in NC15 before gastrulating. This suggests an alteration of the normal transcription-dependent developmental program. In some cases, the cell cycle program and transcriptional program may be decoupled, evidenced by the fact that some abo embryos attempted to gastrulate while still in the process of division. abo embryos that underwent extra divisions exhibited a range of gastrulation defects including expanded mitotic domains and ectopic furrow formation (Wilky, 2019).
Since alterations in histone levels can both decrease and increase the number of divisions before cell cycle slowing, it was reasoned that histone levels might affect activation of checkpoint kinase 1 (Chk1, also known as grp), which is required for cell cycle slowing at the MBT. To test this, a fluorescent biosensor of Chk1 activity was crosses into the Slbp background. Even in Slbp embryos that did not undergo early gastrulation, Chk1 activity was higher than in WT, consistent with the lengthened cell cycle. This result indicates that the observed cell cycle phenotypes in the histone-manipulated embryos are likely mediated through changes in Chk1 activity (Wilky, 2019).
As cellularization and gastrulation require zygotic transcription, it was suspected that embryos with altered development likely have altered gene expression. Single-embryo RNA-seq was performed on staged Slbp embryos that remained in NC13 for more than 30 min. These were compared with either NC-matched (NC13) or time-matched (NC14) WT embryos. To control for maternal effects of Slbp RNAi, pre-blastoderm stage WT and Slbp embryos were compared. The Slbp embryos underwent ZGA one NC earlier than WT. ~5000 genes were identified that were differentially expressed between Slbp and WT NC13, with ~60% being upregulated. The upregulated genes have largely previously been identified as new zygotic transcripts, including cell cycle regulators such as fruhstart (frs, also known as Z600) and signaling molecules such as four-jointed (fj), whereas the downregulated genes are enriched for maternally degraded transcripts. This is thought to represent a coherent change in ZGA timing instead of global transcription dysregulation, as 98% of the genes that are overexpressed in Slbp are expressed before the end of NC14 in the control or previously published datasets. Indeed, the transcriptomes of histone-depleted embryos that stopped in NC13 are more similar to WT NC14 than WT NC13, which suggests a role for cell cycle elongation in ZGA. Nonetheless, ~1500 genes are differentially expressed between Slbp NC13 and WT NC14 without accounting for differences in ploidy. Of these, the majority of the ~1000 overexpressed genes are again associated with zygotic transcription, and downregulated genes associated with maternal products. Thus, ZGA is even further accelerated in the histone knockdown than can be explained by purely time alone (Wilky, 2019).
As ZGA is accelerated by histone depletion, it was asked whether ZGA would be delayed in the histone overexpression mutant. RNA-seq was performed on pools of abo and WT embryos collected 15-30 min into NC14. >1000 genes were identified that were differentially expressed between abo and WT, with approximately equal numbers of genes up- and down-regulated. As expected, the downregulated genes in abo were enriched for previously identified zygotically expressed transcripts, and upregulated transcripts were enriched for maternally deposited genes. Thus, histone overexpression delays the onset of ZGA (Wilky, 2019).
Zygotic genes, the transcription of which is upregulated by histone depletion and downregulated by histone overexpression, contain many important developmental and cell cycle regulators including: frs, hairy (h), fushi tarazu (ftz) and odd-skipped (odd). Conversely, the maternally degraded transcripts that are destabilized by histone depletion and stabilized by histone overexpression include several cell cycle regulators such as Cyclin B (CycB), string (stg, also known as Cdc25string) and Myt1. Therefore, histone concentration can modulate the expression and stability of specific cell cycle regulators, which may contribute to the onset of MBT (Wilky, 2019).
Since histone concentration has previously been implicated in sensing the nuclear-cytoplasmic (N/C) ratio (Amodeo, 2015), this study compared the genes that are changed in both the histone under- and overexpression embryos with those that had previously been found to be dependent on either the N/C ratio or developmental time (Lu, 2009). Both previously identified N/C ratio-dependent and time-dependent genes (Lu, 2009) followed the same general trends as the total zygotic gene sets, indicating that histone availability cannot explain these previous classifications (Wilky, 2019).
Next, attempts were made to disentangle the effects of cell cycle length from transcription in the histone overexpression mutant. Single-embryo time-course RNA-seq was performed on abo and WT embryos collected 3 min before mitosis of NC10-NC13 and 3 min into NC14. In addition, unfertilized embryos (henceforth NC0) of both genotypes were collected to control for differences in maternal contribution. Even with a stringent selection process that accounted for cell cycle time and embryo health, a small set of robustly upregulated (179) and downregulated (260) genes was detected across NC10-NC14. Of the newly transcribed genes, 111 genes were detected with delayed transcription, including frs and only 37 that are upregulated. These results were confirmed using qPCR. When compared with previous datasets, zygotic genes tend to be underexpressed, as was the case for the pooled abo dataset; however, the majority of these enrichments are not statistically significant. Nonetheless the majority of these underexpressed genes are expressed during NC14 in WT. This geneset, in combination with the time-matched Slbp comparison, enables further examination of the chromatin features that underlie histone sensitivity for transcription independent of cell cycle changes (Wilky, 2019).
To identify chromatin features associated with histone sensitivity, the presence was compared of 143 modENCODE chromatin signals near the transcriptional start site (TSS±500 bp) of genes whose expression was altered by changes in histone concentration independent of cell cycle time. A clear pattern was found of unique chromatin features for the histone-sensitive genes, compared with all newly transcribed genes, that was highly similar between the histone over- and underexpression experiments. The pioneer transcription factor Zld, known to be important for nucleosome eviction during ZGA, was enriched in the promoters of histone-sensitive genes. Insulator proteins such as BEAF-32 and CP190 were depleted in histone-sensitive genes. Promoters of histone-sensitive genes also show a strong reduction for SIN3A, a transcriptional repressor associated with cell cycle regulation. SIN3A is known to recruit HDACs to TSSs, and almost all HDACs also show significant de-enrichment at the TSSs of histone-sensitive genes. Taken together, these marks make up a unique chromatin signature that may sensitize a locus to changes in histone concentration, as is likely for pioneer factors such as Zld. Other aspects of this signature may indicate that these genes are subsequently subject to later developmental regulation, as indicated by H3K4me3 and H3K27me3 (Wilky, 2019).
This study has demonstrated that histone concentration regulates the timing of the MBT in Drosophila, resulting in both early gastrulation and extra pre-MBT divisions from histone reduction and increase, respectively. Histone concentration also regulates ZGA. Thousands of genes are prematurely transcribed in histone-depleted embryos and hundreds of genes are delayed in histone-overexpressing embryos. The majority of these genes appear to be downstream of changes in cell cycle duration, suggesting a model in which histones directly regulate cell cycle progression. In other cell types, histone loss halts the cell cycle via accumulation of DNA damage and stalled replication forks. In the early embryo, changes in histone availability may similarly create replication stress to directly or indirectly activate Chk1 as this study has shown. In turn, Chk1 would inhibit Stg and/or Twine to slow the cell cycle. This mechanism is supported by previous observations that loss of zygotic histones causes the downregulation of Stg in the first post-MBT cell cycle. In this case, the observed transcriptional changes would be independent or downstream of the altered cell cycle (Wilky, 2019).
Alternatively, direct changes in transcription downstream of histone availability may feed into the cell cycle. In bulk, histone-sensitive transcripts might underlie the replication stress that has been previously proposed to slow the cell cycle at the MBT. Consistent with this, the cell cycle lengthening and partial arrest phenotypes observed in mutant RNA Pol II embryos occur at a similar frequency to those observed as the result of histone depletion. Another possibility is that specific histone-sensitive transcripts are responsible for cell cycle elongation. One promising candidate for a histone-sensitive cell cycle regulator is the N/C ratio-sensitive CDK inhibitor frs, as zygotic transcription of frs plays a crucial role in stopping the cell cycle at the MBT. In contrast, tribbles, an N/C ratio-dependent inhibitor of Twine that has also been implicated in cell cycle slowing, does not show a consistent response between histone perturbations. In this previously proposed model, maternal histone stores may compete with pioneer transcription factors to set the timing of transcription initiation. Indeed, the central Drosophila pioneer transcription factor Zld is enriched at the promoters of histone-sensitive genes. Moreover, this study has identified a broader set of chromatin features that may sensitize individual loci to changes in histone concentrations. These include less obvious candidates for global early transcriptional regulators, such as SIN3A, HDACs and class I insulator proteins that may protect transcripts from changes in histone concentrations. This work highlights the importance of histone concentration in regulating the timing of MBT and provides evidence that promoters of histone-sensitive genes possess a unique chromatin signature. However, future studies will be required to isolate the specific downstream effectors that respond to changes in histone concentrations in the early embryo (Wilky, 2019).
During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, this study demonstrated that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. It is inferred that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization (Ibarra-Morales, 2021).
The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. This study shows that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. A substantial decrease was observed in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. This study has identified a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process (Torres-Zelada, 2022).
Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. This study explored the novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, transcriptomes of embryos zygotically compromised for either clamp or zelda were examined, and it was found that transcript levels of many zygotically activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, it is proposed that CLAMP acts in concert with Zelda to regulate early zygotic transcription (Colonnetta, 2021).
During the first 2 hours of Drosophila development, precisely orchestrated nuclear cleavages, cytoskeletal rearrangements, and directed membrane growth lead to the formation of an epithelial sheet around the yolk. The newly formed epithelium remains relatively quiescent during the next hour as it is patterned by maternal inductive signals and zygotic gene products. It was discovered that this mechanically quiet period is disrupted in embryos with high levels of dNTPs, which have been recently shown to cause abnormally fast nuclear cleavages and interfere with zygotic transcription. High levels of dNTPs are associated with robust onset of oscillatory two-dimensional flows during the third hour of development. Tissue cartography, particle image velocimetry, and dimensionality reduction techniques reveal that these oscillatory flows are low dimensional and are characterized by the presence of spiral vortices. It is speculated that these aberrant flows emerge through an instability triggered by deregulated mechanical coupling between the nascent epithelium and three-dimensional yolk. These results highlight an unexplored connection between a core metabolic process and large-scale mechanics in a rapidly developing embryo (Dutta, 2020).
The gene products that drive early development are critical for setting up developmental trajectories in all animals. The earliest stages of development are fueled by maternally provided mRNAs until the zygote can take over transcription of its own genome. In early development, both maternally deposited and zygotically transcribed gene products have been well characterized in model systems. Previously, it was demonstrated that across the genus Drosophila, maternal and zygotic mRNAs are largely conserved but also showed a surprising amount of change across species, with more differences evolving at the zygotic stage than the maternal stage. This study used comparative methods to elucidate the regulatory mechanisms underlying maternal deposition and zygotic transcription across species. Through motif analysis, considerable conservation of regulatory mechanisms was discovered associated with maternal transcription, as compared to zygotic transcription. It was also found that the regulatory mechanisms active in the maternal and zygotic genomes are quite different. For maternally deposited genes, many signals were uncovered that are consistent with transcriptional regulation at the level of chromatin state through factors enriched in the ovary, rather than precisely controlled gene-specific factors. For genes expressed only by the zygotic genome, evidence was found for previously identified regulators such as Zelda and GAGA-factor, with multiple analyses pointing toward gene-specific regulation. The observed mechanisms of regulation are consistent with what is known about regulation in these two genomes: during oogenesis, the maternal genome is optimized to quickly produce a large volume of transcripts to provide to the oocyte; after zygotic genome activation, mechanisms are employed to activate transcription of specific genes in a spatiotemporally precise manner. Thus the genetic architecture of the maternal and zygotic genomes, and the specific requirements for the transcripts present at each stage of embryogenesis, determine the regulatory mechanisms responsible for transcripts present at these stages.
At the oocyte-to-embryo transition the highly differentiated oocyte arrested in meiosis becomes a totipotent embryo capable of embryogenesis. Oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest and the trigger for the oocyte-to-embryo transition) serve as prerequisites for this transition, both events being controlled posttranscriptionally. Recently, a comprehensive list of proteins was obtained whose levels are developmentally regulated during these events via a high-throughput quantitative proteomic analysis of Drosophila melanogaster oocyte maturation and egg activation. A targeted screen was conducted for potential novel regulators of the oocyte-to-embryo transition, selecting 53 candidates from these proteins. The function of each candidate gene was reduced using transposable element insertion alleles and RNAi, and defects in oocyte maturation or early embryogenesis were screened. Deletion of the aquaporin gene CG7777 did not affect female fertility. However, CG5003 and nebu (CG10960) were identified as new regulators of the transition from oocyte to embryo. Mutations in CG5003, which encodes an F-box protein associated with SCF-proteasome degradation function, cause a decrease in female fertility and early embryonic arrest. Mutations in nebu, encoding a putative glucose transporter, result in defects during the early embryonic divisions, as well as a developmental delay and arrest. nebu mutants also exhibit a defect in glycogen accumulation during late oogenesis. These findings highlight potential previously unknown roles for the ubiquitin protein degradation pathway and sugar transport across membranes during this time, and paint a broader picture of the underlying requirements of the oocyte-to-embryo transition (Aviles-Pagan, 2020).
How gene expression can evolve depends on the mechanisms driving gene expression. Gene expression is controlled in different ways in different developmental stages; this study asked whether different developmental stages show different patterns of regulatory evolution. To explore the mode of regulatory evolution, this study used the early stages of embryonic development controlled by two different genomes, that of the mother and that of the zygote. During embryogenesis in all animals, initial developmental processes are driven entirely by maternally provided gene products deposited into the oocyte. The zygotic genome is activated later, when developmental control is handed off from maternal gene products to the zygote during the maternal-to-zygotic transition. Using hybrid crosses between sister species of Drosophila (D. simulans, D. sechellia, and D. mauritiana) and transcriptomics, this study finds that the regulation of maternal transcript deposition and zygotic transcription evolve through different mechanisms. Patterns of transcript level inheritance in hybrids, relative to parental species, were found to differ between maternal and zygotic transcripts, and maternal transcript levels are more likely to be conserved. Changes in transcript levels occur predominantly through differences in trans regulation for maternal genes, while changes in zygotic transcription occur through a combination of both cis and trans regulatory changes. Differences in the underlying regulatory landscape in the mother and the zygote are likely the primary determinants for how maternal and zygotic transcripts evolve (Cartwright, 2020).
In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. The maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT (Cao, 2020).
This study has shown that, in Drosophila, an extremely small subset of its maternal proteome is cleared during the MZT. This contrasts with the massive degradation of the maternal mRNA transcriptome that occurs during the MZT of all animals. Previous studies in other animals have suggested that the maternal proteome may behave very differently from the maternal transcriptome during the MZT. For example, in C. elegans, a quarter of the transcriptome is downregulated, whereas only 5% of the proteome shows a similar decrease. In frog embryos, there is also a discordance between the temporal patterns of protein and mRNA (Cao, 2020).
The set of proteins cleared during the Drosophila MZT is enriched for RNP granule components. This is consistent with the importance of post-transcriptional processes during the first ('maternal') phase of the MZT and the possible need to downregulate these processes upon ZGA and the switch to zygotic control of development. By focusing on a subset of these RNP components, which function as post-transcriptional repressors, this study has uncovered precise temporal control of their clearance by two distinct E3 ubiquitin ligase complexes: the SCF E3 ligase governs the degradation of SMG, whereas the CTLH E3 ligase is responsible for the degradation of Cup, TRAL, and ME31B. Intriguingly, SMG is degraded later during the MZT compared with its co-repressors Cup, TRAL, and ME31B. This study also showed that clearance of SMG is essential for appropriate levels of re-expression of a subset of its targets during ZGA. The results raise questions about how temporal specificity of protein degradation is regulated, as well as why at least two temporally distinct mechanisms of protein degradation exist during the MZT (Cao, 2020).
Expression data support the hypothesis that timing of E3 ligase function might, at least in part, be determined by the timing of expression of one or more of their component subunits, notably Muskelin for CTLH and CG14317 for SCF. During the Drosophila MZT, most components of the CTLH complex display constant expression levels, but Muskelin protein is degraded with a similar profile to its target repressors. Mammalian Muskelin has been shown to be auto-ubiquitinated and targeted for degradation. Detection of a ubiquitinated peptide in Muskelin supports the possibility that the Drosophila CTLH complex may be negatively autoregulated through its Muskelin subunit during the MZT. In contrast, activation of CTLH function at the beginning of the MZT may not depend on changes in complex composition: previous studies have shown that going from stage 14 oocytes to activated eggs or early (0-1 h) embryos, there are no significant changes in either the levels of CTLH subunit proteins (including Muskelin) or the ribosome association of their cognate transcripts. Thus, it is speculated that post-translational modification of one or more CTLH subunits may activate CTLH function (Cao, 2020).
Modification of substrates may also play a role: the degradation of Cup, TRAL, and ME31B depends on the PNG kinase, which itself has temporally restricted activity coinciding with degradation of these repressors. PNG-dependent phosphorylation of Cup, TRAL, and ME31B may make them ubiquitination substrates. Furthermore, evidence suggests that temporal regulation of the E2 ubiquitin-conjugating enzyme, UBC-E2H, at this stage depends on the PNG kinase and may also contribute to the timing of ubiquitin ligase complex function during the MZT. Concomitant PNG-dependent activation of the CTLH complex, its cognate E2, and its substrates, coupled with subsequent self-inactivation of the complex through Muskelin degradation, would provide a precise time window for CTLH function and, therefore, for degradation of Cup, TRAL, and ME31B early in the MZT (Cao, 2020).
In contrast with these three co-repressors, degradation of SMG occurs near the end of the MZT and depends on zygotic gene expression. Although the levels of most SCF complex subunits are constant during the MZT, the F-box protein CG14317 displays a unique expression pattern: CG14317 protein and mRNA are absent at the beginning of the MZT, are zygotically synthesized, peak in NC14 embryos, and sharply decline shortly thereafter. Thus, CG14317 expression coincides with the timing of SMG protein degradation and, coupled with the zygotic nature of its accumulation, makes it a strong candidate to be a timer for SCF function. The fact that knockdown of SLMB stabilizes SMG protein suggests that both F-box proteins may be necessary for SMG degradation, with CG14317 serving as the timer. At present there are no forward or reverse genetic reagents available to test this hypothesis. Additionally, the function of SLMB in directing SMG-protein ubiquitination may itself be temporally restricted. Both Drosophila SLMB and its mammalian homolog are known to bind phosphorylated motifs. Phosphorylated residues have been detected in SMG in the embryo, including residues within its C terminus; one of these, S967, resides close to a ubiquitinated lysine, K965. In summary, despite the stable expression of SLMB during the MZT, temporal regulation of phosphorylation of its target proteins, including SMG, through yet uncharacterized mechanisms, may also contribute to temporal control of SMG protein degradation (Cao, 2020).
Because Cup, TRAL, and ME31B are known to function as co-repressors in a complex with SMG, why are the timing of degradation of Cup-TRAL-ME31B and SMG differentially regulated? Although the SMG-Cup-TRAL-ME31B-mRNA complex has been characterized to be extremely stable in vitro, it would be disrupted in vivo by the degradation of Cup, TRAL, and ME31B (or by the degradation of nos and other SMG-target mRNAs). SMG directs translational repression both through AGO1 and through Cup-TRAL-ME31B, as well as transcript degradation through recruitment of the CCR4-NOT deadenylase. CTLH-driven degradation of Cup, TRAL, and ME31B would abrogate SMG-Cup-TRAL-ME31B-dependent translational repression, but not AGO1-dependent repression, because AGO1 levels increase during the MZT. However, the relative contributions of AGO1 versus Cup-TRAL-ME31B to translational repression by SMG are unknown. That said, the CCR4-NOT deadenylase is present both during and after the MZT (Temme et al., 2004); thus, SMG-dependent transcript degradation would occur both before and after clearance of Cup, TRAL, and ME31B. 12% of SMG-associated transcripts are degraded, but not repressed, by SMG. Perhaps this subset is bound and degraded by SMG late in the MZT, after the drop in Cup, TRAL, and ME31B levels (Cao, 2020).
Another possible role for clearance of ME31B and TRAL derives from studies in budding yeast, where it has been shown that their orthologs, respectively, Dhh1p and Scd6p, have a potent inhibitory effect on 'general' translation. If this is also true in Drosophila, then degradation of ME31B and TRAL, which are present at exceedingly high concentrations in embryos, might also serve to permit high-level translation during the second phase of the MZT (Cao, 2020).
Previous work has shown that SMG has both direct and indirect roles in the MZT. SMG's direct role is to bind to a large number of maternal mRNA species and target them for repression and/or degradation. Two indirect effects have been shown in smg mutants. First, if maternal transcripts fail to be degraded and/or repressed, ZGA fails or is significantly delayed, likely because mRNAs encoding transcriptional repressors persist. Second, because zygotically synthesized microRNAs direct a second wave of maternal mRNA decay during the late MZT, in smg mutants, failure to produce those microRNAs results in failure to eliminate a second set of maternal transcripts late in the MZT (Cao, 2020).
This study has uncovered a role for rapid clearance of the SMG protein itself late in the MZT: to permit normal levels of zygotic re-expression of a subset of it targets. Notably, stabilized SMG (SMG767Δ999) rescues both clearance of its maternal targets and ZGA, excluding the possibility that lower-than-normal levels of re-expressed targets are a result of defective SMG function upon deletion of its C terminus. Indeed, in control experiments, SMG's exclusively maternal targets actually dropped to lower levels than normal, likely because SMG767Δ999 continues to direct their decay beyond when SMG normally disappears from embryos. Furthermore, in another control, strictly zygotic transcripts that lack SMG binding sites were expressed at higher levels in SMG767Δ999-rescued mutants than in full-length SMG-rescued mutants. This result is consistent with the hypothesis that clearance of transcriptional repressors by SMG permits ZGA; persistent SMG would clear these repressors to lower levels than normal, hence resulting in higher zygotic expression. The higher-than-normal expression of zygotic transcripts that lack SMG binding sites makes the lower-than-normal levels of SMG's zygotically re-expressed target transcripts by SMG767Δ999 even more striking. Together, these data support a model in which the timing of both SMG synthesis and clearance are important for orderly progression of the MZT (Cao, 2020).
The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, previous work found that three maternally deposited Drosophila RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. This study shows that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme (FlyBase: Ubiquitin conjugating enzyme E2H), and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT (Zavortink, 2020).
Proper embryogenesis is critical for animal development. Many of the earliest events occur prior to the onset of zygotic transcription, and they are instead directed by maternally deposited proteins and messenger RNAs (mRNAs). During the maternal-to-zygotic transition (MZT), genetic control of developmental events changes from these maternally loaded gene products to newly made zygotic ones. Thus, the MZT requires both the activation of zygotic transcription and clearance of maternal transcripts. Failure to mediate either of these processes is lethal for the embryo (Zavortink, 2020).
In contrast to understanding of the transcriptome during the MZT, much less is known about changes in the proteome. Despite the fact that the maternal dowry of proteins plays key roles in embryogenesis, there are only a handful of examples of cleared maternal proteins. Recently, three RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) were found to be rapidly degraded during the MZT in Drosophila melanogaster, at a time point coinciding with the major wave of zygotic transcription. ME31B, TRAL, and Cup form a complex that blocks translation initiation. All three proteins are required for oogenesis, and they appear to bind and repress thousands of deposited maternal mRNAs. The degradation of ME31B, TRAL, and Cup coincides with many of the hallmarks of the MZT, but explorations into this issue have been hindered by a lack of understanding of how their destruction is controlled (Zavortink, 2020).
An intriguing observation has been made that genetically linked the clearance of ME31B, TRAL, and Cup, to the Pan Gu (PNG) kinase (Wang, 2017). Composed of three subunits (PNG, Giant Nuclei [GNU], and Plutonium [PLU]), the PNG kinase is central to the oocyte-to-egg transition and mediates key aspects of embryogenesis, including resumption of the cell cycle, zygotic transcription, and maternal mRNA clearance. Unlike many animals, the oocyte-to-egg transition in Drosophila does not require fertilization but is instead triggered by egg activation. The PNG kinase is activated by mechanical stress as the oocyte passes through the oviduct, and then phosphorylation and degradation of the GNU subunit quickly inactivates the kinase, restricting its activity to the first half hour after egg activation (Hara, 2017). One way that PNG mediates the oocyte-to-embryo transition is by rewiring post-transcriptional gene regulation. Possibly by phosphorylating key RNA-binding proteins such as Pumilio, PNG activity leads to changes in the poly(A)-tail length and translation of thousands of transcripts during egg activation (Hara, 2018). Importantly, two targets induced by PNG activity are the pioneer transcription factor Zelda, which is responsible for initial zygotic transcription, and the RNA-binding protein Smaug, which is responsible for clearance of many maternal transcripts. The PNG kinase also phosphorylates ME31B, Cup, and TRAL (Hara, 2018), but it is unclear what effect phosphorylation has on these proteins. One possibility has been that PNG phosphorylation could lead to the degradation of ME31B, TRAL, and Cup, but this model has been thus far unexplored (Zavortink, 2020).
The ubiquitin-proteasome system is a major protein degradation pathway. A series of ubiquitin activating enzymes, conjugating enzymes, and ligases (E1, E2, and E3, respectively) lead to the post-translational addition of a polyubiquitin chain on a target protein, which then serves as a molecular beacon for degradation by the proteasome. E3 ligases are typically thought to recognize target proteins, while E2 conjugating enzymes provide the activated ubiquitin and in turn recognize the E3 ligase. There are hundreds of different E3 ligases and 29 annotated E2 conjugating enzymes in Drosophila, but most of the client substrates are unknown, and few have been implicated in the MZT (Zavortink, 2020).
Given the key roles of ME31B, Cup, and TRAL in oogenesis and embryogenesis, it was of interest to understand the mechanisms controlling their degradation. In particular, this study sought to answer how PNG activity at egg activation leads to the degradation of these three RNA-binding proteins several hours later, and how their degradation is coordinated with other elements of the MZT, including zygotic transcription and maternal mRNA clearance. To answer these questions, a selective RNAi screen was performed in Drosophila, and the E2 conjugating enzyme was identified as UBC-E2H/Marie Kondo (Kdo) and the E3 ligase as the CTLH complex. Interestingly, structural models based on the S. cerevisiae complex (Qiao, 2020) suggest that the Drosophila version is organized differently than its orthologous complexes. The CTLH complex recognized and bound ME31B and Cup even in the absence of PNG activity, strongly suggesting that phosphorylation is not required for the destruction of these proteins. In contrast, Kdo mRNA is translationally upregulated by more than 20-fold upon egg activation in a PNG-dependent manner. Thus, egg activation through PNG mediates translation upregulation of Kdo and so leads to ME31B, Cup, and TRAL destruction (Zavortink, 2020).
Kdo is conserved from yeast to humans and is known to work through the CTLH E3 ligase, a multicomponent complex. (Note that the S. cerevisiae complex is called the Gid complex.) Using BLAST for the human CTLH components, it was easy to identify putative D. melanogaster homologs: RanBPM (homologous to Hs RanBP9), Muskelin, CG3295 (homologous to Hs RMND5A/GID2), CG7611 (homologous to Hs WDR26), CG6617 (homologous to Hs TWA1/GID8), and CG31357 (homologous to Hs MAEA). Putative homologs for Hs GID5/ARMC8 or Hs GID4 were not found. Notably, none of these genes were annotated as putative E3 components in FlyBase, and thus none were included in an original screen (Zavortink, 2020).
ME31B, Cup, and TRAL are RNA-binding proteins that are degraded during the MZT. Despite occurring several hours after egg laying, degradation of these proteins is triggered by egg activation through the activity of the PNG kinase and appears to be mediated by the ubiquitin-proteasome system. Through a medium-scale RNAi screen, the E2 conjugating enzyme Kdo was identified as being required for the clearance of ME31B, TRAL, and Cup. Kdo is conserved from yeast to humans and, as in those systems, appears to work with the CTLH complex, which acts as the E3 ligase. Components of the CTLH complex physically interact with ME31B and Cup, and the CTLH complex is also required for the degradation of ME31B, TRAL, and Cup during early embryogenesis. Structure-based homology suggests that, despite its conservation from yeast to humans, the Drosophila CTLH complex has an unusual architecture, and it remains unclear how it recognizes its substrate. The association of CTLH with ME31B occurs in the absence of PNG activity, suggesting that, although ME31B (as well as TRAL and Cup) are phosphorylated by the kinase, phosphorylation may not be required for their destruction. Instead, translation of Kdo appears to be suppressed during oogenesis by its short poly(A) tail length and binding of ME31B. Its translation is dramatically upregulated at the oocyte-to-embryo transition, in a process that depends on PNG activity. Together, these data suggest a model that egg activation via the PNG kinase leads to translational activation and production of Kdo, which then allows the CTLH complex to ubiquitinate ME31B, TRAL, and Cup and ultimately leads to their destruction. Interestingly, based on RNA-seq data from FlyBase, Muskelin shows exquisite tissue-specificity and is only strongly expressed in the ovaries. This observation, together with the translational control of Kdo, may partly explain how ME31B, a ubiquitous protein, is specifically destabilized in the early embryo (Zavortink, 2020).
Although the CTLH complex is conserved, it has not yet been studied in Drosophila. The data point to this complex being composed of multiple components (Muskelin, RanBPM, Houki, Souji, and Katazuke), as in other organisms. However, due to a lack of available reagents, the stoichiometry of these components is unknown, and it remains possible that there are additional, Drosophila-specific components. Nonetheless, so far, the CTLH complex in Drosophila appears different than the human and yeast complexes. Although Gid7 and WDR26 are important in the yeast and human versions, respectively, and a Drosophila ortholog (CG7611) was identified, no evidence was found of its association with ME31B or requirement for ME31B degradation; the role of CG7611 in the Drosophila CTLH complex warrants further investigation. Orthologs of Gid4 and Gid5, which are critical for substrate recognition in S. cerevisiae, were not found. Intriguingly, the residues and domains important for the Gid1-Gid4 and Gid8-Gid5 interactions in budding yeast appear absent to be in RanBPM and Hou, raising the fundamental question of how the Drosophila CTLH complex recognizes and positions its substrate proteins. Answering this question will require future investigation and may shed light on other proteins targeted by the Drosophila CTLH complex and the extent to which ME31B, a ubiquitously expressed protein, is targeted outside of the MZT (Zavortink, 2020).
One unexpected result is the role of PNG in mediating the destruction of ME31B, TRAL, and Cup. PNG phosphorylates all three proteins, and so the initial hypothesis was that this modification also stimulated their destruction. However, contrary to expectations, ME31B and Cup interacted with the CTLH complex even in png50 embryos, demonstrating that phosphorylation by PNG was not required for binding of ME31B and Cup by the E3 ligase. An unresolved question, then, is how PNG phosphorylation affects the activities of ME31B, TRAL, and Cup. Intriguing observations from the Orr-Weaver lab suggest that the modification can impact the ability of these proteins to repress gene expression (Hara, 2018). It is tempting to speculate that phosphorylation may then contribute to the MZT by modulating the activities of ME31B, TRAL, and Cup, rather than their stability (Zavortink, 2020).
The link between PNG and the destruction of ME31B, TRAL, and Cup instead appears to be mediated through the translational upregulation of Kdo. Although PNG may contribute through other, as-yet undiscovered, mechanisms as well (such as phosphorylating unknown CTLH adaptor proteins), this link is sufficient to explain the PNG requirement for ME31B degradation: in the absence of Kdo, ME31B is stable during the MZT, and in the absence of PNG, Kdo is not detectably expressed. An important question for the future will be to understand what elements in the Kdo mRNA are responsible for its translational repression during oogenesis. One hint may be that the 3'UTR of Kdo contains several putative Pumilio-binding sites, and translation of Kdo is upregulated in ovaries where Pumilio has been knocked down. Pumilio is also a target of PNG (Hara, 2018), and so a possible model is that translational repressors, such as Pumilio, are phosphorylated and inactivated at egg activation, leading to the production of Kdo (Zavortink, 2020).
PNG also mediates the translational upregulation of key MZT effectors: Zelda, the pioneer transcription factor, and Smaug, an RNA-binding protein that targets nearly two-thirds of the maternal transcriptome for degradation. Together with the current results, a picture is emerging that egg activation stimulates the production of multiple key factors that are important for clearing the maternal RNA and protein dowry and for producing zygotic gene products (Zavortink, 2020).
Although the MZT has typically been considered from the perspective of RNA, a role for maternal protein clearance is becoming clearer. Over the past few years, the list of proteins degraded during the Drosophila MZT has grown and now includes GNU, Matrimony, Cort, Smaug, ME31B, TRAL, and Cup. Unbiased mass spectrometry experiments also suggest that Wispy and Dhd are also robustly degraded. As this list of proteins in Drosophila and other developmental systems increases, a new question is emerging: how many maternally deposited proteins are degraded during the MZT? Understanding the mechanisms controlling protein degradation during the MZT as well as the impact of removing the maternal protein dowry will be key issues to explore in the future (Zavortink, 2020).
Zygotic genome activation (ZGA) is the first transcription event in life. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. By developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), this study investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. Pol II was found to undergo 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation (Liu, 2020).
This study has developed Stacc-seq to interrogate the states of Pol II in early mammalian development. Specifically, protein A/G fused with Tn5 transposases is pre-incubated with antibodies, and then applied to recognize the targeted proteins and cleave chromatin around the binding sites. The released DNA fragments are simultaneously transposed with adaptors for sequencing. This independently developed approach uses similar strategies to CUT&Tag16, ACT-seq17, CoBATCH18 and scChIC19, and can be done in as short a time as 3.5 h. Stacc-seq differs in the pre-incubation of Tn5-protein A/G with antibodies to presumably minimize off-target chromatin tagging by Tn5, and in the optional omission of washing steps for possibly low-affinity antibodies or low-abundance targets. Stacc-seq can profile trimethylation at the 4th or 27th lysine residues of histone H3 (H3K4me3 or H3K27me3, respectively) using as few as 200 mouse embryonic stem cells (mESCs), with comparable or better performance than CUT&Tag16 and CoBATCH18. Stacc-seq can also detect Pol II binding using as few as 500 mESCs, using antibodies against RPB1 (the largest subunit of Pol II), regardless of the phosphorylation state of the CTD. In addition, Pol II Stacc-seq can be robustly applied to mouse tissues. Using spike-in DNA, Stacc-seq can be conducted in a quantitative manner, although it is more effective for samples from the same batch. Therefore, all spike-in normalization and comparison in this study were done only when the related experiments were conducted in parallel. In summary, Stacc-seq is a highly sensitive and efficient method for profiling genome-wide protein binding and histone modifications (Liu, 2020).
It is unclear how the transcription machinery engages mammalian ZGA. This study has shown that, after fertilization, Pol II initially re-engages both CG-rich promoters and distal accessible regions, which appears to reflect neither a history from gametes nor a purpose for major ZGA. This is followed by pre-configuration of Pol II, presumably to ensure accurate synchronization between chromatin maturation and the genome activation clock. One key question is how exactly the pre-configuration of Pol II occurs. Transcription factors might function as pioneering factors to recruit Pol II. On the other hand, Pol II pre-configuration also depends on minor ZGA, and the underlying mechanisms of this dependency are unclear. There are several non-exclusive possibilities: (1) in the presence of DRB or α-amanitin, Pol II disassociates from chromatin during mitosis in one-cell embryos and rebinds to its one-cell targets during the early two-cell stage, suggesting that the chromatin landscape is resistant to pre-configuration when minor ZGA is absent; (2) extended DRB treatment may exhaust free Pol II, preventing sufficient Pol II from being recruited to major ZGA sites. A large amount of Pol II may be essential to tip the transition balance towards its binding to major ZGA sites. This is perhaps analogous to the reprogramming of induced pluripotent stem cells, in which only sufficient amounts of pluripotency factors open pluripotency enhancers; (3) certain transcription products during minor ZGA, although extremely limited, may be essential for guiding Pol II pre-configuration; (4) DRB also partially inhibits the transcription of ribosomal RNA (rRNA) by Pol I, although without affecting the total amount of rRNA. Whether Pol I transcription, as part of minor ZGA, affects Pol II pre-configuration remains to be investigated. The idea that transcription factors recruit Pol II to major ZGA sites, where Pol II or minor ZGA in turn stabilizes the transition, is favored. Together, these findings reveal dynamic engagement of Pol II with chromatin upon the onset of global transcription. These data may define cornerstones for future investigations of mammalian ZGA and early development (Liu, 2020).
Externally deposited eggs begin development with an immense cytoplasm and a single overwhelmed nucleus. Rapid mitotic cycles restore normality as the ratio of nuclei to cytoplasm (N/C) increases. A threshold N/C has been widely proposed to activate zygotic genome transcription and onset of morphogenesis at the mid-blastula transition (MBT). To test whether a threshold N/C is required for these events, N/C increase was blocked by down-regulating cyclin/Cdk1 to arrest early cell cycles in Drosophila. Embryos that were arrested two cell cycles prior to the normal MBT activated widespread transcription of the zygotic genome including genes previously described as N/C dependent. Zygotic transcription of these genes largely retained features of their regulation in space and time. Furthermore, zygotically regulated post-MBT events such as cellularization and gastrulation movements occurred in these cell cycle-arrested embryos. These results are not compatible with models suggesting that these MBT events are directly coupled to N/C. Cyclin/Cdk1 activity normally declines in tight association with increasing N/C and is regulated by N/C. By experimentally promoting the decrease in cyclin/Cdk1, this study uncoupled MBT from N/C increase, arguing that N/C-guided down-regulation of cyclin/Cdk1 is sufficient for genome activation and MBT (Strong, 2020).
Maternal RNA degradation is critical for embryogenesis and is tightly controlled by maternal RNA-binding proteins. Fragile X mental-retardation protein (FMR1) binds target mRNAs to form ribonucleoprotein (RNP) complexes/granules that control various biological processes, including early embryogenesis. However, how FMR1 recognizes target mRNAs and how FMR1-RNP granule assembly/disassembly regulates FMR1-associated mRNAs remain elusive. This study shows that Drosophila FMR1 preferentially binds mRNAs containing m6A-marked 'AGACU' motif with high affinity to contributes to maternal RNA degradation. The high-affinity binding largely depends on a hydrophobic network within FMR1 KH2 domain. Importantly, this binding greatly induces FMR1 granule condensation to efficiently recruit unmodified mRNAs. The degradation of maternal mRNAs then causes granule de-condensation, allowing normal embryogenesis. These findings reveal that sequence-specific mRNAs instruct FMR1-RNP granules to undergo a dynamic phase-switch, thus contributes to maternal mRNA decay. This mechanism may represent a general principle that regulated RNP-granules control RNA processing and normal development (Zhang, 2022).
Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, the behavior of several previously identified N/C ratio-dependent genes was directly assayed using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene examined, nascent RNA output per cycle was found to be delayed in haploid embryos. Moreover, the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. These data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation (Syed, 2021).
During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug (SMG) plays an essential role in progression through the MZT by translationally repressing and destabilizing a large number of maternal mRNAs. The SMG protein itself is rapidly cleared at the end of the MZT by a Skp/Cullin/F-box (SCF) E3-ligase complex. Clearance of SMG requires zygotic transcription and is required for an orderly MZT. This study shows that an F-box protein, which was named Bard (encoded by CG14317), is required for degradation of SMG. Bard is expressed zygotically and physically interacts with SMG at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of SMG. shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of SMG protein, a phenotype that is rescued by transgenes expressing Bard. Bard thus times the clearance of SMG at the end of the MZT (Cao, 2021).
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. This study used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. The screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. It was demonstrated that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, these data identify Nejire and Gcn5 as key regulators of ZGA (Ciabrelli, 2023).
Downregulation of protein phosphatase Cdc25(Twine) activity is linked to remodelling of the cell cycle during the Drosophila maternal-to-zygotic transition (MZT). This study presents a structure-function analysis of Cdc25(Twine). Chimeras were used to show that the N-terminus regions of Cdc25(Twine) and Cdc25(String) control their differential degradation dynamics. Deletion of different regions of Cdc25(Twine) reveals a putative domain involved in and required for its rapid degradation during the MZT. Notably, a very similar domain is present in Cdc25(String) and deletion of the DNA replication checkpoint results in similar dynamics of degradation of both Cdc25(String) and Cdc25(Twine). Finally, this study shows that Cdc25(Twine) degradation is delayed in embryos lacking the left arm of chromosome III. Thus, a model is proposed for the differential regulation of Cdc25 at the Drosophila MZT (Ferree, 2022).
Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, this study focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. Single embryo PCR was developed
and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, increased chromosome segregation errors were found in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI (Warecki, 2022).
The transformative events during early organismal development lay the foundation for body formation and long-term phenotype. The rapid progression of events and the limited material available present major barriers to studying these earliest stages of development. This study reports an operationally simple RNA sequencing approach for high-resolution, time-sensitive transcriptome analysis in early (≤3 h) Drosophila embryos. This method does not require embryo staging but relies on single-embryo RNA sequencing and transcriptome ordering along a developmental trajectory (pseudo-time). The resulting high-resolution, time-sensitive mRNA expression profiles reveal the exact onset of transcription and degradation for thousands of transcripts. Further, using sex-specific transcription signatures, embryos can be sexed directly, eliminating the need for Y chromosome genotyping and revealing patterns of sex-biased transcription from the beginning of zygotic transcription. These data provide an unparalleled resolution of gene expression during early development and enhance the current understanding of early transcriptional processes (Perez-Mojica, 2023).
The maternal-to-zygotic transition (MZT) is a conserved embryonic process in animals where developmental control shifts from the maternal to zygotic genome. A key step in this transition is zygotic transcription, and deciphering the MZT requires classifying newly transcribed genes. However, due to current technological limitations, this starting point remains a challenge for studying many species. This study presents an alternative approach that characterizes transcriptome changes based solely on RNA-seq data. By combining intron-mapping reads and transcript-level quantification, transcriptome dynamics were characterized during the Drosophila melanogaster MZT. This approach provides an accessible platform to investigate transcriptome dynamics that can be applied to the MZT in nonmodel organisms. In addition to classifying zygotically transcribed genes, this analysis revealed that over 300 genes express different maternal and zygotic transcript isoforms due to alternative splicing, polyadenylation, and promoter usage. The vast majority of these zygotic isoforms have the potential to be subject to different regulatory control, and over two-thirds encode different proteins. Thus, this analysis reveals an additional layer of regulation during the MZT, where new zygotic transcripts can generate additional proteome diversity (Riemondy, 2023).
Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. This study profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, deep neural networks were applied to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. Cell lineages were determined; their developmental relationships were infered; and dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs' cognate motifs were linked. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation (Calderon, 2022).
To illustrate the potential of these data to facilitate exploration of specific lineages at finer resolution, 59,012 cells annotated as neuroectoderm were reanalyzed using scRNA data from 6 to 18 hours. This revealed 20 subclusters, including a large group of early cells corresponding to the brain primordium and neural progenitors that express regulators of neurogenesis, such as Notch (N) and Delta (Dl), and neuroblast temporal TFs, such as miranda (mira) and castor (cas). Two additional neural progenitor clusters correspond to sensory progenitors, whereas immature neurons express low levels of both neural progenitor and pan-synaptic genes, including cacophony (cac) and synaptotagmin 1 (syt1). Mature neurons are marked by higher levels of pan- and subtype-specific synaptic genes coupled with low or no expression of earlier developmental genes. Finally, midline cells, consisting of both neurons and glia cluster together, become evident at 6 to 8 hours; using the midline TF single minded (sim) and glial immunoglobulin family member wrapper as markers, it was possible to follow them forward in time as they mature (fig. S7B). It was also follow the maturation of sensory neural progenitors, marked by shaven (sv), from 6 to 16 hours (Calderon, 2022).
To further explore neuronal diversity, 6703 mature neurons were reclustered, revealing 11 neuronal subtypes, which were manually curated. Among these, four clearly separable sensory cell clusters were identified. There are two types of Drosophila sensory neurons based on dendritic morphology: type I sensilla, which include both external sensory (ES) neurons and internal chordotonal (Ch) neurons, and type II multidendritic (MD) neurons. It was possible to clearly distinguish MD neurons on the basis of expression of genes, such as dendritic arbor reduction 1 (dar1), which promotes their characteristic branching dendrites, and the pseudouridine synthase RluA-1, which was recently identified as a marker of MD neurons. Consistent with their nociceptive role, this cluster also specifically expresses the mechanical nociception degenerin/epithelial sodium channel subunits pickpocket (ppk) and ppk26. Mechanosensory ES neurons are specified by the TF hamlet (ham), which is specifically expressed in the middle sensory cluster. The adjacent cluster, likely Ch sensory neurons, is identified by expression of the mechanosensitive nonselective cation channel subunit no mechanoreceptor potential C (nompC) as well as fate-determinant Rfx and a number of as-yet uncharacterized genes specific to this cluster. The final sensory cluster likely corresponds to Ch glial-like support cells based on the expression of glial markers, including moody, and Cbl-associated protein (CAP) and nompA, which promote the development and function of Ch support cells, respectively. On the basis of vesicular neurotransmitter transporter expression, two clusters of central cholinergic neurons were identified, a glutamatergic cluster that likely includes motor neurons, and monoaminergic neurons. Finally, peptidergic neurons cluster separately and were identified on the basis of the expression of neuropeptides [ion transport peptide (ITP)], enzymes involved in their synthesis [amontillado (amon)], and receptors [myosuppressin receptor 1 (MsR1)] (Calderon, 2022).
The expression of uncharacterized long noncoding RNA (lncRNA) CR31451 was validated as enriched in mature neurons as well as two genes, complexin (cpx) and CG4328, identified as enriched in the monoaminergic cluster, which includes midline neurons. This neuronal subtype enrichment is unexpected for cpx, which encodes a presynaptic regulator of synaptic vesicle release, and may point to additional requirements for Cpx in midline monoaminergic neurons. In the course of exploring these fine neuronal subtypes, an unexpected finding was made regarding elav, a classic marker gene for neurons. Specifically, lower-level expression of elav was noticed in clusters annotated as visceral muscle. Performing double fluorescent in situ hybridization with a visceral muscle–specific marker gene (biniou) confirmed this unexpected finding and raises the possibility of a potential previously unknown role of this well-studied gene (Calderon, 2022).
This deeper exploration of the neuroectoderm, validating and extending years of research from many groups, illustrates the depth of information that can be obtained from these data. A more detailed annotation of nonmyogenic mesoderm . A full exploration of all lineages represented in these data will require a community-wide effort by tissue experts (as done in this study for neuronal diversity) (Calderon, 2022).
In addition to delineating developmental trajectories, these data can also capture spatial differences arising during developmental patterning. Previous bulk ATAC-seq on embryo halves has shown variability in the accessibility of enhancers along the anterior-posterior (A-P) axis of the blastoderm embryo. Using label transfer to map anterior or posterior identities from a previous blastoderm dataset onto the 2- to 4-hour data, a positional accessibility skew score was computed for validated enhancers with strict A-P activity. This indicates that accessibility of most A-P enhancers is skewed in the expected anterior or posterior cell group, recapitulating the bulk data. Notably, differences among enhancers of the same gene were identified. For example, in the eve locus, the stripe 1 enhancer has a much stronger skew for anterior accessibility compared with stripe 2, as has also been previously reported. The single-cell data thus capture the biological variability in enhancer accessibility along the A-P axis, extending previous observations. Similarly labels could be transferred from sci-RNA-seq clusters to spatial coordinates from a spatial enhanced resolution omics sequencing (Stereo-seq)–based spatial study of Drosophila embryos at 14 to 16 hours and 16 to 18 hours of development. Using the assigned annotations of tissues from the spatial study, a correspondence was observed with cluster annotations, which again suggests the spatial-relevant variability present in these data (Calderon, 2022).
To further leverage continuous views of unfolding trajectories, the gene regulatory modules active in germ layer–specific development were next explored. Focused was placed on the mesoderm and its derivatives as a complex, well-characterized system that have been studied previously. For this,all cells corresponding to mesoderm-derived cell states were selected, collectively 51,338 (scRNA) and 200,907 (scATAC) profiles across 4 to 20 hours and 2 to 20 hours of inferred developmental age, respectively (Calderon, 2022).
Focusing first on RNA, this study selected the top 2000 most variable genes. After normalizing expression values to be comparable across time, dynamic time warp clustering was used to group genes into four clusters with distinct temporal regulation. These clusters define broad successive waves of gene expression during mesoderm development and notably exhibit similarly ordered waves of chromatin accessibility. Gene pathway enrichment suggests different functional roles for each cluster. Cluster 1 genes are highly expressed from the beginning of mesoderm development (directly after gastrulation; 4 to 9 hours); are enriched for TFs; and likely represent a mixture of genes involved in progenitor cells, mesoderm development, and transcriptional activation. Cluster 2 genes peak at ~9 to 11 hours, during the subdivision of the mesoderm into different muscle primordia and their subsequent specification. This cluster is enriched for genes involved in mesoderm development, including myoblast fusion and myotube differentiation, while losing enrichment for stem cell and self-renewal terms. By contrast, cluster 3 genes (n = 365) initiate expression at ~10 hours and steadily increase to the end of embryogenesis, whereas cluster 4 genes (n = 631) only switch on at ~15 hours, during muscle terminal differentiation. The last cluster lacks enrichment for TFs and rather includes genes involved in myofibril assembly and muscle assembly and maintenance as well as essential contractile proteins for differentiated muscle. The spatiotemporal expression of five poorly characterized genes was validated by in situ hybridization, confirming that they are expressed in the mesoderm or muscle at the inferred time window (Calderon, 2022).
The temporal and cell type–specific nature of these expression signatures for both the downstream effector molecules and their upstream regulators should provide the resolution to order genes into putative regulatory hierarchies. For example, several genes with essential roles in muscle differentiation, such as myosin heavy chain (Mhc), are present in clusters 3 and 4. Mhc protein plays a critical role in providing muscle-contractile force. The scRNA data show increasing Mhc expression along the muscle lineages in cells with later embryonic ages, matching the expression pattern of Mhc. Concomitantly, there is a gradual increase in open chromatin at characterized Mhc enhancers at later stages along multiple muscle trajectories (Calderon, 2022).
Before the expression of Mhc and other muscle differentiation genes, transient expression was observed of mesoderm-associated TFs. One example is Kahuli (Kah), a Snail/Scratch family TF associated with muscle development, which has peak expression at 10 hours of embryogenesis (cluster 2). To investigate the relationship between open chromatin and gene expression, gene activity scores were computed, defined as the sum of sci-ATAC-seq reads in the gene body and the 2 kb flanking the transcription start site (TSS). The gene activity scores for both Mhc and Kah recapitulate their sequential temporal patterns of expression, with Kah's activity signature appearing earlier along the mesodermal trajectories compared with that of Mhc. To determine the extent to which it was possible to map the exact ordering of accessibility and expression changes, the scaled expression values and gene activity scores averaged across bins with equal numbers of cells were overlaid. Notably for Kah, gene expression temporally follows the trajectory of the corresponding gene activity score based on open chromatin, suggesting an ordering where first the gene body becomes accessible followed by accumulating levels of the corresponding transcript; however, this was not the case for Mhc, for which expression and accessibility increased in tandem. Kah binds to several characterized Mhc enhancers near the gene's promoter, as observed in bulk ChIP sequencing (ChIP-seq) data, which suggests a regulatory link between Kah and Mhc expression (Calderon, 2022).
To extend this analysis more globally, TF motifs were sought enriched in putative enhancers (mesoderm-specific scATAC peaks 1 to 10 kb upstream of the TSS) of genes belonging to each of the four scRNA mesoderm expression clusters. This identified 458 TF motif–to-cluster enrichments corresponding with 152 unique TFs. Of these, 31 are TFs whose expression changes along mesoderm differentiation and are thus included in the expression-based clustering. These 31 include many TFs essential for mesoderm development, including a number of direct target genes of the master regulator Twist (the functional ortholog of MyoD) at the beginning of mesoderm development (e.g., hb, en, Ubx, and pb), and concordantly expressed in the first temporal cluster. These factors have many functions, including setting up the segmentation of the mesoderm, regulating the expression of somatic muscle identity genes, establishing midgut constrictions in the visceral mesoderm, and heart patterning. Other examples from the second and third temporal clusters are genes required for cell fate specification of somatic muscle founder cells (e.g., Six4 and ap) and heart development (e.g., tup and Lim3) (Calderon, 2022).
This approach may miss the contribution of important TFs that were not variably expressed in mesoderm. In particular, if a TF is variably expressed and has corresponding variability in motif activity, this TF is likely active. However, this does not imply that all expressed TFs are active (e.g., there may be coactivators or posttranslational modifications that are required). This caveat notwithstanding, these analyses highlight the potential for further discovery of coregulated gene modules related to distinct germ layers or cell types (Calderon, 2022).
Next, an investigation was undertaken about whether it was possible to leverage the diversity of cell states across embryogenesis to infer which TFs drive specific programs of cell type differentiation. For this, all scATAC clusters at all time points (in contrast to the scRNA-focused cluster analysis above) were used and differential enrichment of TF position weight matrices (PWMs) were sought within each cluster's open chromatin regions (Calderon, 2022).
Enrichments across clusters were characterized from the 10- to 12-hour time window based on predicted time. Encouragingly, hierarchical clustering of the enrichment profiles of all associated PWMs grouped each cluster roughly by germ layer (this was also observed in other time windows). The nonmyogenic mesoderm (fat body) and myogenic mesoderm (somatic muscle) cluster together. Open chromatin regions in the myogenic clusters are enriched in motifs for many TFs known to play a role in muscle development, including Mef2 and Fork head (Fkh) TFs. The myogenic clusters also appear close to two neuronal clusters, which is driven by shared motif enrichment with neuroectoderm and glial cells, particularly many C2H2 zinc finger TFs, including Btd, CG7368, Crol, Sr, and Dar1. Many of these factors have known roles in neuronal development (e.g., Dar1), whereas Stripe (Sr) is essential for muscle tendon cell fate and muscle attachment in the epidermis at late stages of embryogenesis (Calderon, 2022).
Because members of the same family of TFs typically recognize similar motif sequences (e.g., GATAe, GATAd, and pnr), it is often difficult from motif analysis alone to pinpoint the responsible TF. To address this, the scRNA data wes leveraged to identify the most likely active TF on the basis of its expression within the clusters among all factors that share the same motif binding pattern. First, a regression-based framework was used to integrate the scATAC and scRNA datasets and identify links between the different cell clusters. Specifically, a nonnegative least square (NNLS) matrix factorization approach was adopted that decomposes expression data as a mixture of components derived from proximal gene activity scores generated from the scATAC data. Despite possible temporal differences between accessibility and expression, NNLS identifies stronger links between clusters from the same 2-hour window compared with those from adjacent 2-hour windows. NNLS links in the opposite direction were also inferred by decomposing proximal gene activity scores by gene expression associated with scRNA clusters. For each cluster of a given data type, the result of NNLS factorization is a mixture proportion of clusters from the other data type, where a higher value represents a stronger association between the scRNA and scATAC cluster. This factor decomposition approach resulted in a strong linkage (NNLS-mixture coefficient of >0.1) of 120 cell state clusters present in the same inferred time windows, with most of the strongly linked clusters being from 4 to 6 hours onward. Upon manual inspection, many linked scATAC and scRNA clusters, which had been independently annotated, are from matching tissues. For example, from the 10- to 12-hour window, the epidermis cluster (cluster 0) in scATAC data was matched to the epidermis in scRNA data. Altogether, of 21 ATAC clusters from the 10- to 12-hour window, 16 had a linked RNA annotation with a NNLS correlation value >0.1, of which 14 were between comparable tissue annotations (Calderon, 2022).
These integrated scRNA and scATAC clusters, which span 0 to 18 hours of embryogenesis, enabled a more direct analysis of the role of specific TFs in different cell types' differentiation. It was reasoned that active TFs should be more highly expressed in cell types for which they have a functional role, and their associated PWM should be more enriched or depleted in accessible regions when the TF is activating or repressing expression. In line with this, correlation values between motif-associated accessibility and gene expression were shifted toward more positive values for TFs annotated [by gene ontology (GO)] as activators and toward more negative values for annotated repressors, a trend also observed in human fetal tissues. This approach of linking TFs' cluster-specific expression and motif enrichments allowed nomination of TFs as active at specific times in specific tissues. For example, this analysis predicts a specific role for Sage in salivary gland development, as the salivary gland is the only cell type exhibiting both high expression of the sage transcript and high accessibility of the Sage-associated PWM. This finding matches the essential role for sage in salivary gland development, as determined by genetic loss-of-function analysis. Similar predictions were made for GATAe in the midgut at 16 to 18 hours and Awh in the epidermis at 14 to 16 hours, matching the functional role for both TFs in midgut endoderm and epidermis development, respectively (Calderon, 2022).
To expand this analysis and systematically nominate TFs that potentially drive germ layer–specific differentiation programs, a linear model was fit that predicts a TF's motif-associated chromatin changes from an estimated effect of an interaction term that includes the expression level of the TF in a specific germ layer and time window. The model's effect estimates can identify TFs with specific motif activity in particular germ layers and suggest time windows from which a TF initiates its activity. For example, the model refined the role of Sage as becoming active in the ectoderm germ layer specifically from 10 to 12 hours onward and the activity of GATAe initiating in the endoderm from 8 to 10 hours onward. Such a model encompassing germ layers across development time may also identify additional likely coactive TFs. For example, in addition to Sage, Fkh was found to be both coexpressed and coactive in the ectoderm-a TF reported to act together with Sage to activate salivary gland–specific genes (Calderon, 2022).
This analysis also generated additional interesting findings for other time points and germ layers [e.g., Fruitless (Fru)]. Altogether, from eight high-level germ layer–associated tissue annotations and 316 TF motifs tested, 1258 significant ( TF-to-tissue relationships having both associated expression and chromatin activity at one or more of the nine time windows assessed. It is noted that in time windows with fewer clusters, the association effect estimates are susceptible to outliers and should be interpreted with caution. Notwithstanding this caveat, these putative assignments represent an extensive resource for future studies (Calderon, 2022).
To demonstrate the potential of this approach to discover previously unknown putative roles for TFs, four genes were selected and whether they were expressed in the linked germ layer was validated by fluorescent in situ hybridization. Although these genes were inferred to have effects in multiple germ layers, their function in either mesoderm (CG5953 and CG11617) or neuroectodermal tissues (Ets65A and CG12605) was poorly characterized. These factors were confirmed to in fact be expressed in the tissue and time window predicted by the data, suggesting potential roles for these TFs in mesoderm and neuronal development (Calderon, 2022).
To complement the NNLS, a recently developed tool, was applied to further facilitate gene regulatory network (GRN) reconstruction. Because multi-omic ATAC-RNA data from the same cell are required for this task, the two independent assays for all cells from 10 to 12 hours were integrated using canonical correlation analysis (CCA), identifying the most likely ATAC-RNA cell pairs using geodesic distance–based pairing (37) within the common CCA space. Using these pairs as input for GRN inference with FigR, ATAC peaks were linked to their target genes based on peak-to-TSS accessibility correlation and then TF motif enrichments were computed for the linked regions, which, together with the TF expression-accessibility correlation, allowed definition of hundreds of putative activators and repressors at this embryonic stage. Ranking the TFs by their regulation score nominated many activators and repressors that were also identified in the NNLS analysis above, including l(3)neo38, Lim3, lola, fkh, and fru. Focusing on the targets of the regulatory networks across all cells at 10 to 12 hours, a large set of genes was found that appear to be extensively regulated (209 genes with >10 linked regulatory regions). The inferred TF activities were used to explore the factors acting on these genes and their mode of regulation. For example, tup, a TF gene required for heart development, undergoes extensive self-regulation (highest motif-RNA correlation) besides being positively regulated by the pan-muscle TF Mef2 and repressed by Run and Opa. Another top-ranking gene, chinmo, an essential neuronal TF, is activated by other nervous system TFs, such as Lim1 and Onecut, and is negatively regulated by Fru, which was also identified as a neuroectoderm-specific repressor in the NNLS-based analysis (Calderon, 2022).
Finally, attempts were made to exploit the fine-grained resolution of inferred nuclear ages to explore the dynamics of an early pioneer TF, Zelda, in regulating chromatin opening followed by transcription during ZGA. The expression of a set of genes was uncovered that are Zelda dependent during ZGA and, for each gene, aggregated accessibility at the linked Zelda-bound regions in intervals of 1 min across 0 to 3 hours of embryogenesis. Clustering of gene expression identified two broad temporal clusters—a first group of early genes and a second group whose expression increases later, after ~1.5 hours of embryogenesis. Notably, although accessibility at the Zelda-bound regions linked to the early cluster seems to mirror the temporal expression, regions linked to the late expression gene cluster gain accessibility much earlier, almost as early as the first cluster, which suggests that Zelda is opening these regions for future activation. To verify whether accessibility is reflective of Zelda binding, Zelda occupancy was retrieved by nuclear cycle, which confirmed that >70% of regions in both temporal clusters are already occupied by Zelda at nuclear cycle 8 to 9, regardless of the associated gene expression. Moreover, a partial Clamp TF motif match was found within the second temporal cluster (and no match for the first cluster of a TF that is also expressed), which corroborates its Zelda-paired role during ZGA. These results suggest that Zelda establishes chromatin accessibility at a large set of regulatory regions in the early embryo, independently of future gene expression, in agreement with its well-known role as a pioneer factor. In some cases, Zelda possibly also functions as the activator of gene expression (cluster 1), whereas in others it retains a pioneering role, and the gene's expression is induced by later TFs (cluster 2) (Calderon, 2022).
The continuum of Drosophila embryogenesis (see Single-cell profiling of chromatin accessibility and gene expression throughout Drosophila embryogenesis.) builds on previous work generating sci-ATAC-seq from three nonoverlapping time windows of embryogenesis and complements other studies performed on specific tissues as well as scRNA from entire embryos at one specific stage or on dissected tissues from adults. Despite the growing use of single-cell assays to generate large-scale atlases, characterizing fine-scale dynamics of chromatin accessibility and gene expression across developmental time remains a challenge. The large number of cell types and even greater number of cell states and branch points during embryogenesis requires extensive cell sampling at continuous stages to capture regulatory transitions, especially for rare cell types. This is very difficult if not essentially impossible to obtain in most model organisms (Calderon, 2022).
In this work, sampling embryo collections from overlapping 2- to 4-hour time windows, coupled with NN-based inference of more precise nuclear ages, enabled continuous representation of Drosophila embryonic development. Other studies have attempted a similar ordering of embryos by developmental time over a 2-day window of mouse development. However, because only dozens rather than thousands of mouse embryos can practically be sampled, reliable inference at the scale of hours or minutes is challenging. Similarly, cell age was inferred in Caenorhabditis elegans using an independent time series of bulk RNA-seq from whole embryos. However, relying on such whole-embryo bulk data to predict developmental age in single cells risks inaccurate aging of rare or transient cell types, especially for more complex organisms (Calderon, 2022).
Computationally, the current neural network-based inference of developmental age bears some similarity to the concept of pseudotime. As originally proposed, pseudotime aims to serve as 'a quantitative measure of progress through a biological process'. Analogously, inferred developmental age tracks the progression of nuclei through development. However, the advantage of pairing an experimental design including overlapping yet tightly defined time windows with temporal ordering is that it is possible to anchor inferred ages to fixed time points, which can potentially lead to a more accurate representation of developmental age for complex cellular trajectories. Put another way, inferred ages are interpretable as units of absolute time that are synchronized across all tissue trajectories. With such a continuum of cellular states, it is possible to begin to infer cell type trajectories that more closely capture the continuous processes of cellular differentiation unfolding within a complex, developing multicellular organism (Calderon, 2022).
There remain further possible improvements to the experimental framework. The alignment or anchoring to real time could be refined with sampling of more tightly staged windows. Multi-omic methods for characterizing multiple data types from the same nuclei may facilitate a joint model that can link paired gene expression and chromatin accessibility (and other modalities) to developmental age inference. There are cases where technical features of the data can lead to increased uncertainty of model predictions. For example, it was found that cells annotated as germ cells, from the first collection time window, or with low read count were associated with greater prediction error. Moving forward, caution is suggested for interpreting findings solely on the basis of inferred nuclear ages from clusters with these features (Calderon, 2022).
The extensive scATAC data, with deep coverage across almost a million cells, likely captured most regulatory elements active during embryonic development and provides a comprehensive resource of potential enhancers for almost any cell type in the embryo. By contrast, the scRNA data had relatively low unique reads per cell and will likely miss some differentially expressed genes in specific cell types. As a result, some delicate analyses remain challenging. For example, transcriptional velocity estimates were found to be unstable with sparse scRNA data, although this issue was mitigated by constructing metacells before velocity analysis , which may be useful for pursuing targeted questions. In scATAC data, it was possible to distinguish XX versus XY nuclei from the proportion of chrX-mapped reads; however, this was challenging for the scRNA data, again as a result of data sparsity. These shortcomings are to some degree compensated by the large number of cells profiled, as shown by the ability to recapitulate aspects of previously documented heterogeneity even for highly dynamic or restricted phenomena—e.g., ZGA (Calderon, 2022).
Overall, this Drosophila embryonic atlas provides broad insights into the orchestration of cellular states during the most dynamic stages in the life cycle of the organism. These results represent a rich resource for understanding precise time points at which genes become active in distinct tissues as well as how chromatin is remodeled across time. The annotation of cell types within these data is an ongoing process and one that is much more challenging at early and mid-stages of embryogenesis as compared with late time points or in adults with differentiated tissues. A comprehensive annotation of embryonic cell states will require a collective effort from the Drosophila community. To support these ongoing efforts, information on expression and peaks are provided from all clusters in addition to all intermediate and raw data for further exploration. Although larval stages remain insufficiently profiled, it is hoped that these data and methods, together with the recently released large-scale adult atlas, bring closer the community-wide goal of a multimodal Drosophila atlas spanning a continuum from zygote to adulthood (Calderon, 2022).
m(5)C is one of the longest-known RNA modifications, however, its developmental dynamics, functions, and evolution in mRNAs remain largely unknown. This study generated quantitative mRNA m(5)C maps at different stages of development in 6 vertebrate and invertebrate species and found convergent and unexpected massive methylation of maternal mRNAs mediated by NSUN2 and NSUN6. Using Drosophila as a model, it was revealed that embryos lacking maternal mRNA m(5)C undergo cell cycle delays and fail to timely initiate maternal-to-zygotic transition, implying the functional importance of maternal mRNA m(5)C. From invertebrates to the lineage leading to humans, two waves of m(5)C regulatory innovations are observed: higher animals gain cis-directed NSUN2-mediated m(5)C sites at the 5' end of the mRNAs, accompanied by the emergence of more structured 5'UTR regions; humans gain thousands of trans-directed NSUN6-mediated m(5)C sites enriched in genes regulating the mitotic cell cycle. Collectively, these studies highlight the existence and regulatory innovations of a mechanism of early embryonic development and provide key resources for elucidating the role of mRNA m(5)C in biology and disease (Liu, 2022).
During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, it was hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. This study demonstrated that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor which plays a targeted yet essential role in ZGA (Duan, 2021).
The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the entire genome. Using 1.5-h-old Drosophila embryos undergoing the first wave of genome activation, This study detected ~110 discrete "speckles" of RNA polymerase II (RNA Pol II) per nucleus, two of which were larger and localized to the histone locus bodies (HLBs). In the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda (Zld) 70% fewer speckles were present; however, the HLBs tended to be larger than wild-type (WT) HLBs, indicating that RNA Pol II accumulates at the HLBs in the absence of robust early-gene transcription. This study observed a uniform distribution of distances between active genes in the nuclei of both WT and zld mutant embryos, indicating that early co-regulated genes do not cluster into nuclear sub-domains. However, in instances whereby transcribing genes did come into close 3D proximity (within 400 nm), they were found to have distinct RNA Pol II speckles. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, the data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model, where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems (Huang, 2021).
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