The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development.

With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.

Immunohistochemistry and Fluorescent Whole Mount RNA In Situ Hybridization in Larval and Adult Brains of Tribolium.

Arthropod brains are fascinating structures that exhibit great complexity but also contain conserved elements that can be recognized between species. There is a long tradition of research in insect neuroanatomy, cell biology, and in studying the genetics of insect brain development. Recently, the beetle Tribolium castaneum has gained attention as a model for insect head and brain development, and many anterior patterning genes have so far been characterized in beetle embryos. The outcome of embryonic anterior development is the larval and, subsequently, the adult brain. A basic requirement to understand genetic cell type diversity within these structures is the ability to localize mRNA and protein of neural genes. Here we detail our protocols for RNA in situ hybridization in combination with immunohistochemistry, optimized for dissected brains of larval and adult beetles.

An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium.

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.

Identification of three site mutations in nonstructural protein 1ß, glycoprotein 3 and glycoprotein 5 that correlate with increased interferon α resistance of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically significant pathogen that has been recognized for its genetic variation, rapid evolution, and immune suppression. Type I interferons (IFNs) play an important role in host defense against viral infection by inducing many antiviral effectors, which might be a selective pressure driving viral evolution towards IFN resistance. To investigate the IFN resistance-related variation of PRRSV genome under IFN selective pressure and explore the molecular mechanism of IFN sensitivity changes, PRRSV strain JXwn06 was serially propagated in porcine pulmonary alveolar macrophages (PAMs) with IFNα treatment for 45 passages and 3 rounds of purification. Four mutant strains named JX-αP51n (n = 1, 2, 3 and 4) with reduced IFNα sensitivity were selected; the strains showed a 100-fold higher titer than the passaging-control strain JX-P51 in IFNα-treated PAMs. IFNα-resistant strains were found to antagonize the IFNα-activated JAK-STAT signaling pathway to a greater extent than the nonresistant strain by down-regulating the expression level of IFNα-activated pJAK1 through interfering with phosphatase. Furthermore, the PRRSV genetic variations interacting with IFNα were identified by full genomic sequencing and alignment. Among these mutations, amino acid substitutions in nsp1ß (E87 G), GP3 (F143 L) and GP5 (Y136 H) were found to correlate with increased IFNα resistance by enhancing the suppression effect on pJAK1, which could be further increased if these three substitution sites were combined. These findings provide some novel evidence for understanding PRRSV genetic variation under host selective pressure and viral evolution strategies to evade the host innate immune response.

Nsp2 and GP5-M of Porcine Reproductive and Respiratory Syndrome Virus Contribute to Targets for Neutralizing Antibodies.

Porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by its genetic variation and limited cross protection among heterologous strains. Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies (NAs) against PRRSV, the mechanism underlying limited cross-neutralization among heterologous strains is still controversial. In the present study, examinations of NA cross reaction between a highly pathogenic PRRSV (HP-PRRSV) strain, JXwn06, and a low pathogenic PRRSV (LP-PRRSV) strain, HB-1/3.9, were conducted with viral neutralization assays in MARC-145 cells. None of the JXwn06-hyperimmuned pigs' sera could neutralize HB-1/3.9 in vitro and vice versa. To address the genetic variation between these two viruses that are associated with limited cross-neutralization, chimeric viruses with coding regions swapped between these two strains were constructed. Viral neutralization assays indicated that variations in nonstructural protein 2 (nsp2) and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9. Furthermore, we substituted the nsp2-, glycoprotein2 (GP2)-, GP3-, and GP4-coding regions together, or nsp2-, GP5-, and membrane (M) protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses. The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells. Taken together, these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.

The insect central complex as model for heterochronic brain development-background, concepts, and tools.

The adult insect brain is composed of neuropils present in most taxa. However, the relative size, shape, and developmental timing differ between species. This diversity of adult insect brain morphology has been extensively described while the genetic mechanisms of brain development are studied predominantly in Drosophila melanogaster. However, it has remained enigmatic what cellular and genetic mechanisms underlie the evolution of neuropil diversity or heterochronic development. In this perspective paper, we propose a novel approach to study these questions. We suggest using genome editing to mark homologous neural cells in the fly D. melanogaster, the beetle Tribolium castaneum, and the Mediterranean field cricket Gryllus bimaculatus to investigate developmental differences leading to brain diversification. One interesting aspect is the heterochrony observed in central complex development. Ancestrally, the central complex is formed during embryogenesis (as in Gryllus) but in Drosophila, it arises during late larval and metamorphic stages. In Tribolium, it forms partially during embryogenesis. Finally, we present tools for brain research in Tribolium including 3D reconstruction and immunohistochemistry data of first instar brains and the generation of transgenic brain imaging lines. Further, we characterize reporter lines labeling the mushroom bodies and reflecting the expression of the neuroblast marker gene Tc-asense, respectively.

A functionalized fluorescent dendrimer as a pesticide nanocarrier: application in pest control.

We report the delivery of a hydrophobic pesticide, thiamethoxam, by water-soluble nanosized cationic dendrimers that contain hydrophobic dendritic polyesters and peripheral amines, demonstrated by DLS, spectral analysis and ITC. The dendrimer-based nanocarrier can efficiently deliver the pesticide into the live cells and largely increase the cytotoxicity of the drug.

Multifunctional magnetic and fluorescent core-shell nanoparticles for bioimaging.

Novel magnetic and fluorescent core-shell nanoparticles have been fabricated, which exhibit superparamagnetic behavior and emit strong near-infrared fluorescence. The nanoparticles are highly biocompatible and can be internalized into cells with nucleic accumulation via strong interaction with nucleic acids, implying potential applications in the biomedical field.

Perylene-cored star-shaped polycations for fluorescent gene vectors and bioimaging.

Two star polycations, poly(2-aminoethyl methacrylate) (PAEMA, P1) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, P2), have been synthesized with perylene diimide (PDI) as the central fluorophore. (1)H NMR and (13)C NMR are used to confirm the successful synthesis of a macromolecular initiator. Using ATRP strategy, P1 and P2 are obtained with narrow molecular weight distribution. The star polymers have good fluorescence properties in aqueous solution, which provides fluorescent tracing and imaging during gene delivery. Both P1 and P2 can efficiently condense DNA into stable nanoparticles. Transfection studies demonstrate that P1 and P2 deliver DNA into live cells with higher efficiency and lower cytotoxicity than polyethylenimine (PEI, 25 kDa). P2 shows higher capacity for gene delivery than P1 due to its better buffering and faster rate of cellular internalization.

Systemic gene silencing in plants triggered by fluorescent nanoparticle-delivered double-stranded RNA.

A cationic fluorescence nanoparticle efficiently enters plants with high transfection efficacy. Applying a mixture of G2/dsRNA to the model plant, Arabidopsis root, leads to significant reduction in the expression of important developmental genes and results in apparent phenotypes. This study reports a non-viral gene nanocarrier which triggers gene silencing in plants and leads to systemic phenotypes.

A multifunctional perylenediimide derivative (DTPDI) can be used as a recyclable specific Hg2+ ion sensor and an efficient DNA delivery carrier.

Multifunctional dithioacetal-modified perylenediimide (DTPDI) is synthesized as a highly sensitive and selective fluorescent chemosensor for recyclable Hg2+ detection and an effective DNA carrier. The central PDI chromophore allows the tracing of cell uptake by fluorescence microscopy, dithioacetals enable the detection of Hg2+, and peripheral amine hydrochloride salts increase the water solubility and also serve as positive charges for noncovalent binding of negatively charged DNA. In addition to serve as a recyclable fluorescent probe for Hg2+ detection, DTPDI can be rapidly internalized into live cells with low cytotoxicity and high DNA delivery efficacy.

Highly water-soluble perylenediimide-cored poly(amido amine) vector for efficient gene transfection.

A highly water-soluble perylenediimide-core poly(amido amine) (PDI-PAmAm) with peripheral amine groups has been synthesized. The central PDI chromophore allows optical monitoring of relevant cellular experiments by fluorescence microscopy. The PAmAm shell provides the steric bulk that notably suppresses the aggregation of the central PDI chromophore in aqueous solution. The peripheral amines provide water solubility and positive charges, and also serve as active sites for the further growth of PAmAm. PDI-PAmAm can be rapidly internalized into live cells with high efficacy of gene delivery and low cytotoxicity. Both in vitro and in vivo experiments demonstrate the high gene transfection efficacy of PDI-PAmAm.

Systemically interfering with immune response by a fluorescent cationic dendrimer delivered gene suppression.

A water-soluble cationic dendrimer with a central fluorescent perylenediimide (PDI) chromophore and many peripheral amines can rapidly penetrate into live hemocytes, gut and fat body. By double fluorescence tracing, the dendrimer is demonstrated to have a high gene-transfection capacity. The synthesized dsRNA targeting at serpin-3, a key immune gene, is systemically delivered by the dendrimer to insect fat bodies and hemocytes outside of midgut. Biological assays, including PCR and immunoblotting, show that the expressions of the target gene and its downstream immunity-related genes are largely suppressed. This study demonstrates for the first time that a PDI-cored, cationic, dendrimer-mediated dsRNA systemically interferes with the immune response in insects. This work provides an insect model for immunology research and a novel strategy for potential pest control.

Fluorescent nanoparticle delivered dsRNA toward genetic control of insect pests.

A fluorescent cationic core-shell nanoparticle efficiently enters into cells with high transfection efficacy. A FNP/CHT10-dsRNA complex is orally fed to insect pests and knocks down a midgut-specific chitinase gene of the Asian corn borer, which leads to death. This is the first report on the genetic control of insect pests through a non-viral gene delivery system to knock down key developmental gene expression.

Fluorescent water-soluble perylenediimide-cored cationic dendrimers: synthesis, optical properties, and cell uptake.

Different generations of perylenediimide-cored dendrimers with peripheral amine groups were synthesized. All these water-soluble dendrimers could rapidly internalize into live cells with high efficacy of gene transfection and low cytotoxicity. Increasing dendrimer generation increased their ability for gene transfection.