\chapter{Material and Methods}
\section{Experimental model and Subject Details}
For the experiments mentioned in this thesis, zebrafish (Danio Rerio) were maintained in the aquatics facility at ISTA with the help of the aquatics facility. Fish were maintained according to local animal ethical regulations regarding space and animal ethics (Breeding 2023-0.288.351). All experiments were done on embryos collected from parents that were set up together the evening before the specified developmental stages. All stages studied in this thesis were within the first 36 hours of development. The embryos were collected in the morning, sieved through a thin mesh and kept in an incubator at $28^\circ C$. Staging was done both according to time from fertilization as well as known developmental stages according to the developmental atlas \cite{kimmel1995stages,westerfield2007zebrafish}. \newline Transgenic lines used in this thesis:
\begin{itemize}
    \item wild-type TL
    \item wild-type AB
    \item Tg(krt18:Krt18GFP)
    \item Tg(actb2:Utrophin-mcherry)
    \item Tg(actb2:Myl-mcherry)
    \item Tg(plakoglobin-GFP)
    \item Tg(actb2:Lifeact- GFP)
    \item Tg(acbt2:Utrophin-mcherry, krt18:Krt18GFP) 
\end{itemize}
\section{Analysis of keratin expression}
Pre-existing single-cell(sc) RNA datasets were analysed using URD dataset portal in the Broad Single-Cell Portal. The expression level and localization were confirmed using qPCR and Fluorescence in situ hybridization (FISH).
For qPCR, total RNA was extracted from 20 embryos at 1k (3.3 hpf), sphere (4 hpf), shield (6 hpf) and bud stage (9.5 hpf) using 750 µl Trizol by dechorionating the embryos followed by homogenizing by vortexing for 60-90 secs. 
The lyzed trizol samples were stored at -70 overnight so that all stages could be processed together. 
The RNA was isolated by adding 250 $\mu$l of methanol and mixing thoroughly. The suspension was incubated at RT for 5 mins before centrifugation at 15000 g for 30 mins at 4°C such that phase separation into 3 phases was seen. The aqueous phase was isolated by tilting the tube while carefully avoiding the interphase and organic phase and transferring it to another tube. The RNA was precipitated using 500 µl  isopropanol and incubation at RT for 10 mins. The suspension was centrifuged at 15,000 × g for 10 minutes at 4°C to pellet the RNA. The supernatant was discarded and the pellet was washed twice with 70 \% ethanol. The pellet was resuspended in 20 $\mu$l Rnase free water. Any contaminations of genomic DNA were cleared using DNA-free™ DNA Removal Kit (Thermo Fisher Scientific) according to the manufacturers protocol. cDNA libraries were then made from 3 $\mu$g of RNA from each sample using Luna RT Supermix kit. cDNA amplification was tested using serial dilution from 1:1-1:10000 to generate standard curves and amplification efficiency was tested by generating a standard curves. Based on 90\%-110\% efficiency 1:100 dilution was used for amplification by qPCR. 
For normalisation, the elongation factor 1 $\alpha$ (EF1$\alpha$), as a housekeeping gene, was amplified but the expression efficiency for EF1$\alpha$was not stable during different stages of development. \newline
The following primers for keratin(krt)4, 5, 8 and 18 were used:\newline
krt4 forward: GCAGTCTATGAGGCTGAACTCC\newline
krt4 reverse: CTCAGCCTTTGTTGAGCGGA\newline
krt5 forward: ACTTCCTTCAAAACCTTCACC\newline
krt5 reverse: CCAGATCCTGCTCCAAAAC\newline
krt8 forward: CCACCTACAGCAAGAAAACC\newline
krt8 reverse: AGAGATGAAGCCACTACCAC\newline
krt18 forward: GTAACATCCAGCATCAGACG\newline
krt18 reverse: CACAACCTTTCCATCCACC\newline
The qPCR runs were performed on  the Bio-Rad C1000 Thermal Cycler in triplicates using the Luna qPCR master mix.


\section{Generation of keratin Crispr Cas9 mutants}
To generate F0 crispant zebrafish embryos with targeted knockouts of keratin4 (krt4) and keratin8 (krt8), we employed a CRISPR-Cas9 approach utilizing the Alt-R CRISPR-Cas9 system from Integrated DNA Technologies (IDT). For each gene, three distinct guide RNAs (gRNAs) were designed to target separate exons, ensuring comprehensive disruption of the gene's coding sequence. The specific gRNA sequences used were as follows:
\begin{enumerate}
    \item Keratin 4 (krt4)
    \newline 5'-[sequence]-3'
    \newline 5'-[sequence]-3'
    \newline 5'-[sequence]-3'
    \item Keratin 8 (krt8)
    \newline 5'-[sequence]-3'
    \newline 5'-[sequence]-3'
    \newline 5'-[sequence]-3'
\end{enumerate}
Each gRNA was prepared by annealing 1 $\mu$L of crRNA (200 µM) with 1 $\mu$L of tracrRNA (200 $\mu$M) in 1.5 $\mu$L of IDT's Duplex Buffer. The mixture was heated to $95^\circ C$ for 5 minutes and then cooled on ice to facilitate annealing. Subsequently, ribonucleoprotein (RNP) complexes were formed by combining the annealed gRNAs with Cas9 protein in equimolar ratios (1:1) and incubating at $37 ^\circ C$ for 15 minutes. For each gene, the injection mixture contained a total of 28.5 fmol (1000 pg) of the gRNA trio (approximately 9.5 fmol per gRNA) and 28.5 fmol (4700 pg) of Cas9 protein.

One-cell stage zebrafish embryos were microinjected with approximately 1–2 nL of the respective RNP complexes targeting krt4 or krt8. Post-injection, embryos were maintained at $28.5^\circ C$, and daily assessments were conducted to monitor survival rates and document phenotypic manifestations indicative of successful gene disruption. This methodological approach was adapted from established protocols for CRISPR-Cas9-mediated gene editing in zebrafish.

\section{Cloning of expression constructs}

Gateway and Gibson assembly were used to create pCS2 expression vectors from cDNA of zebrafish keratin genes. 
Total RNA was extracted from 20 WT embryos at 4 and 8 hpf after dechorionation using 750 $\mu$l Trizol (Invitrogen).  The cDNA library was generated with the Superscript III reverse transcription kit following the manufacturer’s instructions. The coding region of zebrafish krt 18 was isolated using the following primers:
\newline forward: \seqsplit{5'GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGTCTGAGAACAAGCTACAGCG3'}
\newline reverse: \seqsplit{5'GGGGACCACTTTGTACAAAGAAAGCTGGGTTTTAAAGTTTCCTCTCCTTGGTTTCTGTGC3'}\newline
The dominant negative (DN) version of krt18 was generated by mutating the Arginine to Cysteine at position 93 using the following primers:
\newline forward: \seqsplit{5'CATGCAGAACTTGAACGACTGTCTGGCCTCCTATCTGGAG3'}
\newline reverse: \seqsplit{5'CTCCAGATAGGAGGCCAGACAGTCGTTCAAGTTCTGCATG3'}
The template DNA was then digested using dpn1 and the cDNA fragments were cloned into a pDEST plasmid using Gateway cloning. After transformation in NEB 5-alpha Ecoli strain  in a pCS2 plasmid,  the clones with the correct sequences were selected using sequencing.
For krt 8 and 4, the genomic fragments were isolated from cDNA libraries obtained from RNA of 8 hpf embryos as described above. The following primers were used to isolate specific DNA fragments:
\newline krt8 forward: \seqsplit{5'GCATGGACGAGCTGTACAAGAAGACAGAAAACACACAAGGCAGGATGAGTACG3'}
\newline krt8 reverse: \seqsplit{5'GCTGGTTTTCTTACTATACGTACTCATCCTGCCTTGTGTGTTTTCTGTCTTCTTG3'}
\newline krt4 forward: \seqsplit{5'GGCATGGACGAGCTGTACAAGCTCAAAGACACGGGGATCATGTCGACGCGCTCTATCTCT3'}
\newline krt4 reverse: \seqsplit{5'GTAATACGACTCACTATAGTTCTAGAGGCTTAATAGCGTTTACTGCTGACGGTGG3'}
\newline
The PCR products were then integrated to generate entry vectors via recombining with pDONR(P1-P2) (Lawson$\#$208) and the entry clone was further recombined with pCS-N-term-mEmerald (Lawson $\#$223) or pCS-N-term-mCherry (Lawson $\#$362) destination vector (krt4-mcherry, krt8-mcherrry, krt4-mEmerald, krt8-mEmerald) or p3E mNeonGreen, pCS2-Dest (Lawson $\#$444) for C-terminal tagging (DNkrt18).mRNA for expression was trasnscribed using the SP6 mMachine kit (Ambion).
\section{mRNA and morpholino injections}
Morpholinos were obtained from Gene Tools, LLC. Morpholino sequences were obtained from previously published research as described in \cite{pei2007early}.\par
krt4 MO: \seqsplit{5'AGACCTGGTTGACATGATGCCTGTG3'} \par
krt8 MO: \seqsplit{5'GGTTTTCTTGCTGTAGGTGGACATC3'}\par
krt18 MO: \seqsplit{5'TGTAGCTTCTTCTCAGACTCATGGT3'}\par
FoxH1 MO: \seqsplit{5'TACTTAACCCTACCTCTGATAAAGT3'}\par
human beta globin MO: \seqsplit{5'CCTCTTACCTCAGTTACAATTTATA3'}\par
The morpholinos targeting keratins were translation blocking MOs and prevented formation of protein from the spliced sequence. For FoxH1 MO, a splice-disrupting MO was selected. Based on sequence similarity using BLAST, each of these MOs showed specificity to their respective transcripts and the morphology of the keratin network was assessed as an additional factor to control the effect. A gradient of concentrations from 0.5 ng to 3.0 ng of each MO was tested to observe the phenotype and any off-target effects. For experiments involving keratin 4 and keratin 8 MO knockdowns 1ng of each was injected into the embryo at one cell stage (near the animal pole) or at high stage (into the YSL). For experiments involving keratin 18 and FOXH1 knockdown 3ng of MO was injected at one cell stage. The same concentration as the keratin MO of human beta-globin MO was injected as a control. 
For this thesis, keratin 8, keratin 4, keratin 18, and dn-keratin 18 constructs were made from zebrafish cDNA. cDNA was prepared using Trizol (Invitrogen) based total RNA extractions from 20 WT embryos at 4 and 8 hpf after dechorionation. 750 $\mu$l of Trizol per 20 embryos was used for extraction. Luna RT Supermix was used to generate cDNA libraries from the total RNA extracted. The primers used for cloning the  cDNA of splice zebrafish transcripts are:\par
krt18 forward: \seqsplit{5'GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGTCTGAGAACAAGCTACAGCG3'}
\newline
krt18 reverse: \seqsplit{5'GGGGACCACTTTGTACAAAGAAAGCTGGGTTTTAAAGTTTCCTCTCCTTGGTTTCTGTGC3'}
\par
krt8 forward: \seqsplit{5'GCATGGACGAGCTGTACAAGAAGACAGAAAACACACAAGGCAGGATGAGTACG3'}
\newline
krt8 reverse: \seqsplit{5'GCTGGTTTTCTTACTATACGTACTCATCCTGCCTTGTGTGTTTTCTGTCTTCTTG3'}\par
krt4 forward: \seqsplit{5'GGCATGGACGAGCTGTACAAGCTCAAAGACACGGGGATCATGTCGACGCGCTCTATCTCT3'}\newline
krt4 reverse: \seqsplit{5'GTAATACGACTCACTATAGTTCTAGAGGCTTAATAGCGTTTACTGCTGACGGTGG3'}\par
The dominant negative construct for keratin 18 was created from the cDNA for keratin 18 by mutating the arginine at the 93rd position to cysteine. The mutagensis was confirmed by sequencing the construct. Primers used:
\par
dnkrt18 forward: \seqsplit{5'CATGCAGAACTTGAACGACTGTCTGGCCTCCTATCTGGAG3'}
\newline
dnkrt18 reverse: \seqsplit{5' CTCCAGATAGGAGGCCAGACAGTCGTTCAAGTTCTGCATG3'}
\par
The PCR products were then integrated to generate entry vectors via recombining with pDONR(P1-P2) (Lawson $\#$208) and the entry clone was further recombined with pCS-N-term- mEmerald (Lawson $\#$223) or pCS-N-term-mCherry (Lawson $\#$362) destination vector (krt4- mcherry, krt8-mcherrry, krt4-mEmerald, krt8-mEmerald) or p3E mNeonGreen, pCS2-Dest (Lawson $\#$444) for C-terminal tagging (DNkrt18). Expression constructs were created using the pCS2 such that RNA could be produced using the sp6 promoter in the plasmid. RNA for injection was made using the SP6 mMessage mMachine Kit (Ambion). \par
Both mRNA and MO were injected using needles pulled from glass capillaries  (30-0020, Harvard Apparatus) pulled using a needle puller (P-97, Sutter Instruments). Microinjection system (PV820, World Precision Instruments) was used to inject according to the method described before \cite{westerfield2007zebrafish}. For YSL injections, injections were done directly into the newly formed YSL at high-stage (3.3 hpf) through the yolk. For keratin 4 and keratin 8 mRNA injection concentration of mRNA was limited to 25 pg each as anything over 50 pg lead to abnormal keratin network formation with thicker than WT filaments and incomplete divisions being seen.
For CARhoA injections, 0.5-1pg CARhoA together with 2pg H2A-mCherry mRNA were injected into marginal blastomeres at the 128-cell stage. For CAMypt injections, 100pg CAMypt mRNA together with 0.2\% phenol red was injected into the YSL at high-stage (3.3 hpf). For CARhoA injections analysis was restricted to the region where mCherry labelled nuclei were clearly seen.

\section{Sample preparation and fluorescence imaging}
For this thesis, imaging was done on the Leica TCS sp5 (upright and inverted), Leica TCS SP8 (upright and inverted), Andor laser cutter (inverted), Nikon CSU-W1 (inverted) and Leica Stellaris 5 (inverted) systems. Before imaging the embryos were dechorionated and incubated at $28^\circ C$ until they were imaged. The embryos were mounted laterally or animally in  0.3\%–0.5\% low melting point (LMP) agarose in E3 (Invitrogen) on glass bottom dishes (MatTek) for inverted imaging. For upright imaging, the embryos were embedded in wells made from moulds made from 3\% agarose and covered with 0.5\%-0.6\% low-melting point agarose. Fixed samples were used for imaging immunostaining and FISH. Images were acquried with upright microscopes mentioned above using HyD S detectors (410 – 850 nm, tunable detection with 1 nm step, min. range = 5nm). 


\section{UV ablation/cutting}
Two types of cuts were made using the UV ablation set up: UV cortical laser ablation to measure the recoil and UV ablations to study the tissue wound recovery after ablation of a set number of cells. 
UV cortical laser ablations were performed on the YSL actomyosin ring, EVL cell-cell/cell-YSL junction ($<30 \mu m $) and EVL tissue ($>50 \mu m)$ at $28.5^\circ C$ maintained by a stage incubator. UV cortical laser ablation were performed on a Andor SD laser cutter system with a 63x 1.2 NA water immersion lens on Tg(actb1:GFP-utrCH), Tg(actb1:myl12.1-eGFP/mCherry) and Tg(actb1:Utr-mCherry) emrbyos. Cortical laser ablations were carried by applying 25 ultraviolet pulses at 100 Hz at 2 $shots/ \mu m$ along a line parallel and perpendicular to the EVL margin. Fluorescent images were acquired with a iXon DU-897-BV camera (Andor Technology) at frame rate of 0.5 sec/frame. Recoil velocity of the cortex after laser ablations was measured using particle image velocimetry (PIV) and kymographs made on a linear roi with $10 \mu m$ thickness at the centre of the ablation. To asses the initial recoil the temporal average recoil was measured as an average of the PIV flow in a rectangular roi over the length of the wound with a thickness of half the wound length was measured. Under these parameters as observed previously on this setup \cite{behrndt2012forces}, about 60 \% of the cuts were clean cortical openings with minimal changes in the actin/myosin accumulation around the wound while another 40 \% showed an apparent accumulation of actin/myosin or a what is observed as a sinking of the wounded area immediately upon ablation. \par
For EVL tissue $>50 \mu m$ ablation, it was also observed that the wound response was localized to specific cells/cell junctions along the wound length. Junctions showed a higher incidence of wound-responsive actin accumulation, while the surrounding cortex recoiled relatively uniformly. Recoils upon tissue ablations were measured using PIV tissue flow. \par
EVl cell extrusion upon wound healing was used to induce an anisotropic tension actin ring similar to the YSL locally. Line cuts over 3/4 cells were performed at higher than cortical laser ablation UV laser intensities to cause extrusion out of the epithelial layer. The cuts were $80-100 \mu m$ long and led to the simultaneous extrusion of 5-7 EVL cells.  Experiments were performed on the lateral side of the embryo between the EVL margin and the animal pole of Tg(krt18:krt18-GFP, actb2:Utr-mCherry) injected with control MO or Keratin MO at one cell stage. The cuts lasted a total of 2 sec while acquisition was stopped. Actin ring was observed as soon as the acquisition started and up to 3 seconds after the cut the actin ring continued to accumulate. The tissue flow was measured using PIV over the un-ablated region. Cell orientations were measured using the orientation of the longest axis of the cells segmented using Cellpose and Fiji \cite{pachitariu2022cellpose}. A cellpose model was trained using the pre-trained CPx model on the first frame after the ablation of 10 movies and used to segment the movies. 

\section{Pipette aspiration}
Pipette aspirations were used to measure the mechanosensitive properties of the keratin network and the material properties of the EVL. Pipette aspirations were carried out on embryos mounted in 3\% methylcellulose in E3 on an inverted Leica SP5 or a Leica Stellaris 5 confocal microscope equipped with the micropipette aspiration system. Micromanipulators(TransferMan Nk2, Eppendorf) were used to control the micropipette position, and a pump controlled by Microfluidic Flow Control System pump (Fluiwell, Fluigent) and the Dikeria micromanipulation software the pressure applied at the end of the pipette. Fire-polished and heat-inactivated FBS passivated micropipettes (Biomedical Instruments) with an inner diameter of $60 \mu m$, 30° bent positioned by micromanipulators (TransferMan Nk2, Eppendorf), and with a blunt end were placed on the EVL around 3-4 cells away from the EVL margin. For creep-recovery experiments, negative pressure of 200 mbar was applied until the aspirated tissue flowed in the pipette with constant velocity for ~5 mins, followed by pressure release until the tissue reached the end of the pipette. A single z-plane was acquired in the central plane of the pipette every second. The leftmost dark pixel was tracked with a line ROI in Fiji and a custom-made macro script and the length of the aspirated tissue was plotted as a function of time to measure the aspiration and retraction speed \cite{guevorkian2017micropipette}.
\par
Aspirations were performed under constant pressure to measure the mechanosensitive keratin response in Tg(krt18:krt18-GFP) injected with CAAX-mCherry and Tg(krt18:krt18-GFP, actb2:Utr-mCherry). The mean intensity of keratin at the midplane of the pipette and a region of $ 30 \mu m$ around the aspiration site were measured through fluorescence using an ROI defined in Fiji. Intensities before aspiration and upon aspiration were measured for keratin, actin, and membrane intensities. A membrane labeled with CAAX was used as a nonresponsive control for the aspiration, whereas actin was visualized with Utrophin-mCherry.  
\par
For YSL aspirations, a z-stack of $50 \mu m$ was acquired from the pipette mid-plane towards the objective. The intensity of Utrophin as a measure of actin recruitment was measured in an ROI next to the pipette in Tg(krt18:krt18GFP, actb2:Utr-mCherry) injected with control MO or keratin MO at high stage into the YSL.  

\section{Analysis of actomyosin flows}
For high-resolution confocal imaging of YSL actomyosin flows within the YSL Tg(krt18:krt18-GFP,actb2: Utr-mCherry) and Tg(actb1:GFP-utrCH). Imaging of keratin filaments together with F-actin was done on Tg(krt18:krt18-GFP,actb2: Utr-mCherry) at 7 hpf when the keratin network in the YSL was least dense. Imaging was done Nikon CSU W1 spinning disc microscope with a 63x objective at a frame rate of 30 seconds. A 500 × 500-pixel region of interest (ROI) within the YSL was selected with maximum intensity projections centered along the AV axis close to the margin of the EVL. Due to the radial symmetry of epiboly, the 2D flows were reduced to 1D for speed measurement. The flows were measured in this ROI using PIVlab \cite{thielicke2021particle}. The alignment of these flows was analyzed by measuring the angular mean of the flow vectors using a custom Python script.
\section{EVL cell tracking automation}
EVL cells were tracked by using Tg(actb2: Utrophin-mCherry) or Tg(acbt2:Utrophin-mCherry, krt18:Krt18-GFP) transgenic embryos. Maximum intensity projections of Z-stacks approximately $150 \mu m$ depth (for imaging with sp8 25x) or $30 \mu m$ depth (for imaging with Nikon spinning disc microscope) were used to segment apical cell junctions of the EVL cells. Segmentation of the EVL cells were performed using the Cellpose segmentation software using "human in the loop" pipeline described in \cite{pachitariu2022cellpose}.
\par Segmentations obtained from cellpose were verified by visually correcting any mis-segmentations. These 2D segmentations were corrected for their 3D projection artifacts using 3D height maps made with local Z-projector in Fiji. The cell orientations were measured on corrected segmentation using Deproj software in Matlab \cite{herbert2021localzprojector}. Data analysis and plots were made in custom Python scripts. 

\section{Keratin network segmentation}
For measuring the network of the keratin cytoskeleton in EVL cells, Tg(krt18:Krt18GFP,actb2:Utr-mCherry) embryos were imaged on a Nikon CSU-W1 system with a 63x objective. The keratin network distribution was approximated to the apical distribution in z-projected images. The z-projected images were denoised with a gaussain filter and Python-based denoising to obtain a better signal-to-noise ratio for the filaments. These images were thresholded using an adaptive median threshold (Otsu) and the area fraction in Fiji covered by the network was used as an estimate for the network density inside the cell. 
\par Junctional keratin accumulation was measured on the cell margins using the cell outlines with a thickness of 3 pixels obtained from cell segmentations described above. The apical intensity was measured as the intensity of keratin inside the cell outline, excluding the junctional intensity. 

\section{EVL primary cell culture}
EVL primary cell cultures were performed as described in two different stages of keratin maturation at 3.5 hpf and at 6hpf \cite{arslan2024adhesion}. Tg(acbt2:Utrophin-mcherry,krt18:Krt18GFP) or Tg(krt18:Krt18GFP) embryos were transferred to pre-warmed (28.5–31°C) 0.9x DMEM/F12 medium supplemented with GlutaMAX and Penicillin-Streptavidin. The blastoderm caps were dissected from the yolk cells with forceps and transferred to 1.5 ml Eppendorf tubes using glass pipettes. \par 
These explants were dissociated by gentle tapping, seeded at 29°C and imaged for at least 4 hours after seeding. EVL cells were identified by their shape and the presence of keratin fluorescence.

