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In JoVE (1)
Other Publications (3)
Articles by Yiying Luo in JoVE
JoVE General
Principles of Rodent Surgery for the New Surgeon
Charles River, Research Models and Services
Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.
Other articles by Yiying Luo on PubMed
The Zebrafish Pard3 Ortholog is Required for Separation of the Eye Fields and Retinal Lamination
The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.
Molecular Cloning of Three Zebrafish Lin7 Genes and Their Expression Patterns in the Retina
The vertebrate retina develops from an undifferentiated sheet of neuroepithelial cells, whose differentiation requires the generation and maintenance of the correct cellular polarity. To examine the role of cell polarity in retinal development, we cloned three zebrafish lin7 genes (lin7a, lin7b, and lin7c), which each encodes a protein candidate that is required for generation/maintenance of neuroepithelial cell junctions. These three zebrafish Lin7 proteins share over 78% amino acid identity and contain both L27 and PDZ domains that are present in all Lin7 homologs. Immunoblots revealed that the Lin7b and Lin7c proteins were first expressed in the developing eye by 24hr postfertilization (hpf), while Lin7a was not detected in the eye until 72 hpf. At 33 hpf, the Lin7 proteins localized at, or slightly apical of, the actin-associated adherens junctions in the retinal neuroepithelium. This subcellular distribution required the expression of the Nok protein. In the absence of Nok, the Lin7 proteins failed to localize to either the ectopic adherens junctions or the cell membrane. At 4 days postfertilization, in situ hybridisation revealed that all three lin7 genes were expressed in both the ganglion cell layer and the bipolar cell region of the inner nuclear layer. The lin7a gene was also expressed in the amacrine and horizontal cell regions of the inner nuclear layer, while lin7c was also expressed in the outer nuclear layer. In the adult retina, where Lin7a is the predominant form expressed, the Lin7 proteins were localized to the outer and inner plexiform layers, the bipolar and horizontal cells of the inner nuclear layer, and the ganglion cells. These results suggest that the three zebrafish Lin7 proteins possess partially redundant, yet essential, roles in retinal development.
Zebrafish Foxe3: Roles in Ocular Lens Morphogenesis Through Interaction with Pitx3
Foxe3 is a winged helix/forkhead domain transcription factor necessary for mammalian and amphibian lens development. Human FOXE3 mutations cause anterior segment dysgenesis and cataracts. The zebrafish foxe3 cDNA was PCR amplified from 24 h post-fertilization (hpf) embryo cDNA. The zebrafish foxe3 gene consists of a single exon on chromosome 8 and encodes a 422 amino acid protein. This protein possesses 44% and 67% amino acid identity with the human FOXE3 and Xenopus FoxE4 proteins, respectively. A polyclonal antiserum was generated against a bacterial fusion protein containing the Foxe3 carboxyl terminus. The purified antiserum detects zebrafish Foxe3 on immunoblots, in embryo wholemounts, and frozen tissue sections. The zebrafish Foxe3 protein is first detected in the lens at 31hpf and is restricted to the nucleated cell population, including the epithelial and elongating fiber cells. Knockdown of Foxe3 protein using an antisense morpholino results in small lenses with multilayered epithelial cells and fiber cell dysmorphogenesis. The morphants posses normal retinas, although retinal cell proteins, including rhodopsin, are abnormally expressed in the morphant lens tissue. Functional interactions between foxe3 and pitx3 during lens development were assessed by RT-PCR and comparison of Foxe3 and Pitx3 protein expression in both foxe3 and pitx3 morphants. Immunoblots and immunohistochemistry reveal Pitx3 is expressed in the foxe3 morphant lens, while Pitx3 knockdown results in the elimination of Foxe3 expression. These data demonstrate that Foxe3 is necessary for lens development in zebrafish and that foxe3 lies genetically downstream of pitx3 in a zebrafish lens development pathway.
