ABSTRACT Wnt signaling is essential for tissue and organ regeneration in animals such as amphibians, and several Wnt ligands are highly expressed in regenerating tissues. However, little is known about the specific role played by each ligand and their relative contribution to regeneration processes. Here, we show that Wnt5a plays critical roles in cell proliferation and differentiation during Xenopus tadpole tail regeneration. CRISPR/Cas9‐mediated knock‐out (KO) of the wnt5a gene in Xenopus tadpoles caused a delay in tail regeneration. Expression of wnt5a is induced after wound healing in regenerating tails and is positively regulated by the early injury response genes junb and inhba , which are required for the initiation of regenerative cell proliferation. Interestingly, in wnt5a KO tadpoles, cell proliferation initiated by JunB and InhibinβA was reduced at late stages of tail regeneration, suggesting an essential role of Wnt5a in the maintenance of regenerative cell proliferation. Moreover, differentiation of the notochord and neural tissue was also affected in the regenerating tails of wnt5a KO tadpoles. Our findings demonstrate that Wnt5a acts downstream of JunB and InhibinβA to maintain cell proliferation and that it also promotes differentiation in Xenopus tadpole tail regeneration.
ABSTRACT Mouse embryos undergo drastic morphological changes during implantation, but analysis of this process has been challenging because it occurs within the uterus. Various in vitro culture systems supporting development beyond implantation have been established since the 1970s. More recently, 3D‐geec, which supports embryos from the peri‐implantation to egg cylinder stages while preserving their three‐dimensional organization, was developed as a useful platform for studying this critical period. Combining 3D‐geec with genome editing in zygotes or experimental manipulation of embryos should further facilitate mechanistic studies of these stages. In this study, to enable such approaches, we established an in vitro culture protocol that supports continuous development of mouse embryos from the zygote to the egg cylinder stage. Embryos cultured from the zygote to the late blastocyst stage in KSOM AA medium were not compatible with 3D‐geec. These embryos exhibited reduced cell numbers, delayed development, and reduced mTORC1 activity, which regulates cell growth and proliferation. The use of Advanced IVC medium supplemented with 10% fetal bovine serum (FBS) during the blastocyst stage substantially rescued these defects, and the resulting late blastocysts underwent further development into egg cylinders in 3D‐geec. Furthermore, genome‐edited embryos generated from zygotes developed into egg cylinders using this protocol and were successfully subjected to time‐lapse imaging during 3D‐geec. Thus, this culture protocol enables continuous in vitro development from the zygote to the egg cylinder stage and provides a platform for studying gene function and cellular dynamics during peri‐implantation and early postimplantation development.
The stony coral, Acropora tenuis, possesses progenitor/undifferentiated cells similar to other anthozoans; however, their developmental potential and fate remain obscure. The present study initially showed that nerve progenitors and undifferentiated-like cells were both tagged with histone H3 trimethylation at Lys 4 and Lys 27, suggesting that Acropora progenitor/undifferentiated cells might be multipotent. A rabbit anti-reverse transcriptase (anti-AtRTase) antibody recognized undifferentiated-like cells in the middle region of the ectodermal layer in embryos 60-85 h post-fertilization. At the larval stages, AtRTase signals were concentrated around the oral pole where the larval ectoderm invaginated to form the pharyngeal stomodeum. Undifferentiated-like cells, but not the apical-most glandular epithelium in the ectoderm, appeared to enter the pharynx. Consistent with this hypothesis, AtRTase signals were continuous from undifferentiated-like cells to the stomodeum. Nerve progenitors (neuroblasts) expressed nerve-specific tubulin beta-III (TUBB3). They differentiated into nerve cells and expressed a neuroblast differentiation-associated protein around the aboral pole of larvae. On the other hand, non-aboral neuroblasts expressed the autophagy-related protein 10 in addition to TUBB3 and developed into vacuolated hyaline cells (VHCs) that penetrated through the ectoderm apicobasally. The present study suggests strongly that ectodermal undifferentiated cells in A. tenuis have the potential to form the stomodeum. In contrast, nerve progenitors possess the dual potential to differentiate into nerve cells and VHCs, which appears to depend on aboral and non-aboral positions.
Genetically encoded ATP biosensors enable monitoring of cellular energy status, but their application in multicellular organisms remains limited. QUEEN is a ratiometric ATP biosensor consisting of a bacterial ATP-binding protein fused to a circularly permuted fluorescent protein and has been primarily validated in cultured cells. Here, we generated transgenic Drosophila melanogaster lines expressing QUEEN-7μ, enabling tissue-specific expression through the GAL4/UAS system. We characterized its performance in motor neurons and muscles. QUEEN-7μ responses were validated using pharmacological and genetic perturbations of mitochondrial function. Mitochondrial inhibition decreased the QUEEN-7μ ratio, consistent with reduced ATP levels, whereas acute treatment with mitochonic acid-5 (MA-5), a small molecule that enhances mitochondrial ATP synthesis, increased the QUEEN-7μ ratio, with a larger effect at higher concentration. Consistently, genetic manipulations, including the mitochondrial Complex I knockdown and Mitofilin/MIC60 overexpression, produced corresponding decreases and increases in the QUEEN-7μ ratio. These results establish QUEEN-7μ as a reliable ratiometric ATP reporter for quantitative in vivo analysis in Drosophila. This system provides a versatile platform for investigating energy metabolism and mitochondrial function in Drosophila.
The lateral line is a mechanosensory organ found in cyclostomes, teleosts, and amphibians. Hair cells within its neuromasts have kinocilia and stereocilia that sense water flow, and the lateral line nerve transduces this sensory input. Hair cells in the lateral line are analogous to those in the mammalian inner ear. Lateral line development and regeneration have been studied extensively in zebrafish. However, lateral line formation in other species remains poorly understood. The Iberian ribbed newt (Pleurodeles waltl) is a versatile model for regeneration with a short period of sexual maturation and aseasonal/prolific ovulation. Here, we investigated the features, regeneration processes, and additional neuromast formation in the posterior lateral line of Iberian ribbed newts using Brainbow reporters. The lateral line in the Iberian ribbed newts shares common features with other species. Laser ablation of hair cells or transection of the posterior lateral line nerve induced additional neuromast formation, to which epidermal cells contributed at least in part. We refer to it as "replicative neuromast regeneration" to allow distinction from several modes of neuromast formation in normal development. Based on these results, we propose that in response to injury, the posterior lateral line nerve induces epidermal cells to contribute to newly formed neuromasts in a replicative manner. Our results provide insights into lateral line morphogenesis.
