The invariant lineage of Caenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allow routine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increases in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.

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Abstract"RNA bandages" are composed of two 6-12-mer 2′-OMe RNA strands complementary to a mRNA target that are joined by a photocleavable linker. These tandem oligonucleotides typically exhibit much higher affinity for the mRNA than the individual strands. An RNA bandage with binding arms of different lengths and a 4-base gap blocked translation in vitro of GFP mRNA; subsequent near-UV irradiation restored translation. This provides a general method of photomodulating hybridization for a variety of oligonucleotide-based technologies.Photochemical methods for controlling oligonucleotide function have become increasingly important in biological research. 1 Light-activated ("caged") oligonucleotides have been used to regulate DNA hybridization, 2-4 polymerase, 5-9 ribozyme, 10, 11 DNAzyme, 12, 13 aptamer, 14 and RNase H activity, 4, 15 RNA folding, 16 and RNA interference, 17, 18 as well as gene expression in cells and embryos. 17, 19-23 Most caging groups for biological use involve the nitrobenzyl (NB) moiety and its derivatives, which allow for removal at relatively long wavelengths (~365 nm) without harmful side products. Previously, our lab used a NBcontaining photocleavable linker to join an antisense oligodeoxynucleotide (asODN) to a much shorter complementary strand, which controls binding of the asODN to an mRNA target. For example, in human leukemia cells, photoactivation of a caged asODN initiated the degradation of c-myb mRNA by an RNase H-dependent mechanism. 21 And in zebrafish embryos, caged asODNs were shown to block expression of chordin and bozozok, upon irradiation. 20While these caged asODNs succeeded in turning gene expression "off" after photolysis, a related technique for turning gene expression "on" would be equally useful. The timing and location of protein expression within the cell has profound consequences for proper cellular development, and light-activated control of mRNA would allow temporal and spatial analysis of protein function. Herein, we describe the synthesis of an "RNA bandage", in which tandem oligonucleotides are joined by a photocleavable linker. The bandage binds and protects the target mRNA from translation, until UV irradiation cuts the bandage (Fig. 1).Ando et al. generated the first caged mRNA by statistically labeling the phosphodiester backbone with a large number of coumarin photoactive blocking groups, thereby perturbing mRNA structure and preventing protein synthesis in zebrafish. 22 However, UV photolysis yielded relatively little "active" mRNA, due to the low quantum efficiency of removing dozens of blocking groups on a single oligonucleotide. In contrast, RNA bandages seek to cage mRNA function by employing a single, site-specific photoactive group. This strategy has several advantages: shorter irradiation times, more efficient synthesis, and purification of a light-*Corresponding author. Tel./fax: +1 215 898 6459/+ 1 215 898 2037; email: ivandmo@sas.upenn.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for public...

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DNA enzymes (DNAzymes) that catalyze the degradation of complementary RNA molecules have been investigated for many biochemical and sensing applications. Here, we investigated a 10-23 DNAzyme that has been shown previously to possess cellular activity. We determined that it has very low Mg2+ ion dependence, with DNAzyme activity observed at [Mg2+] = 0.01 mM. This metal ion dependence is much lower than is typical for DNAzymes studied to date, and suggests that DNAzymes may be engineered for many additional biological applications. Recently, we demonstrated that this 10-23 DNAzyme can be divided into two parts, which assemble into an active oligonucleotide complex. We investigated in more detail the functionality of the split 10-23 DNAzyme and found that dividing the 15-nucleotide catalytic loop after the 7th or 8th base maximized its activity. The split DNAzymes required higher metal ion concentrations ([Mg2+] = 5 mM), and as we anticipated due to their lower hybridization activity, the split enzymes had the advantage of being more sensitive to single base mismatches in the DNAzyme-RNA duplex. Finally, we demonstrated facile photomodulation of split DNAzyme activity by incorporating a photocleavable biotin moiety bound to streptavidin.