Xenograft of human pluripotent stem cell-derived cardiac lineage cells on zebrafish embryo heart.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are a promising cell source for regenerative medicine and drug discovery. However, the use of animal models for studying human cardiomyocytes derived from hiPSCs in vivo is limited and challenging. Given the shared properties between humans and zebrafish, their ethical advantages over mammalian models, and their immature immune system that is rejection-free against xenografted human cells, zebrafish provide a suitable alternative model for xenograft studies. We microinjected fluorescence-labeled cardiac lineage cells derived from hiPSCs, specifically mesoderm or cardiac mesoderm cells, into the yolk and the area proximal to the outflow tract of the linear heart at 24 hours post-fertilization (hpf). The cells injected into the yolk survived and did not migrate to other tissues. In contrast, the cells injected contiguous with the outflow tract of the linear heart migrated into the pericardial cavity and heart. After 1 day post injection (1 dpi, 22-24 hpi), the injected cells migrated into the pericardial cavity and heart. Importantly, we observed heartbeat-like movements of some injected cells in the zebrafish heart after 1 dpi. These results suggested successful xenografting of hiPSC-derived cardiac lineage cells into the zebrafish embryo heart. Thus, we developed a valuable tool using zebrafish embryos as a model organism for investigating the molecular and cellular mechanisms involved in the grafting process. This is essential in developing cell transplantation-based cardiac therapeutics as well as for drug testing, notably contributing to advancements in the field of cardio-medicine.

Notch signaling modulates the nuclear localization of carboxy-terminal-phosphorylated smad2 and controls the competence of ectodermal cells for activin A.

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps [Coffman, C.R., Skoglund, P., Harris, W.A., Kintner, C.R., 1993. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73, 659-671], although the mechanism by which this modulation occurs remains unknown. Here, we show that Notch signaling also delays the onset of LMC in whole embryos, as it did in animal caps. To better understand this effect and the mechanism of LMC itself, we investigated at which step of activin signal transduction pathway the Notch signaling act to affect the timing of the LMC. In our system, ALK4 (activin type I receptor) maintained the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 could bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. We conclude that LMC was induced because C-terminal-phosphorylated smad2 lost its ability to bind to smad4, and consequently could not accumulate in the nucleus. Notch signal activation restored the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC.

Activin-like signaling activates Notch signaling during mesodermal induction.

Both activin-like signaling and Notch signaling play fundamental roles during early development. Activin-like signaling is involved in mesodermal induction and can induce a broad range of mesodermal genes and tissues from prospective ectodermal cells (animal caps). On the other hand, Notch signaling plays important roles when multipotent precursor cells achieve a specific cell fate. However, the relationship between these two signal pathways is not well understood. Here, we show that activin A induces Delta-1, Delta-2 and Notch expression and then activates Notch signaling in animal caps. Also, in vivo, ectopic activin-like signaling induced the ectopic expression of Delta-1 and Delta-2, whereas inhibition of activin-like signaling abolished the expression of Delta-1 and Delta-2. Furthermore, we show that MyoD, which is myogenic gene induced by activin A, can induce Delta-1 expression. However, MyoD had no effect on Notch expression, and inhibited Delta-2 expression. These results indicated that activin A induces Delta-1, Delta-2 and Notch by different cascades. We conclude that Notch signaling is activated when activin-like signaling induces various tissues from homogenous undifferentiated cells.