Single-cell analysis reveals an Angpt4-initiated EPDC-EC-CM cellular coordination cascade during heart regeneration

Mammals exhibit limited heart regeneration ability,which can lead to heart failure after myocardial infarction.In contrast,zebrafish exhibit remarkable cardiac regeneration capacity.Several cell types and signaling pathways have been reported to participate in this process.However,a comprehensive analysis of how different cells and signals interact and coordinate to regulate cardiac regeneration is unavailable.We collected major cardiac cell types from zebrafish and performed high-precision single-cell transcriptome analyses during both development and post-injury regeneration.We revealed the cellular heterogeneity as well as the molecular progress of cardiomyocytes during these processes,and identified a subtype of atrial cardiomyocyte exhibiting a stem-like state which may transdifferentiate into ventricular cardiomyocytes during regeneration.Furthermore,we identified a regeneration-induced cell(RIC)population in the epicardium-derived cells(EPDC),and demonstrated Angiopoietin 4(Angpt4)as a specific regulator of heart regeneration.angpt4 expression is specifically and transiently activated in RIC,which initiates a signaling cascade from EPDC to endocardium through the Tie2-MAPK pathway,and further induces activation of cathepsin K in cardiomyocytes through RA signaling.Loss of angpt4 leads to defects in scar tissue resolution and cardiomyocyte proliferation,while overexpression of angpt4 accelerates regeneration.Furthermore,we found that ANGPT4 could enhance proliferation of neonatal rat cardiomyocytes,and promote cardiac repair in mice after myocardial infarction,indicating that the function of Angpt4 is conserved in mammals.Our study provides a mechanistic understanding of heart regeneration at single-cell precision,identifies Angpt4 as a key regulator of cardiomyocyte proliferation and regeneration,and offers a novel therapeutic target for improved recovery after human heart injuries.

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Structure of neurofilaments studied with scanning tunneling microscopy

Neurofilaments (NFs) were isolated from bovine spinal cord. The structure of purified NFs was studied by scanning tunneling microscopy (STM). The STM images showed that NF was composed of a long core filament and numerous sidearms flanking the rod regularly. The diameter of the rod was about 10 nm (10.2±0.8 nm). Most of the sidearms were short and the distance between two adjacent sidearms was approximately 10 nm. There were some long sidearms between two proximal core filaments. The distance between two adjacent long sidearms was 21 nm. A three-quarter-staggered fashion of native NF structure was put forward.