\chapter{Results}
This thesis section summarizes the developmental, mechanosensitive, and intercytoskeltal interactions that the keratin network shows during zebrafish early development. Chapter 3.1 contains the publication titled "Keratins coordinate tissue spreading by balancing spreading forces with tissue material properties"
\section{Mechanosensitive regulation of keratin organization co-ordinate tissue spreading}



This section  Chapter 3 section 1 appears in full in the reference \cite{Naik2025.02.14.638262} and is covered by the CC BY ND 4.0 copyright. 

Author Contributions:
S.N., E.H. and C.-P.H. designed the research. S.N. performed the experiments and analyzed the experimental data. S.H. and Y.E.K. performed numerical simulations and modeling. K.P. provided help with reagents, and support with experiments. S.N., Y.E.K, S.H. and C.-P.H. wrote the manuscript. All authors edited the manuscript.

\bibentry{Naik2025.02.14.638262}
\newpage
\input{Results/Paper.tex}
\newpage

\section{Keratin promotes persistence of AJs and TJs}

Keratin morphants and mutants both showed large-scale defects in tissue properties and epithelial integrity. To test whether this effect was due to the loss of junctional components, we observed the localization of some adherens junction and tight junction components in the EVL after knocking down keratin 4/8. We utilized the keratin morpholinos to knock down both  m keratin 4/8 and observed changes in fluorescent protein fused junctional constructs localization in the EVL.
\subsection{Adherens junctions are dependent on keratin accumulation}
\par
For changes in adherens junctions, we utilized an Ecad knock-in line visualizing the endogenous Ecadherin via fluorescence (Fig 3.1 A). Ecadherin fluorescence intensity measured via live imaging Tg(mlanYFP)xt17 cdh1-YFP embryos during epiboly initially decreased until the shield stage (6 hpf) but then steadily increased as epiboly progressed (Fig 3.1 B). We observed that the fluorescence intensity of E-cadherin increased further once blastopore closure occurred (Fig 3.1 B). In keratin morphants, however, this increase was not observed (Fig 3.1 A and B). Instead, the keratin morphants showed an initial decrease similar as in control, followed by maintenance of a lower fluorescence intensity than that of the corresponding time points in the control. However, after blastopore closure, all junctional association of Ecad was lost in the EVL cells (Fig 3.1 B). This loss of Ecad localization preceded the break of epithelial integrity observed in morphants during the somitogenesis stages of development.
\begin{figure}[h!]
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/Ecad intensity.pdf}
    \caption{Ecadherin expression in the EVL upon keratin knockdown}
    A Max intensity projection images of Ecad expression in Tg(mlanYFP)xt17 cdh1-YFP embryos upon control MO (top) and keratin 4/keratin8 MO (bottom) injection over developmental stages. Scale bar 100 µm. \newline
    B Plot of average Ecadherin intensity within the epiblast as a function of time during development (4.5hpf to 20 hpf) in control MO (green) (N=4, n=6 embryos) and keratin MO (orange)(N=4, n=7 embryos.) injected Tg(mlanYFP)xt17 cdh1-YFP embryos. Error bars as ribbons SD of mean.
    \label{fig: Ecadherin intensity}
\end{figure}
\par
The majority of the initial decrease could be attributed to the decrease of Ecad accumulation at the YSl as reported before \cite{schwayer2019MechanosensationTightJunctions}. However, the loss observed in the morphants later in development was specific to the loss of junctional accumulation in the EVL. This decrease did not coincide with the global breakdown of epithelial integrity observed in the EVL, but could potentially play a role in the initiation of local junctional breaks that occured as the epithelium is stretched. 
\par It is interesting, however, that these defects in integrity and loss of adherens junctions are observed only later during development, while the effects observed due to loss of keratin have direct mechanical effects early during development with catastrophic consequences. 
\par
\subsection{Tight junction components require keratin for junctional association}
Tight junctions play a crucial role in maintaining epithelial integrity, forming a barrier that helps maintain homeostasis. Tight junction components accumulate increasingly in the EVL as epiboly progresses. Tight junction proteins accumulate during epiboly, with the expression of their components observed as apico-basal polarity is established in the forming EVL \cite{schwayer2019MechanosensationTightJunctions,koppen2006Coordinatedcellshapechanges}. \par We observed that some tight junction components were mislocalized upon keratin knockdown in the embryo. We observed that OccludinB-GFP fluorescence measured in TgBAC(oclnb-GFP)pd1126 upon control MO injection (Fig 3.2 A). Occludin B fluorescence, during epiboly, increased on EVL-EVL junctions, as epiboly progressed. Occludin B fluorescence was negligible before initiation of epiboly, regarding the maternal deposition observed in the yolk. Some embryos also showed an additional accumulation of Occludin in the nucleus; however, this accumulation was not consistently observed among different clutches of embryos from the same parents (data not shown). In keratin morphant embryos, the accumulation of Occludin B on the junctions was perturbed, and junctions lacked any Occludin B junctional accumulation (Fig 3.2 A and B). The yolk maternal deposition and the nuclear accumulation were unperturbed in the morphants. 
\par
\begin{figure}[h!]
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/OccludinGFP.pdf}
    \caption{Occludin-GFP expression in the EVL upon keratin knockdown}
    A Max intensity projection images of Occludin B expression in TgBAC(oclnb-GFP)pd1126 embryos upon injection of control MO (top) and keratin 4/keratin8 MO (bottom) embryos over epiboly stages. Scale bar 100 µm. \newline
    B Plot of average normalized Occludin B intensity within the epiblast as a function of time during development (4.5hpf to 10 hpf) in control MO (green)(N=3, n=5 embryos) (nand keratin MO (orange)(N=3, n=6 embryos) injected TgBAC(oclnb-GFP)pd1126 embryos.  All curves were normalized to measure the increase over the initial intensity. Error bars as ribbons SD of mean.
    \label{fig:Occludin intensity}
\end{figure}
\par ZO1 localization, on the other hand, was unperturbed upon keratin knockdown.


The lack of occludin but not ZO1 junctional accumulation during epiboly suggested a perturbation of junctional barriers, but not complete loss of barrier integrity upon keratin loss in the EVL. This perturbation underscores the critical role of keratins in maintaining epithelial barrier integrity beyond mechanical resilience. In keratinocytes, it has been suggested that this interaction with barrier proteins depends indirectly through changes in resilience leading to formation of actin stress fibers \cite{karschIntactKeratinNetwork2020}. In the EVL, such fibers are unseen. However, the clear junctional association of the rim network could play spatio-temporally regulated roles in junctional maintenance. 

\newpage

\section{Microtubular and Actin interactions stabilize keratin networks}
Interactions with the microtubular cytoskeleton have been extensively studied for vimentin intermediate filaments, with even evidence for direct transport and interactions with microtubule networks \cite{schaedel2021Vimentinintermediatefilaments}. However, such interactions with microtubules have not been observed for keratin intermediate filaments. In the EVL, we observed evidence for keratin organization and structure dependent on the organization of microtubules. 

\subsection{Nocadazole treatment leads to disorganized keratin network organization}
We detected keratin expression in embryos subjected to 100 µM nocodazole treatment either prior to the onset of epiboly or at the shield stage (6 hpf), when the keratin network was more organized. Disruption of microtubules at early stages (3.5 hpf) impaired the temporal progression of keratin network assembly, interfering with initial minifilament formation as well as keratin enrichment at cell–cell junctions and around the nucleus (Fig 3.3 A). Using Dclk-GFP as a microtubule marker, we confirmed the efficacy of nocodazole treatment, which led to a rapid loss of Dclk signal in the EVL and a more gradual disassembly of the microtubule network in the YSL (Fig 3.3 A). 
\par
\begin{figure}[h!]
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/Nocadazolesp5_2.pdf}
    \caption{Keratin structure upon dissolution of microtubules before epiboly initiation}
    A Max intensity projection images of keratin 18 expression in Tg(krt18:krt18-GFP) embryos upon treatment at 3.5 hours post-fertilization with DMSO control (top) and Nocadazole (middle) embryos over epiboly stages. Max intensity projection of microtubule expression in Tg(XlEef1a1:dclk2-GFP) embryos upon treatment with Nocadazole  (bottom) over epiboly stages. Scale bar 100 µm.\newline
    B Plot of average keratin intensity as a function of time (hours post fertilization) in Tg(krt18:KrtGFP) control (green, N=4, n= 5 embryos) and Nocadazole treatment (orange, N=4, n=6 embryos)
    \label{fig:Keratin Early microtubule disruption} 
\end{figure}
Despite these cytoskeletal perturbations, total keratin expression levels remained comparable to those of untreated controls (Fig 3.3 B). These findings indicate strongly that the increase of keratin during epiboly is largely determined by the mechanical feedback to the stress faced. However, through these experiments, we observed unique changes in the keratin network structure at the filament scale, suggesting that microtubules may be essential for organizing the network into the classical rim and spoke structure of intermediate filaments in the EVL.
\par
Microtubule dissolution after shield stage, however, showed an arrest of network maturation dependent on the level of expression of keratin in the cells (Fig 3.4). Cells exhibiting a higher expression and consequently a higher densly matured network progression was as observed in control cells. However, cells with a sparse network and limited distribution of keratin showed an arrested, delayed maturation, with some cells showing thick bundles that failed to branch and percolate throughout the cell.   
\par

\begin{figure}[h!]
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/Nocadazolelate.pdf}
    \caption{Keratin structure upon dissolution of microtubules after shield stage}
    A Max intensity projection images of keratin 18 expression in Tg(krt18:krt18-GFP) embryos upon treatment at 5.5 hours post-fertilization with DMSO control (top) and Nocadazole 20 µM(middle) embryos over epiboly stages. Scale bar 100 µm.
    \label{fig:Keratin late microtubule disruption}
\end{figure}
Most notably, both of these treatments lead to peculiar holes at the nucleus in the network. These holes were created most prominently after 80\% network density was reached, where the network looked percolated on the apical surface. Additionally, when microtubules were dissociated early in epiboly, the junctional association of keratin was perturbed.
\subsection{Junctional keratin association is perturbed upon microtubule dissociation}
We observed that early dissolution of microtubules leads to perturbed network dynamics, especially during the transition from the minifilament stage to connected filaments and further on to the junctional transition. We observed a keratin expression level-dependent perturbation of filament formation. Cells that would show a greater amount of keratin expression showed a similar initial network density as those seen in similarly expressing control cells. However, cells that were lower in expression showed perturbed minifilament formation at the apical surface. The network progression from minifilaments to longer filaments in higher-expressing cells showed thicker bundles that did not mature into denser networks at the same dynamics as seen in control cells. 
\begin{figure}[h!]
    \centering
    \includegraphics[width=1\linewidth]{graphics/InitialKeratinNetwork.pdf}
    \caption{Keratin structure upon dissolution of microtubules after shield stage}
    \label{fig: Keratin late microtubule disruption}bryos
    A.  Max intensity projection images of keratin expression in Tg(krt18:krt18-GFP) emat early stages with DMSO-treated controls (top) and Nocadazole treatments(bottom). A cluster of cells for each condition are followed from initial minifilaments to network before junctional transition from 4hpf to 5hpf.  Scale bar: 25 µm
\end{figure}
\par
All cells showed keratin expression as lower intensity cells showing bigger filaments without characteristic initial minifilaments at the apical surface being observed. Instead these cells showed a sparse network of thick filaments. On average upon nocadazole treatment the networks showed reduced connectivity and slower network maturation dynamics (Fig 3.5). An interesting observation to note was that the movements of the filaments were exaggerated as compared to the controls, which could arise due to perturbed keratin attachments in a microtubule-dependent process. 
\par
\begin{figure}[h!]
    \centering
    \includegraphics[width=1\linewidth]{graphics/junctionalflow.pdf}
    \caption{Keratin structure upon dissolution of microtubules after shield stage}
    \label{fig: Keratin late microtubule disruption}bryos
    A.  PIV flow graphs on consecutive frames of a junctional flow event of keratin filaments in Tg(krt18:krt18-GFP) upon early Nocadazole treatments. Keratin filaments are shown in grey wherease PIV flow vectors are represted as yellow arrows. Each frame represents the instaneous flow in 2 min intervals as the network clears in the cell.
\end{figure}
\par
Additionally, we also observed perturbed junctional association of keratin filaments when microtubules were disrupted. Junctional filaments showed larger deviations, exhibiting undulating filaments along the junctions upon nocadazole treatment as compared to control embryos. Occasionally, we also observed disruptions of junctionally associated filaments or dynamic movements of the filaments towards the contractile junctions (Fig 3.6). These events lead to a collapse of the formed filaments towards the junction. This, however, was only observed in early treatments, suggesting a temporal dependence on this interaction with the microtubules. Once the network was formed, maintenance was not dependent on microtubules; however, its maturation and localization could be regulated by a microtubule-dependent process. 

\subsection{Nuclear keratin cage regulated by a microtubule dependent process}
To further understand the nuclear hole phenotype, we imaged keratin filaments at high spatiotemporal resolution (Fig 3.7). Interestingly, we observed the nuclear holes in most cells regardless of the amount of keratin. The holes appeared most often after 8 hours of development, when the network was well developed.
\begin{figure}[h!]
    \centering
    \includegraphics[width=0.75\linewidth]{graphics/NuclearClearing.pdf}
    \caption{Keratin structure reorganization leading to clearing around the nucleus upon microtubule dissolution}
    A Maximum intensity projection image of keratin expression in Tg(krt18:Krt18GFP) embryos upon nocadazole treatment, showing the nuclear outline as a dashed line.  Scale bar: 25 µm
    \label{fig:Keratin late microtubule disruption}
\end{figure}
\par
We hypothesize that as the cells flattened, the filaments could be pushed away from the nucleus. As the cells flattened, the nucleus could be seen deforming the filament structure in the vicinity. Filaments were directed to the vicinity with deformations, rearranging filaments to the periphery of the nucleus instead of organizing over the nucleus as in control embryos. The reorganization time scale and tracking movements of the filaments around the nucleus suggested a direct contractility and physical occlusion of filaments around the nucleus. This suggests that currently unknown microtubule-mediated processes are involved in the nuclear lamina attachment of keratin filaments. \par
Intermediate filaments have long been known to work synergistically with other cytoskeletal systems both in the cytoplasm and in the nucleus. In the nucleus, these interactions are mainly made through nesprin proteins that mediate direct interactions with the nuclear lamina\cite{herrmann2016IntermediateFilamentsStructure, noegelRoleNesprinsMultifunctional2011}. We hypothesize that the localization of nesprins could be microtubule-dependent in the later stages of epiboly. 