\chapter{Discussion}
Keratins have always been thought to be important as a "security belt" essential for mechanical resilience functions in cells \cite{kreplakExploringMechanicalBehavior2005}. While intermediate filaments are broadly important in stabilizing cells,  their roles in dynamic interactions with other developmental cytoskeletal systems are largely unknown. Our findings reveal a sophisticated mechanical interplay in which keratins orchestrate a balance between tissue spreading forces and structural resilience during zebrafish epiboly movements. 
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In our work, we elucidate a mechanistic role of keratins in affecting tissue properties as well as a mechanosensitive cross-talk between the keratin network maturation and hitherto unknown functions in actin mechanosensation. We observed that keratins are the first cytoplasmic intermediate filaments that are expressed in the zebrafish embryo. Specifically, they are expressed right at the beginning of gastrulation movements (epiboly) in the newly formed epithelial tissue, the EVL. Generally, keratin proteins form heterodimeric pairs consisting specifically of one type I (acidic) and one type II (basic) keratin. In zebrafish, the keratin gene family displays a notably different genomic organization compared to mammals, especially with respect to the organization of keratin Type II genes. Zebrafish possess at least 17 type I keratin genes distributed across five chromosomes, but only three identified type II keratin genes located on two chromosomes \cite{hoUpdateKeratinGene2022}. Expression data from our studies and previously published RNA transcriptomic analyses indicate that all three type II keratins are actively expressed in the zebrafish's enveloping layer (EVL) during early embryonic development \cite{farrell2018Singlecellreconstructiondevelopmental}. In contrast, only a subset of type I keratins is expressed at this stage with keratin 18 being the most expressed gene. Additional type I keratins become expressed progressively as the epithelium further differentiates after gastrulation is completed, as seen in the periderm of zebrafish \cite{inaba2020keratins}.  These genes, scattered throughout the genome rather than clustered, have seemingly consolidated essential functional roles.  In zebrafish specifically, keratin gene expression peaks prominently in the enveloping layer (EVL) and continues robustly within epidermal lineages during larval developmental stages, underscoring their critical developmental roles \cite{farrell2018Singlecellreconstructiondevelopmental}.
\par A recent intriguing discovery in zebrafish concerns the distinct differences in isoforms of type I and type II keratin proteins compared to mammals such as humans and mice.  In most mammals, these keratin pair genes are tightly correlated in their genomic position and expression patterns even when they are not in the same gene locus \cite{hoUpdateKeratinGene2022,kalabushevaKaleidoscopeKeratinGene2023}. All terrestrial vertebrates possess two distinct keratin gene clusters: one for type I (acidic) keratins and another for type II (basic) keratins. Phylogenetic and genomic analyses support the hypothesis that the distinct type I (acidic) and type II (basic) keratin lineages emerged prior to the evolutionary transition of vertebrates from aquatic to terrestrial environments. Terrestrial vertebrates consistently exhibit two well-defined keratin gene clusters, each highly conserved in structure and orientation. However, mammalian keratin clusters contain an increased gene number, primarily due to the evolution of specialized keratins, such as hair keratins and inner root sheath keratins, which represent mammalian-specific adaptations. In contrast, bird and amphibian keratin clusters contain fewer genes, reflecting their absence of these specialized mammalian keratins. In the human genome, genes encoding all type I keratin genes are clustered on 17q12-q21 except k18, while all keratin type II genes are on 12q11-q13 with the addition of keratin 18 \cite{moll2008human}. This compact organization is thought to be a mechanism for coordinated keratin gene expression, resulting in a 1:1 molar ratio expression of required keratin genes. In zebrafish, however, keratin4 is on chromosome 6 along with keratin 18b, while keratin4 and keratin5, along with keratin18a isoforms, are present on chromosome 23. A strikingly conserved feature across vertebrates, including fish, is the genomic proximity of type I keratin 18 and type II keratin 8. These two keratin genes are widely expressed across various human tissues, a reflection of their fundamental roles in epithelial integrity. Their closely coordinated expression patterns both in humans and zebrafish as we have observed, further underscore their evolutionary significance as ancient, fundamental epithelial keratins, likely directly early diverged from ancestral lamin-like genes.\cite{hoUpdateKeratinGene2022}.
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In teleost fish, such as zebrafish, the type II keratin gene family has undergone significant evolutionary pressures, resulting in fewer gene copies compared to mammals\cite{zimek2005Terrestrialvertebrateshave,hoUpdateKeratinGene2022}.  An intriguing distinction arises when comparing keratin gene structures across zebrafish and other commonly studied model organisms, such as frogs, mice, and humans. Our sequence analysis reveals notable differences in the highly conserved rod domains of type II keratins among these species. This differential structural conservation pattern of keratin isoforms during zebrafish embryogenesis suggests a unique functional requirement and regulation for keratin filament assembly and stability, distinct from that described in terrestrial vertebrates. Affinity and structural data that define the affinity of keratins and their binding partners in other organisms may not be true in zebrafish. This is also of particular interest as these specific rod domains are highly conserved domains in the protein responsible and required for its mechanical function and properties. Post-translational modification of the protein by protein kinases such as ERK and Akt is crucial for their organization and functions. A non-insignificant number of post-translational modifications that are mediated by ERK and Akt are within this sequence of rod forming domains. While zebrafish keratin genes generally share considerable sequence similarity with those of terrestrial vertebrates, specific variations within the rod domain point to evolutionary divergence events\cite{hoUpdateKeratinGene2022,krushnapadhi2006Independentexpansionkeratin}. 
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A potential explanation or evolutionary feature for these modifications could be due to the differences in early development environment requirements. Embryos for mammals with a few notable exceptions develop in-utero with specialized extra-embryonic tissues providing essential nutritional and protective roles. The eggs of zebrafish are in contrast protected by a very thin chorion. The characteristic expression and the evolutionary peculiarities of keratin structures in the developing embryo suggest environmental challenges that could shape the evolutionary and developmental trajectories of keratin genes in teleosts. Intermediate filaments in early metazoans show genes with more significant similarities to lamins than keratin. Keratins from cnidarians (jellyfish) are lost and regained in Testitudes (turtles) \cite{hoUpdateKeratinGene2022}. This supports the idea that keratin genes are lost and can evolve depending on environmental requirements, especially as animals evolved from sea to land. Keratin 24 is a gene that is expressed in the corneal epithelium in the terminally differentiated cells \cite{ehrlich2019Comparativegenomicssuggests}. In multiple aquatic mammals: cetaceans, Otaroidea as well as Camels which have specific environmental requirements. Camels have a thick corneal epithelium which prevents abrasion, potentially requiring the expression of different pattern of keratin genes in their eyes than keratin24. Keratin genes around keratin24 are genes that are involved in hair follicles specifically, which are lost in cetaceans. It has been proposed that keratin24 is associated with the evolution of these specific phenotypic changes.
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It is interesting to consider what evolutionary challenges could have affected the evolution of keratin genes in zebrafish. An interesting evolutionary difference to consider in this respect is the distinction between the mechanics of evolutionarily similar zebrafish and medaka eggs. Medaka eggs are lined with hairlike filaments and a harder chorion than zebrafish eggs. Upon dechorionation, these eggs are also softer and more fragile than zebrafish. EVL cells in medaka are similarly organized but show dramatically elongated cells at the end of epiboly \cite{kageyamaCELLULARBASISEPIBOLY1980}. Keratin architecture in medaka EVL cells, in contrast to the organization in zebrafish EVL, could be helpful in understanding the differential organization of similar keratins in structurally distinct cellular architectures in response to their adaptations to their different environment and development.
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As the EVL spreads during epiboly, we discovered that the keratin network matures and undergoes a transformation of the network from a spare collection of apically distributed unconnected bundles to a dense network that spans throughout the apical surface and lining the junctions. We observed, however, that the density of the network increases at a remarkable rate with a network unlike in any other system/cell and the net density of the network is much larger than any system imaged in this detailed resolution so far. Keratin bundles organize in the cell in a rim and spoke fashion, with a ring of filaments around the cells connecting desmosomal junctions and spoke-like filaments organizing from the periphery to the nucleus\cite{mochKeratinDesmosomeScaffold2020,quinlan2017rimandspokehypothesisexplain}. This entire change and maturation of the network into what has been classically thought of as a rim and spoke organization happens within a few hours, faster than any other model system that can be studied to understand the early maturation and formation of keratin networks. While the structure of keratin networks in different cells is remarkably diverse, the general structure is conserved \cite{windofferQuantitativeMappingKeratin2021}. This architecture of the network is remarkably similar in different cells and is crucial in terms of the response to mechanical deformations. In MDCK cell layers, for example, keratin intermediate filaments show massive reorganization when cells extend at extremely high strains, transforming from a disorganized network spread all over the cell to a bundled spoke arrangement around the nucleus \cite{latorreActiveSuperelasticityThreedimensional2018}.
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In the EVL, however, we noted that the dynamically regulated organization of keratin intermediate filaments during development does not show a clear ring and spoke pattern. Instead, the mini filaments that begin forming the network are distributed on the apical surface in a regularly spaced manner attached to the cortex by an unknown plectin isoform. As the network matured, additional filaments were added that joined together to form longer filaments, forming prominent thick bundles that spanned across the cell, excluding the junction in the beginning. Nuclear association through Linc complex proteins leads to a network of keratin filaments around the nucleus called a nuclear cage \cite{stenvallCytoplasmicKeratinsCouple2022}. In the EVL, we did not observe a clear nuclear organization surrounding the nucleus, suggesting a lack/minimal nuclear cage at the early stages of the network development. EVL cells are extremely squamous, with minimal distance between the nucleus and the cortex. Additionally, the keratin network, once matured, is extremely dense, creating an expansive cover over the entire apical surface. Unlike other developing organisms, we note that the keratin network in zebrafish EVL cells is much more robust and dense \cite{lim2020keratins,bohnekamp2015drosophila,mochKeratinDesmosomeScaffold2020}. We suggest that the "spoke" network that connects the nucleus is modified in the EVL due to these modified physical constraints of the cell, allowing the network to be expansive over the entire tissue rather than limited into a cage at every cell. This is additionally supported by the presence of similar supracellular networks in the extra-embryonic tissues in mice \cite{nahabooKeratinFilamentsMediate2022}. Despite this altered organization, we noted that the mechanical reorganization observed in keratin networks in other systems is also true for keratin networks in zebrafish. This amplification and reorganization of the bundles is most evident after the junctional association of these filaments, suggesting the association with desmosomes is important for this process. We observed that the junctional accumulation of keratin was not equal in all cells. In different EVL cells the amount of mini-filaments seen was unequal, with some cells showing an increased number/density of these initiating filaments. As the network matured, the relative amount of keratin in different cells did not change, i.e., the cells with initially more initiating filaments showed a faster maturation and brighter fluorescence of keratin18 tagged with GFP. We observed that the presence of junctional keratin was closely correlated to the apical network of each cell. While all cells end up showing a junctional network at the end of the shield stage (7hpf), there is a broad transition period where cells with different densities show junctional accumulation at slightly different times. This unequal distribution of keratin in different cells of the same tissue is also seen in the mouse trophectoderm, although the difference is less pronounced due to the lack of a mature, dense keratin network in these cells \cite{lim2020keratins}. 
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Junctional keratin is usually associated with a desmosomal scaffold through desmosomal proteins such as desmoplakin, plakophilin, and plakoglobin to desmosomal cadherins desmoglein and desmocollin. Interestingly, in zebrafish, desmosomal proteins are expressed at shield stage (6hpf) significant levels with a very low level of expression seen at cleavage stages that further decline at the start of epiboly \cite{goonesinghe2012desmosomal}. This correlates very closely with the junctional accumulation we observe with filaments attaching to the junction right as this increased expression of desmosomes is observed. Plakoglobin and plakophilin are armadillo family proteins similar in structure and function to $\beta -$catenin \cite{miravet2003TyrosinePhosphorylationPlakoglobin}. Protein tyrosine kinases (EGFR, Src, Fer, Fyn) phosphorylate distinct tyrosine residues on plakoglobin, differing from the phosphorylation pattern observed in $\beta$-catenin, a vital 
member of the armadillo family proteins. Phosphorylation at these sites affects plakoglobin’s association with E-cadherin and $\alpha$-catenin. This switch changes binding to desmoplakin, shifting plakoglobin from adherens junctions towards desmosomes, hence changing the availability of factors such as $\beta-$catenin in the nucleus and intermediate filament binding to desmosomes. Lack of these plaque linkers leads to not only changes in the organization of these junctional components but also prevents linkage of intermediate filaments across cells \cite{acehan2008PlakoglobinRequiredEffective}. In plakoglobin-deficient cells, the presence of transcellular networks is perturbed. Interestingly, the defects observed by loss of plakoglobin in zebrafish do not show a severe effect during early development with few embryos showing a delayed midbrain-hindbrain border formation \cite{martin2009plakoglobin}. More severe defects are seen as the embryo develops further at 72 hrs with heart oedema and a kinked tail. Desmoplakin mediates interactions with the keratins inside the cell at desmosomal plaque through plakoglobin. Desmoplakin-deficient zebrafish have cardiac abnormalities and altered Wnt/$\beta$-catenin, Yap-Taz, Tgf$\beta$/Smad signalling, impairing cardiac function. \cite{giuliodoriLossCardiacWnt2018}. 
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The keratin-desmosomal scaffold is mechanically crucial for a functional keratin network \cite{delvaDesmosome2009}. However, the most commonly associated desmosomal junctional molecules do not seem to be involved in the precise junction association of keratin that we observed in the EVL early in development. We propose that the molecules involved in the initial cortical attachment and the initial junctional attachment differ from those that are involved later in development. This is further supported by our observation that EVPL and PPL mutants and MO do not show a defective keratin network in the EVL. It is also interesting to consider what signals regulate this precise spatiotemporal regulation of the keratin-desmosomal scaffold, potentially in keratin or an unknown upstream control-mediated manner. In the trophectoderm, it has been suggested that keratin expression is directly correlated to the nuclear accumulation of BAF transcriptional factors \cite{lim2020keratins}. However, a mosaic of transcription factors from the Kruppel-like, p53 family and Sox genes are involved in the regulation of keratin expression \cite{kalabushevaKaleidoscopeKeratinGene2023}. In our work, we did not investigate any direct upstream regulation of keratin expression, but our observations point towards interesting mechanisms of precise expression and translational control for keratin. 
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We do observe that keratin expression is dependent on the force applied by the YSL onto the EVL. This we observed was as possible mechanism that could explain the dynamics of keratin maturation in the embryo. We noted that actomyosin contractility, both in the YSL pulling the EVL and the EVL itself, is responsible for the increased expression and maturation of the keratin network in an epiboly stage-independent manner. Lowering contractility by injecting myosin phosphatase into the ysl or into the EVL led to lower keratin fluorescence observed in the EVL. Additionally, we observed that ectopic force application with a pipette leads to reorganization and an increase in the fluorescence of keratin filaments. This increase is at a timescale that is much faster than any transcriptional or translational increase in keratin in the aspirated cell. Therefore, we propose it results from a direct restructuring of the network in response to stress. Mechanical effects on intermediate filament network maturation are relatively unknown in cells, but insights from their structure and organization give us hints about their functional abilities.
On the filament level, studies have shown the many notable properties of intermediate filaments, especially for the most basic level of its organization, the filament. Among cytoplasmic intermediate filaments, keratins and vimentin are filaments with functions that are similar to each other. Vimentin filaments are hyperelastic, with no plastic deformations seen even under large strains under direct mechanical loads \cite{schepersMultiscaleMechanicsTemporal2021}. Vimentin filaments are stiffer than keratin for strains of up to 0.15 and show no plastic deformation upon strain. Vimentins soften upon repeated stretching due to changes in their molecular structure. Keratins, on the other hand, though they perform similar functions, show very different mechanics already at the single filament level. Keratins undergo plastic deformation but maintain their mechanical properties under stress \cite{lorenz2023Keratinfilamentmechanics}. Even though the secondary structure of these intermediate filaments is conserved, which is valid for most members of the IF family, changes in the amino acid sequence give these filaments different surface charges and interactivity. For example, keratin filaments also do not change their mechanical properties as dramatically as vimentin filaments under physiological levels of ion concentrations\cite{lorenzLateralSubunitCoupling2019}. Vimentin forms compact tetramers that potentially favor $\alpha$helix unfolding rather than subunit sliding when stretched. Keratin filaments, on the other hand, are much more permissive to subunit exchanges and movement upon stress despite performing similar mechanical attenuation as vimentin filaments at the molecular level. It is important to note, although, that the properties of keratin at a filament level may not be similar to the mechanics of a keratin bundle being pulled. However, these observations are useful to understand the dynamics of these filaments under stress.  
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Intermediate filaments, unlike other cytoskeletal proteins, can exchange subunits throughout their length rather than specific sites at the ends of the filament \cite{colakoglu2009Intermediatefilamentsexchange}. Subunit exchange, allowed by the chemical properties of the core protein structure of keratin together with the ability of intermediate filaments to exchange subunits within length, could explain a mechanism for the mechanical recruitment of new subunits within a bundle. As a keratin filament is stretched, new sites are opened up in the filament where subunits in the cytoplasm could exchange and be added into the filament. The ability of keratin intermediate filaments to respond and restructure on force application is crucial to this ability, but examples of how keratin bundles respond to stress in vivo are limited. Pulling forces applied ectopically with a pipette could lead to rapid reorganization of the keratin network, possibly by incorporating new monomers from the cytosol into the bundled filaments upon force application.
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Direct interaction of the actin cytoskeleton and the keratin network is mediated by plakin proteins as well. In mammals, these proteins include BPAG1, desmoplakin, plectin, envoplakin, periplalkin, epiplakin, and MACF1. They act as direct linkers and affect the mechanics of all the cytoskeletons in vivo. Desmoplakin, as discussed above, is a crucial component of the desmosome and forms its most abundant component \cite{mueller1983Biochemicalimmunologicalcharacterization}. In mice, the loss of desmoplakin leads to embryonic developmental failure due to dissociation of extra-embryonic tissues (ectoplacental cone, EPC; extra-embryonic ectoderm, EXE; visceral endoderm, VE; and parietal endoderm, PE). But additionally, in the embryo, it also leads to defects in proliferation and \cite{gallicano2001Rescuingdesmoplakinfunction}. In the skin desmoplakin loss leads to lesser number of desmosomes as well as adherens junction formation. Consequently, actin reorganization to undergo membrane sealng during epithelial sheet formation is affected in these mice \cite{vasioukhin2001Desmoplakinessentialepidermal}. Envoplakin and periplakin are essential for micro-ridge stabilization by forming a ring around the growing actin microridges in the mucosal cells of zebrafish epithelium. The presence of these plakins is regulated such that keratin can be associated to these long epidermal protrusions and stabilize actin filaments in the ridges. Loss of either of these plakins leads to a destabilization of the micro ridges and a shortening of their length \cite{inaba2020keratins}. These proteins are also regulated in a dynamic manner by phosphorylation on all plakin proteins, especially at the C-terminal region, which are important for IF binding \cite{bouameurPlakinsVersatileFamily2014}. As mechanisms and interactions of plakins that could mediate these posttranslational modifications are not well understood, we could not verify the involvement of such interactions/mechanisms playing a dynamic, fast-acting role in keratin mechanosensation.  We did observe that envoplakin and periplakin MO did not have any siginificant effect on the accumulation of keratin filaments during epiboly. However, we did not probe the other plakin family proteins in their roles in keratin regulation, especially by actomyosin-mediated stress. Based on the flows of keratin seen in the YSL it is plausible to consider that keratin and actin cytoskeletons do interact through any of these potential plakin candidates or other unknown interactions.  
In the EVL, before the network is denser than 60\%-70\% filaments show wiggling movements that occur at lengthscales of a few nm. In cell culture such wiggles have been also seen in keratin bundles and have been associated to localized pulling by plectins \cite{nolting2014MechanicsIndividualKeratin}. Dynamic reorganization of keratin binding proteins, especially mediated by phosphorylation changes induced in response to mechanical stress, could be an interesting mechanism that is utilized by the embryo to regulate the maturation of the keratin network during development.
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Intermediate filaments have been shown to be interacting and dependent on other cytoskeletal systems in the cells, but evidence supporting the role of microtubules in organizing keratins had been limited.  In the zebrafish, EVL microtubules may be important in transporting and maintaining keratin on the junctions. We observed that nocadazole treated embryos showed an effect on the recruitment of keratins to the apical surface, with embryos treated with nocadazole before the start of epiboly showing a reduced keratin network. Additionally embryos treated with nocadazole leads to direct loss of keratins from the junctions as a complete network. Evidence supporting the interaction of microtubules with vimentin, on the other hand, is numerous \cite{schepersMultiscaleMechanicsTemporal2021,schaedel2021Vimentinintermediatefilaments}. Surprisingly, vimentin can directly bind to microtubules via electrostatic interactions \cite{schepersMultiscaleMechanicsTemporal2021}.Vimentin intermediate filament networks stabilize microtubules and prevent catastrophe when in culture \cite{schaedel2021Vimentinintermediatefilaments}.  This stabilization occurs due to direct mechanical transient interactions between the two cytoskeletons. In Retinal pigment epithelial cells, this interaction leads to templating for microtubules and more remarkable persistence of cell movements. Microtubules plus end grow on the vimentin filaments and can regrow on existing vimentin filaments if induced to reduce using nocadazole \cite{gan2016Vimentinintermediatefilaments}. Despite the lack of such evidence for keratin maintenance or growth, we provide some evidence suggesting a dual role of microtubules in regulating the keratin network. Microtubules may be involved in the recruitment of unknown plakin linkers on the apical cortex and junctions that regulate the interaction of keratins with these components of the cell.
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The various methods we utilized to study the effect of loss of keratins relied on the limited number of type II keratins in the zebrafish genome \cite{hoUpdateKeratinGene2022}. Keratin knockouts have been of interest in the field due to the direct evidence with keratin defects leading to epidermal lysis effects grouped together in the disease named Epidermla bullosa (EB). A host of different symptoms usually arising out of mutations in the keratin5/keratin17 genes have an epithelial-specific phenotype. A most interesting fact about the symptoms of these diseases is the fact that mutations in particular keratin lead to specific defects, which give further insights into their roles in physiology and development. However, this syndrome exemplifies the ability of keratins to compensate due to a significant amount of redundancy in their structural functions. For example in keratin8/keratin19 negative mice keratin7  and keratin 18 filaments form a compensatory network showcasing the robustness and felxibility of these networks \cite{vijayaraj2007KeratinTransgenicKnockout,tamai2000Cytokeratins819}. Previous reports have shown the ability of zebrafish genome to express homologus genes upon mutant mRNA degradation \cite{el-brolosyGeneticCompensationTriggered2019}.  Additionally given the interactive mechanisms seen in the EVL that show interactions of keratins with other cytoskeletal proteins in the EVL a stable knockout of keratins that abrogated the network entirely was necessary. A major challenge in understanding the specific role of loss of keratins has been the relatively mild effect on development shown by large-scale perturbations of keratins quite often due to extra embryonic tissues that support failing before developmentally critical failure points are reached. Most defects seen in early embryonic death upon knockouts of keratins can be attributed to stress-mediated damage as well as apoptosis triggered due to increased apoptotic signalling in the extra-embryonic tissues such as the placenta and yolk. Therefore that , we utilized methods that lead to an entire locus knockout for keratin or morpholino-mediated loss of keratin mRNA directly. With the large number of keratin Type I isoforms, despite keratin18 MO showing the critical defects due to loss of keratin network in the EVL, we resorted to knocking out keratin Type II. In contrast with the mouse embryonic structure, the extra-embryonic tissues are vital structurally and signaling-wise to zebrafish development starting at the beginning of embryonic development. 
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Loss of type II keratins keratin 8 and keratin 4 early during development in the zebrafish embryo leads to failure of bundle formation and network maturation despite the epithelium spreading and stress applied to the epithelium. A limited amount of keratin 18 filaments seen in the morphant and mutant embryos could be attributed to potential keratin isoforms crossbinding. However, we observed that loss of keratins via morpholinos or via CRISPR-mediated genome knockouts leads to delayed epiboly movements and fragility of the epithelium. Unfortunately, despite various different methods used to create mutants by single spot introductions, as well as whole locus deletions, stable lines could not be generated as none of the embryos survived. This underscores the importance of the keratin cytoskeletal network in early development in zebrafish. We propose that this difference in the crucial requirement of keratin early in development especially arises due to the differential structure of the zebrafish embryo. During epiboly, the EVL undergoes a dramatic increase in the surface area over a very short time, with limited cell divisions seen for most of this process. The yolk and EVL are tissues that directly line the embryo, serving as a physical boundary against which the cells develop. Consequently, the rapid development of the network and the criticality of the loss of keratins could be a direct effect of the unique requirements of the development of the first epithelial tissue in the zebrafish embryo.  
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 We observed that the breaking of the EVL happened most often coincidentally with the convergence of the body axis and the formation of the head. Suggesting that a potential mechanical function plays a critical role in epithelial fragility and developmental defects. This aligns with the idea in the field that keratins function primarily as a mechanical protection for the cell cortex and are most important in tissues under stress \cite{sivaramakrishnanShearStressInduced2009,duqueRuptureStrengthLiving2024}. Using laser cutting experiments to ablate the cell cortex, we observed that both with single keratin and complete network knockdowns, the recoil velocity of the tissue is increased. Keratins have also been known to increase the rigidity of the tissue \cite{lulevich2010single}. With our pipette aspiration experiments, we confirmed that the loss of keratins leads to a less viscous EVL. The recoil after cortical ablation is dependent on the contractility as well as the mechanical properties of the ablated medium \cite{mayerAnisotropiesCorticalTension2010}. Taken together, we hypothesize that the increased recoil could be a direct effect of the reduced viscosity of the EVL upon keratin loss. Hence, the effect on tissue properties is more critical for the mechanical changes in the EVL due to keratins. A question in the field of tissue mechanics relies on whether tissues behave viscously or elastically on a developmental timescale. Actomyosin is the primary force-generating system in embryonic tissues, with the recoils observed in viscoelastic tissues being primarily driven by active myosin force. Degrading myosin in the Drosophila wing hints toward the mechanical properties of tissues at timescales of ablations being elastic \cite{goldner2023Evidencethattissue}. However, our experiments as well as evidence from previous research suggest strongly that tissue properties are dominated largely by their viscous properties \cite{behrndt2012forces,mayerAnisotropiesCorticalTension2010}. A potential explanation for this distinction could be the presence of cytoplasmic intermediate filaments in zebrafish and C. elegans tissues, a distinct difference from Drosophila tissues. Indeed, the difference in tissue properties and recoil suggests that keratins are an essential determinant of tissue viscosity in embryonic tissues.
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 The reduced viscosity of the tissue was not sufficient to explain the delayed movement of the EVL. We also observed an additional effect on the oriented elongation of the EVL, which suggested a defective transmission of forces between EVL cells. During development, the EVL cells align as they stretch in the Animal-vegetal (AV) axis, with the longest axis of cells preferentially aligning towards the AV axis. This is a direct effect of the pulling force on the margin as increased YSL pulling mediated locally by caRhoA led to elongated cells in the region with increased contractility. However the embryos lacking any keratin network formation showed a reduced coordinated movements. In cell culture, the roles of intermediate filaments in collective migration are distributing forces through the migrating sheet\cite{de2018intermediate,gan2016Vimentinintermediatefilaments}. In wound healing assays in the developing embryo, this led us to understand the role of keratins in efficiently transmitting forces throughout the epithelium. In keratin morphants, although the healing epithelium is unperturbed, the forces and stretching of the EVL cells around the cell were limited to the wound edge. While in the control embryos, up to 3 cell layers could respond to the injury and move together to close it, in the keratin morphant embryos, the movements were limited to a single cell layer at the boundary. In stratified epithelia, it has been seen that the tissues can co-ordinate oriented migration as well as proliferation in a Rac dependent manner \cite{park2017Tissuescalecoordinationcellular}. Both of these experimental results we obtained show a direct role of supra-cellular intermediated filament networks in mediating force propagation over a tissue length scale. Desmosomal connections are likely crucial in mediating these force across cell boundaries. Identifying the interactions with the intermediate filament network would be critical in probing the supra-cellular mechanics of keratin intermediate filament networks. In experiments with monolayers which express keratin show the critical features that such a interaction could affect tissues \cite{duqueRuptureStrengthLiving2024,latorreActiveSuperelasticityThreedimensional2018}. In the EVL we see a massive coordinated movement of a squamous epithelium that is dependent on mechanical force transmission by an intermediate filament network.
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YSL is the mechanical driver exerting the majority of the forces driving epiboly movement and spreading of the EVL \cite{behrndt2012forces,campinhoTensionorientedCellDivisions2013}. Specifically the force is generated by flow of actin in the tissue. Keratins are expressed in the YSL at the shield stage (6hpf) once the network in the EVL is around the junctional transitional stage. It is interesting to note that the network initially arises as mini filaments similar to the initial EVL network. We observed a compressive flow that forms a thick band of dense filaments at the EVL, YSL margin. Keratin particles have been shown to travel on actin filaments before they are integrated into a network \cite{kolschActindependentDynamicsKeratin2009}. Our preliminary experiments have also shown an internal YSL (iYSL; under the epiblast) spanning network that forms over the entire yolk cell; however, it is much sparser than the network seen in the EVL or the external YSL (eYSL; the YSL seen at the front of the migrating embryo). The connection at the margin is most likely mediated through segregated desmosomal proteins at the margin
\cite{goonesinghe2012desmosomal}. This could suggest an accumulation of desmosomal plaques at the margin, which is also supported by the blastodisc detachment phenotype exhibited by desmosomal knockout embryos. With keratin morphants, however, we did not observe such detachments. This is despite the numerous pieces of evidence suggesting the bidirectional cross-regulation of desmosomes and keratin recruitment \cite{loschkeKeratinIsotypesControl2016,buchauBidirectionalRegulationDesmosome2022}. Specific keratin isoform binding to desmosomes is associated not just with the strength of binding by desmosomes, but also with their number. An interesting observation seen from the YSL keratin network is unlike the EVL network the filaments don't associate into long bundles rather a denser less ordered filament system. The evident junctional keratin associated with the EVL margin is seen instead as a band similar to the distribution of other junctional components in the YSL such as tight junction proteins. This suggests that desmosomal proteins could be organized in a similar manner in the YSL as ZO proteins. However, this network perturbation by YSL-specific knockout of keratins leads to slower EVL movements. This is due to direct perturbation of the actin flows as keratin are expressed in the YSL. Interstingly what is seen among these flows is a lack of alignment and more variance in their directionality, in direct parallel to the alignment defects seen in the cells. However, the direct cause and hence control of these flows are so far unknown. Keratins may be a key factor playing a role in the mechanics of these flows, perhaps acting as a frictional substrate interacting and affecting the actin cortex via plakin linkers. 
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The Actin band in the YSL increases in intensity and thins as the EVL is pulled during epiboly \cite{behrndt2012forces}. This is mediated through recruitment of actin specifically to the YSL band as the band moves over the yolk. We observed no significant differences between the thickness of the YSL band as would be expected from the defects in the flows. However, our pipette aspirations on the YSL demonstrated that the recruitment of actin in response to force is dependent on keratin.  Without the presence of keratin in the YSL, actin cannot be accumulated in the YSL efficiently, and the intensity of the band after aspiration is reduced. Similar to other cytoskeletal proteins and tight junction proteins, keratins also flowed with actin, but the speed of these movements was halved compared to that of actin. This could suggest a role of keratins as a frictional substrate for the flowing actin cortex.   
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