\chapter{Introduction}
\section{Morphogenesis in multicellularity}
The transition from unicellular to multicellular life marks one of the most significant evolutionary milestones, allowing organisms to overcome the constraints of single-cell systems through specialization and division of labour. Evolution into multicellular life allowed for the development of complex mechanisms that led to the diversity of morphology in the Tree of Life. Guided by both mechanical and chemical signaling pathways, multicellular organization gives rise to cellular patterns whose formation and arrangement can be mathematically described, notably demonstrated by Turing's seminal work on morphogenesis \cite{Turing1952}. A simple interaction between molecules that could diffuse and lead to a reaction with cells could generate complex patterns. Remarkably, these patterns explain diverse complex patterns that metazoans produce in initially uniform tissues. Although metazoan embryos start off as a roughly uniform embryonic state, generally a spherical, symmetric embryo, during development, they undergo massive transformations to acquire diverse shapes. Understanding the process of morphogenesis relies on understanding not just the signals that transform the developing embryo but also the physical changes that accompany the shape changes. Initiating as a single-cell embryo, successive rounds of cell division generate numerous cells that progressively specialize and acquire distinct functions and spatial identities. These cells collectively self-organize into specific, patterned tissue layers—namely, the ectoderm, mesoderm, and endoderm—which subsequently give rise to diverse organs and structures within metazoans. 
\par
Early evidence points to epithelial specialization as a pivotal innovation in the transition from single cellular, multifunctional cells to complex multicellular organisms with a defined division of functional environments \cite{leys2012epithelia}. Epithelial tissues play a central role by providing flexible yet stable structural frameworks, which appear prominently and consistently in developmental tissue atlases across a broad spectrum of organismal body plans. Consequently, epithelia serve as key model systems for investigating the fundamental mechanisms that define and control morphological outcomes in metazoans. Epithelial frameworks effectively mediate morphogenesis by coordinating changes through processes such as cell arrangement and tissue shape. The physical forces exerted by cellular mechanics—including differential adhesion, cortical tension, and cytoskeletal contraction—are critical in influencing morphogenesis, thereby shaping the diverse morphological structures observed in multicellular life \cite{holtfreter1943study,holtfreter1944study,heisenberg2013forces}. 
\par 
\section{Cellular Mechanics driving morphogenesis}
Cellular processes that can exert forces in metazoans driving morphogenesis involve diverse mechanisms that can affect cell number, affinity, contractility, size, shape, and position. A variety of mechanisms exist that allow cells to control and regulate each of these aspects to allow for precise control of morphogenesis. 
\subsection{Growth and Death}
Tissue growth during development is primarily controlled through the control of cell growth, cell divisions, and cell death (apoptosis). Biochemical and mechanical signals spatiotemporally control cell divisions in developing embryos. Maternal RNAs and proteins establish initial patterns, which are further refined by the embryo’s own signaling networks \cite{langdon2011MaternalZygoticControl}. These patterns determine the axes and gradients that guide cell behavior. During most developmental paradigms, the single cellular fertilized embryo undergoes several rounds of division before morphogenetic processes take place. Spatio-temporal control of division can lead to diverse shapes in various organisms, such as the bending of bird beaks, the sizes of fly wings, and the scales of crocodiles \cite{santos-duran2025Selforganizedpatterningcrocodile,wu2006MorphoregulationAvianBeaks,lecuitOrchestratingSizeShape2007}. While cell divisions are a major driver of tissue growth cell growth also plays an equally important role in tissue growth. Cell growth is usually limited early in development until zygotic expression is initiated, but often precedes cell division later in embryonic development \cite{conlon1999SizeControlAnimal}. The molecular basis of the cell size checkpoint is not entirely understood, but some evidence implicates the nuclear-to-cytoplasmic ratio as a critical determinant in this regulation. As the cell grows larger beyond a checkpoint dependent on the cytoplasmic to DNA volume, the cell cycle kinases that regulate cell division can act and initiate mitosis of cells \cite{balachandra2022NucleartoCytoplasmicRatioCoupling}. The growth of cells and an increase in cell volume are primarily controlled through extracellular growth factors. Growth factors, such as epidermal growth factor signalling patterns, are set up by the deposition of factors by the mother or through signal patterns during the organism's development. Cell size is tightly regulated in some tissues, such as the Drosophila wing, and can influence tissue architecture. Growth factor signaling, metabolic nutrients and pathways that metabolize them, control the balance between cell growth and division. Growth factors can stimulate intracellular pathways to stimulate protein and nuclear biosynthesis. The mTOR pathway, as an paradigmatic example, integrates environmental cues to regulate cell growth and division, ensuring that tissues achieve the correct size and shape \cite{lecuitOrchestratingSizeShape2007}. 
\par
In zebrafish, the development of dorso-ventral patterning is dependent on the animal-vegetal patterning set up in the oocyte. The breaking of the germinal vesicle and the balbiani body movement lead to the formation of the animal-vegetal polarity \cite{langdon2011MaternalZygoticControl}. Polarity and axis specification perform a crucial role in setting up gradients and determining cell divisions in the embryo. Cell divisions are a regulated function of tissues in development that are dependent on numerous signals. In certain tissues, such as the Drosophila imaginal discs, the tissue size is regulated regardless of the cell size by regulation of the number of divisions\cite{neufeld1998CoordinationGrowthCell}. Mechanisms that regulate divisions depend on various mechanisms that include but are not limited to a determination of the nuclear vs the cytosplasmic volume of the cell and cell cycle regulating kinases such as cdk. As cell division grows the tissue during development, cell death leads to the opposite effect, reducing the size of the tissue. Various signals can affect the decision between cell division and cell death in a growing tissue, some of which are directly dependent on the state of the tissue. Apoptotic forces can generate forces at the local as well as tissue level due to localized recruitment of the contractile apparatus of the cell, the actomyosin cytoskeleton \cite{rosenblatt2001epithelial,gu2011epithelial}.
\subsection{Cytoskeletal mediated regulation of physical properties}
The mechanics of a cell controlled largely through a set of proteins constituting the cytoskeleton and adhesive machinery, are crucial in allowing single cells to join together and form a tissue (Fig 1.1). 
The adhesive complexes of vertebrates are diverse, with different sets of junctions providing distinct functional elements to cells. The junctional diversity of cellular affinity can be through junctional components: Adherens Junctions (AJs), Tight junctions (TJs), and Desmosomes (Fig 1.1). This diversity enables the different junction components to perform specific functions and interact with specific cytoskeletal elements.  Tight junctions are a critical barrier protein that maintains a fence which prevents the mixing of membrane proteins between the apical and basolateral membranes, and a gate function which controls the paracellular passage of ions and solutes between cells. Adherens junctions are adhesive protein complexes located at cell-cell contact sites, primarily in epithelial and endothelial tissues. They are essential for maintaining tissue structure, mechanical strength, and cellular organization \cite{hartsock2008AdherensTightJunctions}. The core of the complex is made up of the cadherin complex proteins, such as Ecadherin, and catenin proteins such as $\beta$-catenin and $\alpha$-catenin. Both TJs and AJs associate with the actin cortex and regulate the dynamics of actin. Desmosomes, however, form with specific desmosomal cadherins, forming a dense plaque that interacts specifically with intermediate filaments \cite{kowalczyk1998DesmosomesIntercellularAdhesive}. They are calcium-sensitive cadherin proteins that are especially prominent in stratified squamous and simple epithelia, where they play a key role in resisting mechanical stress and maintaining tissue cohesion. The tissue-specific expression of desmosomes in tissues under extreme stress, such as cardiac muscles allows cells to regulate their adhesive fucntion depending on their role and function\cite{kowalczyk2013StructureFunctionRegulation}. Differetial adhesion affects how cells interact with each other and can allow for different cells to attach to each other\cite{tsaiAdhesionCodeEnsures2020,maitreAdhesionFunctionsCell2012}. The adhesion complexes mediate signalling, regulating cell shape, polarity, and mechanical stability in the cells. Armadillo family proteins such as palkoglobin and $\beta$-catenin can shuttle between the adhesive junction and the nucleus, acting as transcriptional regulators. Through this regulation, adhesive junctions can dynamically regulate the cellular cytoskeleton such as the actin cortex and the intermediate filament network\cite{van2008cell,gumbiner1996cell,hay2013cell}.
\par
The cytoskeleton provides structural support, enables intracellular transport, facilitates cell shape changes and motility, and coordinates force generation and signaling essential for the morphogenesis of tissues. The cellular cytoskeleton consists of a diverse array of filamentous protein networks—primarily actin filaments, microtubules, and intermediate filaments—that collectively support cytoskeletal functions. Each of these cytoskeletal systems has diverse, sometimes interdependent functions: from generating force by the activity of myosin in actomyosin networks, transporting materials across the cell, and helping organize organelles by the microtubules, and maintaining mechanical integrity (or so thought of so far) by intermediate filaments. 
\par
Actin forms the quintessential framework of the cell that drives major structural and shape changes in the cell (Fig 1.1). Actin and proteins that bind it form complex networks in the cells, from forming a thin cortex under the plasma membrane to rings that can divide the cells during cytokinesis\cite{schwayer2016actin}. The structures that actin networks are organized into are broadly classified as filamentous networks: linear, aligned filaments most often organized by formin and branched networks: Tree-like, branched filaments often organized by proteins such as Arps\cite{svitkina2020actin}. Actin filaments additionally exist in two forms in the cells: globular g-actin and filamentous f-actin, which exhibit polarity with a fast-growing “plus” (barbed) end and a slower-growing “minus” (pointed) end. Actin networks generate forces by both the association of and action of myosin in the network and the addition of monomers at either end of the actin filament \cite{demoulin2014power,blanchoin2014actin}. This allows the actin cytoskeleton to alter the mechanics of the cell surface, including surface tension, which affects how cells interact with their environment. Differential adhesion and surface tension can allow for cell neighbour exchanges, leading to large-scale cell rearrangements that are necessary for spreading, and compartmentalization of cell populations during morphogenesis \cite{manning2010Coactionintercellularadhesion}. An essential component of this function is myosin, a motor protein that interacts with actin filaments, using the energy from ATP hydrolysis to generate force and movement. In addition to this active motor activity, myosin binding to actin can also act as a cross-linker that changes the stability of the actin cortex \cite{olson2022ActinMyosinCytoskeletonRegulation,laevsky2003Crosslinkingactinfilaments}.
\begin{figure}
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/Epicell.pdf}
    \caption{Cytoskeletal architecture of a cell showing general localization and strucutre of cytoskeletal and junctional elements}
    \label{Cytoskeletal architecture of a cell}
\end{figure}
Microtubules form a network of filaments that are crucial to the transport of intracellular cargo inside the cell and the mechanical stability of the cell (Fig 1.1). Similar to actin, they are polarized filaments with a plus end that grows, which has exposed $\beta$-tubulin, and a minus end that is capped by $\alpha$-tubulin, the two monomers of microtubules. Microtubules are highly dynamic, undergoing cycles of assembly (polymerization) and disassembly (depolymerization), which are regulated by microtubule-associated proteins and cellular signals crucial for cell cycle mechanics \cite{yang2010InhibitionCellMigration}. They are a critical part of the motile machinery of single cells such as cilia and flagella and the spindle apparatus. In addition to large-scale modifications, post-translational modifications such as acetylation are also important responses to stress and cellular metabolic changes in the cell \cite{carmona2023TubulinPostTranslationalModifications}. 
\par
Intermediate filaments(IF) are the most diverse family of cytoskeletal proteins, made up of a wide variety of proteins such as keratins, vimentin, and lamins. Intermediate filaments are made up of fibrous proteins that form apolar homo or heterofilaments. The form networks both inside the cytoplasm and inside the nucleus. No known motors bind to intermediate filaments but there exists a class of proteins that mediate interactions between intermediate filaments and other cytoskeleton elements known as plakin proteins. Similar to microtubules various post translational modifications have been identified for regulating IF assembly, dynamics, and function within the cell. A variety of PTMs such as phosphorylation, glycosylation, sumoylation, acetylation, prenylation, and ubiquitination exist that can influence the mechanical properties, stability, and interactions of IFs \cite{snider2014Posttranslationalmodificationsintermediate}. Understanding the functions of intermediate filaments in the cytoskeleton Intermedaite filaments and their dynamic roles in development have been discussed in detail in section 1.4 of this chapter.
Together, these cytoskeletal elements can affect the properties of the cell such that the shape and the surface tension, which can affect large-scale tissue mechanics, hence affecting the morphogenetic movements of a tissue. These mechanisms have been extensively described in the developmental mechanics of epithelial tissue, which not only serve as an excellent model to study biomechanics but also as a crucial malleable tissue for morphogenesis.

\subsection{Cytoskelton facilitated mechanical properties of cells}
Cytoskeletal proteins affect mechanical properties of cells that can lead to changes in tissue shape size and position of cells. The forces produced by the cytoskeleton and changing properties of the cellular cortex the cytoskeleton can affect the shape, spindle orientations, ionic balances in cells. \par
The cytoskeletal architecture controls the cell morphology critically by forming a variety of structures such as rings and the cortex (for actin), asters (for microtubules) and a rim and spoke network (for intermediate filaments). Dynamic changes in their support and organization affect the membrane and its functions associated with proteins such as ion channels and transporters \cite{hitt1994Membraneinteractionsactin,luna1992CytoskeletonPlasmaMembraneInteractions}. Under osmotonic shocks that cause changes to cell volumes actin cytoskeleton undergoes dynamic reorganization and polymerization that are usually independent of change in total actin in the cell \cite{theodoropoulos1992Hepatocyteswellingleads}. Osmotic shock driven polymerization and depolymerization of the actin cytoskeleton is regulated by signallng pathways that activate actin-binding proteins, kinases, phosphatases, and small GTPases \cite{papakonstanti2000Actincytoskeletonsignalinga}. Additionally actin cytoskeleton is also crucial in responding to mechanical cues from the environment via focal adhesions, integrating extracellular forces into intracellular responses.  Actin cortex organization is central to shape regulation. The filaments and their interacting proteins, such as Arps and formins, can change the organization between branched and filamentous polymerization. At the cell cortex, these can generate protrusive structures like lamellipodia, filopodia, and microvilli. Actin can also form stress fibers that, with antiparallel actin filaments and myosin II motors, generate contractile tension. \par
Microtubules, as well, are essential for maintaining and dynamically regulating cell shape. They indirectly influence cell volume by organizing the cell’s interior, supporting membrane trafficking, and integrating mechanical signals. 
Modulating the mechanical properties of the cell cortex and cytoplasm is a critical driver of shape change, enabling cells to adapt to mechanical cues, migrate, divide, and differentiate.
Microtubules are critical in determining the polarity of the cells. They can also interact with the actin organisation by delivering of signaling proteins and mechanical feedback. These mechanisms act in concert to allow cells to exert forces on their environments. Active force production as well as the regulation of mechanical properties is critical for the movement of cells. Adhesion protein regulation is generally precisely spatiotemporally regulated as cells move, under direct mechanical feedback from the cytoskeleton. 
\par
When cells join together to form tissues, these mechanical components and the interactions are critical in mediating diverse mechanical functions required for morphogenesis.

\newpage

\section{Epithelial morphogenesis in metazoans}
As membranes separate the exterior to the interior of the single cells, epithelia perform a similar function in metazoans. Epithelial tissues possess specialized features that make them uniquely suited for compartmentalization. These attributes emerge as early as the diploblastic metazoans in evolutionary history \cite{tyler2003epithelium}. However, ongoing debate within the field concerns to whether the mono-layered tissues in Cnidarians and Porifera, such as jellyfish and hydra, exhibit all the hallmarks of true epithelia\cite{adler2023tissue,leys2012epithelia}. This debate about classifying tissues into different classes of organisms helps clarify the simple principles that underlie the essential function and physical organization of epithelial tissues.\par
 
Adhesion proteins mediate cellular connections, facilitate signaling, serve as organizational hubs for cytoskeletal elements, and contribute to barrier functions critical for maintaining tissue integrity. The role of barrier functions of epithelia in metazoans can not be understated in its importance to morphogenesis. This intricate interplay between the cytoskeleton and adhesion proteins not only ensures the structural and functional stability of tissues but also enables the dynamic remodelling required for morphogenesis, paving the way for the development of complex multicellular organisms. 
\subsection{Features of epithelia}
A crucial feature that most succinctly defines epithelia is their ability to define and differentiate compartments that make multicellular organisms. This ability allows multicellular organisms to differentiate internal environments from the exterior or between different compartments of the organism. In this section, we explore the features and components of the epithelial architecture that are essential for this function in metazoans.
\par In epithelial tissues, features that mediate critical functions are modulated through mechanisms, such as apicobasal cell polarity, adhesive junctions between their apical and lateral domains, and a basement membrane \cite{belahbib2018new}. Precise cytoskeletal spatiotemporal organization allows for the specification and maintenance of these features defining epithelia \cite{buckley2022apical,vasquez2021cells}. This precise spatiotemporal organization is mediated through conserved principles throughout the tree of life, primarily those that involve the polarization of apical and basal surfaces\cite{campanale2017development}.\par
Apicobasal polarity is a fundamental feature of epithelial cells, enabling them to organize into cohesive sheets that perform specialized functions. This polarity is characterized by the distinct segregation of cellular components into apical and basolateral domains. The specification of apicobasal polarity is necessary for positioning and maintenance of the adhesive contacts between epithelial cells. Additionally, the organization of distinct basal and apical domains allows for polarized exocytosis required for various functions that require the segregation of molecules such as ion channels, signaling factors, and extracellular membrane components\cite{zeng2017polarized}. The apical domain is most often present at the contactless exterior surface (in the case of non-luminal epithelia) or on the hollow lumen (in the case of tubular epithelia). This surface is characterized by the presence of apical protein complexes and lipids such as PI(4,5)P2. The basal surface, on the other hand, is specifically identified by the presence of so-called basal markers such as  SCRB complex proteins, the kinase PAR1, and the lipid PI(3,4,5)P3 \cite{pickett2019polarizing,gibson2003apicobasal}. The lateral contact between cells is, interestingly, the surface that demarcates the boundary and interaction between these mutually antagonistic surfaces. The lateral surfaces of epithelial cells host the junction component, SJs in invertebrates and TJs \& AJs in vertebrate systems that are critical in isolating the specific components that define these domains.\par
This segregation is mediated by so-called polarity proteins such as par proteins, crumbs complex (Crb) and aPKC/ Lethal giant larvae (LGL) complexes \cite{gibson2003apicobasal}. These complexes interact and organize the cytoskeleton depending on the domains \cite{quintin2008epithelial}. The structural organization of the apical domain and the cytoskeleton surrounding it is crucial in the function of epithelial cells. Apical polarization is responsible for the organization of the lateral and apical cell-cell junctions\cite{quintin2008epithelial,shin2006tight}.\par
Although the way apicobasal polarity is mediated in vertebrates vs invertebrates is similar, the organization of adhesion complexes is quite different. In invertebrates such as the model organism Drosophila, the apical cell-cell junction is mediated by adherens junctions. In vertebrates, the apical contact is mediated by Tight junctions (TJs), while the zona adherens, right below these, mediate the adhesive contact. Tight junctions are crucial in maintaining the barrier between cells, and, together with adherence, junctions play an organizer role in mediating connections between the actin and microtubular cytoskeleton. In addition, in vertebrates, macula adherens can be found below the zona adherens, forming the desmosomal contacts between cells. Unlike TJs, these adhesions are known to be major interaction sites for intermediate filaments, important for their subcellular organization \cite{godsel2004molecular}. These adhesion proteins are also crucial in mediating binding to basement membranes, which are extracellular composites of several large glycoproteins that provide structural support and functional inputs to cellular functions\cite{stanley1982structure}.\par Basement membranes are secreted in a diverse tissue-specific fashion, dynamically regulated based on the physiological state of the tissue. Basement membranes are critical for functional tissues, providing a dynamic structural support to most tissues. In mammals such as mice, laminin-111 is among the first ECM proteins expressed, with its three chains ($\alpha$1, $\beta$1, and $\gamma$1) detectable as early as the two-cell stage \cite{horejs2014Biologicallyactivelaminin111fragment}. However, these membranes are usually absent or play a less prominent role in early morphogenesis; hence, epithelial barriers and their mechanics are critical in organizing the mechanics.

\subsection{Mechanical organization of epithelia}
Epithelial tissues exhibit a remarkable diversity in cellular morphology, a characteristic that underpins their varied physiological roles across organ systems. The mechanical properties are critical in regulating the shapes of epithelial tissues \cite{bonnet2012Mechanicalstatematerial,hannezo2014Theoryepithelialsheet,luciano2022Appreciatingrolecell}. Based on the shape of constituent cells and the number of layers they form, epithelia are broadly categorized into several structural types: squamous, cuboidal, columnar, pseudostratified, and stratified epithelia (Fig 1.2). These structural differences are not merely morphological but are tightly correlated with specific functional mechanical adaptations, ranging from passive exchange of substances to active secretion, absorption, and protection.
\begin{figure}
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/EpithelialPacking.pdf}
    \caption{Schematic of epithelial cell shapes observed in different epithelial packing}
    A Schematic representing squamous epithelia (left top) which are formed by a barrier of thin flat cells
    B Schematic representing cuboidal epithelial tissue (left middle), which consists of cube-like cells that are equidimensional. 
    C Schematic representing columnar epithelial tissues (left bottom), which are made up of columnar cells that are longer than their horizontal surfaces.
    D Schematic representing pseudo-stratified epithelial tissues that are made up of epithelial cells that like simple epithelia all share a basal layer but unequal occupation in the two layers giving the impression of being divided in multiple layers
    E Schematic representing stratified epithelial tissues that are made up of mutiple layers of epithelial cells, often with each layer made up of different types of epithelial packing.
    \label{Epithelial cell packing schematic}
\end{figure}
\subsubsection{Simple Squamous Epithelium}
This type consists of a single layer of flattened, thin cells. Due to their minimal thickness, squamous epithelia are highly permeable and are thus ideally suited for facilitating passive processes such as diffusion and filtration. They line surfaces where rapid exchange is essential, such as the alveoli of the lungs, glomeruli of the kidneys, internal linings of organs and the inner walls of blood and lymphatic vessels (endothelium). Their structural simplicity is critical for the trade-off between mechanical barrier and efficient trans-barrier transport.

\subsubsection{Simple Cuboidal Epithelium}
Comprised of cube-shaped cells with roughly equal dimensions in all planes, this epithelium typically performs absorptive and secretory functions. The relatively larger cytoplasmic volume compared to squamous cells allows for the accommodation of organelles involved in active transport and metabolic processes. This epithelial type commonly lines glandular ducts, the surface of ovaries, and renal tubules, where it contributes to the controlled exchange and modification of substances.

\subsubsection{Simple Columnar Epithelium}
Columnar epithelia are composed of tall, rectangular cells arranged in a single layer. These cells frequently exhibit apical specializations such as microvilli (forming a brush border to enhance surface area for absorption) or motile cilia (which aid in the propulsion of mucus and other substances). This type is predominant in the lining of the gastrointestinal tract, from the stomach to the rectum, where it mediates the absorption of nutrients and the secretion of digestive enzymes and mucus. The vertical elongation of the cells provides spatial separation of functional domains—apical, lateral, and basal—enabling finely tuned vectorial transport.

\subsubsection{Pseudostratified Epithelium}
Despite appearing multilayered due to the varying positions of nuclei within the cells, all cells in pseudostratified epithelium contact the basement membrane. This illusion of stratification often accompanies functional complexity. Most notably found in the respiratory tract (pseudostratified columnar ciliated epithelium with goblet cells), it combines mucus production and ciliary movement to protect against inhaled pathogens and particulates. The integrated functions of secretion and clearance are tightly coordinated through this structural arrangement.

\subsubsection{Stratified Epithelium}
Unlike simple epithelia, stratified epithelia consist of multiple cell layers, offering robust mechanical protection. The most common subtype, stratified squamous epithelium, is adapted to resist physical and chemical stress. In areas exposed to abrasion—such as the skin, oral cavity, esophagus, and vagina—it may be keratinized to form a tough, waterproof barrier. Non-keratinized variants, retaining surface nuclei, are found in moist internal linings. Other forms, such as stratified cuboidal or stratified columnar epithelia, are less common and typically found in glandular ducts, contributing to both protection and limited secretion.

In summary, the classification of epithelial tissues by cellular shape and layering reflects a precise tuning of form to function. The structural specialization of each epithelial packing permits a wide range of physiological tasks, from passive exchange to protective insulation and complex secretory dynamics. This morphological and functional diversity is a hallmark of epithelial adaptability across metazoan life.

\subsection{Morphogenetic movements of epithelia}
Epithelial tissues play a crucial role in mediating morphogenetic movements by shaping the organism through co-ordinate cellular behaviors and mechanical forces. Epithelial tissues are essential mechanical elements in developing organisms, serving as crucial barrier structures defending against external environmental challenges while orchestrating intricate cellular interactions essential for embryonic growth and organogenesis \cite{fristrom1988}. Developmental morphogenesis of an organism involves a magnitude of challenges as the embryo takes shape\cite{thomson1917growth,shahbazi2020mechanisms,kimmel1995stages}. This change of form and function is influenced by physical and chemical factors, including the environment and the organism's interactions. Embryonic epithelia are important during this morphogenesis due to their function as a barrier and regulatory tissue to maintain control over these factors\cite{fristrom1988,quintin2008epithelial}.
\par Epithelial cohesion balances cell adhesion and contractile tension, allowing cells to defrom and retain their integrity depending on contextual demands. A critical feature underlying the plasticity and diversity of epithelial function is the tissue fluidity arising from local active forces acting at cell junctions. Epithelial tissue responds in a viscoelastic manner to forces applied, behaving like elastic solids under short-term stress but viscously flowing under longer-term deformations \cite{janshoff2021viscoelastic}. Under sustained stress, epithelial tissues can undergo permanent topological changes through cell rearrangements and junctional remodeling. Collectively these behaviours organize multiscale mechanical frameworks in which tissue-scale outcomes are attained by local cellular activities guided by mechanical and biochemical signals. 
\par
During morphogenesis, epithelial tissues perform complex rearrangements and shape changes of cells within sheets, including movements such as invagination, ingression, involution, intercalation,  and Collective migration. These morphogenetic movements lead to changes in the shapes of epithelial tissues that can organize into varied shapes required for morphogenesis. Invagination is mediated by coordinated movement of the cells such that the apical surfaces move to form tubular inpockets. Apical constriction of the cells, Basal relaxation and wedging, vertical telescoping are some of the mechanisms with which the epithelial cells bend to invaginate. Regulation of cell mechanics and the movement of cells are critical for these mechanisms that lead to invagination \cite{ettensohnMechanismsEpithelialInvagination1985, liEpithelialInvaginationVertical2019}. Similar to invagination of the entire epithelial sheet, individual cells also can ingress into the sheet a process that is critical for straified epithelia formation as well as occurs during epithelial to mesenchymal transition. Apical cell constriction is important for this as well, but unlike in the case of involution, ingression also requires the cells to lose contact with their neighbours and move inwards \cite{davidsonNoStringsAttached2012}. Intercalation of cells is a critical mechanism by which cells can exchange neighbours while maintaining the apico-basal architecture and barrier integrity. Dynamic regulation of actomyosin density at the junctions is critical in mediating this process \cite{tetleyTissueFluidityPromotes2019,rauziCellIntercalationSimple2020,sknepnekGeneratingActiveT12023}. \par
Collective migration in epithelial tissues is crucial for allowing morphogenetic changes while maintaining tissue integrity \cite{tambeCollectiveCellGuidance2011,dasMolecularMechanotransductionPathway2015}. Upon injury or a migratory signal, cells coordinate their movements over multiple cell scales by mechanosensitive signalling by the cells at the edge, called leader cells. Polarization and intracellular pulling forces from the leading cells lead to mechanosensitive changes in the follower cells \cite{tambeCollectiveCellGuidance2011}. This effect is observable through multiple cell distances over the epithelia. Actomyosin contractility plays a critical role in such coordinated movement. During development, actomyosin networks across cells can organize in tandem to generate forces across length scales. For example, in the Drosophila embryo, planar-polarized actomyosin ribbons function as “denoising” mechanisms, ensuring precise formation of tissue folds and boundaries \cite{clarke2021Actinbasedforcegenerationa}. 

\subsubsection{Epithelial morphogenesis in zebrafish}
\begin{figure}
    \centering
    \includegraphics[width=0.85\linewidth]{graphics/EpibolySchematic.pdf}
    \caption{Schematic of EVL spreading during epiboly}
    Schematic represents the spreading of the EVL and YSL starting at 4 hpf (left), to shield stage at 6hpf (center) and to the end of blastopore closure 10 hpf (right).
    \label{EVL spreading}
\end{figure}
    
Teleost embryos serve as an exceptional model system for elucidating morphogenesis during early vertebrate development \cite{bruce2016zebrafish,iwamatsu2004stages,kimmel1995stages}. Unlike mammalian embryos, fish embryos must address the distinctive challenge posed by engulfing and utilizing a substantial anuclear yolk mass\cite{behrndt2012forces}. While notable developmental variations exist between the two prominent teleost model organisms, Danio rerio (zebrafish) and Oryzias latipes (medaka), fundamental cellular movements and developmental challenges remain remarkably conserved \cite{bruce2020mechanisms}. Embryogenesis in teleosts initiates with a polarized distribution of cytoplasm concentrated at the animal pole, covering approximately one-third of the embryonic surface. \par
As development proceeds, the embryo undergoes meroblastic cleavages where the cells divide on top of the yolk.The polarity that drives the segregation of the cytoplasm to the animal pole begins within the oocyte maturation cycle when the germinal vesicle (GV) breaks down. The breakdown of GV is a complex process that ends up breaking symmetry in the oocyte \cite{kondo1997introduction}. This process also leads to an increase in the egg volume and maturation of the egg. The establishment of animal polarity and maturation of the balbiani body that follows GV breakdown is directly associated with the micropyle precursor cell (MPC) formation by mutual competition. This cell is interesting as it forms the micropyle in the mature egg, which is essential for fertilization. Interestingly, this is perhaps the first process in which keratins are expressed and play a mechanical function during zebrafish development. 
\par Krt18 serves as an early marker for MPC differentiation, indicating that intermediate filaments are involved in the cellular differentiation process\cite{xia2019lateral}.  This differentiation leads to rapid growth and distinctive shape of the MPC compared to the surrounding granulosa cells. Buc mutants interestingly show multiple MPC formations that form functional micropyles, suggesting a role of animal polarity in restricting this function specification\cite{marlow2008bucky}. Upon fertilization of the embryo, cytoplasm actively streams towards the animal pole, coinciding with karyokinesis \cite{fuentes2010ooplasmic,shamipour2019bulk}. This leads to increase in the cellular volume, which undergoes meroblastic cleavages that divide the cell from the animal pole to the yolk. \par Unlike in mammalian or other embryos that cleave holoblastically the cytokinetic furrow doesn't extend to the vegetal pole but rather divides the cells over the yolk surface.  Species such as monotremes, avians, reptiles, and fish that contain telolecithal eggs show meroblastic cleavages. This creates an interesting challenge for the organism in terms of further development as the yolk serves as a major source of nutrition and signalling\cite{schauer2020zebrafish}. While embryos where yolk is completely or partially removed retain certain genetically encoded patterns, the loss of certain cell type determinants clearly exemplifies the necessity of the signalling components that are provided by the yolk \cite{schauer2024robust}.  
\par The cell cycles as the embryo develops are initially synchronized but as the divisions develop the durations of the S-phases of cell cycles extend \cite{mishra2025geometry}. This is most obviously seen at division rounds 10 and further, where the cell cycles are almost asynchronous, but starting from round 4, the variance in the timing between cells continuously increases. This variance is driven through the geometry of the embryo that is surrounded by the yolk membrane and the animal plasma membrane.

As the cells continue to divide,  the cells on the surface divide preferentially according to the shape, such that the cells reduce their height-to-radius ratio \cite{xiongInterplayCellShape2014}. This leads to the formation of a thin layer of squamous epithelial cells, which later form the enveloping layer (EVL). Adhesion and proximity to the neighbouring cells is critical for EVL specification \cite{sagerstrom1998determination}.  At the same time, zygotic gene activation occurs where genes specific to epithelial specification express \cite{hongPregastrulaExpressionZebrafish2010}. The marginal cells surrounding the yolk undergo fusion to form a syncytium as the cells continue to divide to form a syncytial layer of tissue on the margin of the embryo called the yolk syncytial layer (YSL). Genes encoding various ion channels and epithelial-specific proteins are expressed in the EVL and YSL, which further drive their specification. FoxH1, Irf1 and Oct4 are critical in the specific expression of the EVL fate and regulate the mechanics of the EVL cells \cite{pei2007early,lachnit2008alterations,sabel2009maternal}.


As gastrulation begins, the YSL serves as a driving motor driven by the retrograde flow of actin \cite{behrndt2012forces}. This flow of actin creats a pull towards the yolk through friction in a direction opposing the direction of the flow (Fig 1.3). Intermediate filaments, especially epithelial keratins, are expressed in these tissues as epiboly progresses, but their roles and interactions during development are so far unknown. 
\newpage
\section{Intermediate filaments in epithelial development}
The cytoskeleton of a cell plays a crucial role in its mechanical functions, through intracellular networks made up of actin, microtubules, and intermediate filaments. These networks are crucially mediated through interactions of specific domains in the monomeric architecture of the filaments. Direct covalent and hydrophobic interactions, weak interactions such as phase separation of the filaments, drive the formation of these networks and provide mechanisms for regulating the function of these networks. As discussed above in section 1.2.2, these cytoskeletal systems perform a myriad of functions to enable cellular movements that drive the morphogenesis of organisms. Among their functions in development, much is known about the role of actin, microtubules, and their interaction proteins; however, the regulation and functions of intermediate filaments are yet understudied. 
\par
Intermediate filaments form a complex class of cytoskeletal networks that perform crucial roles in various developmentally critical functions in tissues. The differences between intermediate filaments and other cytoskeletal networks are evident at the structural level. The expression of intermediate filaments is highly variable and cell-type specific, unlike actin and microtubules, which are expressed ubiquitously in cells. Actin microfilaments and microtubules are polar filaments with motor proteins that attach to them and provide mechanical forces through these interactions. The polarity of these cytoskeletal filaments is crucial to directional movement of the motor proteins, such as myosin, which moves toward the +end of actin filaments, and kinesin, the +end motor on microtubules, while dynein moves toward the -ve end of microtubules. Intermediate filaments, on the other hand, are the only cytoskeletal networks that are apolar and show no direct motor protein associations. The critical reason intermediate filaments are apolar is due to the structural organization of these filaments, which aligns them without a precise polar orientation within the filament. However, various interacting proteins have been identified that can mediate cross-talk and interactions with other cytoskeletal networks. 
\par
Intermediate filaments are of two major types: nuclear intermediate filaments and cytoplasmic intermediate filaments. Both share a similar structure, which is critical for their functions in organisms' developmental programs as mechanical supports of the cells. This classification is dependent on their physical location in the cell, with nuclear intermediate filaments being organized into the nuclear lamina at the nuclear envelope, and cytoplasmic IFs organized from the external nuclear lamina to the cell periphery. However, given their functional and structural diversity, various structural modifications exist in IFs that change distinct domains of the protein. Understanding their structural differences helps understand the organizational differences of these proteins.

\subsection{Structural organization of intermediate filaments}
Intermediate filaments are broadly classified into six types based on their structural and functional distinctions: Type I and Type II form the epithelial cytoskeletal filaments, keratins; Type III is reserved for homo-polymerizing filaments such as desmin, Vimentin and GFAP; Type IV are neurofilaments; Type V are the nuclear localizing lamins and Type VI are the special lens IFs phakinin and Filensin (Fig 1.4). At the molecular level, all intermediate filament proteins are composed of three distinct domains: a flexible N-terminal head, a central $\alpha$-helical rod domain responsible for coiled-coil dimerization, and a variably sized C-terminal tail\cite{steinert1983Completeaminoacid}. These domains represent the minimal structural modules required for filament assembly and are conserved across all IF types, despite substantial sequence variation and functional specialization\cite{herrmann2016IntermediateFilamentsStructure}. While the rod domain has been characterized structurally in detail, the head and tail domains, although structurally elusive, play critical roles in filament dynamics, cellular localization, and regulation through post-translational modifications and phase separation. In this section, we dive into the structural organization of intermediate filaments from their protein structure to the organization in cells.
\begin{figure}[h!]
    \centering
    \includegraphics[width=0.8\textwidth]{graphics/Asset 1.pdf}
    \caption{Basic organization of intermediate filament structures}
    Schematic representing the structural organization of intermediate filament proteins according to their family type. The major coil domains are represented as rectangular boxes; linkers, head and tail domains are shown as green lines connecting these boxes. The relative scale of the average of the amino acid sequences of these proteins is maintained to mark differences between structural organization of different filament types. The schematic is based off structures of IF detailed in \cite{eldiranyRecentInsightIntermediate2021}.
    \label{Schematic representation of the domain architecture of Intermediate filament proteins}
\end{figure}
\par
The rod domain is critical in the dimerization of intermediate filaments, the first step of ULF organization. The rod domain is divided into four helical domains: 1A, 1B, 2A, 2B, which are separated by linker domains: L1, L12, L2. The structural assembly of all intermediate filaments is similar, except that keratin form via obligate heteropolymers of Type I and Type II filaments, whereas other types of IFs form homopolymers of the same filament. These linkers, once thought to be flexible hinges, have now been shown to adopt helical structures with significant bendability, contributing to the filament’s mechanical resilience and adaptability\cite{eldiranyRecentInsightIntermediate2021}. A type I and Type II filament/ homofilament pair to form a parallel heterodimer aligning head to head with each other. 2 heterodimers align antiparallel to each other to form a tetramer. The basic filament is formed by eight tetramers that associate laterally to form a unit length filament (ULF). A conserved symmetric anchoring knob -hydrophobic pocket mechanism at the 1B domain of the rod domain allows the interaction of the heteromeric filament. But the coils 1B and 2B are also involved in higher order IF assemblies above the dimeric forms. Depending on the orientations and exact domain binding different modes of alignment for different intermediate filaments have been observed. 1B and 2B are important in these alignments with alignment of 1B domains giving A11 alignment of the filaments and 2B domain alignment leading to A22 alignment \cite{eldiranyRecentInsightIntermediate2021}. When 1B domains are aligned with 2B A12 alignment is achieved. A12, A11 and A22 alignments are  In different IF types the pathways and mechanisms of elongation can be unique such as the compaction seen in vimentin filaments due to their hydrophobic residues. Lamins show head-to-tail polymerization of dimers before lateral association is seen. However it is clear that the interactions of the rod domains is essential for dimer formation. The head and tail unstructured domains, however, are critical for further network formation as headless filaments do not progress further than dimers \cite{strelkov2003Moleculararchitectureintermediate}. The most evident structural difference between the nuclear and cytoplasmic intermediate filaments is the longer 1B rod and the presence of a nuclear localization signal (NLS) near an immunoglobulin motif at the C-terminal of the nuclear protein. 
\par
The vast structural diversity of intermediate filaments allows for functional and tissue-specific distribution, reflective of their specialized roles in cellular architecture and function \cite{galouImportanceIntermediateFilaments1997}. Keratins, both type I and type II are epithelial-specific and are often used as clear markers for the epithelial fate. Heterodimeric acidic/basic keratin pairs assemble into filamentous networks bound to desmosomes and hemidesmosomes, thereby reinforcing epithelial resilience under mechanical stress and transmitting forces inside the cells. Type III IF proteins include desmin, GFAP, vimentin, and peripherin. Desmin localizes to Z-disks, intercalated discs, and sarcolemmal membranes in striated and smooth muscle, integrating myofibrils and organelles. GFAP is confined to astrocytes and ependymal cells in the central nervous system, with additional expression in specific non‑neural cells such as renal glomeruli and Leydig cells. Vimentin, on the other hand, is a hallmark of mesenchymal cell fates, forming dynamic cytoskeletal scaffolds in fibroblasts, endothelial cells, leukocytes, and during epithelial–mesenchymal transition, anchoring organelles and contributing to cellular elasticity. Neurofilaments form a diverse class of type IV filaments characteristic of neurons, enriched in axons to support long-range transport. Nuclear lamins type V filaments are present in all somatic cells, forming the nuclear lamina beneath the inner nuclear membrane and contributing to nuclear structure and genome organization. They are supposed to be the most ancestral intermediate filament from which most intermediate filaments have evolved \cite{peterEvolutionaryAspectsIntermediate2015,peterEvolutionLaminProtein2012}. Given their critical importance in epithelial tissues, including early developmental epithelia, and similarities with other cytoskeletal intermediate filaments, we discuss the currently proposed model of cytoplasmic intermediate filaments organisation called the rim and spoke model due to their structural organization in the cell (Fig 1.5) \cite{herrmann2016IntermediateFilamentsStructure, quinlan2017rimandspokehypothesisexplain}.
\begin{figure}[h!]
    \centering
    \includegraphics[width=0.75\textwidth]{graphics/Rimspoke_3.pdf}
    \caption{Rim and spoke organization of keratin filaments}
    Schematic representing the rim and spoke architecture of keratin intermediate filaments in a epithelial cell
    \label{Schematic representation of the keratin intermediate filametns}
\end{figure}
\par
\subsection{The Rim}
The Rim intermediate filament network is observed as a ring of intermediate filaments around the junction. Intermediate filaments on the junctional rim are associated with the junctions at desmosomes\cite{quinlan2017rimandspokehypothesisexplain,mochKeratinDesmosomeScaffold2020}. The desmosomal cadherins, desmogleins and desmocollins, associate with intermediate filaments via the plakin family protein desmoplakin. The desmosome can be divided into three zones: the extracellular core (desmoglea), where the cadherin extracellular domains interact, the outer-dense plaque, where the desmosomal cadherins interact with plakoglobin and the N-terminal domain of desmoplakins, and the inner dense plaque, where the desmoplakins interact with intermediate filaments\cite{delvaDesmosome2009,kowalczyk1998DesmosomesIntercellularAdhesive}. Early attachment of intermediate filaments to the junction occurs at distinct points colocalized with desmosomal cadherins. At these focal points, keratin filaments can nucleate and grow further to elongate into a longer filament, hence establishing a ring-like network. The keratin-mediated connection is also proposed to function in positioning and tensioning the desmosomal proteins\cite{quintin2008epithelial}. \par
The connection between the desmosomes and keratin is mediated by desmoplakin. Loss of desmoplakin in different epithelial tissues has different effects on the accumulation of keratin and desmosomes depending on the epithelial tissue \cite{sumigray2012Desmoplakincontrolsmicrovilli,inaba2020keratins,vasioukhin2001Desmoplakinessentialepidermal}. In the intestine, knockout of desmoplakin leads to a mislocalization of adhesion proteins but not of the keratin intermediate filaments \cite{sumigray2012Desmoplakincontrolsmicrovilli}. However, in stratified epithelia from conditional mouse knockouts, desmoplakin knockouts affected keratin organization, especially in the suprabasal layers of the epithelia\cite{vasioukhin2001Desmoplakinessentialepidermal}. Interestingly, in the desmoplakin knockout epithelia, actin filaments showed an increased intracellular organization and decreased cell periphery accumulation, showing a close interaction of the two different cytoskeletal networks. This is in part mediated by the mechanosensitive regulation of these networks through an intricate connection of the rim with the cytoplasmic network of intermediate filaments.
\subsection{The Spoke}
The spoke network is formed as a combination of the nuclear and cytoplasmic network of intermediate filaments. The spoke-like filaments extend from the periphery of the cell to the nucleus, which in some cells look similar to spokes\cite{windofferQuantitativeMappingKeratin2021}. Additionally, their interactions with the nuclear lamina proteins, such as nesprins, lead to a network of keratins that cover the nucleus, called as the nuclear cage \cite{leeStructuralBasisHeteromeric2012a}. Loss of keratin or the interaction leads to a reorganization of nuclear shape, hence proving critical to nuclear mechanics as well as cellular mechanics. Interestingly, it has also been observed that the network can reorganize in certain cells under stress, such that the cytoplasmic network can transition from a disorganized cytoplasmic network into clear spokes that extend to the nucleus \cite{latorreActiveSuperelasticityThreedimensional2018, duqueRuptureStrengthLiving2024}. These dynamic changes are usually observed under extreme stress, where strains of over 150\% are experienced in the cells \cite{meyerKeratinCortexStabilizes2025a}. \par
These interactions are facilitated by the interaction of the filaments with plakin proteins and other cytoskeletal proteins. Plakins are a diverse set of proteins that include plectin, BPAG1, desmoplakin, envoplakin, periplakin, and epiplakin. They are large modular cytolinker proteins that physically connect keratin IFs with other cytoskeletal networks (actin, microtubules), adhesion sites, and the nuclear surface \cite{bouameurPlakinsVersatileFamily2014}. Mechanosensitive interactions of plakins that mediate force mitigation through the keratin intermediate filament network are understudied in developing systems. However, numerous examples point to precise spatiotemporal regulation of the spoke intermediate filament network. 

 