Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Innate Mechanisms of Heart Regeneration.

Heart regeneration is a remarkable process whereby regrowth of damaged cardiac tissue rehabilitates organ anatomy and function. Unfortunately, the human heart is highly resistant to regeneration, which creates a shortage of cardiomyocytes in the wake of ischemic injury, and explains, in part, why coronary artery disease remains a leading cause of death worldwide. Luckily, a detailed blueprint for achieving therapeutic heart regeneration already exists in nature because several lower vertebrate species successfully regenerate amputated or damaged heart muscle through robust cardiomyocyte proliferation. A growing number of species are being interrogated for cardiac regenerative potential, and several commonalities have emerged between those animals showing high or low innate capabilities. In this review, we provide a historical perspective on the field, discuss how regenerative potential is influenced by cardiomyocyte properties, mitogenic signals, and chromatin accessibility, and highlight unanswered questions under active investigation. Ultimately, delineating why heart regeneration occurs preferentially in some organisms, but not in others, will uncover novel therapeutic inroads for achieving cardiac restoration in humans.

Activating transcription factor 3 coordinates differentiation of cardiac and hematopoietic progenitors by regulating glucose metabolism.

The cardiac and hematopoietic progenitors (CPs and HPs, respectively) in the mesoderm ultimately form a well-organized circulation system, but mechanisms that reconcile their development remain elusive. We found that activating transcription factor 3 (atf3) was highly expressed in the CPs, HPs, and mesoderm, in zebrafish. The atf3 -/- mutants exhibited atrial dilated cardiomyopathy and a high ratio of immature myeloid cells. These manifestations were primarily caused by the blockade of differentiation of both CPs and HPs within the anterior lateral plate mesoderm. Mechanistically, Atf3 targets cebpγ to repress slc2a1a-mediated glucose utilization. The high rate of glucose metabolism in atf3 -/- mutants inhibited the differentiation of progenitors by changing the redox state. Therefore, atf3 could provide CPs and HPs with metabolic adaptive capacity to changes in glucose levels. Our study provides new insights into the role of atf3 in the coordination of differentiation of CPs and HPs by regulating glucose metabolism.

[Rapid analysis on chemical constituents in Qige Keli by RRLC-Q-TOF-MS/MS].

The qualitative analysis method of RRLC-Q-TOF-MS/MS was established for determine the chemical constituents in Qige Keli. Kramosil C18 column (4.6 mm×150 mm, 3.5 µm) was used at the temperature of 30 °C. The mobile phase was 0.2% formic acid and acetonitrile by gradient elution, with a flow rate at 1.0 mL·min⁻¹, and the injection volume was 10 µL. The high-resolution quadrupole time-flight mass spectrometry was used as detector with electrospray ion source in both positive and negative models. On the basis of medicinal materials, reference materials, literature reports, and mass spectrometry data, the chemical composition in the Qige Keli was identified. A total of 44 compounds were identified, including 3 flavonoids, 21 flavonoid glycosides, 8 organic acids, 6 lactones, and 3 saponins. The results laid the foundation for the quality control of Qige Keli and the further research on pharmacodynamic materials.

The pro-inflammatory signalling regulator Stat4 promotes vasculogenesis of great vessels derived from endothelial precursors.

Vasculogenic defects of great vessels (GVs) are a major cause of congenital cardiovascular diseases. However, genetic regulators of endothelial precursors in GV vasculogenesis remain largely unknown. Here we show that Stat4, a transcription factor known for its regulatory role of pro-inflammatory signalling, promotes GV vasculogenesis in zebrafish. We find stat4 transcripts highly enriched in nkx2.5+ endothelial precursors in the pharynx and demonstrate that genetic ablation of stat4 causes stenosis of pharyngeal arch arteries (PAAs) by suppressing PAAs 3-6 angioblast development. We further show that stat4 is a downstream target of nkx2.5 and that it autonomously promotes proliferation of endothelial precursors of the mesoderm. Mechanistically, stat4 regulates the emerging PAA angioblasts by inhibiting the expression of hdac3 and counteracting the effect of stat1a. Altogether, our study establishes a role for Stat4 in zebrafish great vessel development, and suggests that Stat4 may serve as a therapeutic target for GV defects.