ABSTRACT
Significance: The reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption. Aim: To prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed. Approach: A deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system. Results: The system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components. Conclusions: An easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.
Subject(s)
Lenses , Optical Devices , Animals , Microscopy/methods , Zebrafish , Optics and Photonics , OxidesABSTRACT
OBJECTIVE: To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. METHODS AND RESULTS: In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. CONCLUSIONS: These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.
Subject(s)
Lymphangiogenesis , Lymphatic System/metabolism , Membrane Proteins/metabolism , Receptors, Notch/metabolism , Signal Transduction , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Biomarkers/metabolism , COS Cells , Cell Movement , Cell Proliferation , Chlorocebus aethiops , Coculture Techniques , Embryo, Nonmammalian/metabolism , Endothelial Cells/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Gene Silencing , Humans , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Lymphangiogenesis/genetics , Lymphatic System/embryology , Membrane Proteins/genetics , RNA, Messenger/metabolism , Receptors, Notch/genetics , Thoracic Duct/embryology , Thoracic Duct/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Muscle regulatory factors activate myogenesis in all vertebrates, but their role has been studied in great detail only in the mouse embryo, where all but myogenin--Myod, Myf5 and Mrf4--are sufficient to activate (albeit not completely) skeletal myogenesis. In the zebrafish embryo, myod and myf5 are required for induction of myogenesis because their simultaneous ablation prevents muscle development. Here we show that mrf4 but not myog can fully rescue myogenesis in the myod/myf5 double morphant via a selective and robust activation of myod, in keeping with its chromatin-remodelling function in vitro. Rescue does not happen spontaneously, because the gene, unlike that in the mouse embryo, is expressed only at the onset of muscle differentiation, Moreover, because of the transient nature of morpholino inhibition, we were able to investigate how myogenesis occurs in the absence of a myotome. We report that in the complete absence of a myotome, subsequent myogenesis is abolished, whereas myogenesis does proceed, albeit abnormally, when the morpholino inhibition was not complete. Therefore our data also show that the early myotome is essential for subsequent skeletal muscle differentiation and patterning in the zebrafish.