We describe a conditional in vivo protein trap mutagenesis system that reveals spatio-temporal protein expression dynamics and assesses gene function in the vertebrate Danio rerio. Integration of pGBT-RP2 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript levels while simultaneously reporting protein expression of each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site blocking morpholinos, thus representing the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines including a diverse array of molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step towards a unified codex of protein expression and direct functional annotation of the vertebrate genome.

Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) is hypothesized to act as a ligand for the CLV1 receptor kinase in the regulation of stem cell specification at shoot and flower meristems. CLV3 is a secreted protein, with an amino-terminal signal sequence and a conserved C-terminal domain of 15 amino acids, termed the CLE (CLV3/ESR-related) domain, based on its similarity to a largely unstudied protein family broadly present in land plants. We have tested the function of 13 Arabidopsis CLEs in vivo and found a significant variability in the ability of CLEs to replace CLV3, ranging from complete to no complementation. The best rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domainswap experiments indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function. Finally, we have detected proteolytic activity in extracts from cauliflower (Brassica oleracea) that process both CLV3 and CLE1 at their C termini. For CLV3, processing appears to occur at the absolutely conserved arginine-70 found at the beginning of the CLE domain. We propose that CLV3 and other CLEs are C-terminally processed to generate an active CLE peptide.

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

Proteins containing a conserved motif known as the CLE domain are found widely distributed across land plants. While the functions of most CLE proteins are unknown, specific CLE proteins have been shown to control shoot meristem, root and vascular development. This has been best studied for CLV3 which is required for stem cell differentiation at shoot and flower meristems. In vivo evidence indicates that the CLE domain is the functional region for CLV3, and that it is proteolytically processed from the CLV3 precursor protein. But the mechanism and activity responsible for this processing is poorly understood. Here we extend analysis of an in vitro CLE processing activity and show that in vitro cleavage occurs at Arg70, exactly matching in vivo maturation. We provide evidence that related processing activities are present in multiple tissues and species. We show that efficient protease recognition can occur with as little as four residues upstream of the CLE domain, and that the conserved arginine at position +1 and conserved acidic residues at positions -2 and/or -3 are required for efficient cleavage. Finally, we provide evidence that the N-terminal processing enzyme is a secreted serine protease while C-terminal processing may occur via a progressive carboxypeptidase.

Hedgehog (Hh) pathway activation and Gli-dependent transcription play critical roles in embryonic patterning, tissue homeostasis, and tumorigenesis. By conducting a genome-scale cDNA overexpression screen, we have identified the Rho GAP family member Arhgap36 as a positive regulator of the Hh pathway in vitro and in vivo. Arhgap36 acts in a Smoothened (Smo)-independent manner to inhibit Gli repressor formation and to promote the activation of full-length Gli proteins. Arhgap36 concurrently induces the accumulation of Gli proteins in the primary cilium, and its ability to induce Gli-dependent transcription requires kinesin family member 3a and intraflagellar transport protein 88, proteins that are essential for ciliogenesis. Arhgap36 also functionally and biochemically interacts with Suppressor of Fused. Transcriptional profiling further reveals that Arhgap36 is overexpressed in murine medulloblastomas that acquire resistance to chemical Smo inhibitors and that ARHGAP36 isoforms capable of Gli activation are upregulated in a subset of human medulloblastomas. Our findings reveal a new mechanism of Gli transcription factor activation and implicate ARHGAP36 dysregulation in the onset and/or progression of GLI-dependent cancers.

Nematodes that parasitize plant roots cause huge economic losses and have few mechanisms for control. Many parasitic nematodes infect plants by reprogramming root development to drive the formation of feeding structures. How nematodes take control of plant development is largely unknown. Here, we identify two host factors involved in the function of a receptor ligand mimic, GrCLE1, secreted by the potato cyst nematode Globodera rostochiensis. GrCLE1 is correctly processed to an active form by host plant proteases. Processed GrCLE1 peptides bind directly to the plant CLE receptors CLV2, BAM1, and BAM2. Involvement of these receptors in the ligand-mimicking process is also supported by the fact that the ability of GrCLE1 peptides to alter plant root development in Arabidopsis (Arabidopsis thaliana) is dependent on these receptors. Critically, we also demonstrate that GrCLE1 maturation can be entirely carried out by plant factors and that the availability of CLE processing activity may be essential for successful ligand mimicry.

Establishing pairwise keys for each pair of neighboring sensors is the first concern in securing communication in sensor networks. This task is challenging because resources are limited. Several random key predistribution schemes have been proposed, but they are appropriate only when sensors are uniformly distributed with high density. These schemes also suffer from a dramatic degradation of security when the number of compromised sensors exceeds a threshold. In this paper, we present a group-based key predistribution scheme, GKE, which enables any pair of neighboring sensors to establish a unique pairwise key, regardless of sensor density or distribution. Since pairwise keys are unique, security in GKE degrades gracefully as the number of compromised nodes increases. In addition, GKE is very efficient since it requires only localized communication to establish pairwise keys, thus significantly reducing the communication overhead. Our security analysis and performance evaluation illustrate the superiority of GKE in terms of resilience, connectivity, communication overhead and memory requirement.

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