Background In the last stage of the C. elegans equivalent of epiboly, an open pocket in the epidermis is closed by marginal epidermal P/pocket cells that express and require VAB-1/Eph and PLX-2/plexin receptors for migration toward and alignment with contralateral partners at the ventral midline. Cellular mechanisms affected by these signaling proteins remain unknown. Results A cellular bridge comprising four neuron cell bodies that spans the open pocket serves as a substratum for migration of contra-lateral P cell pair P9/10 to the midline which can facilitate similar migration of neighboring P cells. This bridge is formed by a stereotypical rearrangement of five sister pairs of PLX-2 and VAB-1 expressing cells, of which three pairs serve as a scaffold for bridge assembly and two pairs form the bridge. Bridge formation requires VAB-1 kinase-dependent extension of presumptive bridge cell protrusions toward the ventral midline. An unassembled mutant bridge obstructs but does not block P cell progression toward the midline, however, cell type-specific rescue experiments show that VAB-1 or a nearly complete cytoplasmic deletion of VAB-1 expressed by scaffold and bridge cells or by P9/10 can facilitate P cell progression to the midline. MAB-20/semaphorin and VAB-1 also exhibit complex redundancies to regulate adhesion and prevent gaps between sister bridge and scaffold forming cells that would otherwise completely block P cell migration. Conclusions The Eph receptor functions to mediate cell extensions required for bridge formation, independently facilitates P cell migration to the ventral midline, and acts redundantly with PLX-2/plexin to prevent gaps between sister plexin band cells that normally serve as a substratum for P9/10 cell migration.

No abstract

In this study, we demonstrate the developmental activation, in the zebrafish embryo, of a surveillance mechanism which triggers apoptosis to remove damaged cells. We determine the time course of activation of this mechanism by exposing embryos to camptothecin, an agent which specifically inhibits topoisomerase I within the DNA replication complex and which, as a consequence of this inhibition, also produces strand breaks in the genomic DNA. In response to an early (pre-gastrula) treatment with camptothecin, apoptosis is induced at a time corresponding approximately to mid-gastrula stage in controls. This apoptotic response to a block of DNA replication can also be induced by early (pre-MBT) treatment with the DNA synthesis inhibitors hydroxyurea and aphidicolin. After camptothecin treatment, a high proportion of cells in two of the embryo's three mitotic domains (the enveloping and deep cell layers), but not in the remaining domain (the yolk syncytial layer), undergoes apoptosis in a cell-autonomous fashion. The first step in this response is an arrest of the proliferation of all deep- and enveloping-layer cells. These cells continue to increase in nuclear volume and to synthesize DNA. Eventually they become apoptotic, by a stereotypic pathway which involves cell membrane blebbing, "margination" and fragmentation of nuclei, and cleavage of the genomic DNA to produce a nucleosomal ladder. Fragmentation of nuclei can be blocked by the caspase-1,4,5 inhibitor Ac-YVAD-CHO, but not by the caspase-2,3,7[, 1] inhibitor Ac-DEVD-CHO. This suggests a functional requirement for caspase-4 or caspase-5 in the apoptotic response to camptothecin. Recently, Xenopus has been shown to display a developmental activation of the capability for stress- or damaged-induced apoptosis at early gastrula stage. En masse, our experiments suggest that the apoptotic responses in zebrafish and Xenopus are fundamentally similar. Thus, as for mammals, embryos of the lower vertebrates exhibit the activation of surveillance mechanisms, early in development, to produce the selective apoptosis of damaged cells.

Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes an axon guiding TGF-beta, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. We present a model for the integrated control of TGF-beta, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail.

SummaryWe address the developmental activation, in the zebrafish embryo, of intrinsic cell-cycle checkpoints which monitor the DNA replication process and progression through the cell cycle. Eukaryotic DNA replication is probably carried out by a multiprotein complex containing numerous enzymes and accessory factors that act in concert to effect processive DNA synthesis (Applegren, N. et al. (1995) J. Cell. Biochem. 59, 91–107). We have exposed early zebrafish embryos to three chemical agents which are predicted to specifically inhibit the DNA polymerase α, topoisomerase I and topoisomerase II components of the DNA replication complex. We present four findings: (1) Before mid-blastula transition (MBT) an inhibition of DNA synthesis does not block cells from attempting to proceed through mitosis, implying the lack of functional checkpoints. (2) After MBT, the embryo displays two distinct modes of intrinsic checkpoint operation. One mode is a rapid and complete stop of cell division, and the other is an ‘adaptive’ response in which the cell cycle continues to operate, perhaps in a ‘repair’ mode, to generate daughter nuclei with few visible defects. (3) The embryo does not display a maximal capability for the ‘adaptive’ response until several hours after MBT, which is consistent with a slow rranscriptional control mechanism for checkpoint activation. (4) The slow activation of checkpoints at MBT provides a window of time during which inhibitors of DNA synthesis will induce cytogenetic lesions without killing the embryo. This could be useful in the design of a deletion-mutagenesis strategy.

SummaryWe have studied the developmental activation of the metaphase checkpoint, and the consequences of activating this checkpoint, in the zebrafish embryo. (1) Treatment with nocodazole (a microtubule destabiliser) before mid-blastula transition (MBT) produces complete destruction of all nuclei in the deep cell layer of the embryo. In contrast, nocodazole treatment after MBT efficiently produces metaphase arrest in this cell layer. Thus, the metaphase checkpoint becomes activated at MBT. (2) Although a metaphase arrest is induced by nocodazole, it is not induced by paclitaxel (a microtubule stabiliser). Thus the metaphase checkpoint appears to sense a destabilisation, but not a stabilisation, of spindle microtubules. (3) Metaphase-arrested cells (in nocodazole) can be driven into the next interphase by adding the Ca2+-specific ionophore A23187. Thus, a Ca2+-signalling pathway lies downstream of, or parallel to, the metaphase checkpoint. (4) After mid-gastrula stage, treatment with nocodazole produces DNA fragmentation in all three cell layers. In the enveloping epithelial monolayer (EVL), this is associated with a classical apoptotic phenotype. In the deep layer, it is associated with an unusual, highly condensed nuclear state that is entered directly from metaphase arrest. Thus, after the mid-gastrula stage, the embryo responds to nocodazle by undergoing apoptosis. (5) Nocodazole-induced apoptosis in the deep cell layer can be blocked by the caspase-1,4,5 inhibitors Ac-YVAD-CHO and Ac-YVAD-CMK. This suggests that a homologue of theC. elegans ced-9—ced-4—ced-3pathway is involved in control over apoptosis in the early zebrafish embryo.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment. Video Link The video component of this article can be found at https://www.jove.com/video/59533/ 1 , and with the development of new tools that allow labeling and continuous imaging of single cells in embryos, biologists can now begin examining different steps in the development of the nematode nervous system from all angles-cell birth; migration and differentiation; neurite formation, targeted outgrowth and fasciculation; synapse formation; and tuning of functional circuits. Capturing neuronal outgrowth dynamics in the C. elegans embryo, by combining stably expressed reporters and fluorescence microscopy, is valuable to the scientific community. Developmental studies in C. elegans often leverage the invariant cell-lineage and cell-fate maps of this species to augment contextual understanding at the single-cell level within the intact organism 1. Auto-lineaging analysis-using StarryNite 2,3,4 and AceTree 5,6,7,8 softwarebenefits from high contrast, high resolution images of fluorescent nuclei. To work optimally, StarryNite/AceTree also depends upon predictable constrained orientation of imaged embryos during development. Confocal microscopy, done in C. elegans embryos compressed between two coverslips, has been the standard auto-lineaging microscopy method for more than a decade because it provides both high contrast/high resolution and a predictable constrained orientation of the embryo 7,8. We previously described the construction and use of a novel light-sheetbased dual-view inverted selective plane illumination microscope (diSPIM) for live sample imaging such as C. elegans embryogenesis 9,10,11,12,13. Light-sheet microscopy, in general, provides low phototoxicity, high speed, and long-term imaging of live 3D specimens 14,15. The diSPIM method, specifically, produces four-dimensional (4D) images with nearly isotropic spatial resolution of approximately 330 nm ...

We describe theoretical and practical advances in algorithm and software design, resulting in ten to several thousand-fold faster deconvolution and multiview fusion than previous methods. First, we adapt methods from medical imaging, showing that an unmatched back projector accelerates Richardson-Lucy deconvolution by at least 10-fold, in most cases requiring only a single iteration. Second, we show that improvements in 3D image-based registration with GPU processing result in speedups of 10-100-fold over CPU processing. Third, we show that deep learning can provide further accelerations, particularly for deconvolution with a spatially varying point spread function. We illustrate the power of our methods from the subcellular to millimeter spatial scale, on diverse samples including single cells, nematode and zebrafish embryos, and cleared mouse tissue. Finally, we show that our methods facilitate the use of new microscopes that improve spatial resolution, including dual-view cleared tissue light-sheet microscopy and reflective lattice light-sheet microscopy.