What is zo 1

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For the detection of the respective 6. Lysates were sonicated and centrifuged at 13, rpm for 20 min to obtain soluble fractions. Two fixation methods were used; paraformaldehyde PFA or methanol fixation.

The sections were quenched with 50 mM ammonium chloride and permeabilized with 0. These were subsequently incubated in primary antibodies and labelled with appropriate fluorophore-conjugated secondary antibodies.

Images were acquired using an Axio Imager. Dehydrated EBs were dried in a critical point dryer before being mounted and sputter coated with gold Leica. The specific flanking-primer sequences for each gene and expected amplicon size are listed in Fig.

EBs were pre-embedded in 1. Images were taken with an Axio Imager. A2 upright microscope coupled to an AxioCam HR camera. The ZO-1 Fig. The ZO-2 WT and mutant alleles were detected as 6. A Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 panel a and ZO-2 showing exons 2—3 panel b in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene green box and a loxP-flanked purple circle Neo cassette white box immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively.

The red bar indicates the position of probe hybridization for Southern blot analysis. B Genotypic analysis by Southern blotting. C Protein expression analysis. GAPDH served as a control for equal lysate input. The development of EBs is illustrated and described in Fig.

A Schematic diagram of EB development. By day 3 of culture, the outer cells differentiate into a circumferential epithelium known as the primitive endoderm PrEn. The EBs at this point are called simple EBs.

The PrEn also further differentiates in the visceral endoderm VEn. In conjunction with this, the interior ICM differentiates into the epiblast. By day 5 or 6, the epiblast in contact with the BM in turn differentiates into the primitive ectoderm PrEc or epiblast epithelium. The rest of the epiblast not in contact with the BM will undergo apoptosis. The apoptotic bodies are removed by autophagy-initiated phagocytosis, leaving a progressively enlarging lumen or Proamniotic-like cavity PAC surrounded by the apical domain of the PrEc and the basal BM.

EBs at this stage are called cystic EBs and equivalent to the egg cylinder stage of the mouse peri-implantation embryo just before gastrulation.

Nuclei are labeled with DAPI blue color. Magnification of image in insets. C Transmission electron micrographs. These results indicate that the apico-lateral localization of ZO-1 and ZO-2 proteins in the epithelium is independent of each other.

The intercellular space at this area appears obliterated, typical for TJ ultrastructure. Furthermore, this area appears to have an expanded intercellular space Fig. Cldn-6 was chosen as a representative of the Cldn family since it is an EB early epithelialization marker [28]. To explore in detail if the ZO gene-deletions have an effect on ExEn morphology, we visualized the ExEn apical surface using scanning electron microscopy SEM at different time points of culture Fig.

Furthermore, the apical cell surface looked distended and microvilli were sparsely present on this surface Fig. S1A and B. A Scanning electron micrographs.

B ExEn permeability assay illustration. To study this, we devised a simple assay in which live EBs where pre-incubated with anti-basement membrane BM antibodies added to the culture medium for 2hrs. Since these antibodies detect basement membrane components, a normal functioning permeability barrier would not be expected to allow IgG to penetrate the ExEn paracellularly, especially since IgG has a large molecular weight of kD [29].

However, if as suspected from the SEM analysis the barrier function is compromised, a large solute like IgG could possibly move paracellularly through the ExEn, reach the underlying BM and, in the case of a specific anti-BM IgG, bind to it. Here, after washing off unbound anti-BM IgG, followed by fixation and permeabilization of the EBs, the anti-BM can then be visualized with fluorophore-tagged secondary antibodies Fig. In Day-7 Fig. In conclusion, SEM and permeability assay studies on EB ExEn organization and integrity show that the absence of both ZO-1 and ZO-2 drastically compromise the paracellular permeability barrier of the ExEn layer, concurrent with less compact organization of ExEn cells.

These phenotypes were intermediate in EBs lacking only ZO-1, especially at early stages Day-5 to -7 of EB culture, but normalcy was restored at later stages Day Epithelial cell-lines are already differentiated when experimentally manipulated, whereas the ExEn of EBs differentiate into epithelia from non-polarized pluripotent inner cell mass ICM cells through an inbuilt developmental mechanism.

We therefore next examined the differentiation of EBs into outer layer primitive and visceral endoderm using molecular markers characteristically expressed at different stages of ExEn differentiation.

These three proteins are essential for the formation of the PrEn on the outer EB layer [30]. The intermediate filament Keratin-8, recognized by the antibody Troma-1, is an epithelial cell marker and is present in early PrEn differentiation [31]. Day 4 EBs were chosen for this semi-quantitative study as the PrEn would have differentiated by this time.

After optimizing the PCR cycle number to coincide with the exponential phase of amplification, the amplicon intensity between WT and the three null mutants for both GATA-4 and -6 amplifications were indistinguishable.

Reverse transcribed cDNA was amplified with specific GATA-4 and -6 primer sets at optimized cycle numbers indicated on right side of panels.

Fixed and permeabilized EB cryosections were treated with antibodies immunoreactive to Dab2 panels a-d and Keratin-8 Troma-1 panels e-h and visualized red color.

Podocalyxin PODXL is a single-pass transmembrane sialoglycoprotein that was first discovered in specialized epithelial cells of the renal glomerulus podocytes [32]. It is localized at the apical membrane of polarized epithelial cells and has a heavily sialylated ectodomain and a cytosolic tail that associates indirectly with actin filaments [33]. Since PODXL positively regulates the formation of microvilli [34] and the apical domain [35] in epithelial cells, we investigated if ZO deficiency had an effect on this protein.

EBs were monitored for PODXL expression and localization continually over a 9 day period of culture by immunofluorescence microscopy. This staining could also be seen on the apical membrane of the primitive ectoderm PrEc surrounding nascent cavities at the EB interior.

GAPDH was used as a lysate loading control panel a. Reverse transcribed cDNA was amplified with specific primer sets at optimized cycle numbers indicated on right side of panels. Ezrin and Pals1 were selected as epithelia polarized controls. Expression levels are presented as average fold-change of three separate experiments normalized to GAPDH and relative to WT control panel c.

C Immunofluorescence staining of Ezrin. Cryosections of EBs harvested at Day-4 panels a—d , Day-7 panels e—h and Day-9 panels i—l of culture were immunostained with antibodies against Ezrin red color. PODXL expression can be detected on immunoblots as post-translationally modified forms of immature glycosylated , mature glycosylated and dimer kD bands [36].

Since we were also interested to evaluate if the changes in PODXL expression were specific for PODXL or a general phenomenon affecting other membrane localized proteins, we looked at two additional membrane polarized epithelial proteins, Ezrin [37] and Pals1 [38] , as indicators. Interestingly, at both protein Fig. Ezrin is a member of the Ezrin-Radixin-Moesin ERM family of submembrane localized proteins, which function in the organization of specialized membrane domains [39]. In polarized epithelial cells, Ezrin localizes at the sub-apical membrane where it interacts directly or indirectly with integral membrane proteins, cross-linking them with the underlying cortical actin network.

Ezrin localization was visualized in EBs by immunostaining at Day-4, -7 and -9 of culture Fig. At Day-4 Fig. A hallmark of epithelial morphogenesis is the deposition of a basal basement membrane BM. Immunofluorescence staining of Nidogen Nid Fig. A Immunofluorescence staining of Nidogen. EB cryosections from Day-9 cultures panels a—d were immunostained for the BM component Nidogen red color, arrow. ExEn and BM are indicated by arrowhead and arrow respectively. B Immunofluorescence staining of PrEc.

C Phase-contrast microscopy. Cavity development in live EBs was tracked by phase-contrast imaging on Day-3 panels a—d , Day-5 panels e—h , Day-7 panels i—l and Day-9 panels m—p of culture. EB histology was analyzed at Day-5 panels a—d , Day-7 panels e—h and Day-9 panels i—l. Cryosections of EBs at Day-9 culture were immunostained with Nidogen Nid to demarcate the BM boundary of cavities panels a,d,g,j, green color.

TUNEL-positive apoptotic cells were visualized in red color panels b,e,h,k. Merged images panels c,f,i,l. The BM is not merely a foundational support that underpins the epithelium but also regulates diverse biological processes like differentiation and survival via cell-surface receptors such as integrins [46].

During EB development, the BM regulates the formation, polarization and survival of the primitive ectoderm PrEc layer [47] [48]. To study this phenomenon in greater detail, phase-contrast imaging Fig. Phase-contrast microscopy of live EBs at various time points indicated no discernible morphological difference between the WT control and null mutants at Day-3 Fig. Small nascent cavities arrows began to form at Day-5 Fig. By Day-9 Fig. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin.

Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures.

In addition, we have identified a amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells.