Establishing an Efficient Electroporation-Based Method to Manipulate Target Gene Expression in the Axolotl Brain.

The tetrapod salamander species axolotl (Ambystoma mexicanum) is capable of regenerating injured brain. For better understanding the mechanisms of brain regeneration, it is very necessary to establish a rapid and efficient gain-of-function and loss-of-function approaches to study gene function in the axolotl brain. Here, we establish and optimize an electroporation-based method to overexpress or knockout/knockdown target gene in ependymal glial cells (EGCs) in the axolotl telencephalon. By orientating the electrodes, we were able to achieve specific expression of EGFP in EGCs located in dorsal, ventral, medial, or lateral ventricular zones. We then studied the role of Cdc42 in brain regeneration by introducing Cdc42 into EGCs through electroporation, followed by brain injury. Our findings showed that overexpression of Cdc42 in EGCs did not significantly affect EGC proliferation and production of newly born neurons, but it disrupted their apical polarity, as indicated by the loss of the ZO-1 tight junction marker. This disruption led to a ventricular accumulation of newly born neurons, which are failed to migrate into the neuronal layer where they could mature, thus resulted in a delayed brain regeneration phenotype. Furthermore, when electroporating CAS9-gRNA protein complexes against TnC (Tenascin-C) into EGCs of the brain, we achieved an efficient knockdown of TnC. In the electroporation-targeted area, TnC expression is dramatically reduced at both mRNA and protein levels. Overall, this study established a rapid and efficient electroporation-based gene manipulation approach allowing for investigation of gene function in the process of axolotl brain regeneration.

Beyond External Light: On-Spot Light Generation or Light Delivery for Highly Penetrated Photodynamic Therapy.

External light sources, such as lasers, light emitting diodes (LEDs) and lamps, are widely applied in photodynamic therapy (PDT); however, their use is severely limited by the nature of shallow tissue penetration depth. The recent exploration of light delivery or local generation on tumor sites has attracted much attention, owing to the fact that these systems are significantly endowed with high tissue penetration. In this review, we briefly introduced the principle of "on-spot light generation or delivery systems" in PDT. These systems are divided into different categories: (1) implantable luminescence, (2) mechanoluminescence, (3) electrochemiluminescence, (4) Cerenkov luminescence, (5) chemiluminescence, and (6) bioluminescence. Finally, their applications, advantages, and disadvantages in PDT will be appropriately summarized and further discussed in detail. We believe that this review will provide general guidance for the further design of light generation or delivery systems and clinical studies for PDT-mediated cancer treatments with unparalleled merits.

Muscles are barely required for the patterning and cell dynamics in axolotl limb regeneration.

Regeneration of a complex appendage structure such as limb requires upstream and downstream coordination of multiple types of cells. Given type of cell may sit at higher upstream position to control the activities of other cells. Muscles are one of the major cell masses in limbs. However, the subtle functional relationship between muscle and other cells in vertebrate complex tissue regeneration are still not well established. Here, we use Pax7 mutant axolotls, in which the limb muscle is developmentally lost, to investigate limb regeneration in the absence of skeletal muscle. We find that the pattern of regenerated limbs is relative normal in Pax7 mutants compared to the controls, but the joint is malformed in the Pax7 mutants. Lack of muscles do not affect the early regeneration responses, specifically the recruitment of macrophages to the wound, as well as the proliferation of fibroblasts, another major population in limbs. Furthermore, using single cell RNA-sequencing, we show that, other than muscle lineage that is mostly missing in Pax7 mutants, the composition and the status of other cell types in completely regenerated limbs of Pax7 mutants are similar to that in the controls. Our study reveals skeletal muscle is barely required for the guidance of other cells, as well the patterning in complex tissue regeneration in axolotls, and provides refined views of the roles of muscle cell in vertebrate appendage regeneration.

Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation.

The differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Gene isoforms allow a single gene diverse functions across different cell types, and isoform dynamics allow different functions over time. However, methods to efficiently identify and quantify RNA isoforms genome-wide in single cells are still lacking. Here, we introduce single cell RNA Cap And Tail sequencing (scRCAT-seq), a method to demarcate the boundaries of isoforms based on short-read sequencing, with higher efficiency and lower cost than existing long-read sequencing methods. In conjunction with machine learning algorithms, scRCAT-seq demarcates RNA transcripts with unprecedented accuracy. We identified hundreds of previously uncharacterized transcripts and thousands of alternative transcripts for known genes, revealed cell-type specific isoforms for various cell types across different species, and generated a cell atlas of isoform dynamics during the development of retinal cones.