Age effects of Moso bamboo on leaf isoprene emission characteristics.

Isoprene is a highly reactive volatile organic compound that significantly affects atmospheric oxidant capacity, regional air quality, and climate change. Moso bamboo (Phyllostachys edulis), a species widely distributed in tropical and subtropical regions, particularly in China, is a strong isoprene emitter with great potential for carbon sequestration. Carbon sequestration is negatively correlated with culm age; however, the effect of this correlation on isoprene emissions remains unknown. In this study, we investigated the photosynthetic and isoprene emission characteristics of Moso bamboo at different culm ages. The results showed that the age effect on isoprene emission was different from that on photosynthesis; the net photosynthesis rate (Pn) was the highest in young, followed by mature, and then old bamboo, whereas the isoprene emission rate (Iso) was the highest in young, followed by old, and then mature bamboo. Moreover, the percentage of carbon loss as isoprene emission (C-loss) during photosynthesis of old bamboo was 35% higher than that of mature bamboo under standard conditions (leaf temperature: 30°C; light intensity: 1000 µmol m-2 s-1). Therefore, we strongly recommend considering the culm age when establishing an isoprene emission model of Moso bamboo. Additionally, because the Iso and C-loss of old bamboo were higher than those of mature bamboo, we suggest that attention should be paid to the management of bamboo age structure and timely felling of aged bamboo to reduce environmental risk.

Dual-Frequency Polarized Reconfigurable Terahertz Antenna Based on Graphene Metasurface and TOPAS.

A hybrid dual-frequency polarized reconfigurable terahertz antenna is designed and studied. Graphene and TOPAS are employed as the polarization conversion metasurface and dielectric substrate, respectively, enabling tunable polarization conversion and circular polarization. TOPAS is a good substrate material for broadband THz components due to its low absorption. By adjusting the chemical potential of graphene between 0 eV and 0.5 eV, the polarization state in the band of 1 THz (0.76-1.02 THz) and 2.5 THz (2.43-2.6 THz) can be reconstructed. Thanks to the multilayer graphene structure and low absorption TOPAS, the graphene metasurface exhibits a broad bandwidth of 0.26 and 0.17 THz, respectively, in the band of 1 THz and 2.5 THz. The working state of the circularly polarized antenna and linearly polarized antenna can be switched in the bands around 1 THz (0.7-0.75 THz, 0.96-1.04 THz) and 2.5 THz (2.42-2.52 THz), respectively, without changing the physical geometry. Moreover, the graphene antenna, metasurface, and hybrid structure are tested, respectively, to verify that the components do not interfere with each other in performance. The hybrid antenna shows great potential in tunable terahertz devices and related applications.

A Neurexin2aa deficiency results in axon pathfinding defects and increased anxiety in zebrafish.

Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.

Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering.

The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish.

During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton.

A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish.

Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease.

The PDZ domain protein Mcc is a novel effector of non-canonical Wnt signaling during convergence and extension in zebrafish.

During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, ß-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation.

Zebrafish prox1b mutants develop a lymphatic vasculature, and prox1b does not specifically mark lymphatic endothelial cells.

BACKGROUND: The expression of the Prospero homeodomain transcription factor (Prox1) in a subset of cardinal venous cells specifies the lymphatic lineage in mice. Prox1 is also indispensible for the maintenance of lymphatic cell fate, and is therefore considered a master control gene for lymphangiogenesis in mammals. In zebrafish, there are two prox1 paralogues, the previously described prox1 (also known as prox1a) and the newly identified prox1b. PRINCIPAL FINDINGS: To investigate the role of the prox1b gene in zebrafish lymphangiogenesis, we knocked-down prox1b and found that depletion of prox1b mRNA did not cause lymphatic defects. We also generated two different prox1b mutant alleles, and maternal-zygotic homozygous mutant embryos were viable and did not show any lymphatic defects. Furthermore, the expression of prox1b was not restricted to lymphatic vessels during zebrafish development. CONCLUSION: We conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish.

Alternative splicing of SMADs in differentiation and tissue homeostasis.

Tissue-specific alternate splicing is an important means of regulating gene expression during development. The effector proteins for the transforming growth factor-beta signaling pathway, the SMADs, encode distinct isoforms generated via alternate splicing, which appear to have distinct tissue-specific expression profiles and functions. Here, we discuss the roles of various SMAD isoforms, and the consequences of mis-regulation of SMAD splicing in development and tissue homeostasis.

The Integrator subunits function in hematopoiesis by modulating Smad/BMP signaling.

Hematopoiesis, the dynamic process of blood cell development, is regulated by the activity of the bone morphogenetic protein (BMP) signaling pathway and by many transcription factors. However, the molecules and mechanisms that regulate BMP/Smad signaling in hematopoiesis are largely unknown. Here, we show that the Integrator complex, an evolutionarily conserved group of proteins, functions in zebrafish hematopoiesis by modulating Smad/BMP signaling. The Integrator complex proteins are known to directly interact with RNA polymerase II to mediate 3' end processing of U1 and U2 snRNAs. We have identified several subunits of the Integrator complex in zebrafish. Antisense morpholino-mediated knockdown of the Integrator subunit 5 (Ints5) in zebrafish embryos affects U1 and U2 snRNA processing, leading to aberrant splicing of smad1 and smad5 RNA, and reduced expression of the hematopoietic genes stem cell leukemia (scl, also known as tal1) and gata1. Blood smears from ints5 morphant embryos show arrested red blood cell differentiation, similar to scl-deficient embryos. Interestingly, targeting other Integrator subunits also leads to defects in smad5 RNA splicing and arrested hematopoiesis, suggesting that the Ints proteins function as a complex to regulate the BMP pathway during hematopoiesis. Our work establishes a link between the RNA processing machinery and the downstream effectors of BMP signaling, and reveals a new group of proteins that regulates the switch from primitive hematopoietic stem cell identity and blood cell differentiation by modulating Smad function.