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.

Applying a Knock-In Strategy to Create Reporter-Tagged Knockout Alleles in Axolotls.

Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the "knocking out" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.

Single-cell Stereo-seq reveals induced progenitor cells involved in axolotl brain regeneration.

The molecular mechanism underlying brain regeneration in vertebrates remains elusive. We performed spatial enhanced resolution omics sequencing (Stereo-seq) to capture spatially resolved single-cell transcriptomes of axolotl telencephalon sections during development and regeneration. Annotated cell types exhibited distinct spatial distribution, molecular features, and functions. We identified an injury-induced ependymoglial cell cluster at the wound site as a progenitor cell population for the potential replenishment of lost neurons, through a cell state transition process resembling neurogenesis during development. Transcriptome comparisons indicated that these induced cells may originate from local resident ependymoglial cells. We further uncovered spatially defined neurons at the lesion site that may regress to an immature neuron-like state. Our work establishes spatial transcriptome profiles of an anamniote tetrapod brain and decodes potential neurogenesis from ependymoglial cells for development and regeneration, thus providing mechanistic insights into vertebrate brain regeneration.

[Clinical value of 24-hour ambulatory electrocardiography in childhood myocarditis].

OBJECTIVE: To investigate the characteristics of 24-hr ambulatory electrocardiography (DCG) of children with myocarditis and to study the clinical value of DCG in the diagnosis of childhood myocarditis. METHODS: 24-hr DCG findings, including abnormal DCG rate, and number, grade and distribution of ventricular premature beat (PVC), as well as heart rate variability, from 59 children with myocarditis were retrospectively reviewed and compared with those detected in 41 children without heart disease. RESULTS: 86.4% of patients with myocarditis showed abnormal DCG, and compound arrhythmia was commonly seen, but only 46.3% showed abnormal DCG (P < 0.01) and single arrhythmia was predominant in the control group. The number and grade of PVC/24 hrs were not significantly different between the two groups. Compared with the control group, the average pattern PVC was predominant in the myocarditis group (84.6% vs 48.7%; P < 0.05). Monopeak pattern PVC was mostly seen (64.4%), followed by multiple-peak pattern (25.4%) and bi-peak pattern (8.4%) in the myocarditis group, which were significantly different from the control group: monopeak pattern 53.6%, bi-peak pattern 36.6% and multiple-peak pattern 7.3% (P < 0.01). CONCLUSIONS: The 24-hr DCG characteristics of children with myocarditis are different from the normal controls, suggesting 24-hr DCG monitoring is useful to the diagnosis of childhood myocaditis.