SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

358

419

The invariant cell lineage and cell fate of Caenorhabditis elegans provide a unique opportunity to decode the molecular mechanisms of animal development. To exploit this opportunity, we have developed a system for automated cell lineage tracing during C. elegans embryogenesis, based on 3D, time-lapse imaging and automated image analysis. Using ubiquitously expressed histone-GFP fusion protein to label cells͞nuclei and a confocal microscope, the imaging protocol captures embryogenesis at high spatial (31 planes at 1 m apart) and temporal (every minute) resolution without apparent effects on development. A set of image analysis algorithms then automatically recognizes cells at each time point, tracks cell movements, divisions and deaths over time and assigns cell identities based on the canonical naming scheme. Starting from the four-cell stage (or earlier), our software, named STARRYNITE, can trace the lineage up to the 350-cell stage in 25 min on a desktop computer. The few errors of automated lineaging can then be corrected in a few hours with a graphic interface that allows easy navigation of the images and the reported lineage tree. The system can be used to characterize lineage phenotypes of genes and͞or extended to determine gene expression patterns in a living embryo at the single-cell level. We envision that this automation will make it practical to systematically decipher the developmental genes and pathways encoded in the genome of C. elegans.embryogenesis ͉ imaging ͉ image analysis algorithms

Section: Methodsmentioning

confidence: 99%

“…We constructed RW10006, a strain that expresses histone H3.3::GFP driven by the H3.3 promoter and histone H2B::GFP driven by the germline promoter pie-1 (21). H3.3::GFP is expressed throughout embryogenesis and provides very strong GFP signals, especially in the AB, MS, and E lineages.…”

mentioning

confidence: 99%

Automated cell lineage tracing in Caenorhabditis elegans

Bao

Murray

Boyle

et al. 2006

358

419

“…The final constructs were introduced in worms by microparticle bombardment as described in ref. 19. All strains used in this study are listed in SI Table 3.…”

Section: Methodsmentioning

confidence: 99%

“…Each construct was used to generate 2-5 individual transgenic strains, using the microparticle bombardment technique (19). To avoid high overexpression of the transgene, in many cases we used the baf-1 promoter to drive gfp::lmn-1 expression, because its expression pattern overlaps that of the lmn-1 promoter (20) (SI Tables 3 and 4).…”

Section: Point Mutations In Conserved Disease-linked Residues Affect mentioning

confidence: 99%

Laminopathic mutations interfere with the assembly, localization, and dynamics of nuclear lamins

Wiesel

Mattout

Melcer

et al. 2008

138

Lamins are nuclear intermediate filament proteins and the major building blocks of the nuclear lamina. Besides providing nuclear shape and mechanical stability, lamins are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly, nuclear positioning, and apoptosis. Mutations in human lamins cause many different heritable diseases, affecting various tissues and causing early aging. Although many of these mutations result in nuclear deformation, their effects on lamin filament assembly are unknown. Caenorhabditis elegans has a single evolutionarily conserved lamin protein, which can form stable 10-nmthick filaments in vitro. To gain insight into the molecular basis of lamin filament assembly and the effects of laminopathic mutations on this process, we investigated mutations in conserved residues of the rod and tail domains that are known to cause various laminopathies in human. We show that 8 of 14 mutant lamins present WT-like assembly into filaments or paracrystals, whereas 6 mutants show assembly defects. Correspondingly, expressing these mutants in transgenic animals shows abnormal distribution of Ce-lamin, abnormal nuclear shape or change in lamin mobility. These findings help in understanding the role of individual residues and domains in laminopathy pathology and, eventually, promote the development of therapeutic interventions. filament assembly ͉ laminopathic diseases ͉ nuclear envelope

“…Although reiterated transgenes are rather effectively suppressed in germ-line cells (38), several potential solutions to this problem are available. These include driving nuclease expression with a natural germ-line promoter (45), reducing the number of transgenes through the use of bombardment (46), eliminating one or more components of the apparatus responsible for suppression (47), and direct protein or mRNA injection. Once germ-line expression is achieved, it is quite likely that deletion alleles and targeted gene replacements can be generated by using engineered ZFNs.…”

Section: Products From Wtmentioning

confidence: 99%

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Morton

Davis

Jørgensen

et al. 2006

169

124

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, Ϸ20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.DNA repair ͉ gene targeting ͉ nematodes ͉ nonhomologous end joining T he ability to make targeted double-strand breaks in chromosomal DNA has several important uses. It allows the detailed study of DNA repair mechanisms; it leads to localized mutagenesis at the break site; and it enhances the efficiency of targeted gene replacement through homologous recombination. We have been exploring the capabilities of one class of targetable cleavage reagents, the zinc-finger nucleases (ZFNs).ZFNs are chimeric proteins composed of DNA-binding Cys 2 His 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme FokI (1). Each finger makes contact primarily with a separate DNA triplet (2, 3). Natural and artificial zinc fingers have been characterized that bind to all 5Ј-GNN-3Ј, many ANN and CNN, and some TNN triplets (4-7). Furthermore, the modular nature of the zinc fingers allows them to be joined in essentially arbitrary combinations. Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the di...