RESUMO
Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened â¼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a â¼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Haploidia , Morfogênese/genética , Mutagênese/genética , Mutação , Neurogênese/genética , Retina , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.
Assuntos
Membrana Basal/embriologia , Olho/embriologia , Crista Neural/embriologia , Alelos , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/fisiologia , Movimento Celular , Eletroforese Capilar , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Mesoderma/embriologia , Microscopia Eletrônica de Transmissão , Morfogênese , Mutação , Crista Neural/citologia , Organogênese , Retina/embriologia , Epitélio Pigmentado da Retina/embriologia , Transdução de Sinais , Fator de Transcrição AP-2/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Epithelial morphogenesis, a fundamental aspect of development, generates 3-dimensional tissue structures crucial for organ function. Underlying morphogenetic mechanisms are, in many cases, poorly understood, but mutations that perturb organ development can affect epithelial cell shape and orientation - difficult features to quantify in three dimensions. The basic structure of the eye is established via epithelial morphogenesis: in the embryonic optic cup, the retinal progenitor epithelium enwraps the lens. We previously found that loss of the extracellular matrix protein laminin-alpha1 (lama1) led to mislocalization of apical polarity markers and apparent misorientation of retinal progenitors. We sought to visualize and quantify this phenotype, and determine whether loss of the apical polarity determinant pard3 might rescue the phenotype. To this end, we developed LongAxis, a MATLAB-based program optimized for the retinal progenitor neuroepithelium. LongAxis facilitates 3-dimensional cell segmentation, visualization, and quantification of cell orientation and morphology. Using LongAxis, we find that retinal progenitors in the lama1-/- optic cup are misoriented and slightly less elongated. In the lama1;MZpard3 double mutant, cells are still misoriented, but larger. Therefore, loss of pard3 does not rescue loss of lama1, and in fact uncovers a novel cell size phenotype. LongAxis enables population-level visualization and quantification of retinal progenitor cell orientation and morphology. These results underscore the importance of visualizing and quantifying cell orientation and shape in three dimensions within the retina.
Assuntos
Forma Celular , Células Epiteliais , Processamento de Imagem Assistida por Computador , Retina , Software , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Laminina/genética , Laminina/metabolismo , Retina/citologia , Retina/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Epithelial wound healing requires a complex orchestration of cellular rearrangements and movements to restore tissue architecture and function after injury. While it is well known that mechanical forces can affect tissue morphogenesis and patterning, how the biophysical cues generated after injury influence cellular behaviors during tissue repair is not well understood. Using time-lapse confocal imaging of epithelial tissues in living zebrafish larvae, we provide evidence that localized increases in cellular crowding during wound closure promote the extrusion of nonapoptotic cells via mechanically regulated stretch-activated ion channels (SACs). Directed cell migration toward the injury site promoted rapid changes in cell number and generated shifts in tension at cellular interfaces over long spatial distances. Perturbation of SAC activity resulted in failed extrusion and increased proliferation in crowded areas of the tissue. Together, we conclude that localized cell number plays a key role in dictating cellular behaviors that facilitate wound closure and tissue repair.
Assuntos
Epitélio/patologia , Cicatrização , Actinas/metabolismo , Animais , Contagem de Células , Proliferação de Células , Canais Iônicos/metabolismo , Miosinas/metabolismo , Peixe-ZebraRESUMO
Epithelia provide a crucial protective barrier for our organs and are also the sites where the majority of carcinomas form. Most studies on epithelia and carcinomas use cell culture or organisms where high-resolution live imaging is inaccessible without invasive techniques. Here, we introduce the developing zebrafish epidermis as an excellent in vivo model system for studying a living epithelium. We developed tools to fluorescently tag specific epithelial cell types and express genes in a mosaic fashion using five Gal4 lines identified from an enhancer trap screen. When crossed to a variety of UAS effector lines, we can now track, ablate or monitor single cells at sub-cellular resolution. Using photo-cleavable morpholino oligonucleotides that target gal4, we can also express genes in a mosaic fashion at specific times during development. Together, this system provides an excellent in vivo alternative to tissue culture cells, without the intrinsic concerns of culture conditions or transformation, and enables the investigation of distinct cell types within living epithelial tissues.
Assuntos
Técnicas Citológicas/métodos , Células Epidérmicas , Peixe-Zebra/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cruzamentos Genéticos , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Epiderme/efeitos dos fármacos , Epiderme/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Imageamento Tridimensional , Masculino , Morfolinos/farmacologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1(UW1)) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1(UW1) mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis.