Development Growth & Differentiation最新文献

Activation of the dopamine type-D₂ receptor in late gastrula of sea urchins is known to decrease the growth rate of post-oral arms of larvae, and, as a result, the phenotype of these larvae mimics that of larvae developing in the abundance of food. Our data indicate that the effect of dopamine on sea urchin larvae is stage-dependent. In our experiment, the early four-armed plutei (96 hours post fertilization, hpf) of Strongylocentrotus intermedius had substantially shorter post-oral arms if they developed from the larvae treated with dopamine at the early pluteus stage (48 hpf), when they had already formed the first dopaminergic neurons, as compared to the plutei from the larvae treated with dopamine at the mid to late gastrula stage (24 hpf), when they did not have any neurons yet. The pre-treatment of larvae in 6-hydroxydopamine, a neurotoxic analog of dopamine that specifically disrupts activity of dopaminergic neurons, prevented the development of the short post-oral arms phenotype in larvae. These results confirm the assumption that dopaminergic neurons play an important role in the development of the short post-oral arms phenotype in sea urchin larvae. Another finding of our study is that the dopamine treatment also affects the growth of the body rods and the overall larval body growth. Based on these observations, we suggest researchers to carefully select the developmental stage, pharmacological agents, and incubation time for experimental manipulation of sea urchin larvae phenotypes through dopaminergic nervous system.

The oxygen environment in African clawed frogs (Xenopus laevis) continuously changes during their development, which involves a rapid increase in the body size, metamorphosis, and transition to adulthood. Nevertheless, there are limited reports on experimental models that are available for studying fluctuations in the oxygen environment in X. laevis. Thus, this study aimed to develop an experimental model on intermittent hypoxia in X. laevis and evaluate hypoxia and oxidative stress in the same. X. laevis were submerged in water with a dissolved oxygen concentration of 2 mg/L for 30 min; they were then removed from the water and allowed to freely absorb oxygen for 5 min. Immunostaining of pimonidazole-containing frozen tissue sections of the lung and liver using anti-pimonidazole antibodies as the hypoxia probes revealed that more than 95% of the submerged X. laevis cells were pimonidazole positive, providing direct evidence of tissue hypoxia. When the amount of oxidative stress in the lungs and liver was evaluated in terms of the amount of lipid peroxides, the diving group showed a 2.08-fold and 3.20-fold increase over the normal group, respectively. Following hypoxia exposure, the dry-to-wet weight ratios of the lung tissues was 1.27 times higher (p < .05), while the liver tissues was 1.06 times higher (although not significant). Thus, the degree of damage depended on the tissues affected. In the future, we believe that this model will be a promising option for analyzing the physiological responses of X. laevis to hypoxia and oxidative stress.

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.

Three-dimensional (3D) registration (i.e., alignment) between two microscopic images is very helpful to study tissues that do not adhere to substrates, such as mouse embryos and organoids, which are often 3D rotated during imaging. However, there is no 3D registration tool easily accessible for experimental biologists. Here we developed an ImageJ-based tool which allows for 3D registration accompanied with both quantitative evaluation of the accuracy and reconstruction of 3D rotated images. In this tool, several landmarks are manually provided in two images to be aligned, and 3D rotation is computed so that the distances between the paired landmarks from the two images are minimized. By simultaneously providing multiple points (e.g., all nuclei in the regions of interest) other than the landmarks in the two images, the correspondence of each point between the two images, i.e., to which nucleus in one image a certain nucleus in another image corresponds, is quantitatively explored. Furthermore, 3D rotation is applied to one of the two images, resulting in reconstruction of 3D rotated images. We demonstrated that this tool successfully achieved 3D registration and reconstruction of images in mouse pre- and post-implantation embryos, where one image was obtained during live imaging and another image was obtained from fixed embryos after live imaging. This approach provides a versatile tool applicable for various tissues and species.

The Drosophila visual center shows columnar structures, basic structural and functional units of the brain, that are shared with the mammalian cerebral cortex. Visual information received in the ommatidia in the compound eye is transmitted to the columns in the brain. However, the developmental mechanisms of column formation are largely unknown. The Irre Cell Recognition Module (IRM) proteins are a family of immunoglobulin cell adhesion molecules. The four Drosophila IRM proteins are localized to the developing columns, the structure of which is affected in IRM mutants, suggesting that IRM proteins are essential for column formation. Since IRM proteins are cell adhesion molecules, they may regulate cell adhesion between columnar neurons. To test this possibility, we specifically knocked down IRM genes in columnar neurons and examined the defects in column formation. We developed a system that automatically extracts the individual column images and quantifies the column shape. Using this system, we demonstrated that IRM genes play critical roles in regulating column shape in a core columnar neuron, Mi1. We also show that their expression in the other columnar neurons, Mi4 and T4/5, is essential, suggesting that the interactions between IRM proteins and multiple neurons shape the columns in the fly brain.

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10–500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.

Maternal microchimerism (MMc) is the phenomenon that a low number of cells from the mother persists within her progeny. Despite their regular presence in mammalian pregnancies, the overall cell type repertoire and roles of maternal cells, especially after birth, remain unclear. By using transgenic mouse strains and human umbilical blood samples, recent studies have for the first time characterized and quantified MMc cell type repertoires in offspring, identified the cross-generational influence on fetal immunity, and determined possible factors that affect their presence in offspring. This review summarizes new findings, especially on the maternal cell type repertoires and their potential role in utero, in postnatal life, and long after birth.

The cortical bones of mammals, birds, and reptiles are composed of a complex of woven bone and lamellar bone (fibrolamellar bone) organized into a variety of different patterns; however, it remains unclear whether amphibians possess similar structures. Importantly, to understand the evolutionary process of limb bones in tetrapods, it is necessary to compare the bone structure of amphibians (aquatic to terrestrial) with that of amniotes (mostly terrestrial). Therefore, this study compared the cortical bones in the long bones of several frog species before and after metamorphosis. Using micro-computed tomography (CT), we found that the cortical bones in the fibrolamellar bone of Xenopus tropicalis (Pipoidea superfamily) and Lithobates catesbeianus (Ranoidea superfamily) froglets are dense, whereas those of Ceratophrys cranwelli (Hyloidea superfamily) are porous. To clarify whether these features are common to their superfamily or sister group, four other frog species were examined. Histochemical analyses revealed porous cortical bones in C. ornata and Lepidobatrachus laevis (belonging to the same family, Ceratophryidae, as C. cranwelli). However, the cortical bones of Dryophytes japonicus (Hylidae, a sister group of Ceratophryidae in the Hyloidea superfamily), Microhyla okinavensis (Microhylidae, independent of the Hyloidea superfamily), and Pleurodeles waltl, a newt as an outgroup of anurans, are dense with no observed cavities. Our findings demonstrate that at least three members of the Ceratophryidae family have porous cortical bones similar to those of reptiles, birds, and mammals, suggesting that the process of fibrolamellar bone formation arose evolutionarily in amphibians and is conserved in the common ancestor of amniotes.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.