The nonsense-mediated mRNA decay (NMD) pathway promotes rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), preventing accumulation of potentially detrimental truncated proteins. In metazoa, seven genes (upf1, upf2, upf3, smg1, smg5, smg6, and smg7) have been identified as essential for NMD; here we show that the zebrafish genome encodes orthologs of upf1, upf2, smg1, and smg5 to smg7 and two upf3 paralogs. We also show that Upf1 is required for degradation of PTC-containing mRNAs in zebrafish embryos. Moreover, its depletion has a severe impact on embryonic development, early patterning, and viability. Similar phenotypes are observed in Upf2-, Smg5-, or Smg6-depleted embryos, suggesting that zebrafish embryogenesis requires an active NMD pathway. Using cultured cells, we demonstrate that the ability of a PTC to trigger NMD is strongly stimulated by downstream exon-exon boundaries. Thus, as in mammals and plants but in contrast to invertebrates and fungi, NMD is coupled to splicing in zebrafish. Our results together with previous studies show that NMD effectors are essential for vertebrate embryogenesis and suggest that the coupling of splicing and NMD has been maintained in vertebrates but lost in fungi and invertebrates.The nonsense-mediated mRNA decay (NMD) pathway is a conserved mRNA quality control mechanism that ensures the fidelity of gene expression by detecting and degrading mRNAs containing premature translation termination codons (PTCs or nonsense codons). In doing so, the NMD pathway protects eukaryotic cells from potentially dominant-negative effects resulting from the accumulation of truncated proteins (3,12,33). In addition to this protective function, NMD regulates 1 to 10% of wild-type transcripts, thereby influencing a broad range of biological processes including development, signal transduction, and cell cycle progression (25,44).NMD is triggered when ribosomes terminate translation prematurely. This event leads to the assembly of what is known as the surveillance complex, which links a premature translation termination event to degradation of a PTC-containing mRNA. To achieve this function, the surveillance complex interacts both with eukaryotic translation termination factors (i.e., eukaryotic release factors 1 and 3) and with the general cellular mRNA decay machinery, thereby accelerating the degradation of mRNAs harboring nonsense codons (8).The components of the surveillance complex (or NMD effectors) were originally identified in genetic screens in Saccharomyces cerevisiae and Caenorhabditis elegans and subsequently by homology searches in other organisms (3,12,33). The surveillance complex components include the Upf1, Upf2, and Upf3 proteins (also known as suppressors with morphogenetic effects on genitalia in C. elegans), which are conserved in all eukaryotes, and the Smg1, Smg5, Smg6, and Smg7 proteins, which are conserved in multicellular organisms (3,12,33).Upf1 is an RNA helicase whose activity is regulated by cycles of phosph...
Epithelial cells are equipped with junctional complexes that are involved in maintaining tissue architecture, providing mechanical integrity and suppressing tumour formation as well as invasiveness. A strict spatial segregation of these junctional complexes leads to the polarisation of epithelial cells. In vertebrate epithelia, basally localised hemidesmosomes mediate stable adhesion between epithelial cells and the underlying basement membrane. Although components of hemidesmosomes are relatively well known, the molecular machinery involved in governing the formation of these robust junctions, remains elusive. Here, we have identified the first component of this machinery using a forward genetic approach in zebrafish as we show that the function of penner (pen)/lethal giant larvae 2(lgl2) is necessary for hemidesmosome formation and maintenance of the tissue integrity in the developing basal epidermis. Moreover, in pen/lgl2 mutant, basal epidermal cells hyper-proliferate and migrate to ectopic positions. Of the two vertebrate orthologues of the Drosophila tumour suppressor gene lethal giant larvae, the function of lgl2 in vertebrate development and organogenesis remained unclear so far. Here, we have unravelled an essential function of lgl2 during development of the epidermis in vertebrates.
Changes in tissue homeostasis, acquisition of invasive cell characteristics, and tumor formation can often be linked to the loss of epithelial cell polarity. In carcinogenesis, the grade of neoplasia correlates with impaired cell polarity. In Drosophila, lethal giant larvae (lgl), discs large (dlg), and scribble, which are components of the epithelial apico-basal cell polarity machinery, act as tumor suppressors, and orthologs of this evolutionary conserved pathway are lost in human carcinoma with high frequency. However, a mechanistic link between neoplasia and vertebrate orthologs of these tumor-suppressor genes remains to be fully explored at the organismal level. Here, we show that the pen/lgl2 mutant phenotype shares two key cellular and molecular features of mammalian malignancy: cell autonomous epidermal neoplasia and epithelial-to-mesenchymal-transition (EMT) of basal epidermal cells including the differential expression of several regulators of EMT. Further, we found that epidermal neoplasia and EMT in pen/lgl2 mutant epidermal cells is promoted by ErbB signalling, a pathway of high significance in human carcinomas. Intriguingly, EMT in the pen/lgl2 mutant is facilitated specifically by ErbB2 mediated E-cadherin mislocalization and not via canonical snail–dependent down-regulation of E-cadherin expression. Our data reveal that pen/lgl2 functions as a tumor suppressor gene in vertebrates, establishing zebrafish pen/lgl2 mutants as a valuable cancer model.
The integrity and homeostasis of the vertebrate epidermis depend on various cellular junctions. How these junctions are assembled during development and how their number is regulated remain largely unclear. Here, we address these issues by analysing the function of Lgl2, E-cadherin and atypical Protein kinase C (aPKC) in the formation of hemidesmosomes in the developing basal epidermis of zebrafish larvae. Previously, we have shown that a mutation in lgl2 (penner) prevents the formation of hemidesmosomes. Here we show that Lgl2 function is essential for mediating the targeting of Integrin alpha 6 (Itga6), a hemidesmosomal component, to the plasma membrane of basal epidermal cells. In addition, we show that whereas aPKCλ seems dispensable for the localisation of Itga6 during hemidesmosome formation, knockdown of E-cadherin function leads to an Lgl2-dependent increase in the localisation of Itga6. Thus, Lgl2 and E-cadherin act antagonistically to control the localisation of Itga6 during the formation of hemidesmosomes in the developing epidermis.
Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that microridges are formed of F-actin networks and require the function of the Arp2/3 complex for their maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.
Fabrication of nanoscale DNA devices to generate 3D nano-objects with precise control of shape, size, and presentation of ligands has shown tremendous potential for therapeutic applications. The interactions between the cell membrane and different topologies of 3D DNA nanostructures are crucial for designing efficient tools for interfacing DNA devices with biological systems. The practical applications of these DNA nanocages are still limited in cellular and biological systems owing to the limited understanding of their interaction with the cell membrane and endocytic pathway. The correlation between the geometry of DNA nanostructures and their internalization efficiency remains elusive. We investigated the influence of the shape and size of 3D DNA nanostructures on their cellular internalization efficiency. We found that one particular geometry, i.e., the tetrahedral shape, is more favored over other designed geometries for their cellular uptake in 2D and 3D cell models. This is also replicable for cellular processes like cell invasion assays in a 3D spheroid model, and passing the epithelial barriers in in vivo zebrafish model systems. Our work provides detailed information for the rational design of DNA nanodevices for their upcoming biological and biomedical applications.
Epithelial cells exhibit apical membrane protrusions, which confer specific functions to epithelial tissues. Microridges are short actin protrusions that are laterally long and form a maze-like pattern in the apical domain. They are widely found on vertebrate squamous epithelia including epidermis and have functions in mucous retention, membrane storage and abrasion resistance. It is largely unknown how the formation of these laterally long actin projections is regulated. Here, we show that antagonistic interactions between aPKC and Lgl–regulators of apical and basolateral domain identity, respectively,–control the length of microridges in the zebrafish periderm, the outermost layer of the epidermis. aPKC regulates the levels of Lgl and the active form of non-muscle myosinII at the apical cortex to prevent actin polymerization-dependent precocious fusion and elongation of microridges. Our data unravels the functional significance of exclusion of Lgl from the apical domain in epithelial cells.
The epidermis is a stratified epithelium, which forms a barrier to maintain the internal milieu in metazoans. Being the outermost tissue, growth of the epidermis has to be strictly coordinated with the growth of the embryo. The key parameters that determine tissue growth are cell number and cell size. So far, it has remained unclear how the size of epidermal cells is maintained and whether it contributes towards epidermal homeostasis. We have used genetic analysis in combination with cellular imaging to show that zebrafish goosepimples/myosin Vb regulates plasma membrane homeostasis and is involved in maintenance of cell size in the periderm, the outermost epidermal layer. The decrease in peridermal cell size in Myosin Vb deficient embryos is compensated by an increase in cell number whereas decrease in cell number results in the expansion of peridermal cells, which requires myosin Vb (myoVb) function. Inhibition of cell proliferation as well as cell size expansion results in increased lethality in larval stages suggesting that this two-way compensatory mechanism is essential for growing larvae. Our analyses unravel the importance of Myosin Vb dependent cell size regulation in epidermal homeostasis and demonstrate that the epidermis has the ability to maintain a dynamic balance between cell size and cell number.
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