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 ␤-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. Developmental Dynamics 236:1025-1035, 2007.

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 chemicalgenetic 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.

Morphogenesis and cell differentiation in multicellular organisms often require accurate control of cell divisions. We show that a novel cell cycle regulator, tribbles, is critical for this control during Drosophila development. During oogenesis, the level of tribbles affects the number of germ cell divisions as well as oocyte determination. The mesoderm anlage enters mitosis prematurely in tribbles mutant embryos, leading to gastrulation defects. We show that Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination.

T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to formation of an “immunological synapse” [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2, 3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell.

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90–140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm2. We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

The role of non-muscle myosin IIA (heavy chain encoded by the non-muscle myosin heavy chain 9 gene, Myh9) in immunological synapse formation is controversial. We have addressed the role of myosin IIA heavy chain protein (MYH9) in mouse T cells responding to MHC-peptide complexes and ICAM-1 in supported planar bilayers – a model for immunological synapse maturation. We found that reduction of MYH9 expression levels using Myh9 siRNA in proliferating mouse CD4+ AND T cell receptor (TCR) transgenic T cells resulted in increased spreading area, failure to assemble the central and peripheral supramolecular activation clusters (cSMAC and pSMAC), and increased motility. Surprisingly, TCR microcluster speed was reduced marginally, however TCR microclusters dissipated prior to forming a cSMAC. TCR microclusters formed in the Myh9 siRNA-treated T cells showed reduced phosphorylation of the Src family kinase (SFK) activation loop and displayed reduced cytoplasmic calcium ion (Ca2+) elevation. In addition, Myh9 siRNA-treated cells displayed reduced phosphorylation of the Cas-L substrate domain – a force-dependent SFK substrate – which was observed in control siRNA-treated cells in foci throughout the immunological synapse except the cSMAC. Cas-L exhibited TCR ligation-dependent induction of phosphorylation. These results provide further evidence that T cell activation is modulated by intrinsic force-generating systems and can be viewed as a mechanically responsive process influenced by MYH9.

Background Because of the strong link between childhood obesity and adulthood obesity comorbidities, and the difficulty in decreasing body mass index (BMI) later in life, effective strategies are needed to address this condition in early childhood. The ability to predict obesity before age five could be a useful tool, allowing prevention strategies to focus on high risk children. The few existing prediction models for obesity in childhood have primarily employed data from longitudinal cohort studies, relying on difficult to collect data that are not readily available to all practitioners. Instead, we utilized real-world unaugmented electronic health record (EHR) data from the first two years of life to predict obesity status at age five, an approach not yet taken in pediatric obesity research. Methods and findings We trained a variety of machine learning algorithms to perform both binary classification and regression. Following previous studies demonstrating different obesity determinants for boys and girls, we similarly developed separate models for both groups. In each of the separate models for boys and girls we found that weight for length z-score, BMI between 19 and 24 months, and the last BMI measure recorded before age two were the most important features for prediction. The best performing models were able to predict obesity with an Area Under the Receiver Operator Characteristic Curve (AUC) of 81.7% for girls and 76.1% for boys. Conclusions We were able to predict obesity at age five using EHR data with an AUC comparable to cohort-based studies, reducing the need for investment in additional data collection. Our results suggest that machine learning approaches for predicting future childhood obesity using EHR data could improve the ability of clinicians and researchers to drive future policy, intervention design, and the decision-making process in a clinical setting.

SUMMARYUnderstanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant -oliver -that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration.