Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration.

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration

Gellan gum capped silver nanoparticle dispersions and hydrogels: cytotoxicity and in vitro diffusion studies

The preparation of highly stable water dispersions of silver nanoparticles using the naturally available gellan gum as a reducing and capping agent is reported. Further, exploiting the gel formation characteristic of gellan gum silver nanoparticle incorporated gels have also been prepared. The optical properties, morphology, zeta potential and long-term stability of the synthesized silver nanoparticles were investigated. The superior stability of the gellan gum–silver nanoparticle dispersions against pH variation and electrolyte addition is revealed. Finally, we studied the cytotoxicity of AgNP dispersions in mouse embryonic fibroblast cells (NIH3T3) and also evaluated the in vitro diffusion of AgNP dispersions/gels across rat skin

Mise en évidence de complexes associés au silicium et à l'hydrogène entrant dans le mécanisme de neutralisation des donneurs dans GaAs:Si(n) hydrogéné

After hydrogen plasma exposure of a n type GaAs:Si crystal, we observe a decrease of the free carrier concentration and a hydrogen diffusion in the near surface region of the material. In bulk crystals, a good correlation has been established between the hydrogen penetration depth and the depth where the free carrier concentration recovers its bulk value. The decrease of the carrier concentration is accompanied by a significant increase of the electron mobility. This increase reveals a neutralization of the active donors and their transformation into electrically neutral complexes. A detailed infrared spectroscopy study on plasma exposed GaAs:Si epilayers shows a very sharp absorption line at 890 cm-1 on hydrogenated samples and 637 cm-1 on deuterated samples. These bands are totally absent in hydrogenated undoped GaAs. The isotopic shift frequency analysis indicates that the 890 cm-1 line could be associated with an arsenic-hydrogen bond where arsenic is supposed to sit as a first nearest neighbour of a silicon donor. Isochronal annealing experiments show a good correlation between the 890 cm-1 absorption band intensity and the neutralized silicon donor concentration. The neutralization would be due to the formation of (SiAs 3)As-H complexes, the extra electron of the silicon donor being trapped in order to participate to the As-H bond.Après exposition d'un cristal de GaAs:Si de type n à un plasma d'hydrogène, on constate une diminution de la concentration de porteurs libres et une diffusion d'hydrogène au voisinage de la surface du matériau. Dans les cristaux massifs, on a pu établir une bonne corrélation entre la profondeur de pénétration de l'hydrogène et la profondeur à laquelle la concentration de porteurs retrouve sa valeur de volume. La réduction de la concentration de porteurs s'accompagne d'une augmentation notable de la mobilité des électrons. Cette augmentation traduit une neutralisation des donneurs actifs par une transformation de ces donneurs en complexes neutres. Une étude détaillée par spectroscopie infrarouge de couches épitaxiées révèle une raie d'absorption étroite à 890 cm -1 dans les échantillons hydrogénés et à 637 cm-1 dans les échantillons deutérés. Ces bandes sont totalement absentes dans l'échantillon hydrogéné non dopé. L'analyse du déplacement isotopique des fréquences montre que la bande à 890 cm-1 est peut-être associée à une liaison arsenic-hydrogène où l'arsenic est supposé être en premier voisin d'un donneur silicium. Une série de recuits isochrones montre une bonne corrélation entre l'intensité de la raie à 890 cm-1 et la concentration de donneurs neutralisés dans le matériau. La neutralisation serait due à la formation de complexes (SiAs3)As-H, l'électron supplémentaire du silicium venant se piéger pour participer à la liaison As-H

Application and optimization of CRISPR–Cas9-mediated genome engineering in axolotl ( Ambystoma mexicanum )

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR–Cas9 system. The protocol, which takes 7–8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)–CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome

Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination.

The axolotl (Mexican salamander, Ambystoma mexicanum) has become a very useful model organism for studying limb and spinal cord regeneration because of its high regenerative capacity. Here we present a protocol for successfully mating and breeding axolotls in the laboratory throughout the year, for metamorphosing axolotls by a single i.p. injection and for axolotl transgenesis using I-SceI meganuclease and the mini Tol2 transposon system. Tol2-mediated transgenesis provides different features and advantages compared with I-SceI-mediated transgenesis, and it can result in more than 30% of animals expressing the transgene throughout their bodies so that they can be directly used for experimentation. By using Tol2-mediated transgenesis, experiments can be performed within weeks (e.g., 5-6 weeks for obtaining 2-3-cm-long larvae) without the need to establish germline transgenic lines (which take 12-18 months). In addition, we describe here tamoxifen-induced Cre-mediated recombination in transgenic axolotls

Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate

Different developmental histories of beta-cells generate functional and proliferative heterogeneity during islet growth.

The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states