# The mechanics behind the Drosophila egg

Megha Maria Jacob, Muriel Grammont

► To cite this version:

Megha Maria Jacob, Muriel Grammont. The mechanics behind the Drosophila egg. Seminars in Cell and Developmental Biology, 2025, 175, pp.103638. 10.1016/j.semcdb.2025.103638. hal-05400845

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Review

# The mechanics behind the *Drosophila* egg

Megha Maria Jacob, Muriel Grammont* ID

<figure><img src="image_3.png" /></figure>

Univ Lyon, ENS de Lyon, Université Lyon 1 Claude Bernard, CNRS UMR 5239, Laboratoire de Biologie et de Modélisation de la Cellule, 15, Parvis Rene Descartes, F, Lyon 69007, France

ARTICLE INFO

Keywords:

Mechanical forces

Morphogenesis

Ovarian follicle development

Drosophila

A B S T R A C T

The formation and the development of the *Drosophila* egg involves multiple mechanical cross-talks between germline cells, somatic cells and the surrounding basement membrane. In this review, we discuss several development stages when the sources, as well as the roles, of mechanical forces in egg shape establishment are well defined. The examples described here illustrate the diversity of these forces as well as of the tools used to measure them and of the outcome each of them generates. We examine their contributions and their integration to morphogenesis. We discuss the limitations of our current knowledge, the importance of developing novel approaches and the support that modelling could bring to tackle some issues. One major future challenge is to understand how robustness in shaping the egg is achieved when the contributors act in different cell types and at different times. Studying *Drosophila* egg formation thus remains an exciting model in developmental biology as it must integrate a variety of biomechanical inputs from its environment, in addition of the biochemical signals discovered in the past.

Making an egg has to be a robust process to ensure species perpet-
uation. This robustness not only applies to gene expression, but is also
relevant to mechanical properties of the different tissues involved in egg
development. Indeed, egg formation never occurs in isolation. It rather
develops within a surrounding environment. How an egg is formed and
shaped in coordination with adjacent tissues is thus a central question in
developmental biology and remains unresolved in any multicellular
organism. Egg formation can be considered through two different per-
spectives (biochemical and mechanical) with both operating at different
scales (organism, tissue, cell and subcellular). A large number of
biochemical components (signals, regulatory pathways, cellular struc-
tural elements) have long been identified as key players in *Drosophila*
oogenesis. Altogether, they provide a good qualitative picture of the
proteins involved in many aspects of egg formation such as cell division,
migration, differentiation, intercalation, elongation, death and eggshell
formation. However, this was clearly insufficient to get a holistic un-
derstanding of how a spherical ovarian follicle emerges and gives rise to
an ovoid egg. The major advances in the field over the last 15 years are
not due to the discovery of new factors, but rather in the identification of
both cell and tissues mechanical properties and in deciphering how
those properties guide the above-mentioned processes. This review will
address this essentially in the three following processes: forming a chain
of follicle, elongating a follicle and controlling border cell migration.

Other processes will be briefly mentioned.

1. Introduction to the *Drosophila* ovarian follicle and its environment (Fig. 1)

Female *Drosophila* have a pair of ovaries connected to the uterus
through oviducts. Each ovary is composed of 16–20 ovarioles protected
by a contractile tube, called the muscle sheath. Each ovariole consists of
a germarium, where the ovarian follicles form, and a vitellarium, where
they mature over 14 stages to give rise to an egg. An ovarian follicle
emerging from the germarium comprises 16 germline cells, including
one posteriorly-localized oocyte and 15 nurse cells (NC), surrounded by
a monolayered follicular epithelium (FC) that adheres to an outer
basement membrane [1,2]. The basement membrane (BM) is a special-
ized extracellular matrix composed of Collagen IV, Laminin, Perlecan
and Nidogen. Collagen IV and Laminin independently self-assemble by
covalent and ionic interaction, respectively, to form sheet-like networks
while Nidogens and Perlecan facilitate interactions between Collagen IV
and Laminin. These interactions ultimately form an interconnected
meshwork that provides mechanical support to the follicle and interacts
with the FC through focal adhesion [3]. About 100 FC are required to
surround the germline in stage 1 (S1) follicles. FC are cuboidal and
undifferentiated, except for polar cells, located at either extremity of the

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: muriel.grammont@ens-lyon.fr (M. Grammont).</footnote>
follicle, and stalk cells, which separate adjacent follicles Fig. 1.

Nascent follicles are round at the beginning, and then gradually elongates along the anterior-posterior (A-P) axis from S4 through multiple mechanisms that are detailed below. During follicle development, the nurse cells endoreplicate their genome and supply mRNA and proteins to the quiescent oocyte through a system of ring canals. In parallel, FC undergo about 3-4 rounds of mitotic division until S6, before

undergoing two to three rounds of endoreplication [1]. Signals from the polar cells and the oocyte cause FC differentiation at the anterior and posterior ends into so-called terminal cells. Anterior terminal cells then further differentiate into border cells, stretched cells, and centripetal cells. FC that do not receive these signals are called main body follicular cells [4,5]. Main steps of FC morphogenesis start at S9, when the border cells and the polar cells migrate towards the oocyte through the nurse

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Schematic representation of the overview of oogenesis: Sagittal view of the follicles across the different developmental stages till the egg.</figcaption></figure>
cells. Concomitantly, the stretched cells become squamous around the nurse cells while the main body follicular cells become columnar around the oocyte. From S10 to S14, the centripetal cells elongate and then migrate between the nurse cells and the oocyte to cover the anterior part of the oocyte. Soon after, the nurse cells transfer their cytoplasmic contents into the oocyte before undergoing apoptosis. The vitelline membrane and the eggshell are also built during this period by the main body follicular cells, the centripetal cells and the border cells before these cells slough off [1,6].

Thus, the immediate environment of the *Drosophila* oocyte encompasses germline cells, multiple somatic cell types, the basement membrane and the muscle sheath. As we will see below, the mechanical properties of each of these elements impact oocyte development. Cellular mechanical properties are usually attributed to two components that may influence each other: surface tension and cytoplasmic pressure. Surface tension is increased by contractility of the actomyosin cortex and reduced by cell-cell/cell-BM adhesion, which is itself mainly controlled by the two adhesive molecules, DE-Cadherin (ECad) and Integrin [7]. BM mechanical properties, regularly understood to be its stiffness, depends on its composition and structure [8–11]. Muscle sheath mechanical properties refers to the amplitude and periodicity of its contractions [12].

## 2. Forming a chain of follicles (Fig. 1)

Follicles are formed in the germarium, which comprise both germline and somatic stem cells. When a germline stem cell divides, it generates a cystoblast, which undergoes four synchronized and incomplete divisions, giving rise to an interconnected group of 16 germline cells, called a cyst. When the cyst reaches the middle of the germarium, it interacts with the somatic stem cells. Their siblings surround the germline cells to form a follicle and a stalk of eight cells connecting it to the next follicle. Thus, follicle formation covers many aspects of early oogenesis, such as the generation of 16-cell cysts, activation of somatic stem cells and polar/stalk differentiation [1]. Here, we will specifically address the mechanisms involved in epithelium formation and in stalk formation. When these mechanisms are impaired, phenotypes range from enlarged germaria to follicles enclosing less or more than 16 germline cells, or to fused follicles with no stalk, or also to follicles separated by an overly long stalk. A large number of mutations have been shown to be generate such phenotypes [5,13–15]. However, none of these studies have provided a clear framework to fully understand how the corresponding genes govern these processes.

Recently, an article described the importance of germline cell contractility and adhesive properties for proper enclosure by the somatic cells [16]. First, the authors discovered that the cortex of germline cells displays waves of actomyosin as the cyst progresses through the germarium and that wave periodicity increases during enclosure. These waves travel from one side of the cell to the other. By using mutations affecting actomyosin contractility in the germline cells, they demonstrate its role for normal packaging by the FC. Cortical contractility of the germline cells provides stiffness to the cysts to counteract the FC convergent-extension movement. An equilibrium between these two forces allows proper cyst enclosure. Second, the authors showed that the adhesive properties of germline cells are essential to preventing the cyst from being cut by FC movement, and further, that interactions between both cell types via Ecad molecules ensure correct cyst positioning during enclosure. As the authors mentioned that cortical contractility waves depend on the level of cyst differentiation, it is interesting to link their results with a recent paper, which demonstrated that the FC convergent-extension movement mechanically controls cyst differentiation [17]. These authors show that FC movement stretches cyst peripheral membranes and modulates levels of cytosolic Ca2+ via Tmc, a conserved mechanosensory channel. Then, they demonstrated that, through calcium/calmodulin-dependent protein kinases, Tmc mediates oxidative phosphorylation activation, which is a known marker of cell

differentiation. Therefore, this leads to a model where germline-soma mechanical interactions in the germarium are crucial to trigger germ-line differentiation, which in turn modulates cyst mechanical properties ensuring proper encapsulation by the FC.

About eight cells intercalate and form a stalk that physically keeps adjacent follicles apart. In 2021, a study showed that the BM is key to structure the stalk [18]. The authors first described the sequence leading to stalk formation with the description of BM layers intercalating between each stalk cell and between the stalk and the anterior and posterior follicles. This leads to the formation of a strong physical structure, likely capable of resisting mechanical movements of the follicles travelling down the ovariole [19]. Since BM components may have different origins (plasmatocyte, FC and fat body) [20], the authors have then elegantly determined the origin of the BM material at each step. By specifically reduced Collagen IV deposition at the poles of the follicles, they observed phenotypes ranging from follicle rupture to follicle fusion/collision in the vitellarium. This demonstrates for the first time the importance of BM mechanical properties in the stalk cells for follicle individualization maintenance.

In summary, these three studies have been key to understand how a chain of follicles is generated. In light of their discoveries, re-analyzing previously identified genes should now provide a much clearer framework about this process.

## 3. Elongation of the follicles

A young follicle has a spherical geometry. Since S4, it elongates along the anterior-posterior (A-P) axis to become an ellipsoid egg [1,21]. This ellipsoid shape is important, not only for embryonic development, but also for female fertility as it facilitates its passage through the narrow oviduct. Thorough investigations into the mechanisms driving follicle elongation point to the multiplicity of actors and processes. In most systems, tissue elongation can come from a restrictive proliferation zone, from oriented cell divisions, from oriented cell elongation or from cell intercalation. While the last three cellular processes are at play, recent works have demonstrated the existence of other mechanisms for follicle elongation, which relies on cell and tissue mechanical properties. We briefly discuss oriented cell division and cell intercalation and then focus on the steps, in which mechanical properties of the environment are key.

### 3.1. Phase I (S3 to S7): apical cortical tension (Fig. 2)

This first phase starts by apical constriction pulses in the FC occurring soon after apical Myosin II recruitment. Over time, the amplitude of these constrictions become more pronounced in both anterior and posterior FC compared to FC in the medio-lateral region. These pulses are driven by a gradient of Jak-STAT signaling emanating from the polar cells [22]. The authors propose that Jak-STAT and Myosin act in parallel to control the spatio-temporal pattern of the pulses, which generate an anisotropic pulling force along the A-P axis. In parallel to the pulses, the authors reported the occurrence of oriented cell intercalation along the A-P axis while others reported a progressive bias in division orientation with more cells dividing in the direction of the axis of elongation [8,23]. Surprisingly, although apical constriction pulses could be responsible for both, neither process is considered essential for elongation. Oriented cell division and cell intercalation could thus be two mechanisms that dampen the A/P anisotropic force, powered at the tissue level Fig. 2.

### 3.2. Phase II (S4 to S8): BM stiffness gradient

#### 3.2.1. Collective cell migration and BM assembly (Fig. 3)

Until S4 the BM is composed of a uniform basal matrix, but thereafter begins incorporating all around 1–3 µm long fibrils, oriented perpendicular to the A/P axis [24]. This is dependent on FC, which undergo a planar polarized migration between S2 and S8, resulting in the
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Follicle elongation (Phase 1): Top view of a follicle with cells undergoing apical constriction. Force (F, orange arrow) and intercalating cells (light blue) are shown.</figcaption></figure>

circumferential rotation of the follicle around the A-P axis within the stationary BM [25,26]. FC migration is motored by lamellipodia and filopodia present at the leading edge of the cells, and by whip-like protrusions at the tricellular junction [24,26,27]. From S4 onwards, the follicular cells structure the BM as they migrate, by secreting BM components either basally, in a Rab8-dependent manner or laterally, in a Rab10-dependent manner [28,29]. When secreted laterally, BM material accumulates in the pericellular space and forms aggregates, which are then incorporated into the BM in the form of fibrils oriented perpendicularly to the A/P axis [29,30]. Abnormal composition or deposition of BM material prevents acquisition of an elongated shape, demonstrating BM role in follicle elongation Fig. 3. Equally, understanding collective cell migration is important in this context, as impaired migration also leads to abnormal BM [25]. FC migration is first governed by focal adhesion, which organizes basal stress fibers [21,24,25,31]. Although basal stress fiber alignment starts before rotation at S1, rotation reinforces their alignment to peak at S7 in a Dystrophin-associated protein complex-dependent manner [26,32]. Second, the formation of the protrusions is regulated by two ligand/receptor couples, the atypical Cadherin Fat2 and Plexin A at the trailing edge and their respective interacting partner Lar and Semaphorin-5c at the leading edge [27,31,33–36].

### 3.2.2. Role and regulation of the BM stiffness gradient (Fig. 3)

Several studies measured BM stiffness from S3 to S8 by Atomic Force Microscopy (AFM) and found that stiffness increases during rotation, that fibrils are stiffer than the non-fibril BM and that BM near the poles is softer than BM in the center of the follicle [9,11]. In parallel, organ swelling assays showed that the resistance to BM rupture is higher in the center than at the poles of the BM. These data, alongside data from mutant analyses in terms of Collagen IV levels, fibril length and density, mechanical properties measurements and overall follicle shape, led to a model proposing that BM mechanical properties generate a corset that constrains isotropic follicle growth, thereby promoting elongation along the A/P axis. Experiments such as reinforcing BM stiffness at the poles or decreasing it in the center confirmed the solidity of this model, as both generate rounder follicles [11]. However, a very recent study has challenged this model, because the authors found that the overall follicle shape of a particular mutant does not correlate with its phenotype in terms of fibril length, fibril density and resistance to BM rupture in an organ swelling assay. Therefore, while most of the data point toward a model in which the BM acts as a physical corset, this study advocates keeping in mind that other unknown mechanisms may also be at play [37].

Despite this, recent studies have provided new insights into the establishment of BM stiffness gradient. First, it may initially arise from

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Follicle elongation (Phase 2): A. Schematic representation of FC migration. B. Deposition of BM components and BM fibrils during follicle rotation. C. Top view of BM with the stiffness gradient represented by a grey color gradient and BM fibrils in red. D. Model of elongation with black cables representing the corset. Anisotropic BM expansion is represented by black arrows.</figcaption></figure>
molecular heterogeneities in BM components. From a comprehensive analysis of individual BM components, data show that both Perlecan and Collagen IV contribute to BM mechanical resistance in the organ swelling assay and that Collagen IV is a strong contributor to BM stiffness [10,11]. In terms of levels of secretion, S6 to S8 follicles present a graded pattern of Collagen IV levels with more Collagen IV in the central part compared to the poles [10,11,38]. Second, BM fibril micro-patterning may also contribute to stiffness anisotropy with high density of short fibrils in anterior compare to low density of long fibrils in posterior [9,10]. These two parameters, Collagen IV levels and BM fibril density have been shown to be regionally controlled by two AdamTS proteases, Stall and AdamTS-A. Both proteins are known to be strongly expressed at the poles and to remodel BM from S6 to S8 [39]. Two recent studies showed that Stall and AdamTS-A are both required in the posterior, with Stall limiting Collagen IV density and Adam-TS-A directing BM fibril micropatterning [38,39]. Moreover, Collagen IV secretion is negatively regulated in the center of the follicle by AdamTS-A, present there at low levels [38]. AdamTS-A interferes with the Rab-10 pathway to prevent excess Collagen IV from being incorporated and thereby regulates A-P differences in Collagen IV levels and BM stiffness. In addition to this patterned regulation in BM component secretion, differences in protein modification may also explain BM stiffness gradient. This has been established for the glycosaminoglycan chondroitin sulfate, a putative regulator of mechanotransduction actors [40]. Third, the metalloprotease MMP1 regulates BM fibril length and density, mainly in the anterior region [41]. While it is currently unknown how MMP1 modifies BM stiffness, it is intriguing that MMP1 is secreted by the anterior polar cells between S6 and S8 depending on the global stiffness of the BM sensed by the focal adhesion. Observations suggest that PCs detect low BM stiffness and subsequently secrete MMP1 at early stages before reducing MMP1 production as follicle develops and BM stiffens. This mechanism would prevent formation of hyper-elongated follicles. In conclusion, several different proteases participate in establishing BM stiffness anisotropy between the poles and the center of the follicle, which raises the question of how their expression is controlled. While the AdamTS A protease is regulated by the JAK-STAT signaling pathway [42], what controls MMP1 remains to be determined [11,41].

### 3.2.3. Role of the FC

An important issue is to determine whether the patterned BM guides elongation only by acting as a mold, or if it also acts by inducing cellular changes. In support of this, recent works showed that anterior FC reorient their long axis from D-V to A-P at S8, as a function of BM stiffness gradient [8]. Similarly, the dPix-Git complex, which is part of the core integrin based adhensive, [43] controls elongation before S8 by regulating Myosin activity in the FC. Follicle elongation is thus not only dependent on the BM stiffness gradient, but also depends on the regulation of force generation within FC. However, apart from their role in follicle rotation, no data support currently a direct role for the basal stress fibers in follicle elongation from S4 to S8.

## 3.3. Phase III (S9 to S10B): anterior StC elongation and posterior molecular corset (Fig. 4)

This third phase of follicle elongation is regionally divided, with stretched cell elongation in anterior and activation of the molecular corset in the main body follicular cells in posterior Fig. 4.

### 3.3.1. Anterior StC elongation

Anterior stretched cell (StC) elongation originates from changes in nurse cell (NC) mechanical properties, measured by AFM and membrane curvature [44]. From S8 to S10, NC display a gradient of cytoplasmic pressure along the A-P axis, with pressure being highest in anterior NC. This gradient depends on individual NC growth, on ring canal diameter and on fluid transfer between the NC or between the NC and oocyte. StC respond to the increase in NC pressure by expressing TGFβ, which then remodels the AJ of the StC. Interestingly, another crucial function for TGFβ is to soften the BM. TGFβ thus adjusts the mechanical properties of the BM as a function of those of the NC, which has been shown to be crucial for their growth [9,44,45]. This has been demonstrated by several experiments. First, BM elongates in an anisotropic manner with an enhanced anterior expansion. Second, if a light reduction of TGFβ signaling causes rounder follicles and eggs, a strong reduction leads to follicle death with NC membrane rupture [46]. This last phenotype is due to the maintenance of a stiff BM, which leads to an increase in NC pressure until membrane rupture. Maintenance of the NC pressure below a certain threshold is thus important for membrane integrity. BM softening allows its anterior expansion, NC growth and follicle elongation. While it is not yet proven, the extreme flattening of the StC is also likely important for NC growth by facilitating gas exchange and nutrient access. To conclude, NC and BM mechanical properties are thus coupled by StC flattening and these three processes are essential for follicle elongation.

### 3.3.2. Posterior molecular corset

During S9, the main body follicular cells (MBFC) become columnar around the oocyte. In all these cells, forces against follicle isotropic growth are governed by the mechanical property of the so-called molecular corset. This corset is composed of FC basal stress fibers and BM fibrils. In contrast to phase II of follicle elongation, the importance of basal stress fibers has been convincingly demonstrated during phase III. From S9 onwards, stress fibers and BM fibrils form a supracellular circumferential network, perpendicular to the A-P axis, that constrains circumferential growth. The MBFC covering the central part of the NC start to exhibit periodic basal contraction and relaxation cycles, which are induced by the assembly and disassembly of basal F actin and Myosin [47-49]. These periodic contractions rapidly spread to the all MBFC in a synchronized manner, generating a global constrictive force at cell level. BM stiffness, sensed through focal adhesions, regulates these actomyosin contractions via the Rho1-Rock pathway [47,48,50]. The result of these oscillations is the generation of a circumferential force in each cell. In addition, a recent analysis demonstrates that long filopodia emerge from

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Follicle elongation (Phase 3): A. 3D representation of a follicle. NC pressure gradient (P in orange), StC flattening and molecular corset are shown. Filopodia mediated supracellular actomyosin network is shown in the inset. B. Model of elongation with black cables representing the corset. Anisotropic BM expansion is represented by black arrows.</figcaption></figure>
basal stress fibers in each cell and couple to actin networks of the
neighboring cells in the D/V orientation. This coupling, probably
mediated through adherens junctions, creates a supracellular actomy-
osin network. By performing laser ablation experiments, the authors
showed that this network is under tension and that a physical link exists
between each cell and their neighbors [51]. In summary, the molecular
corset plays a crucial role at two levels during tissue elongation, by
controlling both stress fiber contraction at cell scale and by the
mechano-sensitive filopodia formation at tissue scale.

3.4. Phase IV (S10B to S12): MBFC expansion

From S10B to S12, the oocyte grows dramatically. Recently it has
been shown that this growth is accompanied by MBFC shape changes
[52]. These changes occur in a wave from posterior to anterior. Posterior
MBFC begin displaying three consecutive phases: first, the “ruffling
phase” is described by a decrease in stress fiber contractility and stress
fiber length and an apparition of multiple randomly oriented large
protrusions. Secondly, they enter the “increasing phase” characterized
by three events: restoration of D-V oriented stress fibers, maintenance of
the A/P-oriented ruffles and rapid extension of the basal area. MBFC
next enter a “spreading phase”, which involves a slow increase of basal
area along the A/P axis. Through time, the ruffling and spreading
progress to more anteriorly located MBFC. While the mechanisms
leading to this MBFC expansion are still unclear, it is manifest that the
mechanical properties of the growing oocyte are a key factor, as
expansion is impaired when oocyte growth is abnormal.

3.5. Role of the muscle sheath

The immediate environment of the ovarioles is a muscle sheath. The
muscle sheath contracts to move the follicles toward the oviduct. One
study has specifically addressed its mechanical role in follicle elongation
[12]. The depletion of Laminin W from the BM of the muscle sheath,
leads to reduced contractility and egg length. The authors demonstrated
that this was due to a low incorporation of the yolk proteins by the
oocyte, suggesting that the ovarian muscle stimulates vitellogenesis by
generating a flow of hemolymph into the abdominal cavity. Interest-
ingly, reduced levels of Laminin W first induce a hyper-contractile phase
before becoming hypo-contractile. When hyper-contractile, the muscle
sheath squeezes the follicles and leads to eggs with reduced circumfer-
ence, but WT length. This suggests that the mechanical constraint
generated by the muscle sheath must remain under a certain threshold to
not impair egg elongation. Thus, the mechanics and dynamics of even
extra-follicular components must be considered to appreciate all aspects
of follicle elongation.

In summary, different mechanisms are used to elongate the follicles and most of these mechanisms require the involvement of at least two different cellular or tissular components. Determining the degree of mechanical contribution of each component is therefore a tricky endeavor. While a wealth of knowledge has been gathered since the development of techniques to monitor FC migration in real time, certain key events remain unelucidated. The first is how stress fibers become oriented in S1, which in turn controls the direction of the FC migration. The second is what stops the collective cell migration. Different possibilities can be put forward. Anterior BM softening combined with StC elongation could generate a break. By modifying BM stiffness and by increasing their size, StC could become increasingly unable to migrate. If StC slow down before the MBFC, it would result in a torsion of the epithelium that would need to be resolved through either topological changes or MBFC migration arrest. Around the time FC migration stops, the MBFC that are close to the StC begin to exhibit changes in contractility on both apical and basal surfaces. It remains to be determined whether or not these changes in contractility depend on a putative torsion, a general change in BM composition/mechanical properties or an increase of germline volume, as suggested by Koride et al. (2014)

[53]. A third key unknown is which mechanisms control BM growth and composition, as well as the role of proteases in modulating its mechanical properties. While the mechanisms that act prior to S9 are currently well studied, much less is known for later stages. For instance, although establishing the relationship between NC pressure, TGFβ expression and BM softening was key to shedding light on anterior elongation, the mechanisms behind each of these steps remain to be decrypted. Similarly, the discovery of the posterior FC expansion raises similar questions about the implication of both BM stiffness and oocyte pressure. Finally, the last question is to what extent the different phases are linked. To conclude, most phases involve an instructive role for BM mechanical properties in follicle elongation, negating the idea that the BM simply provides a passive support for epithelial cells. Nevertheless, the interconnectedness between BM structure, resistance and stiffness, as well as the phenotypes observed when different BM components or BM stiffness regulators are compromised, suggest that BM mechanics depend on numerous as-yet-unknown factors and that we are far from understanding its full complexity.

4. Forming the eggshell

The eggshell is composed of the vitelline membrane and the chorion. Eggshell composition and roles of all the structures are described in a very recent review [6]. Here, we will mainly address border cell migration, with a small paragraph on centripetal cell migration and dorsal appendages. Border cells and centripetal cells are responsible for forming the micropyle and the operculum, respectively.

4.1. Border cell migration (Fig. 5)

Border cell (BC) clusters are an Ecad-dependant group composed of two non-motile polar cells surrounded by about 6 motile BC [54–56]. Once differentiated, BC delaminate from the epithelium and migrate under the guidance of two chemokines: the platelet-derived growth factor and the epidermal growth factor [57–59]. Via chemotaxis, one or two cells at the front of the cluster (the leader cells) form large Rac1-dependant actin-rich protrusions [58,60,61] and conduct the whole group through the NC in the direction of the oocyte. While BC differentiation and guidance cues have long been established, the importance of biomechanical factors controlling BC motility have only recently come to light. Such factors include mechanical coupling within the BC and mechanical constraints emerging from tissue topography Fig. 5.

4.1.1. *Mechanical features of the border cells*

Until recently, one model had been proposed to explain the mecha-
nism behind migratory orientation. The grapple and pull model pro-
posed that the protrusion tip from the leader cell adheres to the NC
surface, and then pulls on its cell body [62,63]. The force generated by
the Rac1-dependent protrusions would be transmitted by an
Ecad-mediated mechanical coupling to the non-leader BC to pull them
[54,55]. New data from several groups are now challenging this model.
First, data show that a supracellular actin cable runs along the periph-
eral BC membranes. Ecad molecules that maintain cohesiveness between
adjacent BC ensure the continuity of these supracellular actin cables.
These cables are under tension and play a role in shaping BC cluster. A
close examination of actin and myosin localization indicates that this
tension is likely not continuous, allowing BC cluster shape to be flexible
[60,63,64]. Second, local inhibition of myosin in any of non-leader cells
is sufficient to generate protrusions, demonstrating the importance of
this peripheral actin cable to restrict protrusion formation by non-leader
cells. In each BC, a dendritic F-actin pool localized near the membrane is
present and can be enriched in actin monomers and small actin filaments
from the peripheral cable when damaged. This observation suggests that
the two actin networks (dendritic and peripheral cable) present in BC
antagonize each other [64,65]. Third, in the leader cells, it has been now
<figure><img src="image_8.png" /><figcaption>Fig. 5. BC migration. A. An overview of Actin distribution in a sagittal view of BC. B. Representation of the topologic factors important for BC migration. Different NC interfaces are represented.</figcaption></figure>

observed that protrusions retract and that myosin accumulates specif-
ically at the base of the protrusion just before retraction, suggesting that
cells move forward, rather than being actively pulled by the protrusion.
The base of the protrusion would also be an exchange zone of actin
between the dendritic pool and a pool close to the starting point of the
peripheric actin cable. Fourth, as the leader cells move forward, the
non-leader cells follow them, not in a passive manner, but instead in a
Rac-dependant manner. This Rac activity leads to actin reorganisation
on one side of the rear cells, allowing them to crawl and enwrap one
another. This maintains cluster cohesion and ensures cluster migration
[60]. Altogether, these data provide a new framework to understand
how migration orientation and efficiency are influenced by the mecha-
nisms controlling acto-myosin networks within BC clusters. In this view,
protrusions from leader cells likely do not pull the group forwards via a
transfer of force from protrusions to actin cables, but instead, that
leader-cell protrusions may act as spears to open a path between the NC.
Then, the protrusions retract and bc cluster migrate into the gap pre-
viously generated by the protrusions. In parallel, actomyosin activity of
the peripheral cable contributes to migration efficiency by preventing
protrusion formation in non-leader cells and by controlling the shape of
the BC cluster.

4.1.2. *Mechanical features of the NC on BC migration*

Cluster shape also depends on a balance between the mechanical
properties of the BC and those of the NC. This was first stated by Ara-
njuez et al., who showed that an increase of NC cortical tension,
generated by the overexpression of RhoGEF2, is sufficient to affect BC
cluster shape and to impair migration [66]. In such follicles, the BC
cluster displays a more stretched shape than WT. Interestingly, BC
clusters respond to this increased compressive NC-dependent force by
upregulating cortical Myosin, indicating that a mechanical stimulus in
NC can elicit a response in BC. Another study reported the comple-
mentary experiment, in which Myosin levels in the NC increase if BC
cortical tension is also enhanced [67]. This increase concerns only the
NC membranes in close proximity to the BC group, indicating that NC
may also modulate locally their cortical tension when interacting with
the BC cluster. These data suggest that physical interactions between BC
and NC generate local modifications of membrane tensions in both cell
types. However, no current techniques can formally demonstrate the
presence of such modifications. While certain techniques, such as AFM
or laser ablation, can indeed measure cortical tension, they cannot be
used to quantify mechanics in NC-BC interacting regions because these
are deep within the follicle. Validation of cortical tension changes in NC
and/or BC when they interact is thus still pending. However, data from
AFM, NC curvature analysis and laser ablation experiments still exist. As
mentioned in 3.3.1, NC cytoplasmic pressure increases during BC

migration and is graded across the follicle, with higher pressure in
anterior cells [44]. Whether this gradient of NC cytoplasmic pressure
plays a role in BC migration remains unknown. From analyses on mu-
tants with increased or reduced NC pressure, Lopez et al. showed that
the speed of BC migration is not significantly affected, but from data in
the literature, it appears that BC migration is often abnormal in mutants
with high pressure [44,68,69]. Thus, the role of the NC cytoplasmic
pressure on BC migration still remains to be clarified. In parallel, data
from AFM and laser ablation indicate that NC cortexes are clearly under
tension and even if the tension is not sufficient to overcome NC cyto-
plasmic pressure, it remains likely possible that NC cortical tension is
locally modified when they are in contact with the BC.

While it is known that Ecad in the NC is important for BC migration, its specific function remained elusive. New data show Ecad molecules in the BC membranes compete with Ecad molecules engaged in NC-NC adhesion. Unzipping NC-NC adhesion allows the BC cluster to invade the NC compartment and allows BC cluster progression [70]. However, lack of Ecad in the NC also leads to guidance defects, with BC migrating away from the center of the follicle. While chemotaxis is responsible for BC migration towards the posterior, the reason they use a central path, rather than a peripheral one, was recently provided by an analysis of NC topography. Due to their stereotypic pattern of divisions, NC present a particular tissue architecture along the A/P axis. While three or four nurse cells come together at the center of the follicle, only two NC come together towards the periphery. Therefore, taking a central path involve BC going through 4-cell interfaces, and not 2-cell interfaces. Data show that protrusions always choose the path going through the highest number of NC interface, suggesting the presence of a wider free spaces at the interface of several nurse cells [70]. BC would, thus, choose such paths possibly because unzipping Ecad-dependant adhesion at such junctions is less costly in energy. This work indicates that the NC topography is an important factor for migration efficiency.

4.1.3. *Mechanical features of the BM on BC migration*

Because the BM is known to constrain the follicle and to dictate NC
shape at this stage, one might wonder about the BM mechanical role on
BC migration. Lopez et al. observed that BC migrate faster when BM
stiffness is reduced whereas they migrate slower when BM stiffness is
increased [68]. By performing laser ablations, they also observed
changes in NC cortical tension in correlation with BM stiffness. The
authors therefore indicate that modifying BM mechanical properties
induces changes in both NC cortical tension and BC migration. One
possible drawback here is that changes in BM stiffness are also known to
modify NC cytoplasmic pressure, which is greater contributor to NC
mechanics than cortical tension. The individual contribution of each
component on BC migration thus remains to be determined.
Interestingly, the authors also showed that BM stiffness regulates FC cortical tension, raising the issue of FC mechanical properties on NC cortical tension and indirectly on BC migration.

Over the last 10 years, studies have demonstrated that physical properties in between the BC are crucial for efficient migration and that BC migration also depends on the mechanical properties of both the NC and the BM. However, it still remains unclear how both the NC and the BM impact the migration. Physical interactions between the leader cells and the NC need to be deciphered more deeply in order to define how this contact is transduced intracellularly in the BC and in the NC in terms of cell contractility and cell adhesion. Results on both fronts will surely help understanding rules behind tissue distortion. Finally, although it is clear that the BM constrains the NC, it would be interesting to determine how this constraint is sensed by the NC and whether NC membranes respond differently to the constraints depending on their distance from the BM. Overall, BC migration remains a model to study cell migration, as it allows the dissection of both biochemical and biomechanical inputs.

## 4.2. *Opening a path for centripetal cell migration*

The centripetal cells (CC) comprise the first three to four rows of columnar cells that are at the border between the NC and the oocyte. They migrate in between these two cell types toward the center of the follicle to cover the anterior part of the oocyte. Recently, Pearson and Raftery have provided a much clearer picture about this process [71]. By examining external factors regulating this migration, they discovered that the NC or the oocyte can physically impair CC migration. In follicles with a reduced level of germline Ecad, the oocyte expands anteriorly or some NC bulge into the oocyte. The CC then bump into one or the other and stall. The mechanical properties of the germline are thus important to leave open a path for CC to migrate and cover the anterior part of the oocyte. Interestingly, this also depends on the BC attachment to the oocyte anterior membrane [72].

## 4.3. *Dorsal appendage formation*

The two dorsal appendages are tubes made of eggshell proteins that allow gas exchange for the embryo. They are located in the dorsal-anterior region of the oocyte. Their formation starts around stage 10B with the differentiation of two patches of about 60 FC on either side of the dorsal midline. Each patch is composed of two types of cells, the roof and the floor cells. Roof cells constrict their apical surfaces, which bends the tissue while floor cells, localized more anteriorly and medially, elongate along the apico-basal axis and dive under the roof cells to close the nascent tube. The tube then elongates anteriorly between the StC and the BM during S12 and S13 (for detailed description, see Berg et al., 2024 and Duhart et al., 2017) [2,6]. Although dorsal appendage formation depends on several well-described morphogenetic processes, such as wrapping tubulogenesis and convergent extension, this process is considered a model of epithelial morphogenesis due to the interplay between several populations of FC, the identification of the signaling pathways that control it, the ability to compare the evolution of the mechanism driving dorsal appendage formation between different species and the use of mathematical modelling [2,73,74]. In terms of mechanics, data show that roof and floor cells display different cell mechanical properties. This is illustrated by the differential expression of a variety of adhesive molecules, such as Ecad, Cad74A, Fas3 or Echinoid, and the implication of some of these in myosin II localization/organization [2]. In regards to the focus of this review, at least one characteristic of dorsal appendage formation must be underlined: the migration of roof cells between the StC and the BM membrane. While this process has not yet been fully deciphered, it is likely to involve a physical interplay between these three components. Investigating this will surely be a source of discoveries that will interest a large community of researchers.

# 5. Dumping the nurse cell cytoplasm in the oocyte

Once the oocyte reaches about 50 % of the size of follicle, it rapidly expends thanks to the transfer of the NC cytoplasmic materials, through the ring canals. While it was long accepted that NC dumping was an acto-myosin driven process, a recent study has used mathematical modelling to challenge this idea [75] and to propose that dumping occurs in two phases. The first is dependent on a physical law called the Young-Laplace law, which posits that the baseline cell surface tension between cells of different sizes is key to allowing cytoplasmic transfer between NC and from NC to the oocyte. More precisely, small NC transfer their contents to larger NC or to the oocyte. During the second phase, the NC exhibit wave-like actomyosin contractions that are necessary at the end of dumping. In parallel, it has been shown that genetic ablation of StC leads to abnormal NC dumping. It is currently unknown whether StC controls NC dumping by sending a biochemical signal or by mechanically squeezing the NC [76]. Finally, two recent studies described the importance of building an actin cage around the NC nuclei to prevent them from moving with the cytoplasm and blocking the ring canals [77,78]. This is achieved with the help of filopodia-like actin cables arising from the NC membrane and extending perpendicular to the cortex toward the nucleus, by cross-linking with the perinuclear actin [79]. Although these actin cables can originate from any part of the NC cortex, a differential actin cable growth is observed depending on its point of origin. The NC cortex is in contact with either the FC, another NC or the oocyte. Logan et al. showed that cables originating from an FC-contacting NC cortex grow slower than cables originating from an NC- or an oocyte-contacting cortex. They also showed that this difference depends on the absence of the actin elongation factor Enabled at the FC-contacting NC cortex and that importantly, this difference in actin cable growth is crucial to physically positioning NC nuclei away from the ring canals [78]. Thus, proper and complete transport of the NC cytoplasm into the oocyte is achieved by regulation of actin cable growth, NC hydraulic properties and cell contractility.

# 6. Inducing ovulation and egg activation

Once the oocyte is mature, it is released from the posterior part of the varioliole into the lateral oviduct, then the common oviduct before reaching the uterus where it is fertilized. Only events that are under a mechanical control will be mentioned here. For a complete description, a recent review is available [6]. When S14 follicles mature, the MBFC surrounding the eggshell are trimmed, leading to follicle rupture with the oocyte being squeezed out of the MBFC. The trimming is under the control of MMP2, which is specifically expressed by the posterior terminal cells and known to alter the BM and the cell-cell junctions. The coordination between the contraction of the muscle sheath and the oviduct muscle also participates in ovulation, by moving the egg through the oviduct. While passing through the tight oviduct, the oocyte is activated by changes in osmotic pressure. Concomitantly, the eggshell is hardening and the vitelline membrane mature through the activity of peroxidase and the generation of covalent bonds. Those chemical modifications fix the final mechanical properties of the eggshell required to ensure egg protection.

# 7. Concluding remarks and perspectives

Live imaging has contributed major improvements in our understanding of morphogenesis. Monitoring living *Drosophila* follicles in real-time is fairly recent [80] and, as expected, has led to a number of discoveries, especially in BC migration and in follicle elongation, as described above. Since a lot was already known about the biochemical regulation of BC migration, *ex-vivo* studies immediately focused on the mechanical aspects of both the BC themselves and of their immediate environment, as well as on their reciprocal interactions. For follicle elongation, the interest was even greater because of the discovery of FC
migration and the major implication of the BM. Such a collective migration, as well as its role to structure the BM, was unexpected. Moreover, although the BM was known to provide mechanical support and to act as a biochemical platform in morphogenesis and wound healing, its involvement in follicle development provided an exciting demonstration of a more instructive role. In this context, although we have gained insight into regional and local stiffness differences, notably through the use of AFM, we are still far from having a holistic understanding of BM mechanical properties. Additionally, other traits, such as BM elastic or viscoelastic properties, remain to be determined. Using Brillouin microscopy, for instance, could shed light on new BM mechanics [81]. To clarify cell mechanical properties, it would be useful to develop a pressure probe to get absolute values of NC cytoplasmic pressure and about the oocyte. The development of optogenetics, of FRET-based sensors and biosensors are also very promising tools to tackle mechanical properties at a sub-cellular level. While optogenetics have already been successfully used in this system [48,64,65,82], a more systematic approach should help us to make clear how the apical, lateral and basal surfaces of the FC are mechanically coupled, an issue the field has been grappling with for many years. This sub-cellular mechanical complexity may stretch even deeper, if we also consider the mechanics of certain cellular elements, such as the nucleus. Two studies have already demonstrated the importance of nuclear mechanics in chromosome pairing [83] and in protrusion stabilization in the leader BC [84]. Another example of the importance of the mechanical properties of cellular elements is shown by the interaction between the microtubules and the oocyte nucleus. From S5, the oocyte nucleus, initially localized near the anterior plasma membrane, moves and docks at the anterior-lateral membrane junction. This nuclear migration is majorly governed by microtubules (MT) from 2 major MT nucleation sites, which regulate migration velocity together. New data now demonstrate the importance of the posterior MT network in oocyte nucleus movement and importantly, show that the force generated by the MT is capable of indenting the oocyte nuclear membrane [85]. Biomechanical interactions also concern molecules. Data have shown that E-Cad and integrin need to form nano-clusters to be active [86,87]. Their formation depends on the lipidic membrane composition, which can encompass fluid or packed domains. In addition to heterogeneities in membrane composition, examination of membrane mechanics points to the existence of a membrane tension that modulates cortical tension and vice-versa. Detecting Ecad or Integrin clusters using HomofRET and monitoring membrane tension using probes such as Laurdan or Flipper TR, whose fluorescence characteristics change in response to plasma membrane tension, will be crucial to fully deciphering the mechanics of the epithelium and its response to environmental forces [88]. Egg shape depends thus on forces generated at tissue scale, but also depends on mechanical properties of elements that transduce those environmental forces at nanoscale.

Integration of all this physical information to existing body of biochemical data, will require mathematical modelling. While some mechanical models have been developed to test the relevance of the biochemical regulation of the molecular corset [47,53], or of NC topology for BC migration [70], no mechanical model currently exists to understand follicle elongation. The involvement of the different layers (germline, soma, BM etc.), as well as the geometry of the tissue are indeed sources of difficulty. Nevertheless, such models could help evaluate the contribution of each component, their reciprocal interactions and the putative hierarchy between them. While mechanical crosstalk is instrumental to synchronize morphogenetic transitions, it remains unclear whether some mechanical events arise independently. For instance, no data currently exist to link the increase in germline cytoplasmic pressure with the active phase of the molecular corset. In parallel, the fact that oocyte elongation is controlled by so many different mechanisms, questions the robustness of this characteristic. To what extent is it important to control BM stiffness, follicle elongation or BC migration efficiency? Each mechanical step may be buffered by other

forces or by the biochemical signals it induces. The importance of the spatio-temporal pattern of mechanical forces or of their levels has thus to be evaluated in regards to the importance of the fidelity in egg shape. Some biological data have already clearly stated that some variations of egg ratio do not compromise fertility or that differences in BM stiffness is more crucial than the absolute value for elongation [11,41]. How tissues self-organize to give rise to a shape that is sufficient to ensure its function is the final step of the global understanding underlying *Drosophila* egg development.

In conclusion, because making an egg has to be an extremely robust process across species, this system provides an important and tractable model to explore the sources of mechanical inputs, the mechanisms of force transduction and associated feedback loops, but also to tackle how the integration of mechanical and biochemical signals is achieved.

## Declaration of Competing Interest

The authors declared no competing interests.

## Acknowledgments

We thank all members of the lab and Pradeep Das for critical comments on the manuscript. This work is supported by the ANR (ForcesOnCell - ANR-22-CE13-0017), the Centre National pour la Recherche Scientifique and the Ecole Normale Supérieure of Lyon.

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