Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue ; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain using scanning Bessel-3PM.

Novel stem cells expressing stage-specific embryonic antigen 3 (SSEA-3) reside among human dermal fibroblasts and are known as multilineage-differentiating stress-enduring (Muse) cells. They enhance the generation efficiency of induced pluripotent stem cells. However, Muse cells have only been found in humans. We aimed to isolate SSEA3-positive cells from terminally differentiated skin fibroblasts of adult goat and determine their pluripotency. Cell clusters from SSEA3(+) populations possessed stem cell-like morphological features and normal karyotypes, were consistently positive for alkaline phosphatase, and expressed stem cell pluripotency markers. These SSEA3(+) cells remained undifferentiated over eight passages in suspension culture and were able to differentiate into cells of all three germ layers in vitro and in vivo. Our combined findings suggest that a subset of adult stem cells expressing SSEA3 also exist among adult goat skin fibroblasts. We are the first to report that multipotent adult goat cells exist among terminally differentiated goat skin in suspension culture. Our results also provide a promising platform for generation of a transgenic goat, because the undifferentiated state of stem cells was thought to be more efficient as donor cells for somatic cell nuclear transfer.

Unlike mammals, zebrafish can regenerate injured hearts even in the adult stage. Cardiac regeneration requires the coordination of cardiomyocyte (CM) proliferation and migration. The TGF-β/Smad3 signaling pathway has been implicated in cardiac regeneration, but the molecular mechanisms by which this pathway regulates CM proliferation and migration have not been fully illustrated. Here, we investigated the function of TGF-β/Smad3 signaling in a zebrafish model of ventricular ablation. Multiple components of this pathway were upregulated/activated after injury. Utilizing a specific inhibitor of Smad3, we detected an increased ratio of unrecovered hearts. Transcriptomic analysis suggested that the TGF-β/Smad3 signaling pathway could affect CM proliferation and migration. Further analysis demonstrated that the CM cell cycle was disrupted and the epithelial–mesenchymal transition (EMT)-like response was impaired, which limited cardiac regeneration. Altogether, our study reveals an important function of TGF-β/Smad3 signaling in CM cell cycle progression and EMT process during zebrafish ventricle regeneration.

Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration.

Previous studies have demonstrated that skin stem cells expressing the pluripotency marker stage-specific embryonic antigen 3 (SSEA3) are easier to reprogram into induced pluripotent stem cells (iPSCs) than skin fibroblasts. Furthermore, it is widely speculated that the undifferentiated state may make stem cells more efficient donor cells for somatic cell nuclear transfer (SCNT). In this study, we isolated SSEA3(+) cells from goat skin fibroblast cells (SFCs) using fluorescence-activated cell sorting (FACS) and examined expression of pluripotency markers and in vitro development of cloned embryos following SCNT. Results showed that cell clusters from SSEA3(+) cells were consistently positive for alkaline phosphatase staining and pluripotency markers, Nanog, Oct4, Sox2, and SSEA3. The cleavage rate of cloned embryos derived from SSEA3(+) cells did not differ compared with SFCs (70.5±0.8% and 68.4±2.1%, respectively), but was significantly higher compared with SSEA3(-) cells (64.9±1.6%, p<0.05). The blastocyst rate was significantly increased in the SSEA3(+) cell group compared with the SFC and SSEA3(-) cell groups (30.3±1.2% vs. 21.2±0.9 and 19.0±1.0%, respectively, p<0.05). The quality of cloned blastocysts from SSEA3(+) cells was higher compared with SFCs and SSEA3(-) cells, based on total cell number and number of apoptotic cells per blastocyst. These findings suggest that using SSEA3(+) cells as donors for SCNT is beneficial for enhancing in vitro development and quality of cloned goat embryos.