Faculty Bibliography

During Drosophila oogenesis, Oskar mRNA is transported to the posterior pole of the oocyte, where it is locally translated and induces germ-plasm assembly. Oskar protein recruits all of the components necessary for the establishment of posterior embryonic structures and of the germline. Tight localization of Oskar is essential, as its ectopic expression causes severe patterning defects. Here, we show that the Drosophila homolog of mammalian Lasp1 protein, an actin-binding protein previously implicated in cell migration in vertebrate cell culture, contributes to the accumulation of Oskar protein at the posterior pole of the embryo. The reduced number of primordial germ cells in embryos derived from lasp mutant females can be rescued only with a form of Lasp that is capable of interacting with Oskar, revealing the physiological importance of the Lasp-Oskar interaction.

Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemical-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR(+) cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.

Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal- and spatial-specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross-linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein-NTR (CFP-NTR) under control of tissue-specific promoters allowed us to induce the death of cardiomyocytes, pancreatic beta-cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration.

Fish and amphibian hearts are known to regenerate after partial resection, but the molecular mechanisms underlying this process remain unclear. In this issue of Cell, Lipilina et al. analyze regeneration in the zebrafish heart. Their work indicates that new cardiomyocytes originate from undifferentiated progenitor cells and reveals a critical role for the epicardium, the cellular layer that covers the heart.

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.